Treatment or preventative agents for HTLV-1-related myelopathy (HAM), and treatment methods for HAM.

Abstract

The present invention provides a treatment or preventive agent for HTLV-1-associated myelopathy (HAM) comprising an RGMa inhibitor; and a treatment method for HAM comprising administering a pharmacologically effective amount of the RGMa inhibitor to a patient with HTLV-1-associated myelopathy (HAM) who requires it.

Description

Technical Field

[0001] The present invention relates to a therapeutic or preventive agent for HTLV-1 associated myelopathy (HAM). The present invention also relates to a method for treating HAM. Prior Art

[0002] HTLV-1 (Human T-cell Lukemia Virus Type 1) is a virus that infects T cells (mainly CD4 positive T cells), one of the white blood cells in the blood. T cells infected with HTLV-1 cause chronic inflammation in the spinal cord, which in turn causes damage and degeneration of spinal nerve cells, leading to spastic myelopathy. Spastic myelopathy caused by cells infected with HTLV-1 is called HTLV-1-associated myelopathy (HAM). Symptoms of HAM include numbness in the legs, pain, urinary difficulties, and persistent constipation caused by nerve damage. If these symptoms progress, patients may become wheelchair-bound or bedridden. HAM is designated as a designated intractable disease in Japan. Currently, there is no established effective treatment for HAM; current treatments primarily focus on symptomatic treatment. As one of the treatment methods, the use of anti-CCR4 antibodies has been shown to reduce HTLV-1 infected cells, alleviate spinal inflammation in HAM, and improve symptoms (Non-Patent Document 1, Patent Document 5).

[0003] RGM is a member of the RGM (repulsive guidance molecule) protein family, involved in axon guidance of retinal and hippocampal neurons, neural tube closure, and other functions. RGM is not limited to these functions and is known to have a variety of other functions.

[0004] For example, Patent Document 1 discloses that bone marrow-derived dendritic cells (BMDCs) express RGM, and CD4+ T cells and CD11b+ macrophages express RGM receptors. Binding of RGM to these RGM receptors enhances the cell adhesion activity of CD4+ T cells and CD11b+ macrophages. Furthermore, Patent Document 1 discloses that neutralizing anti-RGM antibodies can simultaneously alleviate clinical symptoms and tissue lesions in multiple sclerosis model mice, attenuating both antigen-specific and nonspecific T cell activation in spleen cells isolated from these mice.

[0005] Patent Documents 2 and 3 disclose neutralizing monoclonal antibodies against RGMa that selectively inhibit the binding of RGMa to its receptors, regenerative protein and bone morphogenetic proteins 2 and 4 (BMP-2 and BMP-4). These neutralizing monoclonal antibodies are believed to promote neural regeneration and regrowth in damaged and inflamed human central nervous systems, specifically in neurodegenerative diseases such as multiple sclerosis, acute spinal cord injury, post-traumatic brain syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

[0006] Patent Document 4 discloses that RGMa is concentrated in the myelin, fresh lesions, and mature scar tissue of the central nervous system of people who have suffered traumatic brain injury or ischemic stroke, and discloses a method for detecting and quantifying RGMa fragments for the purpose of diagnosing these neurodegenerative diseases. Furthermore, Patent Document 4 lists as targets for the detection of RGMa fragments neurodegenerative diseases and disorders multiple sclerosis, Parkinson's disease, Alzheimer's disease, Tay-Sachs disease, Niemann-Pick disease, Gaucher disease, Hurler syndrome, Huntington's disease, amyotrophic lateral sclerosis, idiopathic inflammatory demyelinating disease, vitamin B12 deficiency, central pontine myelinolysis, tabes dorsalis, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis, spinal cord injury, traumatic brain injury, stroke, glaucoma, diabetic retinopathy, age-related macular degeneration, and leukodystrophy. Prior Art Literature Patent Literature

[0007] Patent Document 1: International Publication No. 2011 / 071059 Patent Document 2: Japanese Patent Application Laid-Open No. 2014-138599 Patent Document 3: Japanese Patent Application Laid-Open No. 2016-175897 Patent Document 4: Japanese Patent No. 2017-526930 Patent Document 5: Japanese Patent Laid-Open No. 2010-100578 Non-patent literature

[0008] Non-patent literature 1: N Engl J Med, 2018, 378, 529-538. Summary of the Invention

[0009] [Problems to be solved by the invention]

[0010] While the methods disclosed in Non-Patent Document 1 and Patent Document 5 using anti-CCR4 antibodies can improve HAM symptoms, their effectiveness is limited for HAM patients whose nerve damage has progressed. Therefore, a better method for treating HAM is sought.

[0011] The present invention has been made in view of the above-mentioned problems, and its object is to provide a therapeutic agent and a therapeutic method capable of treating HAM. [Technical means to solve the problem]

[0012] To solve the above-mentioned problems, the present inventors conducted repeated and intensive research and found that substances that inhibit RGMa are effective in treating HAM, thereby completing the present invention.

[0013] That is, the present invention is as follows. [1] A therapeutic or preventive agent for HTLV-1 associated myelopathy (HAM), comprising an RGMa inhibitory substance. [2] The therapeutic or preventive agent for HAM as described in [1], wherein the RGMa inhibitory substance is an antibody that recognizes RGMa. [3] A method for treating HAM comprises administering a pharmacologically effective amount of an RGMa inhibitory substance to a patient with HTLV-1 associated myelopathy (HAM) in need thereof. [4] The method for treating HAM as described in [3], wherein the RGMa inhibitory substance is an antibody that recognizes RGMa. [Effects of the Invention]

[0014] According to the present invention, HAM, which is an intractable disease, can be treated. Simple diagram description

[0015] FIG1 shows the P, D, and N groups obtained by classifying CD4-positive T cells based on CADM1 and CD7, which are indicators of HTLV-1 infected cells. Figure 2 shows the expression levels of the RGMa gene in normal T cells (Normal.CD4), CD4-positive T cells from HAM patients (HAM.CD4), CD4-positive / CADM1-negative / CD7-positive T cells from healthy individuals (Normal.P), CD4-positive / CADM1-negative / CD7-positive T cells from HTLV-1-infected individuals (Group P), CD4-positive / CADM1-positive / CD7-positive T cells from HTLV-1-infected individuals (Group D), CD4-positive / CADM1-positive / CD7-negative T cells from HTLV-1-infected individuals (Group N), CD4-positive / CADM1-positive / CD7-negative T cells from acute ATL patients (Acute.N), CD4-positive T cells from healthy individuals (Normal.CD4.1), PBMCs (Peripheral Blood Mononuclear Cells) from smoldering ATL patients (Smoldering), PBMCs from chronic ATL patients (Chronic), and PBMCs from acute ATL patients (Acute). FIG3 is a graph showing the expression levels of RGMa in CD4-positive T cells of HAM patients and healthy controls. FIG4 is a graph showing the results of analysis of RGMa expression levels among different cell types of PBMCs from HAM patients. FIG5 is a graph showing changes in RGMa expression accompanying the expression of HTLV-1 virus in culture of PBMC from HAM patients. FIG6 is a graph showing the H3K27me3 level near -2,916 bp upstream of the transcription start site of the RGMa gene. FIG7 is a graph showing the level of RGMa gene mRNA when a lentiviral vector carrying cDNA encoding HTLV-1 Tax was introduced into the human CD4-positive T-cell leukemia cell line Jurkat. FIG8 is a graph showing the analysis results of Tax and RGMa protein expression in the HTLV-1-tax expression-induced cell line JPX-9. [ Fig. 9 ] is a graph showing the results of the effects of RGMa antibodies on PBMCs from HAM patients and the effects of RGMa antibodies on spontaneous proliferation activity. FIG. 10 is a graph showing the results of the effect of RGMa antibody on PBMCs of HAM patients and the effect of RGMa antibody on changes in the amount of HTLV-1 provirus. FIG. 11 is a graph showing the effects of RGMa antibodies on PBMCs from HAM patients and the results of the effects of RGMa antibodies on the production of CXCL10 (CXC motif chemokine 10). FIG. 12 is a graph showing the results of the effects of RGMa antibodies on PBMCs from HAM patients and the effects of RGMa antibodies on cytokines produced by PBMCs from HAM patients. FIG. 13 is a graph showing the induction of apoptosis in neural cell lines by HAM-PBMC. Figure 14 shows the results of the inhibitory effect of RGMa antibodies on apoptosis in neural cell lines induced by HTLV-1-tax. (a) A FACS (Fluorescence Activated Cell Sorting) image of NB-1 cells co-cultured with JPX-9 cells (unstimulated). (b) A FACS image of NB-1 cells co-cultured with CdCl₂-stimulated JPX-9 cells (HTLV-1-tax expressing cells). (c) A FACS image of the cells co-cultured with a control antibody under the conditions described in (b). (d) A FACS image of the cells co-cultured with an anti-RGMa antibody under the conditions described in (b). Implementation Method

[0016] Hereinafter, the embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and various modifications can be made within the scope of the gist of the present invention.

[0017] The present invention is a therapeutic or preventive agent for HTLV-1 associated myelopathy (HAM) comprising an RGMa inhibitory substance. The RGMa inhibitory substance may act on RGMa itself to inhibit RGMa activity, or may inhibit RGMa expression. Examples of RGMa inhibitory substances include compounds having an activity of inhibiting RGMa and antibodies that recognize RGMa.

[0018] Furthermore, examples of RGMa inhibitory substances include substances that inhibit the expression of RGMa, such as siRNA (short interfering RNA), shRNA (short hairpin RNA), and antisense oligonucleotides, which are directed against the gene expressing RGMa. Examples of RGMa genes include, but are not limited to, the human RGMa gene comprising the base sequence shown in SEQ ID NO: 1 and the RGMa gene comprising the base sequence shown in SEQ ID NO: 2. Information on the base sequences of RGMa genes from various organisms can be obtained from databases such as GenBank.

[0019] siRNA is a double-stranded RNA that can inhibit the expression of the target RGMa gene. The length of the base sequence in the siRNA is not particularly limited, but is preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases. shRNA refers to a molecule of approximately 20 base pairs or more, consisting of a double-stranded structure formed by partially containing a palindromic base sequence within a single-stranded RNA. It also has a small hairpin structure at the 3' end that overhangs the shRNA. After introduction into cells, shRNA is broken down into approximately 20 bases in length and can, like siRNA, inhibit the expression of the target RGMa gene.

[0020] siRNA and shRNA can be artificially synthesized chemically. Alternatively, siRNA and shRNA can be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and a T7 promoter to generate antisense and sense RNA.

[0021] Antisense oligonucleotides can be complementary to or hybridize with a contiguous sequence of approximately 30 bases or less within the DNA sequence of the RGMa gene. They can be either DNA or RNA. Furthermore, modified forms are acceptable as long as they do not impair the function of the gene. Antisense oligonucleotides can be synthesized by conventional methods, for example, using commercially available DNA synthesis equipment.

[0022] The RGMa inhibitory substance included in the therapeutic or preventive agent for HAM of the present invention is preferably an antibody that recognizes RGMa. Hereinafter, an antibody that recognizes RGMa is also referred to as an RGMa antibody. The RGMa antibody herein can be any antibody that binds to RGMa and inhibits its activity. Examples include antibodies that bind to RGMa and prevent RGMa from binding to RGMa receptors.

[0023] The RGMa antibodies of the present invention may be monoclonal or polyclonal antibodies. Furthermore, the antibodies of the present invention may be of any isotype, including IgG, IgM, IgA, IgD, and IgE. The RGMa antibodies of the present invention may be, for example, mouse antibodies, human CDR-grafted antibodies, human chimeric antibodies, humanized antibodies, or fully human antibodies, or may be low-molecular-weight antibodies. These antibodies may be used alone or in combination of two or more.

[0024] Humanized CDR-grafted antibodies are antibodies created by replacing the CDRs of non-human antibodies with those of human antibodies. Humanized chimeric antibodies are antibodies that contain the variable regions of non-human antibodies and the constant regions of human antibodies. Furthermore, humanized antibodies are antibodies created by retaining a safer portion of a non-human antibody and incorporating a portion from a human antibody. This concept encompasses both humanized chimeric antibodies and humanized CDR-grafted antibodies.

[0025] In this specification, the term "low-molecular-weight antibody" refers to an antibody fragment or an antibody fragment bound to an arbitrary molecule, and recognizes the same epitope as the original antibody. Specifically, examples include: Fab, which comprises the VL, VH, CL, and CH1 regions; F(ab')2, which is two Fabs linked by a disulfide bond at the hinge region; Fv, which comprises VL and VH; single-chain antibodies (scFv), which link VL and VH via an artificial polypeptide linker; sdFv, diabodies, and sc(Fv)2, but are not limited to these.

[0026] The RGMa antibody used in the present invention can be prepared by known methods using RGMa or a fragment thereof as an immunogen. RGMa activity can be used as an indicator to confirm that the obtained antibody is an RGMa antibody. Examples of RGMa include human RGMa comprising the amino acid sequence shown in SEQ ID NO: 3 and RGMa comprising the amino acid sequence shown in SEQ ID NO: 4. RGMa derived from various organisms can be used as immunogens. The amino acid sequence of RGMa can be obtained from publicly known databases such as the Protein Data Bank.

[0027] When the RGMa antibodies used in the present invention are polyclonal antibodies, they can be produced, for example, by the following method. First, the antigen RGMa or fragments thereof are dissolved in phosphate-buffered saline (also known as PBS), optionally mixed with an appropriate amount of a conventional adjuvant, such as Freund's complete adjuvant. The resulting mixture is used as an immunogen to immunize mammals such as mice, rats, rabbits, sheep, and horses. The immunization method is not particularly limited; examples include single subcutaneous or intraperitoneal injections or two or more injections at appropriate intervals. Next, blood is collected from the immunized animal according to conventional methods, the serum is separated, and the polyclonal antibody fraction is purified to obtain the antibody. When the RGMa antibody used in the present invention is a monoclonal antibody, it can be obtained by fusing immune cells, such as spleen cells, obtained from the immunized mammal described above with myeloma cells to produce a fusion tumor, and then collecting the antibody from the culture of the fusion tumor. Alternatively, the monoclonal antibody can be produced by selecting an antibody gene from the fusion tumor, integrating it into a suitable vector, and introducing it into a host cell using genetic recombination techniques to produce a recombinant monoclonal antibody. Furthermore, the monoclonal antibody can also be produced using phage display methods.

[0028] As the RGMa antibody used in the present invention, for example, antibodies disclosed in Yamashita, T., Mueller, BK & Hata, K. Neogenin and repulsive guidance molecule signaling in the central nervous system. Curr. Opin. Neurobiol. 17, 29-34 (2007); Japanese Patent Publication No. 2014-138599; Japanese Patent Publication No. 2016-175897; Japanese Patent Publication No. 2017-526930; International Publication No. 2016 / 175236; Japanese Patent Publication No. 2015-508061, etc. can be used. Furthermore, the RGMa antibody used in the present invention can also be obtained as a commercial product, for example, from Immuno-Biological Research Institute (IBL) Co., Ltd. or R&D Systems.

[0029] The RGMa antibody preferably contains a GTTPDY (SEQ ID NO: 7); FQATHDPLT (SEQ ID NO: 10); ARRNEYYGSSFFDY (SEQ ID NO: 13); LQGYIPPRT (SEQ ID NO: 16); and A modified CDR amino acid sequence having at least 50% sequence identity with one of said sequences; At least one CDR of the amino acid sequence in the group thus formed serves as an antigen-binding region. The sequence identity is preferably 80% or greater, more preferably 90% or greater. Furthermore, in this specification, amino acids are sometimes represented using conventional one-letter or three-letter notation.

[0030] The complementarity-determining region (CDR) refers to the region of the variable region of an immunoglobulin molecule that forms the antigen-binding site, also known as the hypervariable region. It is the portion of the immunoglobulin molecule where the amino acid sequence varies significantly. Regarding CDRs, the light chain and heavy chain each have three CDRs (CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3). In this application, the CDRs of immunoglobulin molecules are identified according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, NIH, USA). Furthermore, in the antibodies defined by the amino acid sequences of the light and heavy chains of the present invention, the amino acid sequences of the CDRs remain unchanged, while the amino acid sequences outside the CDRs may be altered by mutations, etc. When mutations, etc., exist outside the CDRs, preferably, the homology is 90% or greater.

[0031] Furthermore, the RGMa antibody preferably further comprises at least one CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 5, 6, 8, 9, 11, 12, 14, and 15, and modified CDR amino acid sequences having at least 50% sequence identity with any of these sequences. The sequence identity is preferably 80% or greater, more preferably 90% or greater.

[0032] The RGMa antibody preferably comprises at least three CDRs selected from the variable region CDR sets listed in Table 1, or a variable region set in which at least one of the three CDRs is a modified CDR amino acid sequence having at least 50%, preferably 80%, and even more preferably 90% sequence identity with the parent sequence. Furthermore, the RGMa antibody preferably comprises at least two variable region CDR sets listed in Table 1. Furthermore, the at least two variable region CDR sets are preferably a combination of the VH5F9 set and the VL5F9 set, or a combination of the VH8D1 set and the VL8D1 set.

[0033] [Table 1] VH5F9 group VH5F9 CDR-H1 Residues 31-35 of SEQ ID NO: 17 Sequence number 5 VH5F9 CDR-H2 Residues 50-66 of SEQ ID NO: 17 Sequence number 6 VH5F9 CDR-H3 Residues 99-104 of SEQ ID NO: 17 Sequence number 7 VL5F9 group VL5F9 CDR-L1 Residues 24-39 of SEQ ID NO: 18 Sequence number 8 VL5F9 CDR-L2 Residues 55-61 of SEQ ID NO: 18 Sequence number 9 VL5F9 CDR-L3 Residues 94-102 of SEQ ID NO: 18 Sequence number 10 VH8D1 group VH8D1 CDR-H1 Residues 31-35 of SEQ ID NO: 19 Sequence number 11 VH8D1 CDR-H2 Residues 50-66 of SEQ ID NO: 19 Sequence number 12 VH8D1 CDR-H3 Residues 97-110 of SEQ ID NO: 19 Sequence number 13 VL8D1 group VL8D1 CDR-L1 Residues 24-34 of SEQ ID NO: 20 Serial number 14 VL8D1 CDR-L2 Residues 50-56 of SEQ ID NO: 20 Sequence number 15 VL8D1 CDR-L3 Residues 89-97 of SEQ ID NO: 20 Sequence number 16

[0034] RGMa antibodies may also contain a framework region. Examples of amino acid sequences included in the framework region include SEQ ID NOs. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40. These amino acid sequences may be present alone or in combination of two or more. Furthermore, the RGMa antibody preferably contains at least one heavy chain variable region selected from SEQ ID NOs. 41, 42, 43, 44, 45, 46, 47, 48 and 49 and / or at least one light chain variable region selected from SEQ ID NOs. 50, 51 and 52 as the heavy chain variable region and light chain variable region.

[0035] Furthermore, the binding site of the RGMa antibody when binding to RGMa is not particularly limited as long as it is a site that causes RGMa inhibition. For example, in the case of human RGMa, it is preferably bound to a site having EEVVNAVEDWDSQG (SEQ ID NO: 53) NQQIDFQAFHTNAE (SEQ ID NO: 54) PTAPETFPYET (SEQ ID NO: 55) KLPVEDLYYQA (SEQ ID NO: 56) LYERTRDLPGRAAAGL (SEQ ID NO: 57) One or more peptides of the indicated amino acid sequence. The RGMa antibody preferably binds to a peptide having the amino acid sequence shown in sequence number 53 and / or sequence number 54, and more preferably binds to a peptide having the amino acid sequence shown in sequence number 53 and / or sequence number 54, and a peptide having the amino acid sequence shown in sequence number 55 and / or sequence number 56. The RGMa antibody is preferably an antibody that binds to amino acid sequence 250 and later in the human RGMa. More preferably, the RGMa antibody binds to a peptide having SEQ ID NO: 53 and SEQ ID NO: 54, and having the amino acid sequence shown in SEQ ID NO: 55 or SEQ ID NO: 56.

[0036] RGMa antibodies can be polyclonal antibodies and monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or a partial fragment thereof (e.g., a fragment containing one or more of sequence numbers 53 to 57) as an antigen, chimeric antibodies and humanized antibodies produced using genetic recombination technology, and human antibodies produced using genetically modified animals that produce human antibodies. In the present invention, when RGMa antibodies are administered to humans as a medicine, humanized antibodies or human antibodies are more desirable from the perspective of side effects. RGMa antibodies can also be used as partial fragments of polyclonal antibodies and / or monoclonal antibodies that recognize RGMa protein or its partial fragments (e.g., fragments containing one or more of sequence numbers 53 to 57) as antigens, and can also be low-molecular-weight antibodies.

[0037] The RGMa antibody may be, in addition to those shown in Table 1, the amino acid sequences of the light chain complementary determining region 1 (LCDR1), the light chain complementary determining region 2 (LCDR2), the light chain complementary determining region 3 (LCDR3), the heavy chain complementary determining region 1 (HCDR1), the heavy chain complementary determining region 2 (HCDR2), and the heavy chain complementary determining region 3 (HCDR3) respectively comprise: LCDR1:RASQDISSYLN (SEQ ID NO. 58) LCDR2:YTSRLHS (SEQ ID NO: 59) LCDR3:QQLNTLP (SEQ ID NO: 60) HCDR1:DAWMD (SEQ ID NO: 61) HCDR2: EIRSKANNHATYYAESVKG (SEQ ID NO: 62) and HCDR3: RDGAY (SEQ ID NO: 63), or comprising LCDR1:RSSQSLVHSNGNTYLH (SEQ ID NO: 64) LCDR2:KVSNRFS (SEQ ID 65) LCDR3:SQSTHVP (SEQ ID NO: 66) HCDR1:TSYYWN (SEQ ID NO: 67) HCDR2: YISYDGTNNYNPSLKN (SEQ ID NO: 68) and HCDR3:SFG Isolated RGMa antibody, or an antigen-binding fragment thereof. In each CDR sequence, one or several amino acids may be substituted, deleted, and / or added, for example, one or two amino acids may be substituted, deleted, and / or added.

[0038] Examples of RGMa antibodies include those having the amino acid sequence of SEQ ID NO: 73 in the light chain and the amino acid sequence of SEQ ID NO: 74 in the heavy chain. The amino acid sequences represented by these sequence numbers may have one or several amino acid substitutions, deletions, additions, or insertions (1 to 20, 1 to 10, or 1 to 5). Such substitutions, deletions, and additions may be introduced into the CDRs, but are preferably introduced into regions outside the CDRs.

[0039] The constant regions may be mouse / human chimeric antibodies derived from humans. Examples of mouse / human chimeric antibodies include those having the amino acid sequence of SEQ ID NO: 77 in the light chain (amino acids 1 to 107 in the variable region) and the amino acid sequence of SEQ ID NO: 78 in the heavy chain (amino acids 1 to 116 in the variable region). The amino acid sequences represented by these sequence numbers may have one or more amino acid substitutions, deletions, additions, or insertions (1 to 20, 1 to 10, or 1 to 5). Such substitutions, deletions, and additions may be introduced into the CDRs, but are preferably introduced into regions outside the CDRs.

[0040] Humanized antibodies, excluding CDRs, may be derived from humans. Examples of humanized antibodies include those with amino acid sequences from SEQ ID NOs. 70 to 87 (from the N-terminal end to residue 116 in the variable region) in the heavy chain and from SEQ ID NOs. 88 to 94 (from the N-terminal end to residue 107 in the variable region) in the light chain. The amino acid sequences represented by these sequence numbers may contain substitutions, deletions, additions, or insertions of one or more amino acids (1 to 20, 1 to 10, or 1 to 5). Such substitutions, deletions, and additions may be introduced into the CDRs, but are preferably introduced into regions outside the CDRs.

[0041] The heavy chain amino acid sequence and light chain amino acid sequence can be any combination thereof, but preferably, the antibody has the amino acid sequence of SEQ ID NO: 84 in the heavy chain and the amino acid sequence of SEQ ID NO: 88 in the light chain. The amino acid sequence of SEQ ID NO: 84 corresponding to the heavy chain variable region is represented by SEQ ID NO: 95, and the amino acid sequence corresponding to the light chain variable region is represented by SEQ ID NO: 96.

[0042] As RGMa antibody, preferably the heavy chain variable region (VH) comprises EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDW VRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS (serial number 95) or An amino acid sequence having at least 90% identity with the amino acid sequence, the light chain variable region (VL) comprising DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 96) or An isolated RGMa antibody, or an antigen-binding fragment thereof, having an amino acid sequence that is at least 90% identical to the amino acid sequence.

[0043] Examples of RGMa antibodies include those having the amino acid sequence of SEQ ID NO: 75 in the light chain and the amino acid sequence of SEQ ID NO: 76 in the heavy chain. The amino acid sequences represented by these sequence numbers may have one or several amino acid substitutions, deletions, additions, or insertions (1 to 20, 1 to 10, or 1 to 5). Such substitutions, deletions, and additions may be introduced into the CDRs, but are preferably introduced into regions outside of the CDRs. It can be a mouse / human chimeric antibody in which the constant region is derived from human, or a humanized antibody in which all parts except the CDR are derived from human.

[0044] Furthermore, the anti-RGMa antibody may be an isolated RGMa antibody selected from (a1) to (h1) below, or an antigen-binding fragment thereof. (a1) an anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising LCDR1 comprising the amino acid sequence recorded in sequence number 97, LCDR2 comprising the amino acid sequence recorded in sequence number 98, and LCDR3 comprising the amino acid sequence recorded in sequence number 99, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence recorded in sequence number 100, HCDR2 comprising the amino acid sequence recorded in sequence number 101, and HCDR3 comprising the amino acid sequence recorded in sequence number 102; (b1) an anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising an LCDR1 comprising the amino acid sequence recorded in sequence number 97, an LCDR2 comprising the amino acid sequence recorded in sequence number 98, and an LCDR3 comprising the amino acid sequence recorded in sequence number 103, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence recorded in sequence number 100, an HCDR2 comprising the amino acid sequence recorded in sequence number 101, and an HCDR3 comprising the amino acid sequence recorded in sequence number 102; (c1) an anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising an LCDR1 comprising the amino acid sequence recorded in sequence number 97, an LCDR2 comprising the amino acid sequence recorded in sequence number 98, and an LCDR3 comprising the amino acid sequence recorded in sequence number 104, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence recorded in sequence number 100, an HCDR2 comprising the amino acid sequence recorded in sequence number 101, and an HCDR3 comprising the amino acid sequence recorded in sequence number 102; (d1) an anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising an LCDR1 comprising the amino acid sequence recorded in sequence number 97, an LCDR2 comprising the amino acid sequence recorded in sequence number 98, and an LCDR3 comprising the amino acid sequence recorded in sequence number 105, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence recorded in sequence number 100, an HCDR2 comprising the amino acid sequence recorded in sequence number 101, and an HCDR3 comprising the amino acid sequence recorded in sequence number 102; (e1) an anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising an LCDR1 comprising the amino acid sequence recorded in sequence number 97, an LCDR2 comprising the amino acid sequence recorded in sequence number 98, and an LCDR3 comprising the amino acid sequence recorded in sequence number 106, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence recorded in sequence number 100, an HCDR2 comprising the amino acid sequence recorded in sequence number 101, and an HCDR3 comprising the amino acid sequence recorded in sequence number 102; (f1) an anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising an LCDR1 comprising the amino acid sequence recorded in sequence number 97, an LCDR2 comprising the amino acid sequence recorded in sequence number 98, and an LCDR3 comprising the amino acid sequence recorded in sequence number 107, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence recorded in sequence number 100, an HCDR2 comprising the amino acid sequence recorded in sequence number 101, and an HCDR3 comprising the amino acid sequence recorded in sequence number 102; (g1) an anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising LCDR1 comprising the amino acid sequence recorded in sequence number 97, LCDR2 comprising the amino acid sequence recorded in sequence number 98, and LCDR3 comprising the amino acid sequence recorded in sequence number 108, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence recorded in sequence number 100, HCDR2 comprising the amino acid sequence recorded in sequence number 101, and HCDR3 comprising the amino acid sequence recorded in sequence number 102; and (h1) An anti-RGMa antibody or an antigen-binding fragment thereof comprising: a light chain variable region comprising LCDR1 comprising the amino acid sequence recorded in sequence number 97, LCDR2 comprising the amino acid sequence recorded in sequence number 98, and LCDR3 comprising the amino acid sequence recorded in sequence number 109, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence recorded in sequence number 100, HCDR2 comprising the amino acid sequence recorded in sequence number 101, and HCDR3 comprising the amino acid sequence recorded in sequence number 102.

[0045] The RGMa antibody is preferably a humanized antibody having the above-mentioned amino acid sequence, and more preferably has a human IgG constant region.

[0046] HAM can be treated or prevented by the RGMa inhibitory substance of the present invention. As described in the Examples below and shown in FIG13 , in HAM patients, cells derived from HAM patients induce neuronal cell death, ie, induce spinal cord tissue damage and degeneration. The present inventors conducted research and, as shown in the examples below, found that RGMa was significantly expressed in CD4-positive T cells, the primary HTLV-1-infected cells in HAM patients, demonstrating a correlation between HAM and RGMa expression. Furthermore, RGMa inhibition experiments using RGMa antibodies demonstrated that RGMa is associated with the production of CXCL10, IL-2, and IL-10. CXCL10 is a protein produced by T cells infected with HTLV-1, the pathogenic form of HAM, in response to IFN-γ (interferon-γ), inducing HAM symptoms (Brain 2013). It is also known to be closely related to the progression of HAM symptoms (PLoS Negl Trop Dis 2013). RGMa antibodies inhibit CXCL10 production in cells from HAM patients. IL-10 is a cytokine that suppresses inflammation. RGMa antibodies significantly increase IL-10 production in cells from HAM patients. To achieve a true therapeutic effect on HAM, it is necessary to inhibit neuronal damage. It has been shown that HTLV-1-tax expression levels are elevated in HTLV-1-infected cells from HAM patients (Blood 2002), and that HTLV-1-tax plays a key role in the development of the disease (J Clin Invest 2014). The present invention demonstrates that HTLV-1-tax induces RGMa expression, and that cells derived from HAM patients with elevated RGMa expression induce neuronal cell death. It is shown that RGMa-inhibiting substances are crucial for inhibiting neuronal cell death induced by HTLV-1-tax-expressing cells. As described above, RGMa inhibitors can inhibit the induction of inflammatory symptoms in HAM and the inflammatory response induced by HAM patient cells, thereby treating or preventing HAM. Furthermore, RGMa inhibitors not only inhibit the inflammatory response specific to HAM but also inhibit neuronal cell death induced by HAM patient pathogenic cells, namely HTLV-1-tax expressing cells, thereby treating or preventing HAM.

[0047] As described above, RGMa inhibitory substances can treat or prevent HAM. The present invention provides: an RGMa inhibitory substance for treating HAM; a pharmaceutical composition for treating HAM; a use of an RGMa inhibitory substance for treating HAM; a use of an RGMa inhibitory substance for manufacturing a therapeutic drug for HAM; an RGMa inhibitory substance for manufacturing a therapeutic drug for HAM; and a method for treating or preventing HAM, comprising administering an effective amount of an RGMa inhibitory substance to a subject in need thereof.

[0048] The therapeutic or preventive agent for HAM of the present invention comprises an RGMa inhibitory substance, and can be formulated by appropriately formulating with pharmaceutically acceptable carriers and / or additives. Examples of the formulation include oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; and parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The mixing ratio of the carrier or additive can be appropriately set based on the range commonly used in the pharmaceutical field. The carrier is not particularly limited, and examples thereof include water, physiological saline, other aqueous solvents, and aqueous or oily bases. The additives are not particularly limited, and examples thereof include excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavoring agents, and fragrances.

[0049] When the RGMa inhibitory substance in the present invention is an antibody that recognizes RGMa, it is preferably formulated with a pharmaceutically acceptable carrier to form an injection or infusion, and administered via a non-oral route, such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or local administration. Injections or infusions containing RGMa antibodies can be used in the form of solutions, suspensions, or emulsions. Examples of solvents include distilled water for injection, physiological saline, glucose solutions, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). These solvents can be used alone or in combination of two or more.

[0050] The injection or infusion may further contain additives such as stabilizers, solubilizers, suspending agents, emulsifiers, analgesics, buffers, preservatives, antiseptics, and pH adjusters. As stabilizers, for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, butylated hydroxytoluene, etc. can be used. As solubilizing agents, for example, alcohols (such as ethanol), polyols (such as propylene glycol and polyethylene glycol), and nonionic surfactants (such as Polysorbate 80 (registered trademark) and HCO-50) can be used. As the suspending agent, for example, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. can be used. As the emulsifier, for example, gum arabic, sodium alginate, and gum tragacanth can be used. As analgesics, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. As the buffer, for example, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Trsi (Tris(hydroxymethyl)aminomethane) buffer, etc. can be used. As the preservative, for example, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc. can be used. As preservatives, for example, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol, etc. can be used. As the pH adjuster, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. can be used.

[0051] In the present invention, the RGMa inhibitory substance is a substance that inhibits the expression of RGMa, such as siRNA (small interfering RNA), shRNA (small hairpin RNA), and antisense oligonucleotide, which expresses the gene of RGMa. It can be administered in the form of a non-viral vector or a viral vector. When the RGMa inhibitory substance is in the form of a non-viral vector, examples of administration methods include methods for introducing nucleic acid molecules using liposomes (liposome method, HVJ (Hemagglutinating Virus of Japan)-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, and methods for introducing a vector (metal particle) and nucleic acid molecules into cells together using a gene gun. When using viral vectors to deliver siRNA or shRNA to organisms, recombinant adenoviruses, retroviruses, and other viral vectors can be used. DNA expressing siRNA or shRNA can be introduced into a detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, pox virus, pox virus, polio virus, Sindby virus, Sendai virus, SV40, or other DNA or RNA virus, and cells or tissues can be infected with the recombinant virus, thereby introducing the gene into the cells or tissues.

[0052] HAM can be prevented or treated by administering an effective amount of the preparation thus obtained to humans and other mammals such as rats, mice, rabbits, sheep, pigs, cattle, cats, dogs and monkeys. The dosage is appropriately set in consideration of the purpose, severity of the disease, age, weight, sex, medical history of the patient, type of active ingredient, etc. Example

[0053] Example 1: Comprehensive comparative analysis to clarify the pathogenesis and symptoms of HAM and adult T-cell leukemia / lymphoma (ALT) As shown in Figure 1 , for samples from HTLV-1 infected individuals without symptoms, mRNA expression analysis was performed on the CD4-positive cells in groups P, D, and N, which were obtained by classifying them based on CADM1 and CD7, which are indicators of HTLV-1 infected cells. Furthermore, for HAM, CD4-positive cells and CD4-negative cells were analyzed using magnetic beads from peripheral blood mononuclear cells enriched from HAM patients. Similar isolation was performed on healthy individuals (normal) without HTLV-1 infection. Peripheral blood mononuclear cells are also referred to as PBMCs. For ATL (smodesring, chronic, and acute), PBMCs primarily containing CD4-positive cells were used. A single-color microarray gene expression analysis method manufactured by Agilent Technologies was performed.

[0054] A comprehensive comparative analysis of neural-related molecules showed that RGMa was significantly expressed in CD4-positive cells of HAM patients. Targeting 4 normal T cells (Normal.CD4), 4 CD4-positive T cells from HAM patients (HAM.CD4), 3 CD4-positive / CADM1-negative / CD7-positive T cells from healthy individuals (Normal.P), 5 CD4-positive / CADM1-negative / CD7-positive T cells from HTLV-1 infected individuals (P group), 5 CD4-positive / CADM1-positive / CD7-positive T cells from HTLV-1 infected individuals (D group), 5 CD4-positive / CADM1-positive / CD7-negative T cells from HTLV-1 infected individuals (N group), 3 CD4-positive / CADM1-positive / CD7-negative T cells from acute ATL patients (Acute.N), 21 CD4-positive T cells from healthy individuals (Normal.CD4.1), 3 PBMCs from smoldering ATL patients (Smoldering), 20 PBMCs from chronic ATL patients (Chronic), and PBMCs from acute ATL patients. In 26 cases (Acute), full gene expression data were obtained using the Agilent Technologies Human Gene Expression 4×44K Microarray. After normalization by median value, RGMa gene levels were plotted. The Log 2 values ​​of fluorescence intensity from the array are plotted in the graph. The CD4-positive T cell group derived from HAM patients showed significantly higher fluorescence than all other groups (P < 0.05). This graph is shown in Figure 2.

[0055] [Example 2: Gene Expression Analysis in CD4-Positive HAM Patients] PBMCs were isolated from the peripheral blood of five HAM patients. CD4+ T cells were isolated from these PBMCs using a human CD4+ isolation kit (Miltenyi Biotec) to identify a population containing a high number of HTLV-1-infected cells. Similarly, CD4+ T cells were isolated from PBMCs of four healthy individuals as controls. Total RNA was recovered from the isolated CD4-positive T cells and cDNA was produced using ReverTra Ace (Toyobo). The resulting cDNA was used to analyze the difference in RGMa expression between CD4-positive T cells from HAM patients and healthy controls by real-time PCR (PCR). 18s rRNA was used as an internal control. The analysis results are shown in a graph in Figure 3. In the figure, HD-CD4+ refers to the control group, and HAM-CD4+ refers to the HAM patient group.

[0056] [Example 3: Analysis of RGMa-expressing cells in PBMC] After Fc block treatment of PBMCs from HAM patients using Clear Back (MBL), anti-CD3-PECy7 (TONBO), CD4-FITC (eBioscience), and CD14-PE (eBioscience) antibodies were added and stained at 4°C for 30 minutes. After antibody-stained PBMCs were washed, FACS sorting was performed using AriaIIIu (BD), and CD3-positive CD4-negative cells (CD3+CD4-), CD3-positive CD4-positive cells (CD3+CD4+), CD3-negative CD14-negative cells (CD3-CD14-) and CD3-negative CD14-positive cells (CD3-CD14+) were separated and recovered. Total RNA was recovered from each cell type and cDNA was prepared using ReverTra Ace (Toyobo). The resulting cDNA was used to analyze differences in RGMa expression between cell types by real-time PCR. 18s rRNA was used as an internal control. The analysis results are shown in Figure 4. It can be seen that in HAM-PBMC, the expression of RGMa is highest among CD3-positive CD4-positive cells (CD3+CD4+), which are a large number of infected cells.

[0057] [Example 4: Changes in RGMa expression associated with the culture of HAM patient PBMC] PBMCs from two HAM patients were suspended in culture medium (RPMI1640 medium (wako) containing 10% FBS (Fetal Bovine Serum) (GIBCO)) and seeded into 10 wells of a 96-well round bottom plate at 1e5 cells per well. The cells were cultured for 1, 3, 5, and 7 days. Total RNA was extracted from uncultured PBMCs on Day 0 and from PBMCs cultured at various intervals, and cDNA was prepared using ReverTra Ace (Toyobo). Since HTLV-1 is known to be overexpressed in cultured PBMCs from HAM patients, the prepared cDNA was used to analyze changes in RGMa expression associated with HAM patient PBMC culture and viral expression by real-time PCR. 18s rRNA was used as an internal control. The analysis results are shown in Figure 5.

[0058] [Example 5: Analysis of H3K27me3 Levels on All Promoters] Agilent Technologies' SurePrint G3 Human Promoter 2×400K Microarray was used to obtain H3K27me3 levels across all promoters in three normal T cells, four CD4-positive T cells from HAM patients (HAM), and three PBMCs from acute ATL patients (ATL). After normalization, H3K27me3 levels were mapped near -2,916 bp upstream of the transcription start site of the RGMa gene. The graph is shown in Figure 6. The Log 2 values ​​of the fluorescence intensity of the array are plotted in the graph. Furthermore, HAM50 and HAM123 in the figure refer to HAM patients from whom CD4-positive T cells were obtained. This suggests that the repression of RGMa gene expression is relieved in CD4-positive T cells derived from HAM patients.

[0059] [Example 6: Quantification of RGMa gene mRNA levels] A lentiviral vector carrying a cDNA encoding HTLV-1 Tax was introduced into the human CD4-positive T-cell leukemia cell line Jurkat. Quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was used to measure RGMa gene mRNA levels over time three days after introduction. RPL19 gene mRNA was also measured as an internal standard. The quantitative results are shown in a graph in Figure 7. This suggests that RGMa expression is caused by the HTLV-1 virus.

[0060] [Example 7: Tax-dependent RGMa expression induction in HTLV-1-tax expression-inducing cell line JPX-9 cells] JPX9 cells were cultured in RPMI1640 medium (10% FBS) for 24 hours. Cadmium chloride (CdCl2, Nacalai Tesque), which induces HTLV-1-tax expression, was added to a final concentration of 20 μM and cultured for 1, 2, and 3 days. FACS analysis of Tax and RGMa protein expression was performed in CdCl2-treated and untreated JPX9 cells. Cadmium chloride-treated and untreated JPX9 cells were washed separately and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Kit (eBioscience). Anti-Tax-FITC antibody (Lt-4, provided by Professor Tanaka, University of the Ryukyus) was then added and treated at 4°C for 1 hour to stain the Tax protein expressed within the cells. The stained Tax protein was detected by FACS analysis using a Canto II microscope. Cadmium chloride-treated and untreated JPX9 cells were washed separately and treated with anti-RGMa antibody (Immuno-Biological Research Institute (IBL)) at 4°C for 30 minutes. The cells were then washed and treated with anti-mouse IgG-PE antibody (BioLegend) at 4°C for 30 minutes to stain the RGMa protein expressed in JPX9 cells. The stained RGMa protein was detected by FACS analysis using a Canto II microscope. The analysis results of Tax and RGMa protein expressions are shown in FIG8 .

[0061] [Example 8: Study on the Effect of RGMa Antibody on PBMCs of HAM Patients] (Effect of RGMa Antibody on Spontaneous Proliferation Activity) PBMCs from four HAM patients were suspended in culture medium (RPMI1640 medium containing 10% FBS) and seeded in a 96-well round-bottom culture plate at 1e5 cells per well. RGMa antibody (manufactured by R&D Systems) was added to a final concentration of 10 μg / ml, and the cells were cultured in a total of 0.1 ml of culture medium at 37°C, 5% CO2 for 7 days. A group without any additives (medium), a group supplemented with normal goat IgG (Santa Cruz Biotechnology) at the same concentration (normal IgG), and a group supplemented with 1 μg / ml prednisolone (PSL) (Funakoshi) were used as controls. Six days after the start of culture, 1 μCi of 3H-thymidine was added to each well and incubated at 37°C, 5% CO₂ for 16 hours. The cultured cells were then attached to a glass filter (Printed Filtermat A, PerkinElmer) using a cell harvester (Tomtec MH3, PerkinElmer). After drying, the solid scintillant Meltilex-A (PerkinElmer) was infiltrated and the amount of 3H-thymidine incorporated into the cells was measured using a MicroBeta (WALLAC MicroBeta TriLux 1450-021). The average 3H-thymidine count in the culture medium of each HAM patient's PBMC was set as 100%, and the relative values ​​for each group were calculated to determine the average 3H-thymidine incorporation rate for the four HAM patients. The results are shown in Figure 9.

[0062] (Effect of RGMa Antibody on Changes in HTLV-1 Proviral Load) PBMCs from four HAM patients were suspended in culture medium (RPMI1640 medium containing 10% FBS) and seeded in a 96-well round-bottom culture plate at 1e5 cells per well. RGMa antibody (manufactured by R&D Systems) was added to a final concentration of 10 μg / ml, and the cells were cultured in a total of 0.1 ml of culture medium at 37°C, 5% CO2 for 7 days. The group without any additives (culture medium), the group supplemented with normal goat IgG (Santa Cruz Biotechnology) at the same concentration (normal IgG), and the group supplemented with 1 μg / ml prednisolone (PSL) (Funakoshi) were used as controls. Seven days after the start of culture, genomic DNA was extracted from the cell pellet after centrifugation and the supernatant was removed. The extracted genomic DNA was used to determine the HTLV-1 proviral load (infected cell rate) by real-time PCR. The HTLV-1 proviral load in the culture medium of each HAM patient's PBMC was set to 100%, and the relative values ​​of the HTLV-1 proviral load in each group were calculated. The average HTLV-1 proviral load for the four HAM patients was determined. The results are shown in Figure 10.

[0063] (Effect of RGMa antibody on CXCL10 production) To analyze the effect of RGMa antibody on CXCL10 production by PBMCs from HAM patients, PBMCs from four HAM patients were suspended in culture medium (RPMI1640 medium containing 10% FBS) and seeded at 1e5 cells per well in a 96-well round-bottom culture plate. RGMa antibody (manufactured by R&D Systems) was added to a final concentration of 10 μg / ml and cultured in a total of 0.1 ml of culture medium at 37°C, 5% CO2 for 7 days. The group without any additives (culture medium), the group supplemented with normal goat IgG (Santa Cruz Biotechnology) at the same concentration (normal IgG), and the group supplemented with 1 μg / ml prednisolone (PSL) (Funakoshi) were used as controls. After 7 days of culture, the culture medium was centrifuged and only the culture supernatant was recovered. The CXCL10 concentration in the culture supernatant was measured using a Cytokine Beads Array kit (BD Biosciences) and a flow cytometer (FACSCantoII) (BD Biosciences). The CXCL10 concentration in the culture medium of the medium group was set as 100%, and the relative values ​​of CXCL10 concentration in the culture medium of each group were calculated to determine the average CXCL10 concentration of the four HAM patients. The results are shown in Figure 11.

[0064] (Effect of RGMa antibody on cytokine production in PBMCs of HAM patients) To analyze the effects of RGMa antibodies on the production of various cytokines in PBMCs from HAM patients, PBMCs from four HAM patients were suspended in culture medium (RPMI1640 medium containing 10% FBS) and seeded at 1e5 cells per well in a 98-well round-bottom culture plate. RGMa antibodies (manufactured by R&D Systems) were added to a final concentration of 10 μg / ml and cultured in a total of 0.1 ml of culture medium at 37°C and 5% CO2 for 7 days. The group without any additives (culture medium), the group supplemented with normal goat IgG (Santa Cruz Biotechnology) at the same concentration (normal IgG), and the group supplemented with 1 μg / ml prednisolone (PSL) (Funakoshi) were used as controls. After 7 days of culture, the culture medium was centrifuged and only the culture supernatant was recovered. The concentrations of IFNγ, TNF (tumor necrosis factor), IL-2, and IL-10 in the culture supernatant were measured using a cytokine bead array kit (BD Biosciences) and a flow cytometer (FACSCantoII, BD Biosciences). The concentration of each cytokine in the culture medium of the culture medium group was set as 100%, and the relative value of the cytokine concentration under each culture condition was calculated to determine the average value of the 4 HAM patients.

[0065] [Example 9: Induction of Apoptosis in Neural Cell Lines by HAM-PBMC] Neural cell lines NB-1 or SK-N-AS were seeded in a 6-well culture dish and cultured for 24 hours. PBMCs from healthy controls (HD) or HAM patients were then added for co-culture. Forty-eight hours after the start of co-culture, the culture medium and PBMCs were removed, and the cells were washed with PBS and recovered. Each recovered neural cell line was analyzed using the TUNEL method (MEBSTAIN Apoptosis TUNEL Kit Direct (MBL)), which specifically detects cells undergoing DNA fragmentation due to apoptosis. The analysis results are shown in Figure 13. The X-axis of the bar graph represents the intensity of positive DNA fragmentation. Compared with HD-derived cells, HAM-derived cells induce stronger apoptosis in neural cell lines. Specifically, cell death analysis was performed according to the following <Experimental Procedure>. <Experimental Procedure> The neural cell lines NB-1 and SK-N-AS were inoculated into 6-well culture plates and cultured for 24 hours. Then, HD or HAM-PBMC (twice the number of the inoculated neural cell line) were added and cultured for 48 hours. Thereafter, the cells were fixed with 4% paraformaldehyde and permeabilized with 70% ethanol. For DNA nick end labeling, cells were suspended in 20 μL of TdT (Terminal Deoxynucleotidyl Transferase) solution (TdT Buffer II: TdT: FITC-dUTP (Fluorescein Isothiocyanate-Deoxyuridine Triphosphate) = 18:1:1) from the MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) and incubated at 37°C for 60 minutes before FACS analysis.

[0066] Example 10: Inhibitory Effect of RGMa Antibody on Apoptosis of Neural Cell Lines Induced by HTLV-1 Tax-Expressing T Cell Lines The neural cell line NB-1 was seeded in a 6-well culture dish and cultured for 24 hours. Unstimulated JPX9 (JPX9(-)) or JPX9 (JPX9(+CdCl2)) was added for co-culture. Furthermore, normal mouse IgG2b (MBL) or RGMa antibody (IBL) was added to the co-cultured NB-1 cells and JPX9(+CdCl2) for 24 hours to achieve a final concentration of 10 μg / ml. 48 hours after the start of co-culture, the culture medium and the added JPX9 were removed, and the cells were washed with PBS before being collected. Each collected neural cell line was analyzed using the TUNEL assay (MEBSTAIN Apoptosis TUNEL Kit Direct (MBL)), which specifically detects cells undergoing DNA fragmentation due to apoptosis. The results are shown in Figure 14. The X-axis of the bar graph represents the intensity of positive DNA fragmentation. Specifically, cell death analysis was performed according to the following <Experimental Procedure>. <Experimental Procedure> The neural cell line (NB-1) was inoculated into a 6-well culture dish and cultured for 24 hours. Next, JPX9(-) or JPX9(+CdCl2) was added to NB-1 cells for co-culture. The number of JPX9(-) or JPX9(+CdCl2) cells was set at twice the seeding number of NB-1 cells. JPX9(+CdCl2) was washed three times with 10 ml of culture medium to remove cadmium chloride before being added to the NB-1 cells. Then, normal mouse IgG2b (MBL) or anti-RGMa antibody (IBL) was added to the co-culture of NB-1 cells and JPX9 (+CdCl 2 ) at a final concentration of 10 μg / ml and cultured for 48 hours. Subsequently, the cells were fixed with 4% paraformaldehyde, permeabilized with 70% ethanol, and stained with anti-CD45-V450 antibody. For DNA nick end labeling, cells were suspended in 20 μL of TdT solution (TdT buffer II: TdT: FITC-dUTP = 18:1:1) of MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) and incubated at 37°C for 60 minutes before FACS analysis.

[0067] All contents of the publications, patent documents and non-patent documents cited in this specification are incorporated herein by reference.

[0068]

Claims

1. Use of an RGMa inhibitor in the manufacture of a pharmaceutical composition for the treatment or prevention of HTLV-1-associated myelopathy (HAM), wherein the RGMa inhibitor is a substance that acts on RGMa to inhibit the activity of RGMa, the substance that acts on RGMa to inhibit the activity of RGMa is an antibody that recognizes RGMa or an antigen-binding fragment of the aforementioned antibody, and the aforementioned antibody or the antigen-binding fragment of the aforementioned antibody is bound to one or more peptides having an amino acid sequence selected from the group of sequence numbers 53, 54, 55, 56 and 57.

2. Use of an RGMa inhibitor in the manufacture of a pharmaceutical composition for the treatment or prevention of HTLV-1-associated myelopathy (HAM), wherein the RGMa inhibitor is a substance that acts on RGMa to inhibit the activity of RGMa, and the substance that acts on RGMa to inhibit the activity of RGMa is an antibody that recognizes RGMa or an antigen-binding fragment of the aforementioned antibody, wherein the aforementioned antibody or the antigen-binding fragment of the aforementioned antibody binds to amino acid sequence number 250 or later in the human RGMa sequence.

3. Use of an RGMa inhibitor in the manufacture of a pharmaceutical composition for the treatment or prevention of HTLV-1-associated myelopathy (HAM), wherein the RGMa inhibitor is a substance that acts on RGMa to inhibit the activity of RGMa, and the substance that acts on RGMa to inhibit the activity of RGMa is an antibody that recognizes RGMa or an antigen-binding fragment of the aforementioned antibody, wherein the aforementioned antibody or the antigen-binding fragment of the aforementioned antibody is an amino acid sequence comprising complementarity-determining region 1 (LCDR1), complementarity-determining region 2 (LCDR2), complementarity-determining region 3 (LCDR3) of the light chain, complementarity-determining region 1 (HCDR1), complementarity-determining region 2 (HCDR2), and complementarity-determining region 3 (HCDR3) of the heavy chain, and contains LCDR1: RASQDISSYLN (sequence number 58), LCDR2: YTSRLHS (sequence number 59), LCDR3: QQLNTLP (sequence number 60), and HCDR1: DAWMD (sequence number 61). HCDR2: EIRSKANNHATYYAESVKG (serial number 62) and HCDR3: RDGAY (serial number 63), or containing LCDR1: RSSQSLVHSNGNTYLH (serial number 64), LCDR2: KVSNRFS (serial number 65), LCDR3: SQSTHVP (serial number 66), HCDR1: TSYYWN (serial number 67), HCDR2: YISYDGTNNYNPSLKN (serial number 68) and HCDR3: SFG.

4. Use of an RGMa inhibitor in the manufacture of a pharmaceutical composition for the treatment or prevention of HTLV-1-associated myelopathy (HAM), wherein the RGMa inhibitor is a substance that acts on RGMa to inhibit the activity of RGMa, and the substance that acts on RGMa to inhibit the activity of RGMa is an antibody that recognizes RGMa or an antigen-binding fragment of the aforementioned antibody, wherein the amino acid sequences of the complementarity-determining region 1 (LCDR1), complementarity-determining region 2 (LCDR2), complementarity-determining region 3 (LCDR3) of the light chain, complementarity-determining region 1 (HCDR1), complementarity-determining region 2 (HCDR2), and complementarity-determining region 3 (HCDR3) of the heavy chain are: LCDR1: sequence number 8, LCDR2: sequence number 9, LCDR3: sequence number 10, HCDR1: sequence number 5, HCDR2: sequence number 6, and HCDR3: sequence number 7, or LCDR1: sequence number 14. LCDR2: Serial number 15, LCDR3: Serial number 16, HCDR1: Serial number 11, HCDR2: Serial number 12 and HCDR3: Serial number 13.

5. Use of an RGMa inhibitor in the manufacture of a pharmaceutical composition for the treatment or prevention of HTLV-1-associated myelopathy (HAM), wherein the RGMa inhibitor is a substance that acts on RGMa to inhibit the activity of RGMa, the substance that acts on RGMa to inhibit the activity of RGMa is an antibody that recognizes RGMa or an antigen-binding fragment of the aforementioned antibody, the aforementioned antibody or the antigen-binding fragment of the aforementioned antibody is selected from (a1) to (h1) below, (a1) containing a light chain variable region and a heavy chain variable region of an anti-RGMa antibody, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: LCDR1 containing the amino acid sequence described in sequence number 97, LCDR2 containing the amino acid sequence described in sequence number 98, and LCDR3 containing the amino acid sequence described in sequence number 99, and the heavy chain variable region contains: (b) An anti-RGMa antibody containing the amino acid sequence described in sequence number 100, an HCDR2 containing the amino acid sequence described in sequence number 101, and an HCDR3 containing the amino acid sequence described in sequence number 102; (b) An anti-RGMa antibody containing a light chain variable region and a heavy chain variable region, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: an LCDR1 containing the amino acid sequence described in sequence number 97, an LCDR2 containing the amino acid sequence described in sequence number 98, and an LCDR3 containing the amino acid sequence described in sequence number 103, and the heavy chain variable region contains: an HCDR1 containing the amino acid sequence described in sequence number 100, an HCDR2 containing the amino acid sequence described in sequence number 101, and an HCDR3 containing the amino acid sequence described in sequence number 102; (c) An anti-RGMa antibody containing a light chain variable region and a heavy chain variable region, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: The LCDR1 contains the amino acid sequence described in sequence number 97, the LCDR2 contains the amino acid sequence described in sequence number 98, and the LCDR3 contains the amino acid sequence described in sequence number 104. The heavy chain variable region contains: HCDR1 containing the amino acid sequence described in sequence number 100, HCDR2 containing the amino acid sequence described in sequence number 101, and HCDR3 containing the amino acid sequence described in sequence number 102.(d1) An anti-RGMa antibody containing a light chain variable region and a heavy chain variable region, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: an LCDR1 containing the amino acid sequence described in sequence number 97, an LCDR2 containing the amino acid sequence described in sequence number 98, and an LCDR3 containing the amino acid sequence described in sequence number 105; the heavy chain variable region contains: an HCDR1 containing the amino acid sequence described in sequence number 100, an HCDR2 containing the amino acid sequence described in sequence number 101, and an HCDR3 containing the amino acid sequence described in sequence number 102; (e1) An anti-RGMa antibody containing a light chain variable region and a heavy chain variable region, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: an LCDR1 containing the amino acid sequence described in sequence number 97, an LCDR2 containing the amino acid sequence described in sequence number 98, and an LCDR3 containing the amino acid sequence described in sequence number 106; the heavy chain variable region contains: (f1) An anti-RGMa antibody containing the amino acid sequence described in sequence number 100, an HCDR2 containing the amino acid sequence described in sequence number 101, and an HCDR3 containing the amino acid sequence described in sequence number 102; (g1) An anti-RGMa antibody containing a light chain variable region and a heavy chain variable region, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: an LCDR1 containing the amino acid sequence described in sequence number 97, an LCDR2 containing the amino acid sequence described in sequence number 98, and an LCDR3 containing the amino acid sequence described in sequence number 107, and the heavy chain variable region contains: an HCDR1 containing the amino acid sequence described in sequence number 100, an HCDR2 containing the amino acid sequence described in sequence number 101, and an HCDR3 containing the amino acid sequence described in sequence number 102; (g1) An anti-RGMa antibody containing a light chain variable region and a heavy chain variable region, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: The LCDR1 contains the amino acid sequence described in sequence number 97, the LCDR2 contains the amino acid sequence described in sequence number 98, and the LCDR3 contains the amino acid sequence described in sequence number 108. The heavy chain variable region contains: HCDR1 containing the amino acid sequence described in sequence number 100, HCDR2 containing the amino acid sequence described in sequence number 101, and HCDR3 containing the amino acid sequence described in sequence number 102.(h1) An anti-RGMa antibody containing a light chain variable region and a heavy chain variable region, or an antigen-binding fragment of the aforementioned antibody, wherein the light chain variable region contains: LCDR1 containing the amino acid sequence described in sequence number 97, LCDR2 containing the amino acid sequence described in sequence number 98, and LCDR3 containing the amino acid sequence described in sequence number 109; and the heavy chain variable region contains: HCDR1 containing the amino acid sequence described in sequence number 100, HCDR2 containing the amino acid sequence described in sequence number 101, and HCDR3 containing the amino acid sequence described in sequence number 102.

6. Use of an RGMa inhibitor in the manufacture of a pharmaceutical composition for the treatment or prevention of HTLV-1-associated myelopathy (HAM), wherein the RGMa inhibitor is a substance that acts on RGMa to inhibit the activity of RGMa, the substance that acts on RGMa to inhibit the activity of RGMa is an antibody that recognizes RGMa or an antigen-binding fragment of the antibody, wherein the antibody or the antigen-binding fragment of the antibody has a heavy chain variable region (VH) containing sequence number 95 and a light chain variable region (VL) containing sequence number 96.

7. Use of an RGMa inhibitor in the manufacture of a pharmaceutical composition for the treatment or prevention of HTLV-1-associated myelopathy (HAM), wherein the RGMa inhibitor is a substance that acts on RGMa to inhibit the activity of RGMa, the substance that acts on RGMa to inhibit the activity of RGMa is an antibody that recognizes RGMa or an antigen-binding fragment of the antibody, wherein the heavy chain of the antibody or the antigen-binding fragment of the antibody contains an amino acid sequence of sequence number 84 and the light chain contains an amino acid sequence of sequence number 88.

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