Title of invention: HTLV-1-related myelopathy (HAM) treatment or preventive agent, and method for treating HAM
Technical field
[0001]
　The present invention relates to therapeutic or prophylactic agents for HTLV-1-related myelopathy (HAM). The present invention also relates to a method for treating HAM.
Background technology
[0002]
　HTLV-1 (Human T-cell Lukemia Virus Type 1) is a virus that infects T cells, which are one of the leukemias in the blood (mainly CD4-positive T cells).
　T cells infected with HTLV-1 form chronic inflammation of the spinal cord, resulting in damage and degeneration of spinal nerve cells, leading to spastic spinal paralysis. Spastic spinal cord paralysis caused by cells infected with HTLV-1 is called HTLV-1-related myelopathy (also abbreviated as HAM).
　Symptoms of HAM include symptoms such as paralysis of both legs, pain, dysuria, and stubborn constipation due to nerve tissue damage. As these symptoms progress, people become wheelchair-bound or bedridden. HAM is one of the diseases designated as intractable diseases in Japan. Currently, no effective treatment method for HAM has been established, and symptomatic treatment is exclusively performed.
　As one of the therapeutic methods, it has been proved that a therapeutic method using an anti-CCR4 antibody reduces HTLV-1-infected cells, reduces spinal inflammation of HAM, and has a symptom-improving effect (Non-Patent Document 1, Non-Patent Document 1, Patent Document 5).
[0003]
　The RGMa protein is a member of the RGM (Repulsive guidance molecule) family of proteins involved in axon guidance of nerve cells in the retina and hippocampus, closure of neural tubes, and the like. The function of RGM is not limited to these, and it is known that it has various functions.
[0004]
　For example, Patent Document 1 states that RGM is expressed in bone marrow-derived dendritic cells (BMDCs) , RGM receptors are expressed in CD4 + T cells and CD11b + macrophages, and RGM is expressed in the RGM receptors. It is disclosed that binding enhances the cell adhesion activity of CD4 + T cells and CD11b + macrophages. In addition, Patent Document 1 states that anti-RGM neutralizing antibody can reduce both clinical symptoms and tissue lesions in multiple sclerosis model mice, and that splenocytes obtained from the mice are antigen-specific and non-specific. T cell activation has been diminished.
[0005]
　Patent Documents 2 and 3 disclose neutralizing monoclonal antibodies against RGMa that selectively inhibit the binding of RGMa to the receptors neogenin of RGMa and bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4). ing. According to the neutralized monoclonal antibody, the damaged and inflamed human central nervous system, specifically, multiple sclerosis, acute spinal cord injury, after brain trauma, Huntington chorea, Parkinson's disease, Alzheimer's disease, etc. In neurodegenerative diseases, it is said that it is possible to promote nerve regeneration and regrowth of disrupted neuronal connections.
[0006]
　Patent Document 4 states that RGMa is localized in human central nervous system myelin, fresh lesions and mature scar tissue suffering from traumatic brain injury or ischemic stroke, and to diagnose these neurodegenerative diseases. For the purpose, methods for detecting and quantifying RGMa fragments are disclosed. In addition, Patent Document 4 describes multiple sclerosis, Parkinson's disease, Alzheimer's disease, Teisax's disease, Niemann-Pick's disease, Gaucher's disease, Harler's syndrome, and Huntington as neurodegenerative diseases and disorders for which RGMa fragments are detected. Disease, muscular atrophic sclerosis, idiopathic inflammatory demyelinosis, vitamin B12 deficiency, pons central myelopathy, spinal fistula, transverse myelitis, Devic's disease, progressive multifocal leukoencephalopathy, optic neuritis , Spinal cord injury, traumatic brain injury, stroke, glaucoma, diabetic retinopathy, age-related macular degeneration, white dystrophy.
Prior art literature
Patent documents
[0007]
Patent Document 1: International Publication 2011/071059 Patent
Patent Document 2: JP 2014-138599 Patent
Patent Document 3: JP 2016-175897 Patent
Patent Document 4: JP-T 2017-526930 Patent
Patent Document 5: JP 2010-100578
Non-patent literature
[0008]
Non-Patent Document 1: N Engl J Med, 2018, 378, 529-538.
Outline of the invention
Problems to be solved by the invention
[0009]
　The method using the anti-CCR4 antibody disclosed in Non-Patent Documents 1 and 5 can improve the symptoms of HAM, but the effect of the anti-CCR4 antibody is limited to HAM patients with advanced nerve destruction. Is. Therefore, there is a need for a better method that can treat HAM.
[0010]
　The present invention has been made in view of the above problems, and an object of the present invention is to provide a therapeutic agent and a therapeutic method capable of treating HAM.
Means to solve problems
[0011]
　As a result of diligent studies to solve the above problems, the present inventors have found that a substance that inhibits RGMa is effective for the treatment of HAM, and have completed the present invention.
[0012]
　That is, the present invention is as follows.
[1]
　A therapeutic or prophylactic agent for HTLV-1-related myelopathy (HAM), which comprises an RGMa inhibitor.
[2]
　The therapeutic or prophylactic agent for HAM according to [1], wherein the RGMa inhibitor is an antibody that recognizes RGMa.
[3] A
　method for treating HAM, which comprises administering a pharmacologically effective amount of an RGMa inhibitor to a patient with HTLV-1-related myelopathy (HAM) who needs it.
[4] The
　method for treating HAM according to [3], wherein the RGMa inhibitor is an antibody that recognizes RGMa.
Effect of the invention
[0013]
　According to the present invention, HAM, which is an intractable disease, can be treated.
A brief description of the drawing
[0014]
[Fig. 1] Fig. 1 is a diagram showing CADM1 which is an index of HTLV-1-infected cells and groups P, D, and N classified by CD7 in CD4-positive T cells.
[Fig. 2] Normal T cells (Normal.CD4), CD4 positive T cells derived from HAM patients (HAM.CD4), CD4 positive / CADM1 negative / CD7 positive T cells derived from healthy individuals (Normal.P), HTLV-1 infected persons CD4 positive / CADM1 negative / CD7 positive T cells (P group), HTLV-1-infected CD4 positive / CADM1-positive / CD7 positive T cells (D group), HTLV-1-infected CD4 positive / CADM1 positive / CD7 negative T cells (Group N), CD4 positive / CADM1 positive / CD7 negative T cells (Acute.N) in acute ATL patients, CD4 positive T cells in healthy subjects (Normal.CD4.1), PBMC (Smoldering) derived from smoldering ATL patients, chronic It is a figure which shows the expression level of RGMa gene in PBMC (Chronic) derived from type ATL patient, and PBMC (Acute) derived from acute type ATL patient.
FIG. 3 is a diagram showing the expression level of RGMa in CD4 positive T cells of HAM patients and CD4 positive T cells of healthy subjects.
[Fig. 4] Fig. 4 is a diagram showing the results of analysis of the expression level of RGMa among cell types in PBMC of HAM patients.
FIG. 5 is a diagram showing changes in RGMa expression associated with HTLV-1 virus expression in a culture of PBMC in HAM patients.
[Fig. 6] Fig. 6 shows the H3K27me3 level near the transcription start point of the RGMa gene -2,916 bp upstream.
[Fig. 7] Fig. 7 is a diagram showing the level of RGMa gene mRNA when a lentiviral vector into which a cDNA encoding HTLV-1 Tax is inserted is introduced into a human CD4-positive T cell leukemia cell line Jurkat.
[Fig. 8] Fig. 8 is a diagram showing the analysis results of protein expression of Tax and RGMa in the HTLV-1-tax expression-inducing cell line JPX-9.
FIG. 9 shows the results of the action of RGMa antibody on spontaneous growth activity with respect to the action of RGMa antibody on PBMC of HAM patients.
FIG. 10 is a diagram showing the results of the action of the RGMa antibody on changes in the amount of HTLV-1 provirus with respect to the action of the RGMa antibody on PBMC of HAM patients.
FIG. 11 shows the results of the action of RGMa antibody on CXCL10 production with respect to the action of RGMa antibody on PBMC in HAM patients.
FIG. 12 shows the results of the action of RGMa antibody on cytokine production of HAM patient PBMC with respect to the action of RGMa antibody on PBMC of HAM patient.
[Fig. 13] Fig. 13 shows that HAM-PBMC caused apoptosis induction of nerve cell lines.
[Fig. 14] Fig. 14 is a diagram showing the results of the inhibitory effect of RGMa antibody on the induction of apoptosis of a nerve cell line by an HTLV-1-tax-induced cell line. (a) is a FACS plot when NB-1 cells and JPX-9 cells (unstimulated) were co-cultured. (b) shows NB-1 cells and CdCl 2FACS plot when co-cultured with stimulated JPX-9 cells (HTLV-1-tax expressing cells). (c) is a FACS plot when the control antibody is added to the condition of (b). (d) is a FACS plot when the anti-RGMa antibody was added to the condition of (b).
Mode for carrying out the invention
[0015]
　Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist thereof.
[0016]
　The present invention is a therapeutic or prophylactic agent for HTLV-1-related myelopathy (HAM), which comprises an RGMa inhibitor. The RGMa inhibitor may be a substance that acts on RGMa itself to inhibit the activity of RGMa, or may be a substance that suppresses the expression of RGMa.
　Examples of the RGMa inhibitor include a compound having an activity of inhibiting RGMa, an antibody that recognizes RGMa, and the like.
[0017]
　Examples of the RGMa inhibitor include siRNA (short interfering RNA), shRNA (short hairpin RNA), and antisense oligonucleotides of genes expressing RGMa that suppress the expression of RGMa.
　Examples of the RGMa gene include, but are not limited to, the human RGMa gene consisting of the nucleotide sequence shown in SEQ ID NO: 1, the RGMa gene consisting of the nucleotide sequence shown in SEQ ID NO: 2, and the like. Information on the nucleotide sequences of RGMa genes derived from various organisms can be obtained from databases such as GenBank.
[0018]
　siRNA is a double-stranded RNA capable of suppressing the expression of the target RGMa gene. The length (base length) of the base sequence in 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 has a double-stranded structure in the molecule by containing a partially palindromic base sequence in the single-stranded RNA, and consists of about 20 short hairpin structures having a protrusion at the 3'end. Refers to molecules that are more than a base pair. After being introduced into the cell, the shRNA is decomposed into the cell to a length of about 20 bases, and can suppress the expression of the target RGMa gene like siRNA.
[0019]
　siRNA and shRNA can be artificially chemically synthesized. In addition, siRNA and shRNA can synthesize antisense and sense RNA from template DNA in vitro using, for example, T7 RNA polymerase and T7 promoter.
[0020]
　The antisense oligonucleotide may be any nucleotide that is complementary or hybridizes to a contiguous base sequence of less than about 30 bases in the DNA sequence of the RGMa gene, and may be either DNA or RNA. Further, it may be modified as long as it does not interfere with the function. The antisense oligonucleotide can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer.
[0021]
　As the RGMa inhibitor contained in the therapeutic or prophylactic agent for HAM of the present invention, an antibody that recognizes RGMa is preferable. Hereinafter, an antibody that recognizes RGMa is also referred to as an RGMa antibody. The RGMa antibody in the present invention may be an antibody that binds to RGMa and inhibits its activity, and examples thereof include an antibody that binds to RGMa to prevent RGMa from binding to the RGMa receptor.
[0022]
　The RGMa antibody in the present invention may be a monoclonal antibody or a polyclonal antibody. Further, the antibody in the present invention may be any isotype of IgG, IgM, IgA, IgD and IgE.
　The RGMa antibody in the present invention may be, for example, a mouse antibody, a human CDR transplantation antibody, a human chimeric antibody, a humanized antibody, or a fully human antibody, or may be a small molecule antibody. These antibodies may be used alone or in combination of two or more.
[0023]
　A human-type CDR transplanted antibody is an antibody in which the CDR of an antibody of a non-human animal is replaced with the CDR of a human antibody. A human chimeric antibody is an antibody consisting of a variable region derived from an antibody of a non-human animal and a constant region derived from a human antibody. The humanized antibody refers to an antibody of a non-human animal in which a portion derived from the human antibody is incorporated while leaving a part of a highly safe region, and is a human type chimeric antibody and a human type. A concept that includes a CDR-transplanted antibody.
[0024]
　As used herein, the term "small molecule antibody" means an antibody fragment or an antibody fragment bound to any molecule that recognizes the same epitope as the original antibody. Specifically, a Fab consisting of VL, VH, CL and CH1 regions; F (ab') 2 in which two Fabs are linked by a disulfide bond in a hinge region; Fv consisting of VL and VH; VL and VH are artificially produced. In addition to scFv, which is a single-chain antibody linked with the polypeptide linker of the above, sdFv, Diabody, sc (Fv) 2, but are not limited thereto.
[0025]
　The RGMa antibody used in the present invention can be prepared by a known method using RGMa or a fragment thereof as an immunogen.
　It can be confirmed that the obtained antibody is an RGMa antibody by using the RGMa activity as an index.
　Examples of RGMa include human RGMa containing the amino acid sequence shown in SEQ ID NO: 3, RGMa containing the amino acid sequence shown in SEQ ID NO: 4, and the like. RGMa from various organisms can be used as an immunogen. The amino acid sequence of RGMa can be obtained from a known database such as Protein Data Bank.
[0026]
　When the RGMa antibody used in the present invention is a polyclonal antibody, it can be produced, for example, as follows. First, RGMa or a fragment thereof as an antigen is dissolved in phosphate buffered saline (also referred to as PBS), and if necessary, an appropriate amount of a usual adjuvant, for example, Freund's complete adjuvant, is mixed as an immunogen, and the mouse is used as an immunogen. Immunizes mammals such as rats, rabbits, goats, and horses. The immunization method is not particularly limited, and examples thereof include a method of subcutaneous injection or intraperitoneal injection once or twice or more at appropriate intervals. It can then be obtained by collecting blood from the immunized animal, separating the serum and purifying the polyclonal antibody fraction according to a conventional method.
　When the RGMa antibody used in the present invention is a monoclonal antibody, the monoclonal antibody obtains a hybridoma by fusing immune cells obtained from the immunized mammal, for example, splenocytes and myeloma cells, and culturing the hybridoma. It can be obtained by collecting an antibody from an object. Further, the above-mentioned monoclonal antibody can be obtained by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing the antibody gene into a host cell, and producing a recombinant monoclonal antibody by using a gene recombination technique. Furthermore, the monoclonal antibody can also be prepared by using a phage display method.
[0027]
　Examples of the RGMa antibody used in the present invention include 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 Application Laid-Open No. 2014-138599; Japanese Patent Application Laid-Open No. 2016-175897; Japanese Patent Application Laid-Open No. 2017-526930; International Publication No. 2016/175236; Can be done.
　The RGMa antibody used in the present invention can also be obtained as a commercially available product such as that manufactured by Immuno-Biological Laboratory Co., Ltd. (IBL) or R & D Systems.
[0028]
　The RGMa antibody, as an antigen-binding domain, has
　GTTPDY (SEQ ID NO: 7);
　FQATHDPLT (SEQ ID NO: 10);
　ARRNEYYGSSFFDY (SEQ ID NO: 13);
　LQGYIPPRT (SEQ ID NO: 16); and
　at least 50% sequence identity with one of the sequences.
　It is preferable to include at least one CDR containing an amino acid sequence selected from the group consisting of a modified CDR amino acid sequence having . The above sequence identity is preferably 80% or more, more preferably 90% or more. In this specification, amino acids may be indicated in the conventional one-letter notation or three-letter notation.
[0029]
　Complementarity determining regions (CDRs) refer to regions that form antigen-binding sites among the variable regions of immunoglobulin molecules, and are also called hypervariable regions, and refer to regions where the amino acid sequence changes significantly for each immunoglobulin molecule. .. There are three CDRs (CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, CDR-H3) in each of the light chain and the heavy chain. In this application, the CDRs of immunoglobulin molecules are determined according to Kabat's numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).
　Further, in the antibody defined by the amino acid sequences of the light chain and the heavy chain in the present invention, the amino acid sequence of the CDR may be unchanged from the predetermined sequence, and other than the CDR may be changed by mutation or the like. When there is a mutation or the like other than CDR, the homology is preferably 90% or more.
[0030]
　The RGMa antibody is also an amino acid selected from the group consisting of SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15 and a modified CDR amino acid sequence having at least 50% sequence identity with one of the sequences. It is preferable to further include at least one CDR containing the sequence. The above sequence identity is preferably 80% or more, more preferably 90% or more.
[0031]
　The RGMa antibody is a modified CDR amino acid sequence in which the variable domain CDR set shown in Table 1 or at least one of the three CDRs has at least 50%, preferably 80%, more preferably 90% sequence identity with the parent sequence. More preferably, it comprises at least three CDRs selected from the variable domain set. Further, it is more preferable that the RGMa antibody contains at least two variable domain CDR sets shown in Table 1. Further, at least two variable domain CDR sets are preferably a combination of VH5F9 set and VL5F9 set, or a combination of VH8D1 set and VL8D1 set.
[0032]
[table 1]

[0033]
　The RGMa antibody may include a framework region. The 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. Can be mentioned. These amino acid sequences may be one kind alone or a combination of two or more kinds.
　The RGMa antibody also comprises at least one heavy chain variable domain selected from SEQ ID NOs: 41, 42, 43, 44, 45, 46, 47, 48 and 49 as the heavy chain variable domain and the light chain variable domain; and / Alternatively, it preferably comprises at least one light chain variable domain selected from SEQ ID NOs: 50, 51 and 52.
[0034]
　The binding site of the RGMa antibody when it binds 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,
　EEVVNAVEDWDSQG (SEQ ID NO: 53)
　NQQIDFQAFHTNAE (SEQ ID NO: 54)
　PTAPETFPYET ( SEQ ID NO: 55) It is preferable to bind to one or more peptides having the amino acid sequence represented by 　KLPVEDLYYQA (SEQ ID
　NO: 56)
LYERTRDLPGRAAAGL (SEQ ID NO: 57)
.
　The RGMa antibody more preferably binds to a peptide having the amino acid sequence represented by SEQ ID NO: 53 and / or SEQ ID NO: 54, and the peptide having the amino acid sequence represented by SEQ ID NO: 53 and / or SEQ ID NO: 54. More preferably, it binds to a peptide having the amino acid sequence represented by SEQ ID NO: 55 and / or SEQ ID NO: 56.
　The RGMa antibody is preferably an antibody that binds to the 250th or higher amino acid sequence of human RGMa.
　The RGMa antibody is more preferably bound to a peptide having SEQ ID NO: 53 and SEQ ID NO: 54 and the amino acid sequence represented by SEQ ID NO: 55 or SEQ ID NO: 56.
[0035]
　The RGMa antibody is a polyclonal antibody or a monoclonal antibody obtained by immunizing a mammal such as a mouse with the RGMa protein or a partial fragment thereof (for example, a fragment containing one or more of SEQ ID NOs: 53 to 57) as an antigen. It may be a chimeric antibody or a humanized antibody produced by using a gene recombination technique, or a human antibody or the like produced by using a human antibody-producing transgenic animal or the like.
　In the present invention, when the RGMa antibody is administered to a human as a pharmaceutical, a humanized antibody or a human antibody is desirable from the viewpoint of side effects.
　The RGMa antibody may be used as a polyclonal antibody that recognizes the RGMa protein or a partial fragment thereof (for example, a fragment containing one or more of SEQ ID NOs: 53 to 57) as an antigen and / or a partial fragment of a monoclonal antibody, and is a low molecular weight antibody. May be.
[0036]
　In addition to those shown in Table 1, the RGMa antibodies include light chain complementarity determining regions 1 (LCDR1), light chain complementarity determining regions 2 (LCDR2), and light chain complementarity determining regions 3 (LCDR3). The amino acid sequences of the heavy chain complementarity determining regions 1 (HCDR1), heavy chain complementarity determining regions 2 (HCDR2), and heavy chain complementarity determining regions 3 (HCDR3) are
　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: EIRSKANHANHATYYAESVKG (SEQ ID NO: 62) and
　HCDR3: RDGAY (SEQ ID NO: 63),
or
　LCDR1: RSSQSLVHSNGNTYLH ( SEQ ID NO: 63) SEQ ID NO
　: 64) LCDR2: KVSNRFS (SEQ ID NO
　: 65)
　LCDR3: SQSTHVP (SEQ ID NO: 66 ) HCDR1: TSYYWN (SEQ ID NO : 67)
　HCDR2: YISYDGTNNYNPSLKN (SEQ ID NO: 68) and
　HCDR3:
Isolated RGMa antibodies, including SFG . Alternatively, it may be an antigen-binding fragment thereof.
　In each CDR sequence, one or several amino acids may be substituted, deleted, and / or added, eg, one or two amino acids are substituted, deleted, and / or added. May be good.
[0037]
　Examples of the RGMa antibody include an antibody having the amino acid sequence of SEQ ID NO: 73 on the light chain and the amino acid sequence of SEQ ID NO: 74 on the heavy chain. In the amino acid sequence represented by these SEQ ID NOs, one or several amino acids (1 to 20, 1 to 10 or 1 to 5) may be substituted, deleted, added or inserted. Such substitutions, deletions, and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
[0038]
　A mouse / human chimeric antibody derived from a human constant region may be used. As the mouse / human chimeric antibody, the amino acid sequence of SEQ ID NO: 77 (variable regions are 1 to 107) is on the light chain, and SEQ ID NO: 78 is on the heavy chain. An antibody having an amino acid sequence (variable region is 1-116) is exemplified. In the amino acid sequence represented by these SEQ ID NOs, one or several amino acids (1 to 20, 1 to 10 or 1 to 5) may be substituted, deleted, added or inserted. Such substitutions, deletions, and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
[0039]
　It may be a humanized antibody derived from humans other than CDR. As a humanized antibody, the heavy chain has an amino acid sequence of any of SEQ ID NOs: 70 to 87 (the variable region is up to 116 residues on the N-terminal side), and the light chain has SEQ ID NOs: 88 to 94 (the variable region is the N-terminal). An antibody having any of the amino acid sequences (from 1 to 107 residues on the side) is exemplified. The amino acid sequences represented by these SEQ ID NOs may be substituted, deleted, added or inserted with one or several amino acids (1 to 20, 1 to 10 or 1 to 5). Such substitutions, deletions, and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
[0040]
　The heavy chain amino acid sequence and the light chain amino acid sequence may be any combination thereof, but an antibody having the amino acid sequence of SEQ ID NO: 84 on the heavy chain and the amino acid sequence of SEQ ID NO: 88 on the light chain is preferable. Among the amino acid sequences of SEQ ID NO: 84, the amino acid sequence 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.
[0041]
　The RGMa antibody includes a heavy chain variable region (VH) is
IbuikyuerubuiiesujijijierubuikyuPijiaruesueruarueruesushitieiesujiefutiefuesudieidaburyuemudidaburyubuiarukyueiPijikeijieruidaburyubuieiiaiaruesukeieienuenueichieitiwaiwaieiiesubuikeijiaruefutiaiesuarudidiesukeiesuaibuiwaierukyuemuenuesueruarutiiDTALYYCTRRDGAYWGKGTTVTVSS (SEQ ID NO: 95)
comprises at least 90% amino acid sequence of identity or the amino acid sequence,
light chain variable region (VL) is
DiaikyuemutikyuesuPiesuesubuiesueiesubuijidiarubuitiaitishiarueiesukyudiaiesuesuwaieruenudaburyuwaikyukyukeiPijikeieiPikeierueruaiwaiwaitiesuaruerueichiesujibuiPiesuaruefuesujiesujiesujitidiefutierutiaiesuesuerukyuPiidiFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 96 )
Or an isolated RGMa antibody containing an amino acid sequence having at least 90% identity in the amino acid sequence, or an antigen-binding fragment thereof.
[0042]
　Examples of the RGMa antibody include an antibody having the amino acid sequence of SEQ ID NO: 75 on the light chain and the amino acid sequence of SEQ ID NO: 76 on the heavy chain. In the amino acid sequence represented by these SEQ ID NOs, one or several amino acids (1 to 20, 1 to 10 or 1 to 5) may be substituted, deleted, added or inserted. Such substitutions, deletions, and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
　It may be a mouse / human chimeric antibody whose constant region is derived from humans, or it may be a humanized antibody whose constant region is derived from humans other than CDR.
[0043]
　Further, the anti-RGMa antibody may be an isolated RGMa antibody selected from the following (a1) to (h1) or an antigen-binding fragment thereof.
(A1) To LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 99, and SEQ ID NO: 100. An anti-RGMa antibody containing a heavy chain variable region containing HCDR1 containing the amino acid sequence described, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof,
( b1) LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 103, and SEQ ID NO: 100. HCDR1 containing the amino acid sequence of, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and an anti-RGMa antibody containing a heavy chain variable region containing HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof,
(c1). ) LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 104, and SEQ ID NO: 100. An anti-RGMa antibody containing a heavy chain variable region containing HCDR1 containing an amino acid sequence, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof.
(D1) To LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 105, and SEQ ID NO: 100. An anti-RGMa antibody containing a heavy chain variable region containing HCDR1 containing the amino acid sequence described, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof,
( e1) LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 106, and SEQ ID NO: 100. HCDR1 containing the amino acid sequence of, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and an anti-RGMa antibody containing a heavy chain variable region containing HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof,
(f1). ) LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 107, and SEQ ID NO: 100. An anti-RGMa antibody containing a heavy chain variable region containing HCDR1 containing an amino acid sequence, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof.
(G1) To LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 108, and SEQ ID NO: 100. An anti-RGMa antibody containing a heavy chain variable region containing HCDR1 containing the amino acid sequence described, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof, and
(H1) To LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 97, a light chain variable region containing LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 98 and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 109, and SEQ ID NO: 100. An anti-RGMa antibody containing a heavy chain variable region containing HCDR1 containing the amino acid sequence described, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 101, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 102, or an antigen-binding fragment thereof.
[0044]
　The RGMa antibody is an antibody having the above-mentioned amino acid sequence, preferably a humanized antibody, and preferably has a constant region of human IgG.
[0045]
　The RGMa inhibitor in the present invention can treat or prevent HAM.
　As shown in Examples below and FIG. 13, in HAM patients, cells derived from HAM patients cause neuronal cell death, that is, induction of spinal cord tissue damage and degeneration.
　As a result of examination by the present inventors, as shown in Examples described later, RGMa is remarkably expressed in CD4-positive T cells, which are the main HTLV-1-infected cells of HAM patients, and RGMa is expressed in HAM. Expression was found to be involved. In addition, as a result of a test in which RGMa was inhibited using an RGMa antibody, it was found that RGMa is involved in the production of CXCL10, IL-2, and IL-10.
　CXCL10 is a protein produced by HAM-pathogenic HTLV-1-infected T cells in response to IFN-γ and is known to induce the pathology of HAM (Brain 2013) and is a symptom of HAM. It is known to strongly correlate with the rate of progression (PLoS Negl Trop Dis 2013). When RGMa antibody was allowed to act on the cells of HAM patients, the production of this CXCL10 was suppressed. In addition, IL-10 is one of the cytokines and works to suppress inflammation. The action of RGMa antibody on the cells of HAM patients markedly increased IL-10 production.
　In order to bring about a true therapeutic effect on HAM, it is necessary to suppress damage to nerve cells. High levels of HTLV-1-tax expression in HTLV-1-infected cells in HAM patients (Blood 2002) indicate that HTLV-1-tax is important for pathogenesis (J Clin Invest 2014). In the present invention, it was shown that HTLV-1-tax induces RGMa expression, and cells derived from HAM patients with high RGMa expression levels induce neuronal cell death, and importantly, HTLV-1-tax expressing cells. It was shown that RGMa inhibitors suppress neuronal cell death caused by RGMa.
　As described above, the RGMa inhibitor can suppress the induction of the inflammatory pathological condition of HAM and also suppress the inflammatory reaction by the cells of HAM patients, so that HAM can be treated or prevented. Furthermore, RGMa inhibitors not only suppress the inflammatory response peculiar to HAM, but also suppress nerve cell death caused by HTLV-1-tax-expressing cells, which are pathogenic cells of HAM patients, and thus treat or prevent HAM. can do.
[0046]
　As mentioned above, RGMa inhibitors can treat or prevent HAM, and the present invention is an RGMa inhibitor for use in the treatment of HAM; a pharmaceutical composition for use in the treatment of HAM; for treating HAM. Use of RGMa Inhibitors; Use of RGMa Inhibitors in the Production of Therapeutic Drugs for HAM; RGMa Inhibitors for Use in the Production of Therapeutic Drugs for HAM; To provide a method of treating or preventing HAM, including administration to.
[0047]
　The therapeutic or prophylactic agent for HAM of the present invention may be formulated by appropriately blending an RGMa inhibitor and a pharmaceutically acceptable carrier and / or additive.
　Examples of the form of formulation include oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions and emulsions; injections, infusions, suppositories, ointments, patches and the like. Parenteral preparations;
　The blending ratio of the carrier or the additive may be appropriately set based on the range usually adopted in the pharmaceutical field.
　The carrier is not particularly limited, and examples thereof include water, physiological saline, other aqueous solvents, and an aqueous or oily base. Examples of the additive include, but are not limited to, excipients, binders, pH adjusters, disintegrants, absorption promoters, lubricants, colorants, flavoring agents, fragrances and the like.
[0048]
　When the RGMa inhibitor in the present invention is an antibody that recognizes RGMa, the antibody is used as an injection or infusion formulated with a pharmaceutically acceptable carrier in a parenteral route of administration, such as intravenous or intramuscular. It is preferably administered intradermally, intraperitoneally, subcutaneously or topically.
　Injections or infusions containing RGMa antibodies can be used as solutions, suspensions or emulsions. Examples of the solvent include distilled water for injection, physiological saline, glucose solution, and isotonic solution (for example, a solution of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, boar sand, propylene glycol, etc.). Etc. can be used. These solvents may be used alone or in combination of two or more.
[0049]
　Further, the injection or infusion solution may contain additives such as stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, preservatives and pH adjusters.
　As the stabilizer, for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, EDTA sodium, sodium citrate, dibutylhydroxytoluene and the like are used. Can be done.
　Examples of the solubilizing agent include alcohols (eg, ethanol, etc.), polyalcohols (eg, propylene glycol, polyethylene glycol, etc.), nonionic surfactants (eg, polysorbate 80 (registered trademark), HCO-50, etc.). Etc. can be used.
　As the suspending agent, for example, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
　As the emulsifier, for example, gum arabic, sodium alginate, tragant and the like can be used.
　As the pain-relieving agent, for example, benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
　As the buffer, for example, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, a carbonic acid buffer solution, a citrate buffer solution, a Tris buffer solution, or the like can be used.
　Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Sand or the like can be used.
　As the preservative, for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
　As the pH adjuster, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like can be used.
[0050]
　When the RGMa inhibitor in the present invention is a substance that suppresses the expression of RGMa such as siRNA (short interfering RNA), shRNA (short hairpin RNA), and antisense oligonucleotide of a gene expressing RGMa, a non-viral vector or It can be administered in the form of a viral vector.
　When the RGMa inhibitor is in the form of a non-viral vector, the method of administration is a method of introducing a nucleic acid molecule using liposomes (liposomal method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.). , Micro-injection method, method of transferring nucleic acid molecules into cells together with carriers (metal particles) with a gene gun, etc. can be improved.
　When siRNA or shRNA is administered to a living body using a viral vector, a viral vector such as recombinant adenovirus or retrovirus can be used. DNA that expresses siRNA or shRNA is applied to DNA viruses such as detoxified retrovirus, adenovirus, adeno-related virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, sinobis virus, Sendai virus, SV40, etc. By introducing and infecting cells or tissues with this recombinant virus, genes can be introduced into cells or tissues.
[0051]
　The preparation thus obtained is administered in an effective amount to, for example, humans and other mammals such as rats, mice, rabbits, sheep, pigs, cows, cats, dogs, and monkeys. , HAM can be prevented or treated.
　The dose is appropriately set in consideration of the purpose, the severity of the disease, the age, weight, sex, medical history, type of active ingredient, and the like of the patient.
Example
[0052]
[Example 1: Comprehensive comparative analysis for elucidation of the pathogenic mechanism and pathophysiology of HAM and adult T-cell leukemia / lymphoma (ALT)] As
　shown in FIG. 1, HTLV-1 infection has not been performed. Among the CD4-positive cells, the expression analysis of mRNA was performed for CADM1 which is an index of HTLV-1-infected cells and groups P, D, and N classified by CD7.
　HAM also analyzed 4 cases of CD4 positive cells and 4 cases of CD4 negative cells concentrated with magnetic beads from peripheral blood mononuclear cells of HAM patients. HTLV-1 non-infected healthy subjects (Normal) were also isolated in the same manner. Peripheral blood mononuclear cells are also referred to as PBMC. For ATL (smodering, chronic, acute disease types), PBMC consisting mainly of CD4 positive cells was used.
　A one-color microarray gene expression analysis method manufactured by Agilent Technologies Co., Ltd. was performed.
[0053]
　Comprehensive comparative analysis of nerve-related molecules revealed that RGMa was significantly expressed in CD4-positive cells of HAM patients.
　4 normal T cells (Normal.CD4), 4 CD4-positive T cells derived from HAM patients (HAM.CD4), 3 CD4-positive / CADM1-negative / CD7-positive T cells derived from healthy subjects (Normal.P), HTLV-1 Infected person CD4 positive / CADM1 negative / CD7 positive T cells 5 cases (P group), HTLV-1 infected person CD4 positive / CADM1 positive / CD7 positive T cells 5 cases (Group D), HTLV-1 infected person CD4 positive / CADM1 5 positive / CD7 negative T cells (N group), 3 acute / CDM1 positive / CD7 negative T cells (Acute.N), 21 healthy CD4 positive T cells (Normal.CD4.1) , 3 PBMCs derived from smoldering ATL patients (Smoldering), 20 PBMCs derived from chronic ATL patients (Chronic), 26 PBMCs derived from acute ATL patients (Acute), using Human Gene Expression 4x44K Microarray from Azilent Technology Co., Ltd. All gene expression data were obtained and standardized at the median value, and then the RGMa gene level was graphed.
　The graph plots the Log 2 values of the fluorescence intensity of the array . The CD4-positive T cell group from HAM patients was significantly elevated compared to all other groups (P <0.05). The graph is shown in FIG.
[0054]
[Example 2: Gene expression assay in CD4-positive HAM patients]
　CD4-positive T cells were isolated from PBMCs isolated from the peripheral blood of 5 HAM patients using a human CD4 + isolation kit (Miltenyi Biotec), and HTLV-1-infected cells. Was used as a cell population containing a large number of cells. Similarly, CD4-positive T cells were isolated from PBMCs of 4 healthy subjects and used as a control group.
　Total RNA was recovered from the isolated CD4-positive T cells, and cDNA was prepared using ReverTra Ace (Toyobo). Using the prepared cDNA, the difference in the expression level of RGMa between CD4-positive T cells in HAM patients and CD4-positive T cells in healthy subjects was analyzed by real-time PCR. 18s rRNA was used for internal control.
　The graph of the analysis result is shown in FIG. HD-CD4 + in the graph refers to the control group and HAM-CD4 + refers to the group of HAM patients.
[0055]
[Example 3: Analysis of RGMa-expressing cells in
　PBMC ] After treating PBMC in HAM patients with Fc block using Clear Back (MBL), anti-CD3-PECy7 (TONBO), CD4-FITC (eBioscience), CD14-PE ( eBioscience) Antibodies were added and staining was performed at 4 ° C. for 30 minutes.
　After washing the antibody-stained PBMC, 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 negative CD14 negative cells) CD3-CD14-) and CD3-negative CD14-positive cells (CD3-CD14 +) were isolated and recovered.
　Total RNA was recovered from each recovered cell, and cDNA was prepared using ReverTra Ace (Toyobo). Using the prepared cDNA, the difference in the expression level of RGMa between cell types was analyzed by real-time PCR. 18s rRNA was used for internal control. The graph of the analysis result is shown in FIG.
　In HAM-PBMC, it was found that the expression of RGMa was highest in CD3-positive CD4-positive cells (CD3 + CD4 +) in which many infected cells were present.
[0056]
[Example 4: Changes in RGMa expression associated with culture of PBMC in
　HAM patients ] PBMCs in two HAM patients were suspended in a medium (RPMI1640 medium (wako) containing 10% FBS (GIBCO)) and 1e5 cells each 96 well round bottom plate. After sowing for 10 wells, the cells were cultured for 1, 3, 5, and 7 days.
　Total RNA was extracted from PBMCs cultured for each period together with PBMCs on Day 0 that had not been cultured, and cDNA was prepared using ReverTra Ace (Toyobo). It is known that HTLV-1 virus is overexpressed in HAM patient PBMC when cultured, and the changes in RGMa expression associated with HAM patient PBMC culture, that is, with virus expression, using the prepared cDNA Analyzed by real time PCR. 18s rRNA was used for internal control. A graph showing the results of the analysis is shown in FIG.
[0057]
[Example 5: Analysis of H3K27me3 level on all promoters] About
　3 normal T cells (Normal T cells), 4 CD4-positive T cells derived from HAM patients (HAM), and 3 PBMCs derived from acute ATL patients (ATL) H3K27me3 levels on all promoters were obtained using SurePrint G3 Human Promoter 2x400K Microarray from Agilent Technologies, and standardized, and then H3K27me3 levels near -2,916 bp upstream from the transcription start point of the RGMa gene were graphed.
　The graph is shown in FIG. The graph plots the Log 2 values of the fluorescence intensity of the array . HAM50 and HAM123 in the figure refer to HAM patients who obtained CD4-positive T cells, respectively. It was suggested that the suppression of RGMa gene expression was released in CD4-positive T cells derived from HAM patients.
[0058]
[Example 6: Quantification of RGMa gene mRNA level]
　A lentiviral vector into which a cDNA encoding HTLV-1 Tax is inserted was introduced into a human CD4-positive T-cell leukemia cell line Jurkat, and the time was changed for 3 days after the introduction. The level of RGMa gene mRNA was measured by quantitative RT-PCR. RPL19 gene mRNA was also measured and used as an internal standard.
　A graph showing the result of quantification is shown in FIG. It was suggested that HTLV-1 virus causes RGMa expression.
[0059]
[Example 7: Tax-dependent induction of RGMa expression in HTLV-1-tax expression-inducing cell line JPX-9 cells]
　JPX9 cells were cultured in a medium (RPMI1640 medium containing 10% FBS) for 24 hours, and HTLV-1 After adding cadmium chloride (CdCl2; nacalai tesque), which induces -tax expression, to a final concentration of 20 μM, the cells were cultured for 1, 2, and 3 days. Cadmium chloride-treated and untreated JPX9 cells were FACS-analyzed for tax and RGMa protein expression.
　Cadmium chloride-treated and untreated JPX9 cells were washed and subjected to cell permeation treatment using Foxp3 / Transcription Factor Staining Buffer Kit (eBioscience). Then, an anti-Tax-FITC antibody (Lt-4: distributed by Professor Tanaka of Ryukyu University) was added and treated at 4 ° C. for 1 hour to stain the intracellularly expressed Tax protein. The stained Tax protein was detected by FACS analysis using Canto II.
　Cadmium chloride-treated and untreated JPX9 cells were washed, and anti-RGMa antibody (manufactured by Immuno-Biological Laboratory Co., Ltd. (IBL)) was added and treated at 4 ° C. for 30 minutes. Then, the cells were washed, anti-mouse IgG-PE antibody (BioLegend) was added, and the mixture was reacted at 4 ° C. for 30 minutes to stain the RGMa protein expressed in JPX9. The stained RG Ma protein was detected by FACS analysis using Canto II.
　FIG. 8 shows the analysis results of protein expression of Tax and RGMa.
[0060]
[Example 8: Examination of the effect of RPMI antibody on PBMC of HAM patients]
( Effect of RGMa antibody on spontaneous growth activity)
　Four HAM patient PBMCs were suspended in a medium (RPMI1640 medium containing 10% FBS) and 1e5 cells. Each seed was seeded on a 96-well round bottom plate, 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 under 37 ° C. and 5% CO 2 conditions for 7 days.
　The group in which nothing was added (Medium), the group in which Normal Goat IgG (Santa Cruz Biotechnology) was added at the same concentration (Normal IgG), and the group in which 1 μg / ml prednisolone (PSL) (Funakoshi) were added were used as controls.
　Six days after the start of culturing, 1 μ Ci 3 H-Thymidine was added to each well, and culturing was carried out under the conditions of 37 ° C. and 5% CO 2 for 16 hours. After that, the cultured cells are adsorbed on a glass filter (Printed Filtermat A PerkinElmer) using a cell harvester (Tomtec MH3 PerkinElmer), dried, and then impregnated with the solid scintillator Meltilex-A (PerkinElmer), and MicroBeta (WALLAC MicroBeta TriLux 1450). -021) was taken up by cells using 3The amount of H-Thymidine was measured. The relative value of each group was calculated with the average of 3 H-Thymidine counts in the Medium group of PBMC of each HAM patient as 100%, and the average value of the 3 H-Thymidine uptake rate of 4 HAM patients was calculated . The results are shown in FIG.
[0061]
(Effect of RGMa antibody on changes in HTLV-1 provirus amount)
　Four HAM patient PBMCs were suspended in medium (RPMI1640 medium containing 10% FBS) and 1e5 cells were seeded on a 96-well round bottom plate, and the final concentration was 10 μg. RGMa antibody (manufactured by R & D Systems) was added so as to be / ml, and the cells were cultured in a total of 0.1 ml of culture medium at 37 ° C. under 5% CO 2 conditions for 7 days.
　The group in which nothing was added (Medium), the group in which Normal Goat IgG (Santa Cruz Biotechnology) was added at the same concentration (Normal IgG), and the group in which 1 μg / ml prednisolone (PSL) (Funakoshi) were added were used as controls.
　Seven days after the start of the culture, genomic DNA was extracted from the cell mass obtained by centrifuging and removing the supernatant. Using the extracted genomic DNA, the amount of HTLV-1 provirus (infected cell rate) was measured by real-time PCR.
　Assuming that the amount of HTLV-1 provirus in the Medium group of PBMC of each HAM patient is 100%, the relative value of the amount of HTLV-1 provirus in each group is calculated, and the average value of the amount of HTLV-1 provirus in 4 HAM patients is calculated. I asked. The results are shown in FIG.
[0062]
(Effect of RGMa antibody on CXCL10 production) In
　order to analyze the effect of RGMa antibody on CXCL10 production from HAM patient PBMC, 4 HAM patient PBMCs were suspended in medium (RPMI1640 medium containing 10% FBS) and 1e5 cells each. The cells were seeded on a 96-well round bottom plate, 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 under 37 ° C. and 5% CO 2 conditions for 7 days.
　The group in which nothing was added (Medium), the group in which Normal Goat IgG (Santa Cruz Biotechnology) was added at the same concentration (Normal IgG), and the group in which 1 μg / ml prednisolone (PSL) (Funakoshi) were added were used as controls.
　Seven days after the start of the culture, the culture solution was centrifuged to collect only the culture supernatant. The CXCL10 concentration in the culture supernatant was measured by a flow cytometer FACSCantoII (BD Biosciences) using a Cytokine Beads Array kit (BD Biosciences).
　Assuming that the CXCL10 concentration in the culture medium of the Medium group was 100%, the relative value of the CXCL10 concentration in the culture medium of each group in each group was calculated, and the average value of the CXCL10 concentration in 4 HAM patients was calculated. The results are shown in FIG.
[0063]
(Effect of RGMa antibody on cytokine production of
　HAM patient PBMC ) In order to analyze the effect of RGMa antibody on the production of various cytokines in HAM patient PBMC, 4 HAM patient PBMCs were cultivated in medium (RPMI1640 medium containing 10% FBS). Sow 1e5 cells on a 98 well round bottom plate, add RGMa antibody (manufactured by R & D Systems) to a final concentration of 10 μg / ml, and culture in a total of 0.1 ml of culture medium under 37 ° C. and 5% CO 2 conditions for 7 days. did.
　The group in which nothing was added (Medium), the group in which Normal Goat IgG (Santa Cruz Biotechnology) was added at the same concentration (Normal IgG), and the group in which 1 μg / ml prednisolone (PSL) (Funakoshi) were added were used as controls.
　Seven days after the start of the culture, the culture solution was centrifuged to collect only the culture supernatant. The concentrations of IFNγ, TNF, IL-2, and IL-10 in the culture supernatant were measured by a flow cytometer FACSCantoII (BD Biosciences) using a Cytokine Beads Array kit (BD Biosciences).
　The relative value of the cytokine concentration under each culture condition was calculated with each cytokine concentration in the culture medium of the Medium group as 100%, and the average value of 4 HAM patients was calculated.
[0064]
[Example 9: Induction of apoptosis of nerve cell line by HAM-PBMC]
　After seeding the nerve cell line NB-1 or SK-N-AS on a 6-well plate and culturing for 24 hours, a healthy subject (HD) or HAM patient PBMC is added. Co-culture was performed. 48 hours after the start of co-culture, the PBMC added together with the medium was removed, and the nerve cell line was recovered after washing with PBS.
　Each recovered nerve cell line was analyzed by the TUNEL method (MEBSTAIN Apoptosis TUNEL Kit Direct (MBL)), which specifically detects cells that have undergone DNA fragmentation due to apoptosis. FIG. 13 shows the analysis results. The X-axis in the histogram shows the intensity of positive DNA fragmentation. HAM-derived cells more strongly induced apoptosis in nerve cell lines than HD-derived cells.
　Specifically, cell death was analyzed according to the following .

　Nerve cell lines NB-1 and SK-N-AS were seeded on 6-well plates and cultured for 24 hours.
　Next, HD or HAM-PBMC was added (twice the number of seeded nerve cell lines), and the cells were cultured for 48 hours.
　Then, the cells were fixed with 4% paraformaldehyde and subjected to cell permeation treatment with 70% ethanol.
　For DNA nick end labeling, suspend cells in TdT solution 20uL (TdT buffer II: TdT: FITC-dUTP = 18: 1: 1) of MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) and react at 37 ° C for 60 minutes. After that, FACS analysis was performed.
[0065]
[Example 10: Suppressive effect of RGMa antibody on neuronal cell line apoptosis induced by HTLV-1 Tax expressing T cell line]
　Neuronal cell line NB-1 was seeded on a 6-well plate, cultured for 24 hours, and then unstimulated JPX9 ( JPX9 (-)) or 20 μM cadmium chloride was added for 24 hours to induce Tax expression, and JPX9 (JPX9 (+ CdCl2)) was added and co-cultured. In addition, Normal Mouse IgG2b (MBL) or RGMa antibody (IBL) was added to the co-culture of NB-1 cells and (JPX9 (+ CdCl 2 ) to a final concentration of 10 μg / ml. 48 hours after the start of co-culture. JPX9 added together with the medium was removed, and the nerve cell line was recovered after washing with PBS. For each recovered nerve cell line, the TUNEL method (MEBSTAIN Apoptosis) that specifically detects cells causing DNA fragmentation due to apoptosis. The analysis was performed by TUNEL Kit Direct (MBL)). The analysis results are shown in FIG. 14. The X-axis in the histogram shows the intensity of positive DNA fragmentation.
　Specifically, according to the following . Cell death was analyzed.
A
　nerve cell line (NB-1) was seeded on a 6-well plate and cultured for 24 hours.
　Next, JPX9 (-) or JPX9 (+ CdCl 2 ) was NB-1. It was added to cells and co-cultured. The number of JPX9 (-) or JPX9 (+ CdCl 2 ) cells was twice the number of seeded NB-1 cells . JPX9 (+ CdCl 2 ). Regarding 2 ), in order to remove cadmium chloride, the cells were washed 3 times with 10 ml of medium and added to NB-1 cells.
　Subsequently, NB-1 and cells (JPX9 (+ CdCl 2 added) of co-culture in Normal Mouse IgG2b (MBL) or anti RGMa antibodies (IBL) to a final concentration of 10 [mu] g / ml, and cultured for 48 hours.
　After that , Cell-fixed with 4% paraformaldehyde, cell permeabilized with 70% ethanol, stained with anti-CD45-V450 antibody.
　MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) for DNA nick end labeling . Cells were suspended in TdT solution 20uL (TdT buffer II: TdT: FITC-dUTP = 18: 1: 1), reacted at 37 ° C for 60 minutes, and then FACS analysis was performed.
[0066]
　All contents described in the publications, patent documents and non-patent documents cited and described in the present specification are incorporated in the present specification as they are for reference.
The scope of the claims
[Claim 1]
　A therapeutic or prophylactic agent for HTLV-1-related myelopathy (HAM), including RGMa inhibitors.
[Claim 2]
　The therapeutic or prophylactic agent for HAM according to claim 1, wherein the RGMa inhibitor is an antibody that recognizes RGMa.
[Claim 3]
　A method of treating HAM, including administering a pharmacologically effective amount of an RGMa inhibitor to a patient with HTLV-1-related myelopathy (HAM) who requires it.
[Claim 4]
　The method for treating HAM according to claim 3, wherein the RGMa inhibitor is an antibody that recognizes RGMa.