Agent for treatment or prevention of HTLV-1-associated myelopathy (HAM), and ham treatment method

Abstract

To provide a therapeutic or prophylactic agent for HTLV-1-associated myelopathy (HAM).SOLUTION: A therapeutic or prophylactic agent for HTLV-1-associated myelopathy (HAM) comprising an RGMa-inhibiting substance is provided, wherein the RGMa-inhibiting substance is an antibody that recognizes RGMa. A method for treating HAM is also provided, the method comprising administering a pharmacologically effective amount of an RGMa-inhibiting substance to a HAM patient in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic or preventive agent for HTLV-1 associated myelopathy (HAM). It also relates to methods of treatment. [Background technology]

[0002] HTLV-1 (Human T-cell Lukemia Virus Type 1) is a virus that causes leukemia in the blood. It is a virus that infects T cells (mainly CD4-positive T cells), a type of white blood cell. HTLV-1-infected T cells cause chronic inflammation in the spinal cord, resulting in spinal cord neuronal damage. It causes damage and degeneration, leading to spastic spinal cord paralysis. The spastic spinal cord paralysis caused by HTLV-1 is called HTLV-1-associated myelopathy (HAM). . Symptoms of HAM include paralysis of both legs, pain, difficulty urinating, and stubborn stools due to nerve tissue damage. If these symptoms progress, the patient may be confined to a wheelchair or bedridden. HAM is one of the diseases designated as an intractable disease in Japan. Currently, there are no effective treatments for HAM. Therapeutic efficacy has not been established, and symptomatic treatment is the only option. One of the treatment methods is a treatment using anti-CCR4 antibody, which reduces HTLV-1 infected cells. It has been proven that it reduces spinal cord inflammation caused by HAM and improves symptoms (Non-patent Document 1, Patent document 5).

[0003] RGMa protein is a protein involved in axon guidance of neurons in the retina and hippocampus, neural tube closure, etc. It is a member of the protein RGM (Repulsive Guidance Molecule) family. The function of RGM is It is known that the functions are not limited to these and have a variety of other functions.

[0004] For example, Patent Document 1 discloses that RGM is expressed in bone marrow-derived dendritic cells (BMDCs), and that CD4 + T cells and CD11b + RGM receptors are expressed on macrophages, and RGM binds to these receptors. By doing so, CD4 + T cells and CD11b + Enhancement of macrophage cell adhesion activity Patent Document 1 also discloses that anti-RGM neutralizing antibodies can be used in a multiple sclerosis model. The results showed that the IL-1111 gene was able to alleviate both the clinical symptoms and tissue lesions of the mice. It has been disclosed that antigen-specific and non-specific T cell activation was attenuated in splenocytes.

[0005] Patent Documents 2 and 3 disclose the receptor for RGMa, neogenin, and bone morphogenetic proteins 2 and 4 (BM A neutralizing monoclonal antibody against RGMa that selectively inhibits RGMa binding to P-2, BMP-4 The neutralizing monoclonal antibody is capable of inhibiting the growth of damaged and inflamed tissue. Human central nervous system, specifically, multiple sclerosis, acute spinal cord injury, post-traumatic brain injury, Huntington's disease In neurodegenerative diseases such as rheumatoid arthritis, Parkinson's disease, and Alzheimer's disease, It is believed to be able to promote nerve regeneration and regrowth of the lon junctions.

[0006] Patent Document 4 states that RGMa is effective in treating the central nervous system of humans suffering from traumatic brain injury or ischemic stroke. It is localized to myelin, fresh lesions, and mature scar tissue, making it useful for diagnosing these neurodegenerative diseases. In order to achieve this, a method for detecting and quantifying RGMa fragments has been disclosed. 4. Neurodegenerative diseases and disorders for which RGMa fragments are detected include multiple sclerosis, Parkinson's disease, and the like. Johnsson's disease, Alzheimer's disease, Tay-Sachs disease, Niemann-Pick disease, Gaucher disease, Lehler syndrome, Huntington's disease, amyotrophic lateral sclerosis, idiopathic inflammatory demyelinating disease, vitamins B12 deficiency, central pontine myelinolysis, spinal cord fistula, transverse myelitis, Devic's disease, progressive multifocal Leukoencephalopathy, optic neuritis, spinal cord injury, traumatic brain injury, stroke, glaucoma, diabetic retinopathy, age-related yellowing Macular degeneration and leukodystrophy are mentioned. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2011 / 071059 [Patent Document 2] Patent Publication No. 2014-138599 [Patent Document 3] Patent Publication No. 2016-175897 [Patent Document 4] Special table number 2017-526930 [Patent Document 5] Patent Publication No. 2010-100578 [Non-patent literature]

[0008] [Non-Patent Document 1] N Engl J Med, 2018, 378, 529-538. Summary of the Invention [Problem to be solved by the invention]

[0009] According to the method using anti-CCR4 antibody disclosed in Non-Patent Document 1 and Patent Document 5, HAM disease can be cured. Although anti-CCR4 antibodies can improve the condition of HAM patients, their effectiveness is limited in patients with advanced neurodegeneration. Therefore, there is a need for better methods to treat HAM.

[0010] The present invention has been made in view of the above problems, and provides a therapeutic agent capable of treating HAM. The present invention aims to provide a therapeutic agent and a treatment method. [Means for solving the problem]

[0011] As a result of extensive research to solve the above problems, the present inventors have discovered a substance that inhibits RGMa. found that it is effective in treating HAM, and thus completed the present invention.

[0012] That is, the present invention is as follows. [1] A therapeutic or preventive agent for HTLV-1-associated myelopathy (HAM), comprising an RGMa inhibitor. [2] The method for treating or preventing HAM according to [1], wherein the RGMa inhibitor is an antibody that recognizes RGMa. Agent. [3] Pharmacologically effective doses of RGMa inhibitors are administered to patients with HTLV-1-associated myelopathy (HAM) in need thereof. A method for treating HAM, comprising administering [4] The method for treating HAM described in [3], wherein the RGMa inhibitor is an antibody that recognizes RGMa. [Effects of the Invention]

[0013] According to the present invention, HAM, which is an intractable disease, can be treated. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows groups P, D, and N of CD4-positive T cells classified by CADM1, an indicator of HTLV-1-infected cells, and CD7. [Figure 2]Normal T cells (Normal.CD4), HAM patient-derived CD4-positive T cells (HAM.CD4), healthy person-derived CD4-positive / CADM1-negative / CD7-positive T cells (Normal.P), HTLV-1 infected person CD4-positive / CADM1-negative / CD7-positive T cells (P group), HTLV-1 infected person CD4-positive / CAD M1-positive / CD7-positive T cells (group D), HTLV-1 infected CD4-positive / CADM1-positive / CD7-negative T cells (group N), acute ATL patient CD4-positive / CADM1-positive / CD7-negative T cells (Acute.N), healthy human CD4-positive T cells (Normal.CD4.1), PBMCs derived from smoldering ATL patients. (Smoldering), chronic type ATL patient-derived PBMC (Chronic), and acute type ATL patient-derived PBMC (Acute). [Figure 3] FIG. 1 shows the expression levels of RGMa in CD4-positive T cells of HAM patients and healthy individuals. [Figure 4] FIG. 1 shows the results of analyzing the expression levels of RGMa among cell types in PBMCs from HAM patients. [Figure 5] FIG. 1 shows changes in RGMa expression associated with HTLV-1 virus expression in cultured PBMCs from HAM patients. [Figure 6] FIG. 1 shows the H3K27me3 level near −2,916 bp upstream from the transcription start site of the RGMa gene. [Figure 7] FIG. 1 shows the RGMa gene mRNA levels when a lentiviral vector carrying cDNA encoding HTLV-1 Tax was introduced into the human CD4-positive T-cell leukemia cell line Jurkat. [Figure 8] FIG. 1 shows the results of analyzing the protein expression of Tax and RGMa in the HTLV-1-tax expression-inducible cell line JPX-9. [Figure 9] FIG. 10 shows the results of the effect of RGMa antibodies on spontaneous proliferative activity in relation to the effect of RGMa antibodies on PBMCs from HAM patients. [Figure 10]FIG. 10 shows the effect of RGMa antibodies on changes in HTLV-1 proviral load in relation to the effect of RGMa antibodies on PBMCs from HAM patients. [Figure 11] FIG. 10 shows the results of the effect of RGMa antibodies on CXCL10 production in relation to the effect of RGMa antibodies on PBMCs from HAM patients. [Figure 12] Regarding the effect of RGMa antibodies on PBMCs of HAM patients, this figure shows the results of the effect of RGMa antibodies on cytokine production in PBMCs of HAM patients. [Figure 13] FIG. 10 is a diagram showing that HAM-PBMCs induced apoptosis in neuronal cell lines. [Figure 14] These figures show the results of the inhibitory effect of RGMa antibody on apoptosis induction in neuronal cell lines by HTLV-1-tax-induced cell lines. (a) is a FACS plot of NB-1 cells co-cultured with unstimulated JPX-9 cells. (b) is a FACS plot of NB-1 cells co-cultured with CdCl2-stimulated JPX-9 cells (HTLV-1-tax-expressing cells). (c) is a FACS plot of the same results as in (b) but with the addition of a control antibody. (d) is a FACS plot of the same results as in (b) but with the addition of an anti-RGMa antibody. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, the embodiments of the present invention will be described in detail. The present invention is not limited to the above embodiment, and various modifications can be made within the scope of the present invention.

[0016] The present invention relates to a therapeutic or preventive agent for HTLV-1-associated myelopathy (HAM), which comprises an RGMa inhibitor. The GMa inhibitor may be a substance that acts on RGMa itself to inhibit the activity of RGMa, It may also be a substance that suppresses the expression of a. Examples of RGMa inhibitors include compounds that have the activity of inhibiting RGMa and compounds that recognize RGMa. Examples of antibodies include antibodies that bind to the antibody.

[0017] Furthermore, examples of RGMa inhibitors include siRNA (short interfering RNA) for the gene that expresses RGMa. RGs such as ring RNA (SRRNA), shRNA (short hairpin RNA), and antisense oligonucleotides Examples include substances that suppress the expression of Ma. Examples of the RGMa gene include the human RGMa gene consisting of the base sequence shown in SEQ ID NO: 1, Examples include, but are not limited to, the RGMa gene consisting of the base sequence shown in SEQ ID NO: 2. The base sequence information of RGMa genes from various organisms is available in databases such as GenBank. It can be obtained from the

[0018] siRNA is a double-stranded RNA that can suppress the expression of the target RGMa gene. The length of the base sequence in A is not particularly limited, but is preferably less than about 30 bases. More preferably, it is about 19 to 27 bases, and even more preferably about 21 to 25 bases. shRNA contains a partially palindromic base sequence in the single-stranded RNA, forming a double-stranded RNA molecule. It has a short hairpin structure of about 20 base pairs or more and has an overhang at the 3' end. After being introduced into cells, shRNA is degraded into fragments of approximately 20 bases. In the same way as siRNA, it can suppress the expression of the target RGMa gene.

[0019] siRNA and shRNA can be artificially chemically synthesized. For example, antisense and antisense fragments can be synthesized from template DNA using T7 RNA polymerase and a T7 promoter. Both sense and nonsense RNAs can be synthesized in vitro.

[0020] The antisense oligonucleotide is a sequence of approximately 30 consecutive bases in the DNA sequence of the RGMa gene. Any nucleotide that is complementary to or hybridizes with the base sequence of DN It may be either A or RNA. It may also be modified as long as it does not impair its function. Antisense oligonucleotides can be synthesized by conventional methods, for example, It can be easily synthesized using a commercially available DNA synthesizer.

[0021] The RGMa inhibitor contained in the therapeutic or preventive agent for HAM of the present invention is an antibody that recognizes RGMa. Hereinafter, an antibody that recognizes RGMa may also be referred to as an RGMa antibody. The RGMa antibody of the present invention is Any antibody that binds to RGMa and inhibits its activity may be used. For example, Examples include antibodies that prevent RGMa from binding to the RGMa receptor.

[0022] The RGMa antibody of the present invention may be a monoclonal antibody or a polyclonal antibody. The antibody of the present invention may be any of the isotypes IgG, IgM, IgA, IgD, and IgE. It may be a type. The RGMa antibody of the present invention may be, for example, a mouse antibody, a human CDR-grafted antibody, or a human chimeric antibody. The antibody may be an antibody, a humanized antibody, or a fully human antibody, or may be a small molecule antibody. These antibodies may be used singly or in combination of two or more.

[0023] Human CDR-grafted antibodies are antibodies in which the CDRs of non-human animal antibodies have been replaced with the CDRs of human antibodies. Human chimeric antibodies are antibodies that combine the variable region derived from an antibody of a non-human animal with the variable region derived from a human antibody. Humanized antibodies are antibodies that have constant regions derived from antibodies of animals other than humans. In this case, a portion derived from a human antibody is incorporated, leaving some highly safe regions. This concept includes human chimeric antibodies and human CDR-grafted antibodies.

[0024] As used herein, the term "small molecule antibody" refers to a fragment of an antibody or a molecule bound to a fragment of an antibody. It means a product of the synthesis of an antibody that recognizes the same epitope as the original antibody. Fab, consisting of VL, VH, CL, and CH1 regions; two Fabs joined at the hinge region; F(ab')2 linked by a sulfide bond; Fv consisting of VL and VH; V In addition to scFv, a single-chain antibody in which L and VH are linked by an artificial polypeptide linker, Examples include, but are not limited to, sdFv, diabody, and sc(Fv)2.

[0025] The RGMa antibody used in the present invention is produced using RGMa or a fragment thereof as an immunogen. The film can be prepared by a known method. Whether the obtained antibody is an RGMa antibody can be confirmed using RGMa activity as an indicator. Examples of RGMa include human RGMa containing the amino acid sequence shown in SEQ ID NO: 3, and human RGMa containing the amino acid sequence shown in SEQ ID NO: 4. Examples of such RGMa include those containing the amino acid sequence shown in Table 1. RGMa derived from various organisms can be used as immunogens. The amino acid sequence of RGMa can be found in the publicly known Protein Data B It can be obtained from ank etc.

[0026] When the RGMa antibody used in the present invention is a polyclonal antibody, for example, First, RGMa or a fragment thereof is used as an antigen. It is dissolved in acid-buffered saline (also referred to as PBS) and, if necessary, contains a conventional adjuvant, e.g. For example, a mixture of an appropriate amount of Freund's complete adjuvant is used as an immunogen in mice, rats, Mammals such as rabbits, goats, and horses are immunized. The immunization method is not particularly limited, but may be, for example, For example, subcutaneous or intraperitoneal injection may be given once or twice or more at appropriate intervals. Then, blood is collected from the immunized animals and serum is separated according to a conventional method to obtain polyclonal antibodies. The fraction can be purified to obtain the desired product. When the RGMa antibody used in the present invention is a monoclonal antibody, the monoclonal The cloned antibodies are produced by immunoglobulin-derived immunoglobulins (IG-IgG) from the immunized mammal, such as spleen cells and myeloma cells. and fusing them to obtain hybridomas, and collecting antibodies from the culture of the hybridomas. The monoclonal antibody can be obtained by hybridizing the antibody gene. The vector is then cloned from the doma, inserted into an appropriate vector, and then introduced into a host cell. Recombinant monoclonal antibodies can also be produced using gene recombination techniques. Additionally, the monoclonal antibodies can also be produced using phage display methods. .

[0027] The RGMa antibody used in the present invention is, for example, the antibody described in Yamashita, T., Mueller, BK & Hata, K. Neogenin and repulsive guidance molecules ignaling in the central nerv ous system. Curr. Opin. Neurobiol. 17, 29-34(2007); JP 2014-138599 Publication; JP 2016-175897 Publication; JP 2017-526930 Publication; International Publication Antibodies disclosed in Patent Publication No. 2016 / 175236 and Patent Publication No. 2015-508061 You can use your body. Furthermore, the RGMa antibody used in the present invention is, for example, It is also available as a commercially available product from IBL, R&D Systems, etc.

[0028] The RGMa antibody contains the following antigen-binding domains: 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; Preferably, the CDR comprises at least one CDR comprising an amino acid sequence selected from the group consisting of: The sequence identity is preferably 80% or more, more preferably 90% or more. In this specification, amino acids are represented by conventional one-letter or three-letter symbols. This may happen.

[0029] Complementarity determining regions (CDRs) are the variable regions of immunoglobulin molecules that form the antigen binding site. The region that forms the immunoglobulin molecule is also called the hypervariable region, and it is the region that contains the amino acid sequence that is specific to each immunoglobulin molecule. CDRs are the parts with the largest sequence changes. There are three CDRs (CDR -L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, CDR-H3 In this application, the CDRs of an immunoglobulin molecule are numbered according to the Kabat numbering system. (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of nt of Health and Human Services, NIH, USA). In addition, in the antibody of the present invention, which is defined by the amino acid sequences of the light chain and the heavy chain, The amino acid sequence of the DR is unchanged from the specified sequence, and the amino acid sequence other than the CDR is changed by mutation, etc. When there is a mutation other than in the CDR, the homology is preferably 90% or more. .

[0030] RGMa antibodies are also disclosed as SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15 and sequences thereof. a modified CDR amino acid sequence having at least 50% sequence identity with one of the Preferably, the amino acid sequence further comprises at least one CDR comprising the amino acid sequence of The sequence identity is preferably 80% or more, more preferably 90% or more.

[0031] RGMa antibodies may comprise a set of variable domain CDRs shown in Table 1, or at least three of the CDRs. One has at least 50%, preferably 80%, more preferably 90% sequence identity with the parent sequence. at least one variable domain selected from a set of variable domains, each of which is a modified CDR amino acid sequence having the same identity as the original CDR amino acid sequence; More preferably, the RGMa antibody comprises at least three CDRs. More preferably, it comprises at least two sets of CDRs. The variable domain CDR set is a combination of a VH5F9 set and a VL5F9 set, or , a combination of a VH8D1 set and a VL8D1 set.

[0032] [Table 1]

[0033] The RGMa antibody may comprise a framework region. The amino acid sequences are SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40. The amino acid sequence may be one type alone or a combination of two or more types. The RGMa antibody also contains the heavy chain variable domain and the light chain variable domain of SEQ ID NOs: 41 and 42. 2, 43, 44, 45, 46, 47, 48, and 49. variable domain; and / or at least one selected from SEQ ID NOs: 50, 51 and 52 It preferably comprises a light chain variable domain.

[0034] In addition, the binding site of RGMa antibodies that causes RGMa inhibition is For example, in human RGMa, but not limited to, 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) It is preferred that the nucleotide sequence of the nucleotide sequence be linked to one or more peptides having the amino acid sequence represented by the formula: The RGMa antibody has a peptide having the amino acid sequence represented by SEQ ID NO: 53 and / or SEQ ID NO: 54. More preferably, the peptide is bound to a peptide represented by SEQ ID NO: 53 and / or SEQ ID NO: 54. a peptide having the amino acid sequence represented by SEQ ID NO: 55 and / or SEQ ID NO: 56; It is more preferable to bind to a peptide having the amino acid sequence. RGMa antibodies are antibodies that bind to amino acids 250 and above in the amino acid sequence of human RGMa. It is preferable that: RGMa antibodies are represented by SEQ ID NO: 53 and SEQ ID NO: 54, and also SEQ ID NO: 55 or SEQ ID NO: 56. It is more preferable that the antibody binds to a peptide having an amino acid sequence corresponding to the amino acid sequence of the antibody.

[0035] RGMa antibodies can be prepared by combining RGMa proteins or partial fragments thereof (e.g., one of SEQ ID NOS: 53-57). A polyclonal antibody is obtained by immunizing a mammal such as a mouse with the antigen (a fragment containing the above). Clonal and monoclonal antibodies, chimeric antibodies produced using genetic recombination technology and humanized antibodies, as well as human antibodies produced using human antibody-producing transgenic animals, etc. It may be an antibody or the like. In the present invention, when RGMa antibodies are administered to humans as pharmaceuticals, they are considered to be effective in preventing side effects. Preferably, the antibody is humanized or human. RGMa antibodies can be prepared by combining RGMa proteins or partial fragments thereof (e.g., one of SEQ ID NOS: 53-57). Polyclonal and / or monoclonal antibodies that recognize the above fragments as antigens It may be used as a body fragment or a low molecular weight antibody.

[0036] In addition to those listed in Table 1, RGMa antibodies also contain light chain complementarity-determining region 1 (LCDR1), light chain complementarity-determining region 2 (LCDR3), and light chain complementarity-determining region 3 (LCDR4). Complementarity-determining region 2 (LCDR2), light chain complementarity-determining region 3 (LCDR3), heavy chain complementarity-determining region Complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR 3) Each amino acid sequence is 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, LCDR1: RSSQSLVHSNGNTYLH (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 including HCDR3:SFG, It may be an isolated RGMa antibody, or an antigen-binding fragment thereof. In each CDR sequence, one or several amino acids are substituted, deleted, and / or added. For example, one or two amino acids may be substituted, deleted, and / or added. That's fine.

[0037] The RGMa antibody has 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. Examples of such antibodies include those having the amino acid sequences: Substitution, deletion, addition or deletion of 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 CDRs, but not into the CDRs. It is preferred that the ion beam be introduced into the region of

[0038] It may be a mouse / human chimeric antibody in which the constant region is derived from a human. The antibody used in the present invention has the amino acid sequence of SEQ ID NO: 77 (variable region: 1-107) in the light chain and the amino acid sequence of SEQ ID NO: 77 in the heavy chain. An example of such an antibody is an antibody having the amino acid sequence of SEQ ID NO: 78 (variable region is 1 to 116). In the amino acid sequences represented by these SEQ ID NOs, one or several amino acids (1 to 20, There may be 1 to 10 or 1 to 5 substitutions, deletions, additions or insertions. Although the deletions and additions may be introduced into the CDRs, they are preferably introduced into regions other than the CDRs.

[0039] It may also be a humanized antibody, in which the CDRs are human-derived. It has any of the amino acid sequences of numbers 70 to 87 (the variable region is up to 116 residues on the N-terminal side). and the light chain is selected from any of SEQ ID NOS: 88 to 94 (the variable region is from residues 1 to 107 on the N-terminal side). The amino acid sequences represented by these SEQ ID NOs are exemplified by antibodies having the following amino acid sequences: or substitution, deletion, addition or insertion of several amino acids (1 to 20, 1 to 10 or 1 to 5) Such substitutions, deletions, and additions may be introduced into the CDRs, but may also be introduced into regions other than the CDRs. Preferably, it is introduced into the region.

[0040] The heavy chain amino acid sequence and the light chain amino acid sequence may be any combination of these, but preferably The antibody has an amino acid sequence of SEQ ID NO: 84 in its heavy chain and an amino acid sequence of SEQ ID NO: 88 in its light chain. The amino acid sequence of SEQ ID NO: 84 corresponding to the heavy chain variable region is The amino acid sequence corresponding to the light 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. can be.

[0041] The heavy chain variable region (VH) of the RGMa antibody is EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSI VYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS (SEQ ID NO: 95) or an amino acid sequence having at least 90% identity to said amino acid sequence, The light chain variable region (VL) DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQP EDFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 96) or an isolated amino acid sequence having at least 90% identity to said amino acid sequence. RGMa antibodies, or antigen-binding fragments thereof, are preferred.

[0042] The RGMa antibody has 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. Examples of such antibodies include those having the amino acid sequences: Substitution, deletion, addition or deletion of 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 CDRs, but not into the CDRs. It is preferred that the ion beam be introduced into the region of It may be a mouse / human chimeric antibody in which the constant region is human-derived, and the rest of the antibody is human-derived. It may also be a humanized antibody.

[0043] The anti-RGMa antibody is an isolated RGMa antibody selected from the following (a1) to (h1): or an antigen-binding fragment thereof. (a1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, and the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the sequence and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99 HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102 an anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a variable region; (b1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, and the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 103 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 104; HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 103; an anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a chain variable region; (c1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, and the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105; HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 103; an anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a chain variable region; (d1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, and the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 105 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 106; HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 103; an anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a chain variable region; (e1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107; HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 103; an anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a chain variable region; (f1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 107 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 108; HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 103; an anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a chain variable region; (g1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, and the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109; HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 103; an anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a chain variable region; and (h1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, the amino acid sequence set forth in SEQ ID NO: 98 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109; HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a heavy chain comprising an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 102 and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 103; An anti-RGMa antibody, or an antigen-binding fragment thereof, comprising a chain variable region.

[0044] The RGMa antibody is an antibody having the amino acid sequence described above, and is preferably a humanized antibody. It is also preferable that the antibody has a human IgG constant region.

[0045] HAM can be treated or prevented by the RGMa inhibitor of the present invention. As shown in the Examples below and in Figure 13, in HAM patients, neurons were stimulated by cells derived from HAM patients. Cell death, ie, spinal cord tissue damage and degeneration, is induced. As a result of the investigations conducted by the present inventors, as shown in the Examples below, it was found that the main HTLV-1 infection in HAM patients RGMa is significantly expressed in CD4-positive T cells, which are immunoreactive cells, and RGMa expression is associated with HAM. Furthermore, the results of a test to inhibit RGMa using an RGMa antibody showed that RGMa inhibits CX It was found to be involved in the production of CL10, IL-2, and IL-10. CXCL10 is a protein produced by HAM pathogenic HTLV-1-infected T cells in response to IFN-γ It is known to induce the pathology of HAM (Brain 2013) and to promote the progression of HAM symptoms. It is known that the rate of HAM is strongly correlated with the rate of HAM (PLoS Negl TropDis 2013). When RGMa antibody was applied to the cells, the production of CXCL10 was suppressed. It is one of the compounds that acts to suppress inflammation. When the treatment was performed, IL-10 production was significantly increased. To achieve a true therapeutic effect for HAM, it is necessary to suppress damage to nerve cells. HTLV-1-tax expression levels were high in HTLV-1-infected cells from patients with AM (Blood 2002), and HTLV-1-tax It has been shown that HTLV-1 is important in the pathogenesis of the disease (J Clin Invest 2014). -1-tax induces RGMa expression, and HAM patient-derived cells with high RGMa expression levels induce neuronal cell death. Importantly, RGMa inhibitors were shown to prevent neuronal cell death induced by HTLV-1-tax expressing cells. It was shown that quality is suppressed. As described above, RGMa inhibitors suppress the induction of inflammatory pathology in HAM and inhibit the proliferation of HAM patient cells. Furthermore, the inflammatory response caused by HAM can be suppressed, and thus HAM can be treated or prevented. GMa inhibitors not only suppress the inflammatory response specific to HAM, but also inhibit the pathogenic H cells in HAM patients. It can suppress neuronal cell death caused by TLV-1-tax expressing cells, and therefore can be used to treat or prevent HAM. It is possible.

[0046] As described above, RGMa inhibitors can treat or prevent HAM, and the present invention provides a method for treating HAM. RGMa inhibitors for use in treating HAM; pharmaceutical compositions for use in treating HAM; Use of an RGMa inhibitor for the manufacture of a medicament for the treatment of HAM; Use of an RGMa inhibitor in the manufacture of a medicament for the treatment of HAM RGMa inhibitors for use in the manufacture of a therapeutic medicament; The present invention provides a method for treating or preventing HAM, comprising administering to a subject:

[0047] The therapeutic or preventive agent for HAM of the present invention comprises an RGMa inhibitor and further comprises a pharmaceutically acceptable carrier. The composition may be formulated by appropriately blending the composition with an active ingredient and / or additives. Examples of the formulation include tablets, coated tablets, pills, powders, granules, capsules, Oral preparations such as liquids, suspensions, and emulsions; parenteral preparations such as injections, infusions, suppositories, ointments, and patches; Examples include: The blending ratio of carriers or additives is within the range normally used in the pharmaceutical field. It may be set appropriately based on the above. The carrier is not particularly limited, but may be, for example, water, physiological saline, or other aqueous solvents. The additives are not particularly limited, but include: Excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances etc.

[0048] When the RGMa inhibitor of the present invention is an antibody that recognizes RGMa, the antibody can be pharmaceutically acceptable. Parenteral administration routes, e.g., intravenous, as injections or infusions formulated with an acceptable carrier. Preferably, the administration is intravenously, intramuscularly, 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, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, These solvents include solutions of methyl alcohol, borax, propylene glycol, etc. may be used alone or in combination of two or more.

[0049] Furthermore, the injection or infusion solution may contain stabilizers, solubilizing agents, suspending agents, emulsifying agents, soothing agents, The composition may contain additives such as buffers, preservatives, antiseptics, pH adjusters, etc. Examples of stabilizers include albumin, globulin, gelatin, mannitol, and glucose. Dextran, Ethylene glycol, Propylene glycol, Ascorbic acid, Nitrogen Sodium hydrogen sulfate, sodium thiosulfate, sodium EDTA, sodium citrate, Dibutylhydroxytoluene and the like can be used. Examples of solubilizing agents include alcohols (e.g., ethanol, etc.) and polyalcohols. (e.g., propylene glycol, polyethylene glycol, etc.), nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, etc.) can be used. . Examples of suspending agents include glycerin monostearate and aluminum monostearate. cellulose, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, Sodium lauryl sulfate and the like can be used. Examples of emulsifiers include gum arabic, sodium alginate, and tragacanth. It is possible. Examples of soothing agents include benzyl alcohol, chlorobutanol, and sorbitol. can be used. Examples of buffers include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, Cetate buffer, Tris buffer, etc. can be used. Examples of preservatives include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, Propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol Alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid Acid, borax, etc. can be used. Examples of preservatives include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. A roller or the like can be used. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid. can be done.

[0050] The RGMa inhibitor of the present invention is a siRNA (short interfering RNA) inhibitor of the gene expressing RGMa. RGMa expression vectors such as RNAi (RNA), shRNA (short hairpin RNA), and antisense oligonucleotides When the substance suppresses the expression of HIV, it may be administered in the form of a non-viral vector or a viral vector. It is possible. When the RGMa inhibitor is in a non-viral vector form, the method of administration may be liposomal. methods for introducing nucleic acid molecules into cells (liposome method, HVJ-liposome method, cationic liposome method, etc.) method, lipofection method, lipofectamine method, etc.), microinjection method A method of transferring nucleic acid molecules into cells together with carriers (metal particles) using a gene gun. etc. can be increased. When siRNA or shRNA is administered to a living body using a viral vector, recombinant adenovirus Viral vectors such as viruses and retroviruses can be used. Viruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40 DNA that expresses siRNA or shRNA is introduced into DNA or RNA viruses such as HIV-1, HIV-2, and HIV-1-associated viruses. By infecting tissues with this recombinant virus, genes are introduced into cells or tissues. It is possible.

[0051] The preparations thus obtained are suitable for use in, for example, humans, as well as rats, mice, rabbits, and sheep. and administering an effective amount thereof to other mammals such as pigs, cows, cats, dogs, and monkeys. By doing so, HAM can be prevented or treated. The dosage depends on the purpose, severity of the disease, age, weight, sex, medical history of the patient, type of active ingredient, etc. It is set appropriately taking into consideration the above. [Example]

[0052] [Example 1: HAM and adult T-cell leukemia / lymphoma (ALT) ) and comprehensive comparative analysis to clarify the onset mechanism and pathogenesis] As shown in Figure 1, in the samples of HTLV-1 infected asymptomatic individuals, the HTLV- 1. Analysis of mRNA expression for CADM1, an indicator of infected cells, and groups P, D, and N classified by CD7. Analysis was performed. HAM was also performed by separating CD4-positive and CD4-negative cells enriched with magnetic beads from peripheral blood mononuclear cells of HAM patients. Cells from four individuals were also analyzed. Cells from healthy individuals (Normal) who were not infected with HTLV-1 were also isolated in the same manner. Peripheral blood mononuclear cells (PBMCs) are also referred to as PBMCs. ATL (smodering, chronic, and acute disease types) is mainly PBMCs consisting of CD4 positive cells were used. The one-color microarray gene expression analysis method manufactured by Agilent Technologies was used.

[0053] A comprehensive comparative analysis of nerve-related molecules revealed that CD4+ cells from HAM patients It was found that RGMa was significantly expressed in the spleen. 4 cases of normal T cells (Normal.CD4), 4 cases of CD4-positive T cells from HAM patients (HAM.CD4), C from healthy individuals D4 positive / CADM1 negative / CD7 positive T cells 3 cases (Normal.P), HTLV-1 infected CD4 positive / CADM1 negative / CD7 5 cases of positive T cells (group P), 5 cases of HTLV-1 infected CD4 positive / CADM1 positive / CD7 positive T cells (group D), HTLV- 1 infected person CD4 positive / CADM1 positive / CD7 negative T cells 5 cases (N group), acute type ATL patient CD4 positive / CADM1 positive / CD 7 negative T cells 3 cases (Acute.N), healthy CD4 positive T cells 21 cases (Normal.CD4.1), smoldering ATL 3 patient-derived PBMCs (Smoldering), 20 PBMCs from chronic ATL patients (Chronic), acute ATL patients PBMCs from 26 patients (acute) were analyzed using the Human GeneExplorer of Agilent Technologies. All gene expression data were obtained using a 4x44K Microarray and normalized to the median. , RGMa gene levels were graphed. The graph plots the Log2 values ​​of the fluorescence intensity of the array. The increase was significantly different from all other groups (P < 0.05). The graph is shown in Figure 2.

[0054] Example 2: Gene expression assay in CD4-positive HAM patients PBMCs isolated from the peripheral blood of five HAM patients were analyzed using a human CD4+ isolation kit (Miltenyi Biotech). c) was used to separate CD4-positive T cells, which were then used to obtain a cell population containing a large number of HTLV-1-infected cells. Similarly, CD4+ T cells were isolated from PBMCs of four healthy individuals and used as a control group. Total RNA was collected from the isolated CD4-positive T cells, and cDNA was prepared using ReverTra Ace (Toyobo). The cDNA was used to examine the relationship between CD4-positive T cells from HAM patients and CD4-positive T cells from healthy individuals. The difference in the expression level of RGMa in the internal control was analyzed by real-time PCR. 8srRNA was used. The graph showing the analysis results is shown in Figure 3. In the graph, HD-CD4+ indicates the control group. , HAM-CD4+ refers to the group of HAM patients.

[0055] [Example 3: Analysis of RGMa-expressing cells in PBMCs] PBMCs from HAM patients were treated with Clear Back (MBL) for Fc blocking, followed by anti-CD3-PECy7 (TONBO), CD4-FITC (eBioscience) and CD14-PE (eBioscience) antibodies were added and staining was carried out at 4°C for 30 minutes. It was. After washing the antibody-stained PBMCs, FACS sorting was performed using AriaIIIu (BD). 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 collected. Total RNA was extracted from each of the collected cells, and cDNA was prepared using ReverTraAce (Toyobo). Using the cDNA generated, we analyzed the differences in RGMa expression levels between cell types by real-time PCR. 18s rRNA was used as an internal control. The graph of the analysis results is shown in Figure 4. show. In HAM-PBMC, RGMa was detected in CD3+CD4+ cells (CD3+CD4+), which are rich in infected cells. was found to have the highest expression.

[0056] Example 4: Changes in RGMa expression during culture of PBMCs from HAM patients PBMCs from two HAM patients were suspended in RPMI1640 medium (Wako) containing 10% FBS (GIBCO) for 1 e 5 cells were seeded in 10 wells of a 96-well round bottom plate and cultured for 1, 3, 5, and 7 days. Ta. Total RNA was extracted from PBMCs cultured for each period along with PBMCs on Day 0 that had not been cultured. cDNA was prepared using TraAce (Toyobo). When PBMCs from HAM patients were cultured, the HTLV-1 virus was detected. It is known that the gene is overexpressed, and the cDNA was used to culture PBMCs from HAM patients. In other words, changes in RGMa expression associated with virus expression were analyzed by real-time PCR. 18s rRNA was used as an internal control. The graph showing the analysis results is shown in Figure 5. Shown below.

[0057] [Example 5: Analysis of H3K27me3 levels on all promoters] 3 cases of normal T cells (Normal T cells), 4 cases of CD4-positive T cells from HAM patients (HAM), acute ATL patients For PBMCs from three patients (ATL), Agilent Technologies' SurePrint G3 Human Promoter H3K27me3 levels on all promoters were obtained using a 2x400K Microarray and normalized. Specifically, the H3K27me3 level near -2,916 bp upstream from the transcription start site of the RGMa gene was graphed. The graph is shown in Figure 6. The graph plots the Log2 values ​​of the fluorescence intensity of the array. HAM50 and HAM123 in the table refer to HAM patients from whom CD4-positive T cells were obtained, respectively. These results suggest that RGMa gene expression suppression is relieved in positive T cells.

[0058] [Example 6: Quantification of RGMa gene mRNA levels] cDNA encoding HTLV-1 Tax was inserted into the human CD4-positive T-cell leukemia cell line Jurkat. The lentiviral vector was transduced, and the RGMa gene mRNA levels were measured over a 3-day period. Quantitative RT-PCR was used to measure RPL19 gene mRNA, which was also used as an internal control. The graph showing the quantitative results is shown in Figure 7. RGMa expression was induced by HTLV-1 virus. It was suggested that this could be done.

[0059] Example 7: Tax-dependent induction of RGMa expression in HTLV-1-tax expression-inducible cell line JPX-9 cells JPX9 cells were cultured in RPMI1640 medium containing 10% FBS for 24 hours to induce HTLV-1-tax expression. Cadmium chloride (CdCl2; Nacalai Tesque) was added to a final concentration of 20 μM, and then The cells were cultured for 1, 2, and 3 days. The cadmium chloride-treated and untreated JPX9 cells were cultured for 1, 2, and 3 days. The protein expression of GMa was analyzed by FACS. Cadmium chloride-treated and untreated JPX9 cells were washed, and Foxp3 / Transcription Factor was added. Cells were permeabilized using the Tor Staining Buffer Kit (eBioscience). -FITC antibody (Lt-4: provided by Professor Tanaka, University of the Ryukyus) was added and treated at 4°C for 1 hour. The stained Tax protein was expressed in cells by Canto II. Detection was performed by FACS analysis using a fluorochrome-independent method. Cadmium chloride-treated and untreated JPX9 cells were washed, and then incubated with anti-RGMa antibody (Immunohistochemicals, Inc.). The cells were then treated with IgG1 (manufactured by the Institute of Epidemiology and Biological Sciences (IBL)) at 4°C for 30 minutes. The IgG-PE antibody (BioLegend) was added and incubated at 4°C for 30 minutes to detect the RGMa protein expressed in JPX9. The stained RGMa protein was detected by FACS analysis using Canto II. . Figure 8 shows the analysis results of Tax and RGMa protein expression.

[0060] [Example 8: Examination of the effect of RGMa antibody on PBMCs from HAM patients] (Effect of RGMa antibodies on spontaneous proliferation activity) PBMCs from four HAM patients were suspended in RPMI1640 medium containing 10% FBS, and 1e5 cells were collected at 96 wt. RGMa antibody (R&D Systems, Inc.) was added to a well-round bottom plate at a final concentration of 10 μg / ml. ) was added and the mixture was cultured in a total of 0.1 ml of culture medium at 37°C under 5% CO2 conditions for 7 days. A group with no addition (Medium), a group with the same concentration of Normal Goat IgG (Santa Cruz Biotechnolo The control group was a 1 μg / ml prednisolone (PSL) (Funakoshi)-added group. It was a troll. Six days after the start of culture, 1 μCi was added to each well. 3 H-Thymidine was added and the mixture was incubated at 37°C and 5% CO2. The cultured cells were then harvested using a cell harvester (Tomtec MH3 PerkinElmer). The sample was adsorbed onto a glass filter (Printed Filtermat A, PerkinElmer) using a filter pad and dried. After that, the solid scintillator Meltilex-A (PerkinElmer) was soaked in it, and MicroBeta (WALLAC MicroBeta TriLux 1450-021) was used to transfect the cells. 3 The amount of H-thymidine was measured. In the Medium group of PBMC from each HAM patient 3 The average of the H-thymidine counts was set at 100%, and each The relative values ​​of the groups were calculated, and the results of the four HAM patients were 3 The average H-thymidine uptake rate was calculated. Shown in Figure 9.

[0061] (Effect of RGMa antibody on changes in HTLV-1 proviral load) PBMCs from four HAM patients were suspended in medium (RPMI1640 medium containing 10% FBS) and cultured at 1e5 cells per 96 wells. 1) Inoculate a round bottom plate with RGMa antibody (R&D Systems) at a final concentration of 10 μg / ml. The cells were cultured in a total of 0.1 ml of culture medium at 37°C under 5% CO2 conditions for 7 days. A group with no addition (Medium), a group with the same concentration of NormalGoat IgG (Santa Cruz Biotechnol The control group was treated with 1 μg / ml prednisolone (PSL) (Funakoshi) and the control group was treated with 1 μg / ml prednisolone (PSL) (Funakoshi). It was a troll. Seven days after the start of the culture, the cells were centrifuged, the supernatant was removed, and genomic DNA was extracted from the cell mass. Using the genomic DNA, the HTLV-1 proviral load (infected cell rate) was measured by real-time PCR. The determination was made. The HTLV-1 proviral load in the Medium group of PBMCs from each HAM patient was set at 100%, and the HTLV-1 proviral load in each group was The relative value of the HTLV-1 proviral load was calculated, and the mean value of the HTLV-1 proviral load in four HAM patients was calculated. The results are shown in Figure 10.

[0062] (Effect of RGMa antibody on CXCL10 production) To analyze the effect of RGMa antibody on CXCL10 production from PBMCs of four HAM patients, PBMCs from M patients were suspended in medium (RPMI1640 medium containing 10% FBS) and plated at 1e5 cells per well in a 96-well round plate. The cells were seeded onto an ottom plate, and RGMa antibody (R&D Systems) was added to a final concentration of 10 μg / ml. The cells were cultured in a total of 0.1 ml of culture medium at 37°C and 5% CO2 for 7 days. A group with no addition (Medium), a group with the same concentration of NormalGoat IgG (Santa Cruz Biotechnol The control group was treated with 1 μg / ml prednisolone (PSL) (Funakoshi) and the control group was treated with 1 μg / ml prednisolone (PSL) (Funakoshi). It was a troll. Seven days after the start of the culture, the culture medium was centrifuged and the culture supernatant was collected. The L10 concentration was measured using a Cytokine Beads Array kit (BD Biosciences) and a flow cytometer. -Measurement was performed using a FACSCanto II (BD Biosciences). The CXCL10 concentration in the culture medium of the Medium group was set at 100%, and the CXCL10 concentration in the culture medium of each group was The relative CXCL10 concentration was calculated, and the average CXCL10 concentration for the four HAM patients was determined. The results are shown in Figure 11. show.

[0063] (Effect of RGMa antibody on cytokine production in PBMCs from HAM patients) We analyzed the effect of RGMa antibody on the production of various cytokines in PBMCs from HAM patients. To achieve this, PBMCs from four HAM patients were suspended in RPMI1640 medium containing 10% FBS at 1e5 cells per suspension. The cells were seeded in a 98-well round bottom plate and diluted with RGMa antibody (R&D System) at a final concentration of 10 μg / ml. The cells were cultured in a total of 0.1 ml of culture medium at 37°C under 5% CO2 conditions for 7 days. A group with no addition (Medium), a group with the same concentration of NormalGoat IgG (Santa Cruz Biotechnol The control group was treated with 1 μg / ml prednisolone (PSL) (Funakoshi) and the control group was treated with 1 μg / ml prednisolone (PSL) (Funakoshi). It was a troll. Seven days after the start of culture, the culture medium was centrifuged to collect only the culture supernatant. The concentrations of γ, TNF, IL-2, and IL-10 were measured using a Cytokine Beads Array kit (BD Biosciences). Measurement was performed using a flow cytometer FACSCanto II (BD Biosciences). The concentration of each cytokine in the medium culture medium was set at 100%, and the percentage of each cytokine in each culture condition was calculated. The relative values ​​of cytokine concentrations were calculated, and the average values ​​for the four HAM patients were obtained.

[0064] Example 9: Induction of apoptosis in neuronal cell lines by HAM-PBMC Neuronal cell lines NB-1 or SK-N-AS were seeded in 6-well plates and cultured for 24 hours. Afterwards, the cells were cultured in healthy subjects (HD) or HA. PBMCs from M patients were added and co-cultured. 48 hours after the start of co-culture, PBMCs were added together with the medium. C was removed, and the neuronal cell lines were collected after washing with PBS. For each recovered neuronal cell line, cells with DNA fragmentation due to apoptosis were identified. The analysis was performed using the TUNEL method (MEBSTAIN Apoptosis TUNEL Kit Direct (MBL)) which specifically detects apoptosis. The analysis results are shown in Figure 13. The X axis of the histogram represents the intensity of DNA fragmentation positivity. HAM-derived cells induce apoptosis more strongly in neural cell lines than HD-derived cells. Led. Specifically, cell death was analyzed according to the following <Experimental Procedure>. <Experimental Procedure> Neuronal cell lines NB-1 and SK-N-AS were seeded in 6-well plates and cultured for 24 hours. Next, HD or HAM-PBMCs were added (in an amount twice the number of neuronal cell lines seeded) and cultured for 48 hours. Then, cells were fixed with 4% paraformaldehyde and permeabilized with 70% ethanol. Processing was carried out. For DNA nick end labeling, the Td of MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) was used. Suspend the cells in 20uL of T solution (TdT buffer II: TdT: FITC-dUTP = 18:1:1). After reacting at 37°C for 60 minutes, FACS analysis was performed.

[0065] Example 10: Resistance of apoptosis in neuronal cell lines induced by HTLV-1 Tax-expressing T cell lines Inhibitory effect of RGMa antibody on The neuronal cell line NB-1 was seeded in a 6-well plate and cultured for 24 hours, after which unstimulated JPX9 (JPX9(-)) or 20 JPX9 cells were incubated with JPX9 cells in which Tax expression was induced by adding 1 μM cadmium chloride for 24 hours (JPX9(+CdCl2)). In addition, NB-1 cells were co-cultured with JPX9 (+CdCl2) at a final concentration of 10 μg / ml. Normal Mouse IgG2b (MBL) or RGMa antibody (IBL) was added. 48 hours after the start of co-culture The JPX9 added together with the medium was removed, and the neuronal cell lines were collected after washing with PBS. For each neuronal cell line, we specifically identified cells that had undergone DNA fragmentation due to apoptosis. Analysis was performed using the TUNEL method (MEBSTAIN Apoptosis TUNEL Kit Direct (MBL)) to detect The analysis results are shown in Figure 14. The X-axis of the histogram indicates the intensity of positive DNA fragmentation. Specifically, cell death was analyzed according to the following <Experimental Procedure>. <Experimental Procedure> Neuronal cell line (NB-1) was seeded in a 6-well plate and cultured for 24 hours. Next, JPX9(-) or JPX9(+CdCl2) was added to NB-1 cells and co-cultured. The number of cells in JPX9(+CdCl2) was twice the number of NB-1 cells seeded. The cells were washed three times with 10 ml of medium to remove the cadmium chloride and then added to NB-1 cells. Subsequently, NB-1 cells were co-cultured with JPX9 (+CdCl2) and Normal Mouse IgG2b (MBL) or anti-RGM Antibody a (IBL) was added to a final concentration of 10 μg / ml, and the cells were cultured for 48 hours. Then, cells were fixed with 4% paraformaldehyde and permeabilized with 70% ethanol. After processing, the cells were stained with anti-CD45-V450 antibody. For DNA nick end labeling, the Td of MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) was used. The cells were suspended in 20 μL of T solution (TdT buffer II: TdT: FITC-dUTP = 18:1:1) and incubated for 37 After incubation at 0°C for 60 minutes, FACS analysis was performed.

[0066] All of the publications, patents and non-patent documents cited herein are The entire contents of which are incorporated herein by reference.

Claims

1. A therapeutic or preventive agent for HTLV-1-associated myelopathy (HAM), comprising an RGMa inhibitor.

2. The method for treating or preventing HAM according to claim 1, wherein the RGMa inhibitor is an antibody that recognizes RGMa. Antiseptic.

3. Pharmacologically effective doses of RGMa inhibitors are administered to patients with HTLV-1-associated myelopathy (HAM) in need thereof. A method for treating HAM, comprising administering

4. The method for treating HAM according to claim 3, wherein the RGMa inhibitor is an antibody that recognizes RGMa.

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