阅读说明：本技术 Htlv-1相关性脊髓病（ham）治疗或预防剂、及ham的治疗方法 (Therapeutic or prophylactic agent for HTLV-1-associated myelopathy (HAM), and method for treating HAM ) 是由 内丸薰 山岸诚 石崎伊纯 山野嘉久 于 2019-07-19 设计创作，主要内容包括：本发明提供含有RGMa阻遏物质的HTLV-1相关性脊髓病(HAM)的治疗或预防剂、及包括向需要其的HTLV-1相关性脊髓病(HAM)患者施予药理学有效量的RGMa阻遏物质的、HAM的治疗方法。(The present invention provides an agent for treating or preventing HTLV-1-related myelopathy (HAM) containing an RGMa repressor, and a method for treating HAM, which comprises administering a pharmacologically effective amount of an RGMa repressor to a patient suffering from HTLV-1-related myelopathy (HAM) in need thereof.)

A therapeutic or prophylactic agent for HTLV-1-associated myelopathy (HAM), which contains an RGMa repressor substance.

2. The therapeutic or prophylactic agent for HAM according to claim 1, wherein the RGMa repressor substance is an antibody that recognizes RGMa.

A method of treating HAM comprising administering a pharmacologically effective amount of an RGMa repressor substance to an HTLV-1 associated myelopathy (HAM) patient in need thereof.

4. The method of treating HAM according to claim 3, wherein the RGMa repressor substance is an antibody that recognizes RGMa.

Technical Field

The present invention relates to a therapeutic or prophylactic agent for HTLV-1-related myelopathy (HAM). In addition, the invention also relates to methods of treatment of HAM.

Background

HTLV-1 (Human T-cell leukemia Virus Type 1, Human T-cell Lukemia Virus Type 1; Human T-cell leukemia Virus) is a Virus that infects T cells and the like (mainly CD4 positive T cells) which are one of the leukocytes in blood.

T cells infected with HTLV-1 form chronic inflammation in the spinal cord, which results in the disturbance and degeneration of spinal nerve cells, causing spastic paralysis of the spinal cord. Spastic paralysis of the spinal cord caused by infection of cells with HTLV-1 is known as HTLV-1-associated myelopathy (also abbreviated as HAM).

The symptoms of HAM include symptoms such as paralysis of both legs (feet), pain, dysuria, and intractable constipation due to nervous tissue disorder. The progression of these symptoms can lead to wheelchair life, bedridden life. HAM is one of the diseases designated as prescribed stubborn diseases in japan. Currently, no effective therapy for HAM is established, and symptomatic therapy is mainly performed.

As one of the treatment methods, a treatment method using an anti-CCR 4 antibody has been demonstrated to reduce HTLV-1 infected cells, reduce HAM's spinal cord inflammation, and have an effect of improving symptoms (non-patent document 1 and patent document 5).

RGMa protein is one of RGM (Repulsive guidance molecule) family proteins involved in axonal induction of nerve cells of retina, hippocampus, closing of nerve canals, and the like. The role of RGM is not limited to the above, and it is known to have various functions.

For example, patent document 1 discloses that RGM is expressed in bone marrow-derived dendritic cells (BMDCs), and that the RGM receptor is CD4⁺T cells and CD11b⁺Expressed in macrophages, RGM binds to the RGM receptor and thereby renders CD4⁺T cells and CD11b⁺The cell adhesion activity of macrophages is enhanced. Patent document 1 discloses that both clinical symptoms and tissue lesions of multiple sclerosis model mice can be alleviated by using an anti-RGM neutralizing antibody, and that antigen-specific and non-specific T cell activation is reduced in splenocytes obtained from the mice.

Patent documents 2 and 3 disclose neutralizing monoclonal antibodies against RGMa that selectively inhibit binding of RGMa to receptor regenerating protein (neogenin) of RGMa and to bone morphogenetic protein 2 and bone morphogenetic protein 4 (BMP-2, BMP-4). It is considered that the use of the neutralizing monoclonal antibody can promote nerve regeneration and regrowth of damaged neuron couplings in the central nervous system of a human who has been injured or caused inflammation, specifically, in neurodegenerative diseases such as multiple sclerosis, acute spinal cord injury, sequelae of brain trauma, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

Patent document 4 discloses that RGMa is locally concentrated on myelin, neogenetic lesion, and mature scar tissue of the central nervous system of a human suffering from traumatic brain injury or ischemic stroke, and discloses a method for detecting and quantifying an RGMa fragment for the purpose of diagnosing the neurodegenerative disease. In addition, in patent document 4, as a target neurodegenerative disease or disorder to which an RGMa fragment is detected, multiple sclerosis, parkinson's disease, alzheimer's disease, Tay-saxophone's disease, Niemann-Pick disease, Gaucher's disease, Hurler's syndrome, huntington's disease, amyotrophic lateral sclerosis, idiopathic inflammatory demyelinating disease, vitamin B12 deficiency, central pontine myelinolysis, tuberculosis (locomotor ataxia), transverse myelitis, devick's disease, progressive multifocal leukoencephalopathy, optic neuritis, spinal cord injury, traumatic brain injury, stroke, glaucoma, diabetic retinopathy, age-related macular degeneration, white matter nutrition (ukleotrophy) are mentioned.

Documents of the prior art

Patent document

Patent document 1: international publication No. 2011/071059

Patent document 2: japanese patent laid-open No. 2014-138599

Patent document 3: japanese laid-open patent publication No. 2016-175897

Patent document 4: japanese Kohyo Table 2017-526930

Patent document 5: japanese laid-open patent publication No. 2010-100578

Non-patent document

Non-patent document 1: n Engl J Med,2018,378, 529-.

Disclosure of Invention

Problems to be solved by the invention

Although the use of the method using an anti-CCR 4 antibody disclosed in non-patent document 1 or patent document 5 can improve the symptoms of HAM, the effect of the anti-CCR 4 antibody is limited for HAM patients whose nerve destruction has progressed. Therefore, there is a need for more superior methods of treating HAM.

The present invention has been made in view of the above problems, and an object thereof is to provide a therapeutic agent and a therapeutic method capable of treating HAM.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a substance that represses RGMa is effective for the treatment of HAM, thereby completing the present invention.

Namely, the present invention is as follows.

[1]

An agent for treating or preventing HTLV-1-associated myelopathy (HAM), which contains an RGMa repressor.

[2]

The therapeutic or prophylactic agent for HAM according to [1], wherein the RGMa repressor substance is an antibody that recognizes RGMa.

[3]

A method of treating HAM comprising administering a pharmacologically effective amount of an RGMa repressor substance to an HTLV-1 associated myelopathy (HAM) patient in need thereof.

[4]

The method for treating HAM according to [3], wherein the RGMa repressor substance is an antibody that recognizes RGMa.

Effects of the invention

According to the present invention, HAM, which is a refractory disease, can be treated.

Drawings

FIG. 1 is a graph showing a group P, D, N classified based on CADM1 and CD7 as indicators of HTLV-1-infected cells among CD 4-positive T cells.

FIG. 2 shows normal T cells (normal. CD4), HAM patient-derived CD4 positive T cells (HAM. CD4), healthy human-derived CD4 positive/CADM 1 negative/CD 7 positive T cells (normal. P), HTLV-1-infected CD4 positive/CADM 1 negative/CD 7 positive T cells (group P), HTLV-1-infected CD4 positive/CADM 1 positive/CD 7 positive T cells (group D), HTLV-1-infected CD4 positive/CADM 1 positive/CD 7 negative T cells (group N), Acute ATL patient CD4 positive/CADM 1 positive/CD 7 negative T cells (Acute. N), healthy human CD4 positive T cells (normal. CD4.1), depressed ATL patient-derived PBMC (Smoldering), Chronic ATL patient-derived Acic (Chronic) and Acute ATL-derived PBMC (Acute ATL) in Chronic ATL patients (Acute ATL)) Graph of expression levels of RGMa gene.

FIG. 3 is a graph showing the expression levels of RGMa in HAM patient CD 4-positive T cells and healthy CD 4-positive T cells.

FIG. 4 is a graph showing the results of analyzing the expression level of RGMa between cell types in PBMCs of HAM patients.

FIG. 5 is a graph showing the change in the expression of RGMa accompanying the expression of HTLV-1 virus in the culture of PBMC of HAM patients.

FIG. 6 is a graph showing the level of H3K27me3 in the vicinity of-2,916 bp upstream from the transcription initiation site of the RGMa gene.

FIG. 7 shows the level of RGMa gene mRNA when a lentiviral vector into which a cDNA encoding HTLV-1Tax was inserted was introduced into human CD 4-positive T-cell leukemia cell line Jurkat.

FIG. 8 shows the results of analysis of protein expression of Tax and RGMa in HTLV-1-Tax expression-inducing cell line JPX-9.

FIG. 9 is a graph showing the results of the effect of an RGMa antibody on the autonomously proliferating activity with respect to the effect of the RGMa antibody on PBMCs of HAM patients.

FIG. 10 is a graph showing the results of the effect of RGMa antibodies on HTLV-1 proviral volume changes with respect to the effect of RGMa antibodies on PBMC of HAM patients.

FIG. 11 is a graph showing the results of the effect of an RGMa antibody on CXCL10 production with respect to the effect of the RGMa antibody on PBMCs of HAM patients.

FIG. 12 is a graph showing the results of the effect of an RGMa antibody on cytokine production by HAM patient PBMC, with respect to the effect of the RGMa antibody on HAM patient PBMC.

FIG. 13 shows that HAM-PBMC induces apoptosis in a neural cell line.

[ FIG. 14 ]]Is a graph showing the results of inhibition of the induction of apoptosis of a neural cell line based on an HTLV-1-tax-inducing cell line by an RGMa antibody. (a) FACS mapping (plot) for co-culture of NB-1 cells and JPX-9 cells (unstimulated). (b) For NB-1 cells and CdCl₂FACS mapping when JPX-9 cells (HTLV-1-tax expressing cells) were stimulated for co-culture. (c) FACS mapping was performed under the conditions (b) with the addition of control antibody. (d) FACS mapping was performed under the conditions (b) with the addition of anti-RGMa antibody.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be implemented by being variously modified within the scope of the gist thereof.

The present invention is an agent for treating or preventing HTLV-1-related myelopathy (HAM) which contains an RGMa repressor substance. The RGMa repressor substance may act on RGMa itself to repress the activity of RGMa, or may inhibit the expression of RGMa.

Examples of the RGMa suppressor include a compound having an activity of suppressing RGMa, an antibody recognizing RGMa, and the like.

Examples of the RGMa suppressor include substances that suppress the expression of RGMa, such as siRNA (short interfering RNA), shRNA (short hairpin RNA), and antisense oligonucleotide, which express the RGMa gene.

Examples of the RGMa gene include, but are not limited to, a human RGMa gene having a base sequence represented by seq id No. 1, and an RGMa gene having a base sequence represented by seq id No. 2. Information on the base sequences of RGMa genes from various organisms is available from databases such as GenBank.

siRNA is double-stranded RNA capable of inhibiting the expression of RGMa gene as a target. The length (base length) of the base sequence in the siRNA is not particularly limited, but is preferably less than about 30 bases, more preferably about 19 to 27 bases, and still more preferably about 21 to 25 bases.

The shRNA is a molecule of about 20 base pairs or more formed of a short hairpin structure having a double-stranded structure in the molecule and a protrusion at the 3' -end by partially including a palindromic base sequence in a single-stranded RNA. After being introduced into a cell, the shRNA is broken down into about 20 bases in length in the cell, and can suppress the expression of the RGMa gene as a target in the same manner as the siRNA.

siRNA and shRNA can be artificially and chemically synthesized. In addition, siRNA and shRNA can be synthesized in vitro from a template DNA using, for example, T7RNA polymerase and T7 promoter.

The antisense oligonucleotide may be either DNA or RNA as long as it is complementary to or hybridizes to a less than continuous nucleotide sequence of about 30 bases in the DNA sequence of the RGMa gene. In addition, it may be a modified nucleotide 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, for example, by using a commercially available DNA synthesizer.

The RGMa suppressor contained in the therapeutic or prophylactic agent for HAM of the present invention is preferably an antibody that recognizes RGMa. Hereinafter, an antibody recognizing RGMa is also referred to as RGMa antibody. The RGMa antibody in the present invention may be any antibody that binds to RGMa and suppresses its activity, and examples thereof include an antibody that binds to RGMa and thereby prevents RGMa from binding to an RGMa receptor.

The RGMa antibody of the present invention may be a monoclonal antibody or a polyclonal antibody. The antibody of the present invention may be of any isotype (isotype) of IgG, IgM, IgA, IgD, and IgE.

The RGMa antibody of the present invention may be, for example, a mouse antibody, a human CDR-grafted antibody, a human chimeric antibody, a humanized antibody, or a fully humanized antibody, or may be a small molecule antibody. These antibodies may be used alone in 1 kind, or in combination of 2 or more kinds

The human CDR-grafted antibody is an antibody obtained by replacing the CDRs of an antibody of an animal other than a human with the CDRs of a human antibody. The human-type chimeric antibody is an antibody comprising a variable region derived from an antibody derived from an animal other than a human and a constant region derived from a human antibody. The humanized antibody is an antibody obtained by incorporating a part of a human-derived antibody into an antibody of an animal other than a human, while retaining a part of a region with high safety, and is a concept including a human-type chimeric antibody and a human-type CDR-grafted antibody.

The term "small molecule antibody" as used herein refers to a fragment of an antibody or a substance obtained by binding an arbitrary molecule to a fragment of an antibody, and recognizes the same epitope as that of the original antibody. Specifically, there may be mentioned Fab comprising VL, VH, CL and CH1 regions; 2 Fab fragments are linked to each other at the hinge region via a disulfide bond to form F (ab') 2; fv consisting of VL and VH; a single-chain antibody scFv in which VL and VH are connected by an artificial polypeptide linker; and sdFv, Diabody, sc (Fv)2, but are not limited thereto.

The RGMa antibody used in the present invention can be prepared by a known method using RGMa or a fragment thereof as an immunogen.

The obtained antibody can be confirmed to be an RGMa antibody using RGMa activity as an index.

Examples of the RGMa include human RGMa containing an amino acid sequence shown in seq id No. 3, RGMa containing an 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 Protein Data Bank and the like as a known database.

When the RGMa antibody used in the present invention is a polyclonal antibody, it can be prepared, for example, in the following manner. First, RGMa or a fragment thereof is dissolved in phosphate buffered saline (also referred to as PBS) as an antigen, and a suitable amount of a usual adjuvant, for example, freund's complete adjuvant is mixed as necessary to prepare an immunogen, which is immunized against mammals such as mice, rats, rabbits, goats, and horses. The immunization method is not particularly limited, and examples thereof include a method in which subcutaneous injection or intraperitoneal injection is performed 1 time, or 2 times or more at appropriate intervals. Then, blood is collected from the immunized animal and serum is separated, and the polyclonal antibody component is purified, according to a conventional method, thereby obtaining a polyclonal antibody.

When the RGMa antibody used in the present invention is a monoclonal antibody, the monoclonal antibody can be obtained by the following method: an immune cell, for example, a spleen cell obtained from the immunized mammal is fused with a myeloma cell to obtain a hybridoma, and an antibody is collected from a culture of the hybridoma. In addition, the monoclonal antibody may be produced by cloning an antibody gene from a hybridoma, incorporating an appropriate vector into a host cell, and using a gene recombination technique to produce a recombinant monoclonal antibody. In addition, the monoclonal antibody can also be prepared by phage display method.

As the RGMa antibody used in the present invention, for example, Yamashita, t., Mueller, B.K. & Hata, k.neogenin and repulsive guiding polypeptide signaling in the central nervous system, curr.opin.neurobiol.17, 29-34 (2007); japanese patent laid-open No. 2014-138599; japanese patent laid-open publication No. 2016-175897; japanese Kohyo publication No. 2017-526930; international publication No. 2016/175236; an antibody disclosed in Japanese patent laid-open publication No. 2015-508061 and the like.

The RGMa antibody used in the present invention can be obtained as a commercially available product such as those manufactured by immunobiology research Institute (IBL) of japan and R & D Systems, inc.

The RGMa antibody preferably comprises at least 1 CDR comprising an amino acid sequence selected from the group consisting of seq id no:

GTTPDY (serial number 7);

FQATHDPLT (serial number 10);

ARRNEYYGSSFFDY (serial No. 13);

LQGYIPPRT (serial number 16); and

a modified CDR amino acid sequence having at least 50% sequence identity to one of the above sequences.

The sequence identity is preferably 80% or more, more preferably 90% or more. In the present specification, amino acids are sometimes indicated by the conventional one-letter or three-letter references.

Complementarity Determining Regions (CDRs) refer to the regions of the variable regions of immunoglobulin molecules that form the antigen binding site, also known as hypervariable regions, and refer to portions of each immunoglobulin molecule in which the amino acid sequence varies particularly greatly. For the CDRs, there are 3 CDRs in each of the light and heavy chains (CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, CDR-H3). In the present application, the CDRs of immunoglobulin molecules are determined based on the numbering system of Kabat (Kabat et al, 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH USA).

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 sequences of the CDRs may be unchanged from the predetermined sequences, and the other regions except the CDRs may be changed by mutation or the like. When a mutation or the like exists outside the CDR, the homology is preferably 90% or more.

In addition, the RGMa antibody preferably further comprises at least 1 CDR comprising an amino acid sequence selected from the group consisting of seq id nos 5, 6, 8, 9, 11, 12, 14, 15 and modified CDR amino acid sequences having at least 50% sequence identity to one of these sequences. The sequence identity is preferably 80% or more, more preferably 90% or more.

More preferably, the RGMa antibody comprises at least 3 CDRs selected from the group of variable domain CDRs set forth in table 1, or at least 3 CDRs from the group of variable domains wherein at least 1 of the 3 CDRs is a modified CDR amino acid sequence having at least 50%, preferably 80%, more preferably 90% sequence identity to the parent sequence. In addition, the RGMa antibody further preferably comprises at least 2 variable domain CDR sets as shown in table 1. In addition, the at least 2 variable domain CDR groups are preferably a combination of VH5F9 group and VL5F9 group, or a combination of VH8D1 group and VL8D1 group.

[ Table 1]

The RGMa antibody may comprise framework regions. The amino acid sequence included in the framework region includes seq id nos 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40. These amino acid sequences may be 1 kind alone or 2 or more kinds in combination.

In addition, the RGMa antibody preferably includes, as a heavy chain variable domain and a light chain variable domain: at least 1 heavy chain variable domain selected from the group consisting of seq id nos 41, 42, 43, 44, 45, 46, 47, 48 and 49; and/or at least 1 light chain variable domain selected from the group consisting of SEQ ID Nos. 50, 51 and 52.

In addition, the binding site to RGMa antibody is not particularly limited as long as it is a site that can cause suppression of RGMa, and for example, in the case of human RGMa, it is preferable to bind to one or more peptides having an amino acid sequence represented by the following sequence:

EEVVNAVEDWDSQG (Serial number 53)

NQQIDFQAFHTNAE (Serial number 54)

PTAPETFPYET (Serial number 55)

KLPVEDLYYQA (Serial number 56)

LYERTRDLPGRAAAGL (serial number 57).

The RGMa antibody more preferably binds to a peptide having an amino acid sequence represented by seq id No. 53 and/or 54, and more preferably binds to a peptide having an amino acid sequence represented by seq id No. 53 and/or 54 and a peptide having an amino acid sequence represented by seq id No. 55 and/or 56.

The RGMa antibody is preferably an antibody that binds after position 250 in the amino acid sequence of human RGMa.

It is more preferable for the RGMa antibody to bind to a peptide having an amino acid sequence represented by seq id nos 53 and 54 and having an amino acid sequence represented by seq id nos 55 or 56.

The RGMa antibody may be a polyclonal antibody and 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, a chimeric antibody and a humanized antibody produced by using a gene recombination technique, a human antibody produced by using a transgenic animal that produces a human antibody, or the like.

In the present invention, when the RGMa antibody is administered to a human in a pharmaceutical form, a humanized antibody or a human antibody is desirable from the viewpoint of side effects.

The RGMa antibody may be used in the form of a partial fragment of a polyclonal antibody and/or a monoclonal antibody that recognizes RGMa protein or a partial fragment thereof (for example, a fragment including one or more of seq id nos 53 to 57) as an antigen, or may be a small molecule antibody.

As the RGMa antibody, in addition to those shown in table 1, an isolated RGMa antibody or an antigen-binding fragment thereof may be used, in which the amino acid sequences of each of complementarity determining region 1 of the light chain (LCDR1), complementarity determining region 2 of the light chain (LCDR2), complementarity determining region 3 of the light chain (LCDR3), complementarity determining region 1 of the heavy chain (HCDR1), complementarity determining region 2 of the heavy chain (HCDR2), and complementarity determining region 3 of the heavy chain (HCDR3) include the following sequences:

LCDR 1: RASQDISSYLN (Serial number 58)

LCDR 2: YTSRLHS (SEQ ID NO. 59)

LCDR 3: QQLNTLP (Serial number 60)

HCDR 1: DAWMD (Serial number 61)

HCDR 2: EIRSKANNHATYYAESVKG (Serial number 62) and

HCDR 3: RDGAY (serial No. 63);

or

LCDR 1: RSSQSLVHSNGNTYLH (Serial number 64)

LCDR 2: KVSNRFS (Serial number 65)

LCDR 3: SQSTHVP (Serial number 66)

HCDR 1: TSYYWN (Serial number 67)

HCDR 2: YISYDGTNNYNPSLKN (Serial number 68) and

HCDR3：SFG。

1 or more amino acids may be substituted, deleted, and/or added in each CDR sequence, for example, 1 or 2 amino acids may be substituted, deleted, and/or added.

As the RGMa antibody, an antibody having an amino acid sequence of sequence No. 73 in the light chain and an amino acid sequence of sequence No. 74 in the heavy chain can be exemplified. 1 or more amino acids (1 to 20, 1 to 10, or 1 to 5) may be substituted, deleted, added, or inserted in the amino acid sequences represented by these sequence numbers. Such substitution, deletion, or addition may be introduced into the CDR, but is preferably introduced into a region other than the CDR.

The constant region may be a human-derived mouse/human chimeric antibody, and examples of the mouse/human chimeric antibody include an antibody having an amino acid sequence of SEQ ID NO. 77 in the light chain (variable regions 1 to 107) and an amino acid sequence of SEQ ID NO. 78 in the heavy chain (variable regions 1 to 116). 1 or more amino acids (1 to 20, 1 to 10, or 1 to 5) may be substituted, deleted, added, or inserted in the amino acid sequences represented by these sequence numbers. Such substitution, deletion, or addition may be introduced into the CDR, but is preferably introduced into a region other than the CDR.

The antibody may be a humanized antibody of human origin other than the CDR. Examples of the humanized antibody include an antibody having an amino acid sequence of any one of SEQ ID Nos. 70 to 87 (variable region is up to 116 residues on the N-terminal side) in the heavy chain and an amino acid sequence of any one of SEQ ID Nos. 88 to 94 (variable region is up to 1 to 107 residues on the N-terminal side) in the light chain. The amino acid sequences represented by these sequence numbers may be substituted, deleted, added or inserted with 1 or more amino acids (1 to 20, 1 to 10 or 1 to 5). Such substitution, deletion, or addition may be introduced into the CDR, but is preferably introduced into a region other than the CDR.

The heavy chain amino acid sequence and the light chain amino acid sequence may be any combination of the above, and preferably an antibody having the amino acid sequence of SEQ ID NO. 84 in the heavy chain and the amino acid sequence of SEQ ID NO. 88 in the light chain. 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.

As the RGMa antibody, the following isolated RGMa antibodies, or antigen-binding fragments thereof, are preferred, in which the heavy chain variable region (VH) comprises the following amino acid sequence, or an amino acid sequence having at least 90% identity thereto:

EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAP GKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMN SLRTEDTALYYCTRRDGAYWGKGTTVTVSS (serial number 95),

the light chain variable region (VL) comprises the following amino acid sequence, or an amino acid sequence having at least 90% identity to the amino acid sequence:

DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGK APKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQ QLNTLPWTFGGGTKVEME (serial number 96).

As the RGMa antibody, an antibody having an amino acid sequence of sequence No. 75 in the light chain and an amino acid sequence of sequence No. 76 in the heavy chain can be exemplified. 1 or more amino acids (1 to 20, 1 to 10, or 1 to 5) may be substituted, deleted, added, or inserted in the amino acid sequences represented by these sequence numbers. Such substitution, deletion, or addition may be introduced into the CDR, but is preferably introduced into a region other than the CDR.

The antibody may be a mouse/human chimeric antibody in which the constant region is derived from human, or a humanized antibody in which other than the CDR is derived from human.

The anti-RGMa antibody may be an isolated RGMa antibody selected from the following (a1) to (h1), or an antigen-binding fragment thereof.

(a1) An anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: an LCDR1 comprising the amino acid sequence of seq id No. 97, an LCDR2 comprising the amino acid sequence of seq id No. 98, and an LCDR3 comprising the amino acid sequence of seq id No. 99, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of seq id No. 100, HCDR2 comprising the amino acid sequence of seq id No. 101, and HCDR3 comprising the amino acid sequence of seq id No. 102;

(b1) an anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: an LCDR1 comprising the amino acid sequence set forth in seq id No. 97, an LCDR2 comprising the amino acid sequence set forth in seq id No. 98, and an LCDR3 comprising the amino acid sequence set forth in seq id No. 103, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of seq id No. 100, HCDR2 comprising the amino acid sequence of seq id No. 101, and HCDR3 comprising the amino acid sequence of seq id No. 102;

(c1) an anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: an LCDR1 comprising the amino acid sequence set forth in seq id No. 97, an LCDR2 comprising the amino acid sequence set forth in seq id No. 98, and an LCDR3 comprising the amino acid sequence set forth in seq id No. 104, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of seq id No. 100, HCDR2 comprising the amino acid sequence of seq id No. 101, and HCDR3 comprising the amino acid sequence of seq id No. 102;

(d1) an anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: an LCDR1 comprising the amino acid sequence set forth in seq id No. 97, an LCDR2 comprising the amino acid sequence set forth in seq id No. 98, and an LCDR3 comprising the amino acid sequence set forth in seq id No. 105, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of seq id No. 100, HCDR2 comprising the amino acid sequence of seq id No. 101, and HCDR3 comprising the amino acid sequence of seq id No. 102;

(e1) an anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: an LCDR1 comprising the amino acid sequence set forth in seq id No. 97, an LCDR2 comprising the amino acid sequence set forth in seq id No. 98, and an LCDR3 comprising the amino acid sequence set forth in seq id No. 106, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of seq id No. 100, HCDR2 comprising the amino acid sequence of seq id No. 101, and HCDR3 comprising the amino acid sequence of seq id No. 102;

(f1) an anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: an LCDR1 comprising the amino acid sequence set forth in seq id No. 97, an LCDR2 comprising the amino acid sequence set forth in seq id No. 98, and an LCDR3 comprising the amino acid sequence set forth in seq id No. 107, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of seq id No. 100, HCDR2 comprising the amino acid sequence of seq id No. 101, and HCDR3 comprising the amino acid sequence of seq id No. 102;

(g1) an anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: an LCDR1 comprising the amino acid sequence set forth in seq id No. 97, an LCDR2 comprising the amino acid sequence set forth in seq id No. 98, and an LCDR3 comprising the amino acid sequence set forth in seq id No. 108, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence of seq id No. 100, HCDR2 comprising the amino acid sequence of seq id No. 101, and HCDR3 comprising the amino acid sequence of seq id No. 102; and

(h1) an anti-RGMa antibody, or antigen-binding fragment thereof, comprising a light chain variable region comprising: LCDR1 comprising the amino acid sequence of seq id No. 97, LCDR2 comprising the amino acid sequence of seq id No. 98 and LCDR3 comprising the amino acid sequence of seq id No. 109, wherein the heavy chain variable region comprises: HCDR1 comprising the amino acid sequence shown in SEQ ID NO. 100, HCDR2 comprising the amino acid sequence shown in SEQ ID NO. 101, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO. 102.

The RGMa antibody is preferably an antibody that has the above-described amino acid sequence and is a humanized antibody, and preferably has a constant region of human IgG.

By using the RGMa repressor substance of the present invention, HAM can be treated or prevented.

As shown in the examples described later and fig. 13, in HAM patients, neuronal cell death, i.e., induction of injury and degeneration of spinal cord tissue, was induced by cells derived from HAM patients.

The inventors of the present application conducted studies and, as shown in examples described below, found that RGMa is significantly expressed in CD 4-positive T cells, which are major HTLV-1-infected cells of HAM patients, and that the expression of RGMa is correlated with HAM. In addition, RGMa antibodies were used to perform RGMa repression experiments, and as a result, RGMa was found to be associated with CXCL10, IL-2, and IL-10 production.

CXCL10 is known to be a protein produced from HAM-pathogenic HTLV-1 infected T cells in response to IFN- γ, which induces the condition of HAM (Brain 2013), and CXCL10 is also known to be strongly correlated with the rate of progression of symptoms of HAM (PLoS Negl Trop Dis 2013). When RGMa antibodies were allowed to act on cells of HAM patients, the production of CXCL10 was inhibited. In addition, IL-10 is one of cytokines, in order to inhibit inflammation of the way. When RGMa antibodies were allowed to act on cells of HAM patients, IL-10 production was significantly increased.

In order to provide a true therapeutic effect to HAM, it is required to suppress damage of nerve cells. HTLV-1-tax is expressed at high levels in HAM patients' HTLV-1-tax infected cells (Blood 2002), suggesting that HTLV-1-tax is important for the development of the condition (J Clin Invest 2014). In the present invention, it was shown that HTLV-1-tax induces the expression of RGMa, and that cells derived from HAM patients with high RGMa expression levels induce nerve cell death, and importantly, that nerve cell death caused by HTLV-1-tax expressing cells can be inhibited by RGMa repressor substances.

As described above, the RGMa repressor substance can inhibit the induction of an inflammatory condition of HAM and inhibit an inflammatory response based on the cells of HAM patients, and thus, can treat or prevent HAM. In addition, the RGMa repressor substance not only inhibits the inflammatory response specific to HAM, but also inhibits neuronal cell death caused by HTLV-1-tax expressing cells, which are pathogenic cells in HAM patients, and thus, can treat or prevent HAM.

As described above, the RGMa repressor substance can treat or prevent HAM, and the present invention provides: an RGMa repressor substance for use in the treatment of HAM; a pharmaceutical composition for use in the treatment of HAM; use of an RGMa repressor substance for treating HAM; use of an RGMa repressor substance in the manufacture of a medicament for the treatment of HAM; an RGMa repressor substance for use in the production of a therapeutic drug for HAM; a method of treatment or prevention of HAM comprising administering an effective amount of an RGMa repressor substance to a subject in need thereof.

The therapeutic or prophylactic agent for HAM of the present invention contains an RGMa repressor substance, and can be formulated with a pharmaceutically acceptable carrier and/or additive.

Examples of the formulation form include oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquid preparations, suspensions, and emulsions; parenteral preparations such as injections, infusions, suppositories, ointments and patches.

The blending ratio of the carrier or the additive may be appropriately set based on the range generally used in the field of pharmaceuticals.

The carrier is not particularly limited, and examples thereof include water, physiological saline, other aqueous solvents, and aqueous or oily bases. The additives are not particularly limited, and examples thereof include excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavors, and the like.

When the RGMa suppressor of the present invention is an antibody recognizing RGMa, the antibody is preferably administered to a non-oral administration route, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically, in the form of an injection or infusion solution prepared together with a pharmaceutically acceptable carrier.

Injections or infusions containing the RGMa antibody can be used in the form of solutions, suspensions or emulsions. Examples of the solvent include distilled water for injection, physiological saline, glucose solution, and isotonic solution (e.g., solution of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). These solvents may be used alone in 1 kind, or may be used in combination of 2 or more kinds.

The injection or infusion solution may contain additives such as a stabilizer, a solubilizer, a suspension, an emulsifier, an analgesic, a buffer, a preservative, and a pH adjuster.

Examples of the stabilizer include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium hydrogen sulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene.

As the cosolvent, for example, alcohol (e.g., ethanol, etc.), polyol (e.g., propylene glycol, polyethylene glycol, etc.), nonionic surfactant (e.g., polysorbate 80 (registered trademark), HCO-50, etc.), and the like can be used.

As the suspending agent, for example, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate, or the like can be used.

As the emulsifier, for example, gum arabic, sodium alginate, tragacanth gum, and the like can be used.

Examples of the analgesic agent include benzyl alcohol, chlorobutanol, and sorbitol.

As the buffer, for example, a phosphate buffer, an acetate buffer, a borate buffer, a carbonate buffer, a citrate buffer, a Tris buffer, or the like can be used.

Examples of the preservative include methyl paraben, ethyl paraben, propyl paraben, butyl paraben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax.

As the preservative, for example, benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, or the like can be used.

As the pH adjuster, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, or the like can be used.

The RGMa suppressor of the present invention may be administered in the form of a non-viral vector or a viral vector when the substance inhibiting the expression of RGMa is a substance that inhibits the expression of RGMa, such as sirna (short interfering rna), shrna (short hairpin rna), or antisense oligonucleotide that expresses the gene of RGMa.

When the RGMa suppressor is in the form of a non-viral vector, examples of administration methods include a method of introducing a nucleic acid molecule using a liposome (liposome method, HVJ-liposome method, cationic liposome (cationic liposome) method, lipofection method, Lipofectamine method, and the like), a microinjection method, a method of transferring a nucleic acid molecule to a cell together with a carrier (metal particle) using a Gene Gun (Gene Gun), and the like.

When the siRNA or shRNA is administered to an organism using a viral vector, a viral vector such as a recombinant adenovirus or retrovirus can be used. The recombinant virus can be used to infect cells or tissues by introducing DNA expressing siRNA or shRNA into a detoxified DNA virus or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, etc. Thereby introducing the gene into the cell or tissue.

The thus-obtained preparation can be administered to other mammals such as humans, as well as rats, mice, rabbits, sheep, pigs, cows, cats, dogs, and monkeys in an effective amount to prevent or treat HAM.

The administration amount can be suitably set in consideration of the purpose, severity of disease, age, body weight, sex, past medical history of the patient, kind of the active ingredient, and the like.

Examples

Example 1: comprehensive comparative analysis for elucidating pathogenesis and condition of HAM and adult T-cell leukemia lymphoma (ALT)

As shown in FIG. 1, in a sample of a patient who did not develop HTLV-1 infection, mRNA expression analysis was performed on a group P, D, N classified based on CADM1 and CD7, which are indicators of HTLV-1-infected cells, among CD 4-positive cells.

In addition, 4 cases of HAM were also analyzed for CD 4-positive cells and CD 4-negative cells, each of which was obtained by concentrating peripheral blood mononuclear cells of HAM patients using magnetic beads. HTLV-1 was isolated in a similar manner in non-infected healthy subjects (Normal). Peripheral blood mononuclear cells are also described as PBMCs. ATL (stasis, chronic, acute forms of the disease) uses PBMCs composed mainly of CD4 positive cells.

A single color microarray gene expression analysis method manufactured by Agilent Technologies, Ltd. was performed.

Comparative analyses were performed synthetically on the nerve-associated molecules, and as a result, RGMa was found to be significantly expressed in CD4 positive cells of HAM patients.

The anti-tumor drug is used for 4 cases of normal T cells (normal. CD4), 4 cases of CD4 positive T cells derived from HAM patients (HAM. CD4), 3 cases of CD4 positive/CADM 1 negative/CD 7 positive T cells derived from healthy people (normal. P), 5 cases of CD4 positive/CADM 1 negative/CD 7 positive T cells derived from HTLV-1 infected people (group P), 5 cases of CD4 positive/CADM 1 positive/CD 7 positive T cells derived from HTLV-1 infected people (group N), 5 cases of CD4 positive/CADM 1 positive/CD 7 negative T cells derived from HTLV patients (group N), 3 cases of CD4 positive/CADM 1 positive/CD 7 negative T cells derived from acute type ATL patients (acute. N), 21 cases of healthy people CD4 positive T cells derived from healthy people (normal. CD4.1), 3 cases of PBMC derived from acute type ATL patients (chronic type ATL), and 20 cases of chronic PBMC derived from chronic type ATL patients (chronic type ATL), PBMC from acute ATL patients in 26 cases (acute), whole Gene Expression data were obtained using Human Gene Expression 4 x 44K Microarray from Agilent Technologies, ltd, and plotted against RGMa Gene levels after median normalization.

In the diagram, the position of the line is shown,log of fluorescence intensity for array₂Values are plotted. The group of CD4 positive T cells derived from HAM patients rose in a manner significantly different from all other groups (P)<0.05). The graph is shown in fig. 2.

Example 2: analysis of Gene expression in CD 4-Positive HAM patients

CD4 positive T cells were isolated as a cell population comprising a large number of HTLV-1 infected cells from PBMCs isolated from peripheral blood of 5 HAM patients using a human CD4+ isolation kit (Miltenyi Biotec). CD4 positive T cells were similarly isolated from PBMCs of 4 healthy subjects as a control group.

Total RNA was recovered from the isolated CD 4-positive T cells, and cDNA was prepared using ReverTra Ace (Toyo Boseki Co.). The difference in the expression amount of RGMa between the HAM patient CD 4-positive T cells and the healthy patient CD 4-positive T cells was analyzed by real-time PCR using the prepared cDNA. The internal reference used 18 srna.

The results of the analysis are graphically shown in fig. 3. HD-CD4+ in the graph refers to the control group, and HAM-CD4+ refers to the group of HAM patients.

Example 3: analysis of RGMa-expressing cells in PBMC ]

After Fc blocking treatment with Clear Back (MBL), anti-CD 3-PECy7(TONBO), CD4-FITC (eBioscience), CD14-PE (eBioscience) antibodies were added to HAM patient PBMC and stained at 4 ℃ for 30 minutes.

The antibody-stained PBMC were washed and then subjected to FACS sorting using AriaIIIu (BD), and CD 3-positive CD 4-negative cells (CD3+ CD4-), CD 3-positive CD 4-positive cells (CD3+ CD4+), CD 3-negative CD 14-negative cells (CD3-CD14-), and CD 3-negative CD 14-positive cells (CD3-CD14+) were separated and recovered.

Total RNA was recovered from each of the recovered cells, and cDNA was prepared using ReverTra Ace (Toyo Boseki Co.). The differences in the expression amounts of RGMa among the cell types were analyzed by real-time PCR using the prepared cDNAs. The internal reference used 18s rRNA. The graph of the results of the analysis is shown in fig. 4.

Among HAM-PBMCs, the expression of RGMa was found to be highest among CD 3-positive CD 4-positive cells (CD3+ CD4+) in which a large number of infected cells were present.

Example 4: changes in RGMa expression accompanying culture of PBMC from HAM patients ]

PBMCs from 2 HAM patients were suspended in a medium (RPMI 1640 medium (wako) containing 10% FBS (GIBCO)), and cultured for 1, 3, 5, and 7 days after 10-well aliquots of 1e5 cells per well were seeded into a 96-well round bottom plate.

Total RNA was extracted from PBMC on day 0 without culture and PBMC cultured at each time, and cDNA was prepared using reverta Ace (toyobo). It is known that HTLV-1 virus is overexpressed in culture of HAM patient PBMC, and the change in RGMa expression accompanying the culture of HAM patient PBMC, that is, the expression of virus, is analyzed by real-time PCR using the prepared cDNA. The internal reference used 18s rRNA. Fig. 5 shows a graph representing the analysis result.

Example 5: analysis of H3K27me3 levels on full promoter ]

For 3 cases of normal T cells (normal T cells), 4 cases of CD4 positive T cells derived from HAM patients (HAM), and 3 cases of PBMC derived from acute ATL patients (ATL), the H3K27me3 level on the full Promoter was obtained using SurePrint G3 Human Promoter 2 × 400K Microarray of Agilent Technologies, and after normalization, the H3K27me3 level was graphed from the transcription initiation site of RGMa gene at about-2,916 bp upstream.

The graph is shown in fig. 6. Log of fluorescence intensity of the array in the graph₂Values are plotted. HAM50 and HAM123 in the figure indicate HAM patients who obtained CD4 positive T cells, respectively. This means that the inhibition of gene expression of RGMa was released in CD 4-positive T cells derived from HAM patients.

Example 6: quantification of mRNA levels of the RGMa Gene ]

A cDNA lentiviral vector encoding HTLV-1Tax was inserted into human CD 4-positive T cell leukemia cell line Jurkat, and the mRNA level of RGMa gene was measured 3 days after the introduction by quantitative RT-PCR. The RPL19 gene mRNA was also measured and used as an internal reference.

FIG. 7 is a graph showing the results of the quantification. Means that RGMa expression is caused by HTLV-1 virus.

Example 7: HTLV-1-Tax expression Induction of expression of Tax-dependent RGMa in cell line JPX-9 cells

JPX9 cells were cultured in a medium (RPMI 1640 medium containing 10% FBS) for 24 hours, and cadmium chloride (CdCl) for inducing HTLV-1-tax expression was added to the cells so that the final concentration became 20. mu.M₂(ii) a Nacalai Tesque), followed by culture for 1, 2, and 3 days. FACS analysis was performed for protein expression of Tax and RGMa for JPX9 cells treated with cadmium chloride and untreated.

The JPX9 cells treated with cadmium chloride and untreated were washed separately and cell permeabilized using the Foxp3/Transcription Factor stabilizing Buffer Kit (eBioscience). Then, an anti-Tax-FITC antibody (Lt-4: issued by professor in Youkou university field) was added and treated at 4 ℃ for 1 hour, thereby staining the Tax protein expressed in the cells. Stained Tax proteins were detected by FACS analysis using cantonii.

JPX9 cells treated with cadmium chloride and untreated were washed, and then treated with an anti-RGMa antibody (manufactured by immunobiologies research laboratory (IBL) Co., Ltd.) at 4 ℃ for 30 minutes. Then, the cells were washed, and an anti-mouse IgG-PE antibody (BioLegentd) was added thereto, and the cells were reacted at 4 ℃ for 30 minutes to stain RGMa protein expressed in JPX 9. Stained RGMa protein was detected by FACS analysis using cantonii.

FIG. 8 shows the results of analysis of protein expression of Tax and RGMa.

Example 8: study of the Effect of RGMa antibodies on PBMCs of HAM patients ]

(effect of RGMa antibody on autonomous proliferative Activity)

PBMC of 4 HAM patients were suspended in a medium (RPMI 1640 medium containing 10% FBS), 1e5 cells were seeded into a 96-well round bottom plate for each well, and an RGMa antibody (R) was added to the plate so that the final concentration became 10. mu.g/ml&D Systems) was added to the culture medium in a total volume of 0.1ml at 37 ℃ with 5% CO₂Cultured for 7 days under the condition.

The group without any addition (Medium), the group with the addition of normal goat IgG (santa Cruz biotechnology) at the same concentration (normal IgG), and the group with the addition of 1 μ g/ml Prednisolone (PSL) (Funakoshi co., Ltd.) were used as controls.

6 days after the start of the culture, 1. mu. Ci was added to each well³H-thymidine, 5% CO at 37 ℃₂Incubated under conditions for 16 hours. Then, the cultured cells were adsorbed to a glass filter (Printed Filter A PerkinElmer) using a cell harvester (cell harvester) (Tomtec MH3 PerkinElmer), dried, stained with a solid scintillator Meltlex-A (PerkinElmer), and the uptake of the cells into the cultured cells was measured using Microbeta (WALLAC Microbeta TriLux 1450-³Amount of H-thymidine. In a culture medium group of PBMC of each HAM patient³The average value of H-thymidine readings (count) was taken as 100%, and relative values were calculated for each group to obtain 4 patients with HAM³Average value of H-thymidine uptake. The results are shown in FIG. 9.

(effect of RGMa antibody on changes in HTLV-1 proviral amounts)

PBMC of 4 HAM patients were suspended in a medium (RPMI 1640 medium containing 10% FBS), 1e5 cells were seeded into a 96-well round bottom plate for each well, and an RGMa antibody (R) was added to the plate at a final concentration of 10. mu.g/ml&D Systems) was added to the culture medium in a total volume of 0.1ml at 37 ℃ with 5% CO₂Cultured for 7 days under the condition.

The group without any addition (Medium), the group with the addition of normal goat IgG (santa Cruz biotechnology) at the same concentration (normal IgG), and the group with the addition of 1 μ g/ml Prednisolone (PSL) (Funakoshi co., Ltd.) were used as controls.

After 7 days from the start of the culture, centrifugation was performed and the supernatant was removed, and genomic DNA was extracted from the resultant cell mass. HTLV-1 proviral amount (infected cell rate) was determined by real-time PCR using the extracted genomic DNA.

The relative amount of HTLV-1 provirus in each group was calculated with the amount of HTLV-1 provirus in the culture medium group of PBMC of 4 HAM patients as 100%, and the average of the amounts of HTLV-1 provirus in the 4 HAM patients was determined. The results are shown in FIG. 10.

(effect of RGMa antibody on CXCL10 production)

To analyze the effect of RGMa antibodies on CXCL10 production from PBMCs of HAM patients, we will analyzePBMC of 4 HAM patients were suspended in a medium (RPMI 1640 medium containing 10% FBS), 1e5 cells were seeded into a 96-well round bottom plate for each well, and an RGMa antibody (R) was added to the plate so that the final concentration became 10. mu.g/ml&D Systems) was added to the culture medium in a total volume of 0.1ml at 37 ℃ with 5% CO₂Cultured for 7 days under the condition.

The group without any addition (Medium), the group with the addition of normal goat IgG (santa Cruz biotechnology) at the same concentration (normal IgG), and the group with the addition of 1 μ g/ml Prednisolone (PSL) (Funakoshi co., Ltd.) were used as controls.

After 7 days from the start of the culture, the culture broth was centrifuged, and only the culture supernatant was collected. The CXCL10 concentration in the culture supernatant was determined using a Cytokine Beads Array kit (BD Biosciences) using a flow cytometer facscan ii (BD Biosciences).

The relative value of CXCL10 concentration in the culture solution of each group was calculated with CXCL10 concentration in the culture solution of the culture medium group as 100%, and the average value of CXCL10 concentration of 4 HAM patients was determined. The results are shown in FIG. 11.

(effects of RGMa antibodies on cytokine production by PBMC of HAM patients)

To analyze the effect of the RGMa antibody on the production of various cytokines in the PBMC of HAM patients, 4 cases of PBMC of HAM patients were suspended in a medium (RPMI 1640 medium containing 10% FBS), 1e5 cells were seeded into a 96-well round bottom plate per well, and the RGMa antibody (R) was added to a final concentration of 10. mu.g/ml&D Systems) was added to the culture medium in a total volume of 0.1ml at 37 ℃ with 5% CO₂Cultured for 7 days under the condition.

The group without any addition (Medium), the group with the addition of normal goat IgG (santa Cruz biotechnology) at the same concentration (normal IgG), and the group with the addition of 1 μ g/ml Prednisolone (PSL) (Funakoshi co., Ltd.) were used as controls.

After 7 days from the start of the culture, the culture broth was centrifuged, and only the culture supernatant was collected. IFN γ, TNF, IL-2, IL-10 concentrations in culture supernatants were determined using the Cytokine Beads Array kit (BD Biosciences) using a flow cytometer FACSCAntoII (BD Biosciences).

The relative values of the cytokine concentrations under the respective culture conditions were calculated with the cytokine concentrations in the culture solutions of the medium groups as 100%, and the average value of 4 HAM patients was determined.

Example 9: apoptosis induction of neural cell line based on HAM-PBMC ]

The neural cell line NB-1 or SK-N-AS was inoculated into a 6-well plate, cultured for 24 hours, and then co-cultured with PBMC from healthy subjects (HD) or HAM patients. After 48 hours from the start of co-culture, the culture medium was removed together with the added PBMC, washed with PBS, and the neural cell line was collected.

The recovered neural cell lines were analyzed by the TUNEL method (MEBSTAIN Apoptosis TUNEL Kit Direct (MBL)) for specifically detecting cells in which DNA was fragmented by Apoptosis. Fig. 13 shows the analysis results. The X-axis in the histogram shows the intensity of DNA fragmentation positivity. Cells derived from HAM induce apoptosis more strongly in the neural cell line than cells derived from HD.

Specifically, the analysis of cell death was performed according to the following < experimental procedure >.

< Experimental step >

The neural cell strains NB-1 and SK-N-AS were inoculated into 6-well plates, and cultured for 24 hours.

Then, HD or HAM-PBMC (2 times the number of the neural cell lines inoculated) was added thereto and cultured for 48 hours.

Then, cells were fixed with 4% paraformaldehyde, and cell-permeabilization was performed with 70% ethanol.

For DNA nick end labeling, cells were suspended in 20uL of a TdT solution of MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) (TdT buffer II: TdT: FITC-dUTP ═ 18:1:1), reacted at 37 ℃ for 60 minutes, and then FACS analysis was performed.

Example 10: inhibitory Effect of RGMa antibody on apoptosis of neural cell line induced by HTLV-1 Tax-expressing T cell line ]

The neural cell line NB-1 was inoculated into a 6-well plate, cultured for 24 hours, and then unstimulated JPX9(JPX9(-)), or 20. mu.M cadmium chloride was added thereto for 24 hoursThus, JPX9(JPX9(+ CdCl) of Tax expression was induced₂) Co-cultivation is performed). In addition, in NB-1 cells and (JPX9(+ CdCl)₂) Normal mouse IgG2b (MBL) or RGMa antibody (IBL) was added to the co-culture of (1). 48 hours after the start of co-culture, the medium was removed together with JPX9 added thereto, washed with PBS, and the neural cell line was collected. The recovered neural cell lines were analyzed by the TUNEL method (MEBSTAIN Apoptosis TUNEL Kit Direct (MBL)) for specifically detecting cells in which DNA was fragmented by Apoptosis. Fig. 14 shows the analysis results. The X-axis in the histogram shows the intensity of DNA fragmentation positivity.

Specifically, the analysis of cell death was performed according to the following < experimental procedure >.

< Experimental step >

The neural cell line (NB-1) was inoculated into 6-well plates and cultured for 24 hours.

Then, JPX9(-) or JPX9(+ CdCl) was added to NB-1 cells₂) Co-cultivation is carried out. Note that JPX9(-) or JPX9(+ CdCl)₂) The number of cells of (a) is 2 times the number of NB-1 cells seeded. For JPX9(+ CdCl)₂) To remove cadmium chloride, the cells were washed 3 times with 10ml of the medium and added to NB-1 cells.

Then, in NB-1 cells and (JPX9(+ CdCl)₂) Normal mouse IgG2b (MBL) or anti-RGMa antibody (IBL) was added to the co-culture of (1) in such a manner that the final concentration became 10. mu.g/ml, and the mixture was cultured for 48 hours.

Then, cells were fixed with 4% paraformaldehyde, cell-permeabilized with 70% ethanol, and stained with anti-CD 45-V450 antibody.

For DNA nick end labeling, cells were suspended in 20uL of a TdT solution of MEBSTAIN Apoptosis TUNEL Kit Direct (MBL) (TdT buffer II: TdT: FITC-dUTP ═ 18:1:1), reacted at 37 ℃ for 60 minutes, and then FACS analysis was performed.

The entire contents of the publications, patent documents and non-patent documents cited and described in the present specification are incorporated directly into the present specification by reference.