Agent for preventing or treating acute-phase neuromyelitis optica

Abstract

Provided is an agent for preventing or treating acute-phase neuromyelitis optica and pain symptoms of neuromyelitis optica, the agent containing an RGMa-inhibiting substance.

Description

[Technical Field]

[0001] This invention relates to a preventive or therapeutic agent for acute neuromyelitis optica containing an RGMa inhibitor and a preventive or therapeutic agent for pain symptoms of neuromyelitis optica. [Previous Technology]

[0002] Neuromyelitis optica (NMO), also known as Devic disease, is an inflammatory central nervous system disease characterized by severe optic neuritis and transverse myelitis involving more than three vertebral segments. In 2004, an IgG specific for neuromyelitis optica (NMO-IgG) was discovered (Non-Patent Literature 1), and it was reported that aquaporin-4 (AQP4), an aquaporin highly expressed on the foot processes of stellate cells, is its target antigen, i.e., NMO-IgG is an anti-AQP4 antibody (Non-Patent Literature 2).

[0003] Therefore, unlike multiple sclerosis, a demyelinating disease, neuromyelitis optica is a stellate cell lesion caused by the destruction of stellate cells by anti-AQP4 antibodies. Regarding the symptoms of neuromyelitis optica, it is generally believed that the central inflammation or blood-spinal cord barrier (BSCB) caused by the activation of cellular immunity (in the brain, due to increased permeability of the blood-brain barrier (BBB)) allows anti-AQP4 antibodies to flow into the brain / spinal cord and produce complement activation, resulting in widespread AQP4 deficiency, stellate cell destruction, or shedding. It is believed that due to stellate cell damage (complement-dependent and antibody-dependent cell damage) and stellate cell shedding leading to destruction of the blood-brain barrier or abnormal glutamate metabolism, secondary inflammatory cell infiltration or demyelination / axonal abnormalities, phagocytosis and tissue softening caused by macrophages or microglia may occur, resulting in necrosis of nerve tissue (Non-Patent Literature 3, 4).

[0004] Traditionally, the diagnostic criteria for typical neuromyelitis optica (NMO) have been based on the criteria published by Wingerchuk et al. in 2006 (Non-Patent Literature 5). In 2007, as a neuromyelitis optica-associated disease (NMOSD), in addition to typical NMO, cases of long myelitis involving only optic neuritis (recurrent or bilateral) or more than three vertebral segments were also considered to fall under the same category (Non-Patent Literature 6). Therefore, in recent years, NMO has been considered a broader disease concept than the generally accepted concept of simple NMO. Consequently, clinical and imaging findings such as the length of the spinal cord lesion are not sufficient for a definitive diagnosis of NMO; therefore, anti-AQP4 antibody testing is extremely important for determining the diagnosis and treatment strategy for NMO.

[0005] The symptoms of acute neuromyelitis optica are often more severe than those of multiple sclerosis. A single relapse can lead to blindness if it is optic neuritis, and confinement to a wheelchair if it is myelitis. Therefore, prompt initiation of treatment is crucial. Furthermore, both acute and chronic neuromyelitis optica are accompanied by severe pain; therefore, treatment to alleviate this pain is also extremely important.

[0006] Cholesterol pulse therapy is the first-line treatment for acute neuromyelitis optica (Non-Patent Literature 7), and its effectiveness has been widely observed in clinical practice. For multiple sclerosis, this therapy has been shown to promote recovery from the worsening of clinical symptoms in the acute phase. Therefore, it is anticipated that similar effects can be achieved for neuromyelitis optica (Non-Patent Literature 8-10). Cholesterol pulse therapy is the same as the treatment for multiple sclerosis, with the standard being methylpeniscorticoids administered intravenously at 1,000 mg / day for 3 consecutive days. If symptom improvement is insufficient, an additional 1-2 treatment cycles (kur) are added. When cholesterol pulse therapy is ineffective, plasma exchange therapy is actively considered as a second-line treatment (Non-Patent Literature 11, 12), with early plasma exchange therapy expected in severe cases. As mentioned above, regarding the treatment of acute phase neuromyelitis optica, currently, there are only accumulated case reports based on their potential usefulness for treatments that have shown efficacy in multiple sclerosis or neuromyelitis optica. Furthermore, there are currently no effective treatments available for acute neuromyelitis optica.

[0007] RGM (repulsive guidance molecule) was initially identified as a membrane protein that induces axonal activity in the visual system (Non-Patent Document 13). The RGM family includes three members: RGMa, RGMb, and RGMc (Non-Patent Document 14). It is known that at least RGMa and RGMb function through the same signaling mechanism (Non-Patent Document 15). RGMc plays an important role in iron metabolism. Subsequent research has shown that RGM has functions such as inducing axonal formation and lamellar formation in Xenopus and chicken embryos, and controlling the closure of the neural tube in the head of mouse embryos (Non-Patent Document 16). Patent Document 1 discloses an axonal regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0008] In addition to its function during the production phase, RGMa is considered an inhibitor of axonal regeneration after central nervous system injury because it can recover its function after injury in adults and rats. Furthermore, in rats, inhibiting RGMa can promote axonal growth after spinal cord injury and thus promote functional recovery (Non-Patent Document 17). Specific antibodies that neutralize RGMa are described, for example, in Patent Document 2 (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3). Moreover, it is known that anti-RGMa neutralizing antibodies are effective in inhibiting the pathogenesis of neuromyelitis optica (Non-Patent Document 18). Thus, the role of RGMa in central nervous system injury has been elucidated, and its effect in inhibiting the pathogenesis of neuromyelitis optica has been known; in particular, no one has identified RGMa's involvement in the treatment of acute neuromyelitis optica, and such a therapeutic agent is not yet known. [Previous Technical Documents] [Patent Documents]

[0009] [Patent Document 1] International Publication WO2005 / 087268 [Patent Document 2] International Publication WO2009 / 106356 [Patent Document 3] International Publication WO2013 / 112922 [Patent Document 4] International Publication WO2016 / 175236 [Non-Patent Documents]

[0010] [Non-Patent Literature 1] Lancet 364: 2106-2112, 2004 [Non-Patent Literature 2] J Exp Med 202: 473-477, 2005 [Non-Patent Literature 3] J. Clin. Immunol. 3582, 129-135(2012) [Non-Patent Literature 4] Journal of the Neurological Sciences 306 (2011) 183-187 [Non-Patent Literature 5] Neurology 66: 1485-1489, 2006 [Non-Patent Literature 6] Lancet Neurol 6: 805-815, 2007 [Non-Patent Literature 7] Curr Treat Options Neurol 12: 244-255, 2010 [Non-Patent Literature 8] Neurology 53: 1107-1114, 1999 [Non-Patent Literature 9] Magn Reson Med Sci 7 : 55-58, 2008 [Non-Patent Literature 10] Tohoku J Exp Med 215 : 55-59, 2008 [Non-Patent Literature 11] Neurology 63 : 1081-1083, 2004 [Non-Patent Literature 12] Mult Sclr 13 : 128-132, 2007 [Non-Patent Literature 13] Neuron 5, 735-743 (1990) [Non-Patent Literature 14] Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513‐29, 2006 [Non-Patent Literature 15] Biochem. Biophys. Res. Commun. 382,​​ 795-800 (2009) [Non-Patent Literature 16] Curr. Opin. Neurobiol. 17, 29-34 (2007) [Non-Patent Literature 17] J. Cell Biol. 173, 47-58 (2006) [Non-Patent Literature 18] Scientific Reports 8:34 1-9 (2018) [Summary of the Invention]

[0011] [The problem that the invention aims to solve]

[0012] The inhibitory effect of anti-RGMa neutralizing antibodies on the pathogenesis of neuromyelitis optica is described in Non-Patent Literature 18. In this literature, the inhibitory effect on the pathogenesis of neuromyelitis optica was demonstrated by directly administering IgG derived from anti-AQP4 antibody-positive NMO patients to the spinal cord of healthy animals, along with simultaneous administration of anti-RGMa neutralizing antibodies. However, this literature does not describe the effect of anti-RGMa neutralizing antibodies on an acute neuromyelitis optica model reflecting human symptoms, and based solely on this literature, it remains unclear whether anti-RGMa neutralizing antibodies have a preventive or therapeutic effect on acute neuromyelitis optica. Furthermore, since severe pain accompanies the onset of neuromyelitis optica, there is an urgent need to develop a preventive or therapeutic drug for neuromyelitis optica that can simultaneously alleviate or treat the pain symptoms associated with the disease. The present invention aims to provide a drug effective against acute neuromyelitis optica and its symptoms. [Means for Solving the Problem]

[0013] The inventors of this case, through dedicated research to solve the aforementioned problems, discovered that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, have a restorative effect on acute exacerbations of neuromyelitis optica, an early repair effect on the damage to the blood-spinal cord barrier caused by myelitis, and an inhibitory effect on the granulocyte infiltration visible in the symptoms of acute neuromyelitis optica. Thus, they found excellent effects in the prevention or treatment of acute neuromyelitis optica. Furthermore, they discovered that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, can treat, reduce, or alleviate the pain symptoms of neuromyelitis optica, ultimately completing this invention. That is, this invention is as follows.

[0014] 1. A preventive or therapeutic agent for acute neuromyelitis optica, comprising an RGMa inhibitor. 2. The preventive or therapeutic agent of claim 1, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 3. The preventive or therapeutic agent of claim 2, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 4. The preventive or therapeutic agent of claim 2 or 3, wherein the anti-RGMa neutralizing antibody is an antibody capable of recognizing an amino acid sequence selected from sequence number 16, sequence number 36, sequence number 37, sequence number 38, and sequence number 39. 5. The preventive or therapeutic agent of any one of items 2 to 4, wherein the anti-RGMa neutralizing antibody system is selected from the following (a) to (l): (a) comprising: a light chain variable region containing LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and a heavy chain variable region containing HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10; (b) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12, and LCDR3 containing the amino acid sequence described in sequence number 13; and a heavy chain variable region of antiRGMa containing HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15, and HCDR3 containing SFG in its amino acid sequence. (c) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22; (d) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28; (e) an anti-RGMa neutralizing antibody containing the light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (f) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (g) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (h) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (i) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 42.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (j) containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (k) comprising: a light chain variable region containing LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44, and a heavy chain variable region containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34, and ( l) An antibody containing: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 45; and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

[0015] 6. A preventive or therapeutic agent for pain symptoms of neuromyelitis optica, comprising an RGMa inhibitor. 7. The preventive or therapeutic agent of claim 6, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 8. The preventive or therapeutic agent of claim 7, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 9. The preventive or therapeutic agent of claim 7 or 8, wherein the anti-RGMa neutralizing antibody is an antibody capable of recognizing an amino acid sequence selected from sequence number 16, sequence number 36, sequence number 37, sequence number 38, and sequence number 39. 10. The preventive or therapeutic agent of any one of items 7 to 9, wherein the anti-RGMa neutralizing antibody system is selected from the following (a) to (l): (a) comprising: a light chain variable region containing LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and a heavy chain variable region containing HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10; (b) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12, and LCDR3 containing the amino acid sequence described in sequence number 13; and a heavy chain variable region of antiRGMa containing HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15, and HCDR3 containing SFG in its amino acid sequence. (c) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22; (d) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28; (e) an anti-RGMa neutralizing antibody containing the light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (f) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (g) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (h) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (i) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 42.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (j) containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (k) comprising: a light chain variable region containing LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44, and a heavy chain variable region containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34, and ( 1) An antibody comprising: a light chain variable region comprising LCDR1 comprising the amino acid sequence described in sequence number 29, LCDR2 comprising the amino acid sequence described in sequence number 30, and LCDR3 comprising the amino acid sequence described in sequence number 45, and a heavy chain variable region comprising an anti-RGMa neutralizing antibody comprising HCDR1 comprising the amino acid sequence described in sequence number 32, HCDR2 comprising the amino acid sequence described in sequence number 33, and HCDR3 comprising the amino acid sequence described in sequence number 34. 11. A method for preventing or treating pain symptoms of acute neuromyelitis optica or neuromyelitis optica, comprising administering an effective amount of an RGMa inhibitor to a mammal requiring treatment. 12. The method for preventing or treating neuromyelitis optica as described in item 11, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 13. Use of an RGMa inhibitor in the manufacture of a preventive or therapeutic agent for acute neuromyelitis optica. [Effects of the Invention]

[0016] According to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, exhibit effects such as early repair of damage to the blood-spinal cord barrier (blood-brain barrier in the brain) visible in acute neuromyelitis optica, and are therefore used as preventive or therapeutic agents for acute neuromyelitis optica. Furthermore, according to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, exhibit effects such as inhibiting the infiltration of granulocytes into the spinal cord visible in acute neuromyelitis optica, and are therefore used as preventive or therapeutic agents for acute neuromyelitis optica. Moreover, according to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, can treat, reduce, or alleviate pain symptoms visible in neuromyelitis optica, and are therefore used as preventive or therapeutic agents for such pain symptoms.

Implementation Method

[0017] [Form of the Invention]

[0018] The following explains the terminology used in this invention. [Neutralization] Neutralization as used in this invention refers to a substance that can bind to a target and inhibit any function of that target. For example, an RGMa inhibitor refers to a substance that, after binding to RGMa, exhibits an inhibitory effect on the biological activity of RGMa.

[0019] [Antigenic Determinant Site] As used in this application, an antigenic determinant site comprises a polypeptide determinant capable of specifically binding to immunoglobulin or T-cell receptors. In one embodiment, the antigenic determinant site comprises a chemically active surface group of a molecule (e.g., an amino acid, a sugar side chain, a phosphoryl group, or a sulfonylurea group); in another embodiment, it may have specific three-dimensional structural characteristics and / or specific charge characteristics. An antigenic determinant site is an antigenic region that binds to an antibody.

[0020] [isolated] In this case, "isolated" in the context of RGMa inhibitors (such as antibodies) means that they have been identified and separated, and / or recovered from components in their natural state. Impurities in their natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, such as enzymes, hormones, and other protein- or non-protein-based solutes. Generally, to isolate RGMa inhibitors, purification can be performed through at least one purification step; RGMa inhibitors purified through at least one purification step can be referred to as "isolated RGMa inhibitors".

[0021] [Antibody] In this case, antibody, in a broad sense, refers to an Ig molecule composed of four polypeptide chains: two heavy chains (H chains) and two light chains (L chains), which maintain the characteristic of an immunoglobulin (Ig) molecule that is substantially bound to an antigenic determinant.

[0022] [Human Antibodies] The term "human antibodies" as used in this case refers to antibodies that, along with the light and heavy chains, originate from human immunoglobulins. Based on differences in the constant regions of the heavy chain, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) with a γ-chain heavy chain, IgM with a μ-chain heavy chain, IgA (including IgA1 and IgA2) with an α-chain heavy chain, IgD with a δ-chain heavy chain, or IgE with an ε-chain heavy chain. Furthermore, in principle, the light chain includes either the κ or λ chain.

[0023] [Anthropomorphic antibody] The anthropomorphic antibody referred to in this case refers to an antibody composed of a variable region consisting of a complementarity-determining region derived from an antibody derived from a non-human animal and a structural region derived from a human antibody, and a constant region derived from a human antibody.

[0024] [Chimeric Antibody] The chimeric antibody referred to in this case is an antibody consisting of a light chain, a heavy chain, or both, composed of a variable region derived from non-human and a constant region derived from human.

[0025] [Monospecific antibody] As used in this document, a monospecific antibody refers to an antibody that has a single antigen specificity and a single, independent antigen recognition site. For example, in this specification, a monospecific antibody that can recognize RGMa is referred to as an RGMa monospecific antibody.

[0026] [Multispecific antibody] The multispecific antibody referred to in this case refers to an antibody that has two or more different antigen specificities and two or more independent antigen recognition sites. Examples include bispecific antibodies with two antigen specificities and trispecific antibodies with three antigen specificities.

[0027] [Complementarity Determining Region (CDR)] The complementarity determining region (CDR) refers to the region within the variable region of an immunoglobulin molecule that forms the antigen-binding site. Also known as the hypervariable region, it refers to the part where the amino acid sequence varies significantly depending on the immunoglobulin molecule. There are three CDRs in each of the light and heavy chains. The three CDRs in the light chain are sometimes referred to as LCDR1, LCDR2, and LCDR3, and the three CDRs in the heavy chain are sometimes referred to as HCDR1, HCDR2, and HCDR3. For example, the CDRs of immunoglobulin molecules are determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0028] [Effective dose] An effective dose refers to the amount of a preventive or therapeutic agent sufficient to reduce or improve the severity and / or duration of an abnormality or one or more of its symptoms, prevent the worsening of an abnormality, slow down an abnormality, prevent the recurrence, occurrence, onset or progression of one or more symptoms related to an abnormality, detect an abnormality, or enhance or improve the preventive or therapeutic effect of one or more other treatments (such as preventive or therapeutic drugs).

[0029] [Percentage (%) identity of amino acid sequences] The "percentage (%) identity" of the amino acid sequence of a candidate polypeptide sequence, such as a variable region, relative to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a specific reference polypeptide sequence after the sequences are aligned, with gaps introduced if necessary to obtain maximum % identity, and any retained substitutions are not considered part of the sequence identity. Alignment for determining % identity can be achieved using various methods within the scope of the craft, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). The craft can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment relative to the entire length of the sequences to be compared. However, for this purpose, the % identity value, in pairwise alignment, can be obtained using the sequence comparison computer program BLAST. When BLAST is used for amino acid sequence comparison, the % identity score between provided amino acid sequence A and provided amino acid sequence B is calculated as follows: X / Y = 100 times this, where X is the number of amino acid residues that, when corrected by the BLAST sequence correction program for A and B, represent the same score, and Y is the total number of amino acid residues in B. When the lengths of amino acid sequence A and amino acid sequence B are different, it can be understood that the % identity score of A relative to B is different from the % identity score of B relative to A. Unless otherwise specified, all % identity values ​​here are obtained using the BLAST computer program as shown in the previous paragraph.

[0030] [Retention Substitution] Retention substitution refers to replacing an amino acid residue with another chemically similar amino acid residue in a manner that does not substantially alter the activity of the peptide. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue carrying the same charge. Examples of functionally similar amino acids that can undergo this substitution include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, aspartic acid, and cysteine. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Furthermore, examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0031] The embodiments of the present invention will be described in detail below. The present invention provides a preventive or therapeutic agent for acute neuromyelitis optica, which is a novel use of an RGMa inhibitor. Furthermore, the present invention provides a method for preventing or treating acute neuromyelitis optica, which comprises administering a preventive or therapeutic agent containing an effective amount of an RGMa inhibitor to a mammal requiring treatment.

[0032] <RGMa Inhibitor> The RGMa inhibitor of the present invention can be any substance that can act on RGMa itself to inhibit or weaken the activity of RGMa (hereinafter referred to as "RGMa activity" in this specification). Substances having, for example, the activity of directly inhibiting (weakening) RGMa activity by binding to RGMa, or the activity of indirectly inhibiting (weakening) RGMa activity by inhibiting the binding of RGMa to a receptor (such as compounds or antibodies described later) are referred to as the RGMa inhibitor of the present invention. Furthermore, the RGMa inhibitor of the present invention can also be a substance that inhibits the expression of RGMa, such as a substance that inhibits (weakens) RGMa activity by inhibiting the expression of RGMa (such as nucleic acid molecules described later), which are also included in the RGMa inhibitor of the present invention.

[0033] RGMa is identified as a protein that inhibits the growth of neurites in the central nervous system. The human RGMa protein, as shown in sequence number 1, is biosynthesized as a precursor protein composed of 450 amino acids. The N-terminal signaling peptides Met1 to Pro47 (meaning the peptides from the 1st methionine residue to the 47th proline residue on the N-terminal side, as described below) are removed, and the peptide bond between Asp168 and Pro169 is cleaved to generate the N-terminal region. Then, the C-terminal peptides Ala425 to Cys450 are generated by removing the C-terminal fragment from Pro169. At the same time, the C-terminal carboxyl group of Ala424, which is the C-terminal, is added to form a GPI (polysaccharide phosphatidylinositol) anchor to generate the C-terminal region. Human RGMa protein is a mature protein that is expressed on the cell membrane via GPI anchors, consisting of N-terminal regions (Cys48-Asp168) linked to C-terminal regions (Pro169-Ala424) by disulfide bonds.

[0034] In this invention, RGMa can be derived from any animal, but is preferably human RGMa. The precursor protein of human RGMa is composed of the amino acid sequence shown in Sequence Number 1 of the Sequence Listing. The precursor protein of mouse RGMa is composed of the amino acid sequence shown in Sequence Number 2 of the Sequence Listing, and the precursor protein of rat RGMa is composed of the amino acid sequence shown in Sequence Number 3 of the Sequence Listing; however, since the C-terminal peptide is removed, the amino acid sequence is the same for the mature protein. Examples of RGMa genes include, for example, the human RGMa gene composed of the base sequence shown in Sequence Number 4, but it is not limited thereto. The base sequences of RGM genes derived from various organisms can be easily obtained from well-known databases (GenBank, etc.).

[0035] Specifically, examples of RGMa inhibitors of the present invention include low molecular weight compounds, anti-RGMa neutralizing antibodies, their functionally altering antibodies, their binding antibodies, or their antigen-binding fragments; examples also include siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense oligonucleotides belonging to RGMa nucleic acid molecules. Among these RGMa inhibitors, anti-RGMa neutralizing antibodies, their functionally altering antibodies, their binding antibodies, and their antigen-binding fragments are preferred, and anti-RGMa neutralizing antibodies or their antigen-binding fragments are even more preferred, especially anti-RGMa neutralizing antibodies.

[0036] <Anti-RGMa Neutralizing Antibody> In this invention, the anti-RGMa neutralizing antibody can be any antibody that can bind to RGMa and neutralize RGMa activity, and can be a multi-strain antibody or a monoclonal antibody. In this invention, a monoclonal antibody is preferred. Furthermore, the anti-RGMa neutralizing antibody of this invention can be an RGMa monospecific antibody or a multispecific antibody that can recognize multiple RGMa and other antigens, and is preferably an RGMa monospecific antibody.

[0037] Furthermore, as a specific antigenic determinant, for human RGMa, it is preferably one or more of the following: sequence number 16 (amino acid numbers 47-69 of sequence number 1), sequence number 36 (amino acid numbers 298-311 of sequence number 1), sequence number 37 (amino acid numbers 322-335 of sequence number 1), sequence number 38 (amino acid numbers 349-359 of sequence number 1), and sequence number 39 (amino acid numbers 367-377 of sequence number 1), more preferably a combination of sequence numbers 36 and 37, and even more preferably a combination of sequence numbers 36, 37, and 39.

[0038] The anti-RGMa neutralizing antibody system of the present invention comprises polyclonal antibodies and monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or a partial fragment thereof (e.g., the antigenic determinant fragment mentioned above) as antigens, chimeric antibodies and anthropomorphic antibodies produced using gene recombination technology, and human antibodies produced by generating genetically modified animals using human antibodies. When the antibodies of the present invention are administered to humans as medicine, from the viewpoint of side effects, it is preferable to use anthropomorphic antibodies or human antibodies.

[0039] Specifically, the following antibodies (a) to (l) can be cited as the anti-RGMa neutralizing antibody of the present invention, and the individual manufacturing methods can be the methods described in Patent Documents 2-4.

[0040] Examples include anti-RGMa neutralizing antibodies selected from (a) comprising: light chain variable regions containing LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and heavy chain variable regions containing HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10 (the anti-RGMa neutralizing antibody further comprises antibodies with sequence numbers 36, 37, and 39 as antigenic determinants). (b) Containing: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12, and LCDR3 containing the amino acid sequence described in sequence number 13; and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15, and HCDR3 containing SFG in the amino acid sequence (the anti-RGMa neutralizing antibody further comprises antibodies with sequence numbers 36, 37, and 38 as antigenic determinants). (c) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19; and a heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22. (d) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28; (e) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31.(f) Contains: a light chain variable region of an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further contains an antibody with sequence number 16 as the antigenic determinant); and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 29, HCDR2 containing the amino acid sequence described in sequence number 30, and HCDR3 containing the amino acid sequence described in sequence number 35, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further contains an antibody with sequence number 16 as the antigenic determinant). (g) Containing: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as the antigenic determinant). (h) comprising: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as the antigenic determinant); (i) comprising: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 42.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further includes an antibody with sequence number 16 as the antigenic determinant), (j) containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further includes an antibody with sequence number 16 as the antigenic determinant). (k) comprising: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44; and a heavy chain variable region comprising an anti-RGMa neutralizing antibody comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as the antigenic determinant), and ( l) comprising: an antibody containing the light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 45, and an antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34 (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as the antigenic determinant). Of these, the antibody described in (a) is particularly preferred.

[0041] The method for manufacturing the anti-RGMa neutralizing antibody of the present invention can employ existing commonly used manufacturing methods. The antigen can be used directly for immunization or can be used by forming a complex with a carrier protein. The complex of the antigen and the carrier protein can be modulated using condensing agents such as glutaraldehyde, carbodiimide, and maleimide active ester. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[0042] Examples of mammals that can be immunized include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, sheep, horses, or cattle; methods of administration include subcutaneous, intramuscular, or intraperitoneal administration. At the time of administration, it can be mixed with complete or incomplete Freund's adjuvant, and administration is usually performed once every 2 to 5 weeks. Antibody-producing cell lines obtained from the spleen or lymph nodes of immunized animals are fused with myeloma (bone marrow cancer) cells to form fusion tumors. Myeloma cell lines derived from mammals, such as mice, rats, or humans, are used.

[0043] <Multi-strain antibody> A multi-strain antibody can be obtained, for example, by immunizing a mammal as described above with the antigen as described above, and then, as needed, with Freund's adjuvant, and obtaining the serum from the immunized animal.

[0044] <Monoclonal Antibody> Specifically, monoclonal antibodies can be obtained as follows. That is, using the antigen described above as an immunogen, the immunogen is injected or transplanted, as needed, with Freund's adjuvant, into the subcutaneous, intramuscular, intravenous, plantar, or intraperitoneal space of the mammal described above for sensitization 1 to several times. Generally, immunization is performed 1 to 4 times every 1 to 14 days from the initial immunization, and antibody-producing cells are obtained from the sensitized mammal about 1 to 5 days after the final immunization.

[0045] Monoclonal antibodies can be obtained using methods well known to the industry (e.g., 'Current Protocols in Molecular Biology' (John Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0046] The modulation of "fusion tumors" that secrete monoclonal antibodies can be carried out by following the method of Köhler and Milstein et al. (Nature, 256, 495, 1975) and modification methods based on it. That is, it is modulated by fusing antibody-producing cells, such as those from the spleen of an immune-sensitized mammal, with bone marrow cancer cells derived from mammals, preferably mice, rats, or humans, that do not have the ability to produce their own antibodies.

[0047] The bone marrow cancer cells used for cell fusion may be, for example, mouse bone marrow cancer P3 / X63-AG8.653(653), P3 / NSI / 1-Ag4-1(NS-1), P3 / X63-Ag8.U1(P3U1), SP2 / 0-Ag14(Sp2 / O, Sp2), PAI, F0 or BW5147, rat bone marrow cancer 210RCY3-Ag.2.3, human bone marrow cancer U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15, etc.

[0048] Examples of fusion promoters include polyethylene glycol. Cell fusion can usually be achieved by using polyethylene glycol (average molecular weight 1000-4000) at a concentration of about 20-50% at a temperature of 20-40°C, preferably 30-37°C, with the ratio of antibody-producing cells to myeloma cells usually being about 1:1 to 10:1, and allowing the reaction to proceed for about 1-10 minutes.

[0049] Screening for fusion tumors that produce monoclonal antibodies can be performed by culturing the fusion tumors in, for example, a microtiter plate and measuring the reactivity of the culture supernatant of the wellbore to the immunoantigen by immunochemical methods such as ELISA.

[0050] In the screening of antibody-generating fusion tumors, in addition to the binding assay to RGMa protein, an assessment is also performed to determine whether the antibody can inhibit the RGMa activity of the present invention. Based on these screening methods, the anti-RGMa neutralizing antibody of the present invention can be selected.

[0051] Further selection and propagation of fusion tumors containing target antibodies can be carried out by limiting dilution. Screening and breeding of fusion tumors are usually carried out by adding HAT (hypoxanthine, aminopterin, thymidine) in animal cell culture medium containing 10-20% fetal bovine serum.

[0052] Monoclonal antibodies are produced from fusion tumors by culturing the fusion tumors in vitro or by proliferating them in vivo in the ascites fluid of mammals such as mice and rats, and then isolating them from the culture supernatant or the ascites fluid of the mammals.

[0053] When culturing in vitro, various conditions, such as the characteristics of the cell species to be cultured and the culture method, can be used to promote the proliferation, maintenance, and preservation of the fusion tumor, and a nutrient medium suitable for producing monoclonal antibodies in the culture supernatant can be used. Examples of nutrient media include well-known nutrient media or nutrient media prepared from a basic culture medium.

[0054] The basic culture medium may include low-calcium culture media such as Ham'F12 medium, MCDB153 medium or low-calcium MEM medium, and high-calcium culture media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium. The basic culture medium may contain, depending on the purpose, substances such as serum, hormones, cytokines and / or various inorganic or organic substances.

[0055] The isolation and purification of monoclonal antibodies can be performed by feeding the culture supernatant or ascites to saturated ammonium sulfate, euglobulin precipitation, hexanoic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as anti-immunoglobulin column or protein A column, etc. Specifically, the purification of monoclonal antibodies, as a method for purifying immunoglobulins, can be easily achieved by using known methods, such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, using anion exchangers, or even affinity chromatography of RGMa proteins.

[0056] Monoclonal antibodies can also be obtained using phage presentation. In phage presentation, phages selected from any phage antibody library are screened using a target immunogen to select phages with the desired binding affinity to the immunogen. Next, the antibody-corresponding sequence contained within the phage is isolated or sequence-determined, and based on the isolated or determined sequence information, an expression vector containing a nucleic acid molecule encoding an antibody or antigen-binding region is constructed. Then, by culturing cell lines transfected with the expression vector, monoclonal antibodies can be produced. As a phage antibody library, human antibodies with the desired binding affinity can be generated by using a human antibody library.

[0057] <Nucleic Acid Molecule> The nucleic acid molecule encoding the anti-RGMa neutralizing antibody or its antigen-binding fragment of the present invention can be obtained, for example, by the following method. First, whole RNA is modulated from cells such as fusion tumors using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using random primers. Second, the cDNA encoding the antibody is amplified by PCR using oligonucleotides of preserved sequences as primers in the variable regions of known human antibody heavy chain and light chain genes. For sequences encoding constant regions, known sequences can be amplified by PCR. The base sequence of the DNA can be determined by embedding in sequence-determining plastids or the like, according to common methods. Alternatively, the DNA encoding the monoclonal antibody of the present invention can also be obtained by chemically synthesizing the variable region or a portion thereof and binding it to the sequence containing the constant region. The nucleic acid molecule can encode both the constant and variable regions of the heavy and light chains, or it can encode only the variable regions of the heavy and light chains. The preferred base sequences for encoding all constant and variable regions of the heavy and light chains in the constant regions are those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0058] <Functional Modifying Antibody> The function-modifying antibody of the anti-RGMa neutralizing antibody can be modulated as follows. For example, if the anti-RGMa neutralizing antibody of this invention is used as the host cell and manufactured using CHO cells with the α1,6-fucosyltransferase (FUT8) gene destroyed, an antibody with reduced fucose content in the glycan chain and enhanced cell-killing function can be obtained; if CHO cells with the FUT8 gene introduced are used as the host cell, an antibody with low cell-killing function can be obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). Furthermore, by altering the amino acid residues in the Fc region, complement activation function can be regulated (US Patent No. 6737056, US Patent No. 7297775, US Patent No. 7317091). Furthermore, by using mutants of the Fc region that enhance binding to one FcRn of the Fc receptor, the blood half-life can be prolonged (Hashiguchi et al., Biochemistry, 2010, Vol.82(8), p710). Antibodies with these altered functions can be manufactured through genetic engineering.

[0059] <Linked Antibody> The modified molecule of the anti-RGMa neutralizing antibody of the present invention can be a linked antibody. Examples of linked antibodies include those that chemically or genetically bind non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other functional molecules other than the anti-RGMa neutralizing antibody of this invention.

[0060] When PEG is used as a functional molecule, it can be of a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and can be linear or branched. By using, for example, an NHS active group, PEG can bind to the N-terminal amino group of the amino acid of an anti-RGMa neutralizing antibody.

[0061] When using radioactive materials for functional molecules, 131I, 125I, 90Y, 64Cu, 99Tc, 77Lu, or 211At can be used. Radioactive materials can be directly bound to anti-RGMa neutralizing antibodies via methods such as the chloramine-T method.

[0062] When functional molecules use toxins, they may use bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin), low molecular weight toxins (e.g., gerdromycin), maytansine, and cazithromycin, etc.

[0063] When functional molecules use low molecular weight compounds, examples include daunorubicin, doxorubicin, methotrexate, mitomycin, neocastatin, vincristine, and FITC fluorescent pigments.

[0064] When functional molecules use enzymes, they may use fluorescent enzymes (e.g., firefly fluorescent enzymes and bacterial fluorescent enzymes; US Patent No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., wasabi peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc.

[0065] The linkers used in chemically binding toxins, low molecular weight compounds or enzymes may include divalent free radicals (e.g., alkyl, aryl, heteroaryl), linkers represented by -(CR2)nO(CR2)n- (R is any substituent and n is a positive integer) or repeating units of alkoxy groups (e.g., polyoxyethylene, PEG, polymethyleneoxy, etc.) and alkylamine groups (e.g., polyoxyethylene, Jeffamine (trademark)), as well as esters and acetamides (e.g., succinate, succinamide, diethylene glycol ester, malonic acid ester and hexamethylene amine, etc.). Chemical modification methods for binding functional molecules have been established in this field (DJ King., Applications and Engineering of Monoclonal antibodies., 1998; TJ International Ltd., Monoclonal Antibody-Based Therapy of Cancer., 1998; Marcel Dekker Inc.; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0066] <Antigen-binding fragment> The "antigen-binding fragment" of the antibody referred to in the embodiments of the present invention refers to the antigen-binding portion of the antibody as described above, specifically including F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide bond Fv, single-stranded antibody (scFv) and polymers such as these; furthermore, the antigen-binding fragment includes binding fragments that bind non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, other antibodies and other functional molecules other than the anti-RGMa neutralizing antibody of this case, in a chemical or genetic engineering manner.

[0067] "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulins with proteolytic enzymes such as pepsin or papain, and generated by digestion before and after the disulfide bond between the two heavy chains in the hinge region. For example, if IgG is treated with papain, the upstream of the disulfide bond between the two heavy chains in the hinge region is cleaved, producing two identical antibody fragments: a light chain composed of VL (light chain variable region) and CL (light chain constant region), and a heavy chain fragment composed of VH (heavy chain variable region) and CHγ1 (γ1 region in the heavy chain constant region), which are bound together by disulfide bonds at the C-terminal region. These two identical antibody fragments are each called Fab. Furthermore, if IgG is treated with pepsin, the downstream of the disulfide bond between the two heavy chains in the hinge region is cleaved, producing an antibody fragment slightly larger than the aforementioned two Fab fragments linked by the hinge region. This antibody fragment is called F(ab')2.

[0068] <Chimeric Antibody> A preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a chimeric antibody. "Chimeric antibody" can be exemplified as a chimeric antibody whose variable region is derived from the variable region of an immunoglobulin from a non-human animal (mouse, rat, hamster, chicken, etc.), and whose constant region is derived from the constant region of a human immunoglobulin. For example, a mouse can be immunized with an antigen, and the variable region that binds to the antigen can be excised from the gene of a mouse monoclonal antibody and combined with the constant region of an antibody derived from human bone marrow. The constant region derived from human immunoglobulins has its own inherent amino acid sequence according to isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE; the constant region of the recombinant chimeric antibody of the present invention can be the constant region of any isotype of human immunoglobulin. Preferably, it is the constant region of human IgG. An expression vector can be prepared using the gene of the chimeric antibody thus prepared. The host cells are transformed using the expression vector to obtain chimeric antibodies and transgenic cells. The target chimeric antibody is obtained from the culture supernatant by culturing the transgenic cells.

[0069] <Anthropomorphic Antibody> Other preferred embodiments of the anti-RGMa neutralizing antibody of the present invention include anthropomorphic antibodies. The "anthropomorphic antibody" of the present invention is an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity-determining region) of a non-human animal antibody, such as a mouse, is grafted into a human antibody gene. It can be manufactured by referring to methods described, for example, in Japanese Patent Publication No. 4-506458 and Japanese Patent No. 2912618. Specifically, it refers to an anthropomorphic antibody characterized by a CDR, in which part or all of the CDR is derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the structural region of its variable region is derived from the structural region of a human immunoglobulin, and its constant region is derived from the constant region of a human immunoglobulin.

[0070] The anthropomorphic antibody of the present invention can be manufactured, for example, as follows. However, it is not intended to be limited to this manufacturing method.

[0071] For example, recombinant humanized antibodies derived from mouse monoclonal antibodies can be produced by genetic engineering, as described in Japanese Patent Application Publication No. 4-506458 and Japanese Patent Application Publication No. 62-296890. That is, the DNA of the mouse heavy chain CDR and the DNA of the mouse light chain CDR are isolated from the fusion tumor that produces the mouse monoclonal antibody, and the human heavy chain gene and the human light chain gene (excluding the human heavy chain CDR) are isolated from the human immunoglobulin gene.

[0072] A human heavy chain gene containing the isolated mouse heavy chain CDR portion of DNA is introduced into a suitable expression vector to enable expression. Similarly, a human light chain gene containing the mouse light chain CDR portion of DNA is introduced into another suitable expression vector to enable expression. Alternatively, human heavy and light chain genes containing mouse CDRs can be introduced into the same expression vector to enable expression. Host cells are transformed using the expression vector thus prepared to generate transformed cells with humanized antibodies, and the target humanized antibody is obtained from the culture supernatant by culturing the transformed cells.

[0073] <Human Antibody> Other preferred embodiments of the anti-RGMa neutralizing antibody of the present invention include human antibodies. A human antibody refers to an antibody whose entire region comprising the variable region and constant region of the heavy chain and the variable region and constant region of the light chain of an immunoglobulin is derived from a gene encoding a human immunoglobulin. This antibody can be produced by introducing the human antibody gene into a mouse. Specifically, for example, a genetically modified animal can be produced by inserting at least the human immunoglobulin gene into a gene locus in a mammal other than a human, such as a mouse, and this animal can be sensitized with an antigen, manufactured in the same manner as the aforementioned methods for producing polyclonal or monoclonal antibodies.

[0074] For example, genetically modified mice that produce human antibodies can be produced according to the methods described in Nature Genetics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; Japanese Patent Application Publication No. 4-504365; Japanese Patent Application Publication No. 7-509137; International Publication Manual No. WO94 / 25585; Nature, Vol.368, p.856-859, 1994; and Japanese Patent Application Publication No. 6-500233. More specifically, examples include HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), and KM (FCγRIIb-KO) mice.

[0075] Specifically, examples of the anti-RGMa neutralizing antibodies of the present invention include CDRs having a heavy chain variable region containing a specific amino acid sequence and a light chain variable region containing a specific amino acid sequence (preferably the anti-RGMa neutralizing antibodies of (a) to (l) above). Furthermore, as long as the antibody of the present invention possesses the ability to bind to RGMa and maintains the characteristic of inhibiting (neutralizing) RGMa activity, the amino acid sequence of the anti-RGMa neutralizing antibody (preferably the anti-RGMa neutralizing antibodies of (a) to (l) above) may be substituted, deleted, added, or inserted by one or more amino acids (1 to 20, 1 to 10, or 1 to 5, preferably 1 or 2). Such substitution, deletion, or addition may be introduced into the CDR, but is preferably introduced into regions outside the CDR. Furthermore, to maintain the characteristics of the present invention, the amino acid substitution is preferably a retention substitution.

[0076] The amino acid sequence of the anti-RGMa neutralizing antibody of the present invention (preferably the anti-RGMa neutralizing antibody of (a) to (l) above) containing substitution, deletion, etc. is, for example, an amino acid sequence in which the heavy chain variable region after the amino acid sequence is modified has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) of identity with the amino acid sequence before the modification, and an amino acid sequence in which the light chain variable region after the amino acid sequence is modified has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) of identity with the amino acid sequence before the modification.

[0077] In this invention, siRNA refers to a short double-stranded RNA that can inhibit the expression of a target gene (RGMa gene in this invention). The base sequence or length (base length) is not particularly limited as long as it can function as siRNA to inhibit the activity of RGMa in this invention; preferably, it is less than 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases. In this invention, shRNA refers to a molecule of about 20 base pairs or more, which forms a double-stranded structure within the molecule by including a palindrome-like base sequence in the single-stranded RNA portion, consisting of a short hairpin structure with a protrusion at the 3' end. After being introduced into a cell, this shRNA is broken down into lengths of about 20 bases (representatively, for example, 21, 22, or 23 bases), and similarly inhibits the expression of the target gene. In this invention, the siRNA and shRNA mentioned above can be in any form as long as they can suppress the expression of the RGMa gene.

[0078] In this invention, siRNA or shRNA can be synthesized artificially using chemical methods. Alternatively, for example, T7 RNA polymerase and the T7 promoter can be used to synthesize antisense and sense RNA from template DNA in vitro. Antisense oligonucleotides are any nucleotides that are complementary to or hybridize with a consecutive 5 to 100 base sequence in the DNA sequence of the RGMa gene, and can be either DNA or RNA. Furthermore, they can be modified as long as their function is not affected. Antisense oligonucleotides can be synthesized using common methods, such as easily using commercially available DNA synthesis equipment. Preferred sequences can be selected using general selection methods; for the siRNA or shRNA in this invention, the functional inhibition of RGMa expression can be assessed.

[0079] <Acute Phase of Neuromyelitis Optic> Clinically, the stages of neuromyelitis optica can be broadly divided into two types: the "acute phase" (which includes the concept of acute exacerbation in this invention) and the "chronic phase." The "acute phase" referred to here is the period when symptoms of neuromyelitis optica, such as optic neuritis and myelitis, appear and persist or worsen. During this phase, MRI can show follicular contrast at some lesions; or cerebrospinal fluid examination can show an increase in cell count or protein levels, which can be used as a reference to determine the acute phase. On the other hand, the "chronic phase" refers to the period when treatment improves the symptoms, leading to a stable condition. During this phase, follicular contrast disappears on MRI, which can be used as a reference to determine the chronic phase. The acute phase of neuromyelitis optica in this invention refers to the aforementioned "acute phase," and is not limited to the symptoms of the acute phase during the first episode of neuromyelitis optica; it also includes the symptoms of the acute phase during subsequent relapses of neuromyelitis optica. Furthermore, for the treatment subjects (preferably mammals, especially humans), the peak of symptoms in neuromyelitis optica typically occurs around 8.5 days (range: 2 to 63 days) (Reference: Flanagan et al. Ann Neurol. 2016 Mar; 79(3): 437-47, etc.). Therefore, the acute phase in this invention, from the onset of neuromyelitis optica, is usually within one month.

[0080] In this invention, neuromyelitis optica spectrum disorder (NMOSD) refers to neuromyelitis optica spectrum disorder, encompassing both anti-AQP4 antibody-positive and anti-AQP4 antibody-negative NMOSD as listed in the international diagnostic criteria for neuromyelitis optica (Wingerchuk et al. Neurology, 2015; 8582: 177-189). In this invention, anti-AQP4 antibody-positive NMOSD is preferred. The treatment subjects (preferably mammals, especially humans) in this invention are patients suffering from neuromyelitis optica spectrum disorder (NMOSD), particularly those suffering from anti-AQP4 antibody-positive NMOSD, to whom the prophylactic or therapeutic agent for acute-phase neuromyelitis optica of this invention can be administered.

[0081] Furthermore, in the acute phase of neuromyelitis optica of the present invention, pain is often present as one of the main symptoms of the patient; therefore, in the present invention, an RGMa inhibitor, preferably an anti-RGMa neutralizing antibody, can be used as a preventive or therapeutic agent for the common pain symptoms in neuromyelitis optica, and applied to patients with such pain. In addition, all matters described in the preventive and therapeutic agents and methods for preventing or treating acute phase neuromyelitis optica of the present invention are applicable to the description of the preventive or therapeutic agents and methods for preventing or treating the common pain symptoms in neuromyelitis optica of the present invention.

[0082] The term "treatment" as used herein includes any treatment of a disease of a subject of treatment, preferably a mammal, especially a human, including stopping the worsening of the disease and symptoms, and eliminating, curing, reducing or alleviating such disease and symptoms.

[0083] Furthermore, "prevention" includes preventing or inhibiting the occurrence of the aforementioned diseases in the treatment subject, preferably a mammal, especially a human. Moreover, "prevention" in this invention includes "prevention of recurrence" in alleviating or preventing the recurrence of the aforementioned diseases in the treatment subject, preferably a mammal, especially a human.

[0084] <Pharmaceutical Composition> The preventive or therapeutic agent for acute neuromyelitis optica of the present invention is usually administered systemically or locally in an oral or non-oral form. The preventive or therapeutic agent for acute neuromyelitis optica of the present invention can be formulated with an RGMa inhibitor as the active ingredient and can be pharmaceutically permissible by mixing with a carrier or additive. Such formulated pharmaceutical composition can be administered orally or non-oral. Specifically, it can be made into oral dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, and into non-oral dosage forms such as injections, infusions, suppositories, ointments, and patches. The mixing ratio of the carrier or additive can be appropriately set based on the range commonly used in the pharmaceutical field. The carriers or additives that can be blended are not particularly limited, and examples include various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oil-based bases, as well as various additives such as excipients, binders, pH adjusters, disintegrants, absorption promoters, lubricants, colorants, flavorings, and fragrances.

[0085] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, its functionally altering antibody, its binding antibody, or its antigen-binding fragment, it is preferable to formulate it into an injectable or infusion package co-formulated with a pharmaceutically permissible carrier for non-oral administration, such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, or local administration. For example, injectables or infusions containing anti-RGMa neutralizing antibodies can be used as solutions, suspensions, or emulsions. The solvent can be, for example, distilled water for injection, physiological saline, glucose solution, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). Furthermore, injectables or infusions containing such anti-RGMa neutralizing antibodies may also contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, pH adjusters, etc. Stabilizers can include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, butylated hydroxytoluene, etc. Solubilizers can include alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50, etc.). Suspensors can include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. Emulsifiers can include gum arabic, sodium alginate, fragrances, etc. Soothing agents can include benzyl alcohol, chlorobutanol, sorbitol, etc. Buffers can include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, TRIS buffer, etc. Preservatives can include, for example, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, chlorobutanol, benzyl alcohol, benzyl ammonium chloride, sodium dehydroacetate, sodium ethylenediaminetetraacetate, boric acid, borax, etc. Corrosive agents can include, for example, benzyl ammonium chloride, parabens, chlorobutanol, etc. pH adjusters can include, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.

[0086] When the RGMa inhibitor is a nucleic acid (siRNA, shRNA, antisense oligonucleotide, etc.), it can be delivered in the form of a non-viral vector or a viral vector. If it is in the form of a non-viral vector, methods such as: introducing nucleic acid molecules using liposomes (liposome method, HVJ-liposome method, cationic liposome method, liposome transfection method, lipoamine method, etc.), microinjection, and using a gene gun to transfer nucleic acid molecules and a vector (metal particles) together into cells can be employed. For example, when using a viral vector to deliver siRNA or shRNA to an organism, recombinant adenovirus, retrovirus, or other viral vectors can be used. By introducing DNA expressing siRNA or shRNA into devirulent retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, poxviruses, polioviruses, positive double-stranded viruses, Sendai viruses, SV40, and other DNA or RNA viruses, and infecting cells or tissues with this recombinant virus, genes can be introduced into cells or tissues.

[0087] The preparation thus obtained can prevent or treat acute neuromyelitis optica by administering an effective dose to, for example, humans or other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.). The dosage can be appropriately set considering the purpose, the severity of the disease, the patient's age, weight, sex, medical history, and the type of active ingredient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, the preferred dosage for an average human with a weight of approximately 65-70 kg is about 0.02 mg to 4000 mg daily, more preferably about 0.1 mg to 200 mg. The total daily dosage can be a single dose or a divided dose.

[0088] <Combined use with other drugs or treatments> In this invention, the preventive or therapeutic agent for acute neuromyelitis optica may be administered in combination with other drugs or treatments for the treatment of neuromyelitis optica.

[0089] Other agents or treatments that can be used in conjunction with the preventive or therapeutic agents for acute neuromyelitis optica of the present invention include, for example, intravenous administration of plasma exchange and / or immunoglobulin preparations, administration of mycophenolic acid, rituximab, eculizumab, and / or satralizumab. Such other agents or treatments may be biologically active agents or treatments that can effectively treat central nervous system abnormalities such as neuromyelitis optica, or effectively delay the progression of central nervous system abnormalities.

[0090] For example, other biologically active agents may be corticosteroids, (intravenous) immunoglobulin preparations, or antilymphocyte preparations, mycophenolic acids, rituximab, eculizumab, and / or saltuzumab. In a preferred embodiment, the patient is treated with intravenous immunotherapy (e.g., corticosteroids, such as (synthetic) glucocorticoids like methylpeniscortine). Thus, other biologically active agents may also be corticosteroids, such as (synthetic) glucocorticoids like methylpeniscortine. Other biologically active agents are administered intravenously. Plasma exchange may also be performed for other agents or treatments, such as in patients unresponsive to steroid infusions (e.g., those who have only inadequately suppressed central nervous system inflammation after steroid treatment). Thus, patients unresponsive to steroid infusions may also receive plasma exchange arbitrarily.

[0091] The other drugs or treatments described above may be administered or implemented before or after the administration of the preventive or therapeutic agents for neuromyelitis optica in the acute phase of the present invention, or simultaneously. [Example]

[0092] The present invention will be described in more detail below with reference to embodiments, but the present invention is not limited thereto.

[0093] [Example 1] Effect of anti-RGMa neutralizing antibody on acute neuromyelitis optica: The therapeutic effect of anti-RGMa neutralizing antibody on clinical symptom exacerbation was evaluated using an acute-phase severe NMO rat model. Furthermore, the anti-RGMa neutralizing antibody used in the experiment was an anti-RGMa neutralizing antibody containing the amino acid sequence (sequence numbers 5-10) described in this specification (a).

[0094] (1-1) The procedure for inducing NMO in rats was based on existing reports (Kurosawa K, et al., Acta Neuropathol Commun. 2015;3:82) to establish an acute-phase severe NMO rat model. Female Lewis rats were used in the experiment. As a pre-inflammatory environment to induce and disrupt the blood-spinal barrier (BSCB), an immunotherapy for the central antigen MBP was performed. MBP was prepared using Guinea pig brain myelin basic protein, dissolved in PBS to a concentration of 1 mg / mL, and mixed in an equal volume with Freund's complete adjuvant containing 1 mg / mL of dead tuberculosis bacteria H37Ra. The mixture was then ultrasonically vibrated to form a latex. The prepared latex (200 μl / head) was subcutaneously injected into two sites on the back. Ten days later, individuals with a neurological score below 1 were given a single intraperitoneal injection of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) at a dose of 3 mg / kg, which induced the deterioration of anti-AQP4 antibody-dependent clinical symptoms.

[0095] (1-2) Neurological score assessment was based on the following criteria for scoring neurological symptoms. The total neurological score (0-6 points) was used for assessment, calculated by summing the neurological scores of the tail and both hind limbs. Tail score: 0: no paralysis symptoms; 1: incomplete paralysis; 2: complete paralysis; Hind limb score: 0: no symptoms; 1: incomplete paralysis; 2: complete paralysis of the hind limb after dragging. Neurological scoring was performed blinded, with assessment of neurological symptoms conducted once daily until day 13 after administration of anti-RGMa neutralizing antibody (day 14 after administration of anti-AQP4 antibody).

[0096] (1-3) Grouping and administration of anti-RGMa neutralizing antibody: The animals were divided into two groups based on the deviation between the average neurological score and body weight the day after administration of anti-AQP4 antibody. The animals were administered anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) via a single intravenous tail vein at a dose of 10 mg / kg. Both the anti-RGMa neutralizing antibody group and the isotype control antibody group consisted of 6 animals.

[0097] (1-4) Results The effect of a single dose of anti-RGMa neutralizing antibody on the acute exacerbation of the acute phase severe NMO rat model is shown in Figure 1. Anti-RGMa neutralizing antibody or isotype control antibody was administered intravenously the day after the administration of anti-AQP4 antibody, and neurological symptoms were assessed once daily until day 14 after the administration of anti-AQP4 antibody. Neurological scores in the group receiving anti-AQP4 antibody followed by anti-RGMa neutralizing antibody the day after administration were consistently lower than those in the isotype control group throughout the observation period. The mean scores on days 2-8 and 2-14 after anti-AQP4 antibody administration were significantly lower (mean scores on days 2-8: 2.13±0.41 vs. 3.70±0.20 for isotype control IgG-treated rats, p<0.01, Mann-Whitney U test); mean scores on days 2-14: 1.90±0.43 vs. 3.05±0.19 for isotype control IgG-treated rats, p<0.05, Mann-Whitney U test). These results indicate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, demonstrate therapeutic efficacy in reducing the clinical deterioration of acute neuromyelitis optica.

[0098] [Example 2] Effect of anti-RGMa neutralizing antibody on destruction of the blood-spinal cord barrier: tEAE mice with BSCB destruction limited to the vertebral segments were used. The therapeutic effect of anti-RGMa neutralizing antibody on BSCB destruction was investigated by MRI. Furthermore, as the anti-RGMa neutralizing antibody, an anti-RGMa neutralizing antibody containing the amino acid sequence (sequence numbers 5-10) described in this specification was used in the experiment.

[0099] (2-1) The tEAE mice were prepared using female C57 / BL6J mice. Myelin Oligodendrocyte Glycoprotein Peptide Fragment 35-55, rat, mouse (MEVGWYRSPFSRVVHLYRNGK (sequence number 46); MOG35-55, Sigma-Aldrich) was dissolved in PBS to a concentration of 2 mg / mL and mixed with an equal volume of Freund's complete adjuvant containing 5 mg / mL of dead tuberculosis bacteria H37Ra. The mixture was then ultrasonically vibrated to prepare a latex. 100 μL of the prepared latex was subcutaneously injected into two sites on the back of each mouse (200 μL / head) for MOG immunization. Approximately 21 days later, 1.5 μL of a cytokine mixture (750 ng Tumor Necrosis Factor-α, 1 μg Interferon-γ) was injected into the thoracic spinal cord at a depth of 0.5-0.8 mm below the 8th thoracic vertebra. Subsequently, 200 ng pertussis toxin was administered into the tail vein two days later to induce tEAE.

[0100] (2-2) Neurological score assessment The neurological score was assessed under blind testing based on the existing reported judgment criteria (refer to Tanabe S, Fujita Y, Ikuma K, Yamashita T. Inhibiting repulsive guidance molecule-a suppresses secondary progression in mouse models of multiple sclerosis. Cell Death Dis. 2018;9(11):1061).

[0101] (2-3) Assessment of BSCB damage using Magnetic Resonance Imaging (MRI): Using a BioSpec 117 / 11 (Bruker), dynamic contrast-enhanced MRI (DCE-MRI) images were obtained before and after administration of oxaliscan (Daiichi Sankyo). Leakage of oxalis into the spinal cord, an indicator of BSCB damage, was quantitatively assessed by changes in T1 signal intensity. Under sevoflurane anesthesia, a body temperature maintenance device was installed, and oxaliscan was rapidly administered at a dose of 0.25 mmol / kg via a catheter placed in the tail vein. DCE-MRI was performed with an effective field of view (FoV) of 26 × 26 mm and an acquisition matrix of 200 × 200 mm. Eleven axial cross-sectional images were obtained with a slice thickness of 0.8 mm and intervals, centered on the site of cytokine injection in the thoracic spinal cord. Furthermore, the repetition time (TR) was set to 500 ms, the echo time (TE) to 18 ms, and the number of excitations (NEX) to 4 times, resulting in 6 images (each approximately 100 seconds) acquired over approximately 10 minutes. The images were output in DICOM format and analyzed using Fiji (http: / / fiji.sc / ). To eliminate the influence of the T1 signal from the cerebrospinal fluid, a region of interest (ROI) was defined within the spinal cord, and the T1 signal enhancement ratio (SER) at each time point was calculated based on equation (1). The total Gd influx rate for each individual was calculated as shown in equation (2), using the slope of SER per unit time as the influx rate, obtained using the SLOPE function in Microsoft Excel 2016 (Microsoft), and calculated as the sum of 11 slices.

[0102]

[0103] Based on the results of the study on normal mice, when the value of SLOPE(SER0):SER(10), 0:10) in Equation (2) is less than 0.02, it is considered as an analytical error and is excluded from the calculation objects in Equation (2).

[0104] (2-4) Grouping and administration of anti-RGMa neutralizing antibody: Seven days after injection of cytokines, the mice were divided into two groups in a homogeneous manner. Anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously via the tail vein twice weekly at a dose of 10 mg / kg. The anti-RGMa neutralizing antibody group consisted of 10 mice, and the isotype control antibody group consisted of 9 mice. Neurological score assessment and MRI analysis were performed on the same individual.

[0105] (2-5) Results: The effects of anti-RGMa neutralizing antibody on the recovery of BSCB damage and neurological symptoms in tEAE mice during the acute phase are shown in Figure 2. Using the quantitative value after 7 days as a baseline, the rate of change in BSCB leakage intensity after 7, 14, and 21 days of cytokine injection in the same individual was calculated longitudinally to analyze the recovery effect of anti-RGMa neutralizing antibody on BSCB damage. The BSCB leakage occurring 7 days after cytokine injection was significantly inhibited by repeated administration of anti-RGMa neutralizing antibody (14 days after cytokine injection, p < 0.001, Bonferroni multiple comparison test), indicating early recovery of BSCB damage (Figure 2A). Furthermore, the neurological score obtained in the same individual was also significantly inhibited, indicating early recovery of neurological symptoms (Figure 2B). The correlation between the intensity of BSCB leakage and the severity of neurological symptoms in tEAE mice during the acute phase is shown in Figure 3. A strong positive correlation was observed between the intensity of BSCB leakage 7 days after cytokine injection and the severity of neurological symptoms (r = 0.831, p < 0.001), and this positive correlation was also observed 14 days later (r = 0.549, p < 0.05). This indicates that the severity of BSCB destruction modifies the severity of neurological symptoms. These results suggest that RGMa inhibitors, especially anti-RGMa neutralizing antibodies, can demonstrate efficacy against acute neuromyelitis optica by promoting the repair of BSCB damage.

[0106] [Example 3] The therapeutic effect of anti-RGMa neutralizing antibody on blood-spinal cord barrier destruction was evaluated by immunohistochemical staining to analyze the intraspinal leakage of rat IgG caused by blood-spinal cord barrier destruction.

[0107] (3-1) The rat model of acute severe NMO was established using female Lewis rats. On the 10th day after MBP immunization, individuals with a neurological score below 1 were given a single intraperitoneal injection of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) at a dose of 3 mg / kg to induce NMO symptoms.

[0108] (3-2) Grouping and administration of anti-RGMa neutralizing antibody: The animals were divided into two groups based on the deviation between the mean neurological score and body weight the following day, which minimized the difference between the groups. Anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously via the tail vein at a dose of 10 mg / kg. The anti-RGMa neutralizing antibody treatment group and the isotype control antibody treatment group each consisted of 6 animals, while the healthy untreated group consisted of 4 animals.

[0109] (3-3) Immunohistochemical staining was performed on the 4th day after administration of anti-AQP4 antibody. After blood removal, the spinal cord was harvested and post-fixed in 4% paraformaldehyde at 4°C for 1 day. Following fixation, the tissue was cryoprotected with sucrose and embedded in OCT complex. Thin sections of 30 μm were prepared to form frozen sections, which were then immunostained using Alxa488-labeled donkey anti-rat IgG antibody (1:500, Thermo Fisher Scientific). The stained sections were photographed using an inverted fluorescence microscope (Olympus IX83), and the percentage of rat IgG positive area relative to the spinal cord section (% rat IgG positive area) was determined using ImageJ software.

[0110] (3-4) The effect of anti-RGMa neutralizing antibody on spinal cord leakage of IgG in rats is shown in Figure 4. By administering anti-RGMa neutralizing antibody the day after the onset of NMO, the intraspinal leakage of rat IgG, an indicator of blood-spinal cord barrier damage, was significantly inhibited. The above results indicate that the effect of RGMa inhibitors, especially anti-RGMa neutralizing antibody, on acute neuromyelitis optica is due to its promoting effect on the repair of blood-spinal cord barrier damage.

[0111] [Example 4] Effect of anti-RGMa neutralizing antibody on pain symptoms of NMO syndrome: Anti-RGMa neutralizing antibody was repeatedly administered to rats with acute severe NMO to evaluate its effect on persistent pain.

[0112] (4-1) The acute-phase severe NMO rat model was established using female Lewis rats. On the 10th day after MBP immunization, individuals with a neurological score below 1 were administered a single intraperitoneal injection of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) at a dose of 3 mg / kg to induce NMO symptoms.

[0113] (4-2) Assessment of pain-related behaviors: Pain was assessed using the von Frey stimulus up-down method (see Chaplan, SR, Bach, FW, Pogrel, JW, Chung, JM, Yaksh, TL, Quantitative assessment of tactile allodynia in the rat paw, J. Neurosci. Methods, 53, 55-63 (1994)) to determine the 50% escape response threshold (g) for raising the hind limb. The analysis used the average of the 50% escape response threshold (g) of both hind limbs.

[0114] (4-3) Grouping and Administration of Anti-RGMa Neutralizing Antibody: The groups were divided into two groups based on minimizing the deviation between the mean neurological score and body weight the day after administration of anti-AQP4 antibody. Anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously via the tail vein once weekly at a dose of 10 mg / kg. The analysis group consisted of three groups: the anti-RGMa neutralizing antibody treatment group, the isotype control antibody treatment group, and the healthy untreated group, each consisting of six individuals. During the period of worsening neurological symptoms, individuals unable to undergo von Frey stimulation due to hind limb weakness were observed. Therefore, the data groups on day 4 after anti-AQP4 antibody administration included: the anti-RGMa neutralizing antibody treatment group (n=5), the isotype control antibody treatment group on day 7 after anti-AQP4 antibody administration (n=2), and the anti-RGMa neutralizing antibody treatment group (n=3).

[0115] (4-4) The results show the inhibitory effect of anti-RGMa neutralizing antibodies on pain in Figure 5. The group given anti-RGMa neutralizing antibodies the day after the onset of NMO recovered from the 50% escape response threshold more quickly compared to the group given isotype control antibodies. Furthermore, a significant increase in the 50% escape response threshold was observed on days 18 and 21 after administration of anti-AQP4 antibodies. These results indicate that RGMa inhibitors, especially anti-RGMa neutralizing antibodies, are effective in treating pain symptoms in acute neuromyelitis optica.

[0116] [Example 5] The inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord was analyzed by immunohistochemical staining.

[0117] (5-1) The acute-phase severe NMO rat model was established using female Lewis rats. On the 10th day after MBP immunization, individuals with a neurological score below 1 were administered a single intraperitoneal injection of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) at a dose of 3 mg / kg to induce NMO symptoms.

[0118] (5-2) Grouping and administration of anti-RGMa neutralizing antibody: The animals were divided into two groups based on the deviation between the mean neurological score and body weight the day after administration of anti-AQP4 antibody. The animals were administered anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) via the tail vein at a dose of 10 mg / kg. The anti-RGMa neutralizing antibody treatment group and the isotype control antibody treatment group each consisted of 6 animals, while the healthy untreated group consisted of 4 animals.

[0119] (5-3) Immunohistochemical staining was performed on the 4th day after administration of anti-AQP4 antibody. After blood removal, the spinal cord was harvested and post-fixed in 4% paraformaldehyde at 4°C for 1 day. After fixation, the tissue was cryoprotected by sucrose substitution, and then embedded in OCT complexes. Thin sections of 30 μm were prepared to form frozen sections. For the primary antibody system, rabbit anti-rat granulocyte serum (1:5000, LifeSpan BioSciences) was used; for the secondary antibody system, Alxa488-labeled donkey anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific) was used for immunohistochemical staining. The stained sections were photographed using an inverted fluorescence microscope (Olympus IX83), and the percentage of granulocyte-positive area relative to the spinal cord section (% granulocyte-positive area) was determined using ImageJ software.

[0120] (5-4) The results show the inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord of rats with acute severe NMO in Figure 6. Administration of anti-RGMa neutralizing antibody the day after the onset of NMO significantly inhibited granulocyte infiltration in the spinal cord of NMO rats. These results indicate that RGMa inhibitors, especially anti-RGMa neutralizing antibody, can inhibit granulocyte infiltration seen in the symptoms of acute neuromyelitis optica. Since the study of inhibitory effects on granulocyte infiltration is helpful in assessing the efficacy against acute neuromyelitis optica, it is concluded that RGMa inhibitors, especially anti-RGMa neutralizing antibody, demonstrate efficacy against acute neuromyelitis optica through inhibition of granulocyte infiltration.

[0121] [Example 6] The expression of RGMa at the site of AQP4 detachment in the spinal cord was detected by immunohistochemical staining of the spinal cord of NMO rats in the acute phase of severe NMO rat model.

[0122] (6-1) The acute-phase severe NMO rat model was established using female Lewis rats. Guinea pig brain myelin basic protein was dissolved in PBS to a concentration of 1 mg / mL, and mixed with an equal volume of Freund's complete adjuvant containing 1 mg / mL of dead tuberculosis bacteria H37Ra. The mixture was then ultrasonically vibrated to form a latex. MBP latex (200 μl / head) was administered subcutaneously (MBP immunization). Ten days later, individuals with a neurological score below 1 were given a single intraperitoneal injection of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) at a dose of 3 mg / kg to induce NMO symptoms.

[0123] (6-2) Immunohistochemical staining was performed on the first day after administration of anti-AQP4 antibody. After blood removal, spinal cord was harvested and post-fixed in 4% paraformaldehyde at 4°C for one day. Following fixation, the tissue was cryoprotected with sucrose and embedded in OCT complex. Frozen sections were prepared by thinning to 30 μm. For the primary antibody system, goat anti-RGMa antibody (1:100, R&D Corporation) and rabbit anti-AQP4 antibody (1:1000, Cell Signaling Technology) were used. For the secondary antibody system, Alxa488-labeled donkey anti-goat IgG antibody (1:500, Thermo Fisher Scientific) and Alxa647-labeled donkey anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific) were used for double immunohistochemical staining. The stained sections were photographed using an inverted fluorescence microscope, Olympus IX83.

[0124] (6-3) Results The immunohistochemical staining images of spinal cord sections from rats with acute severe NMO are shown in Figure 7. Strong RGMa expression can be seen at the sites of AQP4 detachment in NMO lesions.

[0125] <Sequence Listing Description> Sequence Number 1: Amino acid sequence of human RGMa precursor protein; Sequence Number 2: Amino acid sequence of mouse RGMa precursor protein; Sequence Number 3: Amino acid sequence of rat RGMa precursor protein; Sequence Number 4: DNA sequence of human RGMa gene; Sequence Number 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3; Sequence Number 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3; Sequence Number 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3; Sequence Number 8: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r116A3; Sequence Number 9: Anti-RGMa neutralizing antibody The amino acid sequence of r116A3 HCDR2 (Sequence No. 10): Amino acid sequence of anti-RGMa neutralizing antibody r116A3 HCDR3 (Sequence No. 11): Amino acid sequence of anti-RGMa neutralizing antibody r70E LCDR1 (Sequence No. 12): Amino acid sequence of anti-RGMa neutralizing antibody r70E LCDR2 (Sequence No. 13): Amino acid sequence of anti-RGMa neutralizing antibody r70E LCDR3 (Sequence No. 14): Amino acid sequence of anti-RGMa neutralizing antibody r70E HCDR1 (Sequence No. 15): Amino acid sequence of anti-RGMa neutralizing antibody r70E HCDR2 (Sequence No. 16): Amino acid sequence of antigenic determinant of human RGMa. Sequence number 17: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 5F9; Sequence number 18: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 5F9; Sequence number 19: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 5F9; Sequence number 20: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 5F9; Sequence number 21: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 5F9; Sequence number 22: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 5F9; Sequence number 23: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 8D1; Sequence number 24: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 8D1. Amino acid sequence of LCDR2 (Sequence No. 25): Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 8D1 (Sequence No. 26): Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 8D1 (Sequence No. 27): Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 8D1 (Sequence No. 28): Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 8D1 (Sequence No. 29): Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody AE12-1 (Sequence No. 30): Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody AE12-1 (Sequence No. 31): Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1 (Sequence No. 31)Sequence number 32: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody AE12-1 Sequence number 33: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody AE12-1 Sequence number 34: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody AE12-1 Sequence number 35: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1Y Sequence number 36: Amino acid sequence of antigenic determinant site of human RGMa Sequence number 37: Amino acid sequence of antigenic determinant site of human RGMa Sequence number 38: Amino acid sequence of antigenic determinant site of human RGMa Sequence number 39: Amino acid sequence of antigenic determinant site of human RGMa Sequence number 40: Amino acid sequence of LCDR3 in anti-RGMa neutralizing antibody AE12-1F; Sequence number 41: Amino acid sequence of LCDR3 in anti-RGMa neutralizing antibody AE12-1H; Sequence number 42: Amino acid sequence of LCDR3 in anti-RGMa neutralizing antibody AE12-1L; Sequence number 43: Amino acid sequence of LCDR3 in anti-RGMa neutralizing antibody AE12-1V; Sequence number 44: Amino acid sequence of LCDR3 in anti-RGMa neutralizing antibody AE12-1I; Sequence number 45: Amino acid sequence of LCDR3 in anti-RGMa neutralizing antibody AE12-1K; Sequence number 46: Amino acid sequences (35-55) of rat and mouse MOGs [Industrial Applicability]

[0126] In this invention, since the RGMa inhibitor is used for the prevention or treatment of acute neuromyelitis optica and for the prevention or treatment of pain symptoms of neuromyelitis optica, it has high utilization value in the pharmaceutical industry. [Simplified Explanation of the Diagram]

[0127] [Figure 1] shows the therapeutic effect of anti-RGMa neutralizing antibody (some reports simply as anti-RGMa antibody) on the clinical acute exacerbation of a severe NMO rat model in the acute phase. Data are expressed as mean ± SEM. The number of cases in each group is indicated in parentheses in the legend of the figure. [Figure 2] shows the effect of anti-RGMa neutralizing antibody on the recovery of hematospinal cord barrier destruction and neurological symptoms in tEAE mice in the acute phase. Figure 2A shows the recovery effect of anti-RGMa neutralizing antibody on the degree of spondylogenetic leakage into the spinal cord during BSCB destruction in tEAE mice in the acute phase. Data are expressed as mean ± SEM, and the number of cases in each group is indicated in parentheses in the legend of the figure. Individuals in which no spondylogenetic leakage into the spinal cord was observed at the time point 7 days after cytokine injection and in which MRI images could not be obtained 14 days after cytokine injection were excluded from the analytical data. Statistical analysis was performed using the Bonferroni multiple comparison test (***p<0.001). Arrows indicate the antibody administration date. Figure 2B shows the effect of anti-RGMa neutralizing antibody on the recovery of neurological symptoms. Data are expressed as mean ± SEM, and the number of cases in each group is indicated in parentheses in the legend. Arrows indicate the date of antibody administration. Statistical analysis was performed at each time point using the Mann-Whitney U test (*p < 0.05, **p < 0.01). [Figure 3] shows the correlation between the intensity of intraspinal leakage and the severity of neurological symptoms in tEAE mice during the acute phase. Data were plotted for each individual, and statistical analysis was performed using SPEARMAN ranking correlation analysis. The number of cases in each group is indicated in parentheses in the legend. One case (anti-RGMa neutralizing antibody administration group) showed a loss on MRI images 14 days after cytokine injection. [Figure 4] shows immunohistochemical staining images and figures showing the therapeutic effect of anti-RGMa neutralizing antibody on the destruction of the blood-spinal cord barrier in an acute-phase severe NMO rat model. Quantitative analysis data are expressed as mean ± SEM, and statistical analysis was performed using Turkey's multiple comparison test (*p < 0.05). [Figure 5] shows the effect of anti-RGMa neutralizing antibody on pain-related behaviors in an acute-phase severe NMO rat model. Data are expressed as mean ± SEM, and statistical analysis was performed using Turkey's multiple comparison test (***p < 0.001, **p < 0.01, *p < 0.05 vs healthy untreated group; ##p < 0.01, #p < 0.05 vs NMO isotype control group antibody treatment group). Arrows indicate the administration date of anti-RGMa neutralizing antibody or isotype control group antibody. [Figure 6] shows immunohistochemical staining images and figures showing the inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord of an acute-phase severe NMO rat model.The quantitative analysis data are expressed as mean ± SEM, and statistical analysis was performed using Turkey's multiple comparison test (**p < 0.01, *p < 0.05). [Figure 7] shows the immunohistochemical staining image of RGMa at the site of AQP4 detachment in the spinal cord of a rat model of acute severe NMO. [Sequence List]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

Claims

1. A preventive or therapeutic agent for acute neuromyelitis optica, comprising an RGMa inhibitor.

2. The preventive or therapeutic agent as requested in item 1, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

3. The preventive or therapeutic agent as requested in item 2, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

4. The preventive or therapeutic agent as requested in item 2 or 3, wherein the anti-RGMa neutralizing antibody is an antibody that can identify an amino acid sequence selected from sequence number 16, sequence number 36, sequence number 37, sequence number 38 and sequence number 39.

5. The preventive or therapeutic agent as claimed in any of claims 2 to 4, wherein the anti-RGMa neutralizing antibody system is selected from the following (a) to (l): (a) comprising: a light chain variable region containing LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and a heavy chain variable region containing HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10; (b) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12, and LCDR3 containing the amino acid sequence described in sequence number 13; and a heavy chain variable region of antiRGMa containing HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15, and HCDR3 containing SFG in its amino acid sequence. (c) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19; and a heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22. (d) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28; (e) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (f) an anti-RGMa neutralizing antibody containing the light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (g) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (h) Contains: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (i) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 42, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (j) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (k) containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44; and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; and ( l) An antibody containing: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 45; and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

6. A preventive or therapeutic agent for pain symptoms of neuromyelitis optica, comprising an RGMa inhibitor.

7. The preventive or therapeutic agent as requested in item 6, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

8. The preventive or therapeutic agent as requested in item 7, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

9. The preventive or therapeutic agent as requested in paragraph 7 or 8, wherein the anti-RGMa neutralizing antibody is an antibody that can identify an amino acid sequence selected from sequence number 16, sequence number 36, sequence number 37, sequence number 38 and sequence number 39.

10. The preventive or therapeutic agent of any one of claims 7 to 9, wherein the anti-RGMa neutralizing antibody system is selected from the following (a) to (l): (a) comprising: a light chain variable region containing LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and a heavy chain variable region containing HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10; (b) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12, and LCDR3 containing the amino acid sequence described in sequence number 13; and a heavy chain variable region of antiRGMa containing HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15, and HCDR3 containing SFG in its amino acid sequence. (c) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19; and a heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22. (d) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28; (e) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (f) an anti-RGMa neutralizing antibody containing the light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (g) Containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (h) Contains: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41; and a heavy chain variable region of antiRGMa neutralizing antibody containing HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34. (i) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 42, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (j) Contains: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43.And an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; (k) containing: a light chain variable region of LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44; and an anti-RGMa neutralizing antibody containing the heavy chain variable region of HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; and ( l) An antibody containing: a light chain variable region comprising LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 45; and a heavy chain variable region comprising HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

11. A method for the prevention or treatment of pain symptoms in acute neuromyelitis optica or neuromyelitis optica, comprising administering an effective amount of an RGMa inhibitor to the mammal requiring treatment.

12. The prevention or treatment method as requested in item 11, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

13. Use of an RGMa inhibitor in the manufacture of a preventive or therapeutic agent for acute neuromyelitis optica.

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