Agent for preventing or treating acute-phase neuromyelitis optica

Abstract

To provide an effective agent for acute-phase neuromyelitis optica and symptoms of neuromyelitis optica.SOLUTION: Provided is an agent for preventing or treating pain symptoms in acute-phase neuromyelitis optica and neuromyelitis optica, the agent comprising an RGMa inhibiting substance.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a preventive or therapeutic agent for acute neuromyelitis optica, which comprises an RGMa inhibitor, and a preventive or therapeutic agent for pain symptoms associated with said neuromyelitis optica. [Background technology]

[0002] Neuromyelitis optica (NMO), also known as Devic's disease, is an inflammatory central nervous system disease characterized by severe optic neuritis and transverse myelitis affecting three or more pyramidal cones. In 2004, an IgG specific to NMO (NMO-IgG) was discovered (Non-Patent Document 1). It was further reported that its target antigen is aquaporin-4 (AQP4), a water channel protein that is highly expressed in the foot processes of astrocytes. In other words, NMO-IgG is an anti-AQP4 antibody. (Non-patent document 2).

[0003] Therefore, unlike multiple sclerosis, which is a demyelinating disease, NMO is an astrocytopathy in which astrocytes are destroyed by anti-AQP4 antibodies. The pathology of NMO is characterized by central inflammation due to activation of cellular immunity and destruction of the blood spinal cord barrier (BSCB). In the brain, increased permeability of the blood-brain barrier (BBB) ​​leads to the influx of anti-AQP4 antibodies into the brain and spinal cord and complement activation, resulting in widespread loss of AQP4 and destruction and loss of astrocytes. Functional disorders such as astrocytic cell damage (complement-dependent and antibody-dependent cellular cytotoxicity) and breakdown of the blood-brain barrier due to astrocyte loss, as well as abnormal glutamate metabolism, secondary to inflammatory cell infiltration, demyelination, axonal damage, phagocytosis by macrophages / microglia, and tissue softening, resulting in necrosis of neural tissue. It is believed that this is caused by the oxidative stress (Non-Patent Documents 3 and 4).

[0004] The criteria published by Wingerchuk et al. in 2006 have been widely used as diagnostic criteria for typical neuromyelitis optica (NMOSD) (Non-patent Document 5), but in 2007, it was renamed as neuromyelitis optica-related disorder (NMOSD). In addition to typical neuromyelitis optica, optic neuritis (recurrent or bilateral) and optic neuritis extending over three or more vertebrae may also occur. Cases of neuromyelitis alone have also come to be considered to be in the same category of disease (Non-Patent Document 6). Therefore, in recent years, neuromyelitis optica has been considered a broader disease concept than the simple neuromyelitis optica that has been traditionally considered. As such, it is difficult to diagnose neuromyelitis optica solely based on clinical and imaging findings, such as the length of spinal cord lesions, and therefore anti-AQP4 antibodies are an extremely important test for diagnosing neuromyelitis optica and determining treatment strategies.

[0005] Symptoms of acute neuromyelitis optica are often more severe than those of multiple sclerosis, and a single relapse can lead to blindness in optic neuritis and wheelchair use in myelitis, so it is important to start treatment promptly. Neuromyelitis optica is also accompanied by severe pain, both in the acute and chronic stages, so it is important to also provide treatment to relieve that pain.

[0006] The first choice of treatment for acute neuromyelitis optica is steroid pulse therapy (Non-Patent Document 7), and its effectiveness is widely recognized in clinical settings. Since its effectiveness in promoting recovery from the exacerbation of acute clinical symptoms in multiple sclerosis has been proven, it is believed that a similar effect can be achieved in neuromyelitis optica (Non-Patent Documents 8-10). Steroid pulse therapy involves administering 1,000 mg of methylprednisolone per day by intravenous infusion for three consecutive days, similar to the treatment for multiple sclerosis. If steroid pulse therapy is ineffective, plasma exchange therapy should be considered as a second-line treatment (Non-Patent Documents 11 and 12), and in severe cases, plasma exchange therapy should be initiated early. As mentioned above, in acute treatment, only case reports based on the potential of treatments that have been shown to be effective in multiple sclerosis and neuromyelitis optica have been accumulated. Furthermore, there are currently no effective treatments on the market for acute neuromyelitis optica.

[0007] RGM (repulsive guidance molecule) was originally identified as an axon guidance molecule in the visual system. RGM family is a membrane protein that has been identified as a marker for iron metabolism (Non-Patent Document 13). The RGM family includes three members, RGMa, RGMb, and RGMc (Non-Patent Document 14), and it is known that at least RGMa and RGMb function via the same signal transduction mechanism (Non-Patent Document 15). RGMc plays an important role in iron metabolism. Subsequent research has revealed that RGM has functions such as axon guidance and lamina formation in Xenopus and chicken embryos, and regulating the closure of the cranial neural tube in mouse embryos (Non-Patent Document 16). Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0008] In addition to its function during development, RGMa is re-expressed after central nervous system injury in adult humans and rats, and RGMa inhibition enhances axonal growth and promotes functional recovery after spinal cord injury in rats (Non-Patent Document 17). Specific antibodies that neutralize RGMa are described in, for example, 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). In addition, it is known that anti-RGMa neutralizing antibodies are effective in suppressing the onset of neuromyelitis optica (Non-Patent Document 18). Thus, the role of RGMa in central nervous system injury has been clarified, and its effect on suppressing the onset of neuromyelitis optica is known. However, the involvement of RGMa in the treatment of acute neuromyelitis optica has not been identified, and no such therapeutic agent is known. [Advanced Technology Documents] [Chartered documents]

[0009]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Non-licensed literature

[0010]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

[0011] The inhibitory effect of anti-RGMa neutralizing antibodies on the onset of neuromyelitis optica is described in Non-Patent Document 18. This document demonstrates that the onset of neuromyelitis optica was suppressed by directly administering IgG from an anti-AQP4 antibody-positive NMO patient to the spinal cord of a healthy animal and simultaneously administering an anti-RGMa neutralizing antibody. However, the document does not describe the effect of anti-RGMa neutralizing antibodies on an acute neuromyelitis optica model that reflects the human pathology, and it is not clear from the description alone whether anti-RGMa neutralizing antibodies have a preventive or therapeutic effect on acute neuromyelitis optica. Furthermore, since the onset of neuromyelitis optica is accompanied by severe pain, there has been a strong demand for a preventive or therapeutic drug for neuromyelitis optica that can also alleviate or treat the pain symptoms associated with the disease. An objective of the present invention is to provide an effective drug for treating acute neuromyelitis optica and the symptoms of neuromyelitis optica. [Means for solving the problem]

[0012] As a result of intensive research to achieve the above object, the present inventors have found that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, have the effect of reversing clinical acute exacerbation in acute-phase neuromyelitis optica, the effect of rapidly repairing breakdown of the blood-spinal cord barrier caused by myelitis, and the effect of suppressing granulocyte infiltration observed in the pathology of acute-phase neuromyelitis optica, thereby exhibiting excellent effects in the prevention or treatment of acute-phase neuromyelitis optica. Furthermore, the present inventors have found that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, can treat, alleviate, or alleviate pain symptoms in neuromyelitis optica, leading to the completion of the present invention. That is, the present invention is as follows.

[0013] 1. A preventive or therapeutic agent for acute neuromyelitis optica, comprising an RGMa inhibitor. 2. The preventive or therapeutic agent according to Item 1, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 3. The preventive or therapeutic agent according to Item 2, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 4. The preventive or therapeutic agent according to Item 2 or 3, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. 5. The anti-RGMa neutralizing antibody is one of the following (a) to (l): (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; Anti-RG antibody containing a heavy chain variable region having an HCDR2 containing the amino acid sequence SFG and an HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 44 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Item 5. The preventive or therapeutic agent according to any one of Items 2 to 4, wherein the antibody is selected from the group consisting of:

[0014] 6. A preventive or therapeutic agent for pain symptoms in neuromyelitis optica, comprising an RGMa inhibitor. 7. The preventive or therapeutic agent according to Item 6, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 8. The preventive or therapeutic agent according to Item 7, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 9. The preventive or therapeutic agent according to Item 7 or 8, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. 10. The anti-RGMa neutralizing antibody is one of the following (a) to (l): (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; Anti-RG antibody containing a heavy chain variable region having an HCDR2 containing the amino acid sequence SFG and an HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; a heavy chain variant comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Anti-RGMa neutralizing antibodies containing variable regions, (j) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 44 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Item 10. The preventive or therapeutic agent according to any one of Items 7 to 9, wherein the antibody is selected from the group consisting of: 11. A method for preventing or treating acute neuromyelitis optica or pain symptoms in neuromyelitis optica, comprising administering to a mammal in need of treatment an effective amount of an RGMa inhibitor. 12. The preventive or therapeutic method according to Item 11, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 13. Use of an RGMa inhibitor for the manufacture of an agent for the prevention or treatment of acute neuromyelitis optica. [Effects of the Invention]

[0015] According to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, exhibit the effect of rapidly repairing the breakdown of the blood-spinal cord barrier (blood-brain barrier in the brain) seen in acute neuromyelitis optica, and are useful as preventive or therapeutic agents for acute neuromyelitis optica. Furthermore, according to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, exhibit the effect of suppressing granulocyte infiltration into the spinal cord seen in acute neuromyelitis optica, and are useful as preventive or therapeutic agents for acute neuromyelitis optica. Furthermore, according to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, can treat, alleviate, or alleviate the pain symptoms seen in neuromyelitis optica, and are useful as agents for preventing or treating such pain symptoms. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows the therapeutic effect of anti-RGMa neutralizing antibodies (sometimes simply referred to as anti-RGMa antibodies) on clinical exacerbation in a rat model of acute, severe NMO. Data are shown as mean ± SEM. The number of cases in each group is indicated in parentheses in the graph legend. [Figure 2]Figure 2 shows the recovery effect of anti-RGMa neutralizing antibodies on blood-spinal cord barrier disruption and neurological symptoms in acute-stage tEAE mice. Panel A of Figure 2 shows the recovery effect of anti-RGMa neutralizing antibodies on changes in gadolinium leakage into the spinal cord in acute-stage BSCB disruption in tEAE mice. Each data point is shown as mean ± SEM, and the number of animals in each group is indicated in parentheses in the graph legend. Animals in which gadolinium leakage into the spinal cord was not observed 7 days after cytokine injection and animals in which MRI images could not be obtained 14 days after cytokine injection were excluded from the analysis data. Statistical analysis was performed using the Bonferroni multiple comparison test (***p<0.001). The arrow indicates the day of antibody administration. Panel B of Figure 2 shows the recovery effect of anti-RGMa neutralizing antibodies on neurological symptoms. Each data point is shown as mean ± SEM, and the number of animals in each group is indicated in parentheses in the graph legend. The arrow indicates the day of antibody administration. Statistical analysis was performed using the Mann-Whitney U test at each time point (*p<0.05, **p<0.01). [Figure 3] Figure 3 shows the correlation between the intensity of gadolinium leakage into the spinal cord and the severity of neurological symptoms in the acute phase of tEAE mice. Data for each individual was plotted, and statistical analysis was performed using Spearman's rank correlation analysis. The number of cases in each group is shown in parentheses in the graph legend. There was one case in which MRI images were not acquired 14 days after cytokine injection (anti-RGMa neutralizing antibody administration group). [Figure 4] Figure 4 shows immunohistochemical staining images and a diagram showing the therapeutic effect of anti-RGMa neutralizing antibodies on blood-spinal barrier breakdown in an acute, severe NMO rat model. Data from quantitative analyses are shown as mean ± SEM and were statistically analyzed using Turkey's multiple comparison test (*p < 0.05). [Figure 5]Figure 5 shows the effect of anti-RGMa neutralizing antibodies on pain-related behavior in an acute, severe NMO rat model. Data are shown as mean ± SEM and statistical analysis was performed using Turkey's multiple comparison test (***p < 0.001, **p < 0.01, *p < 0.05 vs. untreated healthy control group; ##p < 0.01, #p < 0.05 vs. NMO isotype control antibody-treated group). Arrows indicate the days of administration of anti-RGMa neutralizing antibodies or isotype control antibodies. [Figure 6] Figure 6 shows immunohistochemical staining images and diagrams showing the inhibitory effect of anti-RGMa neutralizing antibodies on granulocyte infiltration in the spinal cord of a rat model of acute, severe NMO. Data from quantitative analyses are shown as mean ± SEM and were statistically analyzed using Turkey's multiple comparison test (**p < 0.01, *p < 0.05). [Figure 7] FIG. 7 shows immunohistochemical staining images showing RGMa expression in the AQP4-depleted area of ​​the spinal cord of a rat model of acute severe NMO. DETAILED DESCRIPTION OF THE INVENTION

[0017] The terms used in the present invention will be explained below. [Neutralization] As used herein, "neutralizing" refers to the ability to bind to a target of interest and inhibit any function of that target. For example, an RGMa inhibitor is a substance that inhibits the biological activity of RGMa as a result of binding to RGMa.

[0018] [epitope] As used herein, an epitope includes a polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope includes a chemically active surface grouping of a molecule (e.g., amino acids, sugar side chains, phosphoryl or sulfonyl) and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody.

[0019] [Isolated] As used herein, the term "isolated" in reference to an isolated RGMa inhibitor (e.g., an antibody) means identified and separated and / or recovered from components in its natural state. Impurities in the natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, an RGMa inhibitor can be isolated by purification through at least one purification step, and an RGMa inhibitor purified through at least one purification step can be referred to as an "isolated RGMa inhibitor."

[0020] [antibody] In this application, the term "antibody" broadly refers to an immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), which substantially retains the epitope-binding property of an Ig molecule.

[0021] [Human antibody] In the present application, a human antibody refers to an antibody in which both the light chain and the heavy chain are derived from human immunoglobulin. Depending on the difference in the constant region of the heavy chain, human antibodies are classified into IgG (including IgG1, IgG2, IgG3, and IgG4) having γ heavy chains, IgM having μ heavy chains, and IgM having α heavy chains. IgA (including IgA1 and IgA2), IgD with a delta heavy chain, or IgE with an epsilon heavy chain. In principle, the light chain includes either a kappa or lambda chain.

[0022] [Humanized antibody] In the present application, a humanized antibody refers to an antibody that comprises a variable region consisting of a complementarity-determining region of an antibody derived from a non-human animal and a framework region derived from a human antibody, and a constant region derived from a human antibody.

[0023] [Chimeric antibody] As used herein, a chimeric antibody refers to an antibody in which the light chain, the heavy chain, or both, are composed of a variable region of non-human origin and a constant region of human origin.

[0024] [Monospecific antibodies] As used herein, a monospecific antibody is an antibody that has a single antigen specificity and a single, independent antigen-recognition site. For example, a monospecific antibody that recognizes RGMa may be referred to as an RGMa monospecific antibody.

[0025] [Multispecific antibodies] In the present application, a multispecific antibody refers to an antibody that has two or more independent antigen recognition sites with two or more different antigen specificities, and examples include a bispecific antibody that has two antigen specificities and a trispecific antibody that has three antigen specificities.

[0026] [Complementarity-determining region (CDR)] Complementarity determining regions (CDRs) refer to the regions of the variable regions of immunoglobulin molecules that form the antigen-binding site, also called hypervariable regions, and refer to the parts where there is particularly large variation in amino acid sequence for each immunoglobulin molecule. There are three CDRs in each of the light chain and heavy chain. The three CDRs in the light chain are sometimes called LCDR1, LCDR2, and LCDR3, respectively, and the three CDRs in the heavy chain are sometimes called HCDR1, HCDR2, and HCDR3. For example, the CDRs of immunoglobulin molecules are numbered according to the Kabat numbering system (K abat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health a and Human Services, NIH, USA).

[0027] [Effective dose] An effective amount refers to the amount of a prophylactic or therapeutic agent sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent progression of a disorder, reverse a disorder, prevent the recurrence, occurrence, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve one or more prophylactic or therapeutic effects of another treatment (e.g., a prophylactic or therapeutic agent).

[0028] [Percent (%) identity of amino acid sequence] "Percent (%) identity" of an amino acid sequence of a candidate polypeptide sequence, such as a variable region, with respect to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the particular reference polypeptide sequence after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent identity can be performed using a variety of methods within the skill of one in the art, e.g., BLAST, BLAST-2, ALIG, etc. N, or publicly available computer programs such as Megalign (DNASTAR) software. This can be achieved by using computer software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, percent identity values ​​are obtained using the sequence comparison computer program BLAST in pairwise alignments. In situations where BLAST is used for amino acid sequence comparison, the percent identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical by a program alignment of A and B using the sequence alignment program BLAST, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the percent identity of A to B will differ from the percent identity of B to A. Unless otherwise specified, all percent identity values ​​herein are obtained using the BLAST computer program as set forth in the immediately preceding paragraph.

[0029] [Conservative substitution] Conservative substitution means substituting an amino acid residue with another chemically similar amino acid residue so as not to substantially modify the activity of the peptide. For example, when substituting one hydrophobic residue with another hydrophobic residue, or substituting one polar residue with another polar residue having the same charge. Examples of functionally similar amino acids for which such substitutions can be made include, as nonpolar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like. Examples of positively charged (basic) amino acids include arginine, histidine, lysine, and the like. Examples of negatively charged (acidic) amino acids include aspartic acid, glutamic acid, and the like.

[0030] Hereinafter, embodiments of the present invention will be described in detail. The present invention provides a prophylactic or therapeutic agent for acute neuromyelitis optica, which is a novel use of an RGMa inhibitor. The present invention also provides a method for preventing or treating acute neuromyelitis optica, which includes the step of administering a prophylactic or therapeutic agent containing an effective amount of an RGMa inhibitor to a mammal requiring treatment.

[0031] <RGMa inhibitor> The RGMa inhibitor of the present invention may be any substance that acts on RGMa itself and inhibits or attenuates the activity of RGMa (hereinafter, may be simply referred to as "RGMa activity" in this specification). For example, a substance having an activity that binds to RGMa and directly inhibits (attenuates) RGMa activity, or a substance having an activity that inhibits the binding of RGMa to a receptor and indirectly inhibits (attenuates) RGMa activity (for example, the compounds and antibodies described later) is referred to as the RGMa inhibitor of the present invention. Also, the RGMa inhibitor of the present invention may be a substance that suppresses the expression of RGMa. For example, a substance that inhibits the expression of RGMa and inhibits (attenuates) RGMa activity (for example, the nucleic acid molecules described later) is also included in the RGMa inhibitor of the present invention.

[0032] RGMa has been identified as a neurite outgrowth inhibitor in the central nervous system. Human RGMa protein is biosynthesized as a precursor protein consisting of 450 amino acids as shown in SEQ ID NO: 1. The signal peptide Met1-Pro47 (referring to the peptide from the first methionine residue to the 47th proline residue from the N-terminus) is present at the N-terminus. The peptide bond between Asp168 and Pro169 is cleaved to form the N-terminal The C-terminal peptide Ala425-Cys450 of the fragment C-terminal to Pro169 is then removed, and a GPI anchor is added to the C-terminal carboxyl group of the resulting C-terminal Ala424 to generate the C-terminal domain. Human RGMa protein is expressed on the cell membrane as a mature protein in which the N-terminal domain (Cys48-Asp168) and the C-terminal domain (Pro169-Ala424) are linked by disulfide bonds via a GPI anchor.

[0033] In the present invention, RGMa may be derived from any animal, but is preferably human RGMa. The precursor protein of human RGMa consists of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. The precursor protein of mouse RGMa consists of the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing, and the precursor protein of rat RGMa consists of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing, but because the C-terminal peptide is removed, the mature proteins have the same amino acid sequences. Examples of RGMa genes include, but are not limited to, the human RGMa gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4. The nucleotide sequences of RGM genes derived from various organisms can be easily obtained from publicly known databases (such as GenBank).

[0034] Specific examples of RGMa inhibitors of the present invention include low-molecular-weight compounds, anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and antigen-binding fragments thereof, as well as RGMa nucleic acid molecules such as short interfering RNA (siRNA), short hairpin RNA (shRNA), and antisense oligonucleotides. Of these RGMa inhibitors, preferred are anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and antigen-binding fragments thereof, more preferred are anti-RGMa neutralizing antibodies or antigen-binding fragments thereof, and particularly preferred are anti-RGMa neutralizing antibodies.

[0035] <Anti-RGMa neutralizing antibody> In the present invention, the anti-RGMa neutralizing antibody may be any antibody that binds to RGMa and neutralizes RGMa activity, and may be a polyclonal or monoclonal antibody. In the present invention, a monoclonal antibody is preferred. Furthermore, the anti-RGMa neutralizing antibody of the present invention may be either a monospecific RGMa antibody or a multispecific antibody that recognizes multiple antigens, including RGMa, but is preferably a monospecific RGMa antibody.

[0036] Specific epitopes in human RGMa are preferably one or more of SEQ ID NO: 16 (amino acid numbers 47-69 of SEQ ID NO: 1), SEQ ID NO: 36 (amino acid numbers 298-311 of SEQ ID NO: 1), SEQ ID NO: 37 (amino acid numbers 322-335 of SEQ ID NO: 1), SEQ ID NO: 38 (amino acid numbers 349-359 of SEQ ID NO: 1), and SEQ ID NO: 39 (amino acid numbers 367-377 of SEQ ID NO: 1), and a combination of SEQ ID NOs: 36 and 37. The combination of SEQ ID NOs: 36, 37 and 39 is particularly preferred.

[0037] The anti-RGMa neutralizing antibodies of the present invention include polyclonal and monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or a partial fragment thereof (for example, the epitope fragments described above), chimeric and humanized antibodies produced using genetic recombination techniques, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibodies of the present invention are administered to humans as pharmaceuticals, humanized or human antibodies are preferred in terms of side effects.

[0038] Specific examples of the anti-RGMa neutralizing antibodies of the present invention include the following antibodies (a) to (l), and the methods described in Patent Documents 2 to 4 can be used to produce each of them.

[0039] (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (the anti-RGMa neutralizing antibody further includes antibodies having epitopes set forth in SEQ ID NOs: 36, 37, and 39); (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; Anti-RG antibody containing a heavy chain variable region having an HCDR2 containing the amino acid sequence SFG and an HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody (the anti-RGMa neutralizing antibody further includes antibodies having epitopes of SEQ ID NOs: 36, 37, and 38), (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (g) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (h) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (i) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; a heavy chain variant comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; an anti-RGMa neutralizing antibody comprising a variable region (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope); (j) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (k) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 44 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); (l) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); Examples of antibodies include those selected from the following: Among these, the antibody described in (a) is particularly preferred.

[0040] The anti-RGMa neutralizing antibody of the present invention can be produced by any commonly used existing production method. The antigen may be used for immunization as is, or may be used as a complex with a carrier protein. Condensing agents such as glutaraldehyde, carbodiimide, and maleimide activated esters can be used to prepare the complex of the antigen and carrier protein. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[0041] Mammals to be immunized include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, and cows, and inoculation methods include subcutaneous, intramuscular, and intraperitoneal administration. The vaccine may be administered by mixing with complete or incomplete Freund's adjuvant, and is usually administered once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of immunized animals are fused with myeloma cells and isolated as hybridomas. Myeloma cells derived from mammals, such as mice, rats, and humans, are used.

[0042] <Polyclonal antibody> Polyclonal antibodies can be produced, for example, by immunizing a mammal as described above with the antigen, optionally together with Freund's adjuvant. It can be obtained from serum obtained from sensitized animals.

[0043] <Monoclonal antibodies> Specifically, monoclonal antibodies can be obtained as follows: The above-mentioned antigen is used as an immunogen, and the immunogen is injected subcutaneously, intramuscularly, intravenously, or into the foot pad of the above-mentioned mammals together with Freund's adjuvant as necessary. Immunization is carried out by intraperitoneal or intraperitoneal injection or transplantation one to several times. Usually, immunization is carried out one to four times at intervals of about 1 to 14 days from the first immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

[0044] Monoclonal antibodies can be obtained using methods well known to those skilled in the art (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)).

[0045] Hybridomas secreting monoclonal antibodies can be prepared according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) or modifications thereof. Specifically, hybridomas are prepared by fusing antibody-producing cells contained in the spleen or other tissues obtained from an immunized mammal with myeloma cells derived from a mammal, preferably a mouse, rat, or human, that are not capable of producing autoantibodies.

[0046] Examples of myeloma cells that can be used in cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0, or BW5147; rat-derived myeloma 210RCY3-Ag.2.3; and human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15.

[0047] Examples of fusion promoters include polyethylene glycol, and cell fusion can be achieved by reacting polyethylene glycol (average molecular weight 1000 to 4000) at a concentration of approximately 20 to 50%, at a temperature of 20 to 40°C, preferably 30 to 37°C, at a ratio of antibody-producing cells to myeloma cells of typically 1:1 to 10:1, for approximately 1 to 10 minutes.

[0048] Screening for hybridoma clones that produce monoclonal antibodies can be carried out by culturing the hybridomas, for example, in a microtiter plate and measuring the reactivity of the culture supernatant in the wells to the immunogen by an immunochemical method such as ELISA.

[0049] In screening for antibody-producing hybridomas, in addition to assaying for binding to RGMa protein, we also evaluate whether the antibody inhibits the RGMa activity of the present invention. These screening methods allow us to select anti-RGMa neutralizing antibodies of the present invention.

[0050] Clones can be obtained from wells containing hybridomas that produce the desired antibody by limiting dilution. Hybridoma selection and breeding are usually carried out in an animal cell medium containing 10-20% fetal bovine serum and supplemented with HAT (hypoxanthine, aminopterin, thymidine).

[0051] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or by growing them in vivo, for example, in ascites of a mammal such as a mouse or rat, and isolating the antibodies from the resulting culture supernatant or from the ascites of the mammal.

[0052] When culturing in vitro, a nutrient medium suitable for growing, maintaining, and preserving hybridomas and producing monoclonal antibodies in the culture supernatant can be used, depending on various conditions such as the characteristics of the cell type being cultured and the culture method, etc. Examples of the nutrient medium include known nutrient media and nutrient media prepared from basal media.

[0053] Examples of the basal medium include low calcium media such as Ham's F12 medium, MCDB153 medium, and low calcium MEM medium, as well as MCDB104 medium, MEM medium, and D-ME medium. Examples of such basal media include high-calcium media such as M medium, RPMI1640 medium, ASF104 medium, and RD medium, and the basal medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

[0054] Monoclonal antibodies can be isolated and purified by subjecting the culture supernatant or ascites fluid to saturated ammonium sulfate, euglobulin precipitation, caproic acid, caprylic acid, ion exchange chromatography (DEAE, DE52, etc.), or affinity column chromatography using an anti-immunoglobulin column or protein A column. Specifically, monoclonal antibodies can be purified using known immunoglobulin purification methods, and can be easily achieved by, for example, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of an anion exchanger, or affinity chromatography using RGMa protein.

[0055] Monoclonal antibodies can also be obtained by phage display. In phage display, phages selected from a phage antibody library are screened with the target immunogen, and phages with the desired binding affinity to the immunogen are selected. Next, the antibody-corresponding sequence contained in the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding domain is constructed based on the isolated or sequence information. Monoclonal antibodies can then be produced by culturing a cell line transfected with such an expression vector. Human antibodies with the desired binding affinity can be generated by using a human antibody library as the phage antibody library.

[0056] <Nucleic acid molecule> Nucleic acid molecules encoding the anti-RGMa neutralizing antibodies or antigen-binding fragments thereof of the present invention can be obtained, for example, by the following method. First, total RNA is prepared from cells such as hybridomas using a commercially available RNA extraction kit, and cDNA is synthesized using reverse transcriptase and random primers. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides with sequences conserved in the variable regions of known human antibody heavy chain and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The DNA nucleotide sequence can be determined by standard methods, such as by incorporating it into a sequencing plasmid. Alternatively, DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the sequence of the variable region or a part thereof and ligating it to a sequence containing the constant region. The nucleic acid molecule may encode both the heavy and light chain constant and variable regions, or may encode only the heavy and light chain variable regions. When encoding both the constant and variable regions, the base sequences of the heavy and light chain constant regions are preferably those described in Nucleic Acids Research, vol. 14, p. 1779, 1986; The Journal of Biological Chemistry, vol. 257, p. 1516, 1982; and Cell, vol. 22, p. 197, 1980.

[0057] <Functionally modified antibodies> Functionally modified anti-RGMa neutralizing antibodies can be prepared by the following methods. For example, when the present anti-RGMa neutralizing antibodies are produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as host cells, antibodies with reduced fucose content in the sugar chains and enhanced cell-killing activity are obtained. When the present anti-RGMa neutralizing antibodies are produced using CHO cells into which the FUT8 gene has been introduced as host cells, antibodies with reduced cell-killing activity are obtained (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739). Furthermore, complement activation function can be regulated by modifying amino acid residues in the Fc region (U.S. Patent No. 6,737,056, U.S. Patent No. 7,297,775, U.S. Patent No. 7,317,091). Furthermore, by using Fc region mutants that enhance binding to FcRn, one of the Fc receptors, These functionally modified antibodies can be produced by genetic engineering. By using a mutant Fc region that enhances binding to FcRn, one of the receptors, it is possible to extend the blood half-life (Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering.

[0058] <Conjugate antibody> Conjugated antibodies are examples of modified molecules of the anti-RGMa neutralizing antibodies of the present invention. Conjugated antibodies include those containing 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 the like. Examples include conjugated antibodies to which functional molecules other than anti-RGMa neutralizing antibodies are chemically or genetically bound.

[0059] When PEG is used as a functional molecule, the molecular weight of the PEG may be, but is not limited to, 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be either linear or branched. PEG can be bound to the N-terminal amino group of an amino acid in an anti-RGMa neutralizing antibody, for example, by using an NHS-activated group.

[0060] When a radioactive substance is used as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 Radioactive substances are used to detect anti-RGM antibodies using the chloramine T method. aCan be directly conjugated to a neutralizing antibody.

[0061] When a toxin is used as a functional molecule, bacterial toxins (eg, diphtheria toxin), plant toxins (eg, ricin), low-molecular-weight toxins (eg, geldanamycin), maytansinoids, calicheamicin, and the like can be used.

[0062] When a low molecular weight compound is used as a functional molecule, examples thereof include daunomycin, doxorubicin, metrorexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0063] When an enzyme is used as the functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. may be used.

[0064] Linkers used to chemically link toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2) n O( CR2) n - (R is an optional substituent, n is a positive integer) Examples of suitable groups include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (e.g., succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for attaching functional molecules have already 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 Vol. 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0065] <Antigen-binding fragment> In an embodiment of the present invention, the "antigen-binding fragment" of an antibody refers to a partial region of the antibody having antigen-binding activity as described above, and specifically includes F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and the like. Furthermore, antigen-binding fragments include 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 substances. The present invention also includes conjugate fragments to which functional molecules other than the anti-RGMa neutralizing antibody of the present invention are chemically or genetically linked.

[0066] "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulin with protease enzymes such as pepsin or papain, resulting in digestion across the disulfide bond between the two heavy chains in the hinge region. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond between the two heavy chains in the hinge region, producing two homologous antibody fragments in which a light chain consisting of a VL (light chain variable region) and a CL (light chain constant region), and a heavy chain fragment consisting of a VH (heavy chain variable region) and a CHγ1 (γ1 region in the heavy chain constant region) are linked by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab. Also, I When IgG is treated with pepsin, it is cleaved downstream of the disulfide bond between the two heavy chains in the hinge region, resulting in an antibody fragment that is slightly larger than the two Fab fragments connected by the hinge region. This antibody fragment is called F(ab')2.

[0067] <Chimeric antibody> A preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a chimeric antibody. Examples of "chimeric antibodies" include those whose variable regions are derived from immunoglobulins of non-human animals (such as mice, rats, hamsters, and chickens) and whose constant regions are derived from human immunoglobulins. For example, chimeric antibodies can be produced by immunizing a mouse with an antigen, excising the variable region that binds to the antigen from the mouse monoclonal antibody gene, and then combining it with an antibody constant region derived from human bone marrow. Constant regions derived from human immunoglobulins have unique amino acid sequences depending on the isotype, such as IgG (IgG1, IgG2, IgG3, and IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE. The constant region of the recombinant chimeric antibody of the present invention may be the constant region of a human immunoglobulin belonging to any isotype. Preferably, it is a human IgG constant region. Expression vectors can be constructed using the genes of chimeric antibodies produced in this manner. Host cells are transformed with the expression vector to obtain transformed cells that produce chimeric antibodies, and the transformed cells are cultured to obtain the desired chimeric antibodies from the culture supernatant.

[0068] <Humanized antibody> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a humanized antibody. The "humanized 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, has been grafted onto a human antibody gene (CDR grafting). For example, it can be prepared by referring to the methods described in JP-A-4-506458 and Japanese Patent No. 2912618. Specifically, this refers to a humanized antibody in which some or all of its CDRs are derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework regions of its variable regions are derived from a human immunoglobulin, and its constant regions are derived from a human immunoglobulin.

[0069] The humanized antibody of the present invention can be produced, for example, as follows: However, it goes without saying that the production method is not limited to this.

[0070] For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be produced by genetic engineering with reference to JP-A-4-506458 and JP-A-62-296890, etc. That is, DNA of the mouse heavy chain CDR region and DNA of the mouse light chain CDR region are isolated from a hybridoma producing a mouse monoclonal antibody, and a human heavy chain gene covering the entire region except for the human heavy chain CDR and a human light chain gene covering the entire region except for the human light chain CDR are isolated from a human immunoglobulin gene.

[0071] The isolated human heavy chain gene grafted with DNA of the mouse heavy chain CDR region is introduced into an appropriate expression vector so as to be expressible, and similarly, the human light chain gene grafted with DNA of the mouse light chain CDR region is introduced into another appropriate expression vector so as to be expressible. Alternatively, the human heavy chain and light chain genes grafted with mouse CDRs can be introduced into the same expression vector so as to be expressible. Host cells are transformed with the expression vector prepared in this way to obtain humanized antibody-producing transformants, and the desired humanized antibody is obtained from the culture supernatant by culturing the transformants.

[0072] <Human antibodies> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a human antibody. A human antibody is an antibody in which all regions constituting the immunoglobulin, including the heavy chain variable region, heavy chain constant region, and light chain variable region, and light chain constant region, are derived from a gene encoding human immunoglobulin, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal produced by incorporating at least a human immunoglobulin gene into the genetic locus of a non-human mammal, such as a mouse, can be produced by immunizing the animal with an antigen in a manner similar to the method for producing polyclonal or monoclonal antibodies described above.

[0073] For example, transgenic mice producing human antibodies are described in Nature Genetics, Vol. 7, pp. 13-21, 1994; Nature Genetics, Vol. 15, pp. 146-156, 1997; JP 4-504365 A; It is prepared according to the methods described in JP-A-7-509137; International Publication WO94 / 25585; Nature, Vol. 368, pp. 856-859, 1994; and JP-A-6-500233. More specifically, examples include HuMab (registered trademark) mice (Medarex, Princeton NJ), KM™ mice (Kirin Pharma Company, Japan), and KM (FCγRIIb-KO) mice.

[0074] Specific examples of anti-RGMa neutralizing antibodies of the present invention include those having a CDR containing a specific amino acid sequence in the heavy chain variable region and a CDR containing a specific amino acid sequence in the light chain variable region (preferably the anti-RGMa neutralizing antibodies (a) to (l) above). As long as the antibody of the present invention maintains its ability to bind to RGMa and inhibit (neutralize) the activity of RGMa, it is possible to use an anti-RGMa neutralizing antibody (preferably, any of the above-mentioned (a) to (l)). The amino acid sequence of the anti-RGMa neutralizing antibody may include substitution, deletion, addition, or insertion of one or several 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 a region other than the CDR. Furthermore, the amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0075] The amino acid sequence of the anti-RGMa neutralizing antibody of the present invention (preferably the anti-RGMa neutralizing antibody (a) to (l) above) containing substitutions, deletions, etc. in the amino acid sequence may be, for example, The heavy chain variable region after sequence modification has an amino acid sequence that is 90% or more (more preferably 95%) different from the amino acid sequence before modification. The light chain variable region after amino acid sequence modification has a percent identity of 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before modification.

[0076] In the present invention, siRNA refers to a short double-stranded RNA capable of suppressing the expression of a target gene (in the present invention, the RGMa gene). The base sequence and length (base length) of the siRNA are not particularly limited as long as it functions as an siRNA that inhibits RGMa activity, but it is preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases. In the present invention, shRNA is a single-stranded RNA that contains a partially palindromic base sequence, forming a double-stranded structure within the molecule, and is derived from a short hairpin structure with a protruding portion at the 3' end. shRNA refers to a molecule of about 20 base pairs or more consisting of a single molecule. After being introduced into a cell, such shRNA is degraded into molecules of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) in length within the cell, and can suppress the expression of the target gene in the same way as siRNA. In the present invention, the above-mentioned siRNA and shRNA may be in any form as long as they are capable of suppressing the expression of the RGMa gene.

[0077] In the present invention, siRNA or shRNA can be artificially chemically synthesized. Alternatively, antisense and sense RNAs can be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and a T7 promoter. Antisense oligonucleotides may be either DNA or RNA, as long as they are complementary to or hybridize with a sequence of 5 to 100 consecutive bases in the DNA sequence of the RGMa gene. Modifications may also be used as long as they do not impair function. Antisense oligonucleotides can be synthesized by conventional methods, for example, easily using a commercially available DNA synthesizer. Preferred sequences can be selected using conventional selection methods and confirmed as siRNA or shRNA in the present invention by assessing inhibition of expression of functional RGMa.

[0078] <Acute neuromyelitis optica> Clinically, the disease stages of neuromyelitis optica are roughly divided into two: the "acute phase" (which in the present invention is a concept that includes the acute exacerbation phase) and the "chronic phase." Here, the "acute phase" refers to the period when symptoms of neuromyelitis optica, such as optic neuritis and myelitis, appear and continue, or worsen (progress). During this period, MRI scans may reveal gadolinium enhancement in some lesions, and cerebrospinal fluid tests may reveal increased cell counts and elevated protein levels, which can be used to determine whether the patient is in the acute phase. On the other hand, the "chronic phase" refers to the period when symptoms have subsided, improved, and stabilized due to treatment. Also, during this period, the gadolinium contrast effect disappears on MRI, so this can be used as a reference to determine the chronic phase. The acute phase of neuromyelitis optica in the present invention means the aforementioned "acute phase," and includes not only the acute phase pathology at the onset of the first episode of neuromyelitis optica, but also the acute phase pathology at the time of recurrence of neuromyelitis optica after the second or subsequent episodes. Furthermore, in patients receiving treatment for neuromyelitis optica (preferably mammals, particularly humans), the symptoms peak in approximately 8.5 days (range: 2 to 63 days) (Reference: Flanagan et al. Ann Neurol. 2016 Mar; 79(3): 437-47, etc.). The period is usually within one month from the onset of neuromyelitis optica.

[0079] Neuromyelitis optica in the present invention refers to neuromyelitis optica spectrum disorder (NMOSD), and is defined as a disease that is diagnosed according to the International Diagnostic Criteria for Neuromyelitis Optica (Wingerchuk et al. NMOSD). Neurology, 2015; 8582): 177-189) The concept includes both anti-AQP4 antibody-positive neuromyelitis optica-associated disease (NMOSD) and anti-AQP4 antibody-negative neuromyelitis optica-associated disease (NMOSD). Among these, anti-AQP4 antibody-positive neuromyelitis optica-associated disease is preferred in the present invention. The subject of treatment in the present invention (preferably a mammal, particularly a human) is a patient who has developed neuromyelitis optica-associated disease (NMOSD), preferably a patient who has developed anti-AQP4 antibody-positive neuromyelitis optica-associated disease (NMOSD), and the prophylactic or therapeutic agent for acute neuromyelitis optica of the present invention can be administered to these patients.

[0080] Furthermore, in the acute phase of neuromyelitis optica in the present invention, pain is often one of the main symptoms experienced by patients. Therefore, in the present invention, an RGMa inhibitor, preferably an anti-RGMa neutralizing antibody, can be used in patients with such pain as a preventive or therapeutic agent for the painful symptoms seen in neuromyelitis optica. The matters explained in the description of the preventive or therapeutic agent and the preventive or therapeutic method for acute neuromyelitis optica of the present invention are all applicable to the explanation of the preventive or therapeutic agent and the preventive or therapeutic method for pain symptoms seen in neuromyelitis optica of the present invention.

[0081] As used herein, "treatment" includes any treatment of a disease in a subject, preferably a mammal, particularly a human, including preventing the progression of the disease and symptoms, and eliminating, curing, alleviating or alleviating such disease and symptoms.

[0082] Furthermore, "prevention" includes preventing or suppressing the onset of the above-mentioned diseases in a subject to be treated, preferably a mammal, particularly a human. Furthermore, "prevention" in the present invention also includes "recurrence prevention," which prevents the recurrence of the above-mentioned diseases that repeatedly undergo remission and relapse in a subject to be treated, preferably a mammal, particularly a human.

[0083] <Pharmaceutical Composition> The agent for preventing or treating acute neuromyelitis optica of the present invention is generally administered systemically or locally, orally or parenterally. The agent for preventing or treating acute neuromyelitis optica of the present invention can be formulated by appropriately blending an RGMa inhibitor as an active ingredient with pharmaceutically acceptable carriers or additives. Such a pharmaceutical composition can be administered orally or parenterally. Specifically, oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions can be used. Parenteral preparations such as injections, infusions, suppositories, ointments, and patches can also be used. The proportion of the carrier or additive may be appropriately determined based on the range commonly used in the pharmaceutical field. The carrier or additive that can be used is not particularly limited, and examples include various carriers such as water, saline, other aqueous solvents, and aqueous or oily bases, as well as various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0084] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, a functionally modified antibody thereof, a conjugated antibody thereof, or an antigen-binding fragment thereof, it is preferably administered parenterally, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically, as an injection or infusion formulated together with a pharmaceutically acceptable carrier. For example, injections or infusions containing anti-RGMa neutralizing antibodies can be used as solutions, suspensions, or emulsions, and solvents such as distilled water for injection, physiological saline, glucose solutions, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used. Furthermore, injections or infusions containing such anti-RGMa neutralizing antibodies may contain stabilizers, solubilizers, The pharmaceutical composition may contain additives such as suspending agents, emulsifying agents, soothing agents, buffering agents, preservatives, antiseptics, pH adjusters, etc. Examples of stabilizers that can be used include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol, etc.), polyalcohols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, etc.). As the suspending agent, for example, glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. can be used. As the emulsifier, for example, gum arabic, sodium alginate, tragacanth, etc. can be used. As the soothing agent, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, and Tris buffer. Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Examples of preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.

[0085] When the RGMa inhibitor is a nucleic acid (e.g., siRNA, shRNA, or antisense oligonucleotide), it can be administered in the form of a non-viral or viral vector. In the case of a non-viral vector, methods for introducing nucleic acid molecules using liposomes (e.g., liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method), microinjection, or gene gun (Gene Gun) can be used. Methods such as (n) above can be used to transfer nucleic acid molecules into cells together with carriers (metal particles). For example, when administering siRNA or shRNA to a living body using a viral vector, viral vectors such as recombinant adenoviruses and retroviruses can be used. DNA that expresses siRNA or shRNA can be introduced into DNA or RNA viruses such as detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, poxviruses, polioviruses, Sindbis viruses, Sendai viruses, and SV40, and then cells or tissues can be infected with this recombinant virus to introduce genes into cells or tissues.

[0086] The formulation obtained in this manner can be administered in an effective amount to, for example, humans or other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) to prevent or treat acute neuromyelitis optica. The dosage is determined appropriately taking into consideration the purpose, severity of the disease, the patient's age, weight, sex, medical history, and type of active ingredient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, the daily dosage for an average human weighing approximately 65 to 70 kg is preferably approximately 0.02 mg to 4000 mg, and more preferably approximately 0.1 mg to 200 mg. The total daily dosage may be a single dose or divided doses.

[0087] <Combination with other drugs or treatments> In the present invention, the agent for preventing or treating acute neuromyelitis optica can be administered in combination with other agents or therapies useful for treating neuromyelitis optica.

[0088] Examples of other drugs or treatments that can be used in combination with the agent for the prophylaxis or treatment of acute neuromyelitis optica of the present invention include plasma exchange and / or intravenous administration of immunoglobulin preparations, and administration of mycophenolate, rituximab, eculizumab, and / or satralizumab. Such other drugs or treatments may be other biologically active drugs or treatments that are effective in treating central nervous system disorders such as neuromyelitis optica or delaying the progression of central nervous system disorders.

[0089] For example, the other biologically active agent may be a corticosteroid, an (intravenous) immunoglobulin preparation, or an antilymphocyte preparation, mycophenolate, rituximab, eculizumab, and / or satralizumab. In a preferred embodiment, the patient is treated with intravenous immunotherapy (e.g., a corticosteroid, e.g., a (synthetic) glucocorticoid such as methylprednisolone). Thus, the other biologically active agent may be a corticosteroid, e.g., a (synthetic) glucocorticoid such as methylprednisolone. The other biologically active agent is administered intravenously. The other agent or treatment may be, for example, plasma exchange in patients who do not respond to steroids (e.g., when there is insufficient suppression of central nervous system inflammation after a course of steroid treatment). Thus, the patient may be a steroid-refractory patient and may optionally undergo plasma exchange.

[0090] The other drug or treatment may be administered or performed before or after the administration of the agent for preventing or treating acute neuromyelitis optica of the present invention, or may be administered or performed simultaneously. [Example]

[0091] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0092] Example 1: Effect of anti-RGMa neutralizing antibodies on acute neuromyelitis optica Using a rat model of acute severe NMO, we investigated the therapeutic effect of anti-RGMa neutralizing antibodies on the exacerbation of clinical symptoms. The therapeutic effect of the anti-RGMa neutralizing antibody was evaluated. Anti-RGMa neutralizing antibodies containing the sequences (SEQ ID NOs: 5 to 10) were used in the experiments.

[0093] (1-1) Procedure for NMO induction in rats A rat model of acute severe NMO was created according to a previous report (Kurosawa K, et al., Acta Neuropathol Commun. 2015;3:82). Female Lewis rats were used for the experiment. Immunization against the central antigen MBP was used as a stimulus to induce a proinflammatory environment and disrupt the blood-spinal cord barrier (BSCB). Guinea pig brain myelin basic protein was used as MBP, and the concentration was 1 mg / mL. The tuberculosis bacillus was dissolved in PBS and then mixed with Freund's complete adjuvant containing 1 mg / mL killed tuberculosis H37Ra. The emulsion (200 μl / head) was administered subcutaneously to two sites on the back, and 10 days later, mice with a neurological score of 1 or less were given a single intraperitoneal administration of an anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) at a dose of 3 mg / kg. induced anti-AQP4 antibody-dependent clinical exacerbation.

[0094] (1-2) Neurological score evaluation Neurological symptoms were scored based on the following criteria, and the total score of the neurological scores for the tail and both hind limbs was used for evaluation as a neurological score (score of 0 to 6). Tail score: 0: no paralysis Each hind limb was scored as follows: 0: no symptoms, 1: incomplete paralysis, 2: complete paralysis with hind limbs dragging. Neurological scoring was performed blinded, and neurological symptoms were assessed once daily until day 13 after anti-RGMa neutralizing antibody administration (day 14 after anti-AQP4 antibody administration).

[0095] (1-3) Grouping and administration of anti-RGMa neutralizing antibody The mice were divided into two groups to minimize the bias in the mean neurological scores and body weights on the day after anti-AQP4 antibody administration. Anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously at a single dose of 10 mg / kg to the tail vein. Each group consisted of six mice.

[0096] (1-4) Results Effect of a single administration of anti-RGMa neutralizing antibody on clinical exacerbation in a rat model of acute severe NMO The results are shown in Figure 1. The day after administration of anti-AQP4 antibody, anti-RGMa neutralizing antibody or isotype control The antibody was administered intravenously, and neurological symptoms were evaluated once daily until 14 days after anti-AQP4 antibody administration. The neurological scores of the group administered with anti-RGMa neutralizing antibody the day after anti-AQP4 antibody administration were lower throughout the observation period compared to the group administered with isotype control antibody, and the mean scores on days 2-8 and 2-14 after anti-AQP4 antibody administration were significantly lower (mean score 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 score 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 demonstrate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are effective in treating acute clinical exacerbations of neuromyelitis optica.

[0097] [Example 2] Effect of anti-RGMa neutralizing antibody on blood-spinal cord barrier breakdown Using tEAE mice, in which BSCB disruption limited to the spinal cord segment can be induced, the therapeutic effect of anti-RGMa neutralizing antibodies on BSCB disruption was examined using gadolinium-enhanced MRI. As the neutralizing antibody, an anti-RGMa neutralizing antibody comprising the amino acid sequence (a) (SEQ ID NOs: 5 to 10) described herein was used in the experiment.

[0098] (2-1) Generation of tEAE mice Female C57 / BL6J mice were used. Myelin Oligodendrocyte Glycoprotein Peptide Fragment 35-55, rat, mouse (MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 46)); MOG 35-55 (Sigma-Aldrich) was dissolved in PBS to a concentration of 2 mg / mL, and a 5 mg / mL solution of killed tuberculosis bacteria H37Ra was added. The emulsion was mixed with an equal volume of complete adjuvant and sonicated to prepare an emulsion. 100 μL of the emulsion was administered subcutaneously to two sites on the back (200 μL / head). Approximately 21 days later, tEAE was induced by injecting 1.5 μL of a cytokine mixture (750 ng Tumor Necrosis Factor-α, 1 μg Interferon-γ) 0.5–0.8 mm below the eighth thoracic vertebra into the thoracic spinal cord. Immediately after and 2 days later, 200 ng pertussis toxin was administered intravenously through the tail vein.

[0099] (2-2) Neurological score evaluation The neurological score was calculated based on the previously published criteria (Tanabe S, Fujita Y, Ikuma K, Yamashita T. Inhibiting The evaluation was conducted in a blinded manner based on the repulsive guidance molecule—a suppresses secondary progression in mouse models of multiple sclerosis. Cell Death Dis. 2018;9(11):1061).

[0100] (2-3) Gadolinium-enhanced magnetic resonance imaging (MRI) BSCB bankruptcy assessment using Using BioSpec 117 / 11 (Bruker), time-dependent T1-weighted images (Dynamic contrast-enhanced MRI; DCE-MRI) were obtained before and after administration of gadolinium contrast agent (Omniscan, Daiichi Sankyo). Gadolinium leakage into the spinal cord, an indicator of BSCB disruption, was quantitatively evaluated based on changes in T1 signal intensity. Under sevoflurane anesthesia, a body temperature maintenance device was attached, and gadolinium contrast agent was rapidly administered at a dose of 0.25 mmol / kg via a catheter tube placed in the tail vein. DCE-MRI was performed. The field of view (FoV) was set to 26 × 26 mm, and an acquisition matrix of 200 × 200 was set. The slice thickness was 0.8 mm, centered on the thoracic spinal cord cytokine injection site. A total of 11 axial images were acquired at intervals of 100 and 150°C. The echo time (TE) was set to 500 ms, the echo time (TE) to 18 ms, and the number of excitations (NEX) to 4. A total of six images were acquired over a period of approximately 10 minutes (approximately 100 seconds per image). . The images were exported 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 set within the spinal cord and the formula Based on (1), the T1 signal enhancement ratio (SER) in the spinal cord at each time point was calculated. The gadolinium leakage intensity (total Gd influx rate) in each individual's spinal cord was calculated as the sum of 11 slices, using the SLOPE function in Microsoft Excel 2016 (Microsoft) to determine the time gradient of the SER as the gadolinium influx rate, as shown in Equation (2). I put it out.

[0101]

number

[0102] From the results of the study on normal mice, when the value of SLOPE(SER0):SER(10), 0:10) in formula (2) was less than 0.02, it was considered an analytical error and was excluded from the calculation of formula (2).

[0103] (2-4) Grouping and administration of anti-RGMa neutralizing antibody Patients were divided into two groups so that the quantitative value of gadolinium leakage 7 days after cytokine injection would be uniform between groups, and they were administered 10 mg / kg of anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab). The anti-RGMa neutralizing antibody group consisted of 10 mice, and the isotype control group was administered twice a week via the tail vein. The antibody-treated group consisted of nine animals, and neurological score evaluation and MRI analysis were performed on the same animals.

[0104] (2-5) Results The recovery effect of anti-RGMa neutralizing antibodies on BSCB disruption and neurological symptoms in acute-phase tEAE mice is shown in Figure 2. The rate of change in gadolinium leakage intensity 7, 14, and 21 days after cytokine injection was calculated longitudinally in the same individual, using the quantitative value 7 days later as the base, to analyze the recovery effect of anti-RGMa neutralizing antibodies on BSCB disruption. Gadolinium leakage in the spinal cord 7 days after cytokine injection was significantly reduced by anti-RGMa Repeated administration of neutralizing antibodies significantly suppressed the progression (14 days after cytokine injection, p < 0.001). , Bonferroni multiple comparison test), and early recovery of BSCB breakdown was observed (Figure 2 A). Furthermore, the neurological scores obtained from the same animals were significantly suppressed, and early recovery from neurological symptoms was observed (Fig. 2B). The correlation between the intensity of gadolinium leakage into the spinal cord and the severity of neurological symptoms in the acute phase of tEAE mice is shown in Figure 3. There was a strong positive correlation between the intensity of gadolinium leakage into the spinal cord and the severity of neurological symptoms 7 days after cytokine injection. A positive correlation (r = 0.831, p < 0.001) was observed, and a positive correlation (r = 0.549, p < 0.05) was observed. It was shown that the severity of BSCB disruption determines the severity of neurological symptoms. . These results demonstrate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, exert their therapeutic effects against acute neuromyelitis optica by promoting repair of BSCB disruption.

[0105] [Example 3] Therapeutic effect of anti-RGMa neutralizing antibody on blood-spinal cord barrier breakdown The leakage of rat IgG into the spinal cord due to the breakdown of the blood-spinal cord barrier was analyzed by immunohistochemical staining. We evaluated the therapeutic effect of anti-RGMa neutralizing antibody on blood-spinal barrier disruption in rats with severe NMO. .

[0106] (3-1) Creation of a rat model of acute severe NMO Female Lewis rats were used in the experiment. Ten days after MBP immunization, rats with a neurological score of 1 or less were Anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) was administered intraperitoneally once at a dose of 3 mg / kg. This induced NMO pathology.

[0107] (3-2) Grouping and administration of anti-RGMa neutralizing antibody The subjects were divided into two groups so that the bias in the mean values ​​of neurological scores and body weights on the day after anti-AQP4 antibody administration would be minimized between the groups. The subjects were then administered either an anti-RGMa neutralizing antibody or an isotype control antibody (Palivizumab). The anti-RGMa neutralizing antibody treatment group and the isotype control antibody treatment group each consisted of 6 mice, and the healthy untreated group consisted of 4 mice.

[0108] (3-3) Immunohistochemical staining Dissection was performed 4 days after anti-AQP4 antibody administration. After blood removal, the spinal cord was collected and post-fixed in 4% paraformaldehyde at 4°C for 1 day. After post-fixation, the tissue was cryoprotected by sucrose substitution and embedded in OCT compound. Frozen sections were cut at 30 μm and immunohistochemically stained using Alxa488-labeled donkey anti-rat IgG antibody (1:500, Thermo Fisher Scientific). An inverted Olympus IX83 fluorescence microscope was used to image the stained sections, and the percentage of rat IgG-positive area relative to the spinal cord cross section (% rat IgG) was calculated using image analysis software Image J software. The area of ​​the IgG positive area was measured.

[0109] (3-4) Results The effect of anti-RGMa neutralizing antibodies on spinal cord leakage of rat IgG is shown in Figure 4. Administration of anti-RGMa neutralizing antibodies on the day after the onset of NMO significantly reduced the spinal cord leakage of rat IgG, an indicator of blood-spinal cord barrier breakdown. Intramedullary leakage was significantly suppressed. These results suggest that the effect of RGMa inhibitors, especially anti-RGMa neutralizing antibodies, on acute neuromyelitis optica is due to the promotion of repair of blood-spinal cord barrier breakdown.

[0110] [Example 4] Effect of anti-RGMa neutralizing antibody on pain symptoms caused by NMO pathology Rats with severe acute NMO were repeatedly administered anti-RGMa neutralizing antibody, and the effect of anti-RGMa neutralizing antibody on persistent pain was evaluated.

[0111] (4-1) Creation of a rat model of acute severe NMO Female Lewis rats were used in the experiment. Ten days after MBP immunization, rats with a neurological score of 1 or less were The anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) was administered intraperitoneally at a dose of 3 mg / kg. The drug was administered intraluminally to induce NMO pathology.

[0112] (4-2) Assessment of pain-related behavior The 50% withdrawal threshold (g) for hind paw lift was determined using the von Frey up-down stimulation 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)). Pain was assessed by determining the 50% withdrawal threshold (g) for both hind paws. The mean of the 50% withdrawal thresholds (g) for both hind paws was used for analysis.

[0113] (4-3) Grouping and administration of anti-RGMa neutralizing antibody The subjects were divided into two groups so that the bias in the mean values ​​of neurological scores and body weights on the day after anti-AQP4 antibody administration would be minimized between the groups. The subjects were then administered either an anti-RGMa neutralizing antibody or an isotype control antibody (Palivizumab). A dose of 10 mg / kg was administered intravenously once weekly into the tail vein. Three groups, each consisting of six mice, were analyzed: an anti-RGMa neutralizing antibody-treated group, an isotype control antibody-treated group, and a healthy untreated group. During the phase of neurological symptom exacerbation, some animals were unable to perform von Frey stimulation due to hindlimb weakness. Therefore, the data set included five animals in the anti-RGMa neutralizing antibody-treated group on day 4 after anti-AQP4 antibody administration, two animals in the isotype control antibody-treated group on day 7 after anti-AQP4 antibody administration, and three animals in the anti-RGMa neutralizing antibody-treated group.

[0114] (4-4) Results The pain-suppressing effect of anti-RGMa neutralizing antibodies is shown in Figure 5. The group administered the neutralizing antibody had a 50% greater escape rate than the group administered the isotype control antibody. Recovery from the lowered response threshold was rapid, with 50% escape on days 18 and 21 after anti-AQP4 antibody administration. A significant increase in the response threshold was observed. These results demonstrate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are effective against pain symptoms in acute neuromyelitis optica.

[0115] [Example 5] Inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord The inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord of acute severe NMO rats was investigated by immunohistochemistry. The tissue was analyzed by histochemical staining.

[0116] (5-1) Creation of a rat model of acute severe NMO Female Lewis rats were used in the experiment. Ten days after MBP immunization, rats with a neurological score of 1 or less were Anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) was administered intraperitoneally once at a dose of 3 mg / kg. This induced NMO pathology.

[0117] (5-2) Grouping and administration of anti-RGMa neutralizing antibody The subjects were divided into two groups so that the bias in the mean values ​​of neurological scores and body weights on the day after anti-AQP4 antibody administration would be minimized between the groups. The subjects were then administered either an anti-RGMa neutralizing antibody or an isotype control antibody (Palivizumab). The anti-RGMa neutralizing antibody treatment group and the isotype control antibody treatment group each consisted of 6 mice, and the healthy untreated group consisted of 4 mice.

[0118] (5-3) Immunohistochemical staining Dissections were performed 4 days after anti-AQP4 antibody administration. After blood removal, spinal cords were harvested and post-fixed in 4% paraformaldehyde at 4°C for 1 day. After post-fixation, tissue sections were cryoprotected by sucrose substitution and embedded in OCT compound. Frozen sections were cut at 30 μm and immunohistochemically stained using rabbit anti-rat granulocyte serum (1:5000, LifeSpan BioSciences) as the primary antibody and Alxa488-conjugated donkey anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific) as the secondary antibody. Stained sections were imaged using an Olympus IX83 inverted fluorescence microscope, and the percentage of granulocyte-positive area relative to the spinal cord cross section (% granulocyte-positive area) was measured using Image J software.

[0119] (5-4) Results The inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord of acute severe NMO rats is shown in Figure 6. Administration of anti-RGMa neutralizing antibodies on the day after the onset of NMO significantly suppressed granulocyte infiltration in the spinal cord of NMO rats. These results demonstrate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, suppress the infiltration of granulocytes seen in the pathology of acute neuromyelitis optica. The inhibitory effect on granulocyte infiltration is thought to contribute to the manifestation of the effect on acute neuromyelitis optica. Therefore, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, may suppress the infiltration of granulocytes, thereby preventing acute neuromyelitis optica. It was suggested that it may be effective against myelitis.

[0120] [Example 6] RGMa expression in AQP4-depleted areas of the spinal cord Immunohistochemical staining of the spinal cord of NMO rats detected the expression of RGMa in the acute phase of a severe NMO rat model.

[0121] (6-1) Creation of a rat model of acute severe NMO Female Lewis rats were used in the experiment. 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 killed Mycobacterium tuberculosis H37Ra, followed by sonication to prepare an emulsion. Ten days later, mice with a neurological score of 1 or less were given a single intraperitoneal injection of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) at a dose of 3 mg / kg. This induced NMO pathology.

[0122] (6-2) Immunohistochemical staining Dissection was performed one day after administration of anti-AQP4 antibody. After blood removal, the spinal cord was collected and post-fixed in 4% paraformaldehyde at 4°C for 1 day. After post-fixation, the tissue was cryoprotected by sucrose substitution and then embedded in OCT compound. Frozen sections were cut at 30 μm and analyzed using goat anti-RGMa antibody (1:100, R&D) and rabbit anti-AQP4 antibody (1:1000, Cell Signaling Technology) as primary antibodies, and Alxa488-labeled donkey anti-goat IgG antibody (1:1000, Cell Signaling Technology) as secondary antibody. Double immunohistochemistry was performed using a donkey anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific) and an Alxa647-conjugated donkey anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific). Stained sections were imaged using an inverted Olympus IX83 fluorescence microscope.

[0123] (6-3)Result Immunohistochemical staining images of spinal cord sections from acute, severe NMO rats are shown in Figure 7. Strong RGMa expression was observed in the AQP4-depleted areas of NMO lesions.

[0124] <Description of Sequence Listing> SEQ ID NO: 1: Amino acid sequence of human RGMa precursor protein SEQ ID NO: 2: Amino acid sequence of mouse RGMa precursor protein SEQ ID NO: 3: Amino acid sequence of rat RGMa precursor protein SEQ ID NO: 4: DNA sequence of the human RGMa gene SEQ ID NO: 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 8: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 9: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 10: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 11: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 12: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 13: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 14: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 15: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 16: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 17: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 18: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 19: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 20: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 21: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 22: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 23: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 24: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 25: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 26: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 27: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 28: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 29: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 30: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 31: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 32: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 33: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 34: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 35: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1Y SEQ ID NO: 36: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 37: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 38: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 39: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 40: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1F SEQ ID NO: 41: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1H SEQ ID NO: 42: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1L SEQ ID NO: 43: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1V SEQ ID NO: 44: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1I SEQ ID NO: 45: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1K SEQ ID NO: 46: Amino acid sequence of rat and mouse MOG (35-55) [Industrial Applicability]

[0125] The present invention is highly useful in the pharmaceutical industry because RGMa inhibitors are useful for preventing or treating acute neuromyelitis optica and for preventing or treating pain symptoms associated with neuromyelitis optica.

Claims

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

2. The preventive or therapeutic agent according to claim 1, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

3. The preventive or therapeutic agent according to claim 2, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

4. The preventive or therapeutic agent according to claim 2 or 3, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:

39.

5. The anti-RGMa neutralizing antibody is selected from the following (a) to (l): (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and Anti-RG containing a heavy chain variable region including HCDR2 containing the amino acid sequence SFG and HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; The preventive or therapeutic agent according to any one of claims 2 to 4, which is an antibody selected from the group consisting of:

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

7. The preventive or therapeutic agent according to claim 6, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

8. The preventive or therapeutic agent according to claim 7 , wherein the anti-RGMa neutralizing antibody is a humanized antibody.

9. The preventive or therapeutic agent according to claim 7 or 8, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:

39.

10. The anti-RGMa neutralizing antibody is selected from the following (a) to (l): (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and Anti-RG containing a heavy chain variable region including HCDR2 containing the amino acid sequence SFG and HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; an anti-RGMa neutralizing antibody comprising the region; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; The preventive or therapeutic agent according to any one of claims 7 to 9, which is an antibody selected from the group consisting of:

11. A method for preventing or treating acute neuromyelitis optica or pain symptoms associated with neuromyelitis optica, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of treatment.

12. The method for prevention or treatment according to claim 11, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

13. Use of an RGMa inhibitor for the manufacture of an agent for the prevention or treatment of acute neuromyelitis optica.

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