Prophylactic or therapeutic agent for acute neuromyelitis optica

Abstract

The present invention provides a prophylactic or therapeutic agent for acute-phase neuromyelitis optica and pain symptoms of neuromyelitis optica, said prophylactic or therapeutic agent containing an RGMa inhibitor.

Description

[0001] This application is a divisional application of the invention patent application with Chinese application number 202080090100.1 (the invention name of the original application is "Preventive or therapeutic agent for acute neuromyelitis optica", and the application date of the original application is December 25, 2020). Technical Field

[0002] The present invention relates to a preventive or therapeutic agent for neuromyelitis optica in the acute phase comprising an RGMa inhibitory substance, and to a preventive or therapeutic agent for the pain symptoms of neuromyelitis optica. Background Art

[0003] Neuromyelitis optica (NMO), also known as Devic's disease, is an inflammatory central nervous system disease characterized by severe optic neuritis and transverse myelitis reaching more than three vertebrae. In 2004, IgG specific for neuromyelitis optica (NMO-IgG) was discovered (Non-Patent Document 1), and further, it was reported that aquaporin-4 (AQP4), a water channel protein expressed at high density on the foot processes of astrocytes, is its target antigen, that is, NMO-IgG is an anti-AQP4 antibody (Non-Patent Document 2).

[0004] Therefore, unlike multiple sclerosis, which is a demyelinating disease, neuromyelitis optica is an astroglial cell disease caused by the destruction of astrocytes by anti-AQP4 antibodies. The symptoms of neuromyelitis optica are believed to be central inflammation caused by the activation of cellular immunity or the hyperpermeability of the blood spinal cord barrier (BSCB), which in the brain is caused by the blood-brain barrier (BBB), which causes the anti-AQP4 antibodies to flow into the brain / spinal cord and produce complement activation, resulting in widespread loss of AQP4, destruction of astrocytes, and shedding. It is believed that due to functional damage such as cell damage to astrocytes (complement-dependent, antibody-dependent cell damage) and destruction of the blood-brain barrier caused by shedding of astrocytes or abnormal glutamate metabolism, secondary inflammatory cell infiltration, demyelination / axonal damage, phagocytosis by macrophages / microglia, and tissue softening occur, resulting in necrosis of neural tissue (Non-Patent Documents 3, 4).

[0005] As a typical diagnostic criterion for neuromyelitis optica, the criterion published by Wingerchuk et al. in 2006 (non-patent document 5) has been widely adopted; in 2007, as a neuromyelitis optica-related disease (NMOSD), in addition to typical neuromyelitis optica, only cases of optic neuritis (recurrent or binocular) or long myelitis of more than 3 vertebrae were considered as diseases of the same category (non-patent document 6). Therefore, in recent years, neuromyelitis optica is considered to be a broader disease concept than the simple neuromyelitis optica previously believed. In this way, it is sometimes difficult to diagnose neuromyelitis optica based only on clinical and image observations such as the length of spinal cord lesions, and anti-AQP4 antibodies are extremely important tests for the diagnosis of neuromyelitis optica and the determination of treatment guidelines.

[0006] The symptoms of neuromyelitis optica in the acute stage are more severe than those of multiple sclerosis. When a relapse occurs, optic neuritis may cause blindness, and myelitis may force patients to live in a wheelchair, so it is important to start treatment quickly. In addition, neuromyelitis optica is accompanied by severe pain in both the acute and chronic stages, so it is also important to provide treatment to relieve the pain.

[0007] The first choice for the treatment of neuromyelitis optica in the acute phase is steroid pulse therapy (non-patent document 7), and its effectiveness is widely recognized clinically. For multiple sclerosis, it has been demonstrated that this therapy promotes recovery from the worsening of clinical symptoms in the acute phase, and therefore, it is believed that the same effect can be obtained for neuromyelitis optica (non-patent documents 8-10). Steroid pulse therapy is the same as the treatment for multiple sclerosis. Its standard is to administer methylprednisolone by drip at 1000 mg / day for 3 consecutive days. When the symptom improvement is not sufficient, 1-2 additional courses of treatment are added. When steroid pulse therapy lacks therapeutic effect, plasma exchange therapy is actively considered as a second choice of treatment (non-patent documents 11, 12). In severe cases, it is expected to perform plasma exchange therapy from an early stage.

[0008] As described above, in the treatment of the acute phase, only case reports based on the possibility of therapeutic methods showing usefulness in multiple sclerosis or neuromyelitis optica have been accumulated. In addition, at present, effective therapeutic drugs related to neuromyelitis optica in the acute phase have not yet been marketed.

[0009] RGM (repulsive guidance molecule) is a membrane protein that was originally identified as an axon-inducing molecule of the visual system (Non-patent Document 13). It is known that the RGM family includes three members called RGMa, RGMb and RGMc (Non-patent Document 14), and at least RGMa and RGMb function by the same signal transduction mechanism (Non-patent Document 15). RGMc plays an important role in iron metabolism.

[0010] Subsequent studies have shown that RGM has the functions of inducing axons and forming lamina in Xenopus and chicken embryos, and controlling the closure of the head 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.

[0011] In addition to its function in the developmental stage, RGMa is considered to be an axon regeneration inhibitory substance after central nervous system injury because it is re-expressed after central nervous system injury in adult humans and rats, and in rats, RGMa inhibition can promote axon growth after spinal cord injury and promote functional recovery (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).

[0012] In addition, it is known that anti-RGMa neutralizing antibodies are effective in suppressing the onset of neuromyelitis optica (Non-Patent Document 18).

[0013] Thus, the role of RGMa in central nervous system damage has been clarified, and its effect on suppressing the onset of neuromyelitis optica is known. In particular, the involvement of RGMa in the treatment of neuromyelitis optica in the acute phase has not been identified, and a therapeutic drug for the same is not known.

[0014] Prior art literature

[0015] Patent Literature

[0016] Patent Document 1: International Publication No. WO2005 / 087268

[0017] Patent document 2: International Publication No. WO2009 / 106356

[0018] Patent Document 3: International Publication No. WO2013 / 112922

[0019] Patent document 4: International Publication No. WO2016 / 175236

[0020] Non-patent literature

[0021] Non-patent document 1: Lancet 364: 2106-2112, 2004

[0022] Non-patent document 2: J Exp Med 202:473-477, 2005

[0023] Non-patent document 3: Jpn. J. Clin. Immunol., 35(2), 129-135 (2012)

[0024] Non-patent document 4: Journal of the Neurological Sciences 306 (2011) 183-187

[0025] Non-patent document 5: Neurology 66:1485-1489, 2006

[0026] Non-patent literature 6: Lancet Neurol 6:805-815, 2007

[0027] Non-patent literature 7: Curr Treat Options Neurol 12:244-255,2010

[0028] Non-patent document 8: Neurology 53:1107-1114,1999

[0029] Non-patent literature 9: Magn Reson Med Sci 7:55-58,2008

[0030] Non-patent document 10: Tohoku J Exp Med 215:55-59, 2008

[0031] Non-patent document 11: Neurology 63:1081-1083, 2004

[0032] Non-patent document 12: Mult Sclr 13:128-132, 2007

[0033] Non-patent document 13: Neuron 5, 735-743 (1990)

[0034] Non-patent document 14: Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513-29, 2006

[0035] Non-patent document 15: Biochem. Biophys. Res. Commun. 382, ​​795-800 (2009)

[0036] Non-patent document 16: Curr. Opin. Neurobiol. 17, 29-34 (2007)

[0037] Non-patent document 17: J. Cell Biol. 173, 47-58 (2006)

[0038] Non-patent literature 18: Scientific Reports 8:34 1-9 (2018) Summary of the invention

[0039] The inhibitory effect of anti-RGMa neutralizing antibodies on the onset of neuromyelitis optica is described in Non-Patent Document 18. In this document, IgG from anti-AQP4 antibody-positive NMO patients is directly administered to the spinal cord of healthy animals, and anti-RGMa neutralizing antibodies are simultaneously administered, thereby showing the effect of inhibiting the onset of neuromyelitis optica. However, not only is there no record of the effect of anti-RGMa neutralizing antibodies on the acute phase neuromyelitis optica model that reflects human symptoms, but also, it is unclear from the description of this document alone whether anti-RGMa neutralizing antibodies show a preventive or therapeutic effect on acute phase neuromyelitis optica.

[0040] In addition, since neuromyelitis optica is accompanied by severe pain at the onset of the disease, there is 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.

[0041] An object of the present invention is to provide a drug effective for neuromyelitis optica in the acute stage and the symptoms of neuromyelitis optica.

[0042] The present inventors conducted intensive research to solve the above problems and found that RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies, have a recovery effect on acute clinical deterioration, an early repair effect on the destruction of the blood-spinal cord barrier caused by myelitis, and an effect of inhibiting the infiltration of granulocytes seen in the symptoms of acute neuromyelitis optica, thereby finding that they have excellent effects on the prevention or treatment of acute neuromyelitis optica. In addition, it was found that RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies, can treat, reduce or alleviate the pain symptoms of neuromyelitis optica, and thus the present invention was completed.

[0043] That is, the present invention is as follows.

[0044] 1. A preventive or therapeutic agent for acute neuromyelitis optica, comprising an RGMa inhibitory substance.

[0045] 2. The preventive or therapeutic agent according to item 1, wherein the RGMa inhibitory substance is an anti-RGMa neutralizing antibody.

[0046] 3. The preventive or therapeutic agent according to item 2, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

[0047] 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 sequence number 16, sequence number 36, sequence number 37, sequence number 38 and sequence number 39.

[0048] 5. The preventive or therapeutic agent according to any one of items 2 to 4, wherein the anti-RGMa neutralizing antibody is an antibody selected from the following (a) to (l):

[0049] (a) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10,

[0050] (b) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a HCDR3 comprising SFG in the amino acid sequence,

[0051] (c) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22,

[0052] (d) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28,

[0053] (e) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0054] (f) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0055] (g) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0056] (h) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0057] (i) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0058] (j) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0059] (k) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and

[0060] (l) An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region contains LCDR1 comprising the amino acid sequence recorded in sequence number 29, LCDR2 comprising the amino acid sequence recorded in sequence number 30, and LCDR3 comprising the amino acid sequence recorded in sequence number 45, and the heavy chain variable region contains HCDR1 comprising the amino acid sequence recorded in sequence number 32, HCDR2 comprising the amino acid sequence recorded in sequence number 33, and HCDR3 comprising the amino acid sequence recorded in sequence number 34.

[0061] 6. A preventive or therapeutic agent for the pain symptoms of neuromyelitis optica, comprising an RGMa inhibitory substance.

[0062] 7. The preventive or therapeutic agent according to item 6, wherein the RGMa inhibitory substance is an anti-RGMa neutralizing antibody.

[0063] 8. The preventive or therapeutic agent according to item 7, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

[0064] 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 sequence number 16, sequence number 36, sequence number 37, sequence number 38 and sequence number 39.

[0065] 10. The preventive or therapeutic agent according to any one of items 7 to 9, wherein the anti-RGMa neutralizing antibody is an antibody selected from the following (a) to (l):

[0066] (a) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10,

[0067] (b) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a HCDR3 comprising SFG in the amino acid sequence,

[0068] (c) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22,

[0069] (d) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28,

[0070] (e) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0071] (f) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0072] (g) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0073] (h) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0074] (i) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0075] (j) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0076] (k) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and

[0077] (l) An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region contains LCDR1 comprising the amino acid sequence recorded in sequence number 29, LCDR2 comprising the amino acid sequence recorded in sequence number 30, and LCDR3 comprising the amino acid sequence recorded in sequence number 45, and the heavy chain variable region contains HCDR1 comprising the amino acid sequence recorded in sequence number 32, HCDR2 comprising the amino acid sequence recorded in sequence number 33, and HCDR3 comprising the amino acid sequence recorded in sequence number 34.

[0078] 11. A method for preventing or treating acute neuromyelitis optica or the painful symptoms of neuromyelitis optica, comprising administering an effective amount of an RGMa inhibitory substance to a mammal in need of treatment.

[0079] 12. The method for prevention or treatment according to item 11, wherein the RGMa inhibitory substance is an anti-RGMa neutralizing antibody.

[0080] 13. Use of an RGMa inhibitory substance for the manufacture of a preventive or therapeutic agent for acute neuromyelitis optica.

[0081] According to the present invention, RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies, show effects such as early repair of the destruction 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.

[0082] Furthermore, according to the present invention, RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, exhibit an effect of inhibiting the infiltration of granulocytes into the spinal cord seen in acute neuromyelitis optica, and are useful as a preventive or therapeutic agent for acute neuromyelitis optica.

[0083] Furthermore, according to the present invention, RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, can treat, alleviate or relieve pain symptoms seen in neuromyelitis optica, and are useful as preventive or therapeutic agents for such pain symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The figure shows the therapeutic effect of anti-RGMa neutralizing antibody (sometimes referred to as anti-RGMa antibody) on the acute clinical exacerbation of the acute phase severe NMO rat model. Each data represents mean±SEM. The number of cases in each group is indicated in brackets in the legend of the graph.

[0085] Figure 2 The graph shows the effect of anti-RGMa neutralizing antibody on the recovery of blood-spinal cord barrier destruction and neurological symptoms in acute stage tEAE mice. Figure 2 A represents the restorative effect of anti-RGMa neutralizing antibody on the level change of gadolinium leakage to the spinal cord during BSCB destruction in the acute phase of tEAE mice. Each data represents mean±SEM, and the number of cases in each group is indicated in brackets in the chart legend. Individuals in which no gadolinium leakage to the spinal cord was confirmed at the time point 7 days after cytokine injection and individuals in which MRI images could not be obtained 14 days after cytokine injection were excluded from the analysis data, and statistical analysis was performed using the Bonferroni multiple comparison test (***p < 0.001). Arrows indicate the day of antibody administration. Figure 2 B represents the recovery effect of anti-RGMa neutralizing antibody on neurological symptoms. Each data represents mean±SEM, and the number of cases in each group is indicated in brackets in the graph legend. The arrow indicates the day of antibody administration. Statistical analysis was performed by Mann-Whitney U test at each time point (*p<0.05, **p<0.01).

[0086] Figure 3This is a graph showing the correlation between the intensity of gadolinium spinal cord leakage and the severity of neurological symptoms in the acute phase of tEAE mice. Each data was plotted for each individual, and Spearman's rank correlation analysis was used for statistical analysis. The number of cases in each group is indicated in brackets in the chart legend. There was one case of loss in MRI image acquisition 14 days after cytokine injection (anti-RGMa neutralizing antibody administration group).

[0087] Figure 4 The immunohistochemical staining images and graphs show the therapeutic effect of anti-RGMa neutralizing antibody on blood-spinal cord barrier disruption in the acute severe NMO rat model. Each data in quantitative analysis is represented by mean±SEM, and statistical analysis was performed using Turkey's multiple comparison test (*p<0.05).

[0088] Figure 5 This is a graph showing the effect of anti-RGMa neutralizing antibodies on pain-related behaviors in the acute phase severe NMO rat model. Each data represents mean±SEM, and statistical analysis was performed using Turkey's multiple comparison test (***p<0.001, **p<0.01, *p<0.05 vs healthy untreated group; ##p<0.01, #p<0.05 vs NMO_isotype control antibody treated group). Arrows indicate the day of administration of anti-RGMa neutralizing antibodies or isotype control antibodies.

[0089] Figure 6 The immunohistochemical staining images and graphs show the inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord of acute severe NMO rat model. Each data of quantitative analysis is represented by mean±SEM, and statistical analysis was performed using Turkey's multiple comparison test (**p<0.01, *p<0.05).

[0090] Figure 7 The images are immunohistochemical staining images showing the expression of RGMa in the AQP4 loss site of the spinal cord in the acute phase severe NMO rat model. DETAILED DESCRIPTION

[0091] Hereinafter, the terms used in the present invention will be described.

[0092] [Neutralization]

[0093] In the present application, neutralization refers to an action that binds to a target and is able to inhibit any function of the target. For example, an RGMa inhibitory substance refers to a substance that binds to RGMa and, as a result, inhibits the biological activity of RGMa.

[0094] [Epitope]

[0095] In the present application, an epitope comprises a polypeptide determinant capable of specific binding to an immunoglobulin or T cell receptor. In one embodiment, an epitope comprises a molecular chemically active surface group (e.g., an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group), and in one embodiment, may have specific 3-dimensional structural properties and / or specific charge properties. An epitope is a region of an antigen that is bound by an antibody.

[0096] [Separated]

[0097] In the present application, the "isolated" of isolated RGMa inhibitory substances (such as antibodies, etc.) means identified and separated, and / or recovered from components in the natural state. Impurities in the natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, and examples include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In general, when isolating RGMa inhibitory substances, it is sufficient to purify them through at least one purification step, and the RGMa inhibitory substances purified through at least one purification step can be referred to as "isolated RGMa inhibitory substances".

[0098] [Antibody]

[0099] In the present application, an antibody refers in a broad sense to an immunoglobulin (Ig) molecule consisting of four polypeptide chains, namely, two heavy chains (H chains) and two light chains (L chains), which retains the characteristic of the Ig molecule to substantially bind to an epitope.

[0100] [Human Antibodies]

[0101] In the present application, human antibody refers to an antibody in which both the light chain and the heavy chain are from human immunoglobulin. Human antibody includes IgG (including IgG1, IgG2, IgG3 and IgG4) with a heavy chain of γ chain, IgM with a heavy chain of μ chain, IgA (including IgA1, IgA2) with a heavy chain of α chain, IgD with a heavy chain of δ chain or IgE with a heavy chain of ε chain, according to the difference in the constant region of the heavy chain. In addition, in principle, the light chain includes either one of κ chain and λ chain.

[0102] [Humanized Antibodies]

[0103] In the present application, a humanized antibody refers to an antibody composed of a variable region composed 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.

[0104] [Chimeric antibodies]

[0105] In the present application, a chimeric antibody refers to an antibody in which the light chain, the heavy chain, or both chains are composed of a non-human variable region and a human constant region.

[0106] [Monospecific Antibodies]

[0107] In the present application, a monospecific antibody refers to an antibody having a single antigen specificity and a single independent antigen recognition site. In the present specification, for example, a monospecific antibody that recognizes RGMa is referred to as an RGMa monospecific antibody.

[0108] [Multispecific Antibodies]

[0109] In the present application, a multispecific antibody refers to an antibody having two or more different antigen specificities and simultaneously having two or more independent antigen recognition sites, and examples thereof include bispecific antibodies having two antigen specificities and trispecific antibodies having three antigen specificities.

[0110] [Complementarity Determining Region (CDR)]

[0111] Complementarity determining region (CDR) refers to the region forming the antigen binding site in the variable region of the immunoglobulin molecule, also known as the hypervariable region, which is the part in each immunoglobulin molecule where the amino acid sequence changes particularly greatly. For CDR, there are 3 CDRs in the light chain and the heavy chain, respectively. Sometimes the 3 CDRs contained in the light chain are referred to as LCDR1, LCDR2 and LCDR3, respectively, and the 3 CDRs contained in the heavy chain are referred to as HCDR1, HCDR2 and HCDR3, respectively. For example, the CDR of an immunoglobulin molecule can be determined according to Kabat's numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0112] [Effective amount]

[0113] An effective amount refers to an amount of a preventive or therapeutic agent sufficient to reduce or improve the severity and / or duration of a disorder or one or more of its symptoms, prevent the disorder from getting worse, slow down the disorder, prevent the recurrence, occurrence, onset or worsening of one or more symptoms associated with the disorder, detect the disorder, or enhance or improve one or more preventive or therapeutic effects of other treatments (e.g., preventive or therapeutic drugs).

[0114] [Percentage (%) homology of amino acid sequences]

[0115] "Percentage (%) homology" of the amino acid sequence of the candidate polypeptide sequence of the variable region etc. and the amino acid sequence of the reference polypeptide sequence is defined as to align the sequences, to obtain the maximum % homology, gaps are introduced as required, and any conservative substitution is not considered as the percentage of the amino acid residues in the candidate sequence identical with the amino acid residues in the specific reference polypeptide sequence after a part of the sequence homology. The alignment for the purpose of measuring % homology can be achieved by using various methods within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. As long as it is a person skilled in the art, it is possible to determine the appropriate parameters for aligning sequences including any algorithm necessary for realizing maximum alignment for the full length of the sequence to be compared. However, for the purpose here, % homology values ​​are obtained by using the sequence comparison computer program BLAST in pairwise alignments.

[0116] In the case of using BLAST in amino acid sequence comparison, the % homology between a provided amino acid sequence A and a provided amino acid sequence B is calculated as follows:

[0117] 100 times the fraction X / Y

[0118] Here, X is the number of amino acid residues that are scored as identical when A and B are aligned by the sequence alignment program BLAST, and Y is the total number of amino acid residues in B. In the case where the length of amino acid sequence A is different from the length of amino acid sequence B, it can be understood that the % homology of A to B is different from the % homology of B to A. Unless otherwise specified, all % homology values ​​here are obtained using the BLAST computer program as described in the previous paragraph.

[0119] [Conservative substitution]

[0120] Conservative substitution refers to replacing an amino acid residue with another chemically similar amino acid residue in a manner that does not substantially change the activity of the peptide. For example, a hydrophobic residue is replaced by another hydrophobic residue, a polar residue is replaced by another polar residue with the same charge, etc. As examples of functionally similar amino acids that can undergo such substitutions, as non-polar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. can be mentioned. As polar (neutral) amino acids, glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. can be mentioned. As positively charged (basic) amino acids, arginine, histidine, lysine, etc. can be mentioned. In addition, as negatively charged (acidic) amino acids, aspartic acid, glutamic acid, etc. can be mentioned.

[0121] Hereinafter, embodiments of the present invention will be described in detail.

[0122] The present invention provides a novel use of an RGMa inhibitory substance, namely, a preventive or therapeutic agent for acute neuromyelitis optica.

[0123] In addition, the present invention provides a method for preventing or treating neuromyelitis optica in the acute phase, comprising administering a preventive or therapeutic agent comprising an effective amount of an RGMa inhibitory substance to a mammal in need of treatment.

[0124] <RGMa inhibitory substances>

[0125] The RGMa inhibitory substance of the present invention can be any substance as long as it acts on RGMa itself to inhibit or weaken the activity of RGMa (hereinafter, sometimes referred to as "RGMa activity" in this specification). For example, a substance that has the activity of binding to RGMa and directly inhibiting (weakening) the activity of RGMa, or inhibiting the binding of RGMa to a receptor and indirectly inhibiting (weakening) the activity of RGMa (such as the compounds or antibodies described later) is called the RGMa inhibitory substance of the present invention.

[0126] In addition, the RGMa inhibitory substance of the present invention may be a substance that inhibits the expression of RGMa. For example, a substance that inhibits the expression of RGMa and thus inhibits (weakens) the activity of RGMa (such as the nucleic acid molecules described below) is also included in the RGMa inhibitory substance of the present invention.

[0127] RGMa has been identified as a neurite outgrowth inhibitory protein in the central nervous system. As shown in SEQ ID NO. 1, human RGMa protein is synthesized as a precursor protein consisting of 450 amino acids. The signal peptide Met1 to Pro47 (a peptide from the 1st methionine residue to the 47th proline residue from the N-terminal side, and described in the same manner hereinafter) existing at the N-terminal is removed, the peptide bond between Asp168 and Pro169 is cut, and the N-terminal domain is generated. The C-terminal peptide Ala425 to Cys450 of the C-terminal fragment is further removed from Pro169, and a GPI anchor is added to the C-terminal carboxyl group of Ala424, which becomes the C-terminal, to generate the C-terminal domain. Human RGMa protein is expressed on the cell membrane via the GPI anchor as a mature protein in which the N-terminal domain (Cys48 to Asp168) and the C-terminal domain (Pro169 to Ala424) are connected by a disulfide bond.

[0128] In the present invention, RGMa may be derived from any animal, but is preferably human RGMa. The precursor protein of human RGMa is composed of the amino acid sequence shown in sequence number 1 of the sequence table. Although the precursor protein of mouse RGMa is composed of the amino acid sequence shown in sequence number 2 of the sequence table, and the precursor protein of rat RGMa is composed of the amino acid sequence shown in sequence number 3 of the sequence table, since the C-terminal peptide is removed, the amino acid sequence becomes the same as the mature protein.

[0129] Examples of the RGMa gene include, but are not limited to, the human RGMa gene having the base sequence shown in SEQ ID NO: 4. The base sequences of RGM genes from various organisms can be easily acquired from known databases (GenBank, etc.).

[0130] As the RGMa inhibitory substances of the present invention, specifically, low molecular weight compounds, anti-RGMa neutralizing antibodies, antibodies with modified functions, coupled antibodies, or antigen-binding fragments thereof, etc., can be cited, and in addition, siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense oligonucleotides, etc., which are nucleic acid molecules of RGMa, can be cited. Among these RGMa inhibitory substances, anti-RGMa neutralizing antibodies, antibodies with modified functions, coupled antibodies, and antigen-binding fragments thereof are preferred, anti-RGMa neutralizing antibodies or antigen-binding fragments thereof are more preferred, and anti-RGMa neutralizing antibodies are particularly preferred.

[0131] <Anti-RGMa neutralizing antibody>

[0132] In the present invention, the anti-RGMa neutralizing antibody can be a polyclonal antibody or a monoclonal antibody as long as it binds to RGMa and neutralizes the activity of RGMa. In the present invention, a monoclonal antibody is preferred. In addition, the anti-RGMa neutralizing antibody of the present invention can be a RGMa monospecific antibody or a multispecific antibody that recognizes multiple RGMa and other antigens, preferably a RGMa monospecific antibody.

[0133] In addition, as a specific epitope, for human RGMa, preferably one or more of sequence number 16 (amino acid number 47-69 of sequence number 1), sequence number 36 (amino acid number 298-311 of sequence number 1), sequence number 37 (amino acid number 322-335 of sequence number 1), sequence number 38 (amino acid number 349-359 of sequence number 1), and sequence number 39 (amino acid number 367-377 of sequence number 1), more preferably a combination of sequence numbers 36 and 37, and particularly preferably a combination of sequence numbers 36, 37 and 39.

[0134] The anti-RGMa neutralizing antibodies of the present invention include polyclonal antibodies and monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or its partial fragments (such as the above-mentioned epitope fragments) as antigens, chimeric antibodies and humanized antibodies produced using genetic recombination technology, and human antibodies produced using transgenic animals that produce human antibodies. When the antibodies of the present invention are administered to humans as drugs, humanized antibodies or human antibodies are preferred from the perspective of side effects.

[0135] Specific examples of the anti-RGMa neutralizing antibody of the present invention include the following antibodies (a) to (l), and the methods for producing each of the antibodies can be the methods described in Patent Documents 2 to 4.

[0136] The antibodies selected from (a) to (l) can be cited: (a) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody with sequence numbers 36, 37 and 39 as epitopes), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1 comprising the amino acid sequence recorded in sequence number 5, LCDR2 comprising the amino acid sequence recorded in sequence number 6, and LCDR3 comprising the amino acid sequence recorded in sequence number 7, the heavy chain variable region comprising HCDR1 comprising the amino acid sequence recorded in sequence number 8, HCDR2 comprising the amino acid sequence recorded in sequence number 9, and HCDR3 comprising the amino acid sequence recorded in sequence number 10,

[0137] (b) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes antibodies with SEQ ID NOs: 36, 37 and 38 as epitopes), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, the heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 comprising SFG in the amino acid sequence,

[0138] (c) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22,

[0139] (d) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and the heavy chain variable region comprises a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28,

[0140] (e) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0141] (f) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody using SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0142] (g) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0143] (h) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0144] (i) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0145] (j) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody using SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0146] (k) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44, the heavy chain variable region comprising a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and

[0147] (l) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody using sequence number 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region containing LCDR1 containing the amino acid sequence recorded in sequence number 29, LCDR2 containing the amino acid sequence recorded in sequence number 30, and LCDR3 containing the amino acid sequence recorded in sequence number 45, and the heavy chain variable region containing HCDR1 containing the amino acid sequence recorded in sequence number 32, HCDR2 containing the amino acid sequence recorded in sequence number 33, and HCDR3 containing the amino acid sequence recorded in sequence number 34.

[0148] Among these, the antibodies described in (a) are particularly preferred.

[0149] The method for producing the anti-RGMa neutralizing antibody of the present invention can use the existing generally used production method. The antigen can be used directly for immunization, or it can be made into a complex with a carrier protein and used. The preparation of the complex of the antigen and the carrier protein can use condensation agents such as glutaraldehyde, carbodiimide, and maleimide active ester. Carrier proteins can exemplify bovine serum albumin, thyroglobulin, hemocyanin, KLH, etc.

[0150] As mammals for immunization, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses or cattle can be mentioned, and the inoculation method can be subcutaneous administration, intramuscular administration or intraperitoneal administration. When administering, it can be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant and administered, and the administration is usually performed once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of the immunized animal are fused with myeloma cells and isolated in the form of hybridomas. As myeloma cells, myeloma cells from mammals, such as mice, rats, humans, etc., can be used.

[0151] <Polyclonal Antibodies>

[0152] The polyclonal antibody can be obtained, for example, by immunizing the above-mentioned mammal with the above-mentioned antigen and, if necessary, Freund's adjuvant, and obtaining the serum obtained from the immunosensitized animal.

[0153] <Monoclonal Antibodies>

[0154] Specifically, monoclonal antibodies can be obtained in the following manner. That is, the above-mentioned antigen is used as an immunogen, and the immunogen is injected into the mammal as described above subcutaneously, intramuscularly, intravenously, intrapedicularly or intraperitoneally together with Freund's Adjuvant as needed, one to multiple times, or transplanted, thereby applying immune sensitization. Usually, 1 to 4 immunizations are performed every about 1 to 14 days from the initial immunization, and antibody-producing cells are obtained from the immune-sensitized mammal about 1 to 5 days after the final immunization.

[0155] Monoclonal antibodies can be obtained using methods 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)).

[0156] The preparation of "hybridomas" that secrete monoclonal antibodies can be carried out according to and Milstein et al. (Nature, 256, 495, 1975) and modified methods thereof. That is, antibody-producing cells contained in the spleen or the like obtained from an immunosensitized mammal are fused with myeloma cells from a mammal, preferably a mouse, rat or human, that do not have the ability to produce their own antibodies.

[0157] As myeloma cells used in cell fusion, for example, Myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0 or BW5147 from mice, Myeloma 210RCY3-Ag.2.3. from rats, Myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 from humans can be used.

[0158] Fusion promoters include polyethylene glycol and the like. Cell fusion can usually be achieved by using polyethylene glycol (average molecular weight 1000-4000) at a concentration of about 20-50% at a temperature of 20-40°C, preferably 30-37°C, with the ratio of the number of antibody-producing cells to the number of myeloma cells usually being about 1:1 to 10:1, and reacting for about 1 to 10 minutes.

[0159] Screening of hybridoma clones producing monoclonal antibodies can be performed by culturing the hybridomas in, for example, microtiter plates and measuring the reactivity of the culture supernatants of the wells with the immunizing antigen by an immunochemical method such as ELISA.

[0160] In screening of antibody-producing hybridoma cells, in addition to analysis of binding to RGMa protein, an evaluation is also performed as to whether the antibody inhibits the activity of RGMa of the present invention. Anti-RGMa neutralizing antibodies of the present invention can be selected by these screening methods.

[0161] From the wells containing hybridomas producing the target antibody, clones can be obtained by cloning by the limiting dilution method. Selection and breeding of hybridomas are usually performed in an animal cell culture medium containing 10 to 20% fetal bovine serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).

[0162] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or growing them in vivo, for example, in the ascites of a mammal such as a mouse or rat, and isolating the hybridomas from the obtained culture supernatant or ascites of the mammal.

[0163] In the case of in vitro culture, hybridomas can be proliferated, maintained and preserved according to various conditions such as the characteristics of the cell species being cultured and the culture method, using a nutrient medium suitable for producing monoclonal antibodies in the culture supernatant. The nutrient medium may include a known nutrient medium or a nutrient medium prepared from a minimal medium.

[0164] Examples of the basic culture medium include low calcium culture media such as Ham'F12 culture medium, MCDB153 culture medium, or low calcium MEM culture medium, and high calcium culture media such as MCDB104 culture medium, MEM culture medium, D-MEM culture medium, RPMI1640 culture medium, ASF104 culture medium, or RD culture medium. The basic culture medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

[0165] The isolation and purification of the monoclonal antibody can be performed by subjecting the above-mentioned culture supernatant or ascites to saturated ammonium sulfate, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as anti-immunoglobulin column or protein A column, etc. Specifically, the purification of the monoclonal antibody can be easily achieved by using a known method as a purification method for immunoglobulins, for example, by ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, using an anion exchanger, and further using affinity chromatography of RGMa protein.

[0166] Monoclonal antibodies can also be obtained by phage display. In the phage display method, the target immunogen is used to screen the phage selected from any phage antibody library, and the phage with the desired binding to the immunogen is selected. Next, the antibody corresponding sequence contained in the phage is separated or sequenced, and an expression vector containing a nucleic acid molecule encoding an antibody or antigen binding domain is constructed based on the separated sequence or the determined sequence information. Then, monoclonal antibodies can be produced by culturing the cell line transfected with the expression vector. By using a human antibody library as a phage antibody library, human antibodies with desired binding can be produced.

[0167] <Nucleic acid molecules>

[0168] The nucleic acid molecule encoding the anti-RGMa neutralizing antibody or its antigen-binding fragment of the present invention can be obtained, for example, by the following method. First, using a commercially available RNA extraction kit, total RNA is prepared from cells of a hybridoma, etc., and cDNA is synthesized by reverse transcriptase using random primers, etc. Next, in the variable regions of known human antibody heavy chain genes and light chain genes, the cDNA encoding the antibody is amplified by using oligonucleotides of the sequences preserved separately as primers by the PCR method. For sequences encoding constant regions, it can be obtained by amplifying known sequences using the PCR method. The base sequence of the DNA can be determined by incorporating it into a plasmid for sequence determination and by conventional methods.

[0169] Alternatively, a DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the variable region or a partial sequence thereof and combining it with a sequence containing the constant region.

[0170] The nucleic acid molecule may be a nucleic acid molecule encoding the constant region and variable region of all heavy and light chains, or may be a nucleic acid molecule encoding only the variable region of the heavy and light chains. The base sequence of the constant region of the heavy and light chains when encoding all the constant and variable regions is preferably the base sequence described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0171] <Function-modified antibodies>

[0172] Functionally modified antibodies of anti-RGMa neutralizing antibodies can be prepared by the following method. For example, if CHO cells with destroyed α1,6-fucosyltransferase (FUT8) gene are used as host cells to produce the anti-RGMa neutralizing antibodies of the present application, antibodies with reduced fucose content in sugar chains and improved cell killing function can be obtained. If CHO cells with FUT8 gene introduced are used as host cells for production, antibodies with low cell killing function can be obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). In addition, by changing the amino acid residues in the Fc region, complement activation function can be regulated (U.S. Patent No. 6737056, U.S. Patent No. 7297775, U.S. Patent No. 7317091). Furthermore, by using a variant of the Fc region that improves the binding to FcRn, which is one type of Fc receptor, it is possible to extend the half-life in the blood (Hashiguchi Zhouping et al., Biochemistry, 2010, Vol.82(8), p710). These functionally modified antibodies can be produced by genetic engineering. By using a variant of the Fc region that improves the binding to FcRn, which is one type of Fc receptor, it is possible to extend the half-life in the blood (Hashiguchi Zhouping et al., Biochemistry, 2010, Vol.82(8), p710). These functionally modified antibodies can be produced by genetic engineering.

[0173] <Conjugated Antibodies>

[0174] As the modified molecule of the anti-RGMa neutralizing antibody of the present invention, a conjugated antibody can be cited. As a conjugated antibody, a conjugated antibody formed by chemically or genetically engineering the anti-RGMa neutralizing antibody to which a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor (TGF-β, NGF, neurotrophin, etc.), albumin, an enzyme, other antibodies, etc., other functional molecules other than the anti-RGMa neutralizing antibody of the present application are conjugated.

[0175] When PEG is used as a functional molecule, PEG having a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, can be used without limitation, and can be linear or branched. PEG can be bound to the N-terminal amino group of the amino acid of the anti-RGMa neutralizing antibody by using, for example, an NHS active group.

[0176] In the case of using radioactive substances as functional molecules, 131 I. 125 I. 90 Y. 64 Cu, 99 Tc, 77 Lu or211 At, etc. Radioactive substances can be directly bound to anti-RGMa neutralizing antibodies by the chloramine T method or the like.

[0177] In the case of using a toxin as the functional molecule, bacterial toxins (eg, diphtheria toxin), plant toxins (eg, ricin), low molecular weight toxins (eg, geldamycin), maytansine, calicheamicin, and the like can be used.

[0178] When a low molecular weight compound is used as the functional molecule, examples thereof include daunomycin, doxorubicin, methotrexate, mitomycin, neostatin, vindesine, and fluorescent dyes such as FITC.

[0179] 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, sugar oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. can be used.

[0180] Examples of linkers used when chemically binding toxins, low molecular weight compounds or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2) n O(CR2) n -(R is an arbitrary substituent, n is a positive integer) represented by a linker, repeating units of alkoxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine (trademark)), as well as diacids and amides (including succinate, succinamide, diglycolate, malonate and hexanamide). Chemical modification methods for binding functional molecules have been established in this field (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 T.J. International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chariet al., Cancer Res., 1992 Vol 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681).

[0181] <Antigen-binding fragment>

[0182] In an embodiment of the present invention, the "antigen-binding fragment" of an antibody refers to a portion of the antibody as described above that has antigen binding properties, specifically, F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide bond Fv, single-chain antibody (scFv) and their polymers, etc. Furthermore, the antigen-binding fragment includes a coupled fragment of functional molecules other than the anti-RGMa neutralizing antibody of the present application, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, neurotrophins, etc.), albumin, enzymes, other antibodies, etc., which are chemically or genetically engineered to bind to them.

[0183] "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulin with pepsin or papain, which are proteolytic enzymes, and digested before and after the disulfide bond between the two heavy chains in the hinge region. For example, if IgG is treated with papain, the disulfide bond between the two heavy chains in the hinge region is cut upstream, and the light chain composed of VL (light chain variable region) and CL (light chain constant region) and the heavy chain fragment composed of VH (heavy chain variable region) and CHγ1 (γ1 region in the heavy chain constant region) can be produced. The same two antibody fragments are respectively referred to as Fab. In addition, if IgG is treated with pepsin, the disulfide bond between the two heavy chains in the hinge region is cut downstream, and an antibody fragment slightly larger than the antibody fragment in which the above two Fabs are connected in the hinge region can be produced. This antibody fragment is referred to as F(ab')2.

[0184] <Chimeric antibodies>

[0185] As a preferred embodiment of the anti-RGMa neutralizing antibody of the present invention, a chimeric antibody can be cited. As a "chimeric antibody", a chimeric antibody in which the variable region is the variable region of an immunoglobulin from a non-human animal (mouse, rat, hamster, chicken, etc.) and the constant region is the constant region from a human immunoglobulin can be exemplified. For example, mice can be immunized with an antigen, and the variable region that binds to the antigen can be cut out from the gene of the mouse monoclonal antibody, and combined with the antibody constant region from human bone marrow to produce it. The constant region from human immunoglobulin has its own inherent amino acid sequence according to the isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD and IgE. The constant region of the recombinant chimeric antibody in the present invention can be a constant region of a human immunoglobulin belonging to any isotype. It is preferably a constant region of human IgG. The gene of the chimeric antibody produced in this way can be used to prepare an expression vector. The host cells are transformed with the expression vector to obtain chimeric antibody-producing transformed cells, and the transformed cells are cultured to obtain the target chimeric antibody from the culture supernatant.

[0186] <Humanized Antibodies>

[0187] As other preferred forms of the anti-RGMa neutralizing antibodies of the present invention, humanized antibodies can be cited. The "humanized antibody" in the present invention is an antibody formed by transplanting (CDR grafting) the DNA sequence of the antigen binding site (CDR, complementary determining region) of non-human animal antibodies such as mice to human antibody genes. For example, it can be prepared by referring to the methods described in Japanese Patent Publication No. 4-506458 and Japanese Patent No. 2912618. Specifically, it refers to a humanized antibody, characterized in that part or all of its CDR is the CDR of a monoclonal antibody from a non-human mammal (mouse, rat, hamster, etc.), the framework region of its variable region is the framework region of the variable region from a human immunoglobulin, and its constant region is the constant region from a human immunoglobulin.

[0188] The humanized antibody of the present invention can be produced, for example, as follows, but of course the method is not limited to this one.

[0189] For example, recombinant humanized antibodies from mouse monoclonal antibodies can be prepared by genetic engineering with reference to Japanese Unexamined Patent Publication No. 4-506458 and Japanese Unexamined Patent Publication No. 62-296890, etc. That is, DNA of mouse heavy chain CDR portion and DNA of mouse light chain CDR portion are isolated from hybridomas producing mouse monoclonal antibodies, and human heavy chain genes of all regions except human heavy chain CDR and human light chain genes of all regions except human light chain CDR are isolated from human immunoglobulin genes.

[0190] The human heavy chain gene of the DNA of the isolated mouse heavy chain CDR portion is transplanted into an appropriate expression vector in an expressible manner, and similarly, the human light chain gene of the DNA of the isolated mouse light chain CDR portion is transplanted into another appropriate expression vector in an expressible manner. Alternatively, the heavy chain and light chain genes of the human transplanted mouse CDR can be imported into the same expression vector in an expressible manner. The host cell is transformed using the expression vector thus made, thereby obtaining a humanized antibody-producing transformed cell, and the target humanized antibody is obtained from the culture supernatant by culturing the transformed cell.

[0191] <Human Antibodies>

[0192] As other preferred forms of the anti-RGMa neutralizing antibodies of the present invention, human antibodies can be cited. Human antibodies are antibodies in which all regions of the variable region of the heavy chain and the constant region of the heavy chain and the variable region of the light chain and the constant region of the light chain constituting the immunoglobulin are from genes encoding human immunoglobulins, and human antibody genes can be introduced into mice to produce them. Specifically, for example, at least human immunoglobulin genes can be incorporated into the loci of mammals other than humans such as mice to produce transgenic animals, and the transgenic animals can be immunosensitized using antigens, thereby producing them in the same manner as the above-mentioned polyclonal antibody or monoclonal antibody production method.

[0193] For example, transgenic mice producing human antibodies can be prepared according to the methods described in Nature Genetics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; Japanese Patent Publication No. 4-504365; Japanese Patent Publication No. 7-509137; International Publication No. WO94 / 25585; Nature, Vol.368, p.856-859, 1994; and Japanese Patent Publication No. 6-500233. More specifically, HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice, etc. can be cited.

[0194] Specifically, the anti-RGMa neutralizing antibodies of the present invention include those having CDRs containing specific amino acid sequences in the heavy chain variable region and CDRs containing specific amino acid sequences in the light chain variable region (preferably the antibodies (a) to (l) above).

[0195] It should be noted that as long as the antibody of the present invention has the ability to bind to RGMa and can maintain the activity of inhibiting (neutralizing) RGMa, one or more amino acids (1 to 20, 1 to 10 or 1 to 5, preferably 1 to 2) may be substituted, deleted, added or inserted in the amino acid sequence of the anti-RGMa neutralizing antibody (preferably the anti-RGMa neutralizing antibody of (a) to (l) above). Such substitutions, deletions, and additions can be introduced into CDRs, but are preferably introduced into regions outside CDRs. In addition, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution.

[0196] The amino acid sequence of the anti-RGMa neutralizing antibody of the present invention (preferably the anti-RGMa neutralizing antibody of (a) to (l) above) containing substitutions, deletions, etc. in the amino acid sequence is, for example, the following amino acid sequence: the heavy chain variable region after the amino acid sequence is changed has a % homology of more than 90% (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before the change, and the light chain variable region after the amino acid sequence is changed has a % homology of more than 90% (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before the change.

[0197] In the present invention, siRNA refers to a short double-stranded RNA that can inhibit the expression of a target gene (RGMa gene in the present invention). As long as it functions as an siRNA that inhibits the activity of RGMa of the present invention, the base sequence and length (base length) are not particularly limited, preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases.

[0198] In the present invention, shRNA refers to a molecule of about 20 base pairs or more, which forms a double-stranded structure in the molecule by partially including a scissor-shaped base sequence in a single-stranded RNA, and is composed of a short hairpin structure (hairbin) with a protrusion at the 3' end. After such shRNA is introduced into cells, it is decomposed into a length of about 20 bases (representatively, for example, 21 bases, 22 bases, and 23 bases) in the cell, and can inhibit the expression of the target gene in the same way as siRNA.

[0199] In the present invention, the siRNA and shRNA may be in any form as long as they can inhibit the expression of the RGMa gene.

[0200] In the present invention, siRNA or shRNA can be artificially chemically synthesized. In addition, for example, T7 RNA polymerase and T7 promoter can be used to synthesize antisense and sense RNA in vitro from template DNA. Antisense oligonucleotides can be either DNA or RNA as long as they are nucleotides that are complementary or hybridized to a continuous sequence of 5 to 100 bases in the DNA sequence of the RGMa gene. In addition, modifications can be made as long as they do not affect the function. Antisense oligonucleotides can be synthesized according to conventional methods, for example, they can be easily synthesized using a commercially available DNA synthesis device.

[0201] Preferred sequences can be selected using a common selection method, and can be confirmed as siRNA or shRNA in the present invention by evaluating the inhibition of expression of functional RGMa.

[0202] <Acute neuromyelitis optica>

[0203] The stages of neuromyelitis optica can be clinically roughly divided into two types: an "acute stage" (a concept that includes an acute exacerbation stage in the present invention) and a "chronic stage".

[0204] Here, the "acute phase" refers to the period when symptoms of neuromyelitis optica, such as optic neuritis and myelitis, appear and persist or worsen (worsen). In addition, during this period, the gadolinium contrast effect can be seen in some lesions by MRI, and the increase in cell count or protein can be seen by cerebrospinal fluid examination, so these can also be used as references to determine the acute phase.

[0205] On the other hand, the "chronic stage" refers to a period when the symptoms are improved and stabilized by treatment. In addition, the gadolinium contrast effect disappears in MRI during this period, so this can also be used as a reference to determine the chronic stage.

[0206] The acute phase of neuromyelitis optica in the present invention refers to the above-mentioned "acute phase", and includes not only the symptoms of the acute phase of the first attack of neuromyelitis optica but also the symptoms of the acute phase of the second or subsequent recurrence of neuromyelitis optica.

[0207] In addition, regarding neuromyelitis optica, for the treatment subject (preferably mammals, especially humans), the duration until the peak of the symptoms is about 8.5 days (range: 2 to 63 days) (references: Flanagan et al. Ann Neurol. 2016 Mar; 79 (3): 437-47, etc.). Therefore, the acute phase in the present invention is usually within 1 month from the onset of neuromyelitis optica.

[0208] Neuromyelitis optica in the present invention refers to neuromyelitis optica spectrum disorder (NMOSD), which is a concept including any one of anti-AQP4 antibody-positive neuromyelitis optica spectrum disorder (NMOSD) and anti-AQP4 antibody-negative neuromyelitis optica spectrum disorder (NMOSD) listed in the International Diagnostic Criteria for Neuromyelitis Optic (Wingerchuk et al. Neurology, 2015; 8582: 177-189). Among them, in the present invention, anti-AQP4 antibody-positive neuromyelitis optica spectrum disorder is preferred.

[0209] The treatment subjects (preferably mammals, especially humans) in the present invention are patients suffering from neuromyelitis optica-related diseases (NMOSD), preferably patients suffering from neuromyelitis optica-related diseases (NMOSD) positive for anti-AQP4 antibodies, and the preventive or therapeutic agent for acute neuromyelitis optica of the present invention can be administered to these patients.

[0210] In addition, for neuromyelitis optica in the acute stage of the present invention, pain is often one of the main symptoms of the patient. Therefore, in the present invention, RGMa inhibitory substances, preferably anti-RGMa neutralizing antibodies, can be used as a preventive or therapeutic agent for the pain symptoms seen in neuromyelitis optica for patients with such pain.

[0211] It should be noted that all matters described in the preventive or therapeutic agent and preventive or therapeutic method for acute neuromyelitis optica of the present invention are applicable to the description of the preventive or therapeutic agent and preventive or therapeutic method for pain symptoms seen in neuromyelitis optica of the present invention.

[0212] Here, "treatment" includes any treatment of a disease of a treatment 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.

[0213] In addition, "prevention" includes preventing or inhibiting the occurrence of the above-mentioned diseases in the treatment object, preferably mammals, especially humans. Furthermore, "prevention" in the present invention includes "preventing recurrence" which is to alleviate or prevent the recurrence of the above-mentioned diseases that have repeatedly recurred in the treatment object, preferably mammals, especially humans.

[0214] <Drug Composition>

[0215] The preventive or therapeutic agent for acute neuromyelitis optica in the present invention is usually administered systemically or topically in an oral or parenteral form.

[0216] The preventive or therapeutic agent for acute neuromyelitis optica in the present invention uses RGMa inhibitory substances as active ingredients, and can be formulated by appropriately combining pharmaceutically acceptable carriers or additives. The pharmaceutical composition formulated in this way can be administered orally or non-orally. Specifically, it can be made into oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., and can also be made into non-oral preparations such as injections, infusions, suppositories, ointments, patches, etc. The proportion of the carrier or additive can be appropriately set based on the range commonly used in the field of pharmaceuticals. There is no particular limitation on the carrier or additive that can be formulated, and examples thereof include various carriers such as water, saline, other aqueous solvents, aqueous or oily bases, and various additives such as excipients, adhesives, pH adjusters, disintegrants, absorption promoters, lubricants, colorants, flavoring agents, and spices.

[0217] When the RGMa inhibitory substance is an anti-RGMa neutralizing antibody, its function-modified antibody, its conjugated antibody or their antigen-binding fragments, it is preferably administered as an injection or infusion formulated together with a pharmaceutically acceptable carrier by a non-oral administration route, such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or local administration.

[0218] For example, an injection or infusion containing an anti-RGMa neutralizing antibody can be used in the form of a solution, suspension or emulsion. As its solvent, for example, distilled water for injection, physiological saline, glucose solution and isotonic solution (such as a solution of sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used.

[0219] Furthermore, the injection or infusion containing such an anti-RGMa neutralizing antibody may also contain a stabilizer, a solubilizing agent, a suspending agent, an emulsifier, a soothing agent, a buffer, a preservative, a preservative, a pH adjuster, and the like.

[0220] As the stabilizer, for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium hydrogen sulfite, sodium thiosulfate, sodium EDTA, sodium citrate, butylated hydroxytoluene and the like can be used.

[0221] As the auxiliary solvent, for example, alcohol (eg, ethanol), polyol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysolvate 80 (registered trademark), HCO-50, etc.) and the like can be used.

[0222] As the suspending agent, for example, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate and the like can be used.

[0223] As the emulsifier, for example, gum arabic, sodium alginate, gum tragacanth, etc. can be used.

[0224] As the soothing agent, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used.

[0225] As the buffer, for example, a phosphate buffer, an acetate buffer, a borate buffer, a carbonate buffer, a citrate buffer, a Tris buffer or the like can be used.

[0226] As the preservative, for example, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax and the like can be used.

[0227] As the preservative, for example, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol, etc. can be used.

[0228] As the pH adjuster, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. can be used.

[0229] When the RGMa inhibitory substance is a nucleic acid (siRNA, shRNA, antisense oligonucleotide, etc.), it can be administered in the form of a non-viral vector or a viral vector. In the case of a non-viral vector, a method of using liposomes to introduce nucleic acid molecules (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine (Lipofectamine) method, etc.), microinjection method, a method of using a gene gun (Gene Gun) to move nucleic acid molecules and carriers (metal particles) into cells, etc. For example, when siRNA or shRNA is administered to an organism using a viral vector, a recombinant adenovirus, retrovirus, and other viral vectors can be used. By introducing DNA expressing siRNA or shRNA into DNA viruses or RNA viruses such as detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, cowpox viruses, poxviruses, polio viruses, Sindbis viruses, Sendai viruses, SV40, etc., cells or tissues are infected with the recombinant virus, so that genes can be introduced into cells or tissues.

[0230] The preparation thus obtained can prevent or treat neuromyelitis optica in the acute phase by administering an effective amount thereof to, for example, humans or other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.). The dosage can be appropriately set in consideration of the purpose, severity of the disease, age, weight, sex, medical history, type of active ingredient, etc. of the patient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, when an average human with a body weight of about 65 to 70 kg is taken as the subject, it is preferably about 0.02 mg to 4000 mg per day, and more preferably about 0.1 mg to 200 mg. The total dosage per day can be a single dosage or a batch dosage.

[0231] <Combination use with other drugs or treatments>

[0232] In the present invention, the preventive or therapeutic agent for neuromyelitis optica in the acute stage can be administered in combination with other drugs or treatments useful for treating neuromyelitis optica.

[0233] As other drugs or treatments that can be used in combination with the preventive or therapeutic agent for acute neuromyelitis optica in the present invention, for example, plasma exchange and / or intravenous administration of immunoglobulin preparations, administration of mycophenolate mofetil, rituximab, eculizumab and / or satelizumab can be mentioned. Such other drugs or treatments can be other drugs or treatments with biological activity that are effective for treating central nervous system damage such as neuromyelitis optica or delaying the deterioration of central nervous system damage.

[0234] For example, other medicaments with biological activity can be corticosteroids, (intravenous) immunoglobulin preparations or antilymphocyte preparations, mycophenolate mofetil, rituximab, eculizumab and / or satelizumab. In a preferred embodiment, the patient is treated by intravenous immunotherapy (for example corticosteroids, for example (synthetic) glucocorticoids such as methylprednisolone). Therefore, other medicaments with biological activity can also be corticosteroids, for example (synthetic) glucocorticoids such as methylprednisolone. Other medicaments with biological activity are administered intravenously. For other medicaments or treatment, for example, in the case of patients who are unresponsive to steroids (for example, only being able to confirm that central nervous system inflammation is not fully suppressed after steroid treatment), plasma exchange can also be performed. Therefore, the patient can be a patient for whom steroid infusion is ineffective, and plasma exchange can also be arbitrarily accepted.

[0235] The above-mentioned other drugs or treatments may be administered or carried out before or after the administration of the preventive or therapeutic agent for neuromyelitis optica in the acute stage of the present invention, or may be administered or carried out simultaneously.

[0236] Example

[0237] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0238] [Example 1] Effect of anti-RGMa neutralizing antibody on acute neuromyelitis optica

[0239] The therapeutic effect of anti-RGMa neutralizing antibody on clinical symptom deterioration was evaluated using an acute severe NMO rat model. It should be noted that as anti-RGMa neutralizing antibody, an anti-RGMa neutralizing antibody comprising the amino acid sequence (a) described in this specification (SEQ ID NOs. 5 to 10) was used in the experiment.

[0240] (1-1) Steps of NMO induction in rats

[0241] According to the existing report (Kurosawa K, et al., Acta Neuropathol Commun. 2015; 3: 82), an acute phase severe NMO rat model was prepared. Female Lewis rats were used in the experiment. As a stimulus for inducing and destroying the blood-spinal cord barrier (BSCB) in the pro-inflammatory environment, immune introduction of the central antigen MBP was carried out. As MBP, Guinea pigbrain myelin basic protein was used, which was dissolved in PBS to 1 mg / mL, and mixed with complete Freund's adjuvant containing 1 mg / mL of dead tuberculosis H37Ra in equal amounts, and an emulsion was prepared by ultrasonic treatment. The prepared emulsion (200 μl / head) was subcutaneously administered to two places on the back. After 10 days, an anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) was intraperitoneally administered once at a dose of 3 mg / kg to individuals with a neurological score of less than 1, inducing an exacerbation of clinical symptoms dependent on the anti-AQP4 antibody.

[0242] (1-2) Neurological score evaluation

[0243] Neurological symptoms were scored based on the following criteria, and the sum of the neurological scores of the tail and both hind limbs was used as the neurological score (0 to 6 points) for evaluation. Tail score: 0: no paralysis symptoms; 1: incomplete paralysis; 2: complete paralysis; scores for each hind limb: 0: no symptoms; 1: incomplete paralysis; 2: complete paralysis of the hind limbs with hind limb dragging. Neurological scoring was performed under blind testing, and neurological symptoms were evaluated once a day until the 13th day after the administration of anti-RGMa neutralizing antibody (the 14th day after the administration of anti-AQP4 antibody).

[0244] (1-3) Grouping and administration of anti-RGMa neutralizing antibody

[0245] The mice were divided into two groups so that the deviation of the mean values ​​of the neurological score and body weight on the day after the anti-AQP4 antibody administration was minimized between the groups, and anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously once at a dose of 10 mg / kg. The anti-RGMa neutralizing antibody administration group and the isotype control antibody administration group each consisted of 6 mice.

[0246] (1-4) Results

[0247] The effect of a single administration of anti-RGMa neutralizing antibody on the clinical acute deterioration of the acute phase severe NMO rat model is shown in Figure 1 Anti-RGMa neutralizing antibody or isotype control antibody was intravenously administered on the day after anti-AQP4 antibody administration, and neurological symptoms were evaluated once a day until the 14th day after anti-AQP4 antibody administration. The neurological scores of the group that received anti-RGMa neutralizing antibody on the day after anti-AQP4 antibody administration showed lower values ​​than those of the group that received isotype control antibody throughout the observation period, and the average scores on days 2-8 and 2-14 after anti-AQP4 antibody administration were significantly reduced (average 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); average 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).

[0248] The above results show that RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, show therapeutic effects on clinical exacerbation of neuromyelitis optica in the acute phase.

[0249] [Example 2] Effect of anti-RGMa neutralizing antibody on the destruction of the blood-spinal cord barrier

[0250] Using tEAE mice with BSCB destruction limited to the spinal cord segment, the therapeutic effect of anti-RGMa neutralizing antibodies on BSCB destruction was studied by gadolinium contrast MRI. It should be noted that as anti-RGMa neutralizing antibodies, anti-RGMa neutralizing antibodies containing the amino acid sequence (a) described in this specification (SEQ ID NOs. 5 to 10) were used in the experiment.

[0251] (2-1) Preparation of tEAE mice

[0252] 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, mixed with complete Freund's adjuvant containing 5 mg / mL of dead tuberculosis H37Ra in equal amounts, and an emulsion was prepared by ultrasonic treatment. The prepared emulsion was subcutaneously administered to 2 places on the back (200 μL / head) at 100 μL each for MOG immune introduction. After about 21 days, 1.5 μL of a cytokine mixed solution (750 ng Tumor Necrosis Factor-α, 1 μg Interferon-γ) was injected into the thoracic cord at a depth of 0.5-0.8 mm under the 8th thoracic vertebra, and 200 ng pertussis toxin was administered into the tail vein just after the injection and 2 days later to induce tEAE.

[0253] (2-2) Neurological score evaluation

[0254] The neurological score was evaluated in a blind test based on the previously reported judgment criteria (see Tanabe S, Fujita Y, Ikuma K, Yamashita T. Inhibiting repulsive guidance molecule-a suppresses secondary progression in mouse models of multiple sclerosis. Cell Death Dis. 2018; 9(11): 1061).

[0255] (2-3) Evaluation of BSCB damage using gadolinium contrast-enhanced magnetic resonance imaging (MRI)

[0256] BioSpec 117 / 11 (Bruker) was used to obtain T1-enhanced images (Dynamic contrast-enhanced MRI; DCE-MRI) before and after the administration of gadolinium contrast agent (Omniscan, Daiichi Sankyo), and the leakage of gadolinium into the spinal cord, which is an indicator of BSCB destruction, was quantitatively evaluated based on the change in T1 signal intensity. Under sevoflurane anesthesia, a body temperature maintenance device was installed, and gadolinium contrast agent was rapidly administered at a dose of 0.25 mmol / kg via a catheter placed in the tail vein. DCE-MRI set an acquisition matrix of 200×200 for an effective field of view (FoV) of 26×26 mm, and acquired a total of 11 axial section images with a slice thickness and interval of 0.8 mm, centered on the site of thoracic spinal cord cytokine injection. The repetition time (TR) was set to 500 ms, the echo time (TE) was set to 18 ms, the number of excitations (NEX) was set to 4, and a total of 6 images (each image was about 100 seconds) were acquired over a period of about 10 minutes.

[0257] The images were exported in DICOM format and analyzed using Fiji (http: / / fiji.sc / ). In order to eliminate the influence of the T1 signal of the cerebrospinal fluid, a region of interest (ROI) was set in the spinal cord, and the T1 signal enhancement ratio (SER) in the spinal cord at each time point was calculated based on formula (1). The gadolinium spinal cord leakage intensity (Total Gd influx rate) of each individual was calculated as the slope of SER per unit time as the gadolinium influx rate using the SLOPE function of Microsoft Excel 2016 (Microsoft) as the sum of 11 slices as shown in formula (2).

[0258]

[0259] According to the research results of normal mice, when the value of SLOPE (SER0): SER (10), 0: 10) in formula (2) is less than 0.02, it is excluded from the addition object of formula (2) as an analysis error.

[0260] (2-4) Grouping and administration of anti-RGMa neutralizing antibodies

[0261] The mice were divided into two groups so that the quantitative value of gadolinium leakage 7 days after cytokine injection was uniform between the groups, and anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously into the tail vein at a dose of 10 mg / kg twice a week. The anti-RGMa neutralizing antibody administration group consisted of 10 mice, and the isotype control antibody administration group consisted of 9 mice. Neurological score evaluation and MRI analysis were performed on the same individual.

[0262] (2-5) Results

[0263] The effects of anti-RGMa neutralizing antibodies on BSCB destruction and recovery of neurological symptoms in acute tEAE mice are shown in Figure 2 . Based on the quantitative value after 7 days, the change rate of gadolinium leakage intensity 7, 14, and 21 days after cytokine injection was calculated longitudinally in the same individual to analyze the recovery effect of anti-RGMa neutralizing antibodies on BSCB damage. The gadolinium spinal cord leakage generated at the time point 7 days after cytokine injection was significantly inhibited by repeated administration of anti-RGMa neutralizing antibodies (14 days after cytokine injection, p < 0.001, Bonferroni multiple comparison test), confirming the early recovery of BSCB damage ( Figure 2 A). In addition, the neurological scores obtained in the same individual were also significantly suppressed, and early recovery of neurological symptoms was confirmed ( Figure 2 B).

[0264] The correlation between the intensity of gadolinium leakage into the spinal cord and the severity of neurological symptoms in acute stage tEAE mice is shown in Figure 3 A high positive correlation was found between the intensity of gadolinium leakage into the spinal cord and the severity of neurological symptoms 7 days after cytokine injection (r=0.831, p<0.001), and a positive correlation was also found 14 days later (r=0.549, p<0.05). This indicates that the severity of BSCB damage determines the severity of neurological symptoms.

[0265] The above results show that RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, show efficacy against neuromyelitis optica in the acute phase by promoting the repair of BSCB damage.

[0266] [Example 3] Therapeutic effect of anti-RGMa neutralizing antibody on blood-spinal cord barrier damage

[0267] Immunohistochemical staining was used to analyze the leakage of rat IgG into the spinal cord caused by blood-spinal cord barrier disruption, and the therapeutic effect of anti-RGMa neutralizing antibody on blood-spinal cord barrier disruption in acute severe NMO rats was evaluated.

[0268] (3-1) Preparation of acute severe NMO rat model

[0269] Female Lewis rats were used for the experiment. On the 10th day after MBP immunization, rats with a neurological score of less than 1 were intraperitoneally administered with a single dose of 3 mg / kg of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) to induce NMO symptoms.

[0270] (3-2) Grouping and administration of anti-RGMa neutralizing antibody

[0271] The mice were divided into two groups in such a way that the deviation of the mean value of the neurological score and body weight on the day after the administration of the anti-AQP4 antibody was minimized between the groups, and the anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously into the tail vein at a dose of 10 mg / kg. The anti-RGMa neutralizing antibody treatment group and the isotype control antibody treatment group consisted of 6 mice each, and the healthy non-treatment group consisted of 4 mice.

[0272] (3-3) Immunohistochemical staining

[0273] Dissection was performed on the 4th day after the administration of anti-AQP4 antibody. The spinal cord was collected after blood removal and post-fixed in 4% paraformaldehyde at 4°C for 1 day. After post-fixation, cryoprotection treatment based on sucrose substitution was performed, and then the tissue slices were embedded in OCT compound. Frozen sections were made by thin slices of 30 μm, and immunohistochemical staining was performed using Alxa488-labeled donkey anti-rat IgG antibody (1:500, Thermo Fisher Scientific). The stained sections were photographed using an inverted fluorescence microscope Olympus IX83, and the ratio of rat IgG-positive area to the spinal cord section (% rat IgG-positive area) was determined using image analysis software Image J software.

[0274] (3-4) Results

[0275] The effect of anti-RGMa neutralizing antibody on spinal cord leakage of rat IgG is shown in Figure 4 The administration of anti-RGMa neutralizing antibody on the day after the onset of NMO significantly suppressed the leakage of rat IgG into the spinal cord, which is an indicator of blood-spinal cord barrier disruption.

[0276] The above results indicate that the effect of RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies, on acute neuromyelitis optica is to promote the repair of the damaged blood-spinal cord barrier.

[0277] [Example 4] Effect of anti-RGMa neutralizing antibody on pain symptoms of NMO

[0278] The anti-RGMa neutralizing antibody was repeatedly administered to rats with acute severe NMO to evaluate its effect on persistent pain.

[0279] (4-1) Preparation of acute severe NMO rat model

[0280] Female Lewis rats were used for the experiment. On the 10th day after MBP immunization, rats with a neurological score of less than 1 were intraperitoneally administered with a single dose of 3 mg / kg of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) to induce NMO symptoms.

[0281] (4-2) Evaluation of pain-related behaviors

[0282] The pain was evaluated by determining the 50% escape threshold (g) of the hind limbs when the hind limbs were lifted using the up-down method based on von Frey stimulation (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)). The average value of the 50% escape threshold (g) of the hind limbs on both sides was used for analysis.

[0283] (4-3) Grouping and administration of anti-RGMa neutralizing antibody

[0284] The subjects were divided into two groups in such a way that the deviation of the mean value of the neurological score and body weight on the day after the administration of anti-AQP4 antibody was minimized between the groups. Anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously to the tail vein once a week at a dosage of 10 mg / kg. The analysis group consisted of three groups, namely, the anti-RGMa neutralizing antibody treatment group, the isotype control antibody treatment group, and the healthy non-treatment group, each consisting of 6 animals. During the period of exacerbation of neurological symptoms, individuals who could not perform von Frey stimulation due to weakness of the hind limbs were confirmed, so the data set consisted of 5 cases in the anti-RGMa neutralizing antibody treatment group on the 4th day after the administration of anti-AQP4 antibody, 2 cases in the isotype control antibody treatment group on the 7th day after the administration of anti-AQP4 antibody, and 3 cases in the anti-RGMa neutralizing antibody treatment group.

[0285] (4-4) Results

[0286] The inhibitory effect of anti-RGMa neutralizing antibody on pain is shown in Figure 5 The group that received the anti-RGMa neutralizing antibody from the day after NMO onset recovered faster from the decrease in the 50% escape threshold than the group that received the isotype control antibody, and a significant increase in the 50% escape threshold was observed on the 18th and 21st days after the anti-AQP4 antibody administration.

[0287] The above results show that RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, show effects on the pain symptoms of neuromyelitis optica in the acute stage.

[0288] [Example 5] Inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord

[0289] Immunohistochemical staining was used to analyze the inhibitory effect of anti-RGMa neutralizing antibody on granulocyte infiltration in the spinal cord of rats with acute severe NMO.

[0290] (5-1) Preparation of acute severe NMO rat model

[0291] Female Lewis rats were used for the experiment. On the 10th day after MBP immunization, rats with a neurological score of less than 1 were intraperitoneally administered with a single dose of 3 mg / kg of anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) to induce NMO symptoms.

[0292] (5-2) Grouping and administration of anti-RGMa neutralizing antibody

[0293] The mice were divided into two groups in such a way that the deviation of the mean value of the neurological score and body weight on the day after the administration of the anti-AQP4 antibody was minimized between the groups, and the anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab) was administered intravenously into the tail vein at a dose of 10 mg / kg. The anti-RGMa neutralizing antibody treatment group and the isotype control antibody treatment group consisted of 6 mice each, and the healthy non-treatment group consisted of 4 mice.

[0294] (5-3) Immunohistochemical staining

[0295] Dissection was performed on the 4th day after the administration of anti-AQP4 antibody. The spinal cord was collected after blood removal and post-fixed in 4% paraformaldehyde at 4°C for 1 day. After post-fixation, cryoprotection treatment based on sucrose substitution was performed, and then the tissue slices were embedded in OCT compound. Frozen sections were prepared by thin slices of 30 μm, and immunohistochemical staining was performed using rabbit anti-rat granulocyte serum (1:5000, LifeSpan BioSciences) as the primary antibody and Alxa488-labeled donkey anti-rabbit IgG antibody (1:500, Thermo Fisher Scientific) as the secondary antibody. The stained sections were photographed using an inverted fluorescence microscope Olympus IX83, and the ratio of the granulocyte-positive area to the spinal cord section (% granulocyte-positive area) was measured using the image analysis software Image J software.

[0296] (5-4) Results

[0297] 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 By administering anti-RGMa neutralizing antibody on the day after NMO onset, granulocyte infiltration in the spinal cord of NMO rats was significantly inhibited.

[0298] The above results show that RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies, inhibit the infiltration of granulocytes that can be seen in the symptoms of acute neuromyelitis optica. It is believed that the inhibitory effect on the infiltration of granulocytes contributes to the expression of the effect on acute neuromyelitis optica, and therefore, it is shown that RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies, show the effect on acute neuromyelitis optica through the inhibitory effect on the infiltration of granulocytes.

[0299] [Example 6] RGMa expression in the AQP4 loss site of the spinal cord

[0300] Immunohistochemical staining of the spinal cord of NMO rats was used to detect the expression of RGMa in the acute severe NMO rat model.

[0301] (6-1) Preparation of acute severe NMO rat model

[0302] Female Lewis rats were used in the experiment. Guinea pig brainmyelin basic protein was dissolved in PBS to 1 mg / mL, and mixed with complete Freund's adjuvant containing 1 mg / mL of killed tuberculosis H37Ra in equal amounts, and then an emulsion was prepared by ultrasonic treatment. MBP emulsion (200 μl / head) was administered subcutaneously (MBP immunization), and 10 days later, anti-AQP4 antibody (mouse anti-AQP4 monoclonal antibody E5415A) was intraperitoneally administered once at a dose of 3 mg / kg to individuals with a neurological score of less than 1 to induce NMO symptoms.

[0303] (6-2) Immunohistochemical staining

[0304] Dissection was performed on the first day after administration of the anti-AQP4 antibody. The spinal cord was collected after blood removal and post-fixed in 4% paraformaldehyde at 4°C for 1 day. After post-fixation, cryoprotection treatment based on sucrose substitution was performed, and then the tissue slices were embedded in OCT compound. Frozen sections were made by thinly cutting at 30 μm, and double immunohistostaining was performed 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 (1:500, Thermo Fisher Scientific) and Alxa647-labeled donkey anti-rabbit IgG antibody (1:500, ThermoFisher Scientific) as secondary antibodies. The stained sections were photographed using an inverted fluorescence microscope Olympus IX83.

[0305] (6-3) Results

[0306] The immunohistochemical staining images of spinal cord sections of acute severe NMO rats are shown in Figure 7 Strong expression of RGMa was confirmed at the sites of AQP4 shedding in NMO lesions.

[0307] <Explanation of Sequence Listing>

[0308] SEQ ID NO: 1: Amino acid sequence of human RGMa precursor protein

[0309] SEQ ID NO. 2: Amino acid sequence of mouse RGMa precursor protein

[0310] SEQ ID NO. 3: Amino acid sequence of rat RGMa precursor protein

[0311] SEQ ID NO. 4: DNA sequence of human RGMa gene

[0312] SEQ ID NO: 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3

[0313] SEQ ID NO: 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3

[0314] SEQ ID NO: 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3

[0315] 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

[0316] SEQ ID NO: 11: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r70E

[0317] SEQ ID NO: 12: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r70E

[0318] 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 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 1: 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 epitope of human RGMa

[0319] SEQ ID NO: 37: Amino acid sequence of human RGMa epitope

[0320] SEQ ID NO: 38: Amino acid sequence of human RGMa epitope

[0321] SEQ ID NO: 39: Amino acid sequence of human RGMa epitope 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

[0322] SEQ ID NO: 45: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1K

[0323] SEQ ID NO: 46: Amino acid sequence of rat and mouse MOG (35-55)

[0324] Industrial Applicability

[0325] In the present invention, the RGMa inhibitory substance is useful for the prevention or treatment of neuromyelitis optica in the acute stage, and is also useful for the prevention or treatment of pain symptoms of neuromyelitis optica, and therefore has high utilization value in the pharmaceutical industry.

Claims

1. Use of an RGMa inhibitory substance in the manufacture of a preventive or therapeutic agent for acute neuromyelitis optica, wherein the RGMa inhibitory substance is an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof, The heavy chain variable region VH comprises: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKG LEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS is the amino acid sequence of sequence number 47, and the light chain variable region VL comprises: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPK LLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLP WTFGGGTKVEME is the amino acid sequence of sequence number 48.

2. The use according to claim 1, wherein In the anti-RGMa neutralizing antibody, the heavy chain contains: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, i.e., the amino acid sequence of sequence number 49, The light chain contains: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEMERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC is the amino acid sequence of sequence number 50.

3. The use according to claim 1 or 2, wherein The anti-RGMa neutralizing antibody is a humanized antibody.

4. The use according to claim 1 or 2, 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, and SEQ ID NO:

39.

5. Use of an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof in the manufacture of a preventive or therapeutic agent for the pain symptoms of neuromyelitis optica, The heavy chain variable region VH comprises: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKG LEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS is the amino acid sequence of sequence number 47, and the light chain variable region VL comprises: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPK LLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLP WTFGGGTKVEME is the amino acid sequence of sequence number 48.

6. The use according to claim 5, wherein In the anti-RGMa neutralizing antibody, the heavy chain contains: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, i.e., the amino acid sequence of sequence number 49, The light chain contains: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEMERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC is the amino acid sequence of sequence number 50.

7. The use according to claim 5 or 6, wherein The anti-RGMa neutralizing antibody is a humanized antibody.

8. The use according to claim 5 or 6, 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, and SEQ ID NO: 39.

Images