Preventive or therapeutic agent for associated symptoms accompanying disease induced by spinal cord or brain disorder

Abstract

Provided is a preventive or therapeutic agent for associated symptoms accompanying a disease induced by a spinal cord or brain disorder, such as spinal cord injury and HTLV-1-associated myelopathy, the preventive or therapeutic agent containing an RGMa inhibition substance. Also provided is a preventive or therapeutic agent for spasm or neurogenic bladder dysfunction, the preventive or therapeutic agent containing an RGMa inhibition substance.

Description

Preventive or therapeutic agent for symptoms associated with diseases caused by spinal cord or brain disorders

[0001] The present invention relates to a preventive or therapeutic agent for symptoms accompanying diseases caused by spinal cord or brain damage, which agent comprises an RGMa inhibitor.

[0002] It is known that after a certain period of time following central nervous system injury, increased spinal reflexes cause excessive muscle contraction, resulting in the development of spasticity. Spasticity can also be caused by various central nervous system disorders, including head trauma and spinal cord injury, as well as multiple sclerosis, spinocerebellar degeneration, HTLV-I-associated myelopathy (HAM), myelopathy, stroke, brain tumor, cerebral palsy, and hypoxic encephalopathy. It has been reported that there are over 80,000 cases of severe spasticity in Japan due to sequelae of head trauma, spinal cord injury, cerebral palsy, and multiple sclerosis (Non-Patent Document 1), but the exact prevalence of spasticity in Japan is unknown. Overseas reports indicate that approximately 70% of patients with spinal cord injury develop spasticity (Non-Patent Documents 2 and 3), affecting rehabilitation and quality of life. Furthermore, spasticity has been reported to occur in 30–90% of patients with multiple sclerosis (Non-Patent Document 4).

[0003] Lesions of the central nervous system (brain or spinal cord) can cause upper motor neuron syndrome (UMS) as positive or negative signs. Positive signs include increased muscle tone, increased tendon reflexes, extension of stretch reflexes to other muscles, signs of spasticity such as clonus, spastic dystonia, pathological synkinesis and co-contraction, and increased flexor reflexes. Negative signs include paralysis and muscle weakness, leading to reduced dexterity and increased effort. While all positive signs of UMS are often referred to as spasticity, Lance's definition (Non-Patent Document 5) is more narrow, stating that spasticity is a positive sign of UMS and a movement disorder characterized by a velocity-dependent increase in tonic stretch reflexes (muscle tone) accompanied by increased tendon reflexes. Spasticity symptoms are diverse and complex, depending on the underlying disease and the site of the injury. Spastic paralysis is a notable complication, but the fact that there are many cases in which the degree of spasticity and paralysis do not parallel suggests that there are differences in the neural mechanisms underlying the manifestation of these symptoms (Non-Patent Document 6). For example, it has been reported that the severity of the injury does not correspond to the degree of spasticity in patients with spinal cord injury and in animal models (Non-Patent Documents 7 and 8). It has also been reported that it takes a certain amount of time for spasticity to develop after the cause has arisen, and that this may be due to the emergence of some kind of neural plasticity or reorganization (Non-Patent Document 9). The detailed pathophysiology of spasticity has yet to be elucidated.

[0004] Physical therapy is the basis of spasticity treatment, and for severe spasticity, muscle relaxants, block therapy, and intrathecal baclofen therapy are considered (Non-Patent Document 10). Specifically, for spastic muscles, physical therapy using manual or mechanical exercises such as orthodontic tables, cold, heat, low-frequency electrical stimulation, and vibration stimulation, as well as orthotic therapy, are used (Non-Patent Document 11). For severe symptoms, drug therapy with muscle relaxants may be used. Drugs such as tizanidine, eperisone, baclofen, diazepam, gabapentin, and dantrolene sodium are used, but it is important to note that these drugs have side effects such as drowsiness and muscle weakness (Non-Patent Documents 12 and 13). When drug therapy is ineffective, block therapy using botulinum injections has recently been used to treat hypertonic muscles (Non-Patent Documents 14 and 15). However, injections must be spaced at least three months apart, and adjusting the injection dose to achieve sustained effects is difficult. For severe spasticity, intrathecal baclofen therapy (ITB) is also considered (Non-Patent Document 16). A continuous infusion pump that allows continuous administration is administered intrathecally on a trial basis, and only if the pump is effective is the pump implanted. As such, existing treatment methods are not necessarily satisfactory as treatments for spasticity, and there is a strong need for better treatments for spasticity to improve quality of life (QOL) by reducing the burden on patients and caregivers.

[0005] Neurogenic bladder, which causes dysuria and other symptoms, is a bladder dysfunction caused by neurological disorders, including spinal cord and other nerve damage, and is characterized by lower urinary tract symptoms. Lower urinary tract functions are divided into bladder storage and voiding, each of which is classified as bladder function and urethral function, and are regulated by coordinated action. During voiding, intravesical pressure is maintained low, the urethra is constantly closed, and urethral closure pressure is maintained higher than intravesical pressure. Meanwhile, during voiding, urination can be initiated voluntarily, and the urethra dilates with bladder contraction, resulting in the expulsion of urine. Dysuria is caused by decreased contractility of the bladder's detrusor smooth muscle and increased urethral resistance, resulting in symptoms such as decreased urine volume, splitting of the urinary stream, intermittent urinary stream, hesitancy, straining, and terminal dribbling. In Japan, medications approved for the treatment of neurogenic bladder include anticholinergics, cholinergic agonists, and α1-adrenergic receptor antagonists. However, anticholinergic drugs require higher-than-normal doses to improve neurogenic bladder, and side effects are problematic. Alpha-1 adrenergic receptor antagonists and cholinergic agonists lack evidence, with no clear efficacy demonstrated in randomized trials, raising concerns about serious adverse events such as cholinergic crisis. While the efficacy of beta-3 adrenergic receptor agonists has been reported, the evidence from randomized trials is lacking, and they are currently not indicated for use (Non-Patent Documents 17 and 18). Given these factors, there is a need for more effective therapeutic agents for neurogenic bladder and its symptoms, which are caused by increased urethral resistance and decreased bladder smooth muscle contractility.

[0006] RGM (repulsive guidance molecule) is a membrane protein originally identified as an axon guidance molecule in the visual system (Non-Patent Document 19). The RGM family includes three members, termed RGMa, RGMb, and RGMc (Non-Patent Document 20). At least RGMa and RGMb are known to function via the same signaling mechanism (Non-Patent Document 21). RGMc plays an important role in iron metabolism. Subsequent research has revealed that RGM functions include axon guidance and lamina formation in Xenopus and chicken embryos, as well as regulating cranial neural tube closure in mouse embryos (Non-Patent Document 22). Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0007] In addition to its function during development, RGMa is re-expressed after central nervous system injury in adult humans and rats, and RGMa inhibition in rats enhances axonal growth and promotes functional recovery after spinal cord injury (Non-Patent Document 23). Therefore, RGMa is believed to be an inhibitor of axon regeneration after central nervous system injury. Specific antibodies that neutralize RGMa are described, for example, in Patent Document 2 (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3). While the role of RGMa in central nervous system injury has been elucidated, the involvement of RGMa in spasticity and neurogenic bladder, in particular, has not been identified, and no therapeutic agents for such treatment are known.

[0008] International Publication No. WO2005 / 087268 International Publication No. WO2009 / 106356 International Publication No. WO2013 / 112922 International Publication No. WO2016 / 175236

[0009] Taira T, Akagawa H, Okada Y et al. Epidemiological survey of spasticity in Japan. Rehabilitation Medicine 2000;37(11):863Archives of Physical Medicine and Rehabilitation 2017;98:1132-8Spinal Cord (2016) 54, 973-979Multiple Sclerosis Journal 2004 Oct;10(5):589-95Spasticity:Disordered Motor Control. Chicago, Year Book Medical Publishers, 1980, pp.485-494Tanaka, K.: Neural Mechanisms of Spasticity - Revisited. Rehabilitation Medicine 1995;32:97.105J Neurophysiol 128:470-479, 2022PLoS ONE 12(2):e0171937. doi:10.1371 / journal.pone.0171937Nat Neurosci 2004;7:269-277 Clinical Guidelines for Multiple Sclerosis and Neuromyelitis Optica 2017 Expert Review of Neurotherapeutics, 13(12 Suppl), 55-59 (2013) Clinical Neuroscience, 32, 1296-1298 (2014) Neurology, 51(2), 609-611 (1998) Journal of Neurology, 253(suppl 1), i16-i20 (2006) Journal of Neurology, 253(suppl 1), i26-i28 (2006) Journal of Neurosurgery, 21(2), e6 (2006) Journal of Pharmacological Sciences, 112, 121-127 (2010) Clinical Guidelines for Lower Urinary Tract Dysfunction in Spinal Cord Injury, 2019 Stahl, B., Muller, B., von Boxberg, Y., Cox, EC & Bonhoeffer, F.Biochemical characterization of a putative axonal guidance molecule of the chick visual system. Neuron 5, 735-743 (1990)Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513-29, 2006Liu, Yamashita, T. Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury. Biochem. Biophys. Res. Commun. 382, ​​795-800 (2009)Yamashita, T., Mueller, BK & Hata, K. Neogenin and repulsive guidance molecule signaling in the central nervous system. Curr. Opin. Neurobiol.17, 29-34 (2007) Hata, K. et al. RGMa inhibition promotes axonal growth and recovery after spinal cord injury. J. Cell Biol. 173, 47-58 (2006).

[0010] An object of the present invention is to provide a drug that is effective against accompanying symptoms associated with diseases caused by spinal cord or brain damage.

[0011] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are expected to be effective in treating and preventing symptoms associated with diseases caused by spinal cord or brain damage, such as spinal cord injury and HTLV (Human T-lymphotropic Virus)-1-associated myelopathy, which are caused by spinal cord or brain damage or inflammation, and have thus completed the present invention.

[0012] 1. A preventive or therapeutic agent for spasticity, comprising an RGMa inhibitor as an active ingredient. 2. The preventive or therapeutic agent according to 1 above, wherein the spasticity is associated with a disease caused by a disorder of the spinal cord or brain. 3. The preventive or therapeutic agent according to 1 above, wherein the spasticity is associated with a spinal cord injury. 4. The preventive or therapeutic agent according to 1 above, wherein the spasticity is associated with HTLV-1-associated myelopathy. 5. The agent according to any one of 1 to 4 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 6. The agent according to 5 above, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 7. The agent according to 5 or 6 above, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39. 8. The anti-RGMa neutralizing antibody is selected from the group consisting of the following (a) to (l): (a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; (b) an anti-RGMa neutralizing antibody comprising a 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, and a 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 its amino acid sequence; (c) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22;(d) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; (e) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34;(i) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 44, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 34, and (l) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 45, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 34. The anti-RGMa neutralizing antibody is selected from the group consisting of the following (a) and (f): (a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10;(f) The agent according to any one of items 5 to 8, which is an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:35, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:34. 10. A method for preventing or treating spasticity, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of treatment. 11. The method for preventing or treating spasticity according to item 10, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 12. Use of an RGMa inhibitor for the manufacture of an agent for preventing or treating spasticity. 13. The use according to item 12, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0013] 14. A preventive or therapeutic agent for spasticity and neurogenic bladder, comprising an RGMa inhibitor as an active ingredient. 15. The preventive or therapeutic agent according to claim 14, wherein the spasticity and neurogenic bladder are spasticity and neurogenic bladder associated with a disease caused by a disorder of the spinal cord or brain. 16. The preventive or therapeutic agent according to claim 14, wherein the spasticity and neurogenic bladder are spasticity and neurogenic bladder associated with a spinal cord injury. 17. The preventive or therapeutic agent according to claim 14, wherein the spasticity and neurogenic bladder are spasticity and neurogenic bladder associated with HTLV-1-associated myelopathy. 18. The agent according to any one of claims 14 to 17, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 19. The agent according to claim 18, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 20. 21. The agent according to 18 or 19 above, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. 21. The anti-RGMa neutralizing antibody is one of the following (a) to (l): (a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 5, LCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 7, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 8, HCDR2 comprising the amino acid sequence of SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 10, (b) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence of SEQ ID NO: 11, LCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HCDR3 comprising SFG in its amino acid sequence, (c) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22;(d) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; (e) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34;(i) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; 21. The agent according to any one of 18 to 20 above, wherein the anti-RGMa neutralizing antibody is an antibody selected from the group consisting of: (k) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and (l) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34. 22. The agent according to any one of 18 to 20 above, wherein the anti-RGMa neutralizing antibody is an antibody selected from the group consisting of the following (a) or (f):(a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO:5, an LCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:8, an HCDR2 comprising the amino acid sequence of SEQ ID NO:9, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:10; (f) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO:29, an LCDR2 comprising the amino acid sequence of SEQ ID NO:30, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:35, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:32, an HCDR2 comprising the amino acid sequence of SEQ ID NO:33, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:34. 23. A method for preventing or treating spasticity and neurogenic bladder, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of such treatment. 24. The preventive or therapeutic method according to 23 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 25. Use of an RGMa inhibitor for the manufacture of an agent for the prevention or treatment of spasticity and neurogenic bladder. 26. The use according to 25 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0014] 27. A preventive or therapeutic agent for neurogenic bladder, comprising an RGMa inhibitor as an active ingredient. 28. The preventive or therapeutic agent according to claim 27, wherein the neurogenic bladder is neurogenic bladder associated with a disease caused by a disorder of the spinal cord or brain. 29. The preventive or therapeutic agent according to claim 27, wherein the neurogenic bladder is neurogenic bladder associated with a spinal cord injury. 30. The preventive or therapeutic agent according to claim 27, wherein the neurogenic bladder is neurogenic bladder associated with HTLV-1-associated myelopathy. 31. The agent according to any one of claims 27 to 30, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 32. The agent according to claim 31, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 33. The agent according to any one of 31 or 32 above, wherein the anti-RGMa neutralizing antibody is an anti-RGMa neutralizing antibody comprising the following (a): (a) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:10. 34. A method for preventing or treating neurogenic bladder, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of treatment. 35. The method for preventing or treating neurogenic bladder according to 34 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 36. Use of an RGMa inhibitor for the manufacture of an agent for preventing or treating neurogenic bladder. 37. The use according to 36 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0015] According to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are useful as prophylactic or therapeutic agents for symptoms associated with spinal cord or brain disorders, for example, diseases caused by spinal cord or brain damage or inflammation. Specific examples of such symptoms include spasticity and neurogenic bladder. According to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are useful as prophylactic or therapeutic agents for spasticity and / or neurogenic bladder.

[0016] Figure 1 shows the ameliorative effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Unasnemab) containing the amino acid sequence of (a) (SEQ ID NOS: 5-10) on swimming-induced spasticity in a rat spinal cord contusion model, as determined by an analysis of the incidence of spasticity. Figure 2 is a table showing the ameliorative effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Unasnemab) containing the amino acid sequence of (a) (SEQ ID NOS: 5-10) on swimming-induced spasticity in a rat spinal cord contusion model, as determined by an analysis of the proportion of individuals with severe spasticity. Figure 3 shows the ameliorative effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Elezanumab) containing the amino acid sequence of (f) (SEQ ID NOS: 29, 30, 35, and 32-34) on swimming-induced spasticity in a rat spinal cord contusion model, as determined by an analysis of the incidence of spasticity. Figure 4 is a table showing the improvement effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Elezanumab) containing the amino acid sequence of (f) (SEQ ID NOS: 29, 30, 35, and 32-34) on swimming-induced spasticity in a rat spinal cord contusion model, based on an analysis of the proportion of individuals with severe spasticity. Figure 5 is a graph showing the improvement effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Unasnemab) containing the amino acid sequence of (a) (SEQ ID NOS: 5-10) on dysuria in a rat spinal cord contusion model, based on the cumulative number of days on which spontaneous urination was achieved. Figure 6 is a table showing the improvement effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Unasnemab) containing the amino acid sequence of (a) (SEQ ID NOS: 5-10) on dysuria in a rat spinal cord contusion model, based on an analysis of the total number of days on which spontaneous urination was achieved. Figure 7 is a graph showing the ameliorative effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Elezanumab) containing the amino acid sequence of (f) (SEQ ID NOS: 29, 30, 35, and 32-34) on dysuria in a rat spinal cord contusion model, as measured over time by the cumulative number of days on which spontaneous urination was achieved. Figure 8 is a table showing the ameliorative effect of repeated administration of an anti-RGMa neutralizing antibody (specifically, Elezanumab) containing the amino acid sequence of (f) (SEQ ID NOS: 29, 30, 35, and 32-34) on dysuria in a rat spinal cord contusion model, as measured over time by the cumulative number of days on which spontaneous urination was achieved.Figure 9 is a graph showing the distribution of MAS in both legs (two limbs) at baseline, 4 weeks, 12 weeks, and 24 weeks in the unasnemab-treated and placebo groups.

[0017] The terms used in the present invention are explained below. [Neutralization] In this application, neutralization refers to the ability to bind to a target of interest and inhibit any of the functions of that target. For example, an RGMa inhibitor refers to a substance that inhibits the biological activity of RGMa as a result of binding to RGMa.

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

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

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

[0021] [Human antibody] In the present application, a human antibody refers to an antibody in which both the light chain and the heavy chain are derived from human immunoglobulin. Depending on the difference in the heavy chain constant region, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having a γ heavy chain, IgM having a μ heavy chain, IgA (including IgA1 and IgA2) having an α heavy chain, IgD having a δ heavy chain, or IgE having an ε heavy chain. In principle, the light chain includes either a κ chain or a λ chain.

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

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

[0024] [Monospecific antibody] In the present application, a monospecific antibody is an antibody that has a single antigen specificity and has a single independent antigen recognition site. For example, in this specification, a monospecific antibody that recognizes RGMa may be referred to as an RGMa monospecific antibody.

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

[0026] [Complementarity-Determining Region (CDR)] Complementarity-determining region (CDR) refers to the region of the variable region of an immunoglobulin molecule that forms the antigen-binding site, also known as a hypervariable region, and refers to the portion where the amino acid sequence varies significantly from one immunoglobulin molecule to another. There are three CDRs in each of the light chain and heavy chain. The three CDRs in the light chain are sometimes referred to as LCDR1, LCDR2, and LCDR3, respectively, and the three CDRs in the heavy chain are sometimes referred to as HCDR1, HCDR2, and HCDR3. For example, the CDRs of an immunoglobulin molecule are determined according to the Kabat numbering system (Kabat et al., 1987 and 1991, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA). Unless otherwise specified, the present specification uses CDR sequences defined according to the Kabat numbering system. Furthermore, the CDR sequences of SEQ ID NOs: 5 to 15 use CDR sequences defined according to the IMGT numbering system (Lefranc M.-P., Immunology Today 18,509 (1997); Lefranc M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V., and Lefranc, Dev. Comp. Immunol, 27, 55-77 (2003)). It is obvious to those skilled in the art to replace the CDR sequences described by the IMGT numbering system with any other numbering system, such as the Kabat numbering system. Therefore, embodiments of the present invention include those in which the CDR sequences described by the IMGT numbering system are replaced with the Kabat numbering system, and those in which the CDR sequences described by the Kabat numbering system are replaced with the IMGT numbering system.

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

[0028] Percent (%) Identity of Amino Acid Sequences: The "percent (%) identity" of an amino acid sequence of a candidate polypeptide sequence, such as a variable region, with respect to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in a particular reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and excluding any conservative substitutions from being considered part of the sequence identity. Alignment for purposes of determining percent identity can be achieved by a variety of methods within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent identity values ​​are obtained using the sequence comparison computer program BLAST in pairwise alignments. In situations where BLAST is used for amino acid sequence comparison, the percent identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in a program alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the percent identity of A to B will differ from the percent identity of B to A. Unless otherwise specified, all percent identity values ​​herein are obtained using the BLAST computer program as set forth in the immediately preceding paragraph.

[0029] Conservative substitution refers to the replacement of an amino acid residue with another chemically similar amino acid residue without substantially altering the activity of the peptide. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue with the same charge. Examples of functionally similar amino acids that can be substituted in this way include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0030] Treatment Treatment includes any treatment of a disease in a subject, preferably a mammal, especially a human, including arresting the progression of the disease and symptoms, eliminating, curing, reducing or alleviating such disease and symptoms.

[0031] [Prevention] Prevention includes preventing or suppressing the onset of the above-mentioned diseases in a subject to be treated, preferably a mammal, particularly a human. Furthermore, "prevention" in the present invention also includes "recurrence prevention," which prevents the recurrence of the above-mentioned diseases that repeatedly undergo remission and relapse in a subject to be treated, preferably a mammal, particularly a human.

[0032] Embodiments of the present invention are described in detail below. The present invention provides a novel use of an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody, as a prophylactic or therapeutic agent for spinal cord or brain disorders, for example, associated symptoms associated with diseases caused by spinal cord or brain damage or inflammation. The present invention also provides a method for preventing or treating spinal cord or brain disorders, for example, associated symptoms associated with diseases caused by spinal cord or brain damage or inflammation, comprising the step of administering to a mammal in need of treatment an effective amount of a prophylactic or therapeutic agent comprising an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody.

[0033] <RGMa Inhibitory Substance> The RGMa inhibitor of the present invention may be any substance that acts on RGMa itself and inhibits or attenuates RGMa activity (hereinafter sometimes simply referred to as "RGMa activity" in this specification), and for example, a substance that binds to RGMa and directly inhibits (attenuates) RGMa activity, or that inhibits the binding of RGMa to a receptor and indirectly inhibits (attenuates) RGMa activity (e.g., a compound or antibody described below) is referred to as the RGMa inhibitor of the present invention. The RGMa inhibitor of the present invention may also be a substance that suppresses the expression of RGMa, and for example, a substance that inhibits the expression of RGMa and inhibits (attenuates) RGMa activity (e.g., a nucleic acid molecule described below) is also included in the RGMa inhibitor of the present invention.

[0034] RGMa was identified as a neurite outgrowth inhibitory protein in the central nervous system, and human RGMa protein is biosynthesized as a precursor protein consisting of 450 amino acids as shown in SEQ ID NO: 1. The N-terminal signal peptide Met1 to Pro47 (referring to the peptide from the first methionine residue from the N-terminus to the 47th proline residue, hereinafter the same) is removed, and the peptide bond between Asp168 and Pro169 is cleaved to generate the N-terminal domain. Furthermore, the C-terminal peptide Ala425 to Cys450 of the fragment C-terminal to Pro169 is removed, and a GPI anchor is added to the C-terminal carboxyl group of the resulting C-terminus, Ala424, to generate the C-terminal domain. Human RGMa protein is expressed on the cell membrane via a GPI anchor as a mature protein in which the N-terminal domain (Cys48 to Asp168) and the C-terminal domain (Pro169 to Ala424) are linked by a disulfide bond.

[0035] In the present invention, RGMa may be derived from any animal, but is preferably human RGMa. The precursor protein of human RGMa consists of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. The precursor protein of mouse RGMa consists of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and the precursor protein of rat RGMa consists of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, but because the C-terminal peptide is removed, the mature proteins have the same amino acid sequence. Examples of RGMa genes include, but are not limited to, the human RGMa gene consisting of the base sequence shown in SEQ ID NO: 4. The base sequences of RGM genes derived from various organisms can be easily obtained from known databases (GenBank, etc.).

[0036] Specific examples of the RGMa inhibitor of the present invention include low molecular weight compounds, anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, or antigen-binding fragments thereof, as well as RGMa nucleic acid molecules such as siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense oligonucleotides. Of these RGMa inhibitors, preferred are anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and antigen-binding fragments thereof, more preferred are anti-RGMa neutralizing antibodies or antigen-binding fragments thereof, with anti-RGMa neutralizing antibodies being particularly preferred.

[0037] <Anti-RGMa Neutralizing Antibody> In the present invention, the anti-RGMa neutralizing antibody may be any antibody that binds to RGMa and neutralizes RGMa activity, and may be a polyclonal antibody or a monoclonal antibody. In the present invention, a monoclonal antibody is preferred. Furthermore, the anti-RGMa neutralizing antibody of the present invention may be an RGMa monospecific antibody or a multispecific antibody that recognizes multiple antigens, including RGMa, but is preferably an RGMa monospecific antibody.

[0038] Furthermore, specific epitopes in human RGMa are preferably one or more of SEQ ID NO: 16 (amino acid numbers 47-69 of SEQ ID NO: 1), SEQ ID NO: 36 (amino acid numbers 298-311 of SEQ ID NO: 1), SEQ ID NO: 37 (amino acid numbers 322-335 of SEQ ID NO: 1), SEQ ID NO: 38 (amino acid numbers 349-359 of SEQ ID NO: 1), and SEQ ID NO: 39 (amino acid numbers 367-377 of SEQ ID NO: 1), more preferably the combination of SEQ ID NOs: 36 and 37, and particularly preferably the combination of SEQ ID NOs: 36, 37, and 39.

[0039] The anti-RGMa neutralizing antibodies of the present invention include polyclonal antibodies and monoclonal antibodies obtained by immunizing a mammal such as a mouse with an RGMa protein or a partial fragment thereof (for example, the above-mentioned epitope fragment) as an antigen, chimeric antibodies and humanized antibodies produced using genetic recombination technology, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibodies of the present invention are administered to humans as pharmaceuticals, humanized antibodies or human antibodies are desirable from the viewpoint of side effects.

[0040] Specific examples of the anti-RGMa neutralizing antibody of the present invention include the following antibodies (a) to (l), and the methods described in Patent Documents 2 to 4 can be used to produce each of them.

[0041] (a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (the anti-RGMa neutralizing antibody also includes antibodies having epitopes represented by SEQ ID NOs: 36, 37, and 39); SEQ ID NO: 5: QDISSY SEQ ID NO: 6: YTS (qualifier: mol_type = protein; organism = Mus musculus) SEQ ID NO: 7: QQLNTLPWT SEQ ID NO: 8: GFTFSDAW SEQ ID NO: 9: IRSKANNHAT SEQ ID NO: 10: TRRDGAY

[0042] (b) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of AGS (the anti-RGMa neutralizing antibody further includes antibodies having epitopes set forth in SEQ ID NOs: 36, 37, and 38); SEQ ID NO: 11: QSLVHSNGNTY SEQ ID NO: 12: KVS (qualifier: mol_type = protein; organism = Mus musculus) SEQ ID NO: 13: SQSTHVPYT SEQ ID NO: 14: GYSITTSYY SEQ ID NO: 15: ISYDGTN

[0043] (c) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; SEQ ID NO: 17: RSSQSLEYSDGYTFLE SEQ ID NO: 18: EVSNRFS SEQ ID NO: 19: FQATHDPLT SEQ ID NO: 20: NYGMN SEQ ID NO: 21: MIYYDSSEKHYADSVKG SEQ ID NO: 22: GTTPDY

[0044] (d) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28, SEQ ID NO: 23: QASQDIDNYLA SEQ ID NO: 24: GATNLAD SEQ ID NO: 25: LQGYIPPRT SEQ ID NO: 26: SYVMH SEQ ID NO: 27: YIIPYNDNTKYNEKFKG SEQ ID NO: 28: ARRNEYYGSSFFDY

[0045] (e) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); SEQ ID NO: 29: TGTSSSVGDSIYVS SEQ ID NO: 30: DVTKRPS SEQ ID NO: 31: CSYAGTDTL SEQ ID NO: 32: SHGIS SEQ ID NO: 33: WISPYSGNTNYAQKLQG SEQ ID NO: 34: VGSGPYYYMDV

[0046] (f) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); SEQ ID NO: 29: TGTSSSVGDSIYVS SEQ ID NO: 30: DVTKRPS SEQ ID NO: 35: YSYAGTDTL SEQ ID NO: 32: SHGIS SEQ ID NO: 33: WISPYSGNTNYAQKLQG SEQ ID NO: 34: VGSGPYYYMDV

[0047] (g) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); SEQ ID NO: 29: TGTSSSVGDSIYVS SEQ ID NO: 30: DVTKRPS SEQ ID NO: 40: FSYAGTDTL SEQ ID NO: 32: SHGIS SEQ ID NO: 33: WISPYSGNTNYAQKLQG SEQ ID NO: 34: VGSGPYYYMDV

[0048] (h) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); SEQ ID NO: 29: TGTSSSVGDSIYVS SEQ ID NO: 30: DVTKRPS SEQ ID NO: 41: HSYAGTDTL SEQ ID NO: 32: SHGIS SEQ ID NO: 33: WISPYSGNTNYAQKLQG SEQ ID NO: 34: VGSGPYYYMDV

[0049] (i) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); SEQ ID NO: 29: TGTSSSVGDSIYVS SEQ ID NO: 30: DVTKRPS SEQ ID NO: 42: LSYAGTDTL SEQ ID NO: 32: SHGIS SEQ ID NO: 33: WISPYSGNTNYAQKLQG SEQ ID NO: 34: VGSGPYYYMDV

[0050] (j) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); SEQ ID NO: 29: TGTSSSVGDSIYVS SEQ ID NO: 30: DVTKRPS SEQ ID NO: 43: VSYAGTDTL SEQ ID NO: 32: SHGIS SEQ ID NO: 33: WISPYSGNTNYAQKLQG SEQ ID NO: 34: VGSGPYYYMDV

[0051] (k) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope), and

[0052] (l) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:45, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO:16), such as an antibody selected from the following: SEQ ID NO:29: TGTSSSVGDSIYVS SEQ ID NO:30: DVTKRPS SEQ ID NO:45: KSYAGTDTL SEQ ID NO:32: SHGIS SEQ ID NO:33: WISPYSGNTNYAQKLQG SEQ ID NO:34: VGSGPYYYMDV. Among these, particularly preferred are antibodies described in (a) or (f).

[0053] Furthermore, examples of the anti-RGMa neutralizing antibody of the present invention include the antibodies described in WO2016 / 175236, WO2009 / 106356, and WO2013 / 112922, and the antibodies described in these publications are also included in the anti-RGMa neutralizing antibody of the present invention. Specifically, examples of the anti-RGMa neutralizing antibody of the present invention include antibodies selected from the following (a') to (i'):

[0054] (a') An anti-RGMa neutralizing antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 46 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 47, SEQ ID NO: 46: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME SEQ ID NO: 47: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS

[0055] (b') An anti-RGMa neutralizing antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 48 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 49 (hereinafter, the antibody is also referred to as Unasnemab), SEQ ID NO: 48: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEMERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 49: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0056] (c') An anti-RGMa neutralizing antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 51, SEQ ID NO: 50: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIK SEQ ID NO: 51: DVKLQESGPGLVKPSQSLSLTCSVTGYSITTSYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKNQFFLRLNSVTTEDTATYYCAGSFGYSQGTLVTVSA

[0057] (d') an anti-RGMa neutralizing antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 52 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 53, SEQ ID NO: 52: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 53: DVKLQESGPGLVKPSQSLSLTCSVTGYSITTSYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKNQFFLRLNSVTTEDTATYYCAGSFGYSQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINP CPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIE RTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0058] (e') An anti-RGMa neutralizing antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 55. SEQ ID NO: 54: DVVLTQTPVSLSVTLGDQASMSCRSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYCFQATHDPLTFGSGTKLEIKR SEQ ID NO: 55: EVQLVESGGGLVQPGSSLKLSCVASGFTFSNYGMNWIRQAPKKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLEMNSLRSEDTAIYYCAKGTTPDYWGQGVMVTVSS

[0059] (f') An anti-RGMa neutralizing antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 56 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 57, SEQ ID NO: 56: DIQMTQSPASLSASLEEIVTITCQASQDIDNYLAWYHQKPGKSPRLLIYGATNLADGVPSRFSGSRSGTQFSLKINRLQIEDLGIYYCLQGYIPPRTFGGGTKLELKR SEQ ID NO: 57: EVQLQQSGPELVKPGTSVKMSCKTSGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDNTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRNEYYGSSFFDYWGQGTTLTVSS

[0060] (g') An anti-RGMa neutralizing antibody comprising a light chain variable region (lambda chain) comprising the amino acid sequence set forth in SEQ ID NO: 58 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 59, SEQ ID NO: 58: QSALTQPRSVSGSPGQSVTISCTGTSSSVGDSIYVSWYQQHPGKAPKLMLYDVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGTDTLFGGGTKVTVL SEQ ID NO: 59: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSHGISWVRQAPGQGLDWMGWISPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELSSLRSEDTAVYYCAR VGSGPYYYMDVWGQGTLVTVSS

[0061] (h') an anti-RGMa neutralizing antibody comprising a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 60 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 59; and SEQ ID NO: 60: QSALTQPRSVSGSPGQSVTISCTGTSSSVGDSIYVSWYQQHPGKAPKLMLYDVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCYSYAGTDTLFGGGTKVTVL SEQ ID NO: 59: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSHGISWVRQAPGQGLDWMGWISPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELSSLRSEDTAVYYCAR VGSGPYYYMDVWGQGTLVTVSS

[0062] (i') An anti-RGMa neutralizing antibody comprising a light chain comprising the amino acid sequence set forth in SEQ ID NO: 61 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 62 (hereinafter, this antibody may be referred to as Elezanumab). SEQ ID NO: 61: QSALTQPRSVSGSPGQSVTISCTGTSSSVGDSIYVSWYQQHPGKAPKLMLYDVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCYSYAGTDTLFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 62: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSHGISWVRQAPGQGLDWMGWISPYSGNTNYAQKLQGRVTMTTDTSTSTAYMELSSLRSEDTAVYYCARVGSGPYYYMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0063] The anti-RGMa neutralizing antibody of the present invention can be produced by an existing commonly used production method. The antigen may be used for immunization as is, or may be used as a complex with a carrier protein. Condensing agents such as glutaraldehyde, carbodiimide, and maleimide active ester can be used to prepare the complex of antigen and carrier protein. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[0064] Mammals to be immunized include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, and cows, and inoculation methods include subcutaneous, intramuscular, and intraperitoneal administration. The vaccine may be administered in a mixture with complete or incomplete Freund's adjuvant, and is typically administered once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of immunized animals are fused with myeloma cells and isolated as hybridomas. Myeloma cells derived from mammals, such as mice, rats, and humans, are used.

[0065] <Polyclonal antibodies> Polyclonal antibodies can be obtained, for example, by immunizing a mammal such as those described above with the antigen, together with Freund's adjuvant as necessary, and then obtaining the serum from the immunized animal.

[0066] <Monoclonal Antibodies> Monoclonal antibodies can be obtained specifically as follows. That is, an antigen such as those described above is used as an immunogen, and the immunogen, together with Freund's adjuvant, if necessary, is injected subcutaneously, intramuscularly, intravenously, into the footpad, or intraperitoneally into a mammal such as those described above, or the immunogen is implanted therein, one to several times, to immunize the mammal. Usually, immunization is performed one to four times at intervals of about 1 to 14 days after the initial immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

[0067] Monoclonal antibodies can be obtained using methods well known to those skilled in the art (e.g., Current Protocols in Molecular Biology (John Wiley & Sons (1987)); Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0068] Hybridomas secreting monoclonal antibodies can be prepared according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) or modifications thereof. Specifically, hybridomas are prepared by fusing antibody-producing cells contained in the spleen or other tissues obtained from an immunized mammal with myeloma cells derived from a mammal, preferably a mouse, rat, or human, that are not capable of producing autoantibodies.

[0069] Examples of myeloma cells that can be used in cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3, and human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15.

[0070] Examples of fusion promoters include polyethylene glycol, and cell fusion can be achieved by reacting polyethylene glycol (average molecular weight 1000 to 4000) at a concentration of about 20 to 50%, at a temperature of 20 to 40°C, preferably 30 to 37°C, at a ratio of antibody-producing cells to myeloma cells of usually about 1:1 to 10:1, for about 1 to 10 minutes.

[0071] Hybridoma clones that produce monoclonal antibodies can be screened by culturing the hybridomas, for example, in a microtiter plate and measuring the reactivity of the culture supernatant in the wells against the immunizing antigen by an immunochemical method such as ELISA.

[0072] In screening for antibody-producing hybridomas, in addition to a binding assay with RGMa protein, an evaluation is also performed to determine whether the antibody inhibits the RGMa activity of the present invention. These screening methods allow the selection of anti-RGMa neutralizing antibodies of the present invention.

[0073] Clones can be obtained from wells containing hybridomas that produce the desired antibody by limiting dilution. Hybridoma selection and breeding are usually carried out in an animal cell medium containing 10-20% fetal bovine serum and supplemented with HAT (hypoxanthine, aminopterin, thymidine).

[0074] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or by growing them in vivo, for example, in ascites of a mammal such as a mouse or rat, and isolating the antibodies from the resulting culture supernatant or from the ascites of the mammal.

[0075] When culturing in vitro, a nutrient medium suitable for growing, maintaining, and preserving hybridomas and producing monoclonal antibodies in the culture supernatant can be used, depending on various conditions such as the characteristics of the cell type being cultured and the culture method, etc. Examples of the nutrient medium include known nutrient media and nutrient media prepared from basal media.

[0076] Examples of basal media include low-calcium media such as Ham's F12 medium, MCDB153 medium, and low-calcium MEM medium, and high-calcium media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, and RD medium. The basal media may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

[0077] Monoclonal antibodies can be isolated and purified by subjecting the culture supernatant or ascites fluid described above to saturated ammonium sulfate, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), or affinity column chromatography such as an anti-immunoglobulin column or protein A column. Specifically, monoclonal antibodies can be purified using known immunoglobulin purification methods, and can be easily achieved by, for example, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of an anion exchanger, or affinity chromatography using RGMa protein.

[0078] Monoclonal antibodies can also be obtained by phage display. In phage display, phages selected from a phage antibody library are screened with the target immunogen, and phages with the desired binding affinity to the immunogen are selected. Next, the antibody-corresponding sequence contained in the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding domain is constructed based on the isolated or sequence information. Monoclonal antibodies can then be produced by culturing a cell line transfected with such an expression vector. Human antibodies with the desired binding affinity can be generated by using a human antibody library as the phage antibody library.

[0079] <Nucleic Acid Molecules> Nucleic acid molecules encoding the anti-RGMa neutralizing antibodies or antigen-binding fragments thereof of the present invention can be obtained, for example, by the following method. First, total RNA is prepared from cells such as hybridomas using a commercially available RNA extraction kit, and cDNA is synthesized using reverse transcriptase with random primers or the like. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides with sequences conserved in the variable regions of known human antibody heavy chain and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The base sequence of the DNA can be determined by conventional methods, such as by incorporating it into a sequencing plasmid. Alternatively, DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the sequence of the variable region or a portion thereof and linking it to a sequence containing the constant region. The nucleic acid molecule may encode all of the constant and variable regions of the heavy and light chains, or it may encode only the variable regions of the heavy and light chains. When encoding both the constant and variable regions, the nucleotide sequences of the heavy and light chain constant regions are preferably those described in Nucleic Acids Research, vol. 14, p. 1779, 1986; The Journal of Biological Chemistry, vol. 257, p. 1516, 1982; and Cell, vol. 22, p. 197, 1980.

[0080] Functionally modified anti-RGMa neutralizing antibodies can be prepared by the following methods. For example, when the present anti-RGMa neutralizing antibodies are produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as host cells, antibodies with reduced fucose content in the sugar chains and enhanced cell-killing activity can be obtained. When the present anti-RGMa neutralizing antibodies are produced using CHO cells into which the FUT8 gene has been introduced as host cells, antibodies with reduced cell-killing activity can be obtained (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739). Furthermore, complement activation function can be regulated by modifying amino acid residues in the Fc region (U.S. Pat. No. 6,737,056, U.S. Pat. No. 7,297,775, U.S. Pat. No. 7,317,091). Furthermore, the use of Fc region mutants that enhance binding to FcRn, one of the Fc receptors, can extend the blood half-life (Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering. The use of Fc region mutants that enhance binding to FcRn, one of the Fc receptors, can extend the blood half-life (Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering.

[0081] <Conjugate Antibody> Examples of modified molecules of the anti-RGMa neutralizing antibody of the present invention include conjugate antibodies. Examples of conjugate antibodies include conjugated anti-RGMa neutralizing antibodies chemically or genetically bound to 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, Neurotrophin, etc.), albumin, enzymes, and other antibodies.

[0082] When PEG is attached as a functional molecule, PEG can have a molecular weight of, but not limited to, 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and can be either linear or branched. PEG can be attached to the N-terminal amino group of an amino acid of an anti-RGMa neutralizing antibody, for example, by using an NHS active group.

[0083] When a radioactive substance is used as a functional molecule, 131 I, 125 I, 90 Y. 64 Cu, 99 Tc, 77 Lu or 211 Radioactive substances can be directly bound to anti-RGMa neutralizing antibodies by the chloramine T method or the like.

[0084] When a toxin is used as a functional molecule, bacterial toxins (eg, diphtheria toxin), plant toxins (eg, ricin), low-molecular-weight toxins (eg, geldanamycin), maytansinoids, calicheamicin, and the like can be used.

[0085] When a low molecular weight compound is used as the functional molecule, examples thereof include daunomycin, doxorubicin, metrorexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0086] When an enzyme is used as the functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. may be used.

[0087] Linkers used to chemically link toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR) ｎ O (CR2) ｎ Examples of suitable linkers include linkers represented by - (R is an optional substituent, and n is a positive integer), repeating alkoxy units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.), alkylamino (e.g., polyethyleneamino, Jeffamine™), and diacid esters and amides (succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for attaching functional molecules have already been established in the field (DJ King., Applications and Engineering of Monoclonal Antibodies., 1998 TJ International Ltd; Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol. 152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol. 93:8681).

[0088] <Antigen-binding fragment> In an embodiment of the present invention, an "antigen-binding fragment" of an antibody refers to a partial region of the antibody as described above that has antigen-binding ability, and specific examples include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof. Furthermore, antigen-binding fragments include conjugated fragments to which functional molecules other than the anti-RGMa neutralizing antibody of the present application are chemically or genetically engineered, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies.

[0089] "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulins with protease enzymes such as pepsin or papain, resulting in digestion across the disulfide bond between the two heavy chains in the hinge region. For example, treating IgG with papain results in cleavage upstream of the disulfide bond between the two heavy chains in the hinge region, producing two homologous antibody fragments consisting of a light chain consisting of a VL (light chain variable region) and a CL (light chain constant region), and a heavy chain fragment consisting of a VH (heavy chain variable region) and a CHγ1 (γ1 region of the heavy chain constant region) linked by a disulfide bond at their C-terminal regions. These two homologous antibody fragments are each called Fab. Furthermore, treating IgG with pepsin results in cleavage downstream of the disulfide bond between the two heavy chains in the hinge region, producing an antibody fragment slightly larger than the two Fab fragments linked by the hinge region. This antibody fragment is called F(ab')2.

[0090] <Chimeric Antibody> A preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a chimeric antibody. Examples of "chimeric antibodies" include chimeric antibodies in which the variable region is derived from immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.) and the constant region is derived from human immunoglobulin. For example, chimeric antibodies can be produced by immunizing a mouse with an antigen, excising the variable region that binds to the antigen from the mouse monoclonal antibody gene, and combining it with an antibody constant region derived from human bone marrow. Constant regions derived from human immunoglobulins have unique amino acid sequences depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE. The constant region of the recombinant chimeric antibody of the present invention may be the constant region of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. The gene encoding the chimeric antibody thus prepared can be used to prepare an expression vector, which is then used to transform host cells to obtain transformed cells producing the chimeric antibody.The transformed cells are then cultured to obtain the desired chimeric antibody from the culture supernatant.

[0091] <Humanized Antibody> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a humanized antibody. The "humanized antibody" of the present invention is an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity-determining region) of a non-human animal antibody such as a mouse is grafted onto a human antibody gene (CDR grafting). For example, it can be produced with reference to the methods described in JP-A-4-506458 and Japanese Patent No. 2912618. Specifically, this refers to a humanized antibody in which some or all of its CDRs are CDRs derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework regions of its variable regions are derived from a human immunoglobulin, and its constant regions are derived from a human immunoglobulin.

[0092] The humanized antibody of the present invention can be produced, for example, as follows: However, it goes without saying that the production method is not limited to this.

[0093] For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be produced by genetic engineering with reference to JP-A-4-506458 and JP-A-62-296890, etc. That is, DNA of the mouse heavy chain CDR region and DNA of the mouse light chain CDR region are isolated from a hybridoma that produces a mouse monoclonal antibody, and a human heavy chain gene covering the entire region excluding the human heavy chain CDR and a human light chain gene covering the entire region excluding the human light chain CDR are isolated from a human immunoglobulin gene.

[0094] The human heavy chain gene grafted with the isolated mouse heavy chain CDR DNA is introduced into an appropriate expression vector so as to be expressible, and similarly, the human light chain gene grafted with the mouse light chain CDR DNA is introduced into another appropriate expression vector so as to be expressible. Alternatively, the human heavy chain and light chain genes grafted with mouse CDRs can be introduced into the same expression vector so as to be expressible. Host cells are transformed with the expression vector prepared in this way to obtain humanized antibody-producing transformants, and the desired humanized antibody is obtained from the culture supernatant by culturing the transformants.

[0095] <Human Antibody> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a human antibody. A human antibody is an antibody in which all regions constituting the immunoglobulin, including the heavy chain variable region, heavy chain constant region, and light chain variable region, and light chain constant region, are immunoglobulins derived from genes encoding human immunoglobulins, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal produced by incorporating at least a human immunoglobulin gene into the gene locus of a non-human mammal such as a mouse can be produced by immunizing the animal with an antigen in the same manner as the above-mentioned method for producing polyclonal or monoclonal antibodies.

[0096] For example, transgenic mice that produce human antibodies can be produced according to the methods described in Nature Genetics, Vol. 7, pp. 13-21, 1994; Nature Genetics, Vol. 15, pp. 146-156, 1997; Published Japanese Translation of PCT International Publication No. H4-504365; Published Japanese Translation of PCT International Publication No. H7-509137; International Publication WO94 / 25585; Nature, Vol. 368, pp. 856-859, 1994; and Published Japanese Translation of PCT International Publication No. H6-500233, etc. More specific examples include HuMab® mice (Medarex, Princeton NJ), KM™ mice (Kirin Pharma Company, Japan), and KM (FCγRIIb-KO) mice.

[0097] Specific examples of anti-RGMa neutralizing antibodies of the present invention include those having a CDR containing a specific amino acid sequence in the heavy chain variable region and a CDR containing a specific amino acid sequence in the light chain variable region (preferably the anti-RGMa neutralizing antibodies (a) to (l) and (a') to (i') above). Note that, as long as the properties of the antibodies of the present invention, such as the ability to bind to RGMa and inhibit (neutralize) RGMa activity, are maintained, the amino acid sequence of the anti-RGMa neutralizing antibodies (preferably the anti-RGMa neutralizing antibodies (a) to (l) and (a') to (i') above) may include substitution, deletion, addition, or insertion of one or several amino acids (1 to 20, 1 to 10, or 1 to 5, preferably 1 or 2). While such substitution, deletion, or addition may be introduced into the CDR, it is preferable that it be introduced into a region other than the CDR. Furthermore, the amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0098] The amino acid sequence of the anti-RGMa neutralizing antibody of the present invention (preferably the anti-RGMa neutralizing antibodies (a) to (l) and (a') to (i') above) containing substitutions, deletions, etc. in the amino acid sequence is, for example, an amino acid sequence in which the heavy chain variable region after amino acid sequence modification has a percent identity of 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before modification, and an amino acid sequence in which the light chain variable region after amino acid sequence modification has a percent identity of 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before modification.

[0099] In the present invention, siRNA refers to a short double-stranded RNA capable of suppressing the expression of a target gene (in the present invention, an RGMa gene). As long as it functions as an siRNA of the present invention that inhibits RGMa activity, the base sequence and length (base length) are not particularly limited; however, they are preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases. In the present invention, shRNA refers to a single-stranded RNA molecule of about 20 base pairs or more that contains a partially palindromic base sequence, thereby forming a double-stranded structure within the molecule and consisting of a short hairpin structure with an overhang at the 3' end. After being introduced into a cell, such shRNA is degraded to a length of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) within the cell and can suppress the expression of the target gene in the same way as siRNA. In the present invention, the above-mentioned siRNA and shRNA may be in any form as long as they are capable of suppressing the expression of an RGMa gene.

[0100] In the present invention, siRNA or shRNA can be artificially chemically synthesized. Alternatively, antisense and sense RNAs can be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and a T7 promoter. Antisense oligonucleotides may be either DNA or RNA, as long as they are complementary to or hybridize with a sequence of 5 to 100 consecutive bases in the DNA sequence of the RGMa gene. Modifications may also be used as long as they do not impair function. Antisense oligonucleotides can be synthesized by conventional methods, for example, easily using a commercially available DNA synthesizer. Preferred sequences can be selected using conventional selection methods, and the siRNA or shRNA of the present invention can be confirmed by evaluating inhibition of functional RGMa expression.

[0101] <Preventive or Therapeutic Agent for Spasticity and / or Neurogenic Bladder> In an embodiment of the present invention, spasticity refers to a disorder of sensorimotor control caused by intermittent or sustained involuntary muscle activity due to upper motor nerve injury (Disabil Rehabil. 2005 Jan;27(1-2):2-6). Spasticity is caused by upper motor neurons, long nerves extending from the brain to the spinal cord that control voluntary movements. Damage or impairment to these neurons results in abnormal messages being sent to muscles. Spasticity is an abnormal reflex movement that occurs when damage or impairment to the central nervous system, which controls voluntary movements, disrupts the complex balance between neural excitation and brain or spinal cord inhibition. For example, when signals from the brain to the lower body are interrupted due to spinal cord injury or spinal cord disease (compression, trauma, blood flow disorders, neurodegeneration, etc.), increased neural excitation leads to unwanted muscle contractions, i.e., spasticity. Symptoms of spasticity include muscle tension, sudden muscle contractions (spasms), increased deep tendon reflexes, muscle cramps (clonus), scissors position (a state in which the hip joint is adducted and internally rotated), and joint stiffness (Pract Neurol. 2012;12(5):289-98). The present invention encompasses a novel use of an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody (preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a) (SEQ ID NOs: 5 to 10) or the amino acid sequence of (f) (SEQ ID NOs: 29, 30, 35, and 32 to 34), more preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a') (SEQ ID NOs: 46 and 47), the amino acid sequence of (b') (SEQ ID NOs: 48 and 49), the amino acid sequence of (h') (SEQ ID NOs: 60 and 59), or the amino acid sequence of (i') (SEQ ID NOs: 61 and 62)).

[0102] In an embodiment of the present invention, neurogenic bladder refers to a condition in which abnormalities in the function of the lower urinary tract (bladder and urethra) occur due to damage or disorders of the central nervous system, autonomic nervous system, or peripheral nervous system (Neurogenic Bladder and Neurogenic Lower Urinary Tract Dysfunction. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan.; 2022 Nov. 28). Neurogenic bladder is caused by various neurological disorders, as urination is controlled by coordination and reflexes between the spinal cord, brainstem, and spinal cord (Transl Androl Urol. 2016 Feb; 5(1): 12-21). It is a common complication, particularly with spinal cord injury. This is because the micturition reflex center is located in the sacral cord at the end of the spinal cord, and damage to any part of the spinal cord disrupts the neural pathway between the sacral cord and the cerebrum for urination (Management of the neurogenic bladder for adults with spinal cord injuries. 2013). Lower urinary tract dysfunction, a symptom of neurogenic bladder, is divided into two categories: storage dysfunction and voiding dysfunction (Neurogenic Bladder and Neurogenic Lower Urinary Tract Dysfunction. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan. 2022 Nov. 28). In storage dysfunction, the bladder contracts involuntarily, resulting in symptoms such as frequent urination, urgency, and urinary incontinence. In voiding dysfunction, bladder contraction is impaired, resulting in difficulty urinating.The present invention includes a novel use of an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody (preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a) (SEQ ID NOs: 5 to 10) or the amino acid sequence of (f) (SEQ ID NOs: 29, 30, 35, and 32 to 34), more preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a') (SEQ ID NOs: 46 and 47), the amino acid sequence of (b') (SEQ ID NOs: 48 and 49), the amino acid sequence of (h') (SEQ ID NOs: 60 and 59), or the amino acid sequence of (i') (SEQ ID NOs: 61 and 62)), as a preventive or therapeutic agent for neurogenic bladder. Also included are RGMa inhibitors, particularly anti-RGMa neutralizing antibodies (preferably anti-RGMa neutralizing antibodies comprising the amino acid sequence of (a) (SEQ ID NOs: 5 to 10) or the amino acid sequence of (f) (SEQ ID NOs: 29, 30, 35, and 32 to 34), more preferably anti-RGMa neutralizing antibodies comprising the amino acid sequence of (a') (SEQ ID NOs: 46 and 47), the amino acid sequence of (b') (SEQ ID NOs: 48 and 49), the amino acid sequence of (h') (SEQ ID NOs: 60 and 59), or the amino acid sequence of (i') (SEQ ID NOs: 61 and 62)) as prophylactic or therapeutic agents for neurogenic bladder or lower urinary tract dysfunction.

[0103] Furthermore, the present invention also includes an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody (preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a) (SEQ ID NOs: 5 to 10) or the amino acid sequence of (f) (SEQ ID NOs: 29, 30, 35, and 32 to 34), more preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a') (SEQ ID NOs: 46 and 47), the amino acid sequence of (b') (SEQ ID NOs: 48 and 49), the amino acid sequence of (h') (SEQ ID NOs: 60 and 59), or the amino acid sequence of (i') (SEQ ID NOs: 61 and 62)) as a preventive or therapeutic agent for spasticity and neurogenic bladder (or lower urinary tract dysfunction).

[0104] <Preventive or therapeutic agent for accompanying symptoms associated with diseases caused by spinal cord or brain damage> In an embodiment of the present invention, the preventive or therapeutic agent for accompanying symptoms associated with diseases caused by spinal cord or brain damage, which is a novel use of an RGMa inhibitor, means a drug or pharmaceutical composition that can prevent or treat accompanying symptoms associated with spinal cord or brain damage, specifically diseases caused by spinal cord or brain damage or inflammation, such as spasticity and / or neurogenic bladder (or lower urinary tract dysfunction). Examples of diseases that can cause spasticity include cerebral palsy, multiple sclerosis, amyotrophic lateral sclerosis, stroke (including cerebral infarction and cerebral hemorrhage), brain injury, spinal cord compression, spinal cord injury, motor neuron disease, spinal vascular malformation, hereditary spastic paraplegia (hereditary spastic paraparesis), subacute combined degeneration of the spinal cord, HTLV-1-associated myelopathy (also known as tropical spastic paraparesis), copper deficiency, syringomyelia, and tethered spinal cord (Crit Rev Phys Rehabil Med. 2013;25(1-2):11-22, Pract Neurol. 2012;12(5):289-98). Examples of the diseases that can cause neurogenic bladder (or lower urinary tract dysfunction) include spinal / spinal cord diseases and brain diseases. Specific examples of spinal / spinal cord diseases include spinal diseases (intervertebral disc herniation, spinal stenosis, ossification of the posterior longitudinal ligament), vascular diseases (spinal hemorrhage / infarction), spinal cord tumors, demyelinating diseases (multiple sclerosis, neuromyelitis optica), infectious diseases (myelitis, HTLV-1-associated myelopathy), inflammatory diseases (sarcoidosis, collagen diseases), congenital diseases (spina bifida, sacral hypoplasia, spinal cord retention syndrome), degenerative diseases (amyotrophic lateral sclerosis), exogenous diseases (spinal cord injury, radiation-related), and hereditary spastic paraplegia.Brain diseases include vascular diseases (cerebral hemorrhage and infarction), brain tumors, degenerative diseases (Parkinson's disease, multiple system atrophy, Alzheimer's disease, spinocerebellar degeneration, olivopontocerebellar degeneration, amyotrophic lateral sclerosis), demyelinating diseases (multiple sclerosis), infectious diseases (encephalitis), inflammatory diseases (sarcoidosis, collagen diseases), traumatic diseases (head trauma), and hereditary spastic paraplegia (Transl Androl Urol. 2016 Feb; 5(1): 12-21, etc.).

[0105] The present invention provides a novel use of an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody (preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a) (SEQ ID NOS: 5-10) or the amino acid sequence of (f) (SEQ ID NOS: 29, 30, 35, and 32-34), more preferably an anti-RGMa neutralizing antibody comprising the amino acid sequence of (a') (SEQ ID NOS: 46 and 47), the amino acid sequence of (b') (SEQ ID NOS: 48 and 49), the amino acid sequence of (h') (SEQ ID NOS: 60 and 59), or the amino acid sequence of (i') (SEQ ID NOS: 61 and 62)). This use is directed to a prophylactic or therapeutic agent for symptoms associated with diseases caused by spinal cord or brain damage. Examples of associated symptoms include spasticity and neurogenic bladder (or lower urinary tract dysfunction). The present invention also provides a prophylactic or therapeutic agent for each of the associated symptoms, including two associated symptoms (spasticity and neurogenic bladder (or lower urinary tract dysfunction)).

[0106] Pharmaceutical Compositions The preventive or therapeutic agent for symptoms associated with diseases caused by spinal cord or brain damage, which contains an RGMa inhibitor, or the preventive or therapeutic agent for spasticity and / or neurogenic bladder, which contains an RGMa inhibitor, of the present invention, is typically administered systemically or locally, orally or parenterally. The preventive or therapeutic agent for symptoms associated with diseases caused by spinal cord or brain damage, which contains an RGMa inhibitor, or the preventive or therapeutic agent for spasticity and / or neurogenic bladder, which contains an RGMa inhibitor, of the present invention, can be formulated by appropriately blending an RGMa inhibitor as an active ingredient with a pharmaceutically acceptable carrier or additive. The pharmaceutical composition thus formulated can be administered orally or parenterally. Specifically, it can be administered in the form of oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, or parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The proportion of the carrier or additive may be appropriately set based on the range commonly used in the pharmaceutical field. The carrier or additive that can be added is not particularly limited, and examples thereof include various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0107] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, a functionally modified antibody thereof, a conjugated antibody thereof, or an antigen-binding fragment thereof, it is preferably administered parenterally, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically, as an injection or infusion formulated with a pharmaceutically acceptable carrier. For example, an injection or infusion containing an anti-RGMa neutralizing antibody can be used as a solution, suspension, or emulsion. Examples of solvents that can be used include distilled water for injection, physiological saline, glucose solution, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). Furthermore, an injection or infusion containing such an anti-RGMa neutralizing antibody may contain a stabilizer, a solubilizer, a suspending agent, an emulsifier, a soothing agent, a buffer, a preservative, an antiseptic, a pH adjuster, etc. Examples of stabilizers that can be used include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, and the like). Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, and tragacanth. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of buffering agents that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, and Tris buffer.Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc. Examples of antiseptics that can be used include benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.

[0108] When the RGMa inhibitor is a nucleic acid (such as siRNA, shRNA, or an antisense oligonucleotide), it can be administered in the form of a non-viral vector or a viral vector. In the case of a non-viral vector, methods for introducing nucleic acid molecules using liposomes (such as the liposome method, HVJ-liposome method, cationic liposome method, lipofection method, or lipofectamine method), microinjection, or a method for transferring nucleic acid molecules into cells together with a carrier (metal particles) using a gene gun, can be used. For example, when siRNA or shRNA is administered to a living body using a viral vector, viral vectors such as recombinant adenovirus and retrovirus can be used. DNA expressing siRNA or shRNA can be introduced into a DNA or RNA virus such as a detoxified retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, or SV40, and the gene can be introduced into cells or tissues by infecting the cells or tissues with this recombinant virus.

[0109] The formulation thus obtained can be administered in an effective amount to, for example, humans or other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) to prevent or treat accompanying symptoms associated with diseases caused by spinal cord or brain disorders, and also to prevent or treat spasticity and / or neurogenic bladder. The dosage is determined appropriately taking into consideration the purpose, severity of the disease, the patient's age, weight, sex, medical history, type of active ingredient, etc. For example, when the active ingredient is an anti-RGMa neutralizing antibody, the dosage is preferably approximately 0.02 mg to 4000 mg per day for an average human weighing approximately 65 to 70 kg. The total daily dosage may be a single dose or divided doses.

[0110] The present invention will be described in more detail below with reference to examples, but these examples are not intended to limit the scope of the present invention. A rat spinal cord contusion model was used as a pathological model of spinal cord injury. Anti-RGMa neutralizing antibodies containing the amino acid sequences (a) (SEQ ID NOS: 5-10) and (f) (SEQ ID NOS: 29, 30, 35, and 32-34) described herein were used in each example, and palivizumab was used as a control antibody.

[0111] Example 1: Effect of anti-RGMa neutralizing antibodies on spasticity and urinary dysfunction in a rat spinal cord contusion model. (1-1) Preparation of a rat spinal cord contusion model. Female SD rats (11 weeks old at the time of model preparation) were used for this experiment. The animals were anesthetized with a triple anesthetic mixture consisting of medetomidine hydrochloride, midazolam, and butorphanol tartrate. The hair from the neck to the back was shaved, and then the area was cleaned with disinfectant ethanol and isodine. Anesthesia was maintained throughout the procedure using isoflurane inhalation anesthesia. The dorsal skin was incised to expose the 6th to 2nd lumbar vertebrae, and the 9th and 10th thoracic vertebrae were then resected. A 2.5 mm diameter, 10 g weight was immediately dropped from a height of 50 mm using a MASCIS Impactor (Rutgers University, USA) to injure the spinal cord. Starting on the day of spinal cord injury, cefazolin sodium was administered subcutaneously to the buttocks for seven days to prevent infection, and saline solution was administered subcutaneously to prevent dehydration. In addition, as a general rule, forced urination was performed twice a day (morning and afternoon) by manual bladder compression from the day after spinal cord injury until the end of the study. After the end of the cefazolin sodium administration period, from 8 days after spinal cord injury, if the urine color during forced urination showed signs of urinary tract infection, enrofloxacin was administered subcutaneously every day until the urine color returned to normal.

[0112] (1-2) Evaluation of Spasticity. The frequency of spasticity was assessed using a swimming test, which involves observing hindlimb and trunk spasms during swimming, according to the method described by Ryu et al. (PLos One 2017;12:e0171937). The swimming test was conducted in a rectangular acrylic tank (13 cm wide, 150 cm long, 40 cm high) with a water depth of 20–23 cm and a water temperature of 20–23°C. A trial was defined as the time from the animal's landing point to the end of the 100 cm straight line. A trial in which at least one clonus or spasm of the hindlimb or trunk was observed was considered a positive trial. Ten trials were conducted per animal at each time point, and the frequency (%) of positive trials was calculated. All swimming test trials were videotaped, and two observers independently assessed the spasticity behavior, blinded to the test substance administered. The average spasticity incidence (%) calculated by each observer was used as the representative value for that evaluation point. Based on the paper by Ryu et al. (PLos One 2017;12:e0171937), individuals with a representative spasticity incidence of 35% or higher were determined to have strong spasticity.

[0113] (1-3) Evaluation of spontaneous urination ability. Spontaneous urination was evaluated based on the records of bladder distension and urination during forced urination every morning. Spontaneous urination was determined to have occurred when there was no bladder distension and no urination was observed even when pressure was applied. Evaluation of spontaneous urination was continued up to 56 days after the creation of the rat spinal cord contusion model.

[0114] (1-4) Grouping and Administration of Anti-RGMa Neutralizing Antibodies Before spinal cord contusion, rats were assigned to each group so that their average body weights were uniform. The anti-RGMa neutralizing antibodies used were an anti-RGMa neutralizing antibody (specifically, Unasnemab) containing the amino acid sequence (a) (SEQ ID NOS: 5-10) and an anti-RGMa neutralizing antibody (specifically, Elezanumab) containing the amino acid sequence (f) (SEQ ID NOS: 29, 30, 35, and 32-34), both prepared by the methods described in International Publication WO 2016 / 175236 and International Publication WO 2013 / 112922, respectively. The anti-RGMa neutralizing antibodies were prepared at a dose of 3 ml / kg and administered as a bolus into the tail vein under isoflurane anesthesia. The first administration was administered immediately after spinal cord contusion, followed by weekly administrations for a total of eight doses. In the unasnemab study, mice were administered unasnemab (30 mg / kg per mouse) or a control antibody (palivizumab, 30 mg / kg per mouse). In the elezanumab study, mice were administered elezanumab (25 mg / kg per mouse) or a control antibody (palivizumab, 25 mg / kg per mouse).

[0115] (1-5) Results Effect on Spasticity Figure 1 shows the effect of repeated administration of Unasnemab on swimming-induced spasticity in a rat spinal cord contusion model, based on an analysis of the incidence of spasticity. At 4 and 6 weeks after spinal cord injury, the incidence of spasticity was lower in the Unasnemab 30 mg / kg group compared to the control antibody group. Figure 2 shows the effect of repeated administration of Unasnemab on swimming-induced spasticity in a rat spinal cord contusion model, based on an analysis of the proportion of individuals with severe spasticity. At 6 weeks after spinal cord injury, the proportion of individuals with severe spasticity in the Unasnemab 30 mg / kg group was significantly lower than in the control antibody group (Fisher's exact test). Figure 3 shows the effect of repeated administration of Elezanumab on swimming-induced spasticity in a rat spinal cord contusion model, based on an analysis of the incidence of spasticity. At 4 and 6 weeks after spinal cord injury, the incidence of spasticity in the elezanumab 25 mg / kg group was lower than in the control antibody group, and was significantly reduced at 6 weeks after spinal cord injury (Student's t-test). The effect of repeated administration of elezanumab on swimming-induced spasticity in a rat spinal cord contusion model is shown in Figure 4 by analyzing the proportion of individuals with severe spasticity. At 6 weeks after spinal cord injury, the proportion of individuals with severe spasticity in the elezanumab 25 mg / kg group was lower than in the control antibody group.

[0116] Effect on urinary dysfunction The effect of repeated administration of unasnemab on urinary dysfunction in a rat spinal cord contusion model is shown in Figure 5 as the cumulative number of days on which spontaneous urination was achieved. The cumulative number of days was higher in the unasnemab (30 mg / kg) group than in the control antibody group. The effect of repeated administration of unasnemab on urinary dysfunction in a rat spinal cord contusion model is shown in Figure 6 as an analysis of the total number of days on which spontaneous urination was achieved. The unasnemab group showed a dose-dependent increase in the number of days on which spontaneous urination was achieved, and the number of days on which spontaneous urination was achieved in the unasnemab 30 mg / kg group was significantly higher than in the control antibody group (Student's t-test). The effect of repeated administration of elezanumab on urinary dysfunction in a rat spinal cord contusion model is shown in Figure 7 as the cumulative number of days on which spontaneous urination was achieved. The cumulative number of days on which spontaneous urination was achieved in the elezanumab 25 mg / kg group was higher than in the control antibody group. The effect of repeated administration of elezanumab on urinary disturbance in a rat spinal cord contusion model is shown in Figure 8 as an analysis of the total number of days on which spontaneous urination was achieved. The number of days on which spontaneous urination was achieved in the elezanumab 25 mg / kg group was significantly increased compared to the control antibody group (Student's t-test).

[0117] Example 2 This clinical trial was a Phase Ib, randomized, double-blind, placebo-controlled study with the primary objective of investigating the safety, tolerability, and pharmacokinetics of 20-week administration of an anti-RGMa neutralizing antibody (specifically, Unasnemab) containing the amino acid sequence of (a) (SEQ ID NOS: 5-10) or a placebo in patients with HTLV-1-associated myelopathy (hereinafter referred to as HAM) with moderate to severe disease activity. The efficacy of Unasnemab was also exploratory investigated using the following indices: (1) Modified Ashworth Scale (hereinafter referred to as MAS) (knee extensor and flexor muscles) (2) Overactive Bladder Symptom Score (hereinafter referred to as OABSS)

[0118] The study enrolled 15 participants (10 in the active drug group and 5 in the placebo group). Participants who were deemed eligible for the treatment period were randomly assigned to either the active drug group or the placebo group in a 2:1 ratio. Cerebrospinal fluid (CSF) neopterin levels at screening were used as a stratification factor to stratify participants into those with moderate disease activity (6-43 pmol / mL) or those with severe disease activity (44 pmol / mL or higher). Active drug or placebo was administered intravenously over 1 hour at the start of the treatment period and at weeks 2, 4, 8, 12, 16, and 20. The study period ran from the date of consent acquisition to the end of the follow-up period. The screening period ran from the date of consent acquisition to the start of study drug administration on the start of the treatment period (the first dose of study drug), with a maximum of 6 weeks. The treatment period was the period from the start of administration of the study drug on the treatment start date (the date of first administration of the study drug) to the treatment end date (24 weeks of treatment or the date of discontinuation of treatment).The follow-up period was the period from the day after the treatment end date (24 weeks of treatment or the date of discontinuation of treatment) to 16 weeks after the final administration of the study drug.

[0119] (1) MAS (knee extensor and flexor muscles) MAS is an index for evaluating muscle tone and is rated on a six-point scale: 0, 1, 1+, 2, 3, and 4 (Kansai Rigaku. 2012;12:1-6, Phys Ther. 1987;67(2):206-7). MAS shown in Table 1 was evaluated for knee extensor and flexor muscles at the start of the treatment period (before administration) (baseline), 4 weeks, 12 weeks, and 24 weeks.

[0120] (2) OABSS: The OABSS is an assessment index used to evaluate overactive bladder (OCB) symptoms associated with urinary disorders, and assessed the condition over the past week. The frequency of each of the four questions in Table 2 was scored, with a total score ranging from 0 to 15. Severity was also assessed on a three-point scale: mild (0-5), moderate (6-11), and severe (12-15). Participants completed the OABSS questionnaire (UROLOGY 68(2): 318-323, 2006) at the start of the treatment period (pre-treatment) (baseline), 4 weeks, 12 weeks, and 24 weeks, and at the 12-week follow-up period, and the results were confirmed.

[0121] Results (1) MAS The distribution of MAS scores for both legs (2 limbs) at each evaluation point is shown in Figure 9. The percentage of both legs (2 limbs) that showed improvement in MAS over the 24-week treatment period was 55.6% (10 / 18 limbs) in the active drug group and 37.5% (3 / 8 limbs) in the placebo group, showing a trend toward improvement in spasticity in the active drug group.

[0122] (2) OABSS: Tables 3-1 and 3-2 show the OABSS score at each evaluation point and the change from baseline, and Table 4 shows the severity shift table. The LS Mean ± SE of the change from baseline in OABSS score after 24 weeks of treatment was -1.6 ± 0.5 in the active drug group and 1.6 ± 0.8 in the placebo group. The active drug group showed a greater improvement from baseline than the placebo group, indicating a trend toward improvement in neurogenic bladder. The percentage of subjects whose severity improved after 24 weeks of treatment was 22.2% (2 / 9) in the active drug group and 0% (0 / 4) in the placebo group. The number of subjects whose symptoms worsened was 25.0% (1 / 4) in the placebo group and none (0 / 9) in the active drug group.

[0123] A subgroup analysis was performed on participants whose baseline HAM-BDSG (HAM's assessment index for the severity of urinary dysfunction) was Grade 0 (no treatment and no lower urinary tract symptoms) or Grade 1 (lower urinary tract symptoms or medical treatment), excluding participants undergoing intermittent catheterization. Tables 5-1 and 5-2 show the OABSS score and change from baseline at each assessment time point, and Table 6 shows a severity shift table. The mean ± SD change from baseline in OABSS at 24 weeks of treatment was -1.4 ± 2.3 in the active drug group and 0.5 ± 0.7 in the placebo group. Even within this subgroup, the active drug group showed a greater improvement from baseline compared with the placebo group, indicating a trend toward improvement in neurogenic bladder. The proportion of participants whose severity improved at 24 weeks of treatment was 25.0% (2 / 8) in the active drug group and 0% (0 / 2) in the placebo group; no worsening symptoms were observed in either group.

[0124] <Explanation of the sequence listing> SEQ ID NO: 1: Amino acid sequence of human RGMa precursor protein SEQ ID NO: 2: Amino acid sequence of mouse RGMa precursor protein SEQ ID NO: 3: Amino acid sequence of rat RGMa precursor protein SEQ ID NO: 4: DNA sequence of human RGMa gene SEQ ID NO: 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 8: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 9: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 10: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 11: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 12: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 13: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 14: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 15: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 16: Amino acid sequence of human RGMa epitope 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 AE12-1SEQ ID NO: 30: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 31: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 32: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 33: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 34: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 35: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1Y SEQ ID NO: 36: Amino acid sequence of epitope of human RGMa SEQ ID NO: 37: Amino acid sequence of epitope of human RGMa SEQ ID NO: 38: Amino acid sequence of epitope of human RGMa SEQ ID NO: 39: Amino acid sequence of epitope of human RGMa SEQ ID NO: 40: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1F SEQ ID NO: 41: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1H SEQ ID NO: 42: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1L SEQ ID NO: 43: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1V SEQ ID NO: 44: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1I SEQ ID NO: 45: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1K SEQ ID NO: 46: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab) SEQ ID NO: 47: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab) SEQ ID NO: 48: Light chain amino acid sequence of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab) SEQ ID NO: 49: Heavy chain amino acid sequence of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab) SEQ ID NO: 50: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 51: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 52: Light chain amino acid sequence of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 53: Heavy chain amino acid sequence of anti-RGMa neutralizing antibody r70E4 SEQ ID NO: 54: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 55: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody 5F9SEQ ID NO: 56: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 57: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 58: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 59: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 60: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody AE12-1Y SEQ ID NO: 61: Light chain amino acid sequence of anti-RGMa neutralizing antibody AE12-1Y-QL (Elezanumab) SEQ ID NO: 62: Heavy chain amino acid sequence of anti-RGMa neutralizing antibody AE12-1Y-QL (Elezanumab)

[0125] The present invention is expected to be useful in preventing or treating accompanying symptoms associated with diseases caused by spinal cord or brain damage, specifically spasticity and / or neurogenic bladder, and is of great utility in the pharmaceutical industry.

Claims

1. A preventive or therapeutic agent for spasticity which contains an RGMa inhibitor as an active ingredient.

2. The preventive or therapeutic agent according to claim 1, wherein the spasticity is associated with a disease caused by a spinal cord or brain disorder.

3. The preventive or therapeutic agent according to claim 1, wherein the spasticity is associated with spinal cord injury.

4. The preventive or therapeutic agent according to claim 1, wherein the spasticity is associated with HTLV-1-associated myelopathy.

5. The agent according to any one of claims 1 to 4, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

6. The agent according to claim 5, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

7. The agent described in 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, SEQ ID NO: 38 and SEQ ID NO:

39.

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

34.

9. The agent according to any one of claims 5 to 8, wherein the anti-RGMa neutralizing antibody is the following (a) or (f): (a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; (f) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:35, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

34.

10. A method for preventing or treating spasticity, comprising administering to a mammal in need of treatment an effective amount of an RGMa inhibitor.

11. The preventive or therapeutic method according to claim 10, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

12. Use of an RGMa inhibitor for the manufacture of an agent for the prevention or treatment of spasticity.

13. The use according to claim 12, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

14. A preventive or therapeutic agent for spasticity and neurogenic bladder, comprising an RGMa inhibitor as an active ingredient.

15. The preventive or therapeutic agent according to claim 14, wherein the spasticity and neurogenic bladder are associated with a disease caused by a spinal cord or brain disorder.

16. The preventive or therapeutic agent according to claim 14, wherein the spasticity and neurogenic bladder are those associated with spinal cord injury.

17. The preventive or therapeutic agent according to claim 14, wherein the spasticity and neurogenic bladder are those associated with HTLV-1-associated myelopathy.

18. The agent according to any one of claims 14 to 17, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

19. The agent according to claim 18, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

20. The agent described in claim 18 or 19, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:

39.

21. An anti-RGMa neutralizing antibody, comprising any one of the following (a) to (l): (a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; (b) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:11, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:12, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:13, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:14, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and an HCDR3 whose amino acid sequence includes SFG; (c) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; (d) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; (e) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34;(f) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34;(k) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:44, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:34; and (l) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:45, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

34.

22. The agent according to any one of claims 18 to 21, wherein the anti-RGMa neutralizing antibody is the following (a) or (f): (a) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:10; (f) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:35, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

34.

23. A method for preventing or treating spasticity and neurogenic bladder, comprising administering to a mammal in need of treatment an effective amount of an RGMa inhibitor.

24. The preventive or therapeutic method according to claim 23, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

25. Use of an RGMa inhibitor for the manufacture of an agent for the prevention or treatment of spasticity and neurogenic bladder.

26. The use according to claim 25, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

27. A preventive or therapeutic agent for neurogenic bladder, comprising an RGMa inhibitor as an active ingredient.

28. The preventive or therapeutic agent according to claim 27, wherein the neurogenic bladder is associated with a disease caused by a spinal cord or brain disorder.

29. The preventive or therapeutic agent according to claim 27, wherein the neurogenic bladder is neurogenic bladder associated with spinal cord injury.

30. The preventive or therapeutic agent according to claim 27, wherein the neurogenic bladder is neurogenic bladder associated with HTLV-1-associated myelopathy.

31. The agent according to any one of claims 27 to 30, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

32. The agent according to claim 31, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

33. The agent described in any one of claims 31 or 32, wherein the anti-RGMa neutralizing antibody is an anti-RGMa neutralizing antibody comprising the following (a): (a) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

10.

34. A method for preventing or treating neurogenic bladder, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of treatment.

35. The preventive or therapeutic method according to claim 34, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

36. Use of an RGMa inhibitor for the manufacture of an agent for the prevention or treatment of neurogenic bladder.

37. The use according to claim 36, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

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