Prophylactic or therapeutic agent for accompanying symptoms associated with diseases caused by spinal marrow or brain disorder

Abstract

The present invention provides a prophylactic or therapeutic agent for accompanying symptoms associated with diseases caused by spinal marrow or brain disorders, such as spinal marrow injury or HTLV-1-associated spinal marrow disease, which contains an RGMa inhibitor. Also provided is a prophylactic or therapeutic agent for spasmodic or neurogenic bladder, said prophylactic or therapeutic agent containing an RGMa inhibitor.

Description

Technical Field

[0001] This invention relates to a preventive or therapeutic agent for accompanying symptoms of diseases caused by disorders of the spinal cord or brain, containing an RGMa inhibitor. Background Technology

[0002] It is known that after a certain period of central nervous system injury, excessive muscle contraction due to hyperactive spinal reflexes can lead to spasticity. Spasticity is not only caused by head trauma and spinal cord injury, but also by various central nervous system disorders of the spinal cord and brain, as well as by multiple sclerosis, spinocerebellar degeneration, HTLV-I-related spinal cord disease (HAM), spinal cord disease, stroke, brain tumors, cerebral palsy, and hypoxic encephalopathy. Reports indicate that in Japan, there are an estimated 80,000 cases of severe spasticity due to sequelae of head trauma, spinal cord injury, cerebral palsy, and multiple sclerosis (Non-Patent Literature 1), but the exact prevalence of spasticity in Japan remains unclear. Overseas reports indicate that approximately 70% of patients with spinal cord injury experience spasticity (Non-Patent Literature 2, Non-Patent Literature 3), impacting rehabilitation and quality of life (QOL). In addition, it has been reported that 30-90% of patients with multiple sclerosis experience spasms (Non-Patent Literature 4).

[0003] Lesions of the central nervous system (brain or spinal cord) can manifest as positive or negative signs in upper motor neuron syndrome. Positive signs include increased muscle tension, hyperactive tendon reflexes, stretch reflexes affecting other muscles, spasticity (clonic movements), spastic dystonia, pathological synkinesis or simultaneous contraction, and hyperactive knee flexor reflexes. Negative signs include paralysis, decreased muscle strength, lack of flexibility, and strenuous movement. While many cases of positive signs in upper motor neuron syndrome are termed spasticity, Lance's definition (Non-Patent Literature 5) is narrower: spasticity is considered a positive sign of upper motor neuron syndrome, defined as a motor disorder characterized by a velocity-dependent increase in tonic stretch reflexes (muscle tension) accompanied by hyperactive tendon reflexes.

[0004] Spasticity presents with a variety of complex symptoms depending on the location of the underlying disease or impairment. Spastic paralysis is a contributing symptom of interest; however, numerous cases have been found where the severity of spasticity and paralysis is not parallel, suggesting that the neural mechanisms underlying the two symptoms differ (Non-Patent Literature 6). For example, reports have shown that the severity of impairment does not correlate with the degree of spasticity in patients or animal models of spinal cord injury (Non-Patent Literature 7, Non-Patent Literature 8).

[0005] In addition, it takes a certain amount of time from the occurrence of the cause to the onset of the spasm. As a reason, it is reported that there may be some kind of neural plasticity and reorganization (Non-Patent Literature 9). The specific pathophysiological mechanism of the spasm has not yet been elucidated.

[0006] Treatment for spasticity is primarily based on physical therapy. For severe spasticity, muscle relaxants, nerve block therapy, and intrathecal baclofen injection therapy may be considered (Non-Patent Literature 10). Specifically, this includes manual or exercise therapy using equipment such as a standing correction platform, physical therapy utilizing cold, heat, low-frequency electric shock, and vibration stimulation, and orthotic therapy (Non-Patent Literature 11). In cases of severe symptoms, drug therapy with muscle relaxant effects may be used. Tizanidine, eperisone, baclofen, diazepam, gabapentin, and dantrolene sodium may be used, but it is important to note that these drugs have side effects such as drowsiness and muscle weakness (Non-Patent Literature 12, Non-Patent Literature 13). In recent years, when drug therapy is insufficient, nerve block therapy based on botulinum toxin injections has been used for overly tense muscles (Non-Patent Literature 14, Non-Patent Literature 15). However, the injection interval must be at least 3 months, making it difficult to adjust the injection dosage to achieve a sustained effect. For severe spasticity, ITB (intrathecal baclofen therapy) (Non-Patent Literature 16) was also investigated. A continuous infusion pump with sustained delivery was administered intracavitarily at the experimental level, with pump implantation performed only if an effect was observed.

[0007] Thus, existing treatments for spasticity may not be satisfactory, and there is an urgent need for better spasticity treatments to reduce the burden on patients and caregivers and improve their quality of life (QOL).

[0008] Neurogenic bladder, which leads to urinary dysfunction, is a bladder dysfunction caused by nerve damage, including to the spinal cord, and is accompanied by lower urinary tract symptoms. Lower urinary tract function can be divided into storage and voiding, which are respectively classified as bladder function and urethral function, and are regulated through coordination. That is, during storage, the intravesical pressure is maintained at a low pressure, the urethra is always closed, and the urethral closure pressure remains higher than the intravesical pressure. On the other hand, during voiding, urination can be initiated voluntarily; the urethra opens simultaneously with bladder contraction, expelling urine.

[0009] Urinary dysfunction is caused by decreased contractility of the bladder's smooth muscle or increased urethral resistance, accompanied by symptoms such as decreased urine volume, urine stream bifurcation, interrupted urine flow, delayed urination, urination due to abdominal pressure, and dribbling at the end of urination. In Japan, anticholinergic drugs, cholinergic drugs, and α1-adrenergic receptor antagonists are used to treat neurogenic bladder. However, to improve neurogenic bladder, anticholinergic drugs require higher doses than usual, leading to concerns about side effects. α1-adrenergic receptor antagonists and cholinergic drugs have not shown clear efficacy in randomized trials, lacking sufficient evidence and raising concerns about serious adverse effects such as cholinergic crisis. Furthermore, while the efficacy of β3-adrenergic receptor agonists has been reported, these are not currently applicable due to a lack of clear evidence obtained through randomized trials or similar methods (Non-Patent Literature 17, Non-Patent Literature 18).

[0010] Based on the above, we hope to find more effective treatments for neurogenic bladder or its symptoms caused by increased urethral resistance or decreased bladder smooth muscle contractility.

[0011] RGM (repulsive guidance molecule) is a membrane protein initially identified as an axon-inducing molecule in the visual system (Non-Patent Literature 19). The RGM family is known to include three members: RGMa, RGMb, and RGMc (Non-Patent Literature 20), with at least RGMa and RGMb operating through the same signaling mechanism (Non-Patent Literature 21). RGMc plays a crucial role in iron metabolism.

[0012] Subsequent studies have shown that RGM has functions such as axon induction and layer formation in Xenopus laevis and chicken embryos, as well as control of the closure of the neural tube in the head of mouse embryos (Non-Patent Document 22). Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0013] In addition to its developmental functions, RGMa is re-expressed after central nervous system injury in adults and rats. In rats, inhibition of RGMa leads to increased axonal growth following spinal cord injury, promoting functional recovery (Non-Patent Document 23). Therefore, RGMa is considered an inhibitor of axonal 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).

[0014] Thus, the role of RGMa in central nervous system injury is clear, but its involvement in spasticity and neurogenic bladder has not been established, and such treatments are unknown.

[0015] Existing technical documents

[0016] Patent documents

[0017] Patent Document 1: International Publication No. WO2005 / 087268

[0018] Patent Document 2: International Publication No. WO2009 / 106356

[0019] Patent Document 3: International Publication No. WO2013 / 112922

[0020] Patent Document 4: International Publication No. WO2016 / 175236

[0021] Non-patent literature

[0022] Non-patent literature 1: Hirai Takayoshi, Akikawa Hiroyuki, Okada Yoshikazu, et al. Epidemiological survey of spasticity in Japan. Rehabilitation Medicine 2000; 37(11): 863

[0023] Non-patent literature 2: Archives of Physical Medicine and Rehabilitation 2017;98:1132-8

[0024] Non-patent literature 3: Spinal Cord (2016) 54, 973-979

[0025] Non-patent literature 4: Multiple Sclerosis Journal 2004 Oct;10(5):589-95

[0026] Non-patent literature 5: Spasticity: Disordered Motor Control. Chicago, Year Book Medical Publishers, 1980, pp. 485-494.

[0027] Non-patent literature 6: Reisaku Tanaka: The neural mechanisms of spasticity - a follow-up study. Rehabilitation Medicine 1995; 32: 97-105.

[0028] Non-patent literature 7: J Neurophysiol 128: 470-479, 2022

[0029] Non-patent literature 8: PLoS ONE 12(2): e0171937. doi:10.1371 / journal.pone.0171937

[0030] Non-patent literature 9: Nat Neurosci 2004; 7: 269-277

[0031] Non-Patent Literature 10: Guidelines for the Diagnosis and Treatment of Multiple Sclerosis and Neurospinal Optic Disease (2017)

[0032] Non-patent literature 11: Expert Review of Neurotherapeutics, 13 (12 Suppl), 55-59 (2013)

[0033] Non-patent literature 12: Clinical Neuroscience, 32, 1296-1298 (2014)

[0034] Non-patent literature 13: Neurology, 51(2), 609-611 (1998)

[0035] Non-patent literature 14: Journal of Neurology, 253 (suppl 1), i16-i20 (2006)

[0036] Non-patent literature 15: Journal of Neurology, 253 (suppl 1), i26-i28 (2006)

[0037] Non-patent literature 16: Journal of Neurosurgery, 21(2), e6 (2006)

[0038] Non-patent literature 17: Journal of Pharmacological Sciences, 112, 121-127 (2010)

[0039] Non-Patent Literature 18: Guidelines for the Diagnosis and Treatment of Lower Urinary Tract Dysfunction in Spinal Cord Injury, 2019

[0040] Non-patent literature 19: 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)

[0041] Non-patent literature 20: Mueller et al., Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513‐29, 2006

[0042] Non-patent literature 21: Liu,

[0043] Non-patent literature 22: Yamashita, T., Mueller, BK & Hata, K. Neogenin and repulsive guidance molecule signaling in the central nervous system. Curr. Opin. Neurobiol. 17, 29-34 (2007).

[0044] Non-patent literature 23: Hata, K. et al. RGMa inhibition promotes axonal growth and recovery after spinal cord injury. J. Cell Biol. 173, 47-58 (2006). Summary of the Invention

[0045] The objective of this invention is to provide a medicament effective for the accompanying symptoms of diseases caused by disorders of the spinal cord or brain.

[0046] In order to solve the above-mentioned problems, the inventors conducted in-depth research and found that RGMa inhibitors, especially anti-RGMa neutralizing antibodies, can be expected to have therapeutic or preventive effects on the accompanying symptoms of diseases caused by spinal cord or brain disorders, such as spinal cord injury, HTLV (Human T-lymphotropic Virus)-1-related spinal cord disease, and other diseases caused by spinal cord or brain injury or inflammation. Thus, the present invention was completed.

[0047] That is, the present invention is as follows.

[0048] 1. A preventive or therapeutic agent for spasticity, with RGMa inhibitory substance as the active ingredient.

[0049] 2. According to the preventive or therapeutic agent of 1 above, wherein the spasm is a spasm associated with a disease caused by a disorder of the spinal cord or brain.

[0050] 3. According to the prevention or treatment agent in 1 above, wherein the spasticity is a spasticity associated with spinal cord injury.

[0051] 4. Based on the prevention or treatment of the above 1, wherein the spasticity is the spasticity associated with HTLV-1 related spinal cord disease.

[0052] 5. The preventive or therapeutic agent according to any one of 1 to 4 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0053] 6. The preventive or therapeutic agent according to 5 above, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

[0054] 7. The preventive or therapeutic agent according to 5 or 6 above, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from serial numbers 16, 36, 37, 38 and 39.

[0055] 8. The preventive or therapeutic agent according to any one of 5 to 7 above, wherein the anti-RGMa neutralizing antibody is an antibody selected from (a) to (l) below:

[0056] (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10;

[0057] (b) An anti-RGMa neutralizing antibody comprising the following light chain variable region and heavy chain variable region, wherein the light chain variable region comprises: LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12 and LCDR3 containing the amino acid sequence described in sequence number 13, and the heavy chain variable region comprises: HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15 and HCDR3 containing SFG in the amino acid sequence;

[0058] (c) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22;

[0059] (d) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28;

[0060] (e) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0061] (f) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0062] (g) An anti-RGMa neutralizing antibody comprising the following light chain variable region and heavy chain variable region, wherein the light chain variable region comprises: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40, and the heavy chain variable region comprises: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0063] (h) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0064] (i) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 42, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0065] (j) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0066] (k) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44; and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; and

[0067] (l) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 45, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

[0068] 9. The agent according to any one of 5 to 8 above, wherein the anti-RGMa neutralizing antibody is one of the following (a) or (f):

[0069] (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10;

[0070] (f) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

[0071] 10. A method for preventing or treating spasms, comprising the steps of administering an effective amount of RGMa inhibitor to a mammal requiring treatment.

[0072] 11. The prevention or treatment method according to 10 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0073] 12. The use of an RGMa inhibitor for the manufacture of a preventative or therapeutic agent for spasticity.

[0074] 13. The use as described in 12 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0075] 14. A preventive or therapeutic agent for spasmodic and neurogenic bladder, with an RGMa inhibitor as the active ingredient.

[0076] 15. The preventive or therapeutic agent according to 14 above, wherein spasm and neurogenic bladder are spasm and neurogenic bladder associated with diseases caused by disorders of the spinal cord or brain.

[0077] 16. According to the preventive or therapeutic agents of 14 above, wherein spasticity and neurogenic bladder are spasticity and neurogenic bladder associated with spinal cord injury.

[0078] 17. According to the prevention or treatment of the above 14, spasticity and neurogenic bladder are spasticity and neurogenic bladder associated with HTLV-1 related spinal cord disease.

[0079] 18. The preventive or therapeutic agent according to any one of 14 to 17 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0080] 19. The preventive or therapeutic agent according to 18 above, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

[0081] 20. The preventive or therapeutic agent according to 18 or 19 above, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from serial numbers 16, 36, 37, 38 and 39.

[0082] 21. The preventive or therapeutic agent according to any one of 18 to 20 above, wherein the anti-RGMa neutralizing antibody is an antibody selected from (a) to (l) below:

[0083] (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10;

[0084] (b) An anti-RGMa neutralizing antibody comprising the following light chain variable region and heavy chain variable region, wherein the light chain variable region comprises: LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12 and LCDR3 containing the amino acid sequence described in sequence number 13, and the heavy chain variable region comprises: HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15 and HCDR3 containing SFG in the amino acid sequence;

[0085] (c) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22;

[0086] (d) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28;

[0087] (e) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0088] (f) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0089] (g) An anti-RGMa neutralizing antibody comprising the following light chain variable region and heavy chain variable region, wherein the light chain variable region comprises: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40, and the heavy chain variable region comprises: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0090] (h) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0091] (i) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 42, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0092] (j) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0093] (k) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44; and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; and

[0094] (l) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 45, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

[0095] 22. The preventive or therapeutic agent according to any one of 18 to 21 above, wherein the anti-RGMa neutralizing antibody is one of the following (a) or (f):

[0096] (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10;

[0097] (f) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

[0098] 23. A method for the prevention or treatment of spasmodic and neurogenic bladder, comprising the step of administering an effective amount of RGMa inhibitor to the mammal requiring treatment.

[0099] 24. The prevention or treatment method described in 23 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0100] 25. The use of an RGMa inhibitor for the manufacture of a preventative or therapeutic agent for spasmodic and neurogenic bladder.

[0101] 26. The use according to 25 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0102] 27. A preventive or therapeutic agent for neurogenic bladder, comprising an RGMa inhibitor as the active ingredient.

[0103] 28. The preventive or therapeutic agent according to 27 above, wherein neurogenic bladder is a neurogenic bladder associated with a disease caused by a disorder of the spinal cord or brain.

[0104] 29. The preventive or therapeutic agents according to 27 above, wherein neurogenic bladder is neurogenic bladder associated with spinal cord injury.

[0105] 30. According to the preventive or therapeutic agents of 27 above, wherein neurogenic bladder is neurogenic bladder associated with HTLV-1-related spinal cord disease.

[0106] 31. The preventive or therapeutic agent according to any one of 27 to 30 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0107] 32. The preventive or therapeutic agent according to 31 above, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

[0108] 33. The preventive or therapeutic agent according to any one of 31 or 32 above, wherein the anti-RGMa neutralizing antibody is as follows (a):

[0109] (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10.

[0110] 34. A method for preventing or treating neurogenic bladder, comprising the steps of administering an effective amount of RGMa inhibitor to a mammal requiring treatment.

[0111] 35. The prevention or treatment method according to 34 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0112] 36. The use of an RGMa inhibitor for the manufacture of a preventive or therapeutic agent for neurogenic bladder.

[0113] 37. The use as described in 36 above, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[0114] According to the present invention, RGMa inhibitors, especially anti-RGMa neutralizing antibodies, are useful as preventive or therapeutic agents for accompanying symptoms of diseases caused by disorders of the spinal cord or brain, such as spinal cord or brain injury or inflammation. Specifically, examples of such accompanying symptoms include spasms or neurogenic bladder.

[0115] According to the present invention, RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies, are useful as preventive or therapeutic agents for spasmodic and / or neurogenic bladder. Attached Figure Description

[0116] Figure 1 This figure illustrates the effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Unasnemab) containing the amino acid sequence (sequence number 5–10) of (a) on improving swimming load-induced spasticity in a rat spinal cord contusion model, using spasticity frequency analysis.

[0117] Figure 2 This table illustrates the ameliorative effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Unasnemab) containing the amino acid sequence (sequence numbers 5–10) of (a) on swimming load-induced spasticity in a rat spinal cord contusion model, based on a proportion analysis of individuals exhibiting severe spasticity.

[0118] Figure 3This figure illustrates the effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Elezanumab) containing the amino acid sequence (sequence numbers 29, 30, 35, and 32–34) of (f) on improving swimming load-induced spasticity in a rat spinal cord contusion model, using spasticity frequency analysis.

[0119] Figure 4 This table shows the effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Elezanumab) containing the amino acid sequence (sequence numbers 29, 30, 35, and 32–34) of (f) on improving swimming load-induced spasticity in a rat spinal cord contusion model, based on a proportion analysis of individuals exhibiting strong spasticity.

[0120] Figure 5 This is a graph illustrating the effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Unasnemab) containing the amino acid sequence (sequence number 5–10) of (a) on urination disorders in a rat spinal cord contusion model, by measuring the cumulative number of days of spontaneous urination.

[0121] Figure 6 This table illustrates the effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Unasnemab) containing the amino acid sequence (sequence numbers 5–10) of (a) on urination disorders in a rat spinal cord contusion model, by analyzing the total number of days of spontaneous urination.

[0122] Figure 7 This is a graph illustrating the effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Elezanumab) containing the amino acid sequence (sequence numbers 29, 30, 35, and 32–34) on urination dysfunction in a rat spinal cord contusion model, by measuring the cumulative number of days of spontaneous urination.

[0123] Figure 8 This table illustrates the effect of repeated administration of an anti-RGMa neutralizing antibody (specifically Elezanumab) containing the amino acid sequence (sequence numbers 29, 30, 35, and 32–34) on urination dysfunction in a rat spinal cord contusion model, based on an analysis of the total number of days of spontaneous urination.

[0124] Figure 9 This is a graph showing the MAS distribution of both feet (two limbs) at baseline, 4 weeks, 12 weeks, and 24 weeks in the Unasnemab-treated group and the placebo group. Detailed Implementation

[0125] The terminology used in this invention will be explained below.

[0126] [Neutralization]

[0127] In this application, neutralization refers to the effect of binding to a target site and inhibiting any function of that target site. For example, an RGMa inhibitor is a substance that binds to RGMa and thus exhibits an inhibitory effect on the biological activity of RGMa.

[0128] [tablet]

[0129] In this application, the epitope comprises a polypeptide determinant that can specifically bind to immunoglobulins or T-cell receptors. In one embodiment, the epitope contains a chemically active surface group of the molecule (e.g., an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group), and in another embodiment, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that binds to an antibody.

[0130] [Separate]

[0131] In this application, "separated" in the context of isolated RGMa inhibitors (e.g., antibodies) means identified and separated and / or recovered from components in their natural state. Impurities in their natural state are substances that can inhibit the diagnostic or therapeutic use of the antibody, such as enzymes, hormones, and other protein- or non-protein-based solutes. Generally, to separate RGMa inhibitors, purification can be performed through at least one purification step, and RGMa inhibitors purified through at least one purification step can be referred to as "separated RGMa inhibitors."

[0132] [Antibody]

[0133] In this application, antibody, in a broad sense, refers to an Ig molecule consisting of four polypeptide chains, namely two heavy chains (H chains) and two light chains (L chains), which retain the substantial epitope-binding characteristic of immunoglobulin (Ig) molecules.

[0134] [Human Antibody]

[0135] In this application, human antibody refers to an antibody whose light chain and heavy chain are both derived from human immunoglobulins. Human antibodies, depending on the constant region of the heavy chain, include IgG with a γ chain (including IgG1, IgG2, IgG3, and IgG4), IgM with a μ chain, IgA with an α chain (including IgA1 and IgA2), IgD with a δ chain, or IgE with an ε chain. Furthermore, in principle, the light chain includes either a κ chain or a λ chain.

[0136] [Humanized Antibody]

[0137] In this application, a humanized antibody refers to an antibody that includes a variable region consisting of a complementarity-determining region of a non-human animal-derived antibody and a framework region of a human antibody, as well as a constant region of a human antibody.

[0138] [Chimeric antibodies]

[0139] In this application, chimeric antibody refers to an antibody consisting of a light chain, a heavy chain, or both, composed of a non-human variable region and a human constant region.

[0140] [Monospecific antibody]

[0141] In this application, a monospecific antibody refers to an antibody that has a single antigen specificity and a single independent antigen recognition site. For example, in this specification, a monospecific antibody that recognizes RGMa may sometimes be referred to as an RGMa monospecific antibody.

[0142] [Multispecific antibody]

[0143] In this application, a multispecific antibody refers to an antibody that has two or more different antigen specificities and two or more independent antigen recognition sites. Examples include bispecific antibodies with two antigen specificities and trispecific antibodies with three antigen specificities.

[0144] [Complementarity Determining Region (CDR)]

[0145] The complementarity-determining region (CDR) is the region within the variable region of an immunoglobulin molecule that forms the antigen-binding site. Also known as the hypervariable region, it is the part of each immunoglobulin molecule where the amino acid sequence varies considerably. Each light and heavy chain has three CDRs. The three CDRs in the light chain are designated LCDR1, LCDR2, and LCDR3, and the three CDRs in the heavy chain are designated HCDR1, HCDR2, and HCDR3. For example, the CDRs of immunoglobulin molecules are determined using the Kabat numbering system (Kabat et al., 1987 and 1991, Sequences of Proteins of Immunological Interest, USDA Department of Health and Human Services, NIH, USA). In this specification, unless otherwise specified, CDR sequences defined according to the Kabat numbering system are used.

[0146] Furthermore, the CDR sequences of serial numbers 5-15 use CDR sequences defined according to the IMGT numbering system (Lefranc M.-P., ImmunologyToday 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 will be clear to those skilled in the art that the CDR sequences described according to the IMGT numbering system can be replaced with any other numbering system, such as the Kabat numbering system. Therefore, the invention of replacing the CDR sequences described according to the IMGT numbering system with the Kabat numbering system is included in the embodiments of this invention. Furthermore, the invention of replacing the CDR sequence recorded according to the Kabat numbering system with the IMGT numbering system is also included in the embodiments of the present invention.

[0147] [Effective Quantity]

[0148] An effective dose refers to a dose of a preventive or therapeutic agent that is sufficient to reduce or improve the severity and / or duration of a disorder or one or more of its symptoms, prevent the development of a disorder, cause a relapse of a disorder, prevent the recurrence, occurrence, onset or progression of one or more of the symptoms associated with a disorder, detect a disorder, or enhance or improve the preventive or therapeutic effect of one or more other treatments (e.g., preventive or therapeutic agents).

[0149] [Percentage (%) of amino acid sequence homology]

[0150] The "percentage (%) homology" of the amino acid sequence of a candidate polypeptide sequence, such as a variable region, relative to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of a specific reference polypeptide sequence, after aligning the sequences and introducing vacancies as needed to obtain maximum % homology, excluding any conserved substitutions. Alignment used to determine % homology can be performed using various methods within the skill scope of those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). Appropriate parameters for sequence alignment can be determined by those skilled in the art, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared. However, for the purpose described here, the % homology value is obtained in paired alignments using the sequence comparison computer program BLAST.

[0151] When using BLAST for amino acid sequence comparison, the % homology between provided amino acid sequence A and provided amino acid sequence B is calculated as follows:

[0152] 100 times the fraction X / Y

[0153] Here, X represents the number of amino acid residues that scored identically in the BLAST sequence alignment program for sequences A and B, and Y represents the total number of amino acid residues in sequence B. When the lengths of amino acid sequences A and B differ, it should be understood that the % homology of A relative to B differs from the % homology of B relative to A. Unless otherwise specified, all % homology values ​​here are obtained using the BLAST computer program as described in the previous paragraph.

[0154] [Conservative Replacement]

[0155] Conservative substitution refers to the replacement of an amino acid residue with another amino acid residue of similar chemical properties in a manner that substantially does not alter the peptide's activity. Examples include replacing a hydrophobic residue with another hydrophobic residue, or replacing a polar residue with another polar residue having the same charge. Examples of functionally similar amino acids that can undergo such substitutions include, as nonpolar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine; as polar (neutral) amino acids, glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine; as positively charged (basic) amino acids, arginine, histidine, and lysine; and as negatively charged (acidic) amino acids, aspartic acid and glutamic acid.

[0156] [treat]

[0157] Treatment includes any treatment of diseases of the target, preferably mammals, especially humans, including stopping the development of diseases and symptoms, eliminating, curing, reducing or alleviating such diseases and symptoms.

[0158] [prevention]

[0159] Prevention includes preventing or inhibiting the onset of the aforementioned diseases in the treatment subject, preferably mammals, and especially humans. Furthermore, "prevention" in this invention includes "preventing relapse" of the aforementioned diseases that involve repeated relapses and improvements in the treatment subject, preferably mammals, and especially humans.

[0160] The embodiments of the present invention will now be described in detail.

[0161] This invention provides novel uses for RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, as preventive or therapeutic agents for accompanying symptoms of diseases caused by spinal cord or brain disorders, such as spinal cord or brain injury or inflammation.

[0162] In addition, the present invention provides a method for preventing or treating accompanying symptoms of diseases caused by disorders of the spinal cord or brain, such as spinal cord or brain injury or inflammation, the method comprising the step of administering to a mammal requiring treatment a preventive or therapeutic agent containing an effective amount of an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody.

[0163] <RGMa Inhibiting Substance>

[0164] The RGMa inhibitor of the present invention can be any substance that acts on RGMa itself to inhibit or weaken the activity of RGMa (hereinafter, in this specification, it is sometimes simply referred to as "RGMa activity"). For example, substances that have the activity of directly inhibiting (weakening) RGMa activity by binding to RGMa, or the activity of indirectly inhibiting (weakening) RGMa activity by inhibiting the binding of RGMa to the receptor (e.g., compounds or antibodies described later) are called the RGMa inhibitors of the present invention.

[0165] In addition, the RGMa inhibitor of the present invention can be a substance that inhibits RGMa expression. For example, substances that inhibit RGMa expression or inhibit (weaken) RGMa activity (e.g., nucleic acid molecules described later) are also included in the RGMa inhibitor of the present invention.

[0166] RGMa is identified as a neurite growth inhibitor protein of the central nervous system. Human RGMa protein, as shown in sequence number 1, is biosynthesized as a precursor protein consisting of 450 amino acids. The N-terminal signal peptides Met1–Pro47 (the peptide consisting of the methionine residue at position 1 to the proline residue at position 47 from the N-terminus, hereinafter referred to as such) are removed. The peptide bond between Asp168 and Pro169 is cleaved to generate an N-terminal domain. Furthermore, the C-terminal peptides Ala425–Cys450 of the C-terminal segment of Pro169 are removed, and a GPI-anchored protein is attached to the C-terminal carboxyl group of Ala424, forming a C-terminal domain. Human RGMa protein is expressed on the cell membrane as a mature protein, consisting of the N-terminal domains (Cys48–Asp168) and the C-terminal domains (Pro169–Ala424) linked by disulfide bonds, via a GPI-anchored protein.

[0167] In this invention, RGMa can be of any animal origin, preferably human RGMa. The precursor protein of human RGMa consists of the amino acid sequence shown in sequence number 1 of the sequence listing. The precursor protein of mouse RGMa consists of the amino acid sequence shown in sequence number 2 of the sequence listing, and the precursor protein of rat RGMa consists of the amino acid sequence shown in sequence number 3 of the sequence listing. Since the C-terminal peptide is removed, the mature protein will have the same amino acid sequence.

[0168] Examples of RGMa genes include, for instance, the human RGMa gene, which consists of the base sequence shown in sequence number 4, but are not limited to this. The base sequences of RGM genes from various biological sources can be readily obtained from well-known databases (GenBank, etc.).

[0169] Specifically, examples of RGMa inhibitors according to the present invention include low molecular weight compounds, anti-RGMa neutralizing antibodies, functionally altered antibodies, conjugated antibodies, or antigen-binding fragments thereof. Additionally, examples include RGMa nucleic acid molecules such as siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense oligonucleotides. Among these RGMa inhibitors, anti-RGMa neutralizing antibodies, functionally altered antibodies, conjugated antibodies, and antigen-binding fragments thereof are preferred, more preferably anti-RGMa neutralizing antibodies or their antigen-binding fragments, and especially preferably anti-RGMa neutralizing antibodies.

[0170] <Anti-RGMa neutralizing antibody>

[0171] In this invention, the anti-RGMa neutralizing antibody can be any antibody that binds to RGMa to neutralize RGMa activity; it can be a polyclonal antibody or a monoclonal antibody. In this invention, a monoclonal antibody is preferred. Furthermore, the anti-RGMa neutralizing antibody of this invention can be an RGMa monospecific antibody or a multispecific antibody that recognizes RGMa and other antigens; an RGMa monospecific antibody is preferred.

[0172] Furthermore, as specific epitopes, in human RGMa, one or more of the following are preferred: sequence number 16 (amino acid numbers 47-69 of sequence number 1), sequence number 36 (amino acid numbers 298-311 of sequence number 1), sequence number 37 (amino acid numbers 322-335 of sequence number 1), sequence number 38 (amino acid numbers 349-359 of sequence number 1), and sequence number 39 (amino acid numbers 367-377 of sequence number 1). A combination of sequence numbers 36 and 37 is more preferred, and a combination of sequence numbers 36, 37, and 39 is particularly preferred.

[0173] The anti-RGMa neutralizing antibodies of the present invention include: polyclonal antibodies and monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or a partial fragment thereof (e.g., the epitope fragment described above) as antigens; chimeric antibodies and humanized antibodies manufactured using gene recombination technology; and human antibodies manufactured using transgenic animals that produce human antibodies. When the antibodies of the present invention are administered to humans in the form of pharmaceutical products, from the viewpoint of side effects, humanized antibodies or human antibodies are preferred.

[0174] Specifically, the antibodies (a) to (l) described below can be cited as the anti-RGMa neutralizing antibodies of the present invention, and the manufacturing methods of each can be the methods described in Patent Documents 2 to 4.

[0175] Antibodies selected from (a) to (l) below can be cited:

[0176] (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions (the anti-RGMa neutralizing antibody further comprises antibodies with epitopes of sequence numbers 36, 37 and 39), wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6 and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9 and HCDR3 containing the amino acid sequence described in sequence number 10;

[0177] Serial Number 5: QDISSY

[0178] Serial number 6: YTS (qualifier: mol_type = protein; organism = Mus musculus)

[0179] Serial Number 7: QQLNTLPWT

[0180] Serial Number 8: GFTFSDAW

[0181] Serial Number 9: IRSKANNHAT

[0182] Serial Number 10: TRRDGAY

[0183] (b) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions (the anti-RGMa neutralizing antibody further comprises antibodies with epitopes of sequence numbers 36, 37 and 38), wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 11, LCDR2 containing the amino acid sequence described in sequence number 12 and LCDR3 containing the amino acid sequence described in sequence number 13, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 14, HCDR2 containing the amino acid sequence described in sequence number 15 and HCDR3 containing the amino acid sequence of AGS;

[0184] Serial Number 11: QSLVHSNGNTY

[0185] Serial number 12: KVS (qualifier: mol_type = protein; organism = Mus musculus)

[0186] Serial Number 13: SQSTHVPYT

[0187] Serial Number 14: GYSITTSYY

[0188] Serial Number 15: ISYDGTN

[0189] (c) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 17, LCDR2 containing the amino acid sequence described in sequence number 18, and LCDR3 containing the amino acid sequence described in sequence number 19, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 20, HCDR2 containing the amino acid sequence described in sequence number 21, and HCDR3 containing the amino acid sequence described in sequence number 22;

[0190] Serial Number 17: RSSQSLEYSDGYTFLE

[0191] Serial Number 18: EVSNRFS

[0192] Serial Number 19: FQATHDPLT

[0193] Serial Number 20: NYGMN

[0194] Serial Number 21: MIYYDSSEKHYADSVKG

[0195] Serial Number 22: GTTPDY

[0196] (d) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 23, LCDR2 containing the amino acid sequence described in sequence number 24, and LCDR3 containing the amino acid sequence described in sequence number 25, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 26, HCDR2 containing the amino acid sequence described in sequence number 27, and HCDR3 containing the amino acid sequence described in sequence number 28;

[0197] Serial Number 23: QASQDIDNYLA

[0198] Serial Number 24: GATNLAD

[0199] Serial Number 25: LQGYIPPRT

[0200] Serial number 26: SYVMH

[0201] Serial Number 27: YIIPYNDNTKYNEKFKG

[0202] Serial Number 28: ARRNEYYGSSFFDY

[0203] (e) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions (the anti-RGMa neutralizing antibody further comprises an antibody with an epitope of sequence number 16), wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 31, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0204] Serial Number 29: TGTSSSVGDSIYVS

[0205] Serial Number 30: DVTKRPS

[0206] Serial Number 31: CSYAGTDTL

[0207] Serial Number 32: SHGIS

[0208] Serial Number 33: WISPYSGNTNYAQKLQG

[0209] Serial number 34: VGSGPYYYMDV

[0210] (f) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as an epitope), wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0211] Serial Number 29: TGTSSSVGDSIYVS

[0212] Serial Number 30: DVTKRPS

[0213] Serial Number 35: YSYAGTDTL

[0214] Serial Number 32: SHGIS

[0215] Serial Number 33: WISPYSGNTNYAQKLQG

[0216] Serial number 34: VGSGPYYYMDV

[0217] (g) An anti-RGMa neutralizing antibody comprising the following light chain variable region and heavy chain variable region (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as an epitope), wherein the light chain variable region comprises: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 40, and the heavy chain variable region comprises: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0218] Serial Number 29: TGTSSSVGDSIYVS

[0219] Serial Number 30: DVTKRPS

[0220] Serial Number 40: FSYAGTDTL

[0221] Serial Number 32: SHGIS

[0222] Serial Number 33: WISPYSGNTNYAQKLQG

[0223] Serial number 34: VGSGPYYYMDV

[0224] (h) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as an epitope), wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 41, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0225] Serial Number 29: TGTSSSVGDSIYVS

[0226] Serial Number 30: DVTKRPS

[0227] Serial Number 41: HSYAGTDTL

[0228] Serial Number 32: SHGIS

[0229] Serial Number 33: WISPYSGNTNYAQKLQG

[0230] Serial number 34: VGSGPYYYMDV

[0231] (i) An anti-RGMa neutralizing antibody comprising the following light chain variable region and heavy chain variable region (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as an epitope), wherein the light chain variable region comprises: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30 and LCDR3 containing the amino acid sequence described in sequence number 42, and the heavy chain variable region comprises: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33 and HCDR3 containing the amino acid sequence described in sequence number 34;

[0232] Serial Number 29: TGTSSSVGDSIYVS

[0233] Serial Number 30: DVTKRPS

[0234] Serial number 42: LSYAGTDTL

[0235] Serial Number 32: SHGIS

[0236] Serial Number 33: WISPYSGNTNYAQKLQG

[0237] Serial number 34: VGSGPYYYMDV

[0238] (j) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as an epitope), wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 43, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34;

[0239] Serial Number 29: TGTSSSVGDSIYVS

[0240] Serial Number 30: DVTKRPS

[0241] Serial Number 43: VSYAGTDTL

[0242] Serial Number 32: SHGIS

[0243] Serial Number 33: WISPYSGNTNYAQKLQG

[0244] Serial number 34: VGSGPYYYMDV

[0245] (k) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions (the anti-RGMa neutralizing antibody further comprising an antibody with sequence number 16 as an epitope), wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 44; and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34; and

[0246] Serial Number 29: TGTSSSVGDSIYVS

[0247] Serial Number 30: DVTKRPS

[0248] Serial Number 44: ISYAGTDTL

[0249] Serial Number 32: SHGIS

[0250] Serial Number 33: WISPYSGNTNYAQKLQG

[0251] Serial number 34: VGSGPYYYMDV

[0252] (l) An anti-RGMa neutralizing antibody comprising the following light chain variable region and heavy chain variable region (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as epitope), wherein the light chain variable region comprises: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30 and LCDR3 containing the amino acid sequence described in sequence number 45, and the heavy chain variable region comprises: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33 and HCDR3 containing the amino acid sequence described in sequence number 34;

[0253] Serial Number 29: TGTSSSVGDSIYVS

[0254] Serial Number 30: DVTKRPS

[0255] Serial Number 45: KSYAGTDTL

[0256] Serial Number 32: SHGIS

[0257] Serial Number 33: WISPYSGNTNYAQKLQG

[0258] Serial number 34: VGSGPYYYMDV.

[0259] Among them, the antibodies described in (a) or (f) are particularly preferred.

[0260] Furthermore, as the anti-RGMa neutralizing antibody of the present invention, examples include the antibodies described in Publications WO2016 / 175236, WO2009 / 106356 and WO2013 / 112922, which are also included in the anti-RGMa neutralizing antibody of the present invention. Specifically, as the anti-RGMa neutralizing antibody of the present invention, antibodies selected from (a') to (i') below can be cited.

[0261] (a') An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence described in sequence number 46 and the heavy chain variable region comprises the amino acid sequence described in sequence number 47;

[0262] Serial number 46: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME

[0263] Serial Number 47: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS

[0264] (b') An anti-RGMa neutralizing antibody (hereinafter also referred to as Unasnemab) comprising the following light and heavy chains, wherein the light chain comprises the amino acid sequence described in sequence number 48 and the heavy chain comprises the amino acid sequence described in sequence number 49;

[0265] Serial number 48: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKV EMERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0266] Serial number 49: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWG KGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0267] (c') An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence described in sequence number 50 and the heavy chain variable region comprises the amino acid sequence described in sequence number 51.

[0268] Serial number 50: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIK

[0269] Serial Number 51: DVKLQESGPGLVKPSQSLSLTCSVTGYSITTSYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKNQFFLRLNSVTTEDTATYYCAGSFGYSQGTLVTVSA

[0270] (d') An anti-RGMa neutralizing antibody comprising the following light and heavy chains, wherein the light chain comprises the amino acid sequence described in sequence number 52 and the heavy chain comprises the amino acid sequence described in sequence number 53;

[0271] Serial number 52: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGG TKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0272] Serial Number 53: DVKLQESGPGLVKPSQSLSLTCSVTGYSITTSYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKNQFFLRLNSVTTEDTATYYCAGSFGYSQGTL VTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPC PPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIE RTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0273] (e') An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence described in sequence number 54 and the heavy chain variable region comprises the amino acid sequence described in sequence number 55;

[0274] Serial number 54: DVVLTQTPVSLSVTLGDQASMSCRSSQSLEYSDGYTFLEWFLQKPGQSPQLLIYEVSNRFSGVPDRFIGSGSGTDFTLKISRVEPEDLGVYYCFQATHDPLTFGSGTKLEIKR

[0275] Serial number 55: EVQLVESGGGLVQPGSSLKLSCVASGFTFSNYGMNWIRQAPKKGLEWIGMIYYDSSEKHYADSVKGRFTISRDNSKNTLYLEMNSLRSEDTAIYYCAKGTTPDYWGQGVMVTVSS

[0276] (f') An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence described in sequence number 56 and the heavy chain variable region comprises the amino acid sequence described in sequence number 57;

[0277] Serial number 56: DIQMTQSPASLSSASLEEIVTITCQASQDIDNYLAWYHQKPGKSPRLLIYGATNLADGVPSRFSGSRSGTQFSLKINRLQIEDLGIYYCLQGYIPPRTFGGGTKLELKR

[0278] Serial number 57: EVQLQQSGPELVKPGTSVKMSCKTSGYTFTSYVMHWVKQKPGQGLEWIGYIIPYNDNTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRNEYYGSSFFDYWGQGTTLTVSS

[0279] (g') is an anti-RGMa neutralizing antibody comprising a light chain variable region (λ chain) and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence described in sequence number 58 and the heavy chain variable region comprises the amino acid sequence described in sequence number 59.

[0280] Serial number 58: QSALTQPRSVSGSPGQSVTISCTGTSSSSVGDSIYVSWYQQHPGKAPKLMLYDVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGTDTLFGGTKVTVL

[0281] Serial number 59: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSHGISWVRQAPGQGLDWMGWISPYSGNTNYAQKLQGRVTMTTDTSSTAYMELSSLRSEDTAVYYCAR VGSGPYYYMDVWGQGTLVTVSS

[0282] (h') an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises the amino acid sequence described in sequence number 60, and the heavy chain variable region comprises the amino acid sequence described in sequence number 59; and

[0283] Serial number 60: QSALTQPRSVSGSPGQSVTISCTGTSSSVGDSIYVSWYQQHPGKAPKLMLYDVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCYSYAGTDTLFGGGTKVTVL

[0284] Serial number 59: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSHGISWVRQAPGQGLDWMGWISPYSGNTNYAQKLQGRVTMTTDTSSTAYMELSSLRSEDTAVYYCAR VGSGPYYYMDVWGQGTLVTVSS

[0285] (i') An anti-RGMa neutralizing antibody (hereinafter referred to as Elezanumab) comprising the following light and heavy chains, wherein the light chain comprises the amino acid sequence described in sequence number 61 and the heavy chain comprises the amino acid sequence described in sequence number 62.

[0286] Serial number 61: QSALTQPRSVSGSPGQSVTISCTGTSSSSVGDSIYVSWYQQHPGKAPKLMLYDVTKRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCYSYAGTDTLFGGT KVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0287] Serial number 62: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSHGISWVRQAPGQGLDWMGWISPYSGNTNYAQKLQGRVTMTTDTSTAYMELSSLRSEDTAVYYCARVGSGPYYYMD VWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0288] The method for manufacturing the anti-RGMa neutralizing antibody of the present invention can use existing commonly used manufacturing methods. The antigen can be used directly for immunization or in the form of a complex with a carrier protein. Condensing agents such as glutaraldehyde, carbodiimide, and maleimide active esters can be used in the preparation of the antigen-carrier protein complex. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[0289] Examples of mammals that can be immunized include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, and cattle. Administration methods include subcutaneous, intramuscular, or intraperitoneal injection. Administration can be done in combination with complete or incomplete Freund's adjuvant, and is typically administered every 2–5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of the immunized animal are fused with myeloma cells and isolated as hybridomas. Myeloma cells can be derived from mammals such as mice, rats, or humans.

[0290] <Polyclonal Antibody>

[0291] Polyclonal antibodies can be obtained, for example, from the serum of a mammal sensitized as described above by immunizing it with the antigen described above along with Freund's adjuvant as needed.

[0292] <Monoclonal Antibodies>

[0293] Monoclonal antibodies can be obtained by the following procedure: Using the antigen described above as an immunogen, the immunogen, along with Freund's adjuvant, is injected subcutaneously, intramuscularly, intravenously, into the aforementioned mammals via the subcutaneous, intramuscular, footpad, or intraperitoneal routes to sensitize the mammals. Typically, 1 to 4 immunizations are administered approximately every 1 to 14 days from the initial immunization, and antibody-producing cells are obtained from the sensitized mammal approximately 1 to 5 days after the final immunization.

[0294] Monoclonal antibodies can be obtained using methods known to those skilled in the art (e.g., “Current Protocols in Molecular Biology” (John Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0295] The preparation of "hybridomas" that secrete monoclonal antibodies can be carried out according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) and the modification methods thereunder. That is, it is prepared by fusing antibody-producing cells, such as those from the spleen of an immunosensitized mammal, with myeloma cells from mammals, preferably mice, rats, or humans, that do not have the ability to produce their own antibodies.

[0296] For example, mouse-derived myeloma cells such as P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / O, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3, human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used for cell fusion.

[0297] Examples of fusion promoters include polyethylene glycol. Cell fusion can usually be achieved by using polyethylene glycol (average molecular weight 1000-4000) at a concentration of about 20-50% at a temperature of 20-40°C, preferably 30-37°C, with the ratio of antibody-producing cells to myeloma cells usually set at about 1:1-10:1, and the reaction time being about 1-10 minutes.

[0298] The screening of hybridoma clones that produce monoclonal antibodies can be carried out by culturing the hybridoma in, for example, a microtiter plate and measuring the reactivity of the culture supernatant of the wells with the immunoantigen using immunochemical methods such as ELISA.

[0299] In the screening of antibody-generated hybridomas, in addition to the binding assay to the RGMa protein, the evaluation also considers whether the antibody inhibits the RGMa activity of the present invention. Through these screening methods, the anti-RGMa neutralizing antibody of the present invention can be selected.

[0300] Cloning is further performed using the limiting dilution method from the wells containing hybridomas that produce the target antibody, resulting in clones. Hybridoma screening and breeding are typically carried out using HAT (hypoxanthine, aminopterin, thymidine) supplemented with animal cell culture medium containing 10–20% fetal bovine serum.

[0301] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or in vivo, such as in the ascites of mammals like mice and rats, and then isolating them from the resulting culture supernatant or the ascites of the mammals.

[0302] In in vitro culture, various conditions, such as the characteristics of the cell species being cultured and the culture method, can be used to promote the proliferation, maintenance, and preservation of hybridomas, employing nutrient media suitable for producing monoclonal antibodies in the culture supernatant. Examples of nutrient media include well-known nutrient media or nutrient media prepared from basal media.

[0303] As a basal culture medium, examples include low-calcium media such as Ham'F12 medium, MCDB153 medium or low-calcium MEM medium, and high-calcium media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium. The basal culture medium may contain, for example, serum, hormones, cytokines and / or various inorganic or organic substances, depending on the purpose.

[0304] The isolation and purification of monoclonal antibodies can be performed by subjecting the culture supernatant or ascites to saturated ammonium sulfate, euglobulin precipitation, hexanoic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as anti-immunoglobulin column or protein A column, etc. Specifically, the purification of monoclonal antibodies can be easily achieved by using any known method for purifying immunoglobulins, such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of anion exchangers, and affinity chromatography using RGMa protein.

[0305] Monoclonal antibodies can also be obtained using phage display. In phage display, phages selected from any phage antibody library are screened using a target immunogen to select those with the desired binding affinity to the immunogen. Next, the antibody-corresponding sequences contained within the phages are isolated or sequenced. Based on the isolated or sequenced sequences, an expression vector containing a nucleic acid molecule encoding an antibody or antigen-binding domain is constructed. Cell lines transfected with this expression vector are then cultured, thereby producing monoclonal antibodies. As a phage antibody library, human antibodies with the desired binding affinity can be generated using a human antibody library.

[0306] <Nucleic Acid Molecules>

[0307] The nucleic acid molecule encoding the anti-RGMa neutralizing antibody or its antigen-binding fragment of the present invention can be obtained, for example, by the following method. First, 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. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides with conserved sequences 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 using PCR. The base sequence of the DNA can be determined using conventional methods, such as integrating it into a sequencing plasmid.

[0308] Alternatively, the DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the variable region or a portion thereof and combining it with a sequence containing the constant region.

[0309] This nucleic acid molecule can encode the entire constant and variable regions of both the heavy and light chains, or it can encode only the variable regions of both the heavy and light chains. When encoding both constant and variable regions, the preferred base sequences for the constant regions of the heavy and light chains are those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0310] <Functional Altered Antibodies>

[0311] The functionally altered anti-RGMa neutralizing antibody is prepared by the following methods. For example, if CHO cells with the α1,6-fucosetransferase (FUT8) gene disrupted are used as host cells to manufacture the anti-RGMa neutralizing antibody of this application, an antibody with reduced fucose content in the glycan chain and enhanced cell-killing function is obtained. When CHO cells with the FUT8 gene introduced are used as host cells for manufacturing, an antibody with low cell-killing function can be obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). In addition, complement activation function can be regulated by modifying amino acid residues in the Fc region (US Patent No. 6,737,056, US Patent No. 7,297,775, US Patent No. 7,317,091). Furthermore, by using Fc region mutants that enhance binding to FcRn, one of the Fc receptors, a prolonged blood half-life can be achieved (Hashiguchi et al., Biochemistry, 2010, Vol. 82(8), p710). These functionally altered antibodies can be manufactured using genetic engineering methods.

[0312] <Conjugated Antibody>

[0313] As a modified molecule of the anti-RGMa neutralizing antibody of the present invention, a conjugated antibody can be cited. Examples of conjugated antibodies include those obtained by chemically or genetically binding non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (TGF-β, NGF, neurotrophic factors, etc.), albumin, enzymes, and other functional molecules other than the anti-RGMa neutralizing antibody of this application to the anti-RGMa neutralizing antibody.

[0314] When PEG is used as a functional molecule, it is not limited to PEG with a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and it can be linear or branched. For example, PEG can be bound to the N-terminal amino group of the amino acid of the anti-RGMa neutralizing antibody by using an NHS active group.

[0315] When using radioactive materials as functional molecules, it is possible to use 131 I, 125 I, 90 Y、 64 Cu、 99 Tc, 77 Lu or211 At, etc. Radioactive substances can be directly bound to anti-RGMa neutralizing antibodies via methods such as the chloramine-T method.

[0316] When using toxins as functional molecules, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin), low molecular weight toxins (e.g., gerdemycin), maytansine, and cazithromycin can be used.

[0317] Examples of fluorescent dyes that use low-molecular-weight compounds as functional molecules include daunorubicin, doxorubicin, methotrexate, mitomycin, neomycin, vinblastine, and FITC.

[0318] When using enzymes as functional molecules, luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc., can be used.

[0319] Examples of linkers used in the chemical binding of toxins, low-molecular-weight compounds, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene) and -(CR2). n O(CR2) n – (R is any substituent, n is a positive integer) represents repeating units of linkers or alkoxy groups (e.g., poly(ethyleneoxy), PEG, poly(methyleneoxy), etc.) and alkylamino groups (e.g., poly(ethyleneoxy), Jeffamine (trademark)), as well as esters and amides (e.g., succinate, succinamide, diethylene glycol ester, malonate, and hexamethylene amide). Chemical modification methods for binding functional molecules have been established in this field (DJKing., Applications and Engineering of Monoclonal antibodies., 1998; TJ International Ltd., Monoclonal Antibody-Based Therapy of Cancer., 1998; MarcelDekker Inc.; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc NatlAcad Sci USA., 1996 Vol 93:8681).

[0320] <Antigen-binding fragment>

[0321] In embodiments of the present invention, the “antigen-binding fragment” of an antibody refers to a portion of the antibody that has antigen-binding properties as described above. Specifically, examples include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide bond Fv, single-chain antibody (scFv), and polymers thereof. In addition, it includes conjugated fragments of functional molecules other than the anti-RGMa neutralizing antibody of this application, which are chemically or genetically engineered to bind non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, neurotrophic factors, etc.), albumins, enzymes, and other antibodies to the antigen-binding fragment.

[0322] "F(ab')2" and "Fab" refer to antibody fragments produced by digesting immunoglobulins using proteolytic enzymes such as pepsin or papain, where the disulfide bonds between the two heavy chains in the hinge region are digested. For example, when IgG is treated with papain, the upstream disulfide bond between the two heavy chains in the hinge region is cleaved, thereby producing two identical antibody fragments: a light chain consisting of VL (variable region of the light chain) and CL (constant region of the light chain), and a heavy chain consisting of VH (variable region of the heavy chain) and CHγ1 (γ1 region in the constant region of the heavy chain), linked by disulfide bonds at the C-terminus. These two identical antibody fragments are respectively called Fab. Conversely, when IgG is treated with pepsin, the downstream disulfide bond between the two heavy chains in the hinge region is cleaved, thereby producing an antibody fragment slightly larger than the fragment formed by the two Fab fragments linked in the hinge region. This antibody fragment is called F(ab')2.

[0323] <Chimeric antibodies>

[0324] As a preferred embodiment of the anti-RGMa neutralizing antibody of the present invention, a chimeric antibody can be cited. Examples of "chimeric antibodies" include those with variable regions derived from immunoglobulins of non-human animals (mice, rats, hamsters, chickens, etc.) and constant regions derived from human immunoglobulins. For example, mice can be immunized with an antigen, and the variable region binding to the antigen can be excised from the gene of a mouse monoclonal antibody and combined with the constant region of an antibody derived from human bone marrow. The constant regions derived from human immunoglobulins have their own inherent amino acid sequences depending on the isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE. The constant region of the recombinant chimeric antibody in the present invention can be a constant region belonging to any isotype of human immunoglobulin. Preferably, it is a constant region of human IgG. An expression vector can be prepared using the gene of the chimeric antibody prepared in this way. Transformed cells producing chimeric antibodies are obtained by transforming host cells with this expression vector, and the target chimeric antibody is obtained from the culture supernatant by culturing the transformed cells.

[0325] <Humanized Antibody>

[0326] 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 transplanted (CDR grafting) into the human antibody gene. For example, it can be manufactured according to the methods described in Japanese Patent Publication No. 4-506458 and Japanese Patent No. 2912618. Specifically, it refers to a humanized antibody characterized in that a portion or all of its CDR is derived from the CDR of a monoclonal antibody from a non-human mammal (mouse, rat, hamster, etc.), its variable region is a frame region derived from a variable region of human immunoglobulin, and its constant region is a constant region derived from human immunoglobulin.

[0327] The humanized antibodies in this invention can be manufactured, for example, as follows. However, they are not limited to such manufacturing methods.

[0328] For example, recombinant humanized antibodies derived from mouse monoclonal antibodies can be produced using genetic engineering methods, referring to Japanese Patent Application Publication No. 4-506458 and Japanese Patent Application Publication No. 62-296890. Specifically, this involves isolating the mouse heavy chain CDR and mouse light chain CDR DNA from a hybridoma that produces mouse monoclonal antibodies, and isolating the human heavy chain gene (excluding the human heavy chain CDR) and the human light chain gene (excluding the human light chain CDR) from the human immunoglobulin gene.

[0329] The human heavy chain gene, transplanted with the isolated mouse heavy chain CDR portion of DNA, is introduced in an expressible manner into a suitable expression vector. Similarly, the human light chain gene, transplanted with the mouse light chain CDR portion of DNA, is introduced in an expressible manner into another suitable expression vector. Alternatively, the human heavy and light chain genes, transplanted with mouse CDRs, can be introduced in an expressible manner into the same expression vector. Humanized antibodies are generated by transforming host cells using these prepared expression vectors. The target humanized antibody is then obtained from the culture supernatant by culturing these transformed cells.

[0330] <Human Antibody>

[0331] As another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention, a human antibody can be cited. A human antibody is an immunoglobulin, i.e., an antibody, in which all regions comprising the variable and constant regions of the heavy chain and the variable and constant regions of the light chain of an immunoglobulin are derived from a gene encoding a human immunoglobulin. It can be produced by introducing a human antibody gene into a mouse. Specifically, for example, it can be manufactured in the same manner as the methods described above for producing polyclonal or monoclonal antibodies, by sensitizing transgenic animals created using an antigen to at least integrate the human immunoglobulin gene into a locus in a mammal other than a human, such as a mouse.

[0332] 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; Japanese Patent Publication No. 4-504365; Japanese Patent Publication No. 7-509137; International Publication No. WO94 / 25585; Nature, Vol. 368, pp. 856-859, 1994; and Japanese Patent Publication No. 6-500233. More specifically, examples include HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), and KM (FCγRIIb-KO) mice.

[0333] Specifically, examples of anti-RGMa neutralizing antibodies of the present invention include anti-RGMa neutralizing antibodies with 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') described above).

[0334] It should be noted that, as long as the characteristics of the antibody of the present invention are maintained, namely, its ability to bind to RGMa and its activity of inhibiting (neutralizing) RGMa, one or more amino acids (1 to 20, 1 to 10, or 1 to 5, preferably 1 to 2) can be substituted, deleted, added, or inserted in the amino acid sequence of the anti-RGMa neutralizing antibody (preferably the anti-RGMa neutralizing antibodies (a) to (l) and (a') to (i') described above). Such substitutions, deletions, and additions can be introduced into the CDR, preferably into regions other than the CDR. In addition, the amino acid substitution is preferably a conservative substitution to maintain the characteristics of the present invention.

[0335] The amino acid sequences of the anti-RGMa neutralizing antibodies of the present invention that include substitutions, deletions, etc., are, for example, amino acid sequences in which the heavy chain variable region after amino acid sequence modification has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) homology with the amino acid sequence before modification, and amino acid sequences in which the light chain variable region after amino acid sequence modification has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) homology with the amino acid sequence before modification.

[0336] In this invention, siRNA refers to a short double-stranded RNA capable of inhibiting the expression of a target gene (RGMa gene in this invention). As long as the siRNA can function as an inhibitor of the RGMa activity of this invention, the base sequence and length (base length) are not particularly limited, preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases.

[0337] In this invention, shRNA refers to a molecule of approximately 20 or more base pairs, which forms a double-stranded structure within the molecule by including a partially palindromic base sequence in single-stranded RNA, and has a short hairpin structure with a protrusion at the 3' end. When such shRNA is introduced into a cell, it is broken down into approximately 20 base pairs (representative examples include 21, 22, and 23 base pairs) in length, and can inhibit the expression of the target gene in the same way as siRNA.

[0338] In this invention, the siRNA and shRNA described above can be in any form as long as they can inhibit the expression of the RGMa gene.

[0339] In this invention, siRNA or shRNA can be synthesized artificially using chemical methods. Alternatively, antisense and sense RNA can be synthesized in vitro from template DNA, for example, using T7 RNA polymerase and the T7 promoter. Antisense oligonucleotides can be any nucleotide that is complementary to or hybridizes to a sequence of 5 to 100 consecutive bases in the DNA sequence of the RGMa gene, and can be either DNA or RNA. Furthermore, modifications can be made as long as they do not impair function. Antisense oligonucleotides can be synthesized using conventional methods, for example, they can be easily synthesized using commercially available DNA synthesis equipment.

[0340] Preferred sequences can be selected using conventional selection methods. As siRNA or shRNA in this invention, they can be confirmed by evaluating the inhibition of functional RGMa expression.

[0341] <Preventive or therapeutic agents for spasmodic and / or neurogenic bladder>

[0342] In the embodiments of the present invention, spasticity refers to sensorimotor control disorder caused by intermittent or continuous involuntary muscle activity due to upper motor nerve damage (Disabil Rehabil. 2005 Jan;27(1-2):2-6).

[0343] As a cause of spasticity, the long nerves extending from the brain to the spinal cord, namely the upper motor neurons, are responsible for voluntary movements. When these neurons are damaged or impaired, the information transmitted to the muscles becomes abnormal. Spasticity is caused by damage or impairment of the central nervous system that controls voluntary movements, which disrupts the complex balance between nerve excitation and inhibition in the brain or spinal cord. It is an abnormal movement of the reflex nerves. For example, when the signal from the brain to the lower body is interrupted due to spinal cord injury or disease (compression, trauma, blood flow obstruction, nerve degeneration, etc.), nerve excitability increases, resulting in unnecessary muscle contraction, i.e., spasticity. Symptoms of spasticity include muscle tension, sudden muscle contraction (spasm), hyperreflexia of deep tendons, muscle spasms (clonic), scissor gait (hip joint in adduction and internal rotation), joint stiffness, etc. (Pract Neurol. 2012;12(5):289-98).

[0344] The present invention includes novel uses of RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies (preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a) (serial numbers 5-10) or (f) (serial numbers 29, 30, 35 and 32-34), more preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a') (serial numbers 46 and 47), (b') (serial numbers 48 and 49), (h') (serial numbers 60 and 59) or (i') (serial numbers 61 and 62)) as preventive or therapeutic agents for spasms and their symptoms.

[0345] In addition, in the embodiments of the present invention, neurogenic bladder refers to a state in which the function of the lower urinary tract (bladder and urethra) is abnormal due to damage or impairment of the central nervous system, vegetative nervous system or peripheral nerves (Neurogenic Bladder and Neurogenic Lower Urinary Tract Dysfunction. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan. 2022 Nov 28).

[0346] As a cause of neurogenic bladder, lower urinary tract dysfunction can occur under various neurological diseases because urination is controlled by the coordination and reflexes of the spinal cord, brainstem, and spinal cord (Transl Androl Urol. 2016 Feb; 5(1):12-21). It is a common complication, especially when the spinal cord is injured. This is because the urination reflex center is located in the sacral spinal cord at the end of the spinal cord, so the neural pathway between the sacral spinal cord and the brain is blocked no matter where the spinal cord is damaged (Management of the neurogenic bladder for adults with spinal cordinjuries. 2013). Lower urinary tract dysfunction as a symptom of neurogenic bladder can be divided into two categories: storage dysfunction and voiding dysfunction (Neurogenic Bladder and Neurogenic Lower Urinary TractDysfunction. In: StatPearls [Internet]. Treasure Island (FL): StatPearlsPublishing; 2023 Jan.2022 Nov 28). When there is a bladder storage dysfunction, the bladder will involuntarily contract, resulting in symptoms such as urinary frequency, urgency, and incontinence. When there is a voiding dysfunction, the bladder contraction is impaired, resulting in difficulty urinating.

[0347] The present invention includes novel uses of RGMa inhibitory substances, especially anti-RGMa neutralizing antibodies (preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a) (serial numbers 5-10) or (f) (serial numbers 29, 30, 35 and 32-34), more preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a') (serial numbers 46 and 47), (b') (serial numbers 48 and 49), (h') (serial numbers 60 and 59) or (i') (serial numbers 61 and 62)) as preventive or therapeutic agents for neurogenic bladder. Additionally, this includes RGMa inhibitors, particularly anti-RGMa neutralizing antibodies (preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a) (serial numbers 5-10) or (f) (serial numbers 29, 30, 35, and 32-34), more preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a') (serial numbers 46 and 47), (b') (serial numbers 48 and 49), (h') (serial numbers 60 and 59), or (i') (serial numbers 61 and 62)) for the prevention or treatment of neurogenic bladder or lower urinary tract dysfunction.

[0348] Furthermore, the present invention also includes RGMa inhibitors, particularly anti-RGMa neutralizing antibodies (preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a) (serial numbers 5-10) or (f) (serial numbers 29, 30, 35 and 32-34), more preferably anti-RGMa neutralizing antibodies containing the amino acid sequence of (a') (serial numbers 46 and 47), (b') (serial numbers 48 and 49), (h') (serial numbers 60 and 59) or (i') (serial numbers 61 and 62)) for the prevention or treatment of spasm and neurogenic bladder (or lower urinary tract dysfunction).

[0349] <Preventive or therapeutic agents for accompanying symptoms of diseases caused by disorders of the spinal cord or brain>

[0350] In embodiments of the present invention, a preventive or therapeutic agent for accompanying symptoms of diseases caused by disorders of the spinal cord or brain, as a novel use of RGMa inhibitors, refers to a drug or pharmaceutical composition capable of preventing or treating accompanying symptoms of diseases caused by disorders of the spinal cord or brain, specifically injuries or inflammation of the spinal cord or brain, such as spasms and / or neurogenic bladder (or lower urinary tract dysfunction).

[0351] Here, the diseases that cause spasticity include, for example, cerebral palsy, multiple sclerosis, amyotrophic lateral sclerosis, stroke (including cerebral infarction, cerebral hemorrhage, etc.), brain injury, spinal cord compression, spinal cord injury, motor neuron disease, spinal cord vascular malformation, hereditary spastic paraplegia, subacute combined degeneration of thespinal cord, HTLV-1 related spinal cord disease (also known as tropical spastic paraplegia), copper deficiency, syringomyelia, and tethered cord syndrome (Crit Rev Phys Rehabil Med. 2013;25(1-2):11-22, Pract Neurol. 2012;12(5):289-98).

[0352] In addition, diseases that lead to neurogenic bladder (or lower urinary tract dysfunction) include, for example, spinal marrow diseases or brain diseases. Specifically, spinal marrow diseases include spinal disorders (intervertebral disc herniation, spinal stenosis, ossification of the posterior longitudinal ligament), vascular diseases (spinal marrow hemorrhage / infarction), spinal marrow tumors, demyelinating diseases (multiple sclerosis, neuromyelopathic disease), infectious diseases (myelopathic disease, HTLV-1-related myelopathic disease), inflammatory diseases (sarcoidosis, collagen vascular disease), congenital diseases (spinal bifida, sacral dysplasia, tethered cord syndrome), degenerative diseases (amyotrophic lateral sclerosis), exogenous diseases (spinal marrow injury, radiation injury), and hereditary spastic paraplegia. In addition, as brain diseases, examples include vascular diseases (cerebral hemorrhage and infarction), brain tumors, degenerative diseases (Parkinson's disease, multiple system atrophy, Alzheimer's disease, spinocerebellar degeneration, olivopontocerebellar atrophy, amyotrophic lateral sclerosis), demyelinating diseases (multiple sclerosis), infectious diseases (encephalitis), inflammatory diseases (sarcoidosis, collagen disease), traumatic diseases (head injury), hereditary spastic paraplegia, etc. (Transl Androl Urol. 2016 Feb; 5(1): 12-21, etc.).

[0353] This invention includes novel uses for RGMa inhibitors, particularly anti-RGMa neutralizing antibodies (preferably those containing amino acid sequences of (a) (serial numbers 5-10) or (f) (serial numbers 29, 30, 35, and 32-34), more preferably those containing amino acid sequences of (a') (serial numbers 46 and 47), (b') (serial numbers 48 and 49), (h') (serial numbers 60 and 59), or (i') (serial numbers 61 and 62)) as preventive or therapeutic agents for accompanying symptoms of diseases caused by spinal cord or brain disorders. These accompanying symptoms include spasms, neurogenic bladder (or lower urinary tract dysfunction), etc. This invention includes individual preventive or therapeutic agents for these accompanying symptoms, as well as preventive or therapeutic agents for two accompanying symptoms (spasms and neurogenic bladder (or lower urinary tract dysfunction)).

[0354] <Pharmaceutical Compositions>

[0355] The preventive or therapeutic agents of the present invention for the accompanying symptoms of diseases caused by disorders of the spinal cord or brain containing RGMa inhibitors, or for the preventive or therapeutic agents of spasms and / or neurogenic bladder containing RGMa inhibitors, are typically administered systemically or locally in oral or non-oral form.

[0356] The present invention relates to a preventive or therapeutic agent for accompanying symptoms of diseases caused by spinal cord or brain disorders containing an RGMa inhibitor, or a preventive or therapeutic agent for spasms and / or neurogenic bladder containing an RGMa inhibitor, which can be formulated with an RGMa inhibitor as the active ingredient and appropriately combined with a pharmaceutically acceptable carrier or additive. Such formulated pharmaceutical compositions can be administered orally or non-orally. Specifically, they can be formulated as oral dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., and as non-oral dosage forms such as injections, infusions, suppositories, ointments, patches, etc. The proportions of the carrier or additive can be appropriately set based on the range commonly used in the pharmaceutical industry. There are no particular limitations on the carriers or additives that can be used, including various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oil-based bases, and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavoring agents, and fragrances.

[0357] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, its functionally altered antibody, its conjugated antibody, or its antigen-binding fragment, it is formulated as an injection or infusion with a pharmaceutically acceptable carrier, preferably via a non-oral route of administration, such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, or local administration.

[0358] For example, injectable or infusion solutions containing anti-RGMa neutralizing antibodies can be used in the form of solutions, suspensions, or emulsions. As solvents, for example, distilled water for injection, physiological saline, glucose solutions, and isotropic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used.

[0359] In addition, such injectable or infusion solutions containing anti-RGMa neutralizing antibodies may contain stabilizers, solubilizers, suspending agents, emulsifiers, analgesics, buffers, preservatives, pH adjusters, etc.

[0360] As stabilizers, substances such as albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and butylated hydroxytoluene can be used.

[0361] As a co-solvent, alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants such as polysorbate 80 (registered trademark), HCO-50, etc., can be used.

[0362] As suspending agents, for example, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium dodecyl sulfate, etc. can be used.

[0363] As emulsifiers, substances such as gum arabic, sodium alginate, and tragacanth gum can be used.

[0364] As pain-relieving agents, substances such as benzyl alcohol, chlorobutanol, and sorbitol can be used.

[0365] As a buffer, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, etc. can be used.

[0366] As preservatives, substances such as methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax can be used.

[0367] As preservatives, substances such as benzalkonium chloride, p-hydroxybenzoic acid, and chlorobutanol can be used.

[0368] As pH adjusters, substances such as hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid can be used.

[0369] When the RGMa inhibitor is a nucleic acid (siRNA, shRNA, antisense oligonucleotide, etc.), it can be administered in either a non-viral or viral vector form. In non-viral vector forms, methods such as liposome delivery of nucleic acid molecules (liposome method, HVJ-liposome method, cationic liposome method, liposome transfection, Lipofectamine transfection, etc.), microinjection, and using a gene gun along with a vector (metal particles) to transfer nucleic acid molecules into cells can be employed. For example, when administering siRNA or shRNA to organisms using viral vectors, recombinant adenoviruses, retroviruses, and other viral vectors can be used. Genes can be introduced into cells or tissues by introducing DNA expressing siRNA or shRNA into non-toxic retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, poxviruses, polioviruses, Sinderby viruses, Sendai viruses, SV40, and other DNA or RNA viruses, allowing the recombinant virus to infect cells or tissues.

[0370] The resulting formulation can be administered in effective amounts to prevent or treat accompanying symptoms of diseases caused by spinal cord or brain disorders, and can also prevent or treat spasms and / or neurogenic bladder. The dosage should be appropriately determined considering the purpose, the severity of the disease, the patient's age, weight, sex, medical history, and the type of active ingredient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, the preferred dosage for a healthy adult weighing approximately 65–70 kg is approximately 0.02 mg to 4000 mg daily. The total daily dose can be a single dose or divided doses.

[0371] Example

[0372] The following examples illustrate the invention in more detail, but they do not limit the scope of the invention. A rat spinal cord contusion model is used as a pathological model of spinal cord injury.

[0373] It should be noted that in each embodiment, an anti-RGMa neutralizing antibody containing the amino acid sequences (sequence numbers 5-10) of (a) and (f) of (f) described in this specification was used, and palizumab was used as the control antibody.

[0374] [Example 1]

[0375] Study on the effect of anti-RGMa neutralizing antibody on spasticity and urinary dysfunction in a rat model of spinal cord contusion

[0376] (1-1) Establishment of a rat spinal cord contusion model

[0377] Female SD rats (11 weeks old at the time of model creation) were used in the experiment. Animals were anesthetized using a mixture of metoprolol hydrochloride, midazolam, and butorphanol tartrate. After shaving the fur from the neck to the back, the animals were cleaned with ethanol and iodine for disinfection. During the surgery, isoflurane was inhaled to maintain anesthesia. The skin on the back was incised to expose the 6th thoracic vertebra to the 2nd lumbar vertebra, and the vertebral arches of the 9th and 10th thoracic vertebrae were removed. Immediately afterward, a 2.5 mm diameter, 10 g weight was dropped from a height of 50 mm using a MASCIS Impactor (Rutgers University, USA), causing spinal cord injury. From the day of spinal cord injury, cefazolin sodium for infection prevention and physiological saline solution for dehydration were administered subcutaneously to the buttocks for 7 days.

[0378] In addition, in principle, from the day after spinal cord injury until the end of the trial, forced urination was performed twice a day (morning and afternoon) by manually compressing the bladder. Eight days after the end of the cefazolin sodium administration period, individuals whose urine color showed signs of urinary tract infection during forced urination were given enrofloxacin subcutaneously for several days until the urine color returned to normal.

[0379] (1-2) Evaluation of spasms

[0380] Following the method of Ryu et al. (PLos One 2017;12:e0171937), a swimming test was used to evaluate the frequency of spasticity by observing spastic movements of the hind limbs or trunk during swimming. The swimming test was conducted in a rectangular acrylic tank (13 cm wide, 150 cm long, 40 cm high) at a depth of 20–23 cm and a temperature of 20–23°C. One test was defined as the observation of movement up to 100 cm from the point of entry to the finish line. A positive spasticity test was defined as observing only one instance of clonic or spastic movement in the hind limbs or trunk. Ten tests were performed per animal at each time point, and the frequency (%) of the positive spasticity test was calculated. All swimming tests were video-recorded, and the evaluation of spasticity was conducted independently in a double-blind state, with two observers unable to distinguish which test substance was administered to each individual. The average spasticity frequency (%) calculated by each observer was used as the representative value for that evaluation time. In addition, referring to the paper by Ryu et al. (PLos One 2017;12:e0171937), individuals exhibiting a spasm frequency of more than 35% of the representative value were identified as individuals with severe spasm.

[0381] (1-3) Evaluation of spontaneous urination ability

[0382] Spontaneous urination ability was evaluated based on records of bladder distension and urination observed during forced urination each morning. Spontaneous urination was defined as the absence of bladder distension and urination due to pressure. Evaluation of spontaneous urination was conducted from the start of the rat spinal cord contusion model until 56 days later.

[0383] (1-4) Grouping and administration of anti-RGMa neutralizing antibodies

[0384] Prior to spinal cord contusion treatment, the weight was distributed in a manner that ensured a uniform average weight among the groups.

[0385] As anti-RGMa neutralizing antibodies, Unasnemab (specifically containing amino acid sequences 5-10, part of amino acid sequence (a)) and Elezanumab (specifically containing amino acid sequences 29, 30, 35, and 32-34, part of amino acid sequence (f)) were prepared according to the methods described in International Publications WO2016 / 175236 and WO2013 / 112922, respectively. The anti-RGMa neutralizing antibodies were prepared for administration at 3 ml / kg via intravenous bolus injection under isoflurane anesthesia. Initial administration was given immediately after spinal cord contusion, followed by eight weekly administrations. In the Unasnemab study, specifically, Unasnemab (30 mg / kg per individual) or the control antibody (palizumab 30 mg / kg per individual) was administered. In the Elezanumab study, patients were given either Elezanumab (25 mg / kg per individual) or the control antibody (palizumab 25 mg / kg per individual).

[0386] (1-5) Results

[0387] Effect on spasms

[0388] The effect of repeated administration of Unasnemab on swimming load-induced spasticity in a rat spinal contusion model was shown by analyzing the frequency of spasticity occurrence. Figure 1 At 4 and 6 weeks post-spinal cord injury, the frequency of spasticity was lower in the Unasnemab 30 mg / kg group compared to the control antibody group.

[0389] The effect of repeated administration of Unasnemab on swimming load-induced spasticity in a rat spinal contusion model was shown by analyzing the proportion of individuals exhibiting strong spasticity. Figure 2At 6 weeks post-spinal cord injury, the proportion of individuals exhibiting severe spasticity in the Unasnemab 30 mg / kg group was significantly lower than in the control antibody group (Fisher's exact test).

[0390] The effect of repeated administration of elezanumab on swimming load-induced spasticity in a rat spinal contusion model was shown by spasticity frequency analysis. Figure 3 At 4 and 6 weeks post-spinal cord injury, the frequency of spasticity in the Elezanumab 25 mg / kg group was lower than that in the control antibody group, and significantly reduced at 6 weeks post-spinal cord injury (Student's t-test).

[0391] The effect of repeated administration of elezanumab on swimming load-induced spasticity in a rat model of spinal contusion was shown by analyzing the proportion of individuals exhibiting strong spasticity. Figure 4 At the 6-week time point following spinal cord injury, the proportion of individuals exhibiting severe spasticity in the Elezanumab 25 mg / kg group was lower compared to the control antibody group.

[0392] Effects on urination disorders

[0393] The effect of repeated administration of Unasnemab on urinary dysfunction in a rat spinal contusion model is presented as a progression of cumulative days to achieve spontaneous urination. Figure 5 The cumulative number of days in the Unasnemab (30 mg / kg) dosing group was higher than that in the control antibody dosing group.

[0394] The effects of repeated administration of Unasnemab on urinary dysfunction in a rat spinal contusion model are presented in the form of an analysis of the total number of days to achieve spontaneous urination. Figure 6 The number of days of spontaneous urination was increased in a dose-dependent manner in the Unasnemab administration group, with the number of days of spontaneous urination in the Unasnemab 30 mg / kg administration group being significantly increased compared to the control antibody administration group (Student's t-test).

[0395] The effect of repeated administration of elezanumab on urinary dysfunction in a rat model of spinal contusion is presented as a progression of cumulative days to achieve spontaneous urination. Figure 7 The cumulative number of days in the Elezanumab 25 mg / kg dosing group was higher than that in the control antibody dosing group.

[0396] The effects of repeated administration of Elezanumab on urinary dysfunction in a rat spinal contusion model are presented in the form of an analysis of the total number of days to achieve spontaneous urination. Figure 8 The number of days of spontaneous urination was significantly increased in the elezanumab 25 mg / kg group compared to the control antibody group (Student's t-test).

[0397] Example 2

[0398] This clinical trial, conducted in patients with moderate to high disease activity of HTLV-1-associated spinal marrow disease (hereinafter referred to as HAM), primarily aimed to investigate the safety, tolerability, and pharmacokinetic characteristics of an anti-RGMa neutralizing antibody (specifically Unasnemab) containing the amino acid sequence (serial numbers 5–10) of (a) or placebo for 20 weeks. It was a phase Ib, randomized, double-blind, placebo-controlled trial. Additionally, the efficacy of Unasnemab was investigated exploratoryly using the following endpoints.

[0399] (1) Modified Ashworth Scale (hereinafter referred to as MAS) (knee extensors and knee flexors)

[0400] (2) Overactive bladder score (hereinafter referred to as OABSS)

[0401] The study included 15 participants (10 in the investigational drug group and 5 in the placebo group). Participants deemed suitable for treatment were randomly assigned to either the investigational drug group or the placebo group at a ratio of 2:1. The concentration of neopterin in the cerebrospinal fluid (hereinafter referred to as CSF) at the time of screening was used as a stratification factor, dividing participants into those with moderate disease activity (6–43 pmol / mL) or those with high disease activity (above 44 pmol / mL).

[0402] The investigational drug or placebo was administered intravenously over an hour at the start of treatment, at 2, 4, 8, 12, 16 and 20 weeks.

[0403] The clinical trial period is the period from the date of consent to the end of the subsequent observation period. The screening period is the period from the date of consent to the start of clinical dosing on the treatment start date (the date of first administration of the clinical trial drug), with a maximum of 6 weeks. The treatment period is the period from the start of clinical dosing on the treatment start date (the date of first administration of the clinical trial drug) to the end date of the treatment period (24 weeks of treatment or the date of treatment termination). The subsequent observation period is the period from the day after the end date of the treatment period (24 weeks of treatment or the date of treatment termination) to 16 weeks after the final administration of the clinical trial drug.

[0404] (1) MAS (knee extensors and knee flexors)

[0405] The MAS (muscle tension index) is used to evaluate muscle tension and is assessed in six phases: 0, 1, 1+, 2, 3, and 4 (Kansai Rigaku. 2012;12:1-6, Phys ther. 1987;67(2):206-7). The MAS of the knee extensors and flexors were evaluated on the first day of treatment (before administration) (baseline), at 4 weeks, 12 weeks, and 24 weeks. Table 1 shows the MAS of the knee extensors and flexors.

[0406] [Table 1]

[0407]

[0408] (2) OABSS

[0409] OABSS is an assessment index for overactive bladder, used to evaluate urinary dysfunction over the past week. For the four items in Table 2, the frequency of each item is recorded as a score, with a total score ranging from 0 to 15. Furthermore, severity is assessed in three stages: mild (0–5), moderate (6–11), and severe (12–15).

[0410] Participants completed the OABSS questionnaire (UROLOGY 68(2): 318-323, 2006) at the start of treatment (before administration) (baseline), 4 weeks, 12 weeks, 24 weeks, and 12 weeks of the follow-up observation period, and confirmed their results.

[0411] [Table 2]

[0412]

[0413] result

[0414] (1) MAS

[0415] The distribution of MAS at each evaluation time point for both feet (two limbs) is shown in the figure. Figure 9 .

[0416] The proportion of MAS that improved during the 24-week treatment period in both feet (2 limbs) was 55.6% (10 / 18 limbs) in the investigational drug group and 37.5% (3 / 8 limbs) in the placebo group, showing a trend toward improvement in spasticity in the investigational drug group.

[0417] (2) OABSS

[0418] The changes in OABSS and from baseline at each evaluation time are shown in Tables 3-1 and 3-2, and the changes in severity are shown in Table 4.

[0419] The least squares mean ± standard error (LS Mean ± SE) of the change in OABSS from baseline over the 24-week treatment period was -1.6 ± 0.5 in the investigational drug group and 1.6 ± 0.8 in the placebo group. The investigational drug group showed a greater improvement from baseline compared to the placebo group, indicating a trend toward improvement in neurogenic bladder.

[0420] The proportion of subjects whose severity improved within 24 weeks of treatment was 22.2% (2 / 9) in the investigational drug group and 0% (0 / 4) in the placebo group. The proportion of subjects whose severity worsened was 25.0% (1 / 4) in the placebo group, but this was not observed in the investigational drug group (0 / 9).

[0421] [Table 3-1]

[0422]

[0423] [Table 3-2]

[0424]

[0425] [Table 4]

[0426]

[0427] A partial cohort analysis was conducted on participants whose baseline HAM-BDSG (a measure of the severity of voiding dysfunction in HAM) scores were Grade 0 (no treatment and no lower urinary tract symptoms) or Grade 1 (lower urinary tract symptoms or those receiving medication), excluding those undergoing intermittent catheterization. The changes in OABSS and from baseline at each assessment time point are shown in Tables 5-1 and 5-2, and the changes in severity are shown in Table 6.

[0428] The mean ± SD of the change from baseline in OABSS over the 24 weeks of treatment was -1.4 ± 2.3 in the investigational drug group and 0.5 ± 0.7 in the placebo group. In the aforementioned subgroups, the investigational drug group also showed a larger improvement from baseline compared to the placebo group, indicating a trend toward improvement in neurogenic bladder.

[0429] The proportion of participants whose severity improved within 24 weeks of treatment was 25.0% (2 / 8) in the investigational drug group and 0% (0 / 2) in the placebo group. No deterioration was observed in either group.

[0430] [Table 5-1]

[0431]

[0432] [Table 5-2]

[0433]

[0434] [Table 6]

[0435]

[0436] <Explanation of the sequence list>

[0437] Sequence No. 1: Amino acid sequence of human RGMa precursor protein

[0438] Serial number 2: Amino acid sequence of mouse RGMa precursor protein

[0439] Serial number 3: Amino acid sequence of rat RGMa precursor protein

[0440] Serial number 4: DNA sequence of the human RGMa gene

[0441] Serial number 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3

[0442] Serial number 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3

[0443] Serial number 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3

[0444] Serial number 8: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r116A3

[0445] Serial number 9: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r116A3

[0446] Serial number 10: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody r116A3

[0447] Serial number 11: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r70E4

[0448] Serial number 12: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r70E4

[0449] Serial number 13: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r70E4

[0450] Serial number 14: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r70E4

[0451] Serial number 15: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r70E4

[0452] Serial number 16: Amino acid sequence of the epitope of human RGMa

[0453] Serial number 17: Amino acid sequence of LCDR1 in anti-RGMa neutralizing antibody 5F9

[0454] Serial number 18: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 5F9

[0455] Serial number 19: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 5F9

[0456] Serial number 20: Amino acid sequence of HCDR1 in anti-RGMa neutralizing antibody 5F9

[0457] Serial number 21: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 5F9

[0458] Serial number 22: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 5F9

[0459] Serial number 23: Amino acid sequence of LCDR1 in anti-RGMa neutralizing antibody 8D1

[0460] Serial number 24: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 8D1

[0461] Serial number 25: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 8D1

[0462] Serial number 26: Amino acid sequence of HCDR1 in anti-RGMa neutralizing antibody 8D1

[0463] Serial number 27: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 8D1

[0464] Serial number 28: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 8D1

[0465] Serial number 29: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody AE12-1

[0466] Serial number 30: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody AE12-1

[0467] Serial number 31: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1

[0468] Serial number 32: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody AE12-1

[0469] Serial number 33: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody AE12-1

[0470] Serial number 34: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody AE12-1

[0471] Serial number 35: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1Y

[0472] Serial number 36: Amino acid sequence of the epitope of human RGMa

[0473] Serial number 37: Amino acid sequence of the epitope of human RGMa

[0474] Serial number 38: Amino acid sequence of the epitope of human RGMa

[0475] Serial number 39: Amino acid sequence of the epitope of human RGMa

[0476] Serial number 40: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1F

[0477] Serial number 41: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1H

[0478] Serial number 42: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1L

[0479] Serial number 43: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1V

[0480] Serial number 44: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1I

[0481] Serial number 45: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1K

[0482] Serial number 46: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab)

[0483] Serial number 47: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab)

[0484] Serial number 48: Light chain amino acid sequence of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab)

[0485] Serial number 49: Heavy chain amino acid sequence of anti-RGMa neutralizing antibody r116A3 (humanized antibody) (Unasnemab)

[0486] Serial number 50: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody r70E4

[0487] Serial number 51: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody r70E4

[0488] Serial number 52: Light chain amino acid sequence of anti-RGMa neutralizing antibody r70E4

[0489] Serial number 53: Heavy chain amino acid sequence of anti-RGMa neutralizing antibody r70E4

[0490] Serial number 54: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody 5F9

[0491] Serial number 55: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody 5F9

[0492] Serial number 56: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody 8D1

[0493] Serial number 57: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody 8D1

[0494] Serial number 58: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody AE12-1

[0495] Serial number 59: Amino acid sequence of the heavy chain variable region of anti-RGMa neutralizing antibody AE12-1

[0496] Serial number 60: Amino acid sequence of the light chain variable region of anti-RGMa neutralizing antibody AE12-1Y

[0497] Serial number 61: Light chain amino acid sequence of anti-RGMa neutralizing antibody AE12-1Y-QL (Elezanumab)

[0498] Serial number 62: Heavy chain amino acid sequence of anti-RGMa neutralizing antibody AE12-1Y-QL (Elezanumab)

[0499] Industrial availability

[0500] The RGMa inhibitor of the present invention is expected to be useful for the prevention or treatment of accompanying symptoms of diseases caused by spinal cord or brain disorders, specifically spasms and / or neurogenic bladder, and has high utilization value in the pharmaceutical industry.

Claims

1. A preventive or therapeutic agent for spasticity, with RGMa inhibitory substance as the active ingredient.

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

3. The preventive or therapeutic agent according to claim 1, wherein, Spasm is a spasm that accompanies spinal cord injury.

4. The preventive or therapeutic agent according to claim 1, wherein, Spasm is a type of spasm associated with HTLV-1-related spinal cord disease.

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

6. The preventive or therapeutic agent according to claim 5, wherein, Anti-RGMa neutralizing antibody is a humanized antibody.

7. The preventive or therapeutic agent according to claim 5 or 6, wherein, The anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from serial numbers 16, 36, 37, 38 and 39.

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

9. The preventive or therapeutic agent according to any one of claims 5 to 8, wherein, The anti-RGMa neutralizing antibody is one of the following (a) or (f): (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10; (f) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

10. A method for preventing or treating spasms, comprising the steps of administering an effective amount of RGMa inhibitor to a mammal requiring treatment.

11. The method of prevention or treatment according to claim 10, wherein, The RGMa inhibitor is an anti-RGMa neutralizing antibody.

12. The use of RGMa inhibitors in the prevention or treatment of spasms.

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

14. A preventive or therapeutic agent for spasmodic and neurogenic bladder, with an RGMa inhibitor as the active ingredient.

15. The preventive or therapeutic agent according to claim 14, wherein, Spasmodic and neurogenic bladder is a condition associated with disorders of the spinal cord or brain.

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

17. The preventive or therapeutic agent according to claim 14, wherein, Spasmodic and neurogenic bladder are associated with HTLV-1-related spinal cord disease.

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

19. The preventive or therapeutic agent according to claim 18, wherein, Anti-RGMa neutralizing antibody is a humanized antibody.

20. The preventive or therapeutic agent according to claim 18 or 19, wherein, The anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from serial numbers 16, 36, 37, 38 and 39.

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

22. The preventive or therapeutic agent according to any one of claims 18 to 21, wherein, The anti-RGMa neutralizing antibody is one of the following (a) or (f): (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10; (f) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 29, LCDR2 containing the amino acid sequence described in sequence number 30, and LCDR3 containing the amino acid sequence described in sequence number 35, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 32, HCDR2 containing the amino acid sequence described in sequence number 33, and HCDR3 containing the amino acid sequence described in sequence number 34.

23. A method for the prevention or treatment of spasmodic and neurogenic bladder, comprising the step of administering an effective amount of RGMa inhibitor to the mammal requiring treatment.

24. The method of prevention or treatment according to claim 23, wherein, The RGMa inhibitor is an anti-RGMa neutralizing antibody.

25. The use of RGMa inhibitors in the prevention or treatment of spasmodic 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 the active ingredient.

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

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

30. The preventive or therapeutic agent according to claim 27, wherein, Neurogenic bladder is a neurogenic bladder associated with HTLV-1-related spinal cord disease.

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

32. The preventive or therapeutic agent according to claim 31, wherein, Anti-RGMa neutralizing antibody is a humanized antibody.

33. The preventive or therapeutic agent according to any one of claims 31 or 32, wherein, The anti-RGMa neutralizing antibody is as follows (a): (a) An anti-RGMa neutralizing antibody comprising the following light chain variable regions and heavy chain variable regions, wherein the light chain variable regions comprise: LCDR1 containing the amino acid sequence described in sequence number 5, LCDR2 containing the amino acid sequence described in sequence number 6, and LCDR3 containing the amino acid sequence described in sequence number 7, and the heavy chain variable regions comprise: HCDR1 containing the amino acid sequence described in sequence number 8, HCDR2 containing the amino acid sequence described in sequence number 9, and HCDR3 containing the amino acid sequence described in sequence number 10.

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

35. The method of prevention or treatment according to claim 34, wherein, The RGMa inhibitor is an anti-RGMa neutralizing antibody.

36. The use of RGMa inhibitors in the manufacture of preventive or therapeutic agents for neurogenic bladder.

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

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