Method for preventing or treating pain associated with peripheral neuropathy or diseases in which peripheral neuropathy or astrocyte disorder is observed

Abstract

To provide novel methods of preventing or treating peripheral neuropathies, and methods of preventing or treating pain symptoms associated with diseases characterized by peripheral neuropathies or astrocytic disorders.SOLUTION: The present invention provides an agent for preventing or treating peripheral neuropathies or pain symptoms associated with diseases characterized by peripheral neuropathies or astrocytic disorders, wherein the agent contains an RGM inhibiting substance.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a preventive or therapeutic agent for pain associated with peripheral neuropathy or a disease in which peripheral neuropathy or astrocyte disorder is observed, characterized by inhibiting RGMa activity, and a preventive or therapeutic method using the same. [Background technology]

[0002] Astrocytes are one of the glial cells present in the central nervous system. They have the following functions: (1) structurally support the neuronal network; (2) transport substances to and from the astrocytes; Astrocytes have a variety of functions, including (1) regulating various conditions around the synapse, (2) performing one synaptic function through the close relationship between presynaptic, postsynaptic, and glial cells, (3) regulating the concentration of extracellular ions, (4) buffering energy, and (5) promoting the myelination activity of oligodendrocytes. The above-mentioned functions of astrocytes in various brain diseases have attracted attention, particularly in relation to the association of aging-related decline in astrocyte function with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and cognitive impairment, cerebrovascular disorders, and psychiatric disorders (Non-Patent Documents 1-3). Peripheral neuropathy is a pathological condition in which normal conduction in peripheral nerves is impaired. The types of nerves affected by peripheral neuropathy include motor nerves, sensory nerves, and autonomic nerves. Clinically, peripheral neuropathy is classified as mononeuropathy (a single nerve disorder), multiple mononeuropathy (a disorder involving two or more nerves in separate areas), or polyneuropathy (a widespread, symmetrical neuropathic disorder). Pathologically, it is classified as axonopathy, in which the axon is centrally affected, or myelinopathy, in which the myelin sheath is degenerated and lost. Peripheral neuropathy damages motor, sensory, and autonomic nerves. As a result, symptoms such as pain, paresthesia, paralysis, numbness, muscle weakness, sweating abnormalities, or urinary disorders worsen over time. Major causes of peripheral neuropathy include physical damage such as deformity, compression, circulatory disorders, genetic factors, and metabolic disorders. Diabetic neuropathy includes polyneuropathy and mononeuropathy, and polyneuropathy includes sensory, motor, and autonomic neuropathy of the peripheral nervous system. Methylvitamin B12 (methylcobalamin) is clinically used as a therapeutic agent for peripheral neuropathy (Non-Patent Document 4), but clinically effective cases are extremely rare. Furthermore, motor paralysis associated with neuropathy seriously affects daily life, but an effective treatment has not yet been established.

[0003] RGM (repulsive guidance molecule) was originally identified as an axon guidance molecule in the visual system. RGM family is a membrane protein that has been identified as a marker for iron metabolism (see Non-Patent Document 5). The RGM family includes three members, called RGMa, RGMb, and RGMc (Non-Patent Document 6), and it is known that at least RGMa and RGMb function via the same signal transduction mechanism (Non-Patent Document 7). RGMc plays an important role in iron metabolism. Subsequent research has revealed that RGM has functions such as axon guidance and lamina formation in Xenopus and chicken embryos, and regulating the closure of the cranial neural tube in mouse embryos (see Non-Patent Document 8). Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0004] RGMa is thought to be an inhibitor of axon regeneration after central nervous system injury, because RGMa not only functions during development but also re-expresses after central nervous system injury in adult humans and rats, and because RGMa inhibition in rats enhances axon growth and promotes functional recovery after spinal cord injury (see Non-Patent Document 9). Specific antibodies that neutralize RGMa include those described in, for example, Patent Document 2. (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3). In addition, it is known that anti-RGMa antibodies are effective against neuromyelitis optica (see Non-Patent Document 10). Although the role of RGMa in central nervous system injury has been clarified, the involvement of RGMa in pain symptoms associated with peripheral neuropathy or diseases in which peripheral neuropathy or astrocytic damage is observed has not been identified. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2005 / 087268 [Patent Document 2] International Publication No. WO2009 / 106356 [Patent Document 3] International Publication No. WO2013 / 112922 [Patent Document 4] International Publication No. WO2016 / 175236 [Non-patent literature]

[0006] [Non-Patent Document 1] Dallerac G et al., Prog Neurobiol. 144: 48-67(2016) [Non-patent document 2] Gerkau NJ et al., J Neurosci Res. 2017 Feb 2. [Non-licensed document 3] Alam Q et al., Curr Pharm Des. 22: 541-8(2016)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0007] An object of the present invention is to provide a novel method for preventing or treating pain symptoms associated with peripheral neuropathy or diseases in which peripheral neuropathy or astrocytic disorder is observed. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they discovered that RGMa inhibitors can be used as prophylactic or therapeutic agents for peripheral neuropathy. Furthermore, they discovered that RGMa inhibitors are effective against pain symptoms by improving not only peripheral neuropathy but also astrocyte damage or hypofunction, and therefore can be used as prophylactic or therapeutic agents for pain symptoms associated with diseases in which peripheral neuropathy or astrocyte damage is observed, leading to the completion of the present invention. That is, the present invention relates to the following inventions.

[0009] 1. A preventive or therapeutic agent for peripheral neuropathy, comprising an RGM inhibitor. 2. The agent according to Item 1, wherein the RGM inhibitor is an RGMa inhibitor. 3. The agent according to Item 2, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody or a fragment thereof. 4. The agent according to Item 3, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 5. The agent according to Item 3 or 4, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. 6. The anti-RGMa neutralizing antibody is one of the following (a1) to (l1): (a1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and Antibody containing a heavy chain variable region including HCDR2 containing the amino acid sequence SFG and HCDR3 containing the amino acid sequence SFG. GMa neutralizing antibody, (c1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising a region, and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; a heavy chain comprising an HCDR1 region comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 region comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 region comprising the amino acid sequence set forth in SEQ ID NO: 34; an anti-RGMa neutralizing antibody containing the variable region; (i1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising a region, and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising a region, and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k1) a light chain variable resistor comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 44; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising a region, and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l1) a light chain variable resistor comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising a region, and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; Item 6. The agent according to any one of Items 3 to 5, wherein the antibody is selected from the group consisting of: 7. Peripheral neuropathy includes diabetic neuropathy, entrapment neuropathy (carpal tunnel syndrome, ulnar neuropathy at the elbow, peroneal nerve palsy, or tarsal tunnel syndrome), familial amyloid polyneuropathy, toxic neuropathy, cancer-related neuropathy, immune-mediated neuropathy (Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy (CIDP)), and neuropathy associated with collagen disease. Item 7. The agent according to any one of Items 1 to 6, wherein the neuropathy is selected from the group consisting of encephalopathy, Cough-Fukase syndrome (POEMS syndrome), hereditary neuropathy (Charcor-Marie-Tooth disease), postherpetic neuralgia, peripheral neuropathy due to AIDS or Lyme disease, uremia, multifocal motor neuropathy, and vasculitic neuropathy. 8. The agent according to any one of items 1 to 6, wherein the peripheral neuropathy is diabetic neuropathy. 9. The agent according to item 8, wherein the diabetic neuropathy is painful diabetic neuropathy and / or asymptomatic diabetic neuropathy. 10. The agent according to any one of items 1 to 6, for use in the prevention or treatment of pain symptoms caused by a disease in which peripheral neuropathy or astrocyte disorder is observed. 11. The patient has peripheral neuropathy or astrocytopathy, and the following diseases are present: diabetic neuropathy, entrapment neuropathy (carpal tunnel syndrome, ulnar neuropathy at the elbow, peroneal nerve palsy, or tarsal tunnel syndrome), familial amyloid polyneuropathy, toxic neuropathy, cancer neuropathy, immune-mediated neuropathy (Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy) CIDP), neuropathy associated with collagen disease, Crow-Fukase syndrome (POEMS syndrome) ), hereditary neuropathy (Charcor-Marie-Tooth disease), postherpetic neuralgia, AIDS or leukemia Item 11. The agent according to Item 10, wherein the neuropathy is selected from peripheral neuropathy due to Immunol disease, uremia, multifocal motor neuropathy, vasculitic neuropathy, neuromyelitis optica, and Alexander disease. 12. The agent according to item 11, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is diabetic neuropathy or neuromyelitis optica. 13. The agent according to item 11, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is diabetic neuropathy. 14. The agent according to item 11, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is neuromyelitis optica. 15. Administering to a mammal in need thereof an effective amount of an RGMa inhibitor; A method for preventing or treating peripheral neuropathy. 16. The method according to item 15, wherein the peripheral neuropathy is diabetic neuropathy. [Effects of the Invention]

[0010] The present invention can provide a preventive or therapeutic agent for peripheral neuropathy. The present invention can also provide a preventive or therapeutic agent for pain symptoms associated with diseases in which peripheral neuropathy or astrocyte damage is observed. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the ameliorative effect of repeated administration of r116A3 on mechanical hyperalgesia in a rat streptozocin (STZ)-induced diabetic neuropathy model. [Figure 2] FIG. 2 shows the ameliorative effect of repeated administration of r116A3 on the decrease in motor nerve conduction velocity in a rat STZ-induced diabetic neuropathy model. [Figure 3] FIG. 3 shows the effect of repeated administration of r116A3 on the percentage of glial fibrillary acidic protein (GFAP) immunostaining-positive area in the spinal cord of a rat STZ-induced diabetic neuropathy model. [Figure 4] FIG. 4 shows the effect of repeated administration of r116A3 on the percentage of Iba1 immunostaining-positive area in the spinal cord of a rat STZ-induced diabetic neuropathy model. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, an embodiment of the present invention will be described. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, but in the event of a potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless otherwise stated, singular terms include pluralities, and plural terms include the singular. As used herein, the use of "or" means "and / or" unless otherwise stated.

[0013] Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods known in the art and described in various general references and the more specific references cited and discussed herein, unless otherwise indicated. Enzymatic reactions and purification techniques are either commonly practiced in the art or are performed according to manufacturer's specifications as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparations, formulation, delivery, and treatment of patients. To facilitate understanding of the present invention, the terms used in the present invention are explained below.

[0014] [Neutralization] As used herein, "neutralizing" refers to the ability to bind to a target of interest and inhibit any function of that target. For example, an RGMa inhibitor refers to a substance that inhibits the biological activity of RGMa as a result of binding to RGMa.

[0015] [epitope] As used herein, an epitope includes a polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In some embodiments, an epitope includes a chemically active surface grouping of a molecule (e.g., an amino acid, a sugar side chain, a phosphoryl, or a sulfonyl). In some embodiments, an epitope may have particular three-dimensional structural characteristics and / or particular charge characteristics. An epitope is the region of an antigen that is bound by an antibody.

[0016] [Isolated] As used herein, the term "isolated" in reference to an isolated RGMa inhibitor (e.g., an antibody) means identified and separated and / or recovered from components in its natural state. Impurities in the natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, an RGMa inhibitor can be isolated by purification through at least one purification step, and an RGMa inhibitor purified through at least one purification step can be referred to as an "isolated RGMa inhibitor."

[0017] [antibody] In this application, the term "antibody" broadly refers to an immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), which substantially retains the epitope-binding property of an Ig molecule.

[0018] [Human antibody] As used herein, the term "human antibody" refers to an antibody in which both the light chain and the heavy chain are derived from human immunoglobulin. Depending on the differences in the heavy chain constant region, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having γ heavy chains, IgM having μ heavy chains, IgA (including IgA1 and IgA2) having α heavy chains, IgD having δ heavy chains, and IgE having ε heavy chains. In principle, the light chain includes either a κ chain or a λ chain.

[0019] [Humanized antibody] As used herein, the term "humanized antibody" refers to an antibody that comprises a variable region consisting of a complementarity-determining region of an antibody derived from a non-human animal and a framework region derived from a human antibody, and a constant region derived from a human antibody.

[0020] [Chimeric antibody] As used herein, the term "chimeric antibody" refers to an antibody in which the light chain, the heavy chain, or both, are composed of variable regions of non-human origin and constant regions of human origin.

[0021] [Monospecific antibodies] As used herein, a "monospecific antibody" refers to an antibody that has a single antigen specificity and a single, independent antigen-recognition site. For example, a monospecific antibody that recognizes RGMa may be referred to as an RGMa monospecific antibody.

[0022] [Multispecific antibodies] As used herein, the term "multispecific antibody" refers to an antibody that has two or more independent antigen recognition sites with two or more different antigen specificities, and examples include bispecific antibodies that have two antigen specificities and trispecific antibodies that have three antigen specificities.

[0023] [Complementarity-determining region (CDR)] "Complementarity determining region (CDR)" refers to the region of the variable region of an immunoglobulin molecule that forms the antigen-binding site, also known as the hypervariable region, which is the part where the amino acid sequence varies greatly from one immunoglobulin molecule to another. There are three CDRs in each of the light chain and heavy chain. The three CDRs in the light chain are referred to as LCDR1, LCDR2, and LCDR3, respectively. The three CDRs in the heavy chain and the heavy chain are sometimes referred to as HCDR1, HCDR2, and HCDR3. For example, the CDRs of an immunoglobulin molecule are numbered according to the Kabat numbering system. (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0024] [Effective dose] An "effective amount" refers to the amount of a prophylactic or therapeutic agent sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent the progression of a disorder, reverse a disorder, prevent the recurrence, occurrence, onset or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve one or more prophylactic or therapeutic effects of another treatment (e.g., a prophylactic or therapeutic agent).

[0025] [Percent (%) identity of amino acid sequence] The "percent (%) identity" of an amino acid sequence of a candidate polypeptide sequence, such as a variable region, with respect to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in a particular reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and excluding any conservative substitutions from being considered part of the sequence identity. Alignment for purposes of determining percent identity can be achieved by a variety of methods within the skill of those in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent identity values ​​are obtained using the sequence comparison computer program BLAST in pairwise alignments. In situations where BLAST is used for amino acid sequence comparison, the percent identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues whose scores match as being identical by the program alignment of programs A and B of the array alignment program BLAST, and Y is the total number of amino acid residues of B. It will be understood that when the length of amino acid sequence A is different from the length of amino acid sequence B, the % identity of A to B will be different from the % identity of B to A. Unless otherwise specified, all % identity values here are obtained using the BLAST computer program as shown in the paragraph immediately above.

[0026] Hereinafter, the present invention will be described in detail. Embodiments of the present invention provide a prophylactic or therapeutic agent for peripheral neuropathy, which is a novel use of an RGM inhibitor, particularly an RGMa inhibitor. Further, embodiments of the present invention provide a prophylactic or therapeutic agent for diabetic neuropathy, for example, an RGMa inhibitor. Furthermore, other embodiments of the present invention provide a prophylactic or therapeutic agent for pain symptoms caused by a disease in which peripheral neuropathy or astrocyte damage is recognized, using an RGMa inhibitor. Also, other embodiments of the present invention provide a method for preventing or treating peripheral neuropathy, such as diabetic neuropathy, which includes the step of administering a prophylactic or therapeutic agent containing an effective amount of an RGMa inhibitor to a mammal requiring treatment.

[0027] <RGM inhibitor> As the RGM inhibitor of the present invention, it inhibits the activity of RGM (hereinafter sometimes simply referred to as "RGM activity") that induces pain symptoms of peripheral neuropathy or a disease in which peripheral neuropathy or astrocyte damage is recognized, or inhibits recovery from the disease or symptoms, which will be described later Any substance that inhibits or a substance that inhibits the expression of RGM may be used. Here, RGM means one or more selected from RGMa, RGMb, and RGMc, and preferably RGMa.

[0028] RGMa has been identified as a neurite outgrowth inhibitor in the central nervous system. Human RGMa protein is biosynthesized as a precursor protein consisting of 450 amino acids as shown in SEQ ID NO: 1. The signal peptide Met1-Pro47 (referring to the peptide from the first methionine residue to the 47th proline residue from the N-terminus) is present at the N-terminus. The peptide bond between Asp168 and Pro169 is cleaved to form the N-terminal The C-terminal peptide Ala425-Cys450 of the fragment C-terminal to Pro169 is then removed, and a GPI anchor is added to the C-terminal carboxyl group of the resulting C-terminal Ala424 to form the C-terminal domain. The mature protein, in which the N-terminal domain (Cys48-Asp168) and the C-terminal domain (Pro169-Ala424) are linked by disulfide bonds, is then expressed on the cell membrane via the GPI anchor.

[0029] In the present invention, RGMa may be derived from any animal, but is preferably human RGMa. The precursor protein of human RGMa consists of the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. The precursor protein of mouse RGMa consists of the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing, and the precursor protein of rat RGMa consists of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing, but because the C-terminal peptide is removed, the mature proteins have the same amino acid sequences. Examples of RGMa genes include, but are not limited to, the human RGMa gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4. The nucleotide sequences of RGM genes derived from various organisms can be easily obtained from publicly known databases (such as GenBank).

[0030] The RGMa inhibitor of the present invention may be either a substance that inhibits (neutralizes) the activity of RGMa that induces pain symptoms due to peripheral neuropathy or diseases in which peripheral neuropathy or astrocyte damage is observed, or that inhibits recovery from such diseases or symptoms (hereinafter, sometimes simply referred to as "RGMa activity" in this specification), or a substance that inhibits the expression of RGMa. For example, the RGMa inhibitor of the present invention may be used to evaluate the effect of the RGMa inhibitor on pain and nerve conduction disorders using the evaluation method described in Example 1, i.e., a rat STZ-induced diabetic neuropathy model. The selection can be made by evaluating the improvement effect, etc.

[0031] The RGMa inhibitor of the present invention refers to, for example, a substance that directly inhibits RGMa activity by binding to RGMa, or indirectly inhibits RGMa activity by inhibiting the binding of RGMa to a receptor, and specific examples thereof include low molecular weight compounds, anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and fragments thereof. Substances that inhibit RGMa activity by inhibiting the expression of RGMa are also RGMa inhibitors, and specific examples thereof include siRNA (short interfering RNA), shRNA (short interfering RNA) of the RGMa gene, and the like. Among these RGMa inhibitors, anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and fragments thereof are preferred, and anti-RGMa neutralizing antibodies or fragments thereof are more preferred, with anti-RGMa neutralizing antibodies being particularly preferred.

[0032] Furthermore, the RGMa inhibitors of the present invention also include substances that do not act directly on RGMa but inhibit the activity of any of the related molecules in the signal transduction system in which RGMa induces symptoms of peripheral neuropathy, such as diabetic neuropathy.

[0033] In an embodiment of the present invention, the anti-RGMa neutralizing antibody may be any antibody that binds to RGMa and neutralizes the RGMa activity of the present invention, and may be a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Furthermore, the RGMa neutralizing antibody of the present invention may be either a monospecific RGMa antibody or a multispecific antibody that recognizes multiple antigens, including RGMa, but is preferably a monospecific RGMa antibody.

[0034] Furthermore, specific epitopes in human RGMa are preferably one or more of SEQ ID NO: 16 (amino acid numbers 47-69 of SEQ ID NO: 1), SEQ ID NO: 36 (amino acid numbers 298-311 of SEQ ID NO: 1), SEQ ID NO: 37 (amino acid numbers 322-335 of SEQ ID NO: 1), SEQ ID NO: 38 (amino acid numbers 349-359 of SEQ ID NO: 1), and SEQ ID NO: 39 (amino acid numbers 367-377 of SEQ ID NO: 1), more preferably the combination of SEQ ID NOs: 36 and 37, and particularly preferably the combination of SEQ ID NOs: 36, 37, and 39.

[0035] The anti-RGMa neutralizing antibodies of the present invention include polyclonal and monoclonal antibodies obtained by immunizing a mammal such as a mouse with an RGMa protein or a partial fragment thereof (for example, the epitope fragment described above), as an antigen, chimeric and humanized antibodies produced using genetic recombination techniques, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibodies of the present invention are administered to humans as pharmaceuticals, humanized or human antibodies are preferred from the viewpoint of side effects.

[0036] Specific examples of the anti-RGMa neutralizing antibody of the present invention include the following antibodies (a1) to (l2), and the methods described in Patent Documents 2 to 4 can be used to produce each of them.

[0037] (a1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, and LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (the anti-RGMa neutralizing antibody further includes antibodies having epitopes set forth in SEQ ID NOs: 36, 37, and 39); (b1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and Antibody containing a heavy chain variable region including HCDR2 containing the amino acid sequence SFG and HCDR3 containing the amino acid sequence SFG. GMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes antibodies having epitopes of SEQ ID NOs: 36, 37, and 38), (c1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27, and an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16 (the anti-RGMa neutralizing antibody further comprises an epitope comprising SEQ ID NO: 16); (including antibodies against the target antigen) (f1) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38; an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 39; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (g1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (h1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (i1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (j1) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (k1) a light chain variable resistor comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 44; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); and (l1) a light chain variable resistor comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); an antibody selected from More preferably, the following (a2) to (l2): (a2) LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7 and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 8; an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 36; 7 and 39 as epitopes), (b2) LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, and LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 15; A heavy chain variable region comprising HCDR2 having the amino acid sequence shown above and HCDR3 having the amino acid sequence of SFG. an anti-RGMa neutralizing antibody comprising the region (the anti-RGMa neutralizing antibody further includes antibodies having epitopes represented by SEQ ID NOs: 36, 37, and 38); (c2) LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, and LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 23; (d2) LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, and LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 25, an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 26, an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and a an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 27 and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 28; (e2) LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, and LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 31, an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (f2) LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, and LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (g2) a light-emitting device comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region, and an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (h2) a light-emitting device comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region, and an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (i2) a light-emitting device comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region, and an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (j2) a light-emitting device comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43 a chain variable region, and an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); (k2) a light-emitting device comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 44; an anti-RGMa neutralizing antibody comprising a heavy chain variable region, and an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); (12) A light-emitting device comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45. an anti-RGMa neutralizing antibody comprising a heavy chain variable region, and an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); Examples of antibodies include those selected from the following: Among these, the antibody described in (a2) is particularly preferred.

[0038] The anti-RGMa neutralizing antibody of the present invention can be produced by any commonly used existing production method. The antigen may be used for immunization as is, or may be used as a complex with a carrier protein. Condensing agents such as glutaraldehyde, carbodiimide, and maleimide activated esters can be used to prepare the complex of the antigen and carrier protein. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[0039] Mammals to be immunized include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses, and cows, and inoculation methods include subcutaneous, intramuscular, and intraperitoneal administration. The antigen may be administered by mixing with complete or incomplete Freund's adjuvant, and is usually administered once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of immunized animals are fused with myeloma cells and isolated as hybridomas. Myeloma cells derived from mammals, such as mice, rats, and humans, are used.

[0040] Polyclonal antibodies can be produced, for example, by immunizing a mammal as described above with the antigen, optionally together with Freund's adjuvant. It can be obtained from serum obtained from sensitized animals.

[0041] Monoclonal antibodies are described, for example, in Current Protocols in Molecular Biology (John Wiley & Sons (1987), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988), and Preparation of "hybridomas" secreting monoclonal antibodies can be carried out according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) or modifications thereof. Specifically, monoclonal antibodies can be obtained as follows. That is, the above-mentioned antigen is used as an immunogen, and the immunogen, together with Freund's adjuvant as necessary, is injected subcutaneously, intramuscularly, intravenously, into the footpad, or intraperitoneally into the mammal described above one to several times, or the immunogen is implanted therein, thereby immunizing the mammal. Usually, immunization is carried out one to four times at intervals of about 1 to 14 days after the initial immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

[0042] The hybridoma is a hybrid of the antibody-producing cells and a mammal, preferably a mouse, rat, or human. The antibody is prepared by cell fusion of mouse-derived myeloma cells lacking the ability to produce autoantibodies, using a fusion promoter as needed.

[0043] Examples of myeloma cells that can be used in cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0, or BW5147; rat-derived myeloma 210RCY3-Ag.2.3; and human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15.

[0044] Examples of fusion promoters include polyethylene glycol, and cell fusion can be achieved by reacting polyethylene glycol (average molecular weight 1000 to 4000) at a concentration of approximately 20 to 50%, at a temperature of 20 to 40°C, preferably 30 to 37°C, at a ratio of antibody-producing cells to myeloma cells of typically 1:1 to 10:1, for approximately 1 to 10 minutes.

[0045] Screening for hybridoma clones that produce monoclonal antibodies can be carried out by culturing the hybridomas, for example, in a microtiter plate and measuring the reactivity of the culture supernatant in the wells to the immunogen by an immunochemical method such as ELISA.

[0046] In screening for antibody-producing hybridomas, in addition to assaying for binding to RGMa protein, we also evaluate whether the antibody inhibits the RGMa activity of the present invention. These screening methods allow us to select anti-RGMa neutralizing antibodies of the present invention.

[0047] Clones can be obtained from wells containing hybridomas that produce the desired antibody by limiting dilution. Hybridoma selection and breeding are usually carried out in an animal cell medium containing 10-20% fetal bovine serum and supplemented with HAT (hypoxanthine, aminopterin, thymidine).

[0048] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or by growing them in vivo, for example, in ascites of a mammal such as a mouse or rat, and isolating the antibodies from the resulting culture supernatant or from the ascites of the mammal.

[0049] When culturing in vitro, a nutrient medium suitable for growing, maintaining, and preserving hybridomas and producing monoclonal antibodies in the culture supernatant can be used, depending on various conditions such as the characteristics of the cell type being cultured and the culture method, etc. Examples of the nutrient medium include known nutrient media and nutrient media prepared from basal media.

[0050] Examples of basal media include low-calcium media such as Ham's F12 medium, MCDB153 medium, and low-calcium MEM medium, and high-calcium media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, and RD medium. Depending on the purpose, the basal medium may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances.

[0051] The monoclonal antibodies can be isolated and purified by subjecting the culture supernatant or ascites fluid to saturated ammonium sulfate, the euglobulin precipitation method, the caproic acid method, the caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), or affinity column chromatography such as an anti-immunoglobulin column or a protein A column. Specifically, monoclonal antibodies can be purified using known immunoglobulin purification methods, and can be easily achieved by, for example, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of an anion exchanger, or affinity chromatography using RGMa protein.

[0052] Monoclonal antibodies can also be obtained by phage display. In phage display, phages selected from a phage antibody library are screened with the target immunogen, and phages with the desired binding affinity to the immunogen are selected. Next, the antibody-corresponding sequence contained in the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding domain is constructed based on the isolated or sequence information. Monoclonal antibodies can then be produced by culturing a cell line transfected with such an expression vector. Human antibodies with the desired binding affinity can be generated by using a human antibody library as the phage antibody library.

[0053] Nucleic acid molecules encoding anti-RGM-like antibodies or fragments thereof can be obtained, for example, by the following method. First, total RNA is prepared from cells such as hybridomas using a commercially available RNA extraction kit, and cDNA is synthesized using reverse transcriptase and random primers. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotide primers with sequences conserved in the variable regions of known human antibody heavy chain and light chain genes. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The DNA base sequence can be determined by standard methods, such as by incorporating it into a sequencing plasmid. Alternatively, DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the sequence of the variable region or a part thereof and ligating it to a sequence containing the constant region. The nucleic acid molecule may encode both the heavy and light chain constant and variable regions, or may encode only the heavy and light chain variable regions. When encoding both the constant and variable regions, the base sequences of the heavy and light chain constant regions are preferably those described in Nucleic Acids Research, vol. 14, p. 1779, 1986, The Journal of Biological Chemistry, vol. 257, p. 1516, 1982, and Cell, vol. 22, p. 197, 1980.

[0054] Functionally modified antibodies are prepared by the following methods. For example, the half-life in blood can be extended by using a mutant Fc region that enhances binding to FcRn, one of the Fc receptors (Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p. 710). Mutant antibodies can be produced by genetic engineering.

[0055] Conjugate antibodies include anti-RGMa neutralizing antibodies that have been chemically or genetically linked to functional molecules other than the anti-RGMa neutralizing antibodies of the present application, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies.

[0056] When PEG is used as a functional molecule, the molecular weight of the PEG may be, but is not limited to, 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be either linear or branched. PEG can be attached to the N-terminal amino group of an amino acid in an RGMa inhibitor, for example, by using an NHS-activated group.

[0057] When a radioactive substance is used as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 Radioactive materials are detected by the chloramine T method, etc. The inhibitor can be directly bound.

[0058] When a toxin is used as a functional molecule, bacterial toxins (eg, diphtheria toxin), plant toxins (eg, ricin), low-molecular-weight toxins (eg, geldanamycin), maytansinoids, calicheamicin, and the like can be used.

[0059] When a low molecular weight compound is used as a functional molecule, examples thereof include daunomycin, doxorubicin, metrorexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0060] When an enzyme is used as the functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. may be used.

[0061] Linkers used to chemically link toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2) n O(CR2) n Linkers represented by - (R is an arbitrary substituent, n is a positive integer), alkoxy repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino repeating units (e.g., polyethyleneamino, Jeffamine TM ), and diacid esters and amides (such as succinate, succinamide, diglycolate, malonate, and caproamide). Chemical modification methods for binding functional molecules have already been established in this field (DJ King., Applications and Engineering of Monoclonal Antibodies, 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer, 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol. 152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol. 93:8681).

[0062] In an embodiment of the present invention, the term "fragment" of an antibody refers to a partial region of an antibody as described above that has antigen-binding activity, and specifically includes F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof.Furthermore, fragments include conjugated fragments to which functional molecules other than the anti-RGMa neutralizing antibody of the present application are chemically or genetically engineered, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies.

[0063] "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulin with protease enzymes such as pepsin or papain, resulting in digestion across the disulfide bond between the two heavy chains in the hinge region. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond between the two heavy chains in the hinge region, producing two homologous antibody fragments in which a light chain consisting of a VL (light chain variable region) and a CL (light chain constant region), and a heavy chain fragment consisting of a VH (heavy chain variable region) and a CHγ1 (γ1 region in the heavy chain constant region) are linked by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab. Also, I When IgG is treated with pepsin, it is cleaved downstream of the disulfide bond between the two heavy chains in the hinge region, resulting in an antibody fragment that is slightly larger than the two Fab fragments connected by the hinge region. This antibody fragment is called F(ab')2.

[0064] A preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a chimeric antibody. Examples of "chimeric antibodies" include those whose variable regions are derived from immunoglobulins of non-human animals (such as mice, rats, hamsters, and chickens) and whose constant regions are derived from human immunoglobulins. For example, chimeric antibodies can be produced by immunizing a mouse with an antigen, excising the variable region that binds to the antigen from the mouse monoclonal antibody gene, and then combining it with an antibody constant region derived from human bone marrow. Constant regions derived from human immunoglobulins have unique amino acid sequences depending on the isotype, such as IgG (IgG1, IgG2, IgG3, and IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE. The constant region of the recombinant chimeric antibody of the present invention may be the constant region of a human immunoglobulin belonging to any isotype. Preferably, it is a human IgG constant region. Expression vectors can be constructed using the genes of chimeric antibodies produced in this manner. Host cells are transformed with the expression vector to obtain transformed cells that produce chimeric antibodies, and the transformed cells are cultured to obtain the desired chimeric antibodies from the culture supernatant.

[0065] Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a humanized antibody. The "humanized antibody" of the present invention is an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity-determining region) of a non-human animal antibody, such as a mouse, has been grafted onto a human antibody gene (CDR grafting). For example, it can be prepared by referring to the methods described in JP-A-4-506458 and Japanese Patent No. 2912618. Specifically, this refers to a humanized antibody in which some or all of its CDRs are derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework regions of its variable regions are derived from a human immunoglobulin, and its constant regions are derived from a human immunoglobulin.

[0066] The humanized antibody of the present invention can be produced, for example, as follows: However, it goes without saying that the production method is not limited to this.

[0067] For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be produced by genetic engineering with reference to JP-A-4-506458 and JP-A-62-296890, etc. That is, DNA of the mouse heavy chain CDR region and DNA of the mouse light chain CDR region are isolated from a hybridoma producing a mouse monoclonal antibody, and a human heavy chain gene covering the entire region except for the human heavy chain CDR and a human light chain gene covering the entire region except for the human light chain CDR are isolated from a human immunoglobulin gene.

[0068] The isolated human heavy chain gene grafted with DNA of the mouse heavy chain CDR region is introduced into an appropriate expression vector so as to be expressible, and similarly, the human light chain gene grafted with DNA of the mouse light chain CDR region is introduced into another appropriate expression vector so as to be expressible. Alternatively, the human heavy chain and light chain genes grafted with mouse CDRs can be introduced into the same expression vector so as to be expressible. Host cells are transformed with the expression vector prepared in this way to obtain humanized antibody-producing transformants, and the desired humanized antibody is obtained from the culture supernatant by culturing the transformants.

[0069] Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a human antibody. A "human antibody" is an antibody in which all regions constituting the immunoglobulin, including the heavy chain variable region, heavy chain constant region, and light chain variable region, and light chain constant region, are derived from a gene encoding human immunoglobulin, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal produced by incorporating at least a human immunoglobulin gene into the gene locus of a non-human mammal such as a mouse can be immunized with an antigen to produce the aforementioned polyclonal antibody. Alternatively, it can be produced in the same manner as monoclonal antibodies.

[0070] For example, transgenic mice producing human antibodies are described in Nature Genetics, Vol. 7, pp. 13-21, 1994; Nature Genetics, Vol. 15, pp. 146-156, 1997; JP 4-504365 A; They can be produced according to the methods described in JP-A-7-509137, WO 94 / 25585, Nature, Vol. 368, pp. 856-859, 1994, and JP-A-6-500233, etc. More specifically, HuMab® mice (Medarex, Princeton, NJ), KM™ mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice, etc.

[0071] Specific examples of anti-RGMa neutralizing antibodies of the present invention include those having a CDR containing a specific amino acid sequence in the heavy chain variable region and a CDR containing a specific amino acid sequence in the light chain variable region (antibodies (a1) to (l2) above). As long as the antibody of the present invention maintains its ability to bind to RGMa and inhibit (neutralize) RGMa activity, the amino acid sequence of the anti-RGMa neutralizing antibody may contain substitutions, deletions, additions, or insertions of one or several amino acids (1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 to 2). While such substitutions, deletions, or additions may be introduced into the CDRs, they are preferably introduced into regions other than the CDRs. Furthermore, the amino acid substitutions are preferably conservative substitutions in order to maintain the properties of the present invention.

[0072] The amino acid sequence of the antibody of the present invention, which contains substitutions, deletions, etc. in the amino acid sequence, is, for example, an amino acid sequence in which the heavy chain variable region after amino acid sequence modification has a percent identity of 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before modification, and an amino acid sequence in which the light chain variable region after amino acid sequence modification has a percent identity of 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before modification.

[0073] siRNA is a short double-stranded RNA that can suppress the expression of a target gene (in the present invention, the RGMa gene). As long as it functions as an siRNA that inhibits RGMa activity in the present invention, the base sequence and length (base length) are not particularly limited, but are preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases. shRNA is a single-stranded RNA that contains a partially palindromic base sequence, thereby forming a double-stranded structure within the molecule, and is approximately a long strand consisting of a short hairpin structure with an overhang at the 3' end. This refers to a molecule of 20 base pairs or more. After being introduced into a cell, such shRNA is degraded into a length of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) within the cell, and can suppress the expression of the target gene in the same way as siRNA. In the present invention, siRNA and shRNA may be in any form as long as they can suppress the expression of the RGMa gene.

[0074] siRNA or shRNA can be artificially chemically synthesized. Alternatively, antisense and sense RNAs can be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and a T7 promoter. Antisense oligonucleotides may be either DNA or RNA, as long as they are complementary to or hybridize with a 5- to 100-base sequence of consecutive bases in the DNA sequence of the RGMa gene. Modifications may also be used as long as they do not impair function. Antisense oligonucleotides can be synthesized by conventional methods, for example, easily using a commercially available DNA synthesizer. Preferred sequences can be selected using conventional selection methods and confirmed as siRNA or shRNA in the present invention by assessing inhibition of expression of functional RGMa.

[0075] In an embodiment of the present invention, peripheral neuropathy includes diabetic neuropathy, entrapment neuropathy (carpal tunnel syndrome, ulnar neuropathy at the elbow, peroneal nerve palsy, tarsal tunnel syndrome, etc.), familial amyloid polyneuropathy, toxic neuropathy, cancer-related neuropathy, immune-mediated neuropathy (Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy (CIDP)), neuropathy associated with connective tissue disease, Crowe-Fukase syndrome (POEMS syndrome), hereditary neuropathy (Charcor-Marie-Tooth disease), postherpetic neuralgia, peripheral neuropathy due to AIDS or Lyme disease, uremia, multifocal motor neuropathy, vasculitic neuropathy, etc.

[0076] In another embodiment of the present invention, the disease in which astrocyte damage is observed means a disease in which astrocyte damage or functional decline is observed, and specific examples include neuromyelitis optica, Alexander disease, and the like. Among these embodiments, the peripheral neuropathy is preferably diabetic neuropathy, and the disease in which astrocytic damage is observed is preferably neuromyelitis optica.

[0077] Furthermore, in another embodiment of the present invention, pain symptoms caused by diseases in which peripheral neuropathy or astrocyte dysfunction is observed include symptoms that develop due to peripheral nerve damage or astrocyte dysfunction or dysfunction in the central nervous system. Here, in the present invention, diseases in which peripheral neuropathy or astrocyte dysfunction is observed that cause pain symptoms are synonymous with the above-mentioned diseases in which peripheral neuropathy or astrocyte dysfunction is observed. For example, among such pain symptoms, in diseases in which peripheral neuropathy is observed, as shown in Example 1 (1-6) below, nerve conduction velocity is improved by an RGMa inhibitor, for example, an anti-RGMa antibody, and therefore, RGMa inhibitors are effective in treating diseases in which peripheral neuropathy is observed. Furthermore, it is suggested that RGMa inhibitors can treat peripheral nerve damage by suppressing pain. It is suggested that it has an analgesic effect against pain caused by diseases in which impairment is present.

[0078] In addition, one possible mechanism of pain symptoms caused by diseases involving astrocyte damage is that astrocyte damage or dysfunction reduces the expression of glutamate transporters in the central nervous system, which in turn enhances the effects of glutamate and causes nerve hypersensitivity or excitation (e.g., Neuron 67, 834-846, Sep. 9, 2010). The pain symptoms are caused by astrocyte damage and include numbness, pain, or hypoesthesia associated with the onset of the disease.

[0079] Therefore, as shown in Example 1 (1-6) below, STZ-induced diabetic neuropathy in rats was In this injury model, peripheral neuropathy and astrocyte damage are induced, resulting in the onset of pain, and RGMa inhibitors, such as anti-RGMa neutralizing antibodies, have been shown to have analgesic effects against this pain. On the other hand, RGMa inhibitors, such as anti-RGMa neutralizing antibodies, have no effect on microglia reduction but exhibit inhibitory effects on astrocyte reduction, suggesting that RGMa inhibitors, such as anti-RGMa neutralizing antibodies, exhibit analgesic effects against pain caused by diseases in which astrocyte damage or dysfunction is observed. Therefore, the experimental results of Example 1 suggest that RGMa inhibitors, preferably anti-RGMa neutralizing antibodies, are expected to be effective against pain symptoms caused by either or both of these diseases in which peripheral neuropathy or astrocyte damage is observed. Non-Patent Document 10 also conducted experiments on astrocyte damage in neuromyelitis optica. However, because this experimental model is an animal model that cannot assess pain, the relationship between diseases in which astrocyte damage is observed and pain symptoms remains unclear from this literature. Therefore, the relationship between diseases in which astrocyte damage is observed and pain symptoms is a novel finding obtained in Example 1 of the present application.

[0080] In a preferred embodiment of the present invention, for example, diabetic neuropathy is one of the most common complications in diabetic patients, and is caused by known risk factors such as poor blood sugar control, long duration of diabetes, hypertension, and dyslipidemia, but its mechanism of onset has not yet been identified. Diabetic neuropathy is divided into distal symmetric polyneuropathy and focal mononeuropathy, and the former in particular is said to be the core symptom of diabetic neuropathy, and includes sensory neuropathy, motor neuropathy, and autonomic neuropathy.

[0081] There are no specific symptoms or tests for diagnosing diabetic neuropathy, and a comprehensive diagnosis is made by carefully listening to the patient's neurological symptoms and conducting neurological tests such as pain sensation, vibration sensation, pressure-tactile sensation, and tendon reflex tests. The main symptoms of diabetic neuropathy can be broadly divided into sensory neuropathy, motor neuropathy, and autonomic neuropathy. Sensory neuropathy is divided into painful neuropathy, in which abnormal pain sensation is prominent, and asymptomatic neuropathy, in which there is no spontaneous sensory abnormality. Sensory neuropathy causes numbness and pain in the extremities of the lower limbs early on in the disease, and as the symptoms progress, numbness becomes more pronounced. Furthermore, as the symptoms progress, symptoms such as numbness, pain, and numbness appear not only in the lower limbs but also in the extremities of the upper limbs. Sensory neuropathy not only leads to a decline in the patient's quality of life, but as the symptoms progress and sensitivity worsens, The onset of hypoesthesia increases the risk of foot gangrene and Charcot joint disease, potentially leading to limb amputation and a worsening prognosis. Therefore, early intervention in diabetic neuropathy is crucial. Meanwhile, motor neuropathy manifests with symptoms such as muscle weakness and atrophy, particularly in the lower limbs, and foot deformity. While these symptoms are usually not noticeable enough to affect daily activities, as the condition progresses, they can affect balance, climbing slopes and stairs, and strenuous walking, such as brisk walking. The agent for preventing or treating diabetic neuropathy of the present invention can be used to improve symptoms found by the above-mentioned tests or to prevent or treat symptoms selected from the above-mentioned symptoms.

[0082] Diabetic neuropathy to be prevented or treated by the diabetic neuropathy preventive or therapeutic agent of the present invention, among those exhibiting the above symptoms, preferably includes painful diabetic neuropathy and asymptomatic diabetic neuropathy, more preferably painful diabetic neuropathy.

[0083] Here, "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes inhibiting disease symptoms, i.e., preventing its progression or eliminating the disease or symptoms, and alleviating disease symptoms, i.e., causing regression of the disease or symptoms or slowing the progression of the symptoms. In the present invention, treatment refers to the direct treatment of diabetic neuropathy, rather than treating diabetic neuropathy based on the treatment of diabetes by blood glucose reduction or the like. In another embodiment, the treatment is a therapeutic neuroregenerative or neuroprotective treatment, local or systemic.

[0084] Furthermore, "prevention" includes preventing the onset of the above-mentioned diseases in mammals, particularly humans.

[0085] The agent of the present invention for preventing or treating peripheral neuropathy or pain symptoms caused by diseases in which peripheral neuropathy or astrocytopathy is observed is usually administered systemically or locally, orally or parenterally. The agent for preventing or treating peripheral neuropathy or the agent for preventing or treating pain symptoms due to diseases in which peripheral neuropathy or astrocyte dysfunction is observed in the present invention can be formulated as a pharmaceutical composition containing an RGMa inhibitor as an active ingredient and appropriately blended with pharmaceutically acceptable carriers or additives. Specifically, it can be formulated as oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc.; or parenteral preparations such as injections, infusions, suppositories, ointments, patches, etc. The blending ratio of carriers or additives can be determined in accordance with the ratios commonly used in the pharmaceutical field. The carriers or additives that can be added are not particularly limited, and examples thereof include various carriers such as water, physiological saline, other aqueous solvents, and aqueous or oily bases, and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0086] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, a functionally modified antibody thereof, a conjugated antibody thereof, or a fragment thereof, it is preferably administered parenterally, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically, as an injection or infusion formulated with a pharmaceutically acceptable carrier. The injection or infusion containing the anti-RGMa neutralizing antibody can be used as a solution, suspension, or emulsion. Examples of solvents that can be used include distilled water for injection, physiological saline, glucose solution, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). Furthermore, the injection or infusion may contain stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, etc. Examples of stabilizers that can be used include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol, etc.), polyalcohols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, etc.). Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, and tragacanth. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, and Tris buffer.Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc. Examples of antiseptics that can be used include benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.

[0087] When the RGMa inhibitor is a nucleic acid (e.g., siRNA, shRNA, antisense oligonucleotide, or nucleic acid molecule encoding an anti-RGMa neutralizing antibody or its fragment), it can be administered in the form of a non-viral or viral vector. In the case of a non-viral vector, methods for introducing nucleic acid molecules using liposomes (e.g., liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method), microinjection, or gene gun (gene gun) to transfer nucleic acid molecules into cells together with a carrier (metal particles) can be used. For example, when administering to a living body using a viral vector, viral vectors such as recombinant adenovirus and retrovirus can be used. Genes can be introduced into cells or tissues by introducing DNA expressing siRNA or shRNA into a detoxified DNA or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, or SV40, and then infecting cells or tissues with this recombinant virus.

[0088] The formulation thus obtained can be administered in an effective amount to subjects in need of treatment, such as humans or other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.), to prevent or treat peripheral neuropathy, such as diabetic neuropathy, or pain symptoms associated with diseases associated with peripheral neuropathy or astrocytopathy. The dosage is determined appropriately, taking into consideration the purpose, severity of the disease, the patient's age, weight, sex, medical history, and type of active ingredient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, the daily dosage for an average human weighing approximately 65 to 70 kg is preferably approximately 0.02 mg to 4000 mg, and more preferably approximately 0.1 mg to 200 mg. The total daily dosage may be a single dose or divided doses.

[0089] The agent for preventing or treating peripheral neuropathy or the agent for preventing or treating pain symptoms in diseases in which peripheral neuropathy or astrocytopathy is observed according to the present invention can be used in combination with or in combination with existing pain treatment drugs. Drugs that can be used in combination with the present invention include pain medications and diabetes medications. Pain medications include tricyclic antidepressants (amitriptyline, imipramine, etc.), α2δ ligands (e.g., pregabalin), and sodium channel blockers (mexiletine). It can be used in combination with or in combination with antiepileptic drugs (gabapentin, carbamazepine, etc.), aldose reductase inhibitors (epalrestat, etc.), and serotonin-norepinephrine reuptake inhibitors (SNRIs, e.g. duloxetine).

[0090] As an antidiabetic drug, it can be used in combination with sulfonylurea antidiabetic drugs, biguanide antidiabetic drugs, insulin sensitizers (such as pioglitazone), alpha-glucosidase inhibitors, DPP-4 inhibitors, SGLT-2 inhibitors, GLP-1 analogs, insulin analogs, etc., or combined with other drugs. [Example]

[0091] The present invention will be explained in more detail below by way of examples, but these examples are not intended to limit the scope of the present invention. [Example 1] Anti-RGMa improves pain and motor nerve conduction impairment in a rat STZ-induced diabetic neuropathy model Examination of the effect of neutralizing antibodies This rat STZ-induced diabetic neuropathy model is useful for detecting peripheral neuropathy, especially diabetic neuropathy. This model can be used to evaluate the effects of the drug on peripheral nerve damage in peripheral neuropathy, particularly diabetic neuropathy, as well as pain symptoms associated with diseases in which astrocyte damage is present. (1-1) Creation of a rat model of STZ-induced diabetic neuropathy Male SD rats were used in the experiment. STZ (streptozocin) (60 mg / kg) dissolved at a concentration of 30% in 0.75 mM citrate buffer (pH 4.5) was administered into the tail vein of the rats. After 3 weeks, blood Rats with glucose levels of 300 mg / dl or higher were used as the STZ experimental group (diabetic group). Rats of the same age as the experimental group but not administered STZ were used as the normal group.

[0092] (1-2) Behavioral pain assessment The von Frey stimulation test was performed using the up-down method (see Chaplan, SR, Bach, FW, Pogrel, JW, Chung, JM, Yaksh, TL, Quantitative assessment of tactile allodynia in the rat paw, J. Neurosci. Methods, 53, 55-63 (1994)). Mechanical hyperalgesia was assessed by determining the threshold (g).

[0093] (1-3) Measurement of motor nerve conduction velocity (MNCV) The rats were anesthetized with continuous inhalation of isoflurane gas and fixed in the prone position. Body temperature (rectal temperature) was maintained at a constant value (37.5-38.5°C) using a body temperature control device (ATB-1100, Nihon Kohden Corporation), and MNCV was measured. The sciatic tubercle of the right sciatic nerve was set as the proximal and distal stimulation point (S1), and the ankle joint of the right tibial nerve was set as the distal and proximal stimulation point (S2). A needle electrode (- pole) was inserted into each point. The indifferent electrode (+ pole) was inserted from S1 to the spine. The electrode was inserted approximately 1 cm toward the medulla. The recording electrode (negative electrode) and the reference electrode (positive electrode) were inserted shallowly into the right plantar muscle. The reference electrode (positive electrode) was placed distal to the negative electrode. Using an evoked potential recording device [Neuropack μ (model number: MEB-9102, Nihon Kohden Corporation)], single rectangular pulses (stimulation frequency: 1 Hz, duration: 0.1 msec, current: supramaximal, number of stimuli: 1) were applied to each of S1 and S2, and the resulting action potential changes were recorded using recording electrodes. For S1 and S2 stimulation, the latency t1 and t2 (msec) from the time of stimulation to the onset of the action potential and the distance d (mm) between S1 and S2 were measured, and MNCV was calculated using the following formula:

[0094] MNCV = d / (t1 - t2)

[0095] (1-4) Grouping and administration of anti-RGMa neutralizing antibody Three weeks after STZ administration, animals with blood glucose levels below 300 mg / dL were excluded from the group, and the weight and 50% of the escape rate were measured. The groups were divided so that the mean values ​​and variances of the withdrawal threshold and MNCV were homogenized in each group. It consisted of 10 animals.

[0096] Anti-RGMa neutralizing antibody or control antibody (mouse IgG) was administered intravenously at a dose of 10 mg / kg. The treatment was repeated once a week from the third week of STZ administration, for a total of four times. The anti-RGMa neutralizing antibody used was r116A3 (see Patent Document 4), which was prepared by a method described in a literature.

[0097] (1-5) Histopathological evaluation of GFAP and Iba1 immunostaining 28 days after the first administration of the test substance (51 days after STZ administration), saline was perfused and blood was exsanguinated. The animals were euthanized and perfused with 10 vol% neutral buffered formalin, after which the spinal cords were harvested and immersion-fixed in 10 vol% neutral buffered formalin. Immunostaining for GFAP (staining for astrocytes) and Iba1 (staining for microglia) was performed, and the expression of GFAP and Iba1 was evaluated histopathologically under a light microscope.

[0098] The slide specimens were photographed (20x magnification) using a virtual slide scanner, Aperio (Aperio AT2, Leica Microsystems), and the entire specimen image was extracted at 100% and converted to a JPEG image. Using the image analysis software, Image-Pro Premier (ver. 9.3.2, Media Cybernetics), the anterior horn (ventral horn) and posterior horn (dorsal horn) of the gray matter were enclosed in an Area of ​​Interest (AOI), and the immunostained area in each region was extracted and measured (size > 1 mm). 2 After the analysis, the percentage of stained areas per total area of ​​each region was calculated. The results are shown as individual values ​​and mean ± standard error. Student's t-tests were performed for each area percentage between the non-diabetic and diabetic groups, and between the diabetic group and the diabetic + test substance administration group.

[0099] (1-6)Result The effect of repeated administration of anti-RGMa neutralizing antibody on mechanical hyperalgesia is shown in Figure 1. The 50% withdrawal threshold, which was lowered by disease challenge, significantly improved from 1 week after administration of anti-RGMa neutralizing antibody. The improvement effect became stronger over time and continued at least until the final evaluation point of 4 weeks.

[0100] The effect of repeated administration of anti-RGMa neutralizing antibody on motor nerve conduction disorders is shown in Figure 2. Anti-RGMa neutralizing antibody also improved motor nerve conduction velocity, which had decreased due to diabetes, with significant improvement observed 4 weeks after the start of administration.

[0101] The calculated GFAP-positive area ratio is shown in Figure 3. In the anterior horn, a significant decrease was observed in the diabetic group compared to the non-diabetic group, and in the anterior and posterior horns, a significant increase was observed in the diabetic + test substance group compared to the diabetic group. The calculated Iba1-positive area ratio is shown in Figure 4. A significant decrease was observed in the diabetic group compared with the non-diabetic group in both the anterior and posterior horns.

[0102] <Description of Sequence Listing> SEQ ID NO: 1: Amino acid sequence of human RGMa precursor protein SEQ ID NO: 2: Amino acid sequence of mouse RGMa precursor protein SEQ ID NO: 3: Amino acid sequence of rat RGMa precursor protein SEQ ID NO: 4: DNA sequence of the human RGMa gene SEQ ID NO: 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 8: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 9: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 10: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 11: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 12: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 13: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 14: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 15: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 16: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 17: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 18: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 19: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 20: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 21: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 22: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 23: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 24: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 25: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 26: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 27: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 28: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 29: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 30: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 31: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 32: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 33: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 34: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 35: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1Y SEQ ID NO: 36: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 37: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 38: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 39: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 40: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1F SEQ ID NO: 41: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1H SEQ ID NO: 42: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1L SEQ ID NO: 43: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1V SEQ ID NO: 44: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1I SEQ ID NO: 45: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1K [Industrial Applicability]

[0103] INDUSTRIAL APPLICABILITY The present invention is useful for preventing or treating peripheral neuropathy, or for preventing or treating pain symptoms associated with diseases in which peripheral neuropathy or astrocytic disorders are observed, and is of great utility in the pharmaceutical industry.

Claims

1. A preventive or therapeutic agent for pain symptoms caused by a disease in which peripheral neuropathy or astrocytic disorder is observed, the disease being selected from diabetic neuropathy, entrapment neuropathy (carpal tunnel syndrome, ulnar neuropathy at the elbow, peroneal nerve palsy or tarsal tunnel syndrome), familial amyloid polyneuropathy, toxic neuropathy, cancer neuropathy, immune-mediated neuropathy (Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy (CIDP)), neuropathy associated with connective tissue disease, Crow-Fukase syndrome (POEMS syndrome), hereditary neuropathy (Charcor-Marie-Tooth disease), postherpetic neuralgia, peripheral neuropathy due to AIDS or Lyme disease, uremia, multifocal motor neuropathy, vasculitic neuropathy, neuromyelitis optica and Alexander disease, comprising an RGMa inhibitor; The RGMa inhibitor is an anti-RGMa neutralizing antibody or a fragment thereof; A preventive or therapeutic agent for pain symptoms.

2. The agent according to claim 1, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is diabetic neuropathy or neuromyelitis optica.

3. The agent according to claim 1, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is diabetic neuropathy.

4. The agent according to claim 1, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is neuromyelitis optica.

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

6. The agent according to any one of claims 1 to 5, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:

39.

7. The anti-RGMa neutralizing antibody is selected from the following (a1) to (l1): (a1) a light-sensitive antibody comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 5, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 6, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 7; an anti-RGMa neutralizing antibody comprising a heavy chain variable region, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; (b1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 comprising SFG in its amino acid sequence; (c1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; (d1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; (e1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k1) an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44; an anti-RGMa neutralizing antibody comprising a light chain variable region comprising: a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33; and a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (11) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; The agent according to any one of claims 1 to 6, which is an antibody selected from the group consisting of:

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