A substance for the prevention or treatment of peripheral neuropathy.

Abstract

The invention relates to a substance for the prevention or treatment of peripheral neuropathy or pain symptoms associated with peripheral neuropathy or astrocytosis, which contains an RGM inhibitor.

Description

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

[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.

[0002] Astrocytes are glial cells present in the central nervous system and possess a variety of functions, including (1) structural support for neuronal networks, (2) regulating various conditions surrounding astrocytes through material transport, (3) performing a single synaptic function through the close relationship between presynaptic, postsynaptic, and glial cells, (4) regulating extracellular ion concentrations, (5) providing energy buffering, and (6) 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, as well as 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, sensory, and autonomic nerves. Peripheral neuropathy is clinically classified as mononeuropathy (damage to a single nerve), multiple mononeuropathy (damage to two or more nerves in separate areas), or polyneuropathy (widespread neuropathy occurring bilaterally). Pathologically, it is classified as axonopathy, in which axons are centrally affected, or myelinopathy, in which myelin sheaths are 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 dysuria are caused, and are characterized by worsening over time. Primary 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 nerve disorders that are part 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) is a membrane protein originally identified as an axon guidance molecule in the visual system (see Non-Patent Document 5). The RGM family includes three members, termed RGMa, RGMb, and RGMc (see Non-Patent Document 6), and it is known that at least RGMa and RGMb function via the same signaling mechanism (see Non-Patent Document 7). RGMc plays an important role in iron metabolism. Subsequent research has revealed that RGM functions include axon guidance and lamina formation in Xenopus and chicken embryos, as well as regulating cranial neural tube closure in mouse embryos (see Non-Patent Document 8). Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0004] In addition to its function during development, RGMa is re-expressed after central nervous system injury in adult humans and rats, and RGMa inhibition in rats enhances axonal growth and promotes functional recovery after spinal cord injury (see Non-Patent Document 9). Therefore, RGMa is considered to be an inhibitor of axon regeneration after central nervous system injury. Specific antibodies that neutralize RGMa are described, for example, in Patent Document 2 (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3). Anti-RGMa antibodies are also known to be effective against neuromyelitis optica (see Non-Patent Document 10). While the role of RGMa in central nervous system injury has been elucidated, the involvement of RGMa in pain symptoms associated with peripheral neuropathy or diseases in which peripheral neuropathy or astrocyte damage is observed has not been identified.

[0005] International Publication No. WO2005 / 087268 International Publication No. WO2009 / 106356 International Publication No. WO2013 / 112922 International Publication No. WO2016 / 175236

[0006] Dallerac G et al., Prog Neurobiol. 144: 48-67(2016)Gerkau NJ et al., J Neurosci Res. 2017 Feb 2.Alam Q et al., Curr Pharm Des. 22: 541-8(2016)Yamazaki et al. Neurosci Lett, 170: 195-197. (1994)Stahl, B., Muller, B., von Boxberg, Y., Cox, E.C. & Bonhoeffer, F. Biochemical characterization of a putative axonal guidance molecule of the chick visual system. Neuron 5, 735-743 (1990)Mueller et al.,Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513‐29, 2006Liu, X., Hashimoto, M., Horii, H., Yamaguchi, A., Naito, K. and Yamashita, T. Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury. Biochem. Biophys. Res. Commun. 382, 795-800 (2009)Yamashita, T., Mueller, B.K. & Hata, K. Neogenin and repulsive guidance molecule signaling in the central nervous system. Curr. Opin. Neurobiol.17, 29-34 (2007)Hata, K. et al. RGMa inhibition promotes axonal growth and recovery after spinal cord injury. J. Cell Biol. 173, 47-58 (2006)Harada K. et al., Inhibition of RGMa alleviates symptoms in a rat model of neuromyelitis optica. Scientific Reports 8:34 1-9 (2018).

[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.

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that RGMa inhibitors can be used as preventive or therapeutic agents for peripheral neuropathy. Furthermore, they have found that RGMa inhibitors are effective not only against peripheral neuropathy but also against pain symptoms by improving astrocyte damage or hypofunction, and therefore can be used as preventive 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 selected from the group consisting of the following (a1) to (l1): (a1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; (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 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: 44, 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; (11) The agent according to any one of Items 3 to 5, wherein the antibody is selected from the group consisting of 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. 7. The agent according to any one of Items 1 to 6, wherein the peripheral neuropathy is 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, oncolytic 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, and vasculitic neuropathy. 8. The agent according to any one of Items 1 to 6, wherein the peripheral neuropathy is diabetic neuropathy. 9. Item 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 preventing or treating pain symptoms caused by a disease in which peripheral neuropathy or astrocyte disorder is observed. 11. Item 10. The agent according to Item 10, wherein the disease accompanied by peripheral neuropathy or astrocytopathy is 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, oncolytic 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. 12. The agent according to Item 11, wherein the disease accompanied by peripheral neuropathy or astrocytopathy is diabetic neuropathy or neuromyelitis optica. 13. The agent according to item 11, wherein the disease accompanied by peripheral neuropathy or astrocytopathy is diabetic neuropathy. 14. The agent according to item 11, wherein the disease accompanied by peripheral neuropathy or astrocytopathy is neuromyelitis optica. 15. A method for preventing or treating peripheral neuropathy, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of treatment. 16. The method according to item 15, wherein the peripheral neuropathy is diabetic neuropathy.

[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.

[0011] Figure 1 shows the effect of repeated administration of r116A3 on improving mechanical hyperalgesia in a rat model of streptozocin (STZ)-induced diabetic neuropathy. Figure 2 shows the effect of repeated administration of r116A3 on improving motor nerve conduction velocity reduction in a rat model of STZ-induced diabetic neuropathy. Figure 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 model of STZ-induced diabetic neuropathy. Figure 4 shows the effect of repeated administration of r116A3 on the percentage of Iba1 immunostaining-positive area in the spinal cord of a rat model of STZ-induced diabetic neuropathy.

[0012] Hereinafter, embodiments 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 must be clear, but in cases where the meaning is potentially unclear, the definitions provided herein take precedence over dictionary or external definitions. Furthermore, unless otherwise stated, singular terms include pluralities, and plural terms include the singular. In this specification, the use of "or" means "and / or" unless otherwise stated.

[0013] In general, 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 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 synthesis, chemical analysis, pharmaceutical preparations, formulation, delivery, and treatment of patients. To facilitate understanding of the present invention, the following definitions are provided for terms used herein.

[0014] [Neutralization] In this application, neutralization 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 certain embodiments, an epitope includes a chemically active surface grouping of a molecule (e.g., amino acids, sugar side chains, phosphoryl or sulfonyl) and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody.

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

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

[0018] [Human antibody] In the present application, 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 difference in the heavy chain constant region, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having a γ heavy chain, IgM having a μ heavy chain, IgA (including IgA1 and IgA2) having an α heavy chain, IgD having a δ heavy chain, or IgE having an ε heavy chain. In principle, the light chain includes either a κ chain or a λ chain.

[0019] [Humanized antibody] In the present application, the term "humanized antibody" refers to an antibody comprising a variable region consisting of a complementarity determining region of an antibody derived from a non-human animal and a framework region derived from a human antibody, and a constant region derived from a human antibody.

[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 a variable region of non-human origin and a constant region of human origin.

[0021] [Monospecific antibody] In the present application, a "monospecific antibody" refers to an antibody that has a single antigen specificity and has a single independent antigen recognition site. In this specification, for example, a monospecific antibody that recognizes RGMa may be referred to as an RGMa monospecific antibody.

[0022] [Multispecific antibody] In the present application, 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 thereof include a bispecific antibody having two antigen specificities and a trispecific antibody having three antigen specificities.

[0023] [Complementarity-Determining Region (CDR)] The term "complementarity-determining region (CDR)" refers to the region of the variable region of an immunoglobulin molecule that forms the antigen-binding site. Also called a hypervariable region, this refers to the portion of the immunoglobulin molecule that exhibits particularly large variations in amino acid sequence. There are three CDRs in each of the light and heavy chains. The three CDRs in the light chain are sometimes referred to as LCDR1, LCDR2, and LCDR3, respectively, and the three CDRs in the heavy chain are sometimes referred to as HCDR1, HCDR2, and HCDR3. For example, the CDRs of an immunoglobulin molecule are determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

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

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

[0026] The present invention is described in detail below. An embodiment of the present invention provides a preventive or therapeutic agent for peripheral neuropathy, which is a novel use of an RGM inhibitor, particularly an RGMa inhibitor. An embodiment of the present invention also provides, for example, a preventive or therapeutic agent for diabetic neuropathy using an RGMa inhibitor. Furthermore, another embodiment of the present invention provides a preventive or therapeutic agent for pain symptoms caused by diseases in which peripheral neuropathy or astrocyte damage is observed using an RGMa inhibitor. Another embodiment of the present invention also provides a method for preventing or treating peripheral neuropathy, for example diabetic neuropathy, which comprises the step of administering to a mammal in need of treatment an effective amount of a preventive or therapeutic agent containing an RGMa inhibitor.

[0027] <RGM Inhibitory Substance> The RGM inhibitor of the present invention may be either a substance that inhibits the activity of RGM (hereinafter sometimes simply referred to as "RGM activity") that induces pain symptoms due to peripheral neuropathy or a disease in which peripheral neuropathy or astrocyte disorder is observed, or that inhibits recovery from the disease or symptoms, as described below, or a substance that inhibits the expression of RGM. Here, RGM means one or more selected from RGMa, RGMb, and RGMc, and is preferably RGMa.

[0028] RGMa was 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 N-terminal signal peptide Met1-Pro47 (referring to the peptide from the first methionine residue to the 47th proline residue from the N-terminus; hereafter, the same applies) is removed, and the peptide bond between Asp168 and Pro169 is cleaved to generate the N-terminal domain. Furthermore, the C-terminal peptide Ala425-Cys450 of the fragment C-terminal to Pro169 is removed, and a GPI anchor is added to the C-terminal carboxyl group of the resulting C-terminal Ala424 to generate the C-terminal domain. Furthermore, the N-terminal domain (Cys48-Asp168) and the C-terminal domain (Pro169-Ala424) are linked by disulfide bonds to form a mature protein, which is 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 shown in SEQ ID NO: 1 in the sequence listing. The precursor protein of mouse RGMa consists of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and the precursor protein of rat RGMa consists of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, but because the C-terminal peptide is removed, the mature proteins have the same amino acid sequence. Examples of RGMa genes include, but are not limited to, the human RGMa gene consisting of the base sequence shown in SEQ ID NO: 4. The base sequences of RGM genes derived from various organisms can be easily obtained from known databases (GenBank, etc.).

[0030] The RGMa inhibitor of the present invention may be either a substance that inhibits (neutralizes) the activity of RGMa, which induces pain symptoms due to peripheral neuropathy or diseases in which peripheral neuropathy or astrocyte damage is observed, or 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 can be selected by the evaluation method described in Example 1, i.e., by evaluating the effect of improving pain and nerve conduction disorders using a rat STZ-induced diabetic neuropathy model.

[0031] The RGMa inhibitor in the present invention refers to, for example, a substance that binds to RGMa to directly inhibit RGMa activity, or that inhibits the binding of RGMa to a receptor to indirectly inhibit RGMa activity, and specifically includes low molecular weight compounds, anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, or fragments thereof. Substances that inhibit RGMa activity by inhibiting the expression of RGMa are also RGMa inhibitors, and specifically include RGMa gene siRNA (short interfering RNA), shRNA (short hairpin RNA), antisense oligonucleotides, etc. Of these RGMa inhibitors, preferred are anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugated antibodies thereof, and fragments thereof, more preferably anti-RGMa neutralizing antibodies or fragments thereof, and particularly preferred are anti-RGMa neutralizing antibodies.

[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, but is preferably a monoclonal antibody. Furthermore, the RGMa neutralizing antibody of the present invention may be an RGMa monospecific antibody or a multispecific antibody that recognizes multiple RGMa and other antigens, but is preferably an RGMa monospecific 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 antibody of the present invention includes polyclonal antibodies and monoclonal antibodies obtained by immunizing a mammal such as a mouse with an RGMa protein or a partial fragment thereof (for example, the above-mentioned epitope fragment) as an antigen, chimeric antibodies and humanized antibodies produced using genetic recombination technology, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibody of the present invention is administered to humans as a pharmaceutical, a humanized antibody or a human antibody is desirable 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) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (the anti-RGMa neutralizing antibody also includes antibodies having SEQ ID NOs: 36, 37, and 39 as epitopes); (b1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and an HCDR3 comprising SFG in its amino acid sequence (the anti-RGMa neutralizing antibody also includes antibodies having SEQ ID NOs: 36, 37, and 38 as epitopes); (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 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (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 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (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 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (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 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16);(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 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (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 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope of SEQ ID NO: 16); (k1) 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: 44, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody which has SEQ ID NO: 16 as an epitope), and (l1) 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 anti-RGMa neutralizing antibody also includes an antibody which has SEQ ID NO: 16 as an epitope), More preferably, the following (a2) to (l2):(a2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 5, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 6, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 8, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 9, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 10 (the anti-RGMa neutralizing antibody also includes antibodies having epitopes of SEQ ID NOs: 36, 37, and 39); (b2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 11, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 12, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 14, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 15, and an HCDR3 consisting of the amino acid sequence of SFG (the anti-RGMa neutralizing antibody also includes antibodies having epitopes of SEQ ID NOs: 36, 37, and 38); (c2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 18, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 20, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 22; (d2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, 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) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region comprising 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); (f2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 35, and a heavy chain variable region comprising 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); (g2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and 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) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and 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) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and 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) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 33, and 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); (k2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:44, and a heavy chain variable region comprising 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 which has an epitope of SEQ ID NO:16), and (l2) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO:29, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO:30, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO:45, and a heavy chain variable region comprising 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 which has an epitope of SEQ ID NO:16). Among these, the antibody described in (a2) is particularly preferred.

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

[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 obtained, for example, by immunizing a mammal such as those described above with the antigen, optionally together with Freund's adjuvant, and then from the serum obtained from the immunized animal.

[0041] Monoclonal antibodies can be obtained, for example, by employing the methods described in "Current Protocols in Molecular Biology" (John Wiley & Sons (1987)) and Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988). Monoclonal antibody-secreting "hybridomas" can be prepared 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: The aforementioned 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 mammals described above, or by implantation, one to several times, to immunize the mammal. Generally, immunization is carried out 1 to 4 times at intervals of about 1 to 14 days from the first immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

[0042] Hybridomas are prepared by cell fusion of the above-mentioned antibody-producing cells with myeloma cells derived from a mammal, preferably a mouse, rat, or human, that are not capable of producing 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 about 20 to 50%, at a temperature of 20 to 40°C, preferably 30 to 37°C, at a ratio of antibody-producing cells to myeloma cells of usually about 1:1 to 10:1, for about 1 to 10 minutes.

[0045] Hybridoma clones that produce monoclonal antibodies can be screened by culturing the hybridomas, for example, in a microtiter plate and measuring the reactivity of the culture supernatant in the wells against the immunizing antigen by an immunochemical method such as ELISA.

[0046] In screening for antibody-producing hybridomas, in addition to a binding assay with RGMa protein, an evaluation is also performed to determine whether the antibody inhibits the RGMa activity of the present invention. These screening methods allow the selection of anti-RGMa neutralizing antibodies of the present invention.

[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. The basal media may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

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

[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 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 oligonucleotides with sequences conserved in the variable regions of known human antibody heavy chain and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence using PCR. The base sequence of the DNA can be determined by conventional methods, such as by incorporating it into a sequencing plasmid. Alternatively, DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the sequence of the variable region or a portion thereof and linking it to a sequence containing the constant region. The nucleic acid molecule may encode both the constant and variable regions of the heavy and light chains, or it may encode only the variable regions of the heavy and light chains. When encoding both the constant and variable regions, the nucleotide sequences of the heavy and light chain constant regions are preferably those described in Nucleic Acids Research, vol. 14, p. 1779, 1986; The Journal of Biological Chemistry, vol. 257, p. 1516, 1982; and Cell, vol. 22, p. 197, 1980.

[0054] Functionally modified antibodies can be prepared by the following methods. For example, the serum half-life can be extended by using an Fc region mutant that enhances binding to FcRn, an Fc receptor (Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p. 710). These functionally modified 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 attached as a functional molecule, the PEG may have a molecular weight of, but not limited to, 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be linear or branched. PEG can be attached to the N-terminal amino group of the amino acid of the RGMa inhibitor, for example, by using an NHS active 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 substances can be directly bound to RGMa inhibitors by the chloramine T method or the like.

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

[0064] A preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a chimeric antibody. Examples of "chimeric antibodies" include those in which the variable region is derived from immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.) and the constant region is derived from human immunoglobulin. For example, chimeric antibodies can be produced by immunizing a mouse with an antigen, excising the antigen-binding variable region from the mouse monoclonal antibody gene, and combining it with an antibody constant region derived from human bone marrow. Constant regions derived from human immunoglobulins have unique amino acid sequences depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE. The constant region of the recombinant chimeric antibody of the present invention may be the constant region of a human immunoglobulin belonging to any isotype. Preferably, it is a human IgG constant region. An expression vector can be produced using the gene for the chimeric antibody 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 produced 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 CDRs derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework regions of its variable regions are derived from a human immunoglobulin, and its constant regions are derived from a human immunoglobulin.

[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 that produces a mouse monoclonal antibody, and a human heavy chain gene covering the entire region excluding the human heavy chain CDR and a human light chain gene covering the entire region excluding the human light chain CDR are isolated from a human immunoglobulin gene.

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

[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 immunoglobulins derived from genes encoding human immunoglobulins, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal produced by incorporating at least a human immunoglobulin gene into the genetic locus of a non-human mammal such as a mouse can be produced by immunizing the animal with an antigen in a manner similar to the method for producing polyclonal or monoclonal antibodies described above.

[0070] For example, transgenic mice that produce human antibodies can be produced according to the methods described in Nature Genetics, Vol. 7, pp. 13-21, 1994; Nature Genetics, Vol. 15, pp. 146-156, 1997; Published Japanese Translation of PCT International Publication No. H4-504365; Published Japanese Translation of PCT International Publication No. H7-509137; International Publication WO94 / 25585; Nature, Vol. 368, pp. 856-859, 1994; and Published Japanese Translation of PCT International Publication No. H6-500233, etc. More specific examples include HuMab® mice (Medarex, Princeton NJ), KM™ mice (Kirin Pharma Company, Japan), and KM (FCγRIIb-KO) mice.

[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). Note that, as long as the properties of the antibodies of the present invention, such as the ability to bind to RGMa and inhibit (neutralize) RGMa activity, are maintained, the amino acid sequence of the anti-RGMa neutralizing antibody may include substitution, deletion, addition, or insertion 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, and additions may be introduced into the CDR, they are preferably introduced into a region other than the CDR. 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 containing substitutions, deletions, etc. in the amino acid sequence is, for example, an amino acid sequence in which the heavy chain variable region after the 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 the modification, and an amino acid sequence in which the light chain variable region after the 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 the modification.

[0073] siRNA is a short double-stranded RNA capable of suppressing the expression of a target gene (in the present invention, an RGMa gene). As long as it functions as an siRNA of the present invention that inhibits RGMa activity, the base sequence and length (base length) are not particularly limited; however, they are preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases. shRNA refers to a single-stranded RNA molecule of about 20 base pairs or more that contains a partially palindromic base sequence, forms a double-stranded structure within the molecule, and is composed of a short hairpin structure with an overhang at the 3' end. After being introduced into a cell, such shRNA is degraded to a length of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) within the cell, and can suppress the expression of the target gene in the same way as siRNA. In the present invention, siRNA and shRNA may be in any form as long as they are capable of suppressing the expression of an 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 interfere with function. Antisense oligonucleotides can be synthesized by conventional methods, for example, easily using a commercially available DNA synthesizer. Preferred sequences can be selected using conventional selection methods, and the siRNA or shRNA of the present invention can be confirmed by evaluating inhibition of functional RGMa expression.

[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, 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, etc.

[0076] In another embodiment of the present invention, the disease in which astrocyte dysfunction is observed refers to a disease in which astrocyte dysfunction or functional decline is observed, and specific examples include neuromyelitis optica, Alexander disease, etc. Among these embodiments, the peripheral neuropathy is preferably diabetic neuropathy. Furthermore, the disease in which astrocyte dysfunction 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, suggesting that an RGMa inhibitor can treat diseases in which peripheral neuropathy is observed. Furthermore, since an RGMa inhibitor also suppresses pain, it is suggested that an RGMa inhibitor has an analgesic effect against pain caused by diseases in which peripheral neuropathy is observed.

[0078] Furthermore, one mechanism of action thought to be that pain symptoms caused by diseases in which astrocyte dysfunction is observed is that astrocyte dysfunction or dysfunction reduces the expression of glutamate transporters in the central nervous system, resulting in enhanced action of glutamate and neuronal hypersensitivity or excitation (e.g., Neuron 67, 834-846, September 9, 2010). Such pain symptoms, such as numbness, pain, or hypoesthesia, are observed due to astrocyte dysfunction and are associated with the onset of the disease.

[0079] Therefore, as shown in Example 1 (1-6) below, in a rat STZ-induced diabetic neuropathy model, peripheral neuropathy and astrocyte damage are induced, resulting in the onset of pain, and RGMa inhibitors, such as anti-RGMa neutralizing antibodies, are shown to have an analgesic effect against the pain. On the other hand, RGMa inhibitors, such as anti-RGMa neutralizing antibodies, are ineffective against microglial depletion but exhibit inhibitory effects against astrocyte depletion, suggesting that RGMa inhibitors, such as anti-RGMa neutralizing antibodies, exhibit an analgesic effect 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. Furthermore, Non-Patent Document 10 conducted experiments on astrocyte damage in neuromyelitis optica. However, because this experimental model is an animal model that cannot evaluate pain, the relationship between diseases in which astrocyte damage is observed and pain symptoms was unclear from the 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 diabetic neuropathy. A comprehensive diagnosis is made through careful auscultation of the patient's neurological symptoms and neurological examinations, including pain, vibration, pressure, and tendon reflex testing. The main symptoms of diabetic neuropathy are broadly classified as sensory neuropathy, motor neuropathy, and autonomic neuropathy. Sensory neuropathy can be broadly divided into painful neuropathy, characterized by pronounced abnormalities in pain sensation, and asymptomatic neuropathy, characterized by the absence of spontaneous sensory abnormalities. Sensory neuropathy initially presents with numbness and pain in the distal lower limbs, followed by progressive loss of sensation. Furthermore, as the condition progresses, symptoms such as numbness, pain, and loss of sensation may appear not only in the lower limbs but also in the distal upper limbs. Sensory neuropathy not only reduces patients' quality of life, but as symptoms progress and loss of sensation appears, it increases the risk of foot gangrene and Charcot joint disease, potentially leading to limb amputation and a worsening prognosis. Therefore, early therapeutic intervention is important for diabetic neuropathy. On the other hand, motor neuropathy is characterized by symptoms such as muscle weakness and atrophy, particularly in the lower limbs, and foot deformities. While these symptoms are usually not noticeable enough to affect activities of daily living, as the disorder progresses, they begin to affect balance maintenance, climbing up and down slopes and stairs, and strenuous walking, which requires constant walking. The prophylactic or therapeutic agent for 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 preventive or therapeutic agent for peripheral neuropathy or the preventive or therapeutic agent for pain symptoms caused by diseases in which peripheral neuropathy or astrocytopathy are observed in the present invention is usually administered systemically or locally, orally or parenterally. The preventive or therapeutic agent for peripheral neuropathy or the preventive or therapeutic agent for pain symptoms caused by diseases in which peripheral neuropathy or astrocytopathy are observed in the present invention can be formulated as a pharmaceutical composition containing an RGMa inhibitor as an active ingredient and appropriately blended with a pharmaceutically acceptable carrier or additive. Specifically, it can be oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; or parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The blending ratio of the carrier or additive may be appropriately set based on the range 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. An injection or infusion containing an anti-RGMa neutralizing antibody can be used as a solution, suspension, or emulsion. Examples of solvents that can be used include distilled water for injection, physiological saline, glucose solution, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.). Furthermore, 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), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, and the like). Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, and tragacanth. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of 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 (such as siRNA, shRNA, antisense oligonucleotides, and nucleic acid molecules encoding anti-RGMa neutralizing antibodies and fragments thereof), it can be administered in the form of a non-viral vector or a viral vector. In the case of a non-viral vector form, methods for introducing nucleic acid molecules using liposomes (such as the liposome method, HVJ-liposome method, cationic liposome method, lipofection method, and lipofectamine method), microinjection, and methods for transferring nucleic acid molecules into cells together with carriers (metal particles) using a gene gun 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 that expresses siRNA or shRNA into a detoxified DNA or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpes virus, 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 a subject in need of treatment, such as a human or other mammal (e.g., rat, mouse, rabbit, sheep, pig, cow, cat, dog, monkey, etc.), to prevent or treat peripheral neuropathy, such as diabetic neuropathy, or pain symptoms associated with diseases in which peripheral neuropathy or astrocytopathy is observed. 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 preventive or therapeutic agent for peripheral neuropathy or the preventive or therapeutic agent for pain symptoms in diseases in which peripheral neuropathy or astrocytic disorder is observed according to the present invention can be used in combination with or combined with existing pain medications. Examples of drugs that can be used in combination with the present invention include pain medications and diabetes medications. Pain medications that can be used in combination with or combined with tricyclic antidepressants (amitriptyline, imipramine, etc.), α2δ ligands (e.g., pregabalin, etc.), Na channel blockers (e.g., mexiletine, etc.), antiepileptic drugs (gabapentin, carbamazepine, etc.), aldose reductase inhibitors (e.g., epalrestat), and serotonin-noradrenaline reuptake inhibitors (SNRIs, e.g., duloxetine) can be used in combination with or combined with the above-mentioned drugs.

[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.

[0091] The present invention will be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention. [Example 1] Examination of the Effect of Anti-RGMa Neutralizing Antibodies on Pain and Motor Nerve Conduction Impairment in a Rat STZ-Induced Diabetic Neuropathy Model This rat STZ-induced diabetic neuropathy model is used to evaluate the effect of anti-RGMa neutralizing antibodies on peripheral neuropathy, particularly on peripheral nerve damage in diabetic neuropathy. In addition, this model can evaluate the effect on pain symptoms associated with peripheral neuropathy, particularly diabetic neuropathy, as well as pain symptoms associated with diseases involving astrocytic damage. (1-1) Preparation of a Rat STZ-Induced Diabetic Neuropathy Model Male SD rats were used for this experiment. STZ (streptozocin) (60 mg / kg) dissolved at a 30% concentration in 0.75 mM citrate buffer (pH 4.5) was administered intravenously to the tail vein of rats. After 3 weeks, rats with blood glucose levels of 300 mg / dL or higher were used as the STZ experimental group (diabetic group). The normal group consisted of rats of the same age as the experimental group but not administered STZ.

[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)) to determine the 50% withdrawal threshold (g) of hind paw lift to assess mechanical hyperalgesia.

[0093] (1-3) Measurement of motor nerve conduction velocity (MNCV). Rats were anesthetized with continuous isoflurane gas inhalation and fixed in the prone position. Rectal temperature was maintained at a constant 37.5–38.5°C using a temperature control device (ATB-1100, Nihon Kohden Corporation). MNCV was measured. The sciatic tubercle of the right sciatic nerve was designated the proximal and distal stimulation point (S1), and the ankle joint of the right tibial nerve was designated the distal and distal stimulation point (S2). A needle electrode (negative electrode) was inserted into each point. An indifferent electrode (positive electrode) was inserted approximately 1 cm from S1 toward the spinal cord. The recording electrode (negative electrode) and reference electrode (positive electrode) were shallowly inserted into the right plantaris 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 stimulations: 1) were applied to S1 and S2, respectively, and the resulting action potential changes were recorded with the 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 grouping, and animals were divided into groups so that the means and variances of body weight, 50% withdrawal threshold, and MNCV were homogenized within each group. Each of the STZ / control antibody administration group, STZ / anti-RGMa neutralizing antibody administration group, and normal / control antibody administration group consisted of 10 animals.

[0096] Anti-RGMa neutralizing antibody or control antibody (mouse IgG) was administered into the tail vein at a dose of 10 mg / kg once a week starting from week 3 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 the literature.

[0097] (1-5) Histopathological Evaluation of GFAP and Iba1 Immunostaining. 28 days after the initial administration of the test substance (51 days after STZ administration), the animals were euthanized by saline perfusion and exsanguination, followed by perfusion fixation with 10 vol% neutral buffered formalin. The spinal cords were then 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 percentage area between the non-diabetic and diabetic groups, and between the diabetic group and the diabetic + test substance administration group.

[0099] (1-6) Results The effect of repeated administration of anti-RGMa neutralizing antibody on mechanical hyperalgesia is shown in Figure 1. The 50% withdrawal threshold, which had been reduced by diabetes induction, showed significant improvement from one 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 four 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 showed an improving effect on motor nerve conduction velocity, which had decreased due to the onset of diabetes, with significant improvement observed 4 weeks after the start of administration.

[0101] The calculated results of the GFAP-positive area ratio are 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 horn, a significant increase was observed in the diabetes + test substance administration group compared to the diabetic group. The calculated results of the Iba1-positive area ratio are shown in Figure 4. In the anterior and posterior horn, a significant decrease was observed in the diabetic group compared to the non-diabetic group.

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

[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 peripheral neuropathy, comprising an RGM inhibitor.

2. The agent according to claim 1, wherein the RGM inhibitor is an RGMa inhibitor.

3. The agent according to claim 2, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody or a fragment thereof.

4. The agent according to claim 3, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

5. The agent described in claim 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. An anti-RGMa neutralizing antibody selected from the following (a1) to (l1): (a1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10; (b1) an anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15, and an HCDR3 comprising SFG in its amino acid sequence; (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 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: 44, 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; and (l1) 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.

7. The agent according to any one of claims 1 to 6, wherein the peripheral neuropathy is 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-related 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, and vasculitic neuropathy.

8. The agent according to any one of claims 1 to 6, wherein the peripheral neuropathy is diabetic neuropathy.

9. The agent according to claim 8, wherein the diabetic neuropathy is painful diabetic neuropathy and / or asymptomatic diabetic neuropathy.

10. The agent according to any one of claims 1 to 6 for use in the prevention or treatment of pain symptoms caused by diseases in which peripheral neuropathy or astrocyte disorder is observed.

11. The agent according to claim 10, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is 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, oncolytic 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.

12. The agent according to claim 11, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is diabetic neuropathy or neuromyelitis optica.

13. The agent according to claim 11, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is diabetic neuropathy.

14. The agent according to claim 11, wherein the disease in which peripheral neuropathy or astrocytopathy is observed is neuromyelitis optica.

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

16. The method of claim 15, wherein the peripheral neuropathy is diabetic neuropathy.

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