Agent for preventing or treating diabetic autonomic neuropathy

Abstract

Provided is an agent for the prevention or treatment of diabetic autonomic neuropathy, said agent containing an RGMa inhibitor.

Description

Technical Field

[0001] The present invention relates to a prophylactic or therapeutic agent for diabetic autonomic neuropathy containing an RGMa inhibitor.

Background Art

[0002] Diabetic neuropathy is one of the three major complications specific to diabetes, with an early onset and high frequency. Among them, diabetic autonomic neuropathy is a disorder of the autonomic nerve fibers that control the body's organs caused by chronic hyperglycemia, and is a complex disease presenting various symptoms and signs. It causes abnormalities in the functions of the cardiovascular system, digestive system, urinary and genital systems, skin, pupils, adrenal glands, etc., presenting orthostatic hypotension, postprandial hypotension, gastric emptying disorder, bladder and sexual dysfunction, abnormal sweating, pupillary abnormalities, and asymptomatic hypoglycemia, respectively (Non-Patent Document 1).

[0003] In addition, diabetic nephropathy, which is one of the three major complications of diabetes, progresses to chronic renal failure as the disease progresses. Diabetic nephropathy is the leading cause of end-stage renal failure requiring dialysis therapy in Japan, and is known to be accompanied by autonomic neuropathy caused by both uremic and diabetic factors (Non-Patent Documents 2 and 3).

[0004] On the other hand, there are cases of renal insufficiency patients with diabetes who have little proteinuria. This is thought to be due to the coexistence of other kidney diseases, especially hypertensive nephrosclerosis, with diabetes, but it is difficult to distinguish from diabetic nephropathy, which is premised on the presence of proteinuria. Therefore, in the United States in 2007, chronic kidney disease (CKD), which is clinically considered to be involved in the onset and progression of diabetes without the need for pathological findings as a necessary condition for diagnosis, was defined as diabetic kidney disease (DKD) (Non-Patent Document 4), and the name diabetic kidney disease is also used in Japan. Diabetic kidney disease is a concept that includes diabetic nephropathy (Non-Patent Document 5). Reflecting such a conceptual change, in the past disease stage classification, it was premised that the progression of diabetic nephropathy was accompanied by urinary protein. However, currently, when renal dysfunction complicates diabetes, the presence or absence of urinary protein is not questioned.

[0005] RGM (repulsive guidance molecule) is a membrane protein initially identified as an axon guidance molecule in the visual system (Non-Patent Document 6). The RGM family includes three members called RGMa, RGMb, and RGMc (Non-Patent Document 7), and at least RGMa and RGMb are known to function through the same signal transduction mechanism (Non-Patent Document 8). RGMc plays an important role in iron metabolism. Subsequent studies have revealed that RGM has functions such as axon guidance and lamina formation in Xenopus and chicken embryos, and control of the closure of the head neural tube in mouse embryos (Non-Patent Document 9). Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0006] In addition to its functions during the developmental stage, it is re-expressed after central nervous system injury in adult humans and rats. Since RGMa inhibition enhances axon growth and promotes functional recovery after spinal cord injury in rats (Non-Patent Document 10), RGMa is considered to be an axon regeneration inhibitor after central nervous system injury. Specific antibodies that neutralize RGMa are described in, for example, Patent Document 2 (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3). Although the role of RGMa has been clarified in central nervous system injury, in particular, the involvement of RGMa in the treatment of diabetic autonomic neuropathy, especially diabetic nephropathy, has not been identified, and such therapeutic agents are not known.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

[0008] [Non-Patent Document 1] Diabetes Care 26: 1553-1579, 2003 [Non-Patent Document 2] Journal of Dialysis, 19(9), 905-909, 1986 [Non-Patent Document 3] Diabetes 27(6): 715-721, 1984 [Non-Patent Document 4] Am J Kidney Dis 2007: 49: S12-154 [Non-Patent Document 5] Evidence-Based CKD Clinical Practice Guidelines 2018, P.104-105 [Non-Patent Document 6] Neuron 5, 735-743 (1990) [Non-Patent Document 7] Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513‐29, 2006 [Non-Patent Document 8] Biochem. Biophys. Res. Commun. 382, 795-800 (2009) [Non-Patent Document 9] Curr. Opin. Neurobiol.17, 29-34 (2007) [Non-Patent Document 10] J. Cell Biol. 173, 47-58 (2006) [Summary of the Invention]

Problems to be Solved by the Invention

[0009] An object of the present invention is to provide an effective agent for diabetic autonomic neuropathy.

Means for Solving the Problems

[0010] As a result of intensive studies to solve the above problems, the present inventors have found that an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody, exhibits an ameliorating effect on diabetic autonomic neuropathy, and thus have completed the present invention. That is, the present invention is as follows.

[0011] 1. A prophylactic or therapeutic agent for diabetic autonomic neuropathy, comprising an RGMa inhibitor. 2. The prophylactic or therapeutic agent according to item 1, wherein the diabetic autonomic neuropathy is an autonomic neuropathy caused by chronic hyperglycemia or an autonomic neuropathy as a cause of renal dysfunction. 3. The prophylactic or therapeutic agent according to item 1 or 2, wherein the diabetic autonomic neuropathy is an autonomic neuropathy as a cause of renal dysfunction. 4. The prophylactic or therapeutic agent according to any one of items 1 to 3, wherein the diabetic autonomic neuropathy is a renal disease involving renal dysfunction. 5. The prophylactic or therapeutic agent according to item 4, wherein the renal disease involving renal dysfunction is chronic kidney disease. 6. The prophylactic or therapeutic agent according to any one of items 1 to 5, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 7. The prophylactic or therapeutic agent according to item 6, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 8. The prophylactic or therapeutic agent according to item 6 or 7, 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. 9. The anti-RGMa neutralizing antibody is the following (a) to (l): (a) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. (b) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing 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 the amino acid sequence containing SFG. (c) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22. (d) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing 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. (e) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing 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. (f) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (g) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (h) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (i) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (j) A neutralizing anti-RGMa antibody comprising a light chain variable region containing 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 containing HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (k) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and (l) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, The prophylactic or therapeutic agent according to any one of items 6 to 8, which is an antibody selected from the above.

[0012] 10. A method for preventing or treating diabetic autonomic neuropathy, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of treatment. 11. The prophylactic or therapeutic method according to item 10, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 12. Use of an RGMa inhibitor in the manufacture of a prophylactic or therapeutic agent for diabetic autonomic neuropathy. 13. The use according to item 12, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. [Effect of the Invention]

[0013] According to the present invention, an RGMa inhibitor, particularly an anti-RGMa neutralizing antibody, is useful as a prophylactic or therapeutic agent for, for example, diabetic autonomic neuropathy. [Brief Description of the Drawings]

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0015] Hereinafter, the terms used in the present invention will be explained. [Neutralization] In the present application, neutralization refers to an action that can bind to a target of interest and inhibit any function of the target. For example, an RGMa inhibitor refers to a substance that exhibits an action of inhibiting the biological activity of RGMa as a result of binding to RGMa.

[0016] [Epitope] In the present application, an epitope includes a polypeptide determinant that can specifically bind to an immunoglobulin or a T cell receptor. In certain embodiments, the epitope includes chemically active surface groups of a molecule (e.g., amino acids, sugar side chains, phosphoryl or sulfonyl), and in certain embodiments, it 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.

[0017] [Isolated] As used herein, "isolated" with respect to an RGMa inhibitor (such as an antibody, etc.) isolated in the present application means identified, separated, and / or recovered from components in their natural state. Impurities in their natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, and examples include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, to isolate an RGMa inhibitor or the like, it may be purified 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".

[0018] [Antibody] As used herein, an antibody generally refers to an Ig molecule consisting of four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), which substantially retain the characteristic of binding to an epitope of an immunoglobulin (Ig) molecule.

[0019] [Human antibody] As used herein, a human antibody refers to an antibody derived from human immunoglobulins for both the light chain and the heavy chain. Human antibodies include IgG having a heavy chain of the γ chain (including IgG1, IgG2, IgG3, and IgG4), IgM having a heavy chain of the μ chain, IgA having a heavy chain of the α chain (including IgA1 and IgA2), IgD having a heavy chain of the δ chain, or IgE having a heavy chain of the ε chain due to differences in the constant region of the heavy chain. Also, in principle, the light chain includes either the κ chain or the λ chain.

[0020] [Humanized antibody] As used herein, a humanized antibody refers to an antibody consisting of a variable region composed of a complementarity-determining region of an antibody derived from a non-human animal and a framework region derived from a human antibody, and a constant region derived from a human antibody.

[0021] [Chimeric antibody] As used herein, a chimeric antibody refers to an antibody in which the light chain, the heavy chain, or both are composed of a variable region derived from a non-human source and a constant region derived from a human source.

[0022] [Monospecific antibody] In the present application, a monoclonal antibody is an antibody that has a single antigen specificity and has a single independent antigen recognition site. In this specification, for example, a monoclonal antibody that recognizes RGMa may be referred to as an RGMa monoclonal antibody.

[0023] [Multispecific antibody] In the present application, a multispecific antibody is an antibody that has two or more independent antigen recognition sites with two or more different antigen specificities, and examples include bispecific antibodies having two antigen specificities, trispecific antibodies having three antigen specificities, and the like.

[0024] [Complementary determining region (CDR)] The complementary determining region (CDR) refers to the region within the variable region of an immunoglobulin molecule that forms the antigen-binding site, also called the hypervariable region, and refers to the portion where the amino acid sequence changes particularly greatly for each immunoglobulin molecule. There are three CDRs each in the light chain and the heavy chain. The three CDRs contained in the light chain may be referred to as LCDR1, LCDR2, and LCDR3, respectively, and the three CDRs contained in the heavy chain may be 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).

[0025] [Effective amount] An effective amount refers to an amount of a prophylactic or therapeutic agent that is sufficient to reduce or improve the severity and / or duration of a disorder or one or more of its symptoms, prevent the 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).

[0026] [Percent (%) Identity of Amino Acid Sequences] The "percent (%) identity" of the 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 of a specific reference polypeptide sequence after aligning the sequences, introducing gaps if necessary to obtain the maximum % identity, and not considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of measuring % identity can be achieved by using a variety of methods within the skill of the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment for the full lengths of the sequences being compared. However, for the purposes herein, the % identity value is obtained by using the sequence comparison computer program BLAST in a pairwise alignment. In situations where BLAST is used for amino acid sequence comparison, the % 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 whose scores are identical as determined by the program alignment of A and B by the sequence alignment program BLAST, 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 % identity of A to B will be different from the % identity of B to A. Unless otherwise specified, all % identity values herein are obtained using the BLAST computer program as shown in the immediately preceding paragraph.

[0027] [Conservative Substitutions] A conservative substitution means substituting an amino acid residue with another chemically similar amino acid residue so as not to substantially modify the activity of the peptide. For example, when substituting one hydrophobic residue with another hydrophobic residue, or substituting one polar residue with another polar residue having the same charge. Examples of functionally similar amino acids for which such substitutions can be made include, as nonpolar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like. Examples of positively charged (basic) amino acids include arginine, histidine, lysine, and the like. Also, examples of negatively charged (acidic) amino acids include aspartic acid, glutamic acid, and the like.

[0028] Hereinafter, embodiments of the present invention will be described in detail. The present invention provides a prophylactic or therapeutic agent for diabetic autonomic neuropathy, which is a novel use of an RGMa inhibitor. The present invention also provides a method for preventing or treating diabetic autonomic neuropathy, which includes the step of administering to a mammal in need of treatment a prophylactic or therapeutic agent containing an effective amount of an RGMa inhibitor.

[0029] <RGMa inhibitor> The RGMa inhibitor of the present invention may be any substance that acts on RGMa itself and inhibits or attenuates the activity of RGMa (hereinafter, sometimes simply referred to as "RGMa activity" in this specification). For example, a substance having an activity that binds to RGMa and directly inhibits (attenuates) RGMa activity, or a substance having an activity that inhibits the binding of RGMa to a receptor and indirectly inhibits (attenuates) RGMa activity (for example, the compounds and antibodies described later) is referred to as the RGMa inhibitor of the present invention. Further, the RGMa inhibitor of the present invention may be a substance that suppresses the expression of RGMa. For example, a substance that inhibits the expression of RGMa and inhibits (attenuates) RGMa activity (for example, the nucleic acid molecules described later) is also included in the RGMa inhibitor of the present invention.

[0030] RGMa has been identified as a neurite growth inhibitory protein in the central nervous system, and the human RGMa protein is biosynthesized as a precursor protein consisting of 450 amino acids as shown in SEQ ID NO: 1. The signal peptide Met1~Pro47 present at the N-terminus (referring to the peptide from the first methionine residue to the 47th proline residue from the N-terminal side, hereinafter described in the same way) is removed, the peptide bond between Asp168 and Pro169 is cleaved to generate the N-terminal domain, and further, the C-terminal peptide Ala425~Cys450 of the fragment on the C-terminal side of Pro169 is removed, and a GPI anchor is added to the C-terminal carboxyl group of Ala424 at the C-terminus to generate the C-terminal domain. The human RGMa protein is expressed on the cell membrane via a GPI anchor as a mature protein in which the above N-terminal domain (Cys48~Asp168) and C-terminal domain (Pro169~Ala424) are connected by a disulfide bond.

[0031] 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. However, since the C-terminal peptide is removed, the mature proteins have the same amino acid sequence. Examples of the RGMa gene include, but are not limited to, the human RGMa gene consisting of the nucleotide sequence shown in SEQ ID NO: 4. The nucleotide sequences of RGM genes derived from various organisms can be easily obtained from known databases (such as GenBank).

[0032] Specific examples of the RGMa inhibitor of the present invention include low molecular weight compounds, anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugate antibodies thereof, or antigen-binding fragments thereof. Further, examples include siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense oligonucleotides, which are nucleic acid molecules of RGMa. Among these RGMa inhibitors, anti-RGMa neutralizing antibodies, functionally modified antibodies thereof, conjugate antibodies thereof, and antigen-binding fragments thereof are preferred, anti-RGMa neutralizing antibodies or antigen-binding fragments thereof are more preferred, and anti-RGMa neutralizing antibodies are particularly preferred.

[0033] <anti-RGMa neutralizing antibody> In the present invention, the anti-RGMa neutralizing antibody may be any antibody that binds to RGMa and neutralizes RGMa activity, and may be a polyclonal antibody or a monoclonal antibody. A monoclonal antibody is preferred in the present invention. Further, the anti-RGMa neutralizing antibody of the present invention may be an RGMa monospecific antibody or a multispecific antibody that recognizes a plurality of RGMa and other antigens, but an RGMa monospecific antibody is preferred.

[0034] Specifically, as epitopes, in human RGMa, it is 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). A combination of SEQ ID NO: 36 and 37 is more preferred, and a combination of SEQ ID NO: 36, 37, and 39 is particularly preferred.

[0035] The anti-RGMa neutralizing antibodies of the present invention include polyclonal antibodies and monoclonal antibodies obtained by immunizing mammals such as mice with an antigen of an RGMa protein or a partial fragment thereof (for example, the epitope fragment described above), chimeric antibodies and humanized antibodies produced using genetic recombination techniques, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibody of the present invention is administered to humans as a medicine, 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 antibodies of the following (a) to (l), and the production method for each can use the methods described in Patent Documents 2-4.

[0037] (a) An anti-RGMa neutralizing antibody comprising a light chain variable region containing LCDR1 having the amino acid sequence set forth in SEQ ID NO: 5, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 6, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region containing HCDR1 having the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 10 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NOs: 36, 37, and 39 as epitopes), (b) An anti-RGMa neutralizing antibody comprising a light chain variable region containing LCDR1 having the amino acid sequence set forth in SEQ ID NO: 11, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region containing HCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 containing the amino acid sequence of SFG (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NOs: 36, 37, and 38 as epitopes), (c) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing 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 containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 22, (d) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing 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 containing 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, (e) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing 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 containing 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 neutralizing anti - RGMa antibody also includes antibodies having SEQ ID NO: 16 as an epitope), (f) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing 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 containing 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 neutralizing anti - RGMa antibody also includes antibodies having SEQ ID NO: 16 as an epitope), (g) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (h) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (i) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (j) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (k) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), and (l) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 45, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), Antibodies selected from the following are included. Among these, the antibody described in (a) is particularly preferred.

[0038] For the method for producing the anti-RGMa neutralizing antibody of the present invention, existing commonly used production methods can be used. The antigen may be used directly for immunization or used as a complex with a carrier protein. For the preparation of the complex of the antigen and the carrier protein, condensing agents such as glutaraldehyde, carbodiimide, and maleimide active ester can be used. Examples of the carrier protein include bovine serum albumin, thyroglobulin, hemocyanin, KLH, etc.

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

[0040] <Polyclonal antibody> Polyclonal antibodies can be obtained, for example, from the serum obtained from an immunized animal by immunizing a mammal as described above with an antigen as described above, together with Freund's Adjuvant if necessary.

[0041] <Monoclonal antibody> Specifically, monoclonal antibodies can be obtained as follows. That is, an antigen as described above is used as an immunogen, and the immunogen is immunized by injecting or transplanting it into the subcutaneous, intramuscular, intravenous, footpad, or intraperitoneal cavity of a mammal as described above 1 to several times, together with Freund's Adjuvant if necessary. Usually, immunization is carried out 1 to 4 times every 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] Monoclonal antibodies can be obtained using methods well-known to those skilled in the art (for example, 'Current Protocols in Molecular Biology' (John Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0043] The preparation of "hybridomas" that secrete monoclonal antibodies can be carried out according to the methods of Köhler and Milstein et al. (Nature, 256, 495, 1975) and modified methods analogous thereto. That is, it is prepared by cell fusion of antibody-producing cells contained in the spleen or the like obtained from an immunized mammal and myeloma cells derived from a mammal, preferably a mouse, rat or human, that do not have the ability to produce autoantibodies.

[0044] Examples of myeloma cells used for 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 / O, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15, etc.

[0045] Examples of fusion promoters include polyethylene glycol, etc. Usually, polyethylene glycol with a concentration of about 20 to 50% (average molecular weight 1000 to 4000) is used at a temperature of 20 to 40°C, preferably 30 to 37°C. The ratio of the number of antibody-producing cells to the number of myeloma cells is usually about 1:1 to 10:1, and cell fusion can be carried out by reacting for about 1 to 10 minutes.

[0046] The screening of hybridoma clones that produce monoclonal antibodies can be carried out by culturing the hybridomas in, for example, a microtiter plate and measuring the reactivity of the culture supernatant of the well against the immunizing antigen by an immunochemical method such as ELISA.

[0047] In the screening of antibody-producing hybridomas, in addition to the binding assay with the RGMa protein, an evaluation is also performed as to whether the antibody inhibits the RGMa activity of the present invention. By these screening methods, the anti-RGMa neutralizing antibody of the present invention can be selected.

[0048] Furthermore, cloning can be performed by the limiting dilution method from the wells containing hybridomas that produce the target antibody to obtain clones. The selection and breeding of hybridomas are usually carried out in a culture medium for animal cells containing 10 - 20% fetal bovine serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).

[0049] The production of monoclonal antibodies from hybridomas can be carried out by culturing the hybridomas in vitro or proliferating them in vivo, such as in the ascites of mammals such as mice and rats, and isolating them from the obtained culture supernatant or the ascites of mammals.

[0050] When culturing in vitro, depending on various conditions such as the characteristics of the cell type to be cultured and the culture method, it is possible to use a nutrient medium suitable for growing, maintaining, and preserving the hybridomas and producing monoclonal antibodies in the culture supernatant. The nutrient medium can include known nutrient media or nutrient media prepared from basal media.

[0051] Examples of the basal medium include low-calcium media such as Ham's F12 medium, MCDB153 medium, or low-calcium MEM medium, and high-calcium media such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basal medium can contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances according to the purpose.

[0052] The isolation and purification of monoclonal antibodies can be carried out by subjecting the above-mentioned culture supernatant or ascites to saturated ammonium sulfate, euglobulin precipitation method, capric acid method, caprylic acid method, ion exchange chromatography (such as DEAE or DE52, etc.), affinity column chromatography using an anti-immunoglobulin column or a protein A column, etc. Specifically, the purification of monoclonal antibodies may use methods known as immunoglobulin purification methods. For example, it can be easily achieved by means such as ammonium sulfate fractionation method, PEG fractionation method, ethanol fractionation method, utilization of anion exchangers, and affinity chromatography using RGMa protein, etc.

[0053] Monoclonal antibodies can also be obtained by the phage display method. In the phage display method, phages selected from an arbitrary phage antibody library are screened using the target immunogen, and phages having the desired binding property 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 an antibody or an antigen-binding domain is constructed based on the isolated sequence or the determined sequence information. Then, by culturing the cell line transfected with such an expression vector, monoclonal antibodies can be produced. By using a human antibody library as the phage antibody library, human antibodies having the desired binding property can be generated.

[0054] <Nucleic acid molecule> The nucleic acid molecule encoding the anti-RGMa neutralizing antibody or its antigen-binding fragment of the present invention can be obtained, for example, by the following method. First, total RNA is prepared from cells such as hybridomas using a commercially available RNA extraction kit, and cDNA is synthesized using a reverse transcriptase with random primers or the like. Next, cDNA encoding the antibody is amplified by the PCR method using oligonucleotides of the conserved sequences in the variable regions of the known human heavy chain gene and light chain gene as primers. The sequence encoding the constant region can be obtained by amplifying the known sequence by the PCR method. The nucleotide sequence of the DNA can be determined by a conventional method, such as by incorporating it into a plasmid for sequencing. Alternatively, DNA encoding the monoclonal antibody of the present invention can also be obtained by chemically synthesizing the variable region or a partial sequence thereof and binding it to the sequence containing the constant region. The nucleic acid molecule may encode all of the constant regions and variable regions of the heavy chain and light chain, or may encode only the variable regions of the heavy chain and light chain. The nucleotide sequences of the constant regions of the heavy chain and light chain in the case of encoding all of the constant regions and variable regions are preferably those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0055] <Functionally modified antibody> The functional modified antibodies of the anti-RGMa neutralizing antibody are prepared by the following methods. For example, when the anti-RGMa neutralizing antibody of the present application is produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as host cells, an antibody with a reduced fucose content in the sugar chain and enhanced cell killing function can be obtained. When produced using CHO cells transfected with the FUT8 gene as host cells, an antibody with a low cell killing function can be obtained (International Publication No. WO2005 / 035586, International Publication No. WO2002 / 31140, International Publication No. WO00 / 61739). In addition, the complement activation function can be regulated by modifying the amino acid residues in the Fc region (U.S. Patent No. 6,737,056, U.S. Patent No. 7,297,775, U.S. Patent No. 7,317,091). Furthermore, by using a variant of the Fc region with enhanced binding to FcRn, one of the Fc receptors, the blood half-life can be prolonged (Shimoguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p710). These functional modified antibodies can be produced by genetic engineering. By using a variant of the Fc region with enhanced binding to FcRn, one of the Fc receptors, the blood half-life can be prolonged (Shimoguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p710). These functional modified antibodies can be produced by genetic engineering.

[0056] <Conjugated antibody> As a modified molecule of the anti-RGMa neutralizing antibody of the present invention, a conjugated antibody can be mentioned. Examples of conjugated antibodies include conjugated antibodies in which a functional molecule other than the anti-RGMa neutralizing antibody of the present application, such as a non-peptidic polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular compound, a cytokine, a growth factor (such as TGF-β, NGF, Neurotrophin), albumin, an enzyme, or another antibody, is chemically or genetically bound to the anti-RGMa neutralizing antibody.

[0057] When binding PEG as a functional molecule, PEG with a molecular weight of 2000 to 100000 Da, more preferably 10000 to 50000 Da, can be used without limitation, and it may be linear or branched. PEG can be bound to, for example, the N-terminal amino group of the amino acids of the anti-RGMa neutralizing antibody by using an NHS active group.

[0058] When using a radioactive substance as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 At, etc. are used. The radioactive substance can be directly bound to the anti-RGMa neutralizing antibody by methods such as the chloramine T method.

[0059] When using a toxin as a functional molecule, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin), low molecular weight toxins (e.g., geldanamycin), maytansinoids, and calicheamicin, etc. are used.

[0060] When using a low molecular weight compound as a functional molecule, examples include daunomycin, doxorubicin, methotrexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0061] When using an enzyme as a functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4737456), 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, etc.), lactoperoxidase, microperoxidase, etc. are used.

[0062] As a linker used when chemically bonding a toxin, a low molecular weight compound, or an enzyme, divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2) n O(CR2) n - (where R is an arbitrary substituent and n is a positive integer), linkers represented by repeating units of alkoxy (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.), and alkylamino (e.g., polyethyleneamino, Jeffamine (trademark)), and diesters and amides (such as succinate, succinamide, diglycolate, malonate, and caproamide, etc.) can be mentioned. Chemical modification methods for binding functional molecules have already been established in this field (D.J. King., Applications and Engineering of Monoclonal antibodies., 1998 T.J. International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0063] <Antigen-binding fragment> In an embodiment of the present invention, the "antigen-binding fragment" of an antibody means a partial region of the antibody having antigen-binding property as described above. Specifically, F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-bonded Fv, single-chain antibody (scFv), and polymers thereof, etc. can be mentioned. Furthermore, the antigen-binding fragment includes conjugate fragments in which functional molecules other than the anti-RGMa neutralizing antibody of the present application, such as non-peptidic polymers (e.g., polyethylene glycol (PEG)), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (such as TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies, are chemically or genetically engineered to be bound.

[0064] "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulins with proteolytic enzymes such as pepsin or papain, and digested before and after the disulfide bond present between the two heavy chains in the hinge region. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond present between the two heavy chains in the hinge region to produce a light chain consisting of VL (variable light chain region) and CL (constant light chain region), and a heavy chain fragment consisting of VH (variable heavy chain region) and CHγ1 (γ1 region in the constant heavy chain region), and two identical antibody fragments bound by a disulfide bond at the C-terminal region. These two identical antibody fragments are each called Fab. Also, when IgG is treated with pepsin, it is cleaved downstream of the disulfide bond present between the two heavy chains in the hinge region to produce an antibody fragment slightly larger than the one in which the two Fabs are connected by the hinge region. This antibody fragment is called F(ab')2.

[0065] <Chimeric antibody> A preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a chimeric antibody. Examples of "chimeric antibodies" include chimeric antibodies in which the variable region is a variable region derived from an immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.) and the constant region is a constant region derived from a human immunoglobulin. For example, an antigen can be immunized into a mouse, and the variable region that binds to the antigen can be excised from the gene of the mouse monoclonal antibody and combined with an antibody constant region derived from human bone marrow to produce it. The constant regions derived from human immunoglobulins have unique amino acid sequences depending on isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE, but the constant region of the recombinant chimeric antibody in the present invention may be the constant region of a human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. An expression vector can be prepared using the gene of the chimeric antibody thus produced. By transforming a host cell with the expression vector, a chimeric antibody-producing transformed cell can be obtained, and the target chimerized antibody can be obtained from the culture supernatant by culturing the transformed cell.

[0066] <Humanized antibody> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a humanized antibody. The "humanized antibody" in the present invention is an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity-determining region) of a non-human animal antibody such as a mouse is transplanted (CDR grafting) into a human antibody gene. For example, it can be prepared with reference to the methods described in Japanese Patent Application Laid-Open No. Hei 4-506458 and Japanese Patent No. 2912618. Specifically, it means a humanized antibody characterized in that part or all of the CDRs are CDRs derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework region of the variable region is the framework region of a variable region derived from a human immunoglobulin, and the constant region is a constant region derived from a human immunoglobulin.

[0067] The humanized antibody in the present invention can be produced, for example, as follows. However, it goes without saying that the production method is not limited to such a method.

[0068] For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be produced by genetic engineering with reference to Japanese Patent Publication No. 4-506458 and Japanese Unexamined Patent Application Publication No. 62-296890, etc. That is, from a hybridoma producing a mouse monoclonal antibody, the DNA of the mouse heavy chain CDR region and the DNA of the mouse light chain CDR region are isolated, and from the human immunoglobulin gene, the human heavy chain gene of all regions other than the human heavy chain CDR and the human light chain gene of all regions other than the human light chain CDR are isolated.

[0069] The isolated DNA of the mouse heavy chain CDR region is introduced into an appropriate expression vector so that the human heavy chain gene into which it is transplanted can be expressed. Similarly, the DNA of the mouse light chain CDR region is introduced into another appropriate expression vector so that the human light chain gene into which it is transplanted can be expressed. Alternatively, the human heavy and light chain genes transplanted with the mouse CDR can also be introduced into the same expression vector so that they can be expressed. By transforming a host cell with the thus-produced expression vector, a humanized antibody-producing transformed cell is obtained, and the target humanized antibody is obtained from the culture supernatant by culturing the transformed cell.

[0070] <Human antibody> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a human antibody. A human antibody is an antibody in which all regions including the variable region of the heavy chain, the constant region of the heavy chain, the variable region of the light chain, and the constant region of the light chain constituting the immunoglobulin 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 integrating at least a human immunoglobulin gene into the locus of a non-human mammal such as a mouse is immunized with an antigen, and can be produced in the same manner as the method for producing the above-described polyclonal antibody or monoclonal antibody.

[0071] For example, transgenic mice that produce human antibodies can be prepared according to the methods described in Nature Genetics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; Japanese Patent Publication No. 04-504365; Japanese Patent Publication No. 07-509137; International Publication WO94 / 25585 pamphlet; Nature, Vol.368, p.856-859, 1994; and Japanese Patent Publication No. 06-500233, etc. More specifically, HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice, etc. can be mentioned.

[0072] Specific examples of the anti-RGMa neutralizing antibody of the present invention include those having a CDR containing a specific amino acid sequence in the heavy chain variable region and a CDR containing a specific amino acid sequence in the light chain variable region (preferably, the anti-RGMa neutralizing antibodies of (a) to (l) described above). As long as the characteristics of the antibody of the present invention, which has the ability to bind to RGMa and inhibits (neutralizes) the activity of RGMa, are maintained, one or several amino acids (1 to 20, 1 to 10, or 1 to 5, preferably 1 or 2) may be substituted, deleted, added, or inserted in the amino acid sequence of the anti-RGMa neutralizing antibody (preferably, the anti-RGMa neutralizing antibodies of (a) to (l) described above). Such substitutions, deletions, and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR. Further, the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention.

[0073] The amino acid sequence of the anti-RGMa neutralizing antibody of the present invention (preferably, the anti-RGMa neutralizing antibody of (a) to (l) above) containing substitutions, deletions, etc. in the amino acid sequence is, for example, an amino acid sequence in which the heavy chain variable region after amino acid sequence modification has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) % identity with the amino acid sequence before modification, and the light chain variable region after amino acid sequence modification has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) % identity with the amino acid sequence before modification.

[0074] In the present invention, siRNA is a short double-stranded RNA capable of suppressing the expression of a target gene (RGMa gene in the present invention). As long as it functions as an siRNA that inhibits RGMa activity in the present invention, the base sequence and length (base length) are not particularly limited, but are preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases. In the present invention, shRNA refers to a molecule of about 20 base pairs or more consisting of a short hairpin structure having a single-stranded RNA containing a partially palindromic base sequence, forming a double-stranded structure within the molecule, and having a protrusion at the 3' end. After such shRNA is introduced into cells, it is degraded to a length of about 20 bases (representatively, for example, 21 bases, 22 bases, 23 bases) in the cells, and can suppress the expression of the target gene in the same manner as siRNA. In the present invention, the above-mentioned siRNA and shRNA may be in any form as long as they can suppress the expression of the RGMa gene.

[0075] In the present invention, siRNA or shRNA can be artificially chemically synthesized. Further, for example, using T7 RNA polymerase and a T7 promoter, antisense and sense RNAs can be synthesized in vitro from template DNA. The antisense oligonucleotide may be a nucleotide that is complementary to or hybridizes with a continuous 5 to 100 base sequence in the DNA sequence of the RGMa gene, and may be either DNA or RNA. Further, it may be modified as long as its function is not impaired. The antisense oligonucleotide can be synthesized by a conventional method, and can be easily synthesized, for example, by a commercially available DNA synthesizer. Preferred sequences can be selected using ordinary selection methods, and as siRNA or shRNA in the present invention, it can be confirmed by evaluating the inhibition of functional RGMa expression.

[0076] <Diabetic autonomic neuropathy> The diabetic autonomic neuropathy in the present invention means the autonomic nerve disorder caused by chronic hyperglycemia such as diabetes and the autonomic nerve disorder as a cause of renal dysfunction. Here, the autonomic nerve disorder caused by chronic hyperglycemia such as diabetes includes various signs and symptoms observed in the whole body organs caused by chronic hyperglycemia. For example, (1) as the autonomic nerve disorder of the cardiovascular system, orthostatic hypotension, arrhythmia, etc., (2) as the autonomic nerve disorder of the digestive system, vomiting and diarrhea due to gastric atony (diabetic diarrhea, etc.), (3) as the autonomic nerve disorder of the urinary and genital systems, neurogenic bladder, erectile dysfunction, etc., (4) as the autonomic nerve disorder of the metabolic system, asymptomatic hypoglycemia, hypoglycemia-related autonomic insufficiency, etc., (5) as the autonomic nerve disorder related to peripheral vascular motor function, the breakdown of the blood pressure regulation mechanism caused by the disorder, the breakdown of body fluid homeostasis due to endocrine disorders, anemia, etc. can be mentioned. In addition, since the renal autonomic nerves are deeply involved in renal function through the regulation of the whole body and renal blood flow, the secretion of neurohumoral factors, and the direct action on renal blood vessels, etc., the protection of the renal autonomic nerves is considered to widely contribute to the improvement of renal function in the pathological conditions of chronic kidney disease (Reference: Front Med. 2018 Mar 29;5:82. doi: 10.3389 / fmed.2018.00082.). Therefore, examples of autonomic neuropathy as a cause of renal dysfunction include renal diseases involved in renal dysfunction such as chronic kidney disease (e.g., diabetic kidney disease including diabetic nephropathy) etc. In the present invention, a preventive or therapeutic effect against the disadvantages (diseases or symptoms) caused by the above-mentioned diabetic autonomic neuropathy is expected. The treatment target in the present invention (preferably a mammal, particularly a human) is a patient who has developed diabetic autonomic neuropathy, and the preventive or therapeutic agent for diabetic autonomic neuropathy of the present invention can be administered to these patients.

[0077] Here, "treatment" includes any treatment of a disease in a treatment target, preferably a mammal, particularly a human, and includes preventing the progression of the disease and symptoms, and eliminating, curing, alleviating or relieving such diseases and symptoms.

[0078] In addition, "prevention" includes preventing or suppressing the onset of the above-mentioned disease in a treatment target, preferably a mammal, particularly a human. Furthermore, "prevention" in the present invention includes "recurrence prevention" of preventing the recurrence of the above-mentioned disease that recurs in remission in a treatment target, preferably a mammal, particularly a human.

[0079] <Pharmaceutical composition> The preventive or therapeutic agent for diabetic autonomic neuropathy in the present invention is usually administered systemically or locally, orally or parenterally. The preventive or therapeutic agent for diabetic autonomic neuropathy in the present invention contains an RGMa inhibitor as an active ingredient, and can be formulated by appropriately blending a pharmaceutically acceptable carrier or additive. The pharmaceutical composition formulated in this way can be administered orally or parenterally. Specifically, it can be made into oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., and can also be made into parenteral preparations such as injections, infusions, suppositories, ointments, patches, etc. The blending ratio of the carrier or additive can be appropriately set based on the range usually adopted in the pharmaceutical field. The carrier or additive that can be blended is not particularly limited, and examples include various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, and various additives such as excipients, binders, pH adjusters, disintegrants, absorption promoters, lubricants, colorants, flavoring agents, fragrances, etc.

[0080] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, its functionally modified antibody, its conjugate antibody or their antigen-binding fragments, it is preferably administered parenterally, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously or locally, as an injection or infusion formulated with a pharmaceutically acceptable carrier. For example, an injection or infusion containing an anti-RGMa neutralizing antibody can be used as a solution, suspension or emulsion. As the solvent, for example, distilled water for injection, physiological saline, glucose solution and isotonic solutions (for example, solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used. Furthermore, such an injection or infusion containing an anti-RGMa neutralizing antibody may contain a stabilizer, solubilizing agent, suspending agent, emulsifying agent, pain reliever, buffer, preservative, antiseptic, pH adjuster, etc. As the stabilizer, for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, dibutylhydroxytoluene, etc. can be used. As dissolution aids, for example, alcohols (such as ethanol, etc.), polyalcohols (such as propylene glycol, polyethylene glycol, etc.), nonionic surfactants (such as polysorbate 80 (registered trademark), HCO-50, etc.) can be used. As suspending agents, for example, glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. can be used. As emulsifiers, for example, gum arabic, sodium alginate, tragacanth, etc. can be used. As soothing agents, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. As buffers, for example, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, tris buffer, etc. can be used. As preservatives, for example, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc. can be used. As antiseptics, for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. can be used. As pH adjusters, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. can be used.

[0081] When the RGMa inhibitor is a nucleic acid (such as siRNA, shRNA, antisense oligonucleotide, etc.), it can be administered in the form of a non-viral vector or a viral vector. In the case of a non-viral vector form, methods for introducing nucleic acid molecules using liposomes (such as liposome method, HVJ-liposome method, cationic liposome method, lipofection method, Lipofectamine method, etc.), microinjection method, and methods for transferring nucleic acid molecules into cells together with a carrier (metal particles) using a gene gun can be utilized. For example, when siRNA or shRNA is administered to a living body using a viral vector, viral vectors such as recombinant adenovirus and retrovirus can be utilized. DNA encoding siRNA or shRNA is introduced into a non-toxic retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, etc., which are DNA viruses or RNA viruses, and the recombinant virus is allowed to infect cells or tissues, whereby a gene can be introduced into the cells or tissues.

[0082] The preparation obtained in this way can prevent or treat diabetic autonomic neuropathy by administering an effective amount thereof to, for example, humans and other mammals (such as rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). The dosage is appropriately set considering the purpose, severity of the disease, age, weight, sex, medical history, type of active ingredient, etc. of the patient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, when targeting an average human having a body weight of about 65 to 70 kg, about 0.02 mg to 4000 mg per day is preferable, and about 0.1 mg to 200 mg per day is more preferable. The total daily dosage may be a single dosage or a divided dosage.

[0083] <Combined use with other drugs or treatments> In the present invention, the prophylactic or therapeutic agent for diabetic autonomic neuropathy can be administered in combination with an antidiabetic agent for the purpose of blood glucose control. Examples of the antidiabetic agent to be combined include, for example, hypoglycemic agents, and specifically, DPP4 inhibitors, SGLT inhibitors, GLP-1 receptor agonists, and the like. In the present invention, the prophylactic or therapeutic agent for diabetic autonomic neuropathy can be administered in combination with an antihypertensive agent for the purpose of blood pressure control. Examples of the antihypertensive agent to be combined include, for example, angiotensin II receptor antagonists (ARBs), ACE inhibitors, etc., and when the antihypertensive effect is insufficient, a calcium antagonist or a diuretic may be combined.

[0084] The above-mentioned other drugs or treatments may be administered or performed before or after the administration of the prophylactic or therapeutic agent for diabetic autonomic neuropathy of the present invention, or may be administered or performed simultaneously.

Examples

[0085] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. As the anti-RGMa neutralizing antibody, the anti-RGMa neutralizing antibody containing the amino acid sequence (SEQ ID NOs: 5 to 10) described in (a) in this specification was used in each example.

[0086] [Example 1] Using a drug-induced diabetic model mouse, the expression analysis of RGMa mRNA in the kidney under diabetic conditions was performed, and then the therapeutic effect of the anti-RGMa neutralizing antibody on renal autonomic neuropathy was histologically examined.

[0087] [Induction of diabetes] C57BL / 6J female mice at 7 - 8 weeks of age were used in the experiment. In the diabetes induction group, streptozotocin (STZ: Sigma - Aldrich) at a dose of 20 mg / ml was administered intraperitoneally once at a dose of 10 ml / kg. In the non - diabetes induction group, the solvent was administered at a dose of 10 ml / kg. Referring to previous studies (References 1, 2), blood glucose levels were measured 1 week after diabetes induction, and individuals with blood glucose levels less than 300 mg / dl were excluded.

[0088] <RGMa mRNA Expression Analysis> After 8 weeks from diabetes induction by STZ, four mice and four control mice were deeply anesthetized. After laparotomy, ice - cold PBS was perfused from the left ventricle to perform bloodletting. The kidneys were immediately excised and collected into a tissue disruption tube (TM - 625S; TOMY SEIKO Co, Ltd.) containing an appropriate amount of TRIzol solution (15596026; Thermo Fisher Scientific) and zirconia beads for disruption (ZB - 10; TOMY SEIKO Co, Ltd.). Disruption was performed using a bead - type cell disruptor (MS - 100R; TOMY SEIKO Co, Ltd.). Subsequently, RNA was extracted and purified (using the RNeasy Mini Kit (74104; QIAGEN)), and then cDNA was prepared by reverse transcription reaction. The reaction was carried out using Fast SYBR Green Master mix (4385612, Thermo Fisher Scientific) and QuantStudio 7 Flex Real - Time PCR System (Thermo Fisher Scientific). Relative quantification was performed by the ΔΔCt method using Gapdh as an endogenous control based on the Ct values measured from these results.

[0089] <Histological Analysis: Antibody Administration Method, Grouping> The purchased mice were randomly divided into four groups: "non-diabetic - anti-RGMa neutralizing antibody administration group", "non-diabetic - isotype control antibody (Palivizumab) administration group", "diabetic - anti-RGMa neutralizing antibody administration group", and "diabetic - isotype control antibody (Palivizumab) administration group". Three days after administration of STZ (diabetic group) or solvent (citric acid buffer (pH = 4.5)), administration of the anti-RGMa neutralizing antibody or control antibody was initiated. After adjusting the concentration of each antibody to 6 mg / ml, it was intravenously administered into the tail vein once a week at a dose of 30 mg / kg for a total of 6 times, and samples were collected 6 weeks later.

[0090] <Tissue Sampling, Kidney Clearing, and Immunohistochemical Staining> After sufficient anesthesia, the kidneys were excised after perfusion fixation with 4% paraformaldehyde (PFA), and after post-fixation, they were left standing at 4°C for 2 - 3 days in a 30% sucrose / PBS solution. Then, clearing was performed according to the CUBIC method. Referring to previous studies (References 3 - 7), the following procedure modified after condition examination was used. After washing the kidneys after 30% sucrose substitution with PBS, they were transferred into a CUBIC-L solution diluted to 50% with ultrapure water and shaken overnight at room temperature. Then, they were transferred into 100% CUBIC-L solution and shaken at 37°C for 5 days. After washing with PBS, they were transferred into a primary antibody solution in which the primary antibody was dissolved in PBS containing 0.1% Triton X-100, 0.5% BSA, and 0.01% sodium azide and shaken at 37°C for 5 days. After washing with PBS-0.5% Triton-X100 for 1 day, they were transferred into a secondary antibody solution in which the secondary antibody was diluted in PBS containing 0.1% Triton X-100, 0.1% BSA, and 0.01% sodium azide and shaken at 37°C for 5 days. After washing with PBS-0.5% Triton X-100 for 1 day, they were immersed in a 1% formaldehyde solution diluted with PB (0.2 M) for 3 hours and washed with PBS. They were immersed in CUBIC-R diluted to 50% with ultrapure water for 6 hours or more and then immersed in 100% CUBIC-R for clearing. Observation and imaging of the cleared tissue were performed using a confocal laser microscope FV-3000 (Olympus). The central part of the cleared kidney and the site from the surface to 200 μm were imaged at intervals of 1 μm in the z-axis, and after projecting the captured images onto a single image, quantification of the density of TH-positive sympathetic nerve fibers per unit area was performed. This operation was performed on 6 individuals in each group for analysis. The reagents used are as follows.

[0091] · CUBIC-L solution Triton X-100 (NACALAI TESQUE, INC., Kyoto, Japan) : 10 w% N-buthyldiethanolamine (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) : 10 w% Dissolve the above reagents in ultrapure water. · CUBIC-R solution 2,3-dimethyl-1-phenyl-5-pyrazolane / antipyrine (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) : 45 w% nicotinamide (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) : 30 w% Dissolve the above reagents in ultrapure water. · Primary antibody Anti-tyrosine hydroxylase (TH) antibody (1:100; abcam, Cambridge, UK) · Secondary antibody Alexa Fluor 488 donkey anti-sheep IgG (H+L) (1:200; Invitrogen, Waltham, MA, USA)

[0092] <Results> Gene expression analysis revealed an increase in the expression of RGMa mRNA in the kidneys in the diabetic state (Figure 1), suggesting the involvement of RGMa in this pathological condition. Representative staining images (Figure 2) of the anti-RGMa antibody and control administration experiments in diabetic mice, and quantitative data on TH fiber density (Figure 3) are then shown. The results of the four groups shown in the quantitative data were obtained with n = 6 for each group. In the non-diabetic group, there was no change in the density of TH fibers regardless of the type of antibody administered. On the other hand, when comparing the non-diabetic group with the "diabetes-isotype control antibody administration group", a tendency for a decrease in TH fiber density was observed. On the other hand, in the "diabetes-anti-RGMa neutralizing antibody administration group", a tendency for improvement in TH fiber density was observed compared to the "diabetes-isotype control antibody administration group". From the above results, it is considered that the anti-RGM neutralizing antibody alleviates the impairment of renal autonomic nerves caused by diabetes.

[0093] [Example 2] Using a drug-induced diabetic model mouse, the therapeutic effect of the anti-RGMa neutralizing antibody on renal dysfunction was examined using the urinary protein amount as an index.

[0094] <Induction of diabetes> C57BL / 6J female mice at 7 - 8 weeks of age were used in the experiment. Streptozotocin (STZ: Sigma - Aldrich) at a dose of 20 mg / ml was administered intraperitoneally once at a dose of 10 ml / kg to the diabetes induction group. The solvent was administered to the non - diabetes induction group at a dose of 10 ml / kg. Referring to previous studies (References 1, 2), blood glucose levels were measured 3 days after diabetes induction, and individuals with blood glucose levels less than 300 mg / dl were excluded.

[0095] <Histological analysis: Antibody administration method, grouping> The purchased mice were randomly divided into three groups: "non - diabetes - saline administration group", "diabetes - anti - RGMa neutralizing antibody administration group", and "diabetes - isotype control antibody (Palivizumab) administration group". Starting 3 days after administration of STZ (diabetes group) or solvent (citric acid buffer (pH = 4.5)), administration of saline, anti - RGMa neutralizing antibody, or control antibody was initiated. Saline was administered intravenously into the tail once a week at a dose of 30 mg / kg for a total of 5 times. Also, after adjusting the concentration of each antibody to 6 mg / ml, it was administered intravenously into the tail once a week at a dose of 30 mg / kg for a total of 5 times, and samples were collected after 5 weeks.

[0096] <Calculation of urinary albumin / urinary creatinine ratio> The urinary albumin / urinary creatinine ratio was used as an index of proteinuria due to kidney damage. At 5 weeks after diabetes induction, individuals in each group were placed in a metabolic cage (Tecniplast Japan Co., Ltd., Tokyo, Japan) and urine was collected under free movement. The samples were stored at - 30°C until measurement. Urinary albumin was quantified using a Mouse Albumin ELISA Kit (FUJIFILM Wako Shibayagi Corporation, Gunma pref., Japan), and urinary creatinine was quantified using Lab Assay Creatinine (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and the urinary albumin / urinary creatinine ratio (UACR) was calculated.

[0097] <Result> Quantitative data of the urinary albumin / creatinine ratio are shown in Fig. 4. The results of the three groups shown quantitatively were obtained with n = 6 in each group. When comparing the non-diabetic - saline administration group and the "diabetes - isotype control antibody administration group", an increasing trend in the urinary albumin / creatinine ratio was observed, which was considered to reflect kidney damage. On the other hand, in the "diabetes - anti-RGMa neutralizing antibody administration group", an improving trend in the urinary albumin / creatinine ratio was observed compared with the "diabetes - isotype control antibody administration group". Therefore, it is considered that the anti-RGMa neutralizing antibody has an effect of alleviating kidney damage caused by diabetes. From the above results, it is considered that the anti-RGMa neutralizing antibody alleviates the disorder of renal autonomic nerves caused by diabetes. From the above, it can be expected that an RGMa inhibitor, preferably an anti-RGMa neutralizing antibody, can be used as a preventive or therapeutic agent for autonomic nerve disorders as a cause of kidney dysfunction, and as a preventive or therapeutic agent for kidney diseases involved in kidney dysfunction such as chronic kidney disease.

[0098] <References> 1. Deeds MC, Anderson JM, Armstrong AS, et al. Single dose streptozotocin-induced diabetes: Considerations for study design in islet transplantation models. LabAnim. 2011;45(3):131-140. doi:10.1258 / la.2010.010090 2. O’brien PD, Sakowski SA, Feldman EL. Mouse models of diabetic neuropathy. ILAR J. 2014;54(3):259-272. doi:10.1093 / ilar / ilt052 3. Hasegawa S, Susaki EA, Tanaka T, et al. Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia / reperfusion injury. Kidney Int. 2019;96(1):129-138. doi:10.1016 / j.kint.2019.02.011 4. Kubota SI, Takahashi K, Nishida J, et al. Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution. Cell Rep. 2017;20(1):236-250. doi:10.1016 / j.celrep.2017.06.010 5. Tainaka K, Murakami TC, Susaki EA, et al. Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents. Cell Rep. 2018;24(8):2196-2210.e9. doi:10.1016 / j.celrep.2018.07.056 6. Richardson DS, Lichtman JW. Clarifying Tissue Clearing. Cell. 2015;162(2):246-257. doi:10.1016 / j.cell.2015.06.067 7. Yokoyama T, Lee JK, Miwa K, et al. Quantification of sympathetic hyperinnervation and denervation after myocardial infarction by three-dimensional assessmentof the cardiac sympathetic network in cleared transparent murine hearts. PLoS One. 2017;12(7):1-13. doi:10.1371 / journal.pone.0182072

[0099] <Description of Sequence Listing> SEQ ID NO: 1: Amino acid sequence of human RGMa precursor protein SEQ ID NO: 2: Amino acid sequence of mouse RGMa precursor protein SEQ ID NO: 3: Amino acid sequence of rat RGMa precursor protein SEQ ID NO: 4: DNA sequence of 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 Epitope of Human RGMa SEQ ID NO: 17: Amino Acid Sequence of LCDR1 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 18: Amino Acid Sequence of LCDR2 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 19: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 20: Amino Acid Sequence of HCDR1 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 21: Amino Acid Sequence of HCDR2 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 22: Amino Acid Sequence of HCDR3 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 23: Amino Acid Sequence of LCDR1 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 24: Amino Acid Sequence of LCDR2 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 25: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 26: Amino Acid Sequence of HCDR1 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 27: Amino Acid Sequence of HCDR2 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 28: Amino Acid Sequence of HCDR3 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 29: Amino Acid Sequence of LCDR1 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 30: Amino Acid Sequence of LCDR2 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 31: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 32: Amino Acid Sequence of HCDR1 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 33: Amino Acid Sequence of HCDR2 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 34: Amino Acid Sequence of HCDR3 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 35: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody AE12-1Y SEQ ID NO: 36: Amino Acid Sequence of Epitope of Human RGMa SEQ ID NO: 37: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 38: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 39: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 40: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1F SEQ ID NO: 41: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1H SEQ ID NO: 42: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1L SEQ ID NO: 43: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1V SEQ ID NO: 44: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1I SEQ ID NO: 45: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1K

Industrial Applicability

[0100] Since the RGMa inhibitor of the present invention is useful for the prevention or treatment of diabetic autonomic neuropathy, it has high utility value in the pharmaceutical industry.

Claims

1. A preventive or therapeutic agent for diabetic autonomic neuropathy comprising an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof, wherein the diabetic autonomic neuropathy is an autonomic neuropathy caused by renal dysfunction.

2. The method according to claim 1, wherein the diabetic autonomic neuropathy is a renal disease associated with renal dysfunction.

3. The method according to claim 2, wherein the renal disease associated with renal dysfunction is chronic kidney disease.

4. The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

5. The preventive or therapeutic agent according to any one of claims 1 to 4, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO:

39.

6. The anti-RGMa neutralizing antibody is one of the following (a) to (l): (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, the amino acid sequence set forth in SEQ ID NO:6 and an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:7, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:8, and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO:

9. an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 containing the amino acid sequence of SEQ ID NO: 10 and an HCDR3 containing the amino acid sequence of SEQ ID NO: 11; (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid sequence set forth in SEQ ID NO:

12. a light chain variable region comprising an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 16; Anti-RG comprising a heavy chain variable region comprising an HCDR2 having an amino acid sequence of SFG and an HCDR3 having an amino acid sequence of SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, and the amino acid sequence set forth in SEQ ID NO:

18. a light chain variable region comprising an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 20, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 21, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 22; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence of SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the amino acid sequence set forth in SEQ ID NO: 24 A light chain variable region comprising LCDR2 containing an array and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 25, and HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 26, the amino An anti-RGMa neutralizing antibody comprising a heavy chain variable region containing HCDR2 containing the acid sequence set forth in SEQ ID NO: 27 and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 28, e) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, the amino acid An LCDR2 containing the sequence and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 31, a light chain variable region, and an HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, the amino An anti-RGMa neutralizing antibody comprising a heavy chain variable region containing an acid sequence set forth in SEQ ID NO: 33 and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, f) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, the amino acid An LCDR2 containing the sequence and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 35, a light chain variable region, and an HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, the amino An anti-RGMa neutralizing antibody comprising a heavy chain variable region containing an acid sequence set forth in SEQ ID NO: 33 and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, g) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 40, a light chain variable An anti-RGMa neutralizing antibody comprising a heavy chain variable region containing an HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, h) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 41, a light chain variable An anti-RGMa neutralizing antibody comprising a heavy chain variable region containing an HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, i) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 42, a light chain variable a domain, and a heavy chain variable region containing 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, an anti-RGMa neutralizing antibody, (j) a light chain variable region containing 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 a domain, and a heavy chain variable region containing 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, an anti-RGMa neutralizing antibody, (k) a light chain variable region containing 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 a domain, and a heavy chain variable region containing 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, an anti-RGMa neutralizing antibody, and (l) a light chain variable region containing 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 a domain, and a heavy chain variable region containing 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, an anti-RGMa neutralizing antibody, The prophylactic or therapeutic agent according to any one of claims 1 to 5, which is an antibody selected from the above.

7. Use of an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof in the manufacture of a prophylactic or therapeutic agent for diabetic autonomic neuropathy, wherein the diabetic autonomic neuropathy is autonomic neuropathy as a cause of renal dysfunction, said use.

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