Agent for prevention or treatment of diabetic autonomic neuropathy

Abstract

To provide an effective agent for diabetic autonomic neuropathy.SOLUTION: This invention provides an agent comprising a RGMa inhibiting substance for preventing or treating diabetic autonomic neuropathy.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a preventive or therapeutic agent for diabetic autonomic neuropathy, which comprises an RGMa inhibitor. [Background technology]

[0002] Diabetic neuropathy is one of the three major complications specific to diabetes, and it develops early and is common. Diabetic autonomic neuropathy, in particular, is a complex disease characterized by a variety of signs and symptoms, resulting from chronic hyperglycemia and resulting in damage to autonomic nerve fibers that control organs throughout the body. It affects the cardiovascular system, digestive system, urinary and reproductive systems, skin, pupils, and adrenal glands, resulting in abnormalities in their functions, including orthostatic hypotension, prandial hypotension, gastric emptying disorder, bladder and sexual dysfunction, sweating abnormalities, pupil abnormalities, and hypoglycemia unawareness (Non-Patent Document 1).

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

[0004] On the other hand, there are cases of diabetic renal failure patients with little proteinuria. This is thought to be due to other kidney diseases, especially hypertensive nephrosclerosis, being associated with diabetes, but it is difficult to differentiate from diabetic nephropathy, which requires the presence of proteinuria. Therefore, in 2007, the United States decided not to require pathological findings for diagnosis, and instead defined chronic kidney disease (CKD), whose onset and progression is thought to be clinically related to diabetes, as diabetic kidney disease (DKD) (Non-Patent Document 4), and Japan also decided to use the term diabetic kidney disease. Diabetic kidney disease is a concept that includes diabetic nephropathy (Non-Patent Document 5). Reflecting this change in concepts, previous stage classifications assumed that the progression of diabetic nephropathy was accompanied by urinary protein, but currently, the presence or absence of urinary protein is not an issue when diabetes is complicated by renal dysfunction.

[0005] RGM (repulsive guidance molecule) was originally identified as an axon guidance molecule in the visual system. RGM family is a membrane protein that has been identified as a marker for iron metabolism (Non-Patent Document 6). The RGM family includes three members, RGMa, RGMb, and RGMc (Non-Patent Document 7), and it is known that at least RGMa and RGMb function via the same signal transduction mechanism (Non-Patent Document 8). RGMc plays an important role in iron metabolism. Subsequent research has revealed that RGM has functions such as axon guidance and lamina formation in Xenopus and chicken embryos, and regulating the closure of the cranial neural tube in mouse embryos (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 function during development, RGMa is re-expressed after central nervous system injury in adult humans and rats, and RGMa inhibition in rats enhances axonal growth and promotes functional recovery after spinal cord injury (Non-Patent Document 10). 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 in central nervous system injury has been clarified, the involvement of RGMa in the treatment of diabetic autonomic neuropathy, especially diabetic nephropathy, has not been identified. No such treatment is known. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. WO2005 / 087268 [Patent Document 2] International Publication No. WO2009 / 106356 [Patent Document 3] International Publication No. WO2013 / 112922 [Patent Document 4] International Publication No. WO2016 / 175236 [Non-patent literature]

[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, pp. 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 [Problem to be solved by the invention]

[0009] An objective of the present invention is to provide an effective drug for diabetic autonomic neuropathy. [Means for solving the problem]

[0010] As a result of intensive research conducted by the inventors to solve the above-mentioned problems, they discovered that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, have an improving effect on diabetic autonomic neuropathy, and thus completed the present invention. That is, the present invention is as follows.

[0011] 1. A preventive or therapeutic agent for diabetic autonomic neuropathy, comprising an RGMa inhibitor. 2. The preventive or therapeutic agent according to Item 1, wherein the diabetic autonomic neuropathy is autonomic neuropathy caused by chronic hyperglycemia or autonomic neuropathy caused by renal dysfunction. 3. The preventive or therapeutic agent according to Item 1 or 2, wherein the diabetic autonomic neuropathy is autonomic neuropathy caused by renal dysfunction. 4. The preventive or therapeutic agent according to any one of items 1 to 3, wherein diabetic autonomic neuropathy is a renal disease associated with renal dysfunction. 5. The preventive or therapeutic agent according to Item 4, wherein the renal disease associated with renal dysfunction is chronic kidney disease. 6. The preventive or therapeutic agent according to any one of items 1 to 5, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. 7. The preventive or therapeutic agent according to Item 6, wherein the anti-RGMa neutralizing antibody is a humanized antibody. 8. The preventive 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 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 a light chain variable region comprising 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; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; P.3, an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 10; (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 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; Anti-RG antibody containing a heavy chain variable region having an HCDR2 containing the amino acid sequence SFG and an HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region 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; Item 9. The preventive or therapeutic agent according to any one of Items 6 to 8, wherein the antibody is selected from the group consisting of:

[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 preventive 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 an agent for the prevention or treatment of diabetic autonomic neuropathy. 13. The use according to item 12, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. [Effects of the Invention]

[0013] According to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are useful, for example, as agents for the prevention or treatment of diabetic autonomic neuropathy. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 shows changes in RGMa gene expression in the kidney under diabetic conditions. [Figure 2] Figure 2 shows representative staining images (photographs substitute for drawings). The top left shows the results for the non-diabetic control antibody-administered group, the top right shows the results for the non-diabetic anti-RGMa neutralizing antibody-administered group, the bottom left shows the results for the diabetic control antibody-administered group, and the bottom right shows the results for the diabetic anti-RGMa neutralizing antibody-administered group. [Figure 3] FIG. 3 shows quantitative data of TH fiber density. [Figure 4] FIG. 4 shows quantitative data of the urinary albumin / urinary creatinine ratio. DETAILED DESCRIPTION OF THE INVENTION

[0015] The terms used in the present invention will be explained below. [Neutralization] As used herein, "neutralizing" refers to the ability to bind to a target of interest and inhibit any function of that target. For example, an RGMa inhibitor is a substance that inhibits the biological activity of RGMa as a result of binding to RGMa.

[0016] [epitope] As used herein, an epitope includes a polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope includes a chemically active surface grouping of a molecule (e.g., amino acids, sugar side chains, phosphoryl or sulfonyl) and, in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody.

[0017] [Isolated] As used herein, "isolated," such as with respect to an isolated RGMa inhibitor (e.g., an antibody), means identified and separated and / or recovered from components of its natural state. Natural impurities are substances that may interfere with diagnostic or therapeutic uses of the antibody. These include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, to isolate an RGMa inhibitor or the like, it is sufficient to purify it by at least one purification step, and an RGMa inhibitor purified by at least one purification step can be referred to as an "isolated RGMa inhibitor."

[0018] [antibody] In this application, the term "antibody" broadly refers to an immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), which substantially retains the epitope-binding property of an Ig molecule.

[0019] [Human antibody] In the present application, a human antibody refers to an antibody in which both the light chain and the heavy chain are derived from human immunoglobulin. Depending on the difference in the heavy chain constant region, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having γ heavy chains, IgM having μ heavy chains, IgA (including IgA1 and IgA2) having α heavy chains, IgD having δ heavy chains, and IgE having ε heavy chains. In principle, the light chain includes either a κ chain or a λ chain.

[0020] [Humanized antibody] In the present application, a humanized antibody refers to an antibody that comprises a variable region consisting of a complementarity-determining region of an antibody derived from a non-human animal and a framework region derived from a human antibody, and a constant region derived from a human antibody.

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

[0022] [Monospecific antibodies] As used herein, a monospecific antibody is an antibody that has a single antigen specificity and a single, independent antigen-recognition site. For example, a monospecific antibody that recognizes RGMa may be referred to as an RGMa monospecific antibody.

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

[0024] [Complementarity-determining region (CDR)] Complementarity determining regions (CDRs) refer to the regions of the variable regions of immunoglobulin molecules that form the antigen-binding site, also called hypervariable regions, and refer to the parts where there is particularly large variation in amino acid sequence for each immunoglobulin molecule. There are three CDRs in each of the light chain and heavy chain. The three CDRs in the light chain are sometimes called LCDR1, LCDR2, and LCDR3, respectively, and the three CDRs in the heavy chain are sometimes called HCDR1, HCDR2, and HCDR3. For example, the CDRs of immunoglobulin molecules are numbered according to the Kabat numbering system (K abat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health a and Human Services, NIH, USA).

[0025] [Effective dose] An effective amount refers to the amount of a prophylactic or therapeutic agent sufficient to reduce or ameliorate the severity and / or duration of a disorder or one or more symptoms thereof, prevent 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 sequence] "Percent (%) identity" of an amino acid sequence of a candidate polypeptide sequence, such as a variable region, with respect to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the particular reference polypeptide sequence after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent identity can be performed using a variety of methods within the skill of one in the art, e.g., BLAST, BLAST-2, ALIG, etc. This can be achieved by using publicly available computer software such as BLAST, BLAST, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, percent identity values are obtained using the sequence comparison computer program BLAST in pairwise alignments. In situations where BLAST is used for amino acid sequence comparison, the percent identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues with a score that matched as identical by the program alignment of programs A and B of the sequence alignment program BLAST, and Y is the total number of amino acid residues of B. When the length of amino acid sequence A is different from the length of amino acid sequence B, it will be understood that the % identity of A to B is 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 paragraph immediately above.

[0027] [Conservative substitution] 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 when substituting one polar residue with another polar residue having the same charge, etc. Examples of functionally similar amino acids for which such substitutions can be made include, as non-polar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of positively charged (basic) amino acids include arginine, histidine, lysine, etc. Also, examples of negatively charged (acidic) amino acids include aspartic acid, glutamic acid, etc.

[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 a prophylactic or therapeutic agent containing an effective amount of an RGMa inhibitor to a mammal requiring treatment.

[0029] <RGMa inhibitor> The RGMa inhibitor of the present invention acts on RGMa itself, and the activity of RGMa (hereinafter, Herein, the term "RGMa inhibitor" may be used simply as long as it is a substance that inhibits or attenuates RGMa activity. For example, substances that bind to RGMa and directly inhibit (attenuate) RGMa activity, or that inhibit the binding of RGMa to a receptor and indirectly inhibit (attenuate) RGMa activity (e.g., compounds and antibodies described below) are referred to as RGMa inhibitors of the present invention. In addition, the RGMa inhibitor of the present invention may be a substance that suppresses the expression of RGMa, and for example, substances that inhibit the expression of RGMa and inhibit (attenuate) RGMa activity (e.g., nucleic acid molecules described below) are also included in the RGMa inhibitor of the present invention.

[0030] RGMa has been identified as a neurite outgrowth inhibitor in the central nervous system. Human RGMa protein is biosynthesized as a precursor protein consisting of 450 amino acids as shown in SEQ ID NO: 1. The signal peptide Met1-Pro47 (referring to the peptide from the first methionine residue to the 47th proline residue from the N-terminus) is present at the N-terminus. The N-terminal domain is then generated by the cleavage of the peptide bond between Asp168 and Pro169, followed by the removal of the C-terminal peptide Ala425-Cys450 from the fragment C-terminal to Pro169, and a GPI anchor is added to the C-terminal carboxyl group of the resulting C-terminal Ala424 to generate the C-terminal domain. Human RGMa protein is expressed on the cell membrane as a mature protein in which the N-terminal domain (Cys48-Asp168) and the C-terminal domain (Pro169-Ala424) are connected by disulfide bonds via a GPI anchor.

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

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

[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 or monoclonal antibody. In the present invention, a monoclonal antibody is preferred. Furthermore, the anti-RGMa neutralizing antibody of the present invention may be either a monospecific RGMa antibody or a multispecific antibody that recognizes multiple antigens, including RGMa, but is preferably a monospecific RGMa antibody.

[0034] Specific epitopes include those in human RGMa, such as 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 (sequence SEQ ID NO: 39 (amino acid numbers 349-359 of SEQ ID NO: 1), SEQ ID NO: 39 (amino acid numbers 367-377 of SEQ ID NO: 1), and a combination of SEQ ID NO: 36 and SEQ ID NO: 37. The combination of SEQ ID NOs: 36, 37 and 39 is particularly preferred.

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

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

[0037] (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 a light chain variable region comprising 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; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11 (the anti-RGMa neutralizing antibody further includes antibodies having epitopes set forth in SEQ ID NOs: 36, 37, and 39); (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 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; Anti-RG antibody containing a heavy chain variable region having an HCDR2 containing the amino acid sequence SFG and an HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody (the anti-RGMa neutralizing antibody further includes antibodies having epitopes of SEQ ID NOs: 36, 37, and 38), (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21, and an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (g) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (h) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (i) a light chain variable region comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (j) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 43 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); (k) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 44 an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NO: 16 as an epitope); (l) a light chain variable region comprising LCDR1 having the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34 (the anti-RGMa neutralizing antibody also includes an antibody having an epitope set forth in SEQ ID NO: 16); Examples of antibodies include those selected from the following: Among these, the antibody described in (a) is particularly preferred.

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

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

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

[0041] <Monoclonal antibodies> Specifically, monoclonal antibodies can be obtained as follows: The above-mentioned antigen is used as an immunogen, and the immunogen is injected subcutaneously, intramuscularly, intravenously, or into the foot pad of the above-mentioned mammals together with Freund's adjuvant as necessary. Immunization is carried out by intraperitoneal or intraperitoneal injection or transplantation one to several times. Usually, immunization is carried out one to four times at intervals of about 1 to 14 days from the first immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

[0042] Monoclonal antibodies can be obtained using methods well known to those skilled in the art (e.g., Current Protocols in Molecular Biology (John Wiley & Sons (1987)); Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0043] Hybridomas secreting monoclonal antibodies can be prepared according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) or modifications thereof. Specifically, hybridomas are prepared by fusing antibody-producing cells contained in the spleen or other tissues obtained from an immunized mammal with myeloma cells derived from a mammal, preferably a mouse, rat, or human, that are not capable of producing autoantibodies.

[0044] Examples of myeloma cells that can be used in cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0, or BW5147; rat-derived myeloma 210RCY3-Ag.2.3; and human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15.

[0045] Examples of fusion promoters include polyethylene glycol, and cell fusion can be achieved by reacting polyethylene glycol (average molecular weight 1000 to 4000) at a concentration of approximately 20 to 50%, at a temperature of 20 to 40°C, preferably 30 to 37°C, at a ratio of antibody-producing cells to myeloma cells of typically 1:1 to 10:1, for approximately 1 to 10 minutes.

[0046] Screening for hybridoma clones that produce monoclonal antibodies can be carried out by culturing the hybridomas, for example, in a microtiter plate and measuring the reactivity of the culture supernatant in the wells to the immunogen by an immunochemical method such as ELISA.

[0047] In screening for antibody-producing hybridomas, in addition to assaying for binding to RGMa protein, we also evaluate whether the antibody inhibits the RGMa activity of the present invention. These screening methods allow us to select anti-RGMa neutralizing antibodies of the present invention.

[0048] The wells containing hybridomas producing the desired antibody can be further cloned by limiting dilution to obtain clones. Hybridoma selection and breeding are usually carried out by Culture is carried out in animal cell medium containing 10-20% fetal bovine serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).

[0049] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or by growing them in vivo, for example, in ascites of a mammal such as a mouse or rat, and isolating the antibodies from the resulting culture supernatant or from the ascites of the mammal.

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

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

[0052] Monoclonal antibodies can be isolated and purified by subjecting the culture supernatant or ascites fluid to saturated ammonium sulfate, euglobulin precipitation, caproic acid, caprylic acid, ion exchange chromatography (DEAE, DE52, etc.), or affinity column chromatography using an anti-immunoglobulin column or protein A column. Specifically, monoclonal antibodies can be purified using known immunoglobulin purification methods, and can be easily achieved by, for example, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of an anion exchanger, or affinity chromatography using RGMa protein.

[0053] Monoclonal antibodies can also be obtained by phage display. In phage display, phages selected from a phage antibody library are screened with the target immunogen, and phages with the desired binding affinity to the immunogen are selected. Next, the antibody-corresponding sequence contained in the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding domain is constructed based on the isolated or sequence information. Monoclonal antibodies can then be produced by culturing a cell line transfected with such an expression vector. Human antibodies with the desired binding affinity can be generated by using a human antibody library as the phage antibody library.

[0054] <Nucleic acid molecule> Nucleic acid molecules encoding the anti-RGMa neutralizing antibodies or antigen-binding fragments thereof of the present invention can be obtained, for example, by the following method. First, total RNA is prepared from cells such as hybridomas using a commercially available RNA extraction kit, and cDNA is synthesized using reverse transcriptase and random primers. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides with sequences conserved in the variable regions of known human antibody heavy chain and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The DNA nucleotide sequence can be determined by standard methods, such as by incorporating it into a sequencing plasmid. Alternatively, DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the sequence of the variable region or a part thereof and ligating it to a sequence containing the constant region. The nucleic acid molecule may encode both the heavy and light chain constant and variable regions, or may encode only the heavy and light chain variable regions. When encoding all of the above, the base sequences of the heavy and light chain constant regions are preferably those described in Nucleic Acids Research, vol. 14, p. 1779, 1986; The Journal of Biological Chemistry, vol. 257, p. 1516, 1982; and Cell, vol. 22, p. 197, 1980.

[0055] <Functionally modified antibodies> Functionally modified anti-RGMa neutralizing antibodies can be prepared by the following methods. For example, when the present anti-RGMa neutralizing antibodies are produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as host cells, antibodies with reduced fucose content in the sugar chains and enhanced cell-killing activity are obtained. When the present anti-RGMa neutralizing antibodies are produced using CHO cells transfected with the FUT8 gene as host cells, antibodies with reduced cell-killing activity are obtained (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739). Furthermore, complement activation function can be regulated by modifying amino acid residues in the Fc region (U.S. Patent Nos. 6,737,056, 7,297,775, and 7,317,091). Furthermore, by using a mutant Fc region that enhances binding to FcRn, one of the Fc receptors, it is possible to extend the blood half-life (Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering. By using a mutant Fc region that enhances binding to FcRn, one of the receptors, it is possible to extend the blood half-life (Shuhei Hashiguchi et al., Biochemistry, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering.

[0056] <Conjugate antibody> Conjugated antibodies are examples of modified molecules of the anti-RGMa neutralizing antibodies of the present invention. Conjugated antibodies include those containing non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (TGF-β, NGF, neurotrophin, etc.), albumin, enzymes, other antibodies, and the like. Examples include conjugated antibodies to which functional molecules other than anti-RGMa neutralizing antibodies are chemically or genetically bound.

[0057] When PEG is used as a functional molecule, the molecular weight of the PEG may be, but is not limited to, 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be either linear or branched. PEG can be bound to the N-terminal amino group of an amino acid in an anti-RGMa neutralizing antibody, for example, by using an NHS-activated group.

[0058] When a radioactive substance is used as a functional molecule, 131I, 125I, 90Y, 64Cu, 99Tc, 77Lu, or 211At is used. The radioactive substance is used for the anti-RGM antibody by the chloramine T method or the like. aCan be directly conjugated to a neutralizing antibody.

[0059] When a toxin is used as a functional molecule, bacterial toxins (eg, diphtheria toxin), plant toxins (eg, ricin), low-molecular-weight toxins (eg, geldanamycin), maytansinoids, calicheamicin, and the like can be used.

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

[0061] When an enzyme is used as the functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc., are used.

[0062] Linkers used to chemically link toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2)nO( CR2)n- (R is an optional substituent, n is a positive integer) Examples of suitable groups include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (e.g., succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for attaching functional molecules have already been established in the field (DJ King., Applications and Engineering of Monoclonal Antibodies., 1998 TJ International Ltd; Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol. 152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol. 93:8681).

[0063] <Antigen-binding fragment> In an embodiment of the present invention, the term "antigen-binding fragment" of an antibody refers to a partial region of the antibody as described above that has antigen-binding activity, and specifically includes F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof. Furthermore, antigen-binding fragments of the present invention may also include non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, other antibodies, and the like. The present invention also includes conjugate fragments to which functional molecules other than the anti-RGMa neutralizing antibody are chemically or genetically engineered.

[0064] "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulin with protease enzymes such as pepsin or papain, resulting in digestion across the disulfide bond between the two heavy chains in the hinge region. For example, when IgG is treated with papain, it is cleaved upstream of the disulfide bond between the two heavy chains in the hinge region, producing two homologous antibody fragments in which a light chain consisting of a VL (light chain variable region) and a CL (light chain constant region), and a heavy chain fragment consisting of a VH (heavy chain variable region) and a CHγ1 (γ1 region in the heavy chain constant region) are linked by a disulfide bond at the C-terminal region. These two homologous antibody fragments are each called Fab. Also, I When IgG is treated with pepsin, it is cleaved downstream of the disulfide bond between the two heavy chains in the hinge region, resulting in an antibody fragment that is slightly larger than the two Fab fragments connected by the hinge region. This antibody fragment is called F(ab')2.

[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 those whose variable regions are derived from immunoglobulins of non-human animals (mouse, rat, hamster, chicken, etc.) and whose constant regions are derived from human immunoglobulins. For example, chimeric antibodies can be produced by immunizing a mouse with an antigen, excising the variable region that binds to the antigen from the mouse monoclonal antibody gene, and combining it with an antibody constant region derived from human bone marrow. Constant regions derived from human immunoglobulins have unique amino acid sequences depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE. The constant region of the recombinant chimeric antibody of the present invention may be the constant region of a human immunoglobulin belonging to any isotype. Preferably, it is a human IgG constant region. An expression vector can be produced using the gene of the chimeric antibody produced in this way. The expression vector Host cells are transformed with the vector to obtain transformed cells that produce chimeric antibodies, and the transformed cells are cultured to obtain the desired chimeric antibodies from the culture supernatant.

[0066] <Humanized antibody> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a humanized antibody. The "humanized antibody" of the present invention is an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity-determining region) of a non-human animal antibody, such as a mouse, has been grafted onto a human antibody gene (CDR grafting). For example, it can be prepared by referring to the methods described in JP-A-4-506458 and Japanese Patent No. 2912618. Specifically, this refers to a humanized antibody in which some or all of its CDRs are derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework regions of its variable regions are derived from a human immunoglobulin, and its constant regions are derived from a human immunoglobulin.

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

[0068] For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be produced by genetic engineering with reference to JP-A-4-506458 and JP-A-62-296890, etc. That is, DNA of the mouse heavy chain CDR region and DNA of the mouse light chain CDR region are isolated from a hybridoma producing a mouse monoclonal antibody, and a human heavy chain gene covering the entire region except for the human heavy chain CDR and a human light chain gene covering the entire region except for the human light chain CDR are isolated from a human immunoglobulin gene.

[0069] The isolated human heavy chain gene grafted with DNA of the mouse heavy chain CDR region is introduced into an appropriate expression vector so as to be expressible, and similarly, the human light chain gene grafted with DNA of the mouse light chain CDR region is introduced into another appropriate expression vector so as to be expressible. Alternatively, the human heavy chain and light chain genes grafted with mouse CDRs can be introduced into the same expression vector so as to be expressible. Host cells are transformed with the expression vector prepared in this way to obtain humanized antibody-producing transformants, and the desired humanized antibody is obtained from the culture supernatant by culturing the transformants.

[0070] <Human antibodies> Another preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a human antibody. A human antibody is an antibody in which all regions constituting the immunoglobulin, including the heavy chain variable region, heavy chain constant region, and light chain variable region, and light chain constant region, are derived from a gene encoding human immunoglobulin, and can be produced by introducing a human antibody gene into a mouse. Specifically, for example, a transgenic animal produced by incorporating at least a human immunoglobulin gene into the genetic locus of a non-human mammal, such as a mouse, can be produced by immunizing the animal with an antigen in a manner similar to the method for producing polyclonal or monoclonal antibodies described above.

[0071] For example, transgenic mice producing human antibodies are described in Nature Genetics, Vol. 7, pp. 13-21, 1994; Nature Genetics, Vol. 15, pp. 146-156, 1997; JP 4-504365 A; It is prepared according to the methods described in JP-A-7-509137; International Publication WO94 / 25585; Nature, Vol. 368, pp. 856-859, 1994; and JP-A-6-500233. More specifically, examples include HuMab (registered trademark) mice (Medarex, Princeton NJ), KM™ mice (Kirin Pharma Company, Japan), and KM (FCγRIIb-KO) mice.

[0072] Specific examples of anti-RGMa neutralizing antibodies of the present invention include those having a CDR containing a specific amino acid sequence in the heavy chain variable region and a CDR containing a specific amino acid sequence in the light chain variable region (preferably the anti-RGMa neutralizing antibodies (a) to (l) above). As long as the antibody of the present invention maintains its ability to bind to RGMa and inhibit (neutralize) the activity of RGMa, it is possible to use an anti-RGMa neutralizing antibody (preferably, any of the above-mentioned (a) to (l)). The amino acid sequence of the anti-RGMa neutralizing antibody may include substitution, deletion, addition, or insertion of one or several amino acids (1 to 20, 1 to 10, or 1 to 5, preferably 1 or 2). Such substitution, deletion, or addition may be introduced into the CDR, but is preferably introduced into a region other than the CDR. Furthermore, the amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

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

[0074] In the present invention, siRNA refers to a short double-stranded RNA capable of suppressing the expression of a target gene (in the present invention, the RGMa gene). The base sequence and length (base length) of the siRNA are not particularly limited as long as it functions as an siRNA that inhibits RGMa activity, but it is 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 is a single-stranded RNA that contains a partially palindromic base sequence, forming a double-stranded structure within the molecule, and is derived from a short hairpin structure with a protruding portion at the 3' end. shRNA refers to a molecule of about 20 base pairs or more consisting of a single molecule. After being introduced into a cell, such shRNA is degraded into molecules of about 20 bases (typically, for example, 21 bases, 22 bases, or 23 bases) in length within the cell, and can suppress the expression of the target gene in the same way as siRNA. In the present invention, the above-mentioned siRNA and shRNA may be in any form as long as they are capable of suppressing the expression of the RGMa gene.

[0075] In the present invention, siRNA or shRNA can be artificially chemically synthesized. Alternatively, antisense and sense RNAs can be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and a T7 promoter. Antisense oligonucleotides may be either DNA or RNA, as long as they are complementary to or hybridize with a sequence of 5 to 100 consecutive bases in the DNA sequence of the RGMa gene. Modifications may also be used as long as they do not impair function. Antisense oligonucleotides can be synthesized by conventional methods, for example, easily using a commercially available DNA synthesizer. Preferred sequences can be selected using conventional selection methods and confirmed as siRNA or shRNA in the present invention by assessing inhibition of expression of functional RGMa.

[0076] <Diabetic autonomic neuropathy> In the present invention, diabetic autonomic neuropathy means autonomic nerve disorder caused by chronic hyperglycemia such as diabetes, and autonomic nerve disorder that is a cause of renal dysfunction. Here, autonomic nervous system disorders caused by chronic hyperglycemia such as diabetes include a variety of signs and symptoms seen in organs throughout the body due to chronic hyperglycemia, such as (1) orthostatic hypotension and arrhythmia as autonomic nervous system disorders of the cardiovascular system, (2) vomiting and diarrhea due to gastroparesis (diabetic diarrhea, etc.) as autonomic nervous system disorders of the digestive system, and (3) urinary and reproductive systems. Examples of autonomic nervous system disorders include neurogenic bladder and erectile dysfunction; (4) examples of metabolic autonomic nervous system disorders include hypoglycemia unawareness and hypoglycemia-related autonomic failure; and (5) examples of peripheral vasomotor function disorders include the breakdown of blood pressure regulation mechanisms caused by such disorders, the breakdown of body fluid homeostasis due to endocrine system disorders, and anemia. Furthermore, because the renal autonomic nervous system is deeply involved in renal function through regulation of systemic and renal blood flow, secretion of neurohumoral factors, and direct action on the renal vasculature, protection of the renal autonomic nervous system is thought to contribute to the improvement of renal function in a wide range of chronic kidney disease conditions (Reference: Front Med. 2018 Mar 29;5:82. doi: 10.3389 / fmed.2018.00082.). Therefore, autonomic neuropathy as a cause of renal dysfunction includes, for example, renal diseases associated with renal dysfunction, such as chronic kidney disease (e.g., diabetic nephropathy (including diabetic nephropathy)). The present invention is expected to have a preventive or therapeutic effect on the disadvantages (diseases or symptoms) caused by the above-mentioned diabetic autonomic neuropathy. The subjects of treatment in the present invention (preferably mammals, particularly humans) are patients who have developed diabetic autonomic neuropathy, and the prophylactic or therapeutic agent for diabetic autonomic neuropathy of the present invention can be administered to these patients.

[0077] As used herein, "treatment" includes any treatment of a disease in a subject, preferably a mammal, particularly a human, including preventing the progression of the disease and symptoms, and eliminating, curing, alleviating or alleviating such disease and symptoms.

[0078] Furthermore, "prevention" includes preventing or suppressing the onset of the above-mentioned diseases in a subject to be treated, preferably a mammal, particularly a human. Furthermore, "prevention" in the present invention also includes "recurrence prevention," which prevents the recurrence of the above-mentioned diseases that repeatedly undergo remission and relapse in a subject to be treated, preferably a mammal, particularly a human.

[0079] <Pharmaceutical Composition> The agent for preventing or treating diabetic autonomic neuropathy of the present invention is generally administered systemically or locally, orally or parenterally. The preventive or therapeutic agent for diabetic autonomic neuropathy of the present invention can be formulated by appropriately blending an RGMa inhibitor as an active ingredient with a pharmaceutically acceptable carrier or additive. Such a formulated pharmaceutical composition can be administered orally or parenterally. Specifically, it can be administered in oral forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, or parenteral forms such as injections, infusions, suppositories, ointments, and patches. The proportion of the carrier or additive may be appropriately determined based on the range commonly used in the pharmaceutical field. The carrier or additive that can be blended is not particularly limited, and examples include various carriers such as water, saline, other aqueous solvents, and aqueous or oily bases, as well as various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0080] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, a functionally modified antibody thereof, a conjugated antibody thereof, or an antigen-binding fragment thereof, it is preferably administered parenterally, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically, as an injection or infusion formulated together with a pharmaceutically acceptable carrier. For example, injections or infusions containing anti-RGMa neutralizing antibodies can be used as solutions, suspensions, or emulsions, and solvents such as distilled water for injection, physiological saline, glucose solutions, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used. Furthermore, such injections or infusions containing anti-RGMa neutralizing antibodies may contain stabilizers, solubilizing agents, suspending agents, emulsifiers, soothing agents, buffers, preservatives, antiseptics, pH adjusters, etc. good. Examples of stabilizers that can be used include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol, etc.), polyalcohols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, etc.). Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, tragacanth, etc. As the soothing agent, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, and Tris buffer. Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Examples of preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.

[0081] When the RGMa inhibitor is a nucleic acid (e.g., siRNA, shRNA, or antisense oligonucleotide), it can be administered in the form of a non-viral or viral vector. In the case of a non-viral vector, methods for introducing nucleic acid molecules using liposomes (e.g., liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method), microinjection, or gene gun (Gene Gun) can be used. Methods such as (n) above can be used to transfer nucleic acid molecules into cells together with carriers (metal particles). For example, when administering siRNA or shRNA to a living body using a viral vector, viral vectors such as recombinant adenoviruses and retroviruses can be used. DNA that expresses siRNA or shRNA can be introduced into DNA or RNA viruses such as detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, poxviruses, polioviruses, Sindbis viruses, Sendai viruses, and SV40, and then cells or tissues can be infected with this recombinant virus to introduce genes into cells or tissues.

[0082] The formulation thus obtained can be administered in an effective amount to, for example, humans or other mammals (e.g., rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.) to prevent or treat diabetic autonomic neuropathy. The dosage is determined appropriately taking into consideration the purpose, severity of the disease, the patient's age, weight, sex, medical history, and type of active ingredient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, the daily dosage for an average human weighing approximately 65 to 70 kg is preferably approximately 0.02 mg to 4000 mg, and more preferably approximately 0.1 mg to 200 mg. The total daily dosage may be a single dose or divided doses.

[0083] <Combination with other drugs or treatments> In the present invention, the agent for preventing or treating diabetic autonomic neuropathy can be administered in combination with an antidiabetic drug for the purpose of blood sugar control. Examples of the antidiabetic drug to be used in combination include hypoglycemic agents, specifically DPP4 inhibitors, SGLT inhibitors, and GLP-1 receptor agonists. Examples include animal drugs. In the present invention, the preventive or therapeutic agent for diabetic autonomic neuropathy can be administered in combination with an antihypertensive drug for the purpose of blood pressure control. Examples of the antihypertensive drug to be combined include, for example, an angiotensin II receptor antagonist (ARB), an ACE inhibitor, etc. 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 preventive or therapeutic agent for diabetic autonomic neuropathy of the present invention, or may be administered or performed simultaneously.

Example

[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 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 examined histologically. <Induction of diabetes>

[0087] C57BL / 6J female mice at 7 to 8 weeks of age were used in the experiment. Streptozocin (STZ: Sigma-Aldrich) at 20 mg / ml was intraperitoneally administered 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 1 week after diabetes induction, and individuals with blood glucose levels less than 300 mg / dl were excluded.

[0088] <RGMa mRNA expression analysis>[[]] ​Four mice 8 weeks after induction of diabetes with STZ and four control mice were thoroughly anesthetized, and then the abdomen was opened. Ice-cold PBS was perfused through the left ventricle to remove blood. The kidneys were removed and collected in a tissue disruption tube (TM-625S; TOMY SEIKO Co., Ltd.) containing an appropriate amount of TRIzol solution (15596026; Thermo Fisher Scientific) and zirconia beads (ZB-10; TOMY SEIKO Co., Ltd.), and then disrupted using a bead-type cell disrupter (MS-100R; TOMY SEIKO Co., Ltd.). Then, RNA was extracted and purified using the RNeasy Mini Kit (74104; QIAGEN). After the PCR reaction, cDNA was generated by reverse transcription and then reacted using Fast SYBR Green Master mix (4385612, Thermo Fisher Scientific) and the QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific). From the Ct values calculated from these results, relative quantification was performed using the ΔΔCt method with Gapdh as an endogenous control.

[0089] <Histological analysis: antibody administration method, group division> 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 STZ (diabetic group) or vehicle (citrate buffer (pH = 4.5)) administration, administration of anti-RGMa neutralizing antibody or control antibody began. All antibodies were administered at a concentration of 6 mg / ml. After preparation, the mixture was administered into the tail vein at a dose of 30 mg / kg once a week for a total of six times, and samples were collected after six weeks.

[0090] <Tissue sampling / kidney clearing / immunological staining> After sufficient anesthesia, the kidneys were perfused with 4% paraformaldehyde (PFA) and then removed. After fixation, the cells were left to stand in 30% sucrose / PBS solution at 4°C for 2-3 days, and then cleared according to the CUBIC method. The following procedure was used, which was modified based on previous research (References 3-7) and after examining the conditions. After 30% sucrose replacement, the kidneys were washed with PBS and then transferred to a CUBIC-L solution diluted to 50% with ultrapure water and shaken overnight at room temperature. They were then transferred to a 100% CUBIC-L solution and shaken at 37°C for 5 days. After washing with PBS, the kidneys were transferred to a primary antibody solution containing 0.1% Triton X-100, 0.5% BSA, and 0.01% sodium azide in PBS and shaken at 37°C for 5 days. After washing with PBS-0.5% Triton X-100 for 1 day, the kidneys were transferred to a secondary antibody solution containing 0.1% Triton X-100, 0.1% BSA, and 0.01% sodium azide in PBS and shaken at 37°C for 5 days. The cells were washed with PBS-0.5% Triton X-100 for 1 day, immersed in 1% formaldehyde solution diluted with PB (0.2 M) for 3 hours, and then washed with PBS. The specimens were immersed in CUBIC-R diluted to 50% with pure water for more than 6 hours, and then immersed in 100% CUBIC-R to make them transparent. The cleared tissue was observed and photographed using a confocal laser microscope FV-3000 (Olympus). Images were taken of the central part of the cleared kidney, from the surface to 200 μm, at a z-interval of 1 μm. The captured images were projected onto a single image, and the density of TH-positive sympathetic nerve fibers per unit area was determined. This procedure was carried out for six individuals in each group, and the analysis was performed. The reagents used are as follows:

[0091] ·CUBIC-L solution Triton 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) :45w%nicotinamide (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) :30w% 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] <Result> Gene expression analysis revealed elevated RGMa mRNA expression in the kidneys in diabetic conditions (Figure 1), suggesting the involvement of RGMa in this pathology. Representative staining images (Figure 2) and quantitative data of TH fiber density (Figure 3) from the subsequent anti-RGMa antibody and control administration experiments in diabetic mice are shown. The results of the four groups shown in the quantitative data were obtained with n=6 in 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 "diabetic-isotype control antibody administered group," the density of TH fibers was significantly increased. On the other hand, in the "diabetes-anti-RGMa neutralizing antibody administration group," Compared with the "diabetes-isotype control antibody administration group," there was a tendency for improvement in TH fiber density. These results suggest that anti-RGM neutralizing antibodies may prevent renal autonomic nerve damage caused by diabetes. It is thought to mitigate harm.

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

[0094] <Induction of diabetes> C57BL / 6J female mice aged 7-8 weeks were used in the experiment. The diabetic group received a single intraperitoneal injection of 20 mg / ml of streptozocin (STZ: Sigma-Aldrich) at a dose of 10 ml / kg. The non-diabetic group received a vehicle injection at a dose of 10 ml / kg. Based on previous studies (References 1 and 2), we investigated the effects of diabetic retinopathy on the development of diabetes. Three days after the induction of the disease, blood glucose levels were measured, and individuals with blood glucose levels below 300 mg / dl were excluded. Ta.

[0095] <Histological analysis: antibody administration method, group division> The purchased mice were randomly divided into three groups: a non-diabetic saline-treated group, a diabetic anti-RGMa neutralizing antibody-treated group, and a diabetic isotype control antibody (Palivizumab)-treated group. Three days after administration of STZ (diabetic group) or vehicle (citrate buffer (pH = 4.5)), administration of saline, anti-RGMa neutralizing antibody, or control antibody was initiated. Saline was administered into the tail vein at a dose of 30 mg / kg once a week for a total of five doses. Each antibody was adjusted to a concentration of 6 mg / ml and then administered into the tail vein at a dose of 30 mg / kg once a week for a total of five doses. Samples were collected after five weeks.

[0096] <Calculation of urinary albumin / urinary creatinine ratio> The urinary albumin / urinary creatinine ratio was used as an index of proteinuria due to renal damage. Five weeks after the induction of the disease, each animal was placed in a metabolic cage (Tecniplast Japan Co., Ltd., Tokyo, Japan) and urine was collected under free movement. The urine albumin was measured using the Lebis Albumin Mouse ELISA Kit. (FUJIFILM Wako Shibayagi Corporation, Gunma pref., Japan), and urinary creatinine Laboratory assay creatinine (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) ) and the urinary albumin / urinary creatinine ratio (UACR) was calculated.

[0097] <Result> The quantitative data of the urinary albumin / urinary creatinine ratio is shown in Figure 4. The results were obtained for n=6 in each group. When comparing the two groups, the urinary albumin / urinary creatinine ratio was significantly higher in the two groups. An increasing trend was observed, which was thought to reflect renal damage. On the other hand, the urinary albumin / urinary creatinine ratio tended to improve in the "diabetes-anti-RGMa neutralizing antibody administration group" compared with the "diabetes-isotype control antibody administration group." Therefore, anti-RGMa neutralizing antibodies are effective in preventing diabetes. These results suggest that anti-RGMa neutralizing antibodies may alleviate renal autonomic nerve damage caused by diabetes. Based on the above, RGMa inhibitors, preferably anti-RGMa neutralizing antibodies, are expected to be useful as preventive or therapeutic agents for autonomic nerve disorders, which are causes of renal dysfunction, and for renal diseases associated with renal 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 the human RGMa gene SEQ ID NO: 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 8: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 9: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 10: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 11: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 12: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 13: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 14: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 15: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 16: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 17: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 18: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 19: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 20: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 21: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 22: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 23: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 24: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 25: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 26: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 27: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 28: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 8D1 SEQ ID NO: 29: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 30: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 31: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 32: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 33: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 34: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 35: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1Y SEQ ID NO: 36: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 37: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 38: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 39: Amino acid sequence of an epitope of human RGMa SEQ ID NO: 40: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1F SEQ ID NO: 41: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1H SEQ ID NO: 42: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1L SEQ ID NO: 43: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1V SEQ ID NO: 44: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1I SEQ ID NO: 45: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1K [Industrial Applicability]

[0100] The present invention is highly useful in the pharmaceutical industry because RGMa inhibitors are useful for preventing or treating diabetic autonomic neuropathy.

Claims

1. A preventive or therapeutic agent for diabetic autonomic neuropathy, comprising an RGMa inhibitor.

2. 2. The preventive or therapeutic agent according to claim 1, wherein the diabetic autonomic neuropathy is autonomic neuropathy caused by chronic hyperglycemia or autonomic neuropathy caused by renal dysfunction.

3. 3. The preventive or therapeutic agent according to claim 1, wherein the diabetic autonomic neuropathy is autonomic neuropathy caused by renal dysfunction.

4. The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the diabetic autonomic neuropathy is a renal disease associated with renal dysfunction.

5. The preventive or therapeutic agent according to claim 4, wherein the renal disease associated with renal dysfunction is chronic kidney disease.

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

7. The preventive or therapeutic agent according to claim 6, wherein the anti-RGMa neutralizing antibody is a humanized antibody.

8. The preventive or therapeutic agent according to claim 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 selected from the following (a) to (l): (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 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; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and Anti-RG containing a heavy chain variable region including HCDR2 containing the amino acid sequence SFG and HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) 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; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; The preventive or therapeutic agent according to any one of claims 6 to 8, which is an antibody selected from the group consisting of:

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 method for prevention or treatment according to claim 10, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

12. Use of an RGMa inhibitor in the manufacture of an agent for the prevention or treatment of diabetic autonomic neuropathy.

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

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