Agent for preventing or treating dementia

Abstract

Provided is a prophylactic or therapeutic agent for a type of dementia selected from diabetic dementia and vascular dementia, the agent containing an RGMa-inhibiting substance.

Description

Technical Field

[0001] The present invention relates to a preventive or therapeutic agent for diabetic dementia and vascular dementia, which contains an RGMa inhibitor.

Background Art

[0002] With the aging of the population, the number of patients with diabetes and dementia has been increasing year by year. It has been clarified that when diabetes develops, the risk of developing Alzheimer's disease or vascular dementia increases, and there is a close pathological relevance between the two diseases (Non-Patent Document 1, Non-Patent Document 2). In addition, in diabetic patients without dementia, there are many reports of a decline in cognitive function compared to non-diabetic patients. As such cognitive dysfunction in diabetic patients, a decline in attention-concentration, a decline in visual or verbal memory, and a decline in the Mini-Mental State Examination (MMSE) have been reported (Non-Patent Document 3).

[0003] In recent years, as a classification of dementia associated with diabetes, in addition to Alzheimer's disease and vascular dementia, a clinical disease type called diabetic dementia in which abnormal glucose metabolism is deeply involved in the onset of dementia has been proposed (Non-Patent Document 4, Non-Patent Document 5). Diabetic dementia rarely shows characteristic brain imaging findings (such as hippocampal atrophy) of Alzheimer's disease, but more cases of coexisting vascular lesions such as microinfarct lesions are observed (Non-Patent Document 6). Clinically, it is slightly older, has poor control of diabetes, shows more prominent decline in attention-concentration and executive function than memory impairment, and has a relatively slow progression.

[0004] On the other hand, vascular dementia is a dementia mainly caused by cerebrovascular disorders. In particular, it is mainly caused by cerebral small vessel diseases such as Binswanger disease and multiple lacunar infarcts, and is defined as having (1) dementia, (2) cerebrovascular disorder, and (3) a causal relationship between the two. As a disease classification according to NINDS-AIREN (National Institute of Neurological Disorders and Stroke-Association International pour la Recherche et l’Enseignement en Neurosciences), which is a clinical diagnostic criterion, there are five types: (1) multiple infarct type, (2) single lesion type, (3) small vessel disease type, (4) low perfusion type, and (5) cerebral hemorrhage type. However, since each of these disease types has problems of being heterogeneous both etiologically and clinically, vascular dementia is considered a heterogeneous disease concept including various pathological conditions (Non-Patent Document 7, Non-Patent Document 8). Also, it is widely known that diabetes is included as a risk factor for vascular dementia (Non-Patent Document 8, Non-Patent Document 9).

[0005] RGM (repulsive guidance molecule) is a membrane protein initially identified as an axon guidance molecule in the visual system (Non-Patent Document 10). The RGM family includes three members called RGMa, RGMb, and RGMc (Non-Patent Document 11), and it is known that at least RGMa and RGMb function through the same signal transduction mechanism (Non-Patent Document 12). RGMc plays an important role in iron metabolism. Subsequent studies have revealed that RGM has functions such as axon guidance and lamina formation in Xenopus and chicken embryos, and control of the closure of the head neural tube in mouse embryos (Non-Patent Document 13). Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.

[0006] In addition to its function at the developmental stage, it reappears after central nervous system injury in adult humans and rats. Since in rats, RGMa inhibition enhances axonal growth and promotes functional recovery after spinal cord injury (Non-Patent Document 14), RGMa is considered to be an axonal regeneration inhibitor after central nervous system injury. Specific antibodies that neutralize RGMa are described, for example, in Patent Document 2 (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3). In addition, Patent Document 2 discloses the therapeutic use of anti-RGMa antibodies for dementia. Although the role of RGMa has been clarified in central nervous system injury, in particular, the involvement of RGMa in the treatment of diabetic dementia and vascular dementia has not been identified, and such therapeutic agents are not known.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Summary of the Invention

Problems to be Solved by the Invention

[0009] An object of the present invention is to provide an effective drug for diabetic dementia and vascular dementia.

Means for Solving the Problems

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

[0011] [1] A prophylactic or therapeutic agent for dementia selected from diabetic dementia and vascular dementia, comprising an RGMa inhibitor. [2] A prophylactic or therapeutic agent for diabetic dementia, comprising an RGMa inhibitor. [3] A prophylactic or therapeutic agent for vascular dementia, comprising an RGMa inhibitor. [4] The prophylactic or therapeutic agent according to any one of [1] to [3], wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. [5] The prophylactic or therapeutic agent according to [4], wherein the anti-RGMa neutralizing antibody is a humanized antibody. [6] The prophylactic or therapeutic agent according to [4] or [5], wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39. [7] The anti-RGMa neutralizing antibody is the following (a) to (l): (a) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 5, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 6, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 10, an anti-RGMa neutralizing antibody; (b) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 11, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 12, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 containing the amino acid sequence of SFG, an anti-RGMa neutralizing antibody; (c) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22, (d) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28, (e) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (f) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (g) An anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, (h) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 41, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 34. (i) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 42, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 34. (j) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 43, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 34. (k) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 44, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 34, and An anti-RGMa neutralizing antibody comprising a light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 45, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34. The prophylactic or therapeutic agent according to any one of [4] to [6], which is an antibody selected from the following.

[0012] [8] A method for preventing or treating dementia selected from diabetic dementia and vascular dementia, comprising administering an effective amount of an RGMa inhibitor to a mammal in need of treatment. [9] The prophylactic or therapeutic method according to [8], wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

[10] Use of an RGMa inhibitor for the manufacture of a prophylactic or therapeutic agent for dementia selected from diabetic dementia and vascular dementia.

[11] The use according to

[10] , wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody. [Advantages of the Invention]

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

[0014]

Figure 1

Figure 2

Figure 3

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

[0016] [Epitope] In the present application, an epitope includes a polypeptide determinant that can specifically bind to an immunoglobulin or a T cell receptor. In certain embodiments, an epitope includes chemically active surface groups 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" with respect to an RGMa inhibitor (such as an antibody, etc.) isolated in the present application means identified, separated, and / or recovered from components in their natural state. Impurities in their natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, and examples include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, to isolate an RGMa inhibitor or the like, it may be purified by at least one purification step, and an RGMa inhibitor purified by at least one purification step can be referred to as an "isolated RGMa inhibitor".

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

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

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

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

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

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

[0024] [Complementary determining region (CDR)] The complementary determining region (CDR) refers to the region in the variable region of an immunoglobulin molecule that forms the antigen-binding site, also called the hypervariable region, which refers to the part where the amino acid sequence changes particularly greatly for each immunoglobulin molecule. There are three CDRs each in the light chain and the heavy chain. The three CDRs contained in the light chain may be referred to as LCDR1, LCDR2, and LCDR3 respectively, and the three CDRs contained in the heavy chain may be referred to as HCDR1, HCDR2, and HCDR3. For example, the CDRs of an immunoglobulin molecule are determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0025] [Effective amount] An effective amount refers to the amount of a prophylactic or therapeutic agent sufficient to reduce or improve the severity and / or duration of a disorder or one or more of its symptoms, prevent the progression of a disorder, reverse a disorder, prevent the recurrence, occurrence, onset, or progression of one or more symptoms associated with a disorder, detect a disorder, or enhance or improve one or more prophylactic or therapeutic effects of another treatment (e.g., a prophylactic or therapeutic agent).

[0026] [Percent (%) Identity of Amino Acid Sequences] The "percent (%) identity" of an amino acid sequence of a candidate polypeptide sequence such as a variable region with respect to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of a specific reference polypeptide sequence, after aligning the sequences and introducing gaps if necessary to obtain the maximum % identity, and not considering any conservative substitutions as part of the sequence identity. Alignments for the purpose of measuring % identity can be achieved by using various methods within the skill of the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment for the full lengths of the sequences being compared. However, for the purposes here, the % identity value is obtained by using the sequence comparison computer program BLAST in a pairwise alignment. In the situation where BLAST is used for amino acid sequence comparison, the % identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues whose scores are matched as identical by the program alignment of A and B by the sequence alignment program BLAST, and Y is the total number of amino acid residues of B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % identity of A to B will be different from the % identity of B to A. Unless otherwise specified, all % identity values here are obtained using the BLAST computer program as shown in the paragraph immediately above.

[0027] [Conservative Substitutions] 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 dementia selected from diabetic dementia and vascular dementia, which is a novel use of an RGMa inhibitor. The present invention also provides a method for preventing or treating dementia selected from diabetic dementia and vascular dementia, which includes the step of administering a prophylactic or therapeutic agent containing an effective amount of an RGMa inhibitor to a mammal in need of treatment.

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

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

[0031] In the present invention, RGMa may be derived from any animal, but is preferably human RGMa. The precursor protein of human RGMa consists of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. The precursor protein of mouse RGMa consists of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, and the precursor protein of rat RGMa consists of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. However, since the C-terminal peptide is removed, the mature proteins have the same amino acid sequence. Examples of the RGMa gene include the human RGMa gene consisting of the nucleotide sequence shown in SEQ ID NO: 4, but are not limited thereto. The nucleotide sequences of RGM genes derived from various organisms can be easily obtained from known databases (such as GenBank).

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

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

[0034] Specific examples of the epitope include one or more of SEQ ID NO: 16 (amino acid numbers 47-69 of SEQ ID NO: 1), SEQ ID NO: 36 (amino acid numbers 298-311 of SEQ ID NO: 1), SEQ ID NO: 37 (amino acid numbers 322-335 of SEQ ID NO: 1), SEQ ID NO: 38 (amino acid numbers 349-359 of SEQ ID NO: 1), and SEQ ID NO: 39 (amino acid numbers 367-377 of SEQ ID NO: 1) in human RGMa. A combination of SEQ ID NO: 36 and 37 is more preferred, and a combination of SEQ ID NO: 36, 37, and 39 is particularly preferred.

[0035] The anti-RGMa neutralizing antibody of the present invention includes polyclonal antibodies and monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or its partial fragment (for example, the epitope fragment described above) as an antigen, chimeric antibodies and humanized antibodies produced using genetic recombination technology, and human antibodies produced using human antibody-producing transgenic animals, etc. When the antibody of the present invention is administered to humans as a medicine, a humanized antibody or a human antibody is desirable from the viewpoint of side effects.

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

[0037] (a) An anti-RGMa neutralizing antibody comprising a light chain variable region containing LCDR1 having the amino acid sequence set forth in SEQ ID NO: 5, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 6, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region containing HCDR1 having the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 10 (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NOs: 36, 37, and 39 as epitopes), (b) An anti-RGMa neutralizing antibody comprising a light chain variable region containing LCDR1 having the amino acid sequence set forth in SEQ ID NO: 11, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and LCDR3 having the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region containing HCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 containing the amino acid sequence of SFG (the anti-RGMa neutralizing antibody also includes an antibody having SEQ ID NOs: 36, 37, and 38 as epitopes), (c) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 17, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 18, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 19, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 20, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 21, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 22, (d) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 23, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 24, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 25, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 26, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 27, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 28, (e) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 31, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 34 (the neutralizing anti - RGMa antibody also includes antibodies having SEQ ID NO: 16 as an epitope), (f) A neutralizing anti - RGMa antibody comprising a light - chain variable region containing LCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 35, and a heavy - chain variable region containing HCDR1 comprising the amino - acid sequence set forth in SEQ ID NO: 32, HCDR2 comprising the amino - acid sequence set forth in SEQ ID NO: 33, and HCDR3 comprising the amino - acid sequence set forth in SEQ ID NO: 34 (the neutralizing anti - RGMa antibody also includes antibodies having SEQ ID NO: 16 as an epitope), (g) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 40, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (h) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 41, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (i) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (j) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 43, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), (k) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 44, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), and (l) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 45, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody having SEQ ID NO: 16 as an epitope), Antibodies selected from the above are included. Among these, the antibody described in (a) is particularly preferred.

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

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

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

[0041] <Monoclonal antibody> Specifically, monoclonal antibodies can be obtained as follows. That is, an antigen as described above is used as an immunogen, and the immunogen is immunized by injecting or transplanting it subcutaneously, intramuscularly, intravenously, into the footpad or intraperitoneally of a mammal as described above one to several times, together with Freund's Adjuvant if necessary. Usually, immunization is carried out 1 to 4 times every about 1 to 14 days from the first immunization, and antibody-producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.

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

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

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

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

[0046] The screening of 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 of the wells against the immunizing antigen by immunochemical methods such as ELISA.

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

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

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

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

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

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

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

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

[0055] <Functionally modified antibody> The functional modified antibodies of the anti-RGMa neutralizing antibody are prepared by the following methods. For example, when the anti-RGMa neutralizing antibody of the present application is produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as host cells, an antibody with a reduced fucose content in the sugar chain and enhanced cell killing function can be obtained. When produced using CHO cells transfected with the FUT8 gene as host cells, an antibody with a low cell killing function can be obtained (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739). In addition, the complement activation function can be regulated by modifying the amino acid residues in the Fc region (US Patent No. 6,737,056, US Patent No. 7,297,775, US Patent No. 7,317,091). Furthermore, by using a mutant of the Fc region with enhanced binding to FcRn, one of the Fc receptors, the blood half-life can be prolonged (Shigekazu Hashiguchi et al., Biochemistry, 2010, Vol. 82(8), p710). These functional modified antibodies can be produced by genetic engineering. By using a mutant of the Fc region with enhanced binding to FcRn, one of the Fc receptors, the blood half-life can be prolonged (Shigekazu Hashiguchi et al., Biochemistry, 2010, Vol. 82(8), p710). These functional modified antibodies can be produced by genetic engineering.

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

[0057] When binding PEG as a functional molecule, PEG with a molecular weight of 2000 to 100,000 Da, more preferably 10,000 to 50,000 Da, can be used without limitation, and it can be linear or branched. PEG can be bound to, for example, the N-terminal amino group of the amino acids of the anti-RGMa neutralizing antibody by using an NHS active group.

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

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

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

[0061] When using an enzyme as a functional molecule, luciferase (for example, firefly luciferase and bacterial luciferase; US Patent No. 4737456), malate dehydrogenase, urease, peroxidase (for example, horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (for example, uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. are used.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0076] <Diabetic dementia> The diabetic dementia in the present invention is a dementia caused by abnormal glucose metabolism, and typically includes dementia associated with diabetes. As its pathogenesis, in addition to vascular factors such as cerebral infarction and arteriosclerosis, promotion of aging changes due to glucotoxicity, oxidative stress, AGE (advanced glycation end-product), etc., and furthermore, hyperinsulinemia, insulin resistance, and impairment of insulin signal transduction are presumed to be involved (Lancet Neurol, 5: 64-74, 2006). Diabetic dementia is clinically shown or suggested to have a close relationship with abnormal glucose metabolism, while no pathological changes or vascular lesions of Alzheimer's disease are observed or it is a milder dementia. Further, such diabetic dementia also includes dementia in which neuropathy due to glucose metabolism disorder (including hyperglycemia, etc.) is involved. The diabetic dementia in the present invention includes diabetic cognitive dysfunction, which is a mild cognitive dysfunction that does not progress to dementia.

[0077] <Vascular dementia> Vascular dementia in the present invention refers to dementia caused by cerebrovascular disorders, meaning those in which there is a causal relationship between cerebrovascular disorders and dementia. As causes of vascular dementia, in addition to cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage, disease types such as cerebral circulatory insufficiency, hypoperfusion, and white matter lesions are also included. Vascular dementia in the present invention also includes vascular cognitive impairment, which is a mild cognitive impairment that does not progress to dementia. Further, in accordance with the diagnostic criteria for vascular dementia, the vascular dementia of the present invention can also be specified (Non-Patent Document 8). The treatment target in the present invention (preferably a mammal, particularly a human) is a patient who has developed diabetic dementia or vascular dementia, and a preventive or therapeutic agent for dementia selected from the diabetic dementia and vascular dementia of the present invention can be administered to these patients.

[0078] Here, "treatment" includes any treatment of a disease in a treatment target, preferably a mammal, particularly a human, and includes preventing the progression of the disease and its symptoms, and eliminating, curing, alleviating, or relieving such a disease and its symptoms.

[0079] Also, "prevention" includes preventing or suppressing the onset of the above-mentioned disease in a treatment target, preferably a mammal, particularly a human. Further, "prevention" in the present invention includes "recurrence prevention" of preventing recurrence of the above-mentioned disease that recurs in remission in a treatment target, preferably a mammal, particularly a human.

[0080] <Pharmaceutical composition> The preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia in the present invention is usually administered systemically or locally, orally or parenterally. The preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia in the present invention contains an RGMa inhibitor as an active ingredient and can be formulated by appropriately blending a pharmaceutically acceptable carrier or additive. The pharmaceutical composition formulated in this way can be administered orally or parenterally. Specifically, it can be made into oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., and can also be made into parenteral preparations such as injections, infusions, suppositories, ointments, patches, etc. The blending ratio of the carrier or additive may be appropriately set based on the range usually adopted in the pharmaceutical field. The carrier or additive that can be blended is not particularly limited, and examples include various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, and various additives such as excipients, binders, pH adjusters, disintegrants, absorption promoters, lubricants, coloring agents, flavoring agents, fragrances, etc.

[0081] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, its functionally modified antibody, its conjugate antibody or their antigen-binding fragments, it is preferably administered parenterally, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously or locally, as an injection or infusion formulated with a pharmaceutically acceptable carrier. For example, an injection or infusion containing an anti-RGMa neutralizing antibody can be used as a solution, suspension or emulsion. As the solvent, for example, distilled water for injection, physiological saline, glucose solution and isotonic solutions (for example, solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used. Furthermore, such an injection or infusion containing an anti-RGMa neutralizing antibody may contain a stabilizer, solubilizing agent, suspending agent, emulsifying agent, pain reliever, buffer, preservative, antiseptic, pH adjuster, etc. As the stabilizer, for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, dibutylhydroxytoluene, etc. can be used. As solubilizing agents, for example, alcohols (such as ethanol, etc.), polyalcohols (such as propylene glycol, polyethylene glycol, etc.), nonionic surfactants (such as polysorbate 80 (registered trademark), HCO-50, etc.) can be used. As suspending agents, for example, glycerol monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. can be used. As emulsifying agents, for example, gum arabic, sodium alginate, tragacanth, etc. can be used. As soothing agents, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. As buffering agents, for example, phosphate buffer solution, acetate buffer solution, borate buffer solution, carbonate buffer solution, citrate buffer solution, Tris buffer solution, etc. can be used. As preservatives, for example, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc. can be used. As antiseptics, for example, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. can be used. As pH adjusters, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. can be used.

[0082] When the RGMa inhibitor is a nucleic acid (such as siRNA, shRNA, antisense oligonucleotide, etc.), it can be administered in the form of a non-viral vector or a viral vector. In the case of a non-viral vector form, methods for introducing nucleic acid molecules using liposomes (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, Lipofectamine method, etc.), microinjection method, method of transferring nucleic acid molecules into cells together with a carrier (metal particles) using a gene gun, etc. can be utilized. For example, when siRNA or shRNA is administered to a living body using a viral vector, viral vectors such as recombinant adenovirus and retrovirus can be utilized. DNA encoding siRNA or shRNA is introduced into a non-toxic retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, etc., which are DNA viruses or RNA viruses, and the recombinant virus is allowed to infect cells or tissues, whereby a gene can be introduced into the cells or tissues.

[0083] The preparation thus obtained can prevent or treat dementia selected from diabetic dementia and vascular dementia by administering an effective amount thereof to, for example, humans and other mammals (such as rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). The dosage is appropriately set in consideration of the purpose, severity of the disease, age, weight, sex, medical history, type of active ingredient, etc. of the patient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, when targeting an average human having a body weight of about 65 to 70 kg, about 0.02 mg to 4000 mg per day is preferable, and about 0.1 mg to 200 mg per day is more preferable. The total daily dosage may be a single dosage or a divided dosage.

[0084] <Combined use with other drugs or treatments> In the present invention, the preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia can be administered in combination with an antidiabetic agent. Examples of the therapeutic agent of the antidiabetic agent to be combined include, for example, insulin resistance improving agents and the like. In the present invention, the preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia can be administered in combination with antithrombotic therapy or an antihypertensive agent. Examples of the antihypertensive agent to be combined include, for example, angiotensin II receptor antagonists (such as candesartan), ACE inhibitors (such as perindopril), and the like.

[0085] The above other drugs or treatments may be administered or carried out before or after the administration of the preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia of the present invention, or may be administered or carried out simultaneously.

Examples

[0086] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. As the anti-RGMa neutralizing antibody, the anti-RGMa neutralizing antibody containing the amino acid sequence (SEQ ID NOs: 5 to 10) described in (a) herein was used in each example.

[0087] [Example 1] Using a drug-induced type 1 diabetes model mouse, the therapeutic effect of the anti-RGMa neutralizing antibody on cognitive dysfunction was examined by a novel object recognition test.

[0088] [Induction of diabetes] 7- to 8-week-old C57BL / 6J female mice were used in the experiment. Streptozocin (STZ: Sigma-Aldrich Co. LLC, St. Louis, MO, USA) at 20 mg / ml was administered intraperitoneally once at a dose of 10 ml / kg to the diabetes induction group, and 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.

[0089] <Grouping, antibody administration schedule> The purchased mice were divided into two groups: the "Diabetes - anti - RGMa neutralizing antibody administration group" and the "Diabetes - isotype control antibody (Palivizumab) administration group". In both groups, antibody administration was started 3 days after diabetes induction. After adjusting the concentration of each antibody to 6 mg / ml, it was intravenously administered into the tail vein once a week at a dose of 30 mg / kg for a total of 4 times. Fourteen mice were used in the "Diabetes - anti - RGMa neutralizing antibody administration group" and 15 mice were used in the "Diabetes - isotype control antibody (Palivizumab) administration group" for the object recognition test. Ten mice in the "Diabetes - anti - RGMa neutralizing antibody administration group" and 8 mice in the "Diabetes - isotype control antibody (Palivizumab) administration group" that met the criteria of the object recognition test were used for analysis. In addition, 7 mice were used to calculate the DI value in non - diabetic mice (healthy mice).

[0090] <Novel object recognition memory test> Handling was performed for 3 days, and the next day, each individual was placed in an open field and acclimated to the field by allowing free exploration for 15 minutes (Handling Trial 1). Acquisition trials were conducted starting from the next day. Prior to the acquisition trials, free exploration of the field for 5 minutes (Handling Trial 2) was performed for each individual. After Performing Handling Trial 2, each individual was temporarily returned to the housing cage, and an object (Object A) with the same shape made using blocks was placed at two locations, upper left and lower right, on the diagonal of the field. Then, each individual was returned to the field and allowed free exploration (Acquisition Trial). After the completion of the acquisition trials, a test trial was conducted after a 12-hour interval (determined based on prior condition examination). At the time of the test trial, a bottle (Object B) containing opaque beads as a novel object was placed instead of Object A, which was placed in the lower right on the diagonal of the field. On the other hand, Object A in the upper left on the diagonal of the field was placed as in the acquisition trials. In the test trial, each individual was returned to the field where Objects A and B were placed and allowed free exploration for 10 minutes. Note that Objects A and B were prepared based on previous studies (Reference 3). All trials were recorded with a video camera, and after the completion of the behavioral test, the exploration time of each individual for both objects in each trial was calculated. The exploration behavior included the behavior of smelling the odor of each object and the behavior of touching the object, excluding the behavior of climbing on the object. For the analysis, the Discrimination Index (DI) in the test trial, calculated with reference to previous studies, was used (Reference 4). DI is defined as {(exploration time for Object B) - (exploration time for Object A)} / {(exploration time for Object B) + (exploration time for Object A)}. Individuals whose total exploration time for Objects A and B in the test trial was less than 30 seconds were not used for the analysis. Note that the DI of healthy control mice was calculated by performing this behavioral test on 16-week-old non-diabetic mice.

[0091] <Results> The DI values obtained from the anti-RGMa neutralizing antibody administration group and the isotype control antibody administration group at 4 weeks after the onset of diabetic conditions, as well as the DI values of healthy mice, are shown in Fig. 1. In the "Diabetes - Isotype Control Antibody Administration Group", a significant decrease in DI was observed compared to healthy mice (p < 0.05, Tukey's multiple comparisons test). On the other hand, in the "Diabetes - Anti-RGMa Neutralizing Antibody Administration Group", a significant increase in DI value was seen compared to the "Diabetes - Isotype Control Antibody Administration Group" (p < 0.05, Tukey's multiple comparisons test), and no difference was found compared to healthy mice (p = 0.8640, Tukey's multiple comparisons test). From these results, it was found that administration of the anti-RGMa neutralizing antibody can improve object memory impairment impaired by diabetes. From the above results, it became clear that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, exhibit a therapeutic effect against diabetic dementia.

[0092] [Example 2] As a cause of hippocampus-dependent cognitive dysfunction in an animal model of diabetes, impaired neurogenesis in the hippocampus has been pointed out (Reference 6). Using a drug-induced type 1 diabetes model mouse, the therapeutic effect of an anti-RGMa neutralizing antibody on the suppression of hippocampal dentate gyrus neurogenesis caused by diabetes was examined by immunohistochemical staining.

[0093] [Induction of Diabetes] C57BL / 6J female mice at 7 - 8 weeks of age were used in the experiment. In the diabetes induction group, streptozotocin (STZ: Sigma-Aldrich Co. LLC, St. Louis, MO, USA) at 20 mg / ml was administered intraperitoneally once at a dose of 10 ml / kg, and in the non-diabetes induction group, the solvent was administered at a dose of 10 ml / kg. Referring to previous studies (References 1, 2), blood glucose levels were measured 1 week after diabetes induction, and individuals with a blood glucose level less than 300 mg / dl were excluded.

[0094] [Antibody Administration] The purchased mice were 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". The diabetic group was administered with STZ solution, and the non-diabetic induction group was administered with solvent. Three days later, the administration of anti-RGMa neutralizing antibody or isotype control antibody was started. After adjusting the concentration of each antibody to 6 mg / ml, it was intravenously administered via the tail vein once a week at a dose of 30 mg / kg for a total of 6 times.

[0095] <Tissue Sampling and Immunohistochemical Staining> Sampling of the mouse brain was performed 6 weeks after diabetes induction. After sufficient anesthesia, perfusion fixation with 4% paraformaldehyde (PFA) was carried out, followed by removal of the brain, which was then post-fixed in 4% PFA. Subsequently, the tissue was transferred to a 30% sucrose solution with PBS as the solvent, left standing at 4°C for 2 to 3 days, and then embedded using OCT compound (Sakura Finetek USA Inc., Torrance, CA, USA). The brain was sectioned at a thickness of 30 μm using a cryostat, and the prepared sections were attached to MAS-coated slide glasses and subjected to immunostaining. Blocking was performed at room temperature for 1 hour with PBS containing 3% Normal Donkey Serum (NDS) and 0.3% Triton X-100 (blocking solution), followed by washing with PBS. Then, as the primary antibody, an anti-mouse doublecortin antibody (1:100; abcam, Cambridge, UK, diluted with the blocking solution) was used and reacted overnight at 4°C. After washing with PBST, as the secondary antibody, an Alexa Fluor 568 donkey anti-rabbit IgG (H+L) antibody (1:500; Invitrogen, Waltham, MA, USA, diluted with the blocking solution) was used and reacted at room temperature for 1 hour. After washing with PBST, nuclear staining was performed using DAPI (1 μg / ml), and then it was sealed. The entire hippocampal dentate gyrus was imaged using a confocal laser microscope FV3000 (Olympus, Tokyo, Japan), and the number of cell bodies of doublecortin-positive neural progenitor cells was counted. The hippocampal dentate gyrus was measured in 6 sections per individual. The number of doublecortin-positive cells was normalized by the area of the hippocampal dentate gyrus measured using ImageJ.

[0096] <Results> The number of doublecortin-positive cells normalized by the hippocampal dentate gyrus area is shown in Figure 2. Among the non-diabetes induction groups, there was no change in the number of doublecortin-positive cells regardless of the type of antibody administered (p > 0.9999, Tukey's multiple comparisons test). Also, in the "Diabetes-Iotype Control Antibody Administration Group", the number of doublecortin-positive cells was significantly decreased compared to the "Non-Diabetes-Anti-RGMa Neutralizing Antibody Administration Group" and the "Non-Diabetes-Iotype Control Antibody Administration Group" (vs "Non-Diabetes-Iotype Control Antibody Administration Group"; p < 0.001, Tukey's multiple comparisons test, vs "Non-Diabetes-Anti-RGMa Neutralizing Antibody Administration Group"; p < 0.001, Tukey's multiple comparisons test). On the other hand, in the "Diabetes-Anti-RGMa Neutralizing Antibody Administration Group", the number of doublecortin-positive cells was significantly improved compared to the "Diabetes-Iotype Control Antibody Administration Group" (p < 0.05, Tukey's multiple comparisons test). There was no significant difference in the number of doublecortin-positive cells in the "Diabetes-Anti-RGMa Neutralizing Antibody Administration Group" compared to the "Non-Diabetes-Iotype Control Antibody Administration Group" and the "Non-Diabetes-Anti-RGMa Neutralizing Antibody Administration Group" (vs Non-Diabetes-Iotype Control Antibody Administration Group; p = 0.1071, Tukey's multiple comparisons test, vs Non-Diabetes-Anti-RGMa Neutralizing Antibody Administration Group; p = 0.1130, Tukey's multiple comparisons test). From these results, it was found that the decrease in neurogenesis by doublecortin-positive neural progenitor cells in the hippocampal dentate gyrus caused by diabetes can be improved by administering an anti-RGMa neutralizing antibody. From the above results, it became clear that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, exhibit a medicinal effect on the impairment of neurogenesis in the hippocampus, which is one of the causes of diabetic dementia.

[0097] [Example 3] Effects of therapeutic intervention with anti-RGMa neutralizing antibody on novel object recognition memory The therapeutic effect of anti-RGMa neutralizing antibody on cognitive dysfunction in a chronic cerebral hypoperfusion model mouse (Bilateral common carotid artery stenosis model: BCAS model) induced by bilateral internal carotid artery microcoil placement was examined by a novel object recognition test.

[0098] <Induction of chronic cerebral hypoperfusion model> C57BL / 6J male mice aged 9 to 10 weeks were used in the experiment. Under isoflurane inhalation anesthesia (induction 4%, maintenance 1.5%), the mice were fixed in the ventral position and cerebral blood flow was measured using laser speckle flowmetry. During the measurement, a heat pad was used to control the rectal temperature at 35.0 - 36.5°C. Subsequently, the mice were fixed in the supine position, the neck was incised, and micro-coils were placed in both internal carotid arteries after releasing the carotid sheath (Reference 5). One day after BCAS induction, cerebral blood flow was measured, and individuals in whom cerebral blood flow did not decrease to 90% or less compared to before the surgery were excluded. OZ-3 (Omegawave, Tokyo, Japan) was used for the measurement of cerebral blood flow.

[0099] <Novel object recognition memory test> Individuals were placed in an open field and acclimated to the field by allowing them to freely explore for 15 minutes (habituation trial 1). The next day, prior to the acquisition trial, each individual was allowed to freely explore the field for 5 minutes (habituation trial 2). After habituation trial 2, the individuals were temporarily returned to their home cages, and an object (object A) with the same shape made using blocks was placed at two locations, upper left and lower right, on the diagonal of the field at a distance of 10 cm from the wall using double-sided tape. Then, the individuals were returned to the field and allowed to freely explore (acquisition trial). After the acquisition trial, a 12-hour interval was given, and a test trial was conducted. In the test trial, a bottle (object B) containing opaque beads as a novel object was placed at a position 10 cm from the wall using double-sided tape instead of object A placed at the lower right on the diagonal of the field. On the other hand, object A at the upper left on the diagonal of the field was placed as in the acquisition trial. After the interval elapsed, each individual was returned to the field where objects A and B were placed and allowed to freely explore for 10 minutes (test trial). Objects A and B used in each trial were fabricated based on previous studies (Reference 3). During the interval, each individual was allowed free access to water and food in their home cages. All trials were recorded with a video camera, and after the behavioral test, the exploration time of each individual for each object in each trial was calculated. The exploration behavior included the behavior of smelling the odor of each object and the behavior of touching the object, and the behavior of climbing on the object was excluded. The Discrimination Index (DI) in the test trial was used for the analysis. DI is defined as {(exploration time for object B) - (exploration time for object A)} / {(exploration time for object B) + (exploration time for object A)}. Individuals whose total exploration time for objects A and B in the test trial was less than 30 seconds were not used for the analysis.

[0100] <Antibody administration> The purchased mice were divided into three groups: the BCAS - anti - RGMa neutralizing antibody administration group, the BCAS - isotype control antibody (Palivizumab) administration group, and the Sham - isotype control antibody (Palivizumab) administration group. In all three groups, antibody administration was started 3 days after BCAS surgery or Sham surgery. After adjusting the concentration of each antibody to 2 mg / ml, it was intraperitoneally administered at a dose of 10 mg / kg twice a week for a total of 7 times, depending on body weight. The number of mice in each group is as follows. 13 mice in the BCAS - anti - RGMa neutralizing antibody administration group 9 mice in the BCAS - isotype control antibody (Palivizumab) administration group 9 mice in the Sham - isotype control antibody (Palivizumab) administration group

[0101] <Results> In the BCAS - isotype control antibody group, a significant decrease in DI was observed compared with the Sham - isotype control antibody (Palivizumab) administration group (p < 0.001, Tukey’s multiple comparisons test). On the other hand, in the BCAS - anti - RGMa neutralizing antibody administration group, DI was significantly increased compared with the BCAS - isotype control antibody group (p < 0.001, Tukey’s multiple comparisons test). From this, it was found that therapeutic intervention by anti - RGMa neutralizing antibody administration significantly improved object recognition memory impairment impaired by chronic ischemia. (Figure 3) From the above results, it became clear that RGMa inhibitors, especially anti - RGMa neutralizing antibodies, exhibit efficacy against vascular dementia.

[0102] [References] 1. Deeds MC, Anderson JM, Armstrong AS, et al. Single dose streptozotocin-induced diabetes: Considerations for study design in islet transplantation models. Lab Anim. 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. Leger M, Quiedeville A, Bouet V, et al. Object recognition test in mice. Nat Protoc. 2013;8(12):2531-2537. doi:10.1038 / nprot.2013.155 4. Grinan-Ferre C, Puigoriol-Illamola D, Palomera-avalos V, et al. Environmental enrichment modified epigenetic mechanisms in SAMP8 mouse hippocampus by reducing oxidative stress and inflammaging and achieving neuroprotection. Front Aging Neurosci. 2016;8(OCT). doi:10.3389 / fnagi.2016.00241 5. Hattori Y et al, Gradual Carotid Artery Stenosis in Mice Closely Replicates Hypoperfusive Vascular Dementia in Humans. Journal of the American Heart Association 2016 2 Feb.22;5(2): e002757. DOI: 10.1161 / JAHA.115.002757 6. Stranahan, A., Arumugam, T., Cutler, R. et al. Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons. Nat Neurosci 11, 309-317 (2008). https: / / doi.org / 10.1038 / nn2055

[0103] <Description of Sequence Listing> SEQ ID NO: 1: Amino acid sequence of human RGMa precursor protein SEQ ID NO: 2: Amino acid sequence of mouse RGMa precursor protein SEQ ID NO: 3: Amino acid sequence of rat RGMa precursor protein SEQ ID NO: 4: DNA sequence of human RGMa gene SEQ ID NO: 5: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 6: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 7: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 8: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 9: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 10: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody r116A3 SEQ ID NO: 11: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody r70E SEQ ID NO: 12: Amino Acid Sequence of LCDR2 of Anti-RGMa Neutralizing Antibody r70E SEQ ID NO: 13: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody r70E SEQ ID NO: 14: Amino Acid Sequence of HCDR1 of Anti-RGMa Neutralizing Antibody r70E SEQ ID NO: 15: Amino Acid Sequence of HCDR2 of Anti-RGMa Neutralizing Antibody r70E SEQ ID NO: 16: Amino Acid Sequence of Epitope of Human RGMa SEQ ID NO: 17: Amino Acid Sequence of LCDR1 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 18: Amino Acid Sequence of LCDR2 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 19: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 20: Amino Acid Sequence of HCDR1 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 21: Amino Acid Sequence of HCDR2 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 22: Amino Acid Sequence of HCDR3 of Anti-RGMa Neutralizing Antibody 5F9 SEQ ID NO: 23: Amino Acid Sequence of LCDR1 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 24: Amino Acid Sequence of LCDR2 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 25: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 26: Amino Acid Sequence of HCDR1 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 27: Amino Acid Sequence of HCDR2 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 28: Amino Acid Sequence of HCDR3 of Anti-RGMa Neutralizing Antibody 8D1 SEQ ID NO: 29: Amino Acid Sequence of LCDR1 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 30: Amino Acid Sequence of LCDR2 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 31: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 32: Amino Acid Sequence of HCDR1 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 33: Amino Acid Sequence of HCDR2 of Anti-RGMa Neutralizing Antibody AE12-1 SEQ ID NO: 34: Amino acid sequence of the HCDR3 of the anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 35: Amino acid sequence of the LCDR3 of the anti-RGMa neutralizing antibody AE12-1Y SEQ ID NO: 36: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 37: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 38: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 39: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 40: Amino acid sequence of the LCDR3 of the anti-RGMa neutralizing antibody AE12-1F SEQ ID NO: 41: Amino acid sequence of the LCDR3 of the anti-RGMa neutralizing antibody AE12-1H SEQ ID NO: 42: Amino acid sequence of the LCDR3 of the anti-RGMa neutralizing antibody AE12-1L SEQ ID NO: 43: Amino acid sequence of the LCDR3 of the anti-RGMa neutralizing antibody AE12-1V SEQ ID NO: 44: Amino acid sequence of the LCDR3 of the anti-RGMa neutralizing antibody AE12-1I SEQ ID NO: 45: Amino acid sequence of the LCDR3 of the anti-RGMa neutralizing antibody AE12-1K

Industrial Applicability

[0104] Since the RGMa inhibitor of the present invention is useful for the prevention or treatment of diabetic dementia or vascular dementia, it has high utility value in the pharmaceutical industry.

Claims

**Claim 1**: A prophylactic or therapeutic agent for dementia selected from diabetic dementia and vascular dementia, comprising an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof. **Claim 2**: A prophylactic or therapeutic agent for diabetic dementia, comprising an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof. **Claim 3**: A prophylactic or therapeutic agent for vascular dementia, comprising an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof. **Claim 4** The prophylactic or therapeutic agent according to any one of claims 1 to 3, wherein the anti-RGMa neutralizing antibody is a humanized antibody. **Claim 5** The prophylactic or therapeutic agent according to any one of claims 1 to 4, wherein the anti-RGMa neutralizing antibody is an antibody that recognizes an amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO:

39. **Claim 6** The anti-RGMa neutralizing antibody is as follows (a) to (l): (a) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 5, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 6, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 8, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 9, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 10, an anti-RGMa neutralizing antibody (b) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 11, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 12, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 13, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 15, and HCDR3 containing the amino acid sequence including SFG, an anti-RGMa neutralizing antibody (c) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 17, LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 18, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 19, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 20, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 21, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 22, an anti-RGMa neutralizing antibody (d) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24, and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain variable region comprising HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 26, HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 27, and HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 28, an anti-RGMa neutralizing antibody (e) A light chain variable region comprising LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, the amino acid A light chain variable region comprising LCDR2 containing an array and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 25, and HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 26, the amino acid sequence containing HCDR2 and an anti-RGMa neutralizing antibody containing a heavy chain variable region containing HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 28, (e) LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, the amino acid A light chain variable region comprising LCDR2 containing the sequence and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 31, and HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, the amino An anti-RGMa neutralizing antibody comprising an acid sequence-containing HCDR2 and a heavy chain variable region containing HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, (f) LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, the amino acid A light chain variable region comprising LCDR2 containing the sequence and LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 35, and HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, the amino An anti-RGMa neutralizing antibody comprising an acid sequence-containing HCDR2 and a heavy chain variable region containing HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, (g) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 40, including a light chain variable Region, and an anti-RGMa neutralizing antibody comprising an HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, including a heavy chain variable region, (h) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 41, including a light chain variable Region, and an anti-RGMa neutralizing antibody comprising an HCDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, including a heavy chain variable region, (i) An LCDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 containing the amino acid sequence set forth in SEQ ID NO: 42, including a light chain variable a heavy chain variable region-containing anti-RGMa neutralizing antibody comprising a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, (j) a light chain variable region comprising a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 43 a heavy chain variable region-containing anti-RGMa neutralizing antibody comprising a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, (k) a light chain variable region comprising a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 44 a heavy chain variable region-containing anti-RGMa neutralizing antibody comprising a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, and (l) a light chain variable region comprising a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 29, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 30, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 45 a heavy chain variable region-containing anti-RGMa neutralizing antibody comprising a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 32, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 33, and a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 34, The prophylactic or therapeutic agent according to any one of claims 1 to 5, which is an antibody selected from the above.

7. Use of an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof for the manufacture of a prophylactic or therapeutic agent for dementia selected from diabetic dementia and vascular dementia.

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