Prophylactic or therapeutic agent for dementia

Abstract

The present invention provides a prophylactic or therapeutic agent for dementia selected from the group consisting of diabetic dementia and vascular dementia, the prophylactic or therapeutic agent comprising an RGMa inhibitor.

Description

[0001] This application is a divisional application of the invention patent application with Chinese application number 202180009256.7 (the invention name of the original application is “Preventive or therapeutic agent for dementia”, and the application date of the original application is January 15, 2021). Technical Field

[0002] The present invention relates to a preventive or therapeutic agent for diabetic dementia and vascular dementia, comprising an RGMa inhibitory substance. Background Art

[0003] The number of patients with diabetes and dementia increases year by year with aging. In the case of diabetes, the risk of developing Alzheimer's disease and vascular dementia increases, and it has been clarified that there is a close pathological correlation between the two diseases (Non-Patent Documents 1 and 2). In addition, many reports indicate that in diabetic patients without dementia, cognitive function is confirmed to be reduced compared with non-diabetic patients. As cognitive dysfunction in such diabetic patients, reduced attention and concentration, reduced visual memory or verbal memory, and reduced Mini-Mental State Examination (MMSE) have been reported (Non-Patent Document 3).

[0004] In recent years, as a classification of dementia associated with diabetes, in addition to Alzheimer's disease and vascular dementia, a clinical disease type such as diabetic dementia, in which abnormal glucose metabolism is closely related to the onset of dementia, has been proposed (Non-Patent Documents 4 and 5). Diabetic dementia rarely presents the characteristic brain imaging findings of Alzheimer's disease (hippocampal atrophy, etc.), but more cases of combined vascular lesions such as microinfarction lesions are seen (Non-Patent Document 6). Clinically, the patient is slightly older, has poor control of diabetes, and has more obvious impairment of attention and concentration and executive function than memory impairment, and walks slightly slowly.

[0005] On the other hand, vascular dementia is a dementia mainly caused by cerebrovascular disorders, especially cerebral small vessel diseases such as subcortical arteriosclerotic encephalopathy (Binswanger disease) and multiple lacunar infarctions. It is defined as a disease in which (1) dementia, (2) cerebrovascular disorders, and (3) the two are causally related.

[0006] As a classification of disease types based on the clinical diagnostic criteria of NINDS-AIREN (National Institute of Neurological Disorders and Stroke-Association International pour la Recherche et l'Enseignement en Neurosciences), there are five types: (1) multi-infarct, (2) single lesion, (3) small vessel lesion, (4) hypoperfusion, and (5) cerebral hemorrhage. However, these disease types are inconsistent in etiology and clinically, so vascular dementia is considered to be a heterogeneous disease concept that includes various symptoms (Non-Patent Documents 7 and 8).

[0007] In addition, diabetes is widely known as a risk factor for vascular dementia (Non-Patent Documents 8 and 9).

[0008] RGM (repulsive guidance molecule) is a membrane protein originally identified as an axon inducing molecule of the visual system (non-patent document 10). It is known that the RGM family includes three members called RGMa, RGMb and RGMc (non-patent document 11), and at least RGMa and RGMb function by the same signal transduction mechanism (non-patent document 12). RGMc plays an important role in iron metabolism.

[0009] Subsequent studies have shown that RGM has the function of inducing axon and layer formation in Xenopus and chicken embryos, and controlling 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.

[0010] In addition to its function in the developmental stage, RGMa is considered to be an inhibitory substance for axon regeneration after central nervous system injury because it is re-expressed after central nervous system injury in adult humans and rats, and in rats, RGMa inhibition leads to hyperplasia of axon growth after spinal cord injury and promotes functional recovery (Non-patent Document 14). Specific antibodies that neutralize RGMa are described in, for example, Patent Document 2 (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3).

[0011] In addition, Patent Document 2 discloses the use of anti-RGMa antibodies for the treatment of dementia.

[0012] Thus, the role of RGMa in central nervous system damage has been clarified, but in particular, the involvement of RGMa in the treatment of diabetic dementia and vascular dementia has not been identified, and such therapeutic drugs are not yet known.

[0013] Prior art literature

[0014] Patent Literature

[0015] Patent Document 1: International Publication No. WO2005 / 087268

[0016] Patent Document 2: International Publication No. WO2009 / 106356

[0017] Patent Document 3: International Publication No. WO2013 / 112922

[0018] Patent Document 4: International Publication No. WO2016 / 175236

[0019] Non-patent literature

[0020] Non-patent literature 1: Neurology, 45: 1161-1168, 1995

[0021] Non-patent document 2: Am. J. Epidemiol. 145(4):301-308, 1997

[0022] Non-patent document 3: Diabetes Care, 20: 438-445, 1997

[0023] Non-patent document 4: Dement Geriatr Cogn Disord, 35: 280-290, 2013

[0024] Non-patent literature 5: J. Neurol. Sci. 349: 45-51, 2015

[0025] Non-patent document 6: Nat Rev Neurol 5:305-306, 2009

[0026] Non-patent document 7: Neurology, 43: 250-260, 1993

[0027] Non-patent literature 8: Dementia Treatment Guidelines 2017 Chapter 14 Vascular Dementia Non-patent literature 9: Neuron 80: 844-866, 2013

[0028] Non-patent document 10: Neuron 5, 735-743 (1990)

[0029] Non-patent document 11: Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513-29, 2006

[0030] Non-patent document 12: Biochem. Biophys. Res. Commun. 382, ​​795-800 (2009)

[0031] Non-patent document 13: Curr. Opin. Neurobiol. 17, 29-34 (2007)

[0032] Non-patent document 14: J. Cell Biol. 173, 47-58 (2006) Summary of the invention

[0033] An object of the present invention is to provide a drug effective for diabetic dementia and vascular dementia.

[0034] The present inventors have conducted intensive studies to solve the above problems and have found that RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, show an improving effect on diabetic dementia and vascular dementia, thereby completing the present invention.

[0035] That is, the present invention is as follows.

[0036] [1] A preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia, comprising an RGMa inhibitory substance.

[0037] [2] A preventive or therapeutic agent for diabetic dementia, comprising an RGMa inhibitory substance.

[0038] [3] A preventive or therapeutic agent for vascular dementia, comprising an RGMa inhibitory substance.

[0039] [4] The preventive or therapeutic agent according to any one of [1] to [3], wherein the RGMa inhibitory substance is an anti-RGMa neutralizing antibody.

[0040] [5] The preventive or therapeutic agent according to [4], wherein the anti-RGMa neutralizing antibody is a humanized antibody.

[0041] [6] The preventive 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 sequence number 16, sequence number 36, sequence number 37, sequence number 38 and sequence number 39.

[0042] [7] The preventive or therapeutic agent according to any one of [4] to [6], wherein the anti-RGMa neutralizing antibody is an antibody selected from the following (a) to (l):

[0043] (a) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10,

[0044] (b) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15, and the HCDR3 comprises SFG in its amino acid sequence,

[0045] (c) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22,

[0046] (d) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28,

[0047] (e) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34,

[0048] (f) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34,

[0049] (g) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34,

[0050] (h) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 41; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34,

[0051] (i) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 42; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34,

[0052] (j) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 43; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34,

[0053] (k) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 44, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, and

[0054] (l) An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence recorded in sequence number 29, the LCDR2 comprises the amino acid sequence recorded in sequence number 30, and the LCDR3 comprises the amino acid sequence recorded in sequence number 45, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence recorded in sequence number 32, the HCDR2 comprises the amino acid sequence recorded in sequence number 33, and the HCDR3 comprises the amino acid sequence recorded in sequence number 34.

[0055] [8] A method for preventing or treating dementia selected from diabetic dementia and vascular dementia, comprising administering an effective amount of an RGMa inhibitory substance to a mammal in need of treatment.

[0056] [9] The method for prevention or treatment according to [8], wherein the RGMa inhibitory substance is an anti-RGMa neutralizing antibody.

[0057]

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

[0058]

[11] The use according to

[10] , wherein the RGMa inhibitory substance is an anti-RGMa neutralizing antibody.

[0059] According to the present invention, the RGMa inhibitory substance, in particular, the anti-RGMa neutralizing antibody is useful as a preventive or therapeutic agent for dementia selected from, for example, diabetic dementia and vascular dementia. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 It is a graph showing the discrimination index (DI) values ​​obtained from the anti-RGMa neutralizing antibody (sometimes simply referred to as anti-RGMa antibody) administration group and the isotype control antibody administration group at 4 weeks after the onset of diabetes (DM) symptoms, and the DI values ​​of healthy mice.

[0061] Figure 2 The graph shows the number of doublecortin-positive cells when the anti-RGMa neutralizing antibody or isotype control antibody was administered to non-diabetic (non-DM) mice or diabetic (DM) mice. The number of doublecortin-positive cells was standardized by the area of ​​the hippocampal dentate gyrus.

[0062] Figure 3 It is a graph showing the discrimination index (DI) values ​​obtained from the anti-RGMa neutralizing antibody-administered group and the isotype control antibody-administered group in the chronic cerebral hypoperfusion model, and the DI value obtained from the isotype control antibody-administered group of sham mice. DETAILED DESCRIPTION

[0063] Hereinafter, the terms used in the present invention will be described.

[0064] [Neutralization]

[0065] In the present application, neutralization refers to an action that binds to a target and is able to inhibit any function of the target. For example, an RGMa inhibitory substance refers to a substance that binds to RGMa and, as a result, inhibits the biological activity of RGMa.

[0066] [Epitope]

[0067] In the present application, an epitope comprises a polypeptide determinant capable of specific binding to an immunoglobulin or T cell receptor. In one embodiment, an epitope comprises a molecular chemically active surface group (e.g., an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group), and in one embodiment, may have specific 3-dimensional structural properties and / or specific charge properties. An epitope is a region of an antigen that is bound by an antibody.

[0068] [Separated]

[0069] In the present application, the "isolated" of isolated RGMa inhibitory substances (such as antibodies, etc.) means identified and separated, and / or recovered from components in the natural state. Impurities in the natural state are substances that may hinder the diagnostic or therapeutic use of the antibody, and examples include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In general, when isolating RGMa inhibitory substances, it is sufficient to purify them through at least one purification step, and the RGMa inhibitory substances purified through at least one purification step can be referred to as "isolated RGMa inhibitory substances".

[0070] [Antibody]

[0071] In the present application, an antibody refers in a broad sense to an immunoglobulin (Ig) molecule consisting of four polypeptide chains, namely, two heavy chains (H chains) and two light chains (L chains), which retains the characteristic of the Ig molecule to substantially bind to an epitope.

[0072] [Human Antibodies]

[0073] In the present application, human antibody refers to an antibody in which both the light chain and the heavy chain are from human immunoglobulin. Human antibody includes IgG (including IgG1, IgG2, IgG3 and IgG4) with a heavy chain of γ chain, IgM with a heavy chain of μ chain, IgA (including IgA1, IgA2) with a heavy chain of α chain, IgD with a heavy chain of δ chain or IgE with a heavy chain of ε chain, according to the difference in the constant region of the heavy chain. In addition, in principle, the light chain includes either one of κ chain and λ chain.

[0074] [Humanized Antibodies]

[0075] In the present application, a humanized antibody refers to an antibody composed 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.

[0076] [Chimeric antibodies]

[0077] In the present application, a chimeric antibody refers to an antibody in which the light chain, the heavy chain, or both chains are composed of a non-human variable region and a human constant region.

[0078] [Monospecific Antibodies]

[0079] In the present application, a monospecific antibody refers to an antibody having a single antigen specificity and a single independent antigen recognition site. In the present specification, for example, a monospecific antibody that recognizes RGMa is referred to as an RGMa monospecific antibody.

[0080] [Multispecific Antibodies]

[0081] In the present application, a multispecific antibody refers to an antibody having two or more different antigen specificities and simultaneously having two or more independent antigen recognition sites, and examples thereof include bispecific antibodies having two antigen specificities and trispecific antibodies having three antigen specificities.

[0082] [Complementarity Determining Region (CDR)]

[0083] Complementarity determining region (CDR) refers to the region forming the antigen binding site in the variable region of an immunoglobulin molecule, also referred to as a hypervariable region, which is a particularly large part of the change in the amino acid sequence in each immunoglobulin molecule. For CDR, there are 3 CDRs in the light chain and the heavy chain, respectively. Sometimes the 3 CDRs contained in the light chain are referred to as LCDR1, LCDR2 and LCDR3, respectively, and the 3 CDRs contained in the heavy chain are referred to as HCDR1, HCDR2 and HCDR3, respectively. For example, the CDR of an immunoglobulin molecule can be determined according to Kabat's numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0084] [Effective amount]

[0085] An effective amount is an amount of a preventive 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 disorder from getting worse, slow down the disorder, prevent the recurrence, occurrence, onset or worsening of one or more symptoms associated with the disorder, detect the disorder, or enhance or improve the preventive or therapeutic effects of one or more other treatments (e.g., preventive or therapeutic drugs).

[0086] [Percentage (%) homology of amino acid sequences]

[0087] The "percent (%) homology" of the amino acid sequence of the candidate polypeptide sequence of the variable region, etc., with respect to the amino acid sequence of the reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific reference polypeptide sequence after the sequences are aligned, gaps are introduced as needed to obtain the maximum % homology, and any conservative substitutions are not considered to be part of the sequence homology. Alignment for the purpose of determining % homology can be achieved by using various methods within the capabilities of those skilled in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. As long as one is skilled in the art, appropriate parameters for aligning sequences including any algorithm necessary to achieve maximum alignment for the full length of the compared sequence can be determined. However, for the purpose here, % homology values ​​are obtained by using the sequence comparison computer program BLAST in pairwise alignments.

[0088] In the case of using BLAST in amino acid sequence comparison, the % homology between a provided amino acid sequence A and a provided amino acid sequence B is calculated as follows:

[0089] 100 times the fraction X / Y

[0090] Here, X is the number of amino acid residues that are scored as identical when A and B are aligned by the sequence alignment program BLAST, and Y is the total number of amino acid residues in B. In the case where the length of amino acid sequence A is different from the length of amino acid sequence B, it can be understood that the % homology of A to B is different from the % homology of B to A. Unless otherwise specified, all % homology values ​​here are obtained using the BLAST computer program as described in the previous paragraph.

[0091] [Conservative substitution]

[0092] Conservative substitution refers to replacing an amino acid residue with another chemically similar amino acid residue in a manner that does not substantially change the activity of the peptide. For example, a hydrophobic residue is replaced by another hydrophobic residue, a polar residue is replaced by another polar residue with the same charge, etc. As examples of functionally similar amino acids that can undergo such substitutions, as non-polar (hydrophobic) amino acids, alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. can be mentioned. As polar (neutral) amino acids, glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. can be mentioned. As positively charged (basic) amino acids, arginine, histidine, lysine, etc. can be mentioned. In addition, as negatively charged (acidic) amino acids, aspartic acid, glutamic acid, etc. can be mentioned.

[0093] Hereinafter, embodiments of the present invention will be described in detail.

[0094] The present invention provides a preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia as a novel use of an RGMa inhibitory substance.

[0095] In addition, the present invention provides a method for preventing or treating dementia selected from diabetic dementia and vascular dementia, comprising administering a preventive or therapeutic agent comprising an effective amount of an RGMa inhibitory substance to a mammal in need of treatment.

[0096] <RGMa inhibitory substances>

[0097] The RGMa inhibitory substance of the present invention can be any substance as long as it acts on RGMa itself to inhibit or weaken the activity of RGMa (hereinafter, sometimes referred to as "RGMa activity" in this specification). For example, a substance that has the activity of binding to RGMa and directly inhibiting (weakening) the activity of RGMa, or inhibiting the binding of RGMa to a receptor and indirectly inhibiting (weakening) the activity of RGMa (such as the compounds or antibodies described later) is called the RGMa inhibitory substance of the present invention.

[0098] In addition, the RGMa inhibitory substance of the present invention may be a substance that inhibits the expression of RGMa. For example, a substance that inhibits the expression of RGMa and thus inhibits (weakens) the activity of RGMa (such as the nucleic acid molecules described below) is also included in the RGMa inhibitory substance of the present invention.

[0099] RGMa has been identified as a neurite outgrowth inhibitory protein in the central nervous system. As shown in SEQ ID NO. 1, human RGMa protein is biosynthesized in the form of a precursor protein consisting of 450 amino acids. The signal peptide Met1 to Pro47 (a peptide from the 1st methionine residue to the 47th proline residue from the N-terminal side, and described in the same manner hereinafter) existing at the N-terminal is removed, the peptide bond between Asp168 and Pro169 is cut, and the N-terminal domain is generated. The C-terminal peptide Ala425 to Cys450 of the C-terminal fragment is further removed from Pro169, and a GPI anchor is added to the C-terminal carboxyl group of Ala424, which becomes the C-terminal, to generate the C-terminal domain. Human RGMa protein is expressed on the cell membrane via the GPI anchor as a mature protein in which the N-terminal domain (Cys48 to Asp168) and the C-terminal domain (Pro169 to Ala424) are connected by a disulfide bond.

[0100] In the present invention, RGMa may be derived from any animal, but is preferably human RGMa. The precursor protein of human RGMa is composed of the amino acid sequence shown in sequence number 1 of the sequence table. Although the precursor protein of mouse RGMa is composed of the amino acid sequence shown in sequence number 2 of the sequence table, and the precursor protein of rat RGMa is composed of the amino acid sequence shown in sequence number 3 of the sequence table, since the C-terminal peptide is removed, the amino acid sequence becomes the same as the mature protein.

[0101] Examples of the RGMa gene include, but are not limited to, the human RGMa gene having the base sequence shown in SEQ ID NO: 4. The base sequences of RGM genes from various organisms can be easily obtained from known databases (GenBank, etc.).

[0102] As the RGMa inhibitory substances of the present invention, specifically, low molecular weight compounds, anti-RGMa neutralizing antibodies, antibodies with modified functions, coupled antibodies, or antigen-binding fragments thereof, etc., can be cited, and in addition, siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense oligonucleotides, etc., which are nucleic acid molecules of RGMa, can be cited. Among these RGMa inhibitory substances, anti-RGMa neutralizing antibodies, antibodies with modified functions, coupled antibodies, 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.

[0103] <Anti-RGMa neutralizing antibody>

[0104] In the present invention, the anti-RGMa neutralizing antibody can be a polyclonal antibody or a monoclonal antibody as long as it binds to RGMa and neutralizes the activity of RGMa. In the present invention, a monoclonal antibody is preferred. In addition, the anti-RGMa neutralizing antibody of the present invention can be a RGMa monospecific antibody or a multispecific antibody that recognizes multiple RGMa and other antigens, preferably a RGMa monospecific antibody.

[0105] In addition, as a specific epitope, for human RGMa, preferably one or more of sequence number 16 (amino acid numbers 47-69 of sequence number 1), sequence number 36 (amino acid numbers 298-311 of sequence number 1), sequence number 37 (amino acid numbers 322-335 of sequence number 1), sequence number 38 (amino acid numbers 349-359 of sequence number 1), and sequence number 39 (amino acid numbers 367-377 of sequence number 1), more preferably a combination of sequence numbers 36 and 37, and particularly preferably a combination of sequence numbers 36, 37 and 39.

[0106] The anti-RGMa neutralizing antibodies of the present invention include polyclonal antibodies and monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or its partial fragments (such as the above-mentioned epitope fragments) as antigens, chimeric antibodies and humanized antibodies produced using genetic recombination technology, and human antibodies produced using transgenic animals that produce human antibodies. When the antibodies of the present invention are administered to humans as drugs, humanized antibodies or human antibodies are preferred from the perspective of side effects.

[0107] Specific examples of the anti-RGMa neutralizing antibody of the present invention include the following antibodies (a) to (l), and the methods described in Patent Documents 2 to 4 can be used for the production of each of the antibodies.

[0108] Examples include antibodies selected from the following (a) to (l):

[0109] (a) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes antibodies with sequence numbers 36, 37 and 39 as epitopes), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in sequence number 5, the LCDR2 comprising the amino acid sequence set forth in sequence number 6, the LCDR3 comprising the amino acid sequence set forth in sequence number 7, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in sequence number 8, the HCDR2 comprising the amino acid sequence set forth in sequence number 9, the HCDR3 comprising the amino acid sequence set forth in sequence number 10,

[0110] (b) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes antibodies with sequence numbers 36, 37 and 38 as epitopes), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in sequence number 11, the LCDR2 comprising the amino acid sequence set forth in sequence number 12, the LCDR3 comprising the amino acid sequence set forth in sequence number 13, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in sequence number 14, the HCDR2 comprising the amino acid sequence set forth in sequence number 15, the HCDR3 comprising SFG in its amino acid sequence,

[0111] (c) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22,

[0112] (d) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, wherein LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24, wherein LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, and wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26, wherein HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, wherein HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28,

[0113] (e) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody with SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 31, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0114] (f) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody with sequence number 16 as the epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence recorded in sequence number 29, the LCDR2 comprising the amino acid sequence recorded in sequence number 30, and the LCDR3 comprising the amino acid sequence recorded in sequence number 35, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence recorded in sequence number 32, the HCDR2 comprising the amino acid sequence recorded in sequence number 33, and the HCDR3 comprising the amino acid sequence recorded in sequence number 34,

[0115] (g) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody with SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0116] (h) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody with SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0117] (i) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody with SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0118] (j) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further comprises an antibody with SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34,

[0119] (k) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further includes an antibody with SEQ ID NO: 16 as an epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34, and

[0120] (l) an anti-RGMa neutralizing antibody (the anti-RGMa neutralizing antibody further comprises an antibody with sequence number 16 as the epitope), comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, the LCDR1 comprising the amino acid sequence recorded in sequence number 29, the LCDR2 comprising the amino acid sequence recorded in sequence number 30, the LCDR3 comprising the amino acid sequence recorded in sequence number 45, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the HCDR1 comprising the amino acid sequence recorded in sequence number 32, the HCDR2 comprising the amino acid sequence recorded in sequence number 33, and the HCDR3 comprising the amino acid sequence recorded in sequence number 34.

[0121] Among these, the antibodies described in (a) are particularly preferred.

[0122] The method for producing the anti-RGMa neutralizing antibody of the present invention can use the existing generally used production method. The antigen can be used directly for immunization, or it can be made into a complex with a carrier protein and used. The preparation of the complex of the antigen and the carrier protein can use condensation agents such as glutaraldehyde, carbodiimide, and maleimide active ester. Carrier proteins can exemplify bovine serum albumin, thyroglobulin, hemocyanin, KLH, etc.

[0123] As mammals for immunization, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses or cattle can be mentioned, and the inoculation method can be subcutaneous administration, intramuscular administration or intraperitoneal administration. When administering, it can be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant and administered, and the administration is usually performed once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of the immunized animal are fused with myeloma cells and isolated in the form of hybridomas. As myeloma cells, myeloma cells from mammals, such as mice, rats, humans, etc., can be used.

[0124] <Polyclonal Antibodies>

[0125] The polyclonal antibody can be obtained, for example, by immunizing the above-mentioned mammal with the above-mentioned antigen and, if necessary, Freund's adjuvant, and obtaining the serum from the immunosensitized animal.

[0126] <Monoclonal Antibodies>

[0127] Specifically, monoclonal antibodies can be obtained in the following manner. That is, the above-mentioned antigen is used as an immunogen, and the immunogen is injected into the mammal as described above subcutaneously, intramuscularly, intravenously, intrapedicularly or intraperitoneally together with Freund's Adjuvant as needed, one to multiple times or transplanted, thereby applying immune sensitization. Usually, 1 to 4 immunizations are performed every about 1 to 14 days from the initial immunization, and antibody-producing cells are obtained from the immune-sensitized mammal about 1 to 5 days after the final immunization.

[0128] Monoclonal antibodies can be obtained using methods known to those skilled in the art (e.g., "Current Protocols in Molecular Biology" (John Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0129] The preparation of "hybridomas" that secrete monoclonal antibodies can be carried out according to and Milstein et al. (Nature, 256, 495, 1975) and modified methods thereof. That is, the antibody-producing cells contained in the spleen or the like obtained from an immunosensitized mammal are prepared by cell fusion with myeloma cells from a mammal, preferably a mouse, rat or human, which have no ability to produce their own antibodies.

[0130] As myeloma cells used for cell fusion, for example, myeloma cells 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 from mice, myeloma cells 210RCY3-Ag.2.3. from rats, myeloma cells U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 from humans can be used.

[0131] Fusion promoters include polyethylene glycol and the like. Cell fusion can usually be achieved by using polyethylene glycol (average molecular weight 1000-4000) at a concentration of about 20-50% at a temperature of 20-40°C, preferably 30-37°C, with the ratio of the number of antibody-producing cells to the number of myeloma cells usually being about 1:1 to 10:1, and reacting for about 1 to 10 minutes.

[0132] Screening of hybridoma clones producing monoclonal antibodies can be performed by culturing the hybridomas in, for example, microtiter plates and measuring the reactivity of the culture supernatants of the wells to the immunizing antigen by immunochemical methods such as ELISA.

[0133] In screening of antibody-producing hybridoma cells, in addition to analysis of binding to RGMa protein, an evaluation is also performed as to whether the antibody inhibits the activity of RGMa of the present invention. Anti-RGMa neutralizing antibodies of the present invention can be selected by these screening methods.

[0134] From the wells containing hybridomas producing the target antibody, clones can be obtained by cloning by the limiting dilution method. Selection and breeding of hybridomas are usually performed in an animal cell culture medium containing 10 to 20% fetal bovine serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).

[0135] Monoclonal antibodies can be produced from hybridomas by culturing the hybridomas in vitro or growing them in vivo, for example, in the ascites of a mammal such as a mouse or rat, and isolating the hybridomas from the obtained culture supernatant or ascites of the mammal.

[0136] In the case of in vitro culture, hybridomas can be proliferated, maintained and preserved according to various conditions such as the characteristics of the cell species being cultured and the culture method, using a nutrient medium suitable for producing monoclonal antibodies in the culture supernatant. The nutrient medium may include a known nutrient medium or a nutrient medium prepared from a minimal medium.

[0137] Examples of basic culture media include low calcium culture media such as Ham'F12 culture media, MCDB153 culture media, or low calcium MEM culture media, and high calcium culture media such as MCDB104 culture media, MEM culture media, D-MEM culture media, RPMI1640 culture media, ASF104 culture media, or RD culture media. The basic culture media may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

[0138] The isolation and purification of the monoclonal antibody can be performed by subjecting the above-mentioned culture supernatant or ascites to saturated ammonium sulfate, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as anti-immunoglobulin column or protein A column, etc. Specifically, the purification of the monoclonal antibody can be easily achieved by using a known method as a purification method for immunoglobulins, for example, by ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, using an anion exchanger, and further using affinity chromatography of RGMa protein.

[0139] Monoclonal antibodies can also be obtained by phage display. In the phage display method, the target immunogen is used to screen the phage selected from any phage antibody library, and the phage with the desired binding to the immunogen is selected. Next, the antibody corresponding sequence contained in the phage is separated or sequenced, and an expression vector containing a nucleic acid molecule encoding an antibody or antigen binding domain is constructed based on the separated sequence or the determined sequence information. Then, monoclonal antibodies can be produced by culturing the cell line transfected with the expression vector. By using a human antibody library as a phage antibody library, human antibodies with desired binding can be generated.

[0140] <Nucleic acid molecules>

[0141] 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, using a commercially available RNA extraction kit, total RNA is prepared from cells of a hybridoma, etc., and cDNA is synthesized by reverse transcriptase using random primers, etc. Next, in the variable regions of known human antibody heavy chain genes and light chain genes, the cDNA encoding the antibody is amplified by using oligonucleotides of the sequences preserved separately as primers by the PCR method. For sequences encoding constant regions, it can be obtained by amplifying known sequences using the PCR method. The base sequence of the DNA can be determined by incorporating it into a plasmid for sequence determination and by conventional methods.

[0142] Alternatively, a DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the variable region or a partial sequence thereof and combining it with a sequence containing the constant region.

[0143] The nucleic acid molecule may be a nucleic acid molecule encoding the constant region and variable region of all heavy and light chains, or may be a nucleic acid molecule encoding only the variable region of heavy and light chains. The base sequence of the constant region of the heavy and light chains when encoding all the constant regions and variable regions is preferably the base sequence described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[0144] <Function-modified antibodies>

[0145] Functionally modified antibodies of anti-RGMa neutralizing antibodies can be prepared by the following method. For example, if CHO cells with a disrupted α1,6-fucosyltransferase (FUT8) gene are used as host cells to produce the anti-RGMa neutralizing antibodies of the present application, antibodies with reduced fucose content in sugar chains and improved cell-killing function can be obtained. If CHO cells with FUT8 gene introduced are used as host cells for production, antibodies with low cell-killing function can be obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). In addition, by changing the amino acid residues in the Fc region, complement activation function can be regulated (U.S. Patent No. 6737056, U.S. Patent No. 7297775, U.S. Patent No. 7317091). Furthermore, by using a variant of the Fc region that improves the binding to FcRn, which is one of the Fc receptors, it is possible to extend the half-life in the blood (Hashiguchi Zhouping et al., Biochemistry, 2010, Vol.82(8), p710). These functionally modified antibodies can be produced by genetic engineering. By using a variant of the Fc region that improves the binding to FcRn, which is one of the Fc receptors, it is possible to extend the half-life in the blood (Hashiguchi Zhouping et al., Biochemistry, 2010, Vol.82(8), p710). These functionally modified antibodies can be produced by genetic engineering.

[0146] <Conjugated Antibodies>

[0147] As the modified molecule of the anti-RGMa neutralizing antibody of the present invention, a conjugated antibody can be cited. As a conjugated antibody, a conjugated antibody formed by chemically or genetically engineering the anti-RGMa neutralizing antibody to which a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor (TGF-β, NGF, neurotrophin, etc.), albumin, an enzyme, other antibodies, etc., other functional molecules other than the anti-RGMa neutralizing antibody of the present application can be cited.

[0148] When PEG is used as a functional molecule, PEG having a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, can be used without limitation, and can be linear or branched. PEG can be bound to the N-terminal amino group of the amino acid of the anti-RGMa neutralizing antibody by using, for example, an NHS active group.

[0149] In the case of using radioactive substances as functional molecules, 131 I. 125 I. 90 Y. 64 Cu, 99 Tc, 77 Lu or211 At, etc. Radioactive substances can be directly bound to anti-RGMa neutralizing antibodies by the chloramine T method or the like.

[0150] In the case of using a toxin as the functional molecule, bacterial toxins (eg, diphtheria toxin), plant toxins (eg, ricin), low molecular weight toxins (eg, geldamycin), maytansine, calicheamicin, and the like can be used.

[0151] When a low molecular weight compound is used as the functional molecule, examples thereof include daunomycin, doxorubicin, methotrexate, mitomycin, neostatin, vindesine, and fluorescent dyes such as FITC.

[0152] When an enzyme is used as the functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. can be used.

[0153] Examples of linkers used when chemically binding toxins, low molecular weight compounds or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2) n O(CR2) n - (R is an arbitrary substituent, n is a positive integer) represented by a linker, an alkoxy repeating unit (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and an alkylamino group (e.g., polyethyleneamino, Jeffamine (trademark)), and a diacid and an amide (e.g., succinate, succinamide, diglycolate, malonate, and hexanamide, etc.). Chemical modification methods for binding functional molecules have been established in this field (DJ 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 Vol 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681).

[0154] <Antigen-binding fragment>

[0155] In an embodiment of the present invention, the "antigen-binding fragment" of an antibody refers to a portion of the antibody as described above that has antigen binding properties, specifically, F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide bond Fv, single-chain antibody (scFv) and their polymers, etc. Furthermore, the antigen-binding fragment includes a coupled fragment of functional molecules other than the anti-RGMa neutralizing antibody of the present application, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, neurotrophins, etc.), albumin, enzymes, other antibodies, etc., which are chemically or genetically engineered to bind.

[0156] "F(ab')2" and "Fab'" refer to antibody fragments produced by treating immunoglobulin with pepsin or papain, which are proteolytic enzymes, and digested before and after the disulfide bond between the two heavy chains in the hinge region. For example, if IgG is treated with papain, the disulfide bond between the two heavy chains in the hinge region is cut upstream, and the light chain composed of VL (light chain variable region) and CL (light chain constant region) and the heavy chain fragment composed of VH (heavy chain variable region) and CHγ1 (γ1 region in the heavy chain constant region) can be produced. The same two antibody fragments are respectively referred to as Fab. In addition, if IgG is treated with pepsin, the disulfide bond between the two heavy chains in the hinge region is cut downstream, and an antibody fragment slightly larger than the antibody fragment in which the above two Fabs are connected in the hinge region can be produced. This antibody fragment is referred to as F(ab')2.

[0157] <Chimeric antibodies>

[0158] As a preferred embodiment of the anti-RGMa neutralizing antibody of the present invention, a chimeric antibody can be cited. As a "chimeric antibody", a chimeric antibody in which the variable region is the variable region of an immunoglobulin from a non-human animal (mouse, rat, hamster, chicken, etc.) and the constant region is the constant region from a human immunoglobulin can be exemplified. For example, mice can be immunized with an antigen, and the variable region that binds to the antigen can be cut out from the gene of the mouse monoclonal antibody, and combined with the antibody constant region from human bone marrow to produce it. The constant region from human immunoglobulin has its own inherent amino acid sequence according to the isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD and IgE. The constant region of the recombinant chimeric antibody in the present invention can be a constant region of a human immunoglobulin belonging to any isotype. It is preferably a constant region of human IgG. The gene of the chimeric antibody produced in this way can be used to prepare an expression vector. The host cells are transformed with the expression vector to obtain chimeric antibody-producing transformed cells, and the transformed cells are cultured to obtain the target chimeric antibody from the culture supernatant.

[0159] <Humanized Antibodies>

[0160] As other preferred forms of the anti-RGMa neutralizing antibodies of the present invention, humanized antibodies can be cited. The "humanized antibody" in the present invention is an antibody formed by transplanting (CDR grafting) the DNA sequence of the antigen binding site (CDR, complementary determining region) of non-human animal antibodies such as mice to human antibody genes. For example, it can be prepared by referring to the methods described in Japanese Patent Publication No. 4-506458 and Japanese Patent No. 2912618. Specifically, it refers to a humanized antibody, characterized in that part or all of its CDR is the CDR of a monoclonal antibody from a non-human mammal (mouse, rat, hamster, etc.), the framework region of its variable region is the framework region of the variable region from a human immunoglobulin, and its constant region is the constant region from a human immunoglobulin.

[0161] The humanized antibody of the present invention can be produced, for example, as follows. However, the production method is not limited to this method.

[0162] For example, recombinant humanized antibodies derived from mouse monoclonal antibodies can be prepared by genetic engineering with reference to Japanese Patent Application Publication No. 4-506458 and Japanese Patent Application Laid-Open No. 62-296890, etc. That is, DNA of the mouse heavy chain CDR portion and DNA of the mouse light chain CDR portion are isolated from hybridomas producing mouse monoclonal antibodies, and human heavy chain genes of all regions except human heavy chain CDRs and human light chain genes of all regions except human light chain CDRs are isolated from human immunoglobulin genes.

[0163] The human heavy chain gene of the DNA of the isolated mouse heavy chain CDR portion is imported into an appropriate expression vector in a manner that can be expressed, and similarly, the human light chain gene of the DNA of the mouse light chain CDR portion is imported into another appropriate expression vector in a manner that can be expressed. Alternatively, the heavy chain and light chain genes of the human CDR of the mouse can be imported into the same expression vector in a manner that can be expressed. The host cell is transformed using the expression vector made in this way, thereby obtaining a humanized antibody to produce a transformed cell, and the target humanized antibody is obtained from the culture supernatant by culturing the transformed cell.

[0164] <Human Antibodies>

[0165] As other preferred forms of the anti-RGMa neutralizing antibodies of the present invention, human antibodies can be cited. Human antibodies are antibodies in which all regions of 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 from genes encoding human immunoglobulins, and human antibody genes can be introduced into mice to produce them. Specifically, for example, at least human immunoglobulin genes can be incorporated into the loci of mammals other than humans such as mice to produce transgenic animals, and the transgenic animals can be immunosensitized using antigens, thereby producing them in the same manner as the above-mentioned polyclonal antibody or monoclonal antibody production method.

[0166] For example, transgenic mice producing human antibodies can be prepared according to the methods described in Nature Genetics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; Japanese Patent Publication No. 4-504365; Japanese Patent Publication No. 7-509137; International Publication No. WO94 / 25585; Nature, Vol.368, p.856-859, 1994; and Japanese Patent Publication No. 6-500233. More specifically, HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice, etc. can be cited.

[0167] Specifically, the anti-RGMa neutralizing antibodies of the present invention include those having a CDR containing a specific amino acid sequence in the heavy chain variable region and a CDR containing a specific amino acid sequence in the light chain variable region (preferably the anti-RGMa neutralizing antibodies of (a) to (l) above).

[0168] It should be noted that as long as the antibody of the present invention has the ability to bind to RGMa and can maintain the activity of inhibiting (neutralizing) RGMa, one or more amino acids (1 to 20, 1 to 10 or 1 to 5, preferably 1 to 2) may be substituted, deleted, added or inserted in the amino acid sequence of the anti-RGMa neutralizing antibody (preferably the anti-RGMa neutralizing antibody of (a) to (l) above). Such substitutions, deletions, and additions can be introduced into CDRs, but are preferably introduced into regions outside CDRs. In addition, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution.

[0169] 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, the following amino acid sequence: the heavy chain variable region after the amino acid sequence is changed has a % homology of more than 90% (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before the change, and the light chain variable region after the amino acid sequence is changed has a % homology of more than 90% (more preferably 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequence before the change.

[0170] In the present invention, siRNA refers to a short double-stranded RNA that can inhibit the expression of a target gene (RGMa gene in the present invention). As long as it functions as an siRNA that inhibits the activity of RGMa of the present invention, the base sequence and length (base length) are not particularly limited, preferably less than about 30 bases, more preferably about 19 to 27 bases, and even more preferably about 21 to 25 bases.

[0171] In the present invention, shRNA refers to a molecule of about 20 base pairs or more, which forms a double-stranded structure in the molecule by partially including a scissor-shaped base sequence in a single-stranded RNA, and is composed of a short hairpin structure (hairbin) with a protrusion at the 3' end. After such shRNA is introduced into cells, it is decomposed into a length of about 20 bases (representatively, for example, 21 bases, 22 bases, and 23 bases) in the cell, and can inhibit the expression of the target gene in the same way as siRNA.

[0172] In the present invention, the siRNA and shRNA may be in any form as long as they can inhibit the expression of the RGMa gene.

[0173] In the present invention, siRNA or shRNA can be artificially chemically synthesized. In addition, for example, T7 RNA polymerase and T7 promoter can be used to synthesize antisense and sense RNA in vitro from template DNA. Antisense oligonucleotides can be either DNA or RNA as long as they are nucleotides that are complementary or hybridized to a continuous sequence of 5 to 100 bases in the DNA sequence of the RGMa gene. In addition, modifications can be made as long as they do not affect the function. Antisense oligonucleotides can be synthesized according to conventional methods, for example, they can be easily synthesized using a commercially available DNA synthesis device.

[0174] Preferred sequences can be selected using a common selection method, and can be confirmed as siRNA or shRNA in the present invention by evaluating the inhibition of expression of functional RGMa.

[0175] <Diabetic dementia>

[0176] Diabetic dementia in the present invention refers to dementia caused by abnormal glucose metabolism, and typically, dementia associated with diabetes can be cited. As its pathogenesis, it is speculated that in addition to vascular factors such as cerebral infarction and arteriosclerosis, it is also related to the promotion of aging changes caused by glucose toxicity, oxidative stress, AGE (advanced glycation end-product), etc., as well as hyperinsulinemia, insulin resistance, and insulin signal transduction disorders (Lancet Neurol, 5: 64-74, 2006). Diabetic dementia clinically shows or implies that it is closely related to abnormal glucose metabolism. In contrast, no pathological changes or vascular lesions of Alzheimer's disease have been confirmed, or it is a milder dementia. In addition, such diabetic dementia also includes dementia in which neurological disorders caused by glucose metabolism disorders (including hyperglycemia, etc.) are involved. Diabetic dementia in the present invention also includes mild cognitive dysfunction that does not reach dementia, that is, diabetic cognitive dysfunction.

[0177] <Vascular dementia>

[0178] The vascular dementia in the present invention refers to dementia caused by cerebrovascular disease, and there is a causal relationship between cerebrovascular disease and dementia. As the cause of vascular dementia, in addition to cerebral infarction, cerebral hemorrhage, and subarachnoid hemorrhage, it also includes cerebral circulatory insufficiency, hypoperfusion, white matter lesions and other disease types. The vascular dementia in the present invention also includes mild cognitive dysfunction that does not reach dementia, namely vascular cognitive dysfunction. In addition, the vascular dementia of the present invention can also be determined based on the diagnostic criteria related to vascular dementia (non-patent document 8).

[0179] The treatment subjects (preferably mammals, particularly humans) of the present invention are patients suffering from diabetic dementia or vascular dementia, and the preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia of the present invention can be administered to these patients.

[0180] Here, "treatment" includes any treatment of a disease of a treatment subject, preferably a mammal, particularly a human, including preventing the progression of the disease and symptoms, and eliminating, curing, alleviating or alleviating such disease and symptoms.

[0181] In addition, "prevention" includes preventing or inhibiting the onset of the above-mentioned diseases in the treatment subject, preferably mammals, especially humans. Furthermore, "prevention" in the present invention includes "preventing recurrence" which is to alleviate or prevent the recurrence of the above-mentioned diseases that have repeatedly recurred in the treatment subject, preferably mammals, especially humans.

[0182] <Drug Composition>

[0183] The preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia in the present invention is usually administered systemically or topically in an oral or parenteral form.

[0184] The preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia in the present invention has an RGMa inhibitory substance as an active ingredient, and can be formulated by appropriately combining a pharmaceutically acceptable carrier or additive. The pharmaceutical composition formulated in this way can be administered orally or non-orally. 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 non-oral preparations such as injections, infusions, suppositories, ointments, patches, etc. The proportion of the carrier or additive can be appropriately set based on the range commonly used in the field of pharmaceuticals. There is no particular limitation on the carrier or additive that can be formulated, and examples thereof include various carriers such as water, saline, other aqueous solvents, aqueous or oily bases, and various additives such as excipients, adhesives, pH adjusters, disintegrants, absorption promoters, lubricants, colorants, flavoring agents, and spices.

[0185] When the RGMa inhibitory substance is an anti-RGMa neutralizing antibody, its function-modified antibody, its conjugated antibody or their antigen-binding fragments, it is preferably administered as an injection or infusion formulated together with a pharmaceutically acceptable carrier by a non-oral administration route, such as intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous or local administration.

[0186] For example, an injection or infusion containing an anti-RGMa neutralizing antibody can be used as a solution, suspension or emulsion. As its solvent, for example, distilled water for injection, physiological saline, glucose solution and isotonic solution (such as sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used.

[0187] Furthermore, the injection or infusion containing such an anti-RGMa neutralizing antibody may also contain a stabilizer, a solubilizing agent, a suspending agent, an emulsifier, a soothing agent, a buffer, a preservative, a preservative, a pH adjuster, and the like.

[0188] As the stabilizer, for example, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium hydrogen sulfite, sodium thiosulfate, sodium EDTA, sodium citrate, butylated hydroxytoluene and the like can be used.

[0189] As the auxiliary solvent, for example, alcohol (eg, ethanol), polyol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, Polysolvate 80 (registered trademark), HCO-50, etc.) and the like can be used.

[0190] As the suspending agent, for example, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate and the like can be used.

[0191] As the emulsifier, for example, gum arabic, sodium alginate, gum tragacanth, etc. can be used.

[0192] As the soothing agent, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used.

[0193] As the buffer, for example, a phosphate buffer, an acetate buffer, a borate buffer, a carbonate buffer, a citrate buffer, a Tris buffer or the like can be used.

[0194] As the preservative, for example, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax and the like can be used.

[0195] As the preservative, for example, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol, etc. can be used.

[0196] As the pH adjuster, for example, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. can be used.

[0197] When the RGMa inhibitory substance is a nucleic acid (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, a method of introducing nucleic acid molecules using liposomes (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), a microinjection method, a method of transferring nucleic acid molecules and carriers (metal particles) into cells using a gene gun, etc. can be used. For example, when siRNA or shRNA is administered to an organism using a viral vector, a viral vector such as a recombinant adenovirus or retrovirus can be used. By introducing DNA expressing siRNA or shRNA into a detoxified retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, SV40, or other DNA virus or RNA virus, and infecting cells or tissues with the recombinant virus, the gene can be introduced into cells or tissues.

[0198] The preparation thus obtained can be used to prevent or treat dementia selected from diabetic dementia and vascular dementia by administering an effective amount thereof to humans, other mammals (such as rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.). The dosage can be appropriately set in consideration of the purpose, the severity of the disease, the patient's age, weight, sex, medical history, the type of active ingredient, etc. For example, when the active ingredient is an anti-RGMa neutralizing antibody, when humans with an average body weight of about 65 to 70 kg are taken as the subject, it is preferably about 0.02 mg to 4000 mg per day, and more preferably about 0.1 mg to 200 mg. The total dosage per day can be a single dosage or a divided dosage.

[0199] <Combination use with other drugs or treatments>

[0200] 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 drug. Examples of therapeutic agents for the antidiabetic drug used in combination include insulin resistance-improving drugs.

[0201] 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 antithrombotic therapy or a hypotensive agent. Examples of the hypotensive agent used in combination include angiotensin II receptor antagonists (candesartan, etc.), ACE inhibitors (perindopril, etc.), and the like.

[0202] The above-mentioned 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.

[0203] Example

[0204] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0205] In addition, as an anti-RGMa neutralizing antibody, the anti-RGMa neutralizing antibody containing the amino acid sequence (a) described in this specification (SEQ ID NOs: 5 to 10) was used in each Example.

[0206] [Example 1]

[0207] Using drug-induced type 1 diabetes model mice, the therapeutic effect of anti-RGMa neutralizing antibody on cognitive dysfunction was studied by a novel object recognition test.

[0208] <Guidelines for Diabetes>

[0209] C57BL / 6J female mice aged 7 to 8 weeks were used for the experiment. For the diabetes-induced group, streptozotocin (STZ: Sigma-Aldrich Co. LLC, St. Louis, MO, USA) 20 mg / ml was administered intraperitoneally at a dose of 10 ml / kg, and for the non-diabetes-induced group, the solvent was administered at a dose of 10 ml / kg. With reference to existing studies (references 1, 2), blood glucose levels were measured 1 week after diabetes induction, and individuals with blood glucose levels below 300 mg / dl were excluded.

[0210] <Grouping and Antibody Administration Schedule>

[0211] The purchased mice were divided into two groups, the "diabetes-anti-RGMa neutralizing antibody administration group" or the "diabetes-isotype control antibody (Palivizumab) administration group", and both groups were given antibodies starting 3 days after diabetes induction. After adjusting the concentration of either antibody to 6 mg / ml, it was administered through the tail vein once a week at a dose of 30 mg / kg for a total of 4 times. In the object recognition test, 14 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". 10 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 of non-diabetic mice (healthy mice).

[0212] <Novel object recognition memory test>

[0213] After 3 days of familiarization (management (handling)), the next day, each individual was placed in an open field and allowed to freely explore for 15 minutes to acclimate to the field (familiarization test 1). The acquisition test was carried out the next day. Before the acquisition test, each individual was allowed to freely explore the field for 5 minutes (familiarization test 2). After the implementation of familiarization test 2, each individual was temporarily returned to the home cage, and objects (object A) with the same shape made of blocks were placed at the upper left and lower right positions on the diagonal of the field, and then each individual was allowed to return to the field for free exploration (acquisition test). After the acquisition test, a test test was carried out at an interval of 12 hours (determined by a preliminary condition study). During the test test, a bottle (object B) filled with opaque beads was configured as a novel object instead of object A configured on the lower right diagonal of the field. On the other hand, object A on the upper left diagonal of the field was configured in the same way as in the acquisition test. In the test test, each individual was returned to the field where the above-mentioned objects A and B were configured and freely explored for 10 minutes. It should be noted that objects A and B were made based on existing research (reference 3). All experiments were recorded with a camera, and the exploration time of the two objects for each individual in each experiment was calculated after the action test was completed. Exploration behavior includes the action of smelling each object and the action of touching the object, excluding the action of climbing the object. The analysis was calculated with reference to existing research, using the discrimination index (DI) in the test experiment (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 experiment was less than 30 seconds were not used in the analysis. It should be noted that the DI of healthy mice used as a benchmark was calculated by implementing this action test in 16-week-old non-diabetic mice.

[0214] <Results>

[0215] The DI values ​​of the anti-RGMa neutralizing antibody-administered group and the isotype control antibody-administered group at 4 weeks after the onset of diabetic symptoms, as well as the DI values ​​of healthy mice are shown in FIG. Figure 1. A significant decrease in DI was observed in the "diabetes-isotype control antibody administration group" compared with healthy mice (p<0.05, Tukey's multiple comparisons test). On the other hand, a significant increase in DI value was observed in the "diabetes-anti-RGMa neutralizing antibody administration group" compared with the "diabetes-isotype control antibody administration group" (p<0.05, Tukey's multiple comparisons test), and no difference was observed when compared with healthy mice (p=0.8640, Tukey's multiple comparisons test). According to the results, the administration of anti-RGMa neutralizing antibodies can improve object memory impairment caused by diabetes.

[0216] The above results indicate that RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, are effective against diabetic dementia.

[0217] [Example 2]

[0218] As the cause of hippocampal-dependent cognitive dysfunction in animal models of diabetes, impairment of neurogenesis in the hippocampus has been suggested (Reference 6). Using drug-induced type 1 diabetes model mice, the therapeutic effect of anti-RGMa neutralizing antibody on the inhibition of diabetes-induced hippocampal dentate gyrus neurogenesis was studied by immunohistochemical staining.

[0219] <Induction of diabetes>

[0220] The experiment used 7-8 week old C57BL / 6J female mice. For the diabetes-induced group, streptozotocin (STZ: Sigma-Aldrich Co. LLC, St. Louis, MO, USA) 20 mg / ml was intraperitoneally administered once at a dose of 10 ml / kg, and for the non-diabetes-induced group, the solvent was administered at a dose of 10 ml / kg. Based on the existing 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.

[0221] <Antibody administration>

[0222] The purchased mice were divided into four groups: "non-diabetes-anti-RGMa neutralizing antibody administration group", "non-diabetes-isotype control antibody (Palivizumab) administration group", "diabetes-anti-RGMa neutralizing antibody administration group" and "diabetes-isotype control antibody (Palivizumab) administration group". The diabetes group was administered with STZ solution, and the non-diabetes induced group was administered with solvent. Anti-RGMa neutralizing antibody or isotype control antibody was administered from 3 days later. After adjusting the concentration of either antibody to 6 mg / ml, the tail vein was administered at a dose of 30 mg / kg once a week for a total of 6 times.

[0223] <Tissue sampling and immunohistochemical staining>

[0224] The brain of the mouse was sampled 6 weeks after the induction of diabetes. After being fully anesthetized, the brain was removed after perfusion fixation with 4% paraformaldehyde (PFA) and post-fixed in 4% PFA. Next, the tissue was transferred to a 30% sucrose solution with PBS as the solvent, left to stand at 4°C for 2 to 3 days, and then embedded in OCT compound (Sakura Finetek USA Inc., Torrance, CA, USA). The brain was sliced ​​into 30 μm thick slices using a cryostat, and the slices were attached to MAS-coated slides for immunostaining. The slices were blocked at room temperature for 1 hour using PBS (blocking solution) containing 3% normal donkey serum (NDS) and 0.3% Triton X-100, and after washing with PBS, the anti-mouse doublecortin antibody (1:100; abcam, Cambridge, UK, diluted with blocking solution) was used as the primary antibody to react overnight at 4°C. After washing with PBST, Alexa Fluor 568 donkey anti-rabbit IgG (H+L) antibody (1:500; Invitrogen, Waltham, MA, USA, diluted with blocking solution) was used as a secondary antibody to react at room temperature for 1 hour. After washing with PBST, nuclear staining was performed with DAPI (1μg / ml) and then sealed. The entire hippocampal dentate gyrus was photographed using a confocal laser microscope FV3000 (Olympus, Tokyo, Japan), and the number of cell bodies of doublecortin-positive neural precursor cells was measured. The dentate gyrus of the hippocampus was measured for 6 sections of each individual. The number of doublecortin-positive cells was normalized to the area of ​​the hippocampal dentate gyrus measured using ImageJ.

[0225] <Results>

[0226] The number of doublecortin-positive cells normalized to the area of ​​the dentate gyrus of the hippocampus is shown in Figure 2 . The number of doublecortin-positive cells did not change between the non-diabetes-induced groups, regardless of the type of antibody administered (p>0.9999, Tukey's multiple comparisons test). In addition, the number of doublecortin-positive cells in the "diabetes-isotype control antibody administration group" was significantly reduced compared with the "non-diabetes-anti-RGMa neutralizing antibody administration group" and the "non-diabetes-isotype control antibody administration group" (vs "non-diabetes-isotype 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, the number of doublecortin-positive cells in the "diabetes-anti-RGMa neutralizing antibody administration group" was significantly improved compared with the "diabetes-isotype control antibody administration group" (p<0.05, Tukey's multiple comparisons test). There was no significant difference in the number of doublecortin-positive cells between the "diabetes-anti-RGMa neutralizing antibody administration group" and the "non-diabetes-isotype control antibody administration group" and the "non-diabetes-anti-RGMa neutralizing antibody administration group" (vs non-diabetes-isotype 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). These results show that the reduction of neurogenesis caused by doublecortin-positive neural progenitor cells in the dentate gyrus of the hippocampus caused by diabetes can be improved by administering anti-RGMa neutralizing antibodies.

[0227] The above results indicate that RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, are effective against impairment of neurogenesis in the hippocampus, which is one of the causes of diabetic dementia.

[0228] [Example 3] Effects of therapeutic intervention using anti-RGMa neutralizing antibodies on novel object recognition memory

[0229] The novel object recognition test was used to study the therapeutic effect of anti-RGMa neutralizing antibody on cognitive dysfunction in the chronic cerebral hypoperfusion model mice induced by bilateral common carotid artery microcoil indwelling (BCAS model).

[0230] <Induction of chronic cerebral hypoperfusion model>

[0231] The experiment used C57BL / 6J male mice aged 9 to 10 weeks. Under isoflurane inhalation anesthesia (4% introduction, 1.5% maintenance), the mice were fixed in a prone position, and cerebral blood flow was measured using a laser speckle flowmeter. During the measurement, the rectal temperature was managed at 35.0 to 36.5°C using a heating pad. Then, the mice were fixed in a supine position, the neck was incised, the carotid sheath was opened, and microcoils were placed in the bilateral internal carotid arteries (reference 5). Cerebral blood flow was measured one day after BCAS induction, and individuals whose cerebral blood flow did not decrease to less than 90% compared with before surgery were excluded. OZ-3 (Omegawave, Tokyo, Japan) was used to measure cerebral blood flow.

[0232] <Novel object recognition memory test>

[0233] Each individual was placed in an open field and allowed to freely explore for 15 minutes to acclimate to the field (familiarization test 1). The next day, each individual was allowed to freely explore the field for 5 minutes before the acquisition test (familiarization test 2). After the implementation of familiarization test 2, each individual was temporarily returned to the home cage, and an object (object A) with the same shape made of a block was set at a distance of 10 cm from the wall using double-sided tape at the upper left and lower right positions on the diagonal of the field, and then each individual was returned to the field for free exploration (acquisition test). After the acquisition test, a test test was implemented after an interval of 12 hours. In the test test, a bottle (object B) filled with opaque beads as a novel object was replaced by an object A configured at the lower right of the diagonal of the field using double-sided tape at a position 10 cm from the wall. On the other hand, the object A on the upper left of the diagonal of the field was configured in the same way as the acquisition test. After the interval, each individual was returned to the field configured with the above-mentioned objects A and B and freely explored for 10 minutes (test test). Objects A and B used in each test were made according to existing research (reference 3). During the interval, each individual was free to take water and food in the home cage. All experiments were recorded with a video camera, and the exploration time of each individual on the object in each experiment was calculated after the action test was completed. Exploration behavior includes the action of smelling the odor of each object and the action of touching the object, excluding the action of climbing the object. The discrimination index (DI) in the test experiment was used for 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 experiment was less than 30 seconds were not used in the analysis.

[0234] <Antibody administration>

[0235] The purchased mice were divided into three groups: BCAS-anti-RGMa neutralizing antibody administration group, BCAS-isotype control antibody (Palivizumab) administration group, and Sham-isotype control antibody (Palivizumab) administration group. All three groups began to receive antibodies 3 days after BCAS surgery or Sham surgery. After adjusting the concentration of each antibody to 2 mg / ml, the body weight-dependent intraperitoneal administration was performed twice a week at a dose of 10 mg / kg for a total of 7 times. The number of mice in each group is as follows.

[0236] BCAS-anti-RGMa neutralizing antibody group: 13

[0237] BCAS-isotype control antibody (Palivizumab) administration group: 9

[0238] Sham-isotype control antibody (Palivizumab) administration group: 9

[0239] <Results>

[0240] Compared with the sham-isotype control antibody (palivizumab) administration group, the BCAS-isotype control antibody group showed a significant decrease in DI (p<0.001, Tukey's multiple comparisons test). On the other hand, the BCAS-anti-RGMa neutralizing antibody administration group showed a significant increase in DI compared with the BCAS-isotype control antibody group (p<0.001, Tukey's multiple comparisons test). This shows that therapeutic intervention based on the administration of anti-RGMa neutralizing antibodies significantly improves object recognition memory impairment caused by chronic ischemia. ( Figure 3 )

[0241] The above results indicate that RGMa inhibitory substances, particularly anti-RGMa neutralizing antibodies, are effective against vascular dementia.

[0242] [References]

[0243] 1.Deeds MC,Anderson JM,Armstrong AS,et al.Single dosestreptozotocin-induced diabetes:Considerations for study design in islettransplantationmodels.Lab Anim.2011;45(3):131-140.doi:10.1258 / la.2010.010090

[0244] 2.O'brien PD, Sakowski SA, Feldman EL. Mouse models ofdiabeticneuropathy.ILAR J.2014;54(3):259-272.doi:10.1093 / ilar / ilt052

[0245] 3.Leger M, Quiedeville A, Bouet V, et al.Object recognition test inmice.Nat Protoc.2013;8(12):2531-2537.doi:10.1038 / nprot.2013.155

[0246] 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

[0247] 5.Hattori Y et al,Gradual Carotid Artery Stenosis in MiceCloselyReplicates Hypoperfusive Vascular Dementia in Humans.Journal of the American Heart Association 2016 2Feb.22;5(2):e002757.DOI:10.1161 / JAHA.115.002757

[0248] 6.Stranahan,A.,Arumugam,T.,Cutler,R.et al.Diabetes impairshippocampalfunction through glucocorticoid-mediated effects on new andmatureneurons.Nat Neurosci 11,309-317(2008).https: / / doi.org / 10.1038 / nn2055

[0249] <Explanation of Sequence Listing>

[0250] 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

[0251] 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 Column 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: HCD of anti-RGMa neutralizing antibody r70E SEQ ID NO: 16: Amino acid sequence of the epitope of human RGMa SEQ ID NO: 17: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 18: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 19: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 20: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody 5F9 SEQ ID NO: 21: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody 5F9 Column 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

[0252] SEQ ID NO: 28: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody 8D1

[0253] SEQ ID NO: 29: Amino acid sequence of LCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 30: Amino acid sequence of LCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 31: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 32: Amino acid sequence of HCDR1 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 33: Amino acid sequence of HCDR2 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 34: Amino acid sequence of HCDR3 of anti-RGMa neutralizing antibody AE12-1 SEQ ID NO: 35: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1Y SEQ ID NO: 36: Amino acid sequence of epitope of human RGMa

[0254] SEQ ID NO: 37: Amino acid sequence of human RGMa epitope

[0255] SEQ ID NO: 38: Amino acid sequence of human RGMa epitope

[0256] SEQ ID NO: 39: Amino acid sequence of the epitope of human RGMa

[0257] SEQ ID NO: 40: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1F SEQ ID NO: 41: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1H SEQ ID NO: 42: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1L SEQ ID NO: 43: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1V SEQ ID NO: 44: Amino acid sequence of LCDR3 of anti-RGMa neutralizing antibody AE12-1I

[0258] SEQ ID NO: 45: Amino Acid Sequence of LCDR3 of Anti-RGMa Neutralizing Antibody AE12-1K Industrial Applicability

[0259] The RGMa inhibitory substance of the present invention is useful for preventing or treating diabetic dementia or vascular dementia, and therefore has high utilization value in the pharmaceutical industry.

Claims

1. Use of an RGMa inhibitory substance for producing a preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia.

2. Use of an RGMa inhibitory substance in the manufacture of a preventive or therapeutic agent for diabetic dementia.

3. Use of an RGMa inhibitory substance in the manufacture of a preventive or therapeutic agent for vascular dementia.

4. The use according to any one of claims 1 to 3, wherein The RGMa inhibitory substance is an anti-RGMa neutralizing antibody or an antigen-binding fragment thereof.

5. The use according to claim 4, wherein The anti-RGMa neutralizing antibody is a humanized antibody.

6. The use according to claim 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.

7. The use according to claim 4, wherein The anti-RGMa neutralizing antibody is an antibody selected from the following (a) to (l): (a) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 5, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 7; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 8, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 9, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10, (b) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence recorded in SEQ ID NO: 11, the LCDR2 comprises the amino acid sequence recorded in SEQ ID NO: 12, and the LCDR3 comprises the amino acid sequence recorded in SEQ ID NO: 13; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence recorded in SEQ ID NO: 14, the HCDR2 comprises the amino acid sequence recorded in SEQ ID NO: 15, and the HCDR3 comprises SFG in its amino acid sequence, (c) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 19; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 20, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22, (d) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 24, the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 25, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 26, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 27, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28, (e) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 31; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, (f) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 35; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, (g) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 40; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, (h) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 41; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, (i) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 42; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, (j) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 43; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, (k) an anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 30, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 44, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 32, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 33, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 34, and (l) An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence recorded in sequence number 29, the LCDR2 comprises the amino acid sequence recorded in sequence number 30, and the LCDR3 comprises the amino acid sequence recorded in sequence number 45; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence recorded in sequence number 32, the HCDR2 comprises the amino acid sequence recorded in sequence number 33, and the HCDR3 comprises the amino acid sequence recorded in sequence number 34.

8. The use according to claim 4, wherein The anti-RGMa neutralizing antibody is the following antibody (a): (a) An anti-RGMa neutralizing antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence recorded in sequence number 5, the LCDR2 comprises the amino acid sequence recorded in sequence number 6, and the LCDR3 comprises the amino acid sequence recorded in sequence number 7; the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence recorded in sequence number 8, the HCDR2 comprises the amino acid sequence recorded in sequence number 9, and the HCDR3 comprises the amino acid sequence recorded in sequence number 10.

Images