Prophylactic or therapeutic agent for dementia

Abstract

To provide an effective agent for diabetic dementia and vascular dementia.SOLUTION: This invention provides an agent comprising a RGMa inhibiting substance for preventing or treating dementia selected from diabetic dementia and vascular dementia.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent for preventing or treating diabetic dementia and vascular dementia, which comprises an RGMa inhibitor. [Background technology]

[0002] As the population ages, the number of patients with diabetes and dementia increases year by year, and it has become clear that people with diabetes are at increased risk of developing Alzheimer's disease and vascular dementia, with a close pathological relationship between the two diseases (Non-Patent Documents 1 and 2). Furthermore, many reports have shown that diabetic patients without dementia exhibit reduced cognitive function compared to non-diabetic patients, and reported cognitive impairments in such diabetic patients include reduced attention and concentration, reduced visual or verbal memory, and reduced Mini-Mental State Examination (MMSE) scores (Non-Patent Document 3).

[0003] In recent years, a clinical type of diabetic dementia has been proposed as a classification of diabetes-associated dementia, in addition to Alzheimer's disease and vascular dementia, in which abnormal glucose metabolism is deeply involved in the onset of dementia (Non-Patent Documents 4 and 5). Diabetic dementia rarely exhibits the characteristic brain imaging findings of Alzheimer's disease (such as hippocampal atrophy), but is more often accompanied by vascular lesions such as microinfarction (Non-Patent Document 6). Clinically, patients are somewhat older, their diabetes is poorly controlled, and they are more likely to be diagnosed with impaired attention and concentration and impaired executive function rather than memory impairment, with a somewhat slower progression.

[0004] On the other hand, vascular dementia is dementia that is mainly caused by cerebrovascular disorders, particularly cerebral small vessel diseases such as Binswanger disease and multiple lacunar infarctions, and is defined as (1) dementia, (2) cerebrovascular disorders, and (3) a causal relationship between the two. The clinical diagnostic criteria of the NINDS-AIREN (National Institute of Neurological Disorders and Stroke-Association International pour la Recherche et l'Enseignement en Neurosciences) classifies the disease into five types: (1) multi-infarct, (2) single-lesion, (3) small vessel lesion, (4) hypoperfusion, and (5) cerebral hemorrhage. However, because each of these disease types is etiologically and clinically heterogeneous, vascular dementia is considered a heterogeneous disease concept that includes various pathologies (Non-patent Document 7, Non-patent Document 8). ). It is also widely known that diabetes is a risk factor for vascular dementia (Non-patent Document 8, Non-patent Document 9).

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

[0006] In addition to its function during development, RGMa is re-expressed after central nervous system injury in adult humans and rats, and inhibition of RGMa in rats enhances axonal growth and promotes functional recovery after spinal cord injury (Non-patent Document 14). Therefore, RGMa is considered to be an inhibitor of axon regeneration after central nervous system injury. Specific antibodies that neutralize RGMa are described in, for example, Patent Document 2 (e.g., 5F9, 8D1), Patent Document 3 (e.g., AE12-1, AE12-1Y), and Patent Document 4 (e.g., r116A3, r70E4, r116A3C, rH116A3). Furthermore, Patent Document 2 discloses the use of anti-RGMa antibodies in the treatment of dementia. Although the role of RGMa in central nervous system injury has been clarified, the involvement of RGMa in the treatment of diabetic and vascular dementia in particular has not been identified, and no such therapeutic agents are known. [Prior art documents] [Patent documents]

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

[0008] [Non-Patent Document 1] Neurology, 45: 1161, 1995 [Non-patent document 2] Am. J. Epidemiol. 1455: 301, 1997 [Non-patent document 3] Diabetes Care, 20: 438, 1997 [Non-patent document 4] Dement Geriatr Cogn Disord, 35: 280-290, 2013 [Non-Patent Document 5] J. Neurol. Sci. 349: 45-51, 2015 [Non-patent document 6] Nat Rev Neurol 5: 305-306, 2009 [Non-Patent Document 7] Neurology, 43: 250-260, 1993 [Non-patent document 8] Dementia Treatment Guidelines 2017 Chapter 14 Vascular Dementia [Non-Patent Document 9] Neuron 83: 844-866, 2013 [Non-Patent Document 10] Neuron 5, 735-743 (1990) [Non-Patent Document 11] Philos. Trans. R. Soc. Lond. B Biol. Sci., 361: 1513-29, 2006 [Non-Patent Document 12] Biochem. Biophys. Res. Commun. 382, ​​795-800 (2009) [Non-Patent Document 13] Curr. Opin. Neurobiol.17, 29-34 (2007) [Non-Patent Document 14] J. Cell Biol. 173, 47-58 (2006) Summary of the Invention [Problem to be solved by the invention]

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

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

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

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

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

[11] The use according to

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

[0013] According to the present invention, RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are useful as agents for preventing or treating dementia selected from, for example, diabetic dementia and vascular dementia. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows 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, as well as the DI values ​​of healthy mice, 4 weeks after the onset of diabetes mellitus (DM). [Figure 2]Figure 2 shows the number of doublecortin-positive cells in non-diabetic (non-DM) mice or diabetic (DM) mice treated with anti-RGMa neutralizing antibodies or isotype control antibodies. The number of doublecortin-positive cells was normalized by the area of ​​the hippocampal dentate gyrus. [Figure 3] Figure 3 shows the Discrimination Index (DI) values ​​obtained from the anti-RGMa neutralizing antibody-administered group and the isotype control antibody-administered group in a chronic cerebral hypoperfusion model, as well as the DI value obtained from the isotype control antibody-administered group in sham mice. DETAILED DESCRIPTION OF THE INVENTION

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

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

[0017] [Isolated] As used herein, the term "isolated" in reference to an isolated RGMa inhibitor (e.g., an antibody) means identified and separated and / or recovered from components in its natural state. Impurities in the natural state are substances that may interfere with the diagnostic or therapeutic use of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, an RGMa inhibitor can be isolated by purification through at least one purification step, and an RGMa inhibitor purified through at least one purification step can be referred to as an "isolated RGMa inhibitor."

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

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

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

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

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

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

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

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

[0026] [Percent (%) identity of amino acid sequence] "Percent (%) identity" of an amino acid sequence of a candidate polypeptide sequence, such as a variable region, with respect to the amino acid sequence of a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the particular reference polypeptide sequence after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent identity can be performed using a variety of methods within the skill of one in the art, e.g., BLAST, BLAST-2, ALIG, etc. This can be achieved by using publicly available computer software such as BLAST, BLAST, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, percent identity values ​​are obtained using the sequence comparison computer program BLAST in pairwise alignments. In situations where BLAST is used for amino acid sequence comparison, the percent identity of a given amino acid sequence A to a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical by a program alignment of A and B using the sequence alignment program BLAST, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the percent identity of A to B will differ from the percent identity of B to A. Unless otherwise specified, all percent identity values ​​herein are obtained using the BLAST computer program as set forth in the immediately preceding paragraph.

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

[0028] Hereinafter, embodiments of the present invention will be described in detail. The present invention provides a preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia, which is a novel use of an RGMa inhibitor. to provide a preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia. The present invention also provides a method for preventing or treating dementia selected from diabetic dementia and vascular dementia, which includes the step of administering to a mammal requiring treatment a preventive or therapeutic agent containing an effective amount of an RGMa inhibitor.

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

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

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

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

[0033] <Anti-RGMa neutralizing antibody> In the present invention, the anti-RGMa neutralizing antibody may be either a polyclonal antibody or a monoclonal antibody, as long as it binds to RGMa and neutralizes RGMa activity. In addition, the anti-RGMa neutralizing antibody of the present invention may be either a monoclonal antibody specific to RGMa or a multispecific antibody that recognizes multiple antigens, including RGMa, but is preferably a monoclonal antibody.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0048] Clones can be obtained from wells containing hybridomas that produce the desired antibody by limiting dilution. Hybridoma selection and breeding are usually carried out in an animal cell medium containing 10-20% fetal bovine serum and supplemented with HAT (hypoxanthine, aminopterin, thymidine).

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

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

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

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

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

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

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

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

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

[0058] When a radioactive substance is used as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211 Radioactive substances are used to detect anti-RGM antibodies using the chloramine T method. aCan be directly conjugated to a neutralizing antibody.

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

[0060] When a low molecular weight compound is used as a functional molecule, fluorescent dyes such as daunomycin, doxorubicin, metrolexate, mitomycin, neocarzinostatin, vindesine, and FITC are used. Examples include pigments.

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

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

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

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

[0065] <Chimeric antibody> A preferred embodiment of the anti-RGMa neutralizing antibody of the present invention is a chimeric antibody. Examples of "chimeric antibodies" include chimeric antibodies whose variable regions are derived from immunoglobulins of non-human animals (mouse, rat, hamster, chicken, etc.) and whose constant regions are derived from human immunoglobulins. For example, a mouse is immunized with an antigen, and the mouse monoclonal antibody is A chimeric antibody can be produced by excising the antigen-binding variable region from a clonal antibody gene and combining it with an antibody constant region derived from human bone marrow. Constant regions derived from human immunoglobulins have unique amino acid sequences depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE. The constant region of the recombinant chimeric antibody of the present invention may be the constant region of a human immunoglobulin belonging to any isotype. Preferably, it is a human IgG constant region. An expression vector can be constructed using the gene for the chimeric antibody produced in this way. Host cells are transformed with the expression vector to obtain chimeric antibody-producing transformed cells, and the desired chimeric antibody is obtained from the culture supernatant by culturing the transformed cells.

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

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

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

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

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

[0071] For example, transgenic mice that produce human antibodies are reported in Nature Genetics, Vol. p.13-21, 1994;Nature Genetics, Vol.15, p.146-156, 1997;Special Publication No. 4-504365; It is prepared according to the methods described in JP-A-7-509137; International Publication WO94 / 25585; Nature, Vol. 368, pp. 856-859, 1994; and JP-A-6-500233. More specifically, examples include HuMab (registered trademark) mice (Medarex, Princeton NJ), KM™ mice (Kirin Pharma Company, Japan), and KM (FCγRIIb-KO) mice.

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

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

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

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

[0076] <Diabetic dementia> Diabetic dementia in the present invention refers to dementia caused by abnormal glucose metabolism, typically including diabetes-related dementia. Its onset mechanism includes vascular factors such as cerebral infarction and arteriosclerosis, as well as glucose toxicity, oxidative stress, and accelerated age-related changes due to AGEs (advanced glycation end-products), hyperinsulinemia, insulin resistance, insulin resistance, and other factors. It is believed that impaired thrombin signaling is involved (Lancet Neurol, 5: 64-74, 2006). Clinically, diabetic dementia is closely related to abnormal glucose metabolism, or has been shown to be associated with this condition. In contrast to diabetic dementia, in which pathological changes or vascular lesions associated with Alzheimer's disease are not observed or the dementia is milder. Furthermore, such diabetic dementia also includes dementia involving neuropathy due to impaired glucose metabolism (including hyperglycemia). The diabetic dementia of the present invention also includes diabetic cognitive dysfunction, which is a mild cognitive impairment that does not progress to dementia.

[0077] <Vascular dementia> Vascular dementia in the present invention refers to dementia caused by cerebrovascular disease, and means that there is a causal relationship between cerebrovascular disease and dementia. Causes of vascular dementia include cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, as well as pathological types such as cerebral circulatory insufficiency, hypoperfusion, and white matter lesions. Vascular dementia in the present invention also includes vascular cognitive dysfunction, which is a mild cognitive dysfunction that does not lead to dementia. In addition, vascular dementia of the present invention can be identified according to the diagnostic criteria for vascular dementia (Non-Patent Document 8). The subjects of treatment in the present invention (preferably mammals, particularly humans) are patients who have developed diabetic dementia or vascular dementia, and the prophylactic or therapeutic agent for dementia selected from diabetic dementia and vascular dementia of the present invention can be administered to these patients.

[0078] As used herein, "treatment" includes any treatment of a disease in a subject, preferably a mammal, particularly a human, including preventing the progression of the disease and symptoms, and eliminating, curing, alleviating or alleviating such disease and symptoms.

[0079] Furthermore, "prevention" includes preventing or suppressing the onset of the above-mentioned diseases in a subject to be treated, preferably a mammal, particularly a human. Furthermore, "prevention" in the present invention also includes "recurrence prevention," which prevents the recurrence of the above-mentioned diseases that repeatedly undergo remission and relapse in a subject to be treated, preferably a mammal, particularly a human.

[0080] <Pharmaceutical Composition> The agent for preventing or treating dementia selected from diabetic dementia and vascular dementia of the present invention is usually administered systemically or locally, orally or parenterally. The preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia of the present invention can be formulated by appropriately blending an RGMa inhibitor as an active ingredient with a pharmaceutically acceptable carrier or additive. Such a formulated pharmaceutical composition can be administered orally or parenterally. Specifically, it can be administered in oral forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, or parenteral forms such as injections, infusions, suppositories, ointments, and patches. The proportion of the carrier or additive may be appropriately determined based on the range commonly used in the pharmaceutical field. The carrier or additive that can be blended is not particularly limited, and examples include various carriers such as water, saline, other aqueous solvents, and aqueous or oily bases, as well as various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances.

[0081] When the RGMa inhibitor is an anti-RGMa neutralizing antibody, a functionally modified antibody thereof, a conjugated antibody thereof, or an antigen-binding fragment thereof, it is preferably administered parenterally, for example, intravenously, intramuscularly, intradermally, intraperitoneally, subcutaneously, or topically, as an injection or infusion formulated together with a pharmaceutically acceptable carrier. For example, injections or infusions containing anti-RGMa neutralizing antibodies can be used as solutions, suspensions, or emulsions, and solvents such as distilled water for injection, physiological saline, glucose solutions, and isotonic solutions (e.g., solutions of sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, propylene glycol, etc.) can be used. Furthermore, injections or infusions containing such anti-RGMa neutralizing antibodies may contain stabilizers, solubilizing agents, suspending agents, emulsifying agents, soothing agents, buffers, preservatives, antiseptics, pH adjusters, etc. Examples of stabilizers that can be used include albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium EDTA, sodium citrate, and dibutylhydroxytoluene. Examples of solubilizing agents that can be used include alcohols (e.g., ethanol, etc.), polyalcohols (e.g., propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50, etc.). As the suspending agent, for example, glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. can be used. As the emulsifier, for example, gum arabic, sodium alginate, tragacanth, etc. can be used. As the soothing agent, for example, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. Examples of buffers that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, and Tris buffer. Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax. Examples of preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid.

[0082] When the RGMa inhibitor is a nucleic acid (e.g., siRNA, shRNA, or antisense oligonucleotide), it can be administered in the form of a non-viral or viral vector. In the case of a non-viral vector, methods for introducing nucleic acid molecules using liposomes (e.g., liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method), microinjection, or gene gun (Gene Gun) can be used. Methods such as (n) above can be used to transfer nucleic acid molecules into cells together with carriers (metal particles). For example, when administering siRNA or shRNA to a living body using a viral vector, viral vectors such as recombinant adenoviruses and retroviruses can be used. DNA that expresses siRNA or shRNA can be introduced into DNA or RNA viruses such as detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, poxviruses, polioviruses, Sindbis viruses, Sendai viruses, and SV40, and then cells or tissues can be infected with this recombinant virus to introduce genes into cells or tissues.

[0083] The preparation thus obtained can be administered in an effective amount to, for example, humans or other mammals (e.g., rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.) to prevent or treat dementia selected from diabetic dementia and vascular dementia. The dosage is appropriately determined taking into consideration the purpose, severity of the disease, the patient's age, weight, sex, medical history, and type of active ingredient. For example, when the active ingredient is an anti-RGMa neutralizing antibody, the daily dosage for an average human weighing approximately 65 to 70 kg is preferably approximately 0.02 mg to 4000 mg, and more preferably approximately 0.1 mg to 200 mg. The total daily dosage may be a single dose or divided doses.

[0084] <Combination with other drugs or treatments> In the present invention, the agent for preventing or treating dementia selected from diabetic dementia and vascular dementia can be administered in combination with an antidiabetic drug, such as an insulin sensitizer. In the present invention, the prophylactic or therapeutic agent for dementia selected from diabetic dementia and vascular dementia can be administered in combination with antithrombotic therapy or an antihypertensive drug, such as an angiotensin II receptor antagonist (e.g., candesartan) or an ACE inhibitor (e.g., perindopril).

[0085] The above-mentioned other drugs or treatments may be administered or performed before or after the administration of the prophylactic or therapeutic agent for dementia selected from diabetic dementia and vascular dementia of the present invention, or may be administered or performed simultaneously. [Example]

[0086] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. As the anti-RGMa neutralizing antibody, the amino acid sequence of (a) described in the present specification (SEQ ID NO: 5 to Anti-RGMa neutralizing antibodies including 10) were used in each example.

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

[0088] <Induction of diabetes> Seven- to eight-week-old C57BL / 6J female mice were used in the experiment. The diabetic group received a single intraperitoneal injection of 20 mg / ml of streptozocin (STZ; Sigma-Aldrich Co. LLC, St. Louis, MO, USA) at a dose of 10 ml / kg. The non-diabetic group received vehicle at a dose of 10 ml / kg. Based on previous studies (1, 2), blood glucose levels were measured one week after diabetic induction, and mice with blood glucose levels below 300 mg / dL were excluded.

[0089] <Grouping and antibody administration schedule> The purchased mice were divided into two groups: a diabetes-anti-RGMa neutralizing antibody group and a diabetes-isotype control antibody (Palivizumab) group. Both groups received the anti-RGMa neutralizing antibody three days after diabetes induction. Both antibodies were adjusted to a concentration of 6 mg / ml and then administered once a week at a dose of 30 mg / kg. The mice were administered a total of four times into the tail vein. Fourteen mice were used in the "diabetes-anti-RGMa neutralizing antibody administration group" and 15 mice in the "diabetes-isotype control antibody (Palivizumab) administration group" for the object recognition test. Ten mice from the "diabetes-anti-RGMa neutralizing antibody administration group" and eight mice from the "diabetes-isotype control antibody (Palivizumab) administration group" that met the study criteria were used for analysis. Seven mice were used to calculate the DI value in normal mice.

[0090] <Novel object recognition memory test> The animals were acclimatized (handled) for 3 days, and the next day, each animal was placed in an open field. The rats were allowed to freely explore the field for 5 minutes to acclimate to the field (acclimatization trial 1). The acquisition trial was conducted the next day. Prior to the acquisition trial, each rat was allowed to freely explore the field for 5 minutes ( After the habituation trial 2, each individual was temporarily returned to its home cage, and an object (object A) with the same shape made of blocks was placed on the diagonal left of the field. After placing them in two locations, at the top and bottom right, each individual was returned to the field and allowed to explore freely (acquisition trial). After the acquisition trial, a 12-hour interval (determined by prior condition review) was allowed, and then a test trial was conducted. During the test, a bottle containing opaque beads (object B) was placed as a novel object in place of object A, which had been placed in the diagonal bottom right of the field. Meanwhile, object A in the diagonal top left of the field was placed in the same position as in the acquisition trial. In the test trial, each individual The individual was returned to the field containing the above-mentioned objects A and B and allowed to explore freely for 10 minutes. Objects A and B were prepared based on previous research (Reference 3). All trials were recorded with a video camera. The video was recorded, and after the behavioral test was completed, the exploration time for each individual to both objects in each trial was calculated. Exploratory behavior included sniffing and touching each object, but excluded climbing on the objects. For analysis, the Discrimination Index (DI) for the test trial was calculated based on previous research (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 to objects A and B in the test trial was less than 30 seconds were not used in the analysis. The DI of normal mice was calculated by performing this behavioral test on 16-week-old non-diabetic mice.

[0091] <Result> Anti-RGMa neutralizing antibody administration group and isotype control group 4 weeks after the onset of diabetic pathology The DI values ​​obtained from each antibody-administered group and the DI values ​​of healthy mice are shown in Figure 1. A significant decrease in DI was observed in the "diabetes-isotype control antibody-administered group" compared to healthy mice. On the other hand, the diabetes-anti-RGMa neutralizing antibody group showed a significant DI increase compared with the diabetes-isotype control antibody group (p < 0.05, Tukey's multiple comparisons test). The values ​​were elevated (p < 0.05, Tukey's multiple comparisons test), which was significantly higher than those of healthy mice. No significant difference was observed in the comparison (p = 0.8640, Tukey's multiple comparisons test). These results indicate that administration of anti-RGMa neutralizing antibodies can improve object memory impairment caused by diabetes. These results demonstrate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are effective against diabetic dementia.

[0092] [Example 2] Impaired hippocampal neurogenesis has been suggested as a cause of hippocampal-dependent cognitive dysfunction in diabetic animal models (Reference 6). Using a drug-induced type 1 diabetes model mouse, we investigated the therapeutic effect of anti-RGMa neutralizing antibodies on diabetes-induced suppression of hippocampal dentate gyrus neurogenesis using immunohistochemical staining.

[0093] <Induction of diabetes> Seven- to eight-week-old C57BL / 6J female mice were used in the experiment. The diabetic group received a single intraperitoneal injection of 20 mg / ml of streptozocin (STZ; Sigma-Aldrich Co. LLC, St. Louis, MO, USA) at a dose of 10 ml / kg. The non-diabetic group received vehicle at a dose of 10 ml / kg. Based on previous studies (1, 2), blood glucose levels were measured one week after diabetic induction, and mice with blood glucose levels below 300 mg / dL were excluded.

[0094] <Antibody administration> The purchased mice were divided into four groups: a non-diabetic group administered anti-RGMa neutralizing antibody, a non-diabetic group administered isotype control antibody (Palivizumab), a diabetic group administered anti-RGMa neutralizing antibody, and a diabetic group administered isotype control antibody (Palivizumab). The diabetic group was administered STZ solution, and the non-diabetic group was administered solvent. Three days later, the mice were administered anti-RGMa neutralizing antibody or isotype control antibody (Palivizumab). The administration of the isotype control antibody was started. Both antibodies were adjusted to a concentration of 6 mg / ml and then administered for 30 minutes. The drug was administered into the tail vein at a dose of mg / kg once a week for a total of six doses.

[0095] <Tissue sampling / immunohistochemical staining> Mouse brains were collected 6 weeks after the induction of diabetes. After sufficient anesthesia, the brains were perfused with 4% paraformaldehyde (PFA) and then removed and post-fixed in 4% PFA. The tissues were then transferred to a 30% sucrose solution in PBS and left at 4°C for 2 to 3 days before undergoing optical coherence tomography (OCT). The tissue was embedded in cryoprotectant compound (Sakura Finetek USA Inc., Torrance, CA, USA). The brains were sliced ​​at a thickness of 30 μm using a CT microscope, and the sections were attached to MAS-coated slides for immunostaining. After blocking with PBS containing 3% Normal Donkey Serum (NDS) and 0.3% Triton X-100 (blocking solution) for 1 hour at room temperature and washing with PBS, the sections were incubated overnight at 4°C with anti-mouse doublecortin antibody (1:100; Abcam, Cambridge, UK, diluted in blocking solution). After washing with PBST, the sections were incubated with Alexa Fluor 568 donkey anti-rabbit IgG (H+L) antibody (1:500; Invitrogen, Waltham, MA) as the secondary antibody. The cells were incubated with 1000kJ / ml doublecortin-positive neural progenitor cells (Diluted in blocking solution, USA) at room temperature for 1 hour. After washing with PBST, nuclear staining was performed with DAPI (1 μg / ml) and then mounted. The entire dentate gyrus was imaged using a confocal laser microscope FV3000 (Olympus, Tokyo, Japan), and the number of doublecortin-positive neural progenitor cells was counted. Six sections of the dentate gyrus were counted from each individual. The number of doublecortin-positive cells was normalized by the area of ​​the dentate gyrus measured using ImageJ.

[0096] <Result> Figure 2 shows the number of doublecortin-positive cells normalized by the area of ​​the hippocampal dentate gyrus. There was no change in the number of doublecortin-positive cells between the non-diabetic groups, regardless of the type of antibody administered (p > 0.9999, Tukey's multiple comparisons test). Furthermore, the number of doublecortin-positive cells was significantly reduced in the "diabetic isotype control antibody-treated group" compared with the "non-diabetic anti-RGMa neutralizing antibody-treated group" and the "non-diabetic isotype control antibody-treated group" (vs. "non-diabetic isotype control antibody-treated group"; p < 0.001, Tukey's multiple comparisons test, vs. "non-diabetic anti-RGMa neutralizing antibody-treated group"; p < 0.001, Tukey's multiple comparisons test). On the other hand, the number of doublecortin-positive cells was significantly improved in the "diabetic anti-RGMa neutralizing antibody-treated group" compared with the "diabetic isotype control antibody-treated group" (p < 0.05, Tukey's multiple comparisons test). The number of doublecortin-positive cells in the diabetic anti-RGMa neutralizing antibody group was not significantly different from that in the non-diabetic isotype control antibody group or the non-diabetic anti-RGMa neutralizing antibody group (vs. non-diabetic isotype control antibody group; p = 0.1071, Tukey's multiple comparisons test; vs. non-diabetic anti-RGMa neutralizing antibody group; p = 0.1130, Tukey's multiple comparisons test). These results demonstrate that the diabetes-induced decrease in neurogenesis by doublecortin-positive neural progenitor cells in the dentate gyrus of the hippocampus can be reversed by administration of anti-RGMa neutralizing antibodies. These results demonstrate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are effective against impaired hippocampal neurogenesis, one of the causes of diabetic dementia.

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

[0098] <Induction of a chronic cerebral hypoperfusion model> Nine- to ten-week-old C57BL / 6J male mice were used in the experiment. Isoflurane inhalation anesthesia (induction 4) was administered. The mice were fixed in a prone position under a 1.5% (maintenance 1.5%) condition, and cerebral blood flow was measured by laser speckle flowmetry. During the measurement, a heat pad was used to maintain the rectal temperature at 35.0 to 36.5°C. After that, the patient was fixed in a supine position, the neck was incised, and after releasing the carotid sheath, micro-coils were placed in both internal carotid arteries. (Reference 5) Cerebral blood flow was measured one day after BCAS induction, and the cerebral blood flow was 9 times lower than before surgery. Cerebral blood flow was measured using an OZ-3 (Omegawave, Tokyo, Japan).

[0099] <Novel object recognition memory test> Each individual was placed in the open field and allowed to freely explore for 15 minutes to acclimate to the field (habituation trial 1). The next day, prior to the acquisition trial, each individual was allowed to freely explore the field for 5 minutes (habituation trial 2). After habituation trial 2, each individual was temporarily returned to its home cage and given an object (object A) with the same shape made from blocks to play with. After placing the object at two points on the diagonal top left and bottom right of the field using double-sided tape, 10 cm from the wall, each individual was returned to the field and allowed to explore freely (acquisition trial). After the acquisition trial, a 12-hour interval was allowed, and then a test trial was conducted. In the test trial, a bottle containing opaque beads was placed as a novel object in place of object A, which had been placed at the diagonal bottom right of the field. The object (object B) was placed 10 cm from the wall using double-sided tape. Object A on the diagonal top left was placed in the same position as in the acquisition trial. After the interval, each individual was placed in the above object. Objects A and B were returned to the field where they had been placed and allowed to explore freely for 10 minutes (test trial). Objects A and B used in each trial were prepared based on previous research (Reference 3). During the interval, each individual had free access to water and food in their living cage. All trials were videotaped, and after the behavioral test was completed, the time each individual spent exploring the objects in each trial was calculated. Exploratory behavior included sniffing and touching each object, but excluded climbing on the object. The Discrimination Index (DI) from the test trial was used for analysis. DI is calculated as {(exploration time for object B) - (exploration time for object A)} / {(exploration time for object B) + (exploration time for object A)} / {(exploration time for object B) + (exploration time for object A)}. The total search time for objects A and B in the test trial was defined as the search time for objects A and B. Individuals whose total search time for objects A and B in the test trial was less than 30 seconds were not used in the analysis.

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

[0101] <Result> The BCAS-isotype control antibody group showed a significant decrease in DI compared with the sham-isotype control antibody (Palivizumab) group (p<0.001, Tukey's multiple comparisons test). The DI increased significantly compared to the anti-RGMa antibody group (p<0.001, Tukey's multiple comparisons test). This indicates that the therapeutic intervention with anti-RGMa neutralizing antibody administration reduces the damage caused by chronic ischemia. It was found that it significantly improved recognition memory impairment (Figure 3). These results demonstrate that RGMa inhibitors, particularly anti-RGMa neutralizing antibodies, are effective against vascular dementia.

[0102] [References] 1. Deeds MC, Anderson JM, Armstrong AS, et al. Single dose streptozotocin-induced diabetes: Considerations for study design in islet transplantation models. Lab Anim. 2011;45(3):131-140. doi:10.1258 / la.2010.010090 2. O'brien PD, Sakowski SA, Feldman EL. Mouse models of diabetic neuropathy. IL AR J. 2014;54(3):259-272. doi:10.1093 / ilar / ilt052 3. Leger M, Quiedeville A, Bouet V, et al. Object recognition test in mice. Nat Protoc. 2013;8(12):2531-2537. doi:10.1038 / nprot.2013.155 4. Grinan-Ferre C, Puigoriol-Illamola D, Palomera-avalos V, et al. Environmental enrichment modified epigenetic mechanisms in SAMP8 mouse hippocampus by reducing oxidative stress and inflammaging and achieving neuroprotection. Front Aging Neurosci. 2016;8(OCT). doi:10.3389 / fnagi.2016.00241 5. Hattori Y et al, Gradual Carotid Artery Stenosis in Mice Closely Replicates Hypoperfusive Vascular Dementia in Humans.Journal of the American Heart Association 2016 2 Feb.22;5(2): e002757.DOI: 10.1161 / JAHA.115.002757 6. Stranahan, A., Arumugam, T., Cutler, R. et al. Diabetes impairs hippocampal function through glucocorticoid-mediated effects on new and mature neurons. Nat Neurosci 11, 309-317 (2008). https: / / doi.org / 10.1038 / nn2055

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

[0104] The present invention is highly useful in the pharmaceutical industry because RGMa inhibitors are useful for preventing or treating diabetic dementia or vascular dementia.

Claims

1. A preventive or therapeutic agent for dementia selected from diabetic dementia and vascular dementia, comprising an RGMa inhibitor.

2. A preventive or therapeutic agent for diabetic dementia, comprising an RGMa inhibitor.

3. A preventive or therapeutic agent for vascular dementia, comprising an RGMa inhibitor.

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

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

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

39.

7. The anti-RGMa neutralizing antibody is selected from the following (a) to (l): (a) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, and the amino acid sequence set forth in SEQ ID NO: 6 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10 and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; (b) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 12 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, and Anti-RG containing a heavy chain variable region including HCDR2 containing the amino acid sequence SFG and HCDR3 containing the amino acid sequence SFG. Ma neutralizing antibody, (c) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 17, the amino acid sequence set forth in SEQ ID NO: 18 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; (d) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, the amino acid sequence set forth in SEQ ID NO: 24 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 25 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26 and an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 27; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 28 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 29; (e) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 31 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 32; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (f) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 35 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 36; an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 37; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (g) LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, the amino acid sequence set forth in SEQ ID NO: 30 a light chain variable region comprising an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 40 and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (h) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 41; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (i) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 42; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (j) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; (k) a light chain variable region comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 44; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; and (l) a light chain variable region comprising LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 29, LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 30, and LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; an anti-RGMa neutralizing antibody comprising a heavy chain variable region comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 33, and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 34; The preventive or therapeutic agent according to any one of claims 4 to 6, which is an antibody selected from the group consisting of:

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

9. The method for prevention or treatment according to claim 8, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

10. Use of an RGMa inhibitor for the manufacture of an agent for the prevention or treatment of dementia selected from diabetic dementia and vascular dementia.

11. The use according to claim 10, wherein the RGMa inhibitor is an anti-RGMa neutralizing antibody.

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