Rgma binding protein and use thereof

Abstract

To obtain an anti-RepulsiveGuidanceMoleculea (RGMa) antibody which can be used as a medicine for preventing, treating or preventing the recurrence of neurological diseases or immunological diseases, has high binding activity and little side effects.SOLUTION: The problem is solved by providing an isolated RGMa-binding protein which does not inhibit the binding of RGMa to neogenin and neutralizes the neurite outgrowth inhibitory activity of RGMa, preferably an anti-RGMa antibody in which the amino acid sequence of a complementarity-determining region is an amino acid sequence having a specific sequence in the sequence table and SFG.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to RGMa binding proteins and uses thereof. [Background technology]

[0002] RGM (repulsive guidance molecule) is a GPI-anchored membrane protein with a molecular weight of approximately 33 kDa, which was initially identified as an axon guidance molecule in the visual system (see Non-Patent Document 1). The RGM family contains three members, RGMa, RGMb, and RGMc. Among these, RGMa is thought to be a neurite inhibitor after central nervous system injury because it is re-expressed not only during development but also after central nervous system injury in adult humans and rats, and RGMa inhibition enhances neurite outgrowth and promotes functional recovery after spinal cord injury in rats (see Non-Patent Document 2).

[0003] RGMa has also been reported to have an effect on the immune system. RGMa is expressed on dendritic cells and binds to T cells. This action enhances T cell adhesion to ICAM-1 and fibronectin, and promotes cytokine production. In multiple sclerosis model mice, administration of anti-RGMa antibodies suppresses the symptoms of encephalomyelitis and also suppresses the onset and recurrence of the disease. Anti-RGMa antibodies bind to RGMa expressed in dendritic cells, thereby suppressing the activation of T cells and preventing the onset of multiple sclerosis. It is believed to be effective against the disease.

[0004] The signal transduction mechanism of RGMa is also being elucidated, and the Neogenin protein has been reported as a receptor for RGMa (Patent Document 3). Neogenin is a single-pass transmembrane protein that is expressed on neurons and T cells. RGMa binds to Neogenin on the cell membrane, inducing intracellular RhoA activation and Ras inactivation. On the other hand, in the developing chick brain, Neogenin is known to induce apoptosis in the absence of RGMa (Matsunaga et al., Dev. Growth Differ. 46, 481, 2004). It is thought to have two opposing effects: a favorable effect for nerve regeneration, which promotes the survival of nerve cells, and a negative effect, which inhibits neurite outgrowth.

[0005] As a pharmaceutical targeting RGM, Patent Document 1 discloses an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient. Patent Documents 2 and 3 disclose a therapeutic agent for mechanical damage to the brain and spinal cord that uses an anti-RGM antibody that regulates the binding of RGM to the neogenin receptor. Patent Document 4 also discloses the medicinal use of an anti-RGM antibody for multiple sclerosis and other conditions. In addition, Patent Document 5 discloses the efficacy of anti-RGM antibodies for multiple sclerosis, brain trauma in mammals, spinal cord injury, stroke, Therapeutic applications for diseases including neurodegenerative diseases and schizophrenia have been disclosed. Furthermore, Patent Document 6 discloses the therapeutic use of RGM modulators such as anti-RGM antibodies for spinal cord injury and multiple sclerosis, and Non-Patent Document 3 discloses the therapeutic use for progressive multiple sclerosis. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication WO2005 / 087268 Pamphlet [Patent Document 2] Special Publication No. 2010-537655 [Patent Document 3] Special Publication No. 2009-510002 [Patent Document 4] International Publication WO2011 / 071059 Pamphlet [Patent Document 5] Special Publication No. 2011-512806 [Patent Document 6] Special Publication No. 2004-525875 [Non-patent literature]

[0007] [Non-Patent Document 1] Neuron 5, 735-743 (1990) [Non-patent document 2] J. Cell Biol. 173, 47-58 (2006) [Non-patent document 3] Cell Reports 10, 1-12 (2015) Summary of the Invention [Problem to be solved by the invention]

[0008] As described above, the therapeutic use of anti-RGMa antibodies for neurological and immunological diseases has been disclosed, but conventional antibodies have insufficient activity or may impair the inherent functions of RGMa, resulting in side effects. In particular, conventional antibodies may inhibit the binding between RGMa and Neogenin, thereby potentially inhibiting the desirable effects of Neogenin bound to RGMa, such as apoptosis inhibition. Therefore, the present invention provides a compound that does not inhibit the RGMa / Neogenin interaction and inhibits neurite outgrowth of RGMa. The objective of the present invention is to provide an RGMa-binding protein that neutralizes the activity. [Means for solving the problem]

[0009] The present inventors conducted extensive research to solve the above problems and succeeded in obtaining an RGMa-binding protein that does not inhibit the binding of RGMa to neogenin and neutralizes the neurite outgrowth inhibitory activity of RGMa, and found that this protein can be used as a medicine for neurological and immunological diseases, leading to the completion of the present invention.

[0010] The present invention is as follows. [1] An isolated RGMa-binding protein that does not inhibit the binding of RGMa to neogenin and neutralizes the neurite outgrowth inhibitory activity of RGMa. [2] The RGMa-binding protein according to [1], which binds to human RGMa, rat RGMa, and / or mouse RGMa. [3] An RGMa-binding protein according to [1] or [2], which binds to the peptides EEVVNAVEDWDSQG (SEQ ID NO: 26 in the Sequence Listing), NQQIDFQAFHTNAE (SEQ ID NO: 27 in the Sequence Listing), PTAPETFPYET (SEQ ID NO: 28 in the Sequence Listing), and / or KLPVEDLYYQA (SEQ ID NO: 29 in the Sequence Listing). [4] An RGMa-binding protein according to any one of [1] to [3], which binds to the peptides of SEQ ID NO: 26 and SEQ ID NO: 27 in the sequence listing. [5] An RGMa-binding protein according to any one of [1] to [4], which binds to the peptides of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28 in the Sequence Listing. [6] An RGMa-binding protein according to any one of [1] to [4], which binds to the peptides of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 29 in the Sequence Listing. [7] The RGMa-binding protein according to any one of [1] to [6], wherein the RGMa-binding protein is a human antibody, a humanized antibody, or a chimeric antibody, or an antigen-binding fragment thereof. [8] A nucleic acid molecule encoding a protein portion of the RGMa-binding protein described in any one of [1] to [7]. [9] A recombinant vector comprising the nucleic acid molecule described in [8].

[10] A host cell containing the recombinant vector described in [9].

[11] A method for producing an RGMa-binding protein according to any one of [1] to [7], comprising the step of culturing the host cell according to

[10] .

[12] A pharmaceutical composition comprising the RGMa-binding protein of any one of [1] to [7].

[13] The pharmaceutical composition of

[12] for use in the prevention, treatment, or recurrence prevention of a neurological or immunological disease.

[14] Neurological diseases include amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barré syndrome, cerebral leukodystrophy, multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), neuromyelitis optica, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal tumor, transverse myelitis, and dementia.

[13] The pharmaceutical composition according to

[13] , wherein the disease is selected from the group consisting of dementia (including senile dementia, mild cognitive impairment, Alzheimer's disease, and Alzheimer's-related dementia), Huntington's chorea, tardive dyskinesia, mania, Parkinson's disease, Steele-Richard syndrome, Down's syndrome, myasthenia gravis, neurotrauma (including optic nerve trauma), vascular amyloidosis, cerebral hemorrhage associated with amyloidosis, cerebral infarction, encephalitis, acute confusional disorder, glaucoma, schizophrenia, and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal dystrophy, age-related macular degeneration, eye diseases characterized by optic disc drusen, eye diseases characterized by genetic determinants of photoreceptor degeneration, autosomal recessive cone-rod dystrophy, and mitochondrial disorders associated with optic neuropathy).

[15] Immunological diseases include multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), Guillain-Barré syndrome, neuro-Behçet's disease, pernicious anemia, type 1 (insulin-dependent) diabetes mellitus, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjögren's syndrome, Good-Bascher's syndrome, Graves' disease, and autoimmune hemolysis. The pharmaceutical composition according to

[13] , wherein the inflammatory bowel disease is selected from the group consisting of chronic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis.

[16] The pharmaceutical composition according to

[13] , wherein the neurological or immunological disease is selected from the group consisting of spinal cord injury, nerve trauma (including optic nerve trauma), and multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis).

[17] The amino acid sequences of the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), the heavy chain complementarity determining region 1 (HCDR1), the heavy chain complementarity determining region 2 (HCDR2), and the heavy chain complementarity determining region 3 (HCDR3) are: LCDR1: RASQDISSYLN (SEQ ID NO: 30 in the sequence listing) LCDR2: YTSRLHS (SEQ ID NO: 31 in the sequence listing) LCDR3: QQLNTLP (SEQ ID NO: 32 in the sequence listing) HCDR1: DAWMD (SEQ ID NO: 33 in the Sequence Listing) HCDR2: EIRSKANNHATYYAESVKG (SEQ ID NO: 34 in the Sequence Listing) and HCDR3: RDGAY (SEQ ID NO: 35 in the sequence listing), or LCDR1: RSSQSLVHSNGNTYLH (SEQ ID NO: 36 in the sequence listing) LCDR2: KVSNRFS (SEQ ID NO: 37 in the sequence listing) LCDR3: SQSTHVP (SEQ ID NO: 38 in the Sequence Listing) HCDR1: TSYYWN (SEQ ID NO: 39 in the sequence listing) HCDR2: YISYDGTNNYNPSLKN (SEQ ID NO: 40 in the Sequence Listing) and Contains HCDR3:SFG, In each CDR sequence, one or several amino acids are substituted, deleted, and / or added. An isolated anti-RGMa antibody, or antigen-binding fragment thereof, which may be

[18] Heavy chain variable region (VH) VH: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS (SEQ ID NO: 41 in the Sequence Listing) or an amino acid sequence having at least 90% identity thereto; The light chain variable region (VL) VL:DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 42 in the Sequence Listing) or a small amino acid sequence An anti-RGMa antibody or antigen-binding fragment thereof according to

[17] , comprising an amino acid sequence that is at least 90% identical to the antibody or antigen-binding fragment thereof.

[19] The anti-RGMa antibody or antigen-binding fragment thereof according to

[17] or

[18] , wherein the anti-RGMa antibody is a humanized antibody.

[20] Any of

[17] to

[19] , in which the anti-RGMa antibody has a human IgG constant region. 2. A method for the treatment of rhesus malabsorption, comprising administering to a subject an anti-RGMa antibody or antigen-binding fragment thereof, comprising administering to said subject an anti-RGMa antibody or antigen-binding fragment thereof.

[21] An RGMa-binding protein whose binding to RGMa competes with the anti-RGMa antibody described in

[17] or

[18] .

[22] A nucleic acid molecule encoding a protein portion of an anti-RGMa antibody or an antigen-binding fragment thereof according to any one of

[17] to

[20] .

[23] A nucleic acid sequence encoding the amino acid sequence of VH and VL VH:gaagtgcagctggtggaatctggcggcggactggtgcagcctggcagatccctgagactgtcctgtaccgcctccggcttcaccttctccgacgcctggatggatgggtgcgacaggctcctggcaagggcctggaatgggtggccgagatccggtccaaggccaacaaccacgccacc and VL: gacatccagatgacccagtccccctcctccgtgtctgcttccgtgggcgacagagtgaccatcacctgtcgggcctcccaggacatctcctcctacctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctactacacctcccggctgcactccggcgtgccctctagattttccggctctggctccggcaccgactttaccctgaccatctccagcctgcagcccgaggacttcgcctcctacttctgtcagcagctgaacaccctgccctggacctttggcggaggcaccaaggtggaaatggaa (SEQ ID NO: 44 in the Sequence Listing). The nucleic acid molecule according to

[22] , which is a nucleic acid sequence comprising:

[24] A recombinant vector comprising the nucleic acid molecule according to

[22] or

[23] .

[25] A host cell containing the recombinant vector described in

[24] .

[26] A method for producing an anti-RGMa antibody or an antigen-binding fragment thereof described in any one of

[17] to

[20] , comprising a step of culturing the host cell described in

[25] .

[27] A pharmaceutical composition comprising an anti-RGMa antibody or an antigen-binding fragment thereof according to any one of

[17] to

[20] .

[28] The pharmaceutical composition according to

[27] for use in the prevention, treatment or recurrence prevention of a neurological or immunological disease.

[29] Neurological diseases include amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barré syndrome, leukodystrophy, multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), neuromyelitis optica, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal tumor, transverse myelitis, dementia (including senile dementia, mild cognitive impairment, Alzheimer's disease, and Alzheimer's-related dementia), Huntington's chorea, tardive dyskinesia, mania, Parkinson's disease, Steele-Richards syndrome, and Dow Jones Infectious Disease (DWD). glaucoma, schizophrenia, glaucoma, myasthenia gravis, neurotrauma (including optic nerve trauma), vascular amyloidosis, cerebral hemorrhage associated with amyloidosis, cerebral infarction, encephalitis, acute confusional disorder, glaucoma, schizophrenia, and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal dystrophy, age-related macular degeneration, eye diseases characterized by optic disc drusen, eye diseases characterized by genetic determinants of photoreceptor degeneration, autosomal recessive cone-rod dystrophy, and mitochondrial disorders associated with optic neuropathy).

[30] Immunological diseases include multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), Guillain-Barré syndrome, neuro-Behçet's disease, pernicious anemia, type 1 (insulin-dependent) diabetes mellitus, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjögren's syndrome, Good-Bascher's syndrome, Graves' disease, and autoimmune hemolysis.

[28] The pharmaceutical composition according to

[28] , wherein the disease is selected from the group consisting of chronic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis.

[31] The pharmaceutical composition according to

[28] , wherein the neurological or immunological disease is selected from the group consisting of spinal cord injury, neurotrauma (including optic nerve trauma), and multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis).

[32] A method for preventing, treating, or administering to a subject in need thereof an effective amount of the RGMa-binding protein according to any one of [1] to [7]. is a method to prevent recurrence.

[33] A method for preventing, treating, or preventing recurrence of a neurological or immunological disease, comprising administering an effective amount of an anti-RGMa antibody or antigen-binding fragment thereof described in any one of

[17] to

[20] to a subject in need thereof. [Effects of the Invention]

[0011] The RGMa-binding protein of the present invention does not inhibit the interaction between RGMa and Neogenin, and therefore can maintain the effects of Neogenin bound to RGMa, such as inhibiting apoptosis in neurons. Therefore, the humanized anti-RGMa antibody of the present invention has a strong protective effect on neurons and is less likely to cause side effects associated with neuronal cell loss. Furthermore, the humanized anti-RGMa antibody of the present invention has superior properties, such as binding affinity to human RGMa and thermal stability, compared to conventional antibodies. Therefore, it can be used as a pharmaceutical for neurological and immunological diseases with excellent efficacy and few side effects. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of an RGMa-Neogenin binding inhibition test using an RGMa polyclonal antibody (AF2459), a comparative antibody (r5F9), and antibodies of the present invention (r70E4, 116A3). [Figure 2] FIG. 1 shows the results of an RGMa-BMP2 binding inhibition test using control mouse IgG and the antibodies of the present invention (B5.70E4, B5.116A3). [Figure 3] FIG. 1 shows the results of an antibody thermal stability test using a comparative antibody (rH5F9), a chimeric antibody of the present invention (r116A3C), and a humanized antibody of the present invention (HE / KA, HA / KC). [Figure 4] FIG. 1 shows the results of a neurite outgrowth assay using the antibodies of the present invention (B5.70E4 (left), B5.116A3 (right)). [Figure 5] Figure 1 shows the results of efficacy tests using control mouse IgG (mo-IgG2bk) and the antibodies of the present invention (r70E4, r116A3) in rat models of spinal cord injury: (A) shows the results of efficacy tests in a spinal cord crush model, and (B) shows the results of efficacy tests in a spinal cord hemisection model. [Figure 6]

[0033] Figure 1 shows the results of an efficacy test of the antibody of the present invention (B5.116A3) using a mouse model of multiple sclerosis induced with the PLP139-151 peptide. The left side shows the EAE score, and the right side shows the change in body weight. The upper panel shows the results when the test antibody was administered 7 and 10 days later, and the lower panel shows the results when the test antibody was administered 18 and 21 days later. DETAILED DESCRIPTION OF THE INVENTION

[0013] To facilitate understanding of the present invention, the terms used in the present invention are explained below.

[0014] [RGMa] RGMa is 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 in the sequence listing. The signal peptide Met1-Pro47 (referring to the peptide from the first methionine residue from the N-terminus to the proline residue just before the 47th residue) is present at the N-terminus. The N-terminal domain (described in the Sequence Listing) is removed, the peptide bond between Asp168 and Pro169 is cleaved, the C-terminal peptide Arg423 to Cys450 is removed, and a GPI anchor is added to the C-terminal carboxyl group of the resulting C-terminus, Gly422. Human RGMa protein is expressed on the cell membrane via a GPI anchor as a mature protein in which the N-terminal domain (Cys48 to Asp168) and the C-terminal domain (Pro169 to Ala424) are connected by a disulfide bond. The mouse RGMa precursor protein has the amino acid sequence shown in SEQ ID NO:2 in the Sequence Listing, and the rat RGMa precursor protein has the amino acid sequence shown in SEQ ID NO:3 in the Sequence Listing. However, because the C-terminal peptide is removed, the mature proteins have the same amino acid sequence. In the present invention, RGMa may refer to either the precursor protein, the mature protein, or an active fragment thereof, or a derivative or mutant thereof, as long as it binds to neogenin (described below) and acts. Furthermore, human RGMa or RGMa derived from other organisms may be used, although human RGMa is preferred.

[0015] [Neogenin] Neogenin is expressed in neurons of the central nervous system and functions as one of the receptors for RGMa. As shown in SEQ ID NO: 10 in the sequence listing, human neogenin protein consists of 1,461 amino acids and is expressed as a mature membrane protein with the signal peptide Met1 to Ala33 removed. In the present invention, neogenin may refer to either a precursor protein, a mature protein, or an RGMa-binding fragment thereof, or a derivative or mutant thereof, as long as it binds to RGMa. Furthermore, either human neogenin or neogenin derived from another organism may be used, but human neogenin is preferred.

[0016] [Neutralization] As used herein, "neutralizing" refers to the ability to bind to a target of interest and inhibit any function of that target. That is, "neutralizing the neurite outgrowth inhibitory activity of RGMa" refers to inhibiting the neurite outgrowth inhibitory activity of RGMa by binding an RGMa-binding protein to RGMa. Neurite outgrowth inhibitory activity can be assessed by one or more of several in vitro or in vivo assays known in the art, such as the neurite outgrowth inhibition assay described herein.

[0017] [Isolated] The term "isolated," as used herein with respect to an isolated RGMa binding protein, means identified and separated and / or recovered from components of 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 binding protein can be isolated by purification using at least one purification step, and an RGMa binding protein purified using at least one purification step can be referred to as an "isolated RGMa binding protein."

[0018] [RGMa binding protein] As used herein, the term "RGMa-binding protein" refers to a molecule containing a protein that binds to RGMa. RGMa-binding proteins include anti-RGMa antibodies and antigen-binding fragments thereof, RGMa-binding scaffold proteins, and soluble RGMa receptor proteins such as the extracellular domain of neogenin, as well as fusion proteins thereof. An RGMa-binding scaffold protein is a protein that achieves binding to RGMa by introducing mutations into the Kunitz domain of a serine protease inhibitor, the extracellular domain of human fibronectin, ankyrin, lipocalin, or the like. Fusion proteins are proteins that combine an RGMa-binding protein with other RGMa receptor proteins of the present invention, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (e.g., TGF-β, NGF, neurotrophin), albumin, enzymes, or other antibodies. An RGMa-binding protein to which a functional molecule other than the Ma-binding protein has been chemically or genetically linked.

[0019] [Human antibody] A human antibody is an antibody in which both the light and heavy chains are derived from human immunoglobulins. Depending on the constant region of the heavy chain, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) with γ heavy chains, IgM with μ heavy chains, IgA (including IgA1 and IgA2) with α heavy chains, IgD with δ heavy chains, and IgE with ε heavy chains. In principle, the light chain contains either a κ chain or a λ chain.

[0020] [Humanized antibody] A humanized antibody is 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] A chimeric antibody is an antibody in which the light chain, the heavy chain, or both, are composed of variable regions of non-human origin and constant regions of human origin.

[0022] [Anti-RGMa antibody] As used herein, an anti-RGMa antibody refers to an immunoglobulin molecule that binds to RGMa, or a modified molecule thereof. Modified molecules include multispecific antibodies, chimeric antibodies, humanized antibodies, functionally modified antibodies, and conjugated antibodies.

[0023] [Multispecific antibodies] A multispecific antibody is an asymmetric antibody that has two or more independent antigen recognition sites with two or more different antigen specificities. Examples include bispecific antibodies, which have two antigen specificities, and trispecific antibodies, which have three antigen specificities.

[0024] [Functionally modified antibodies] In the present application, a functionally modified antibody refers to an antibody whose functions other than the antigen-binding function, such as its cell-killing function, complement activation function, or blood half-life extension function, have been modified primarily by modifying the amino acids or sugar chains in the Fc region of the antibody.

[0025] [Conjugated antibody] In this application, a conjugated antibody is an antibody to which a functional molecule other than an antibody, such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low-molecular-weight compound, a cytokine, a growth factor (TGF-β, NGF, Neurotrophin, etc.), albumin, or an enzyme, has been added. An antibody to which a specific antibody has been chemically or genetically linked.

[0026] [Antigen-binding fragment] In the present application, an antigen-binding fragment refers to a protein containing a portion of an antibody and capable of binding to an antigen. Examples of antigen-binding fragments include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and their derivatives. Furthermore, the antigen-binding fragment may be a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor (TGF-β, NGF, Neurotrophin, etc.), albumin, an enzyme, another antibody, or the like, as described herein. It is intended to include conjugated antigen-binding fragments to which functional molecules other than anti-RGMa antibodies are chemically or genetically engineered.

[0027] [Complementarity-determining region] Complementarity determining regions (CDRs) refer to the regions of the variable regions of immunoglobulin molecules that form the antigen-binding site, and are also called hypervariable regions, which are portions of immunoglobulin molecules that exhibit particularly large variations in amino acid sequence. There are three CDRs (LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3) in each of the light and heavy chains. In this application, the CDRs of immunoglobulin molecules are numbered according to the Kabat numbering system. at et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and The NIH Guidelines for Human Services (NIH, USA) determine the risk of heart failure.

[0028] [Percent (%) identity of amino acid sequence] As used herein, "percent (%) identity" with respect to an identified reference polypeptide sequence, such as a variable region, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the particular reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and excluding any conservative substitutions from being considered part of the sequence identity. Alignment for purposes of determining percent identity can be performed by a variety of methods within the skill of one in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. It is achievable. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the maximum alignment with respect to the full length of the compared sequences. However, for the purposes here, the % identity value is obtained by using the sequence comparison computer program BLAST in pairwise alignment. In the situation where BLAST is used for amino acid sequence comparison, the % identity of a given amino acid sequence A with a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues whose scores are agreed to be identical by the program alignment of A and B of the sequence alignment program BLAST, and Y is the total number of amino acid residues of B. It will be understood that when the length of amino acid sequence A is different from the length of amino acid sequence B, the % identity of A to B is different from the % identity of B to A. Unless otherwise specified, all % identity values here are obtained using the BLAST computer program as shown in the immediately preceding paragraph.

[0029] [Competing] In the present application, "competing" with the anti-RGMa antibody of the present invention means that when measured by the surface plasmon resonance (SPR) method described in this specification, the binding of the anti-RGMa antibody or its antigen-binding fragment results in a significant decrease in the binding between the anti-RGMa antibody of the present invention and RGMa.

[0030] Hereinafter, the present invention will be described in detail. <RGMa-binding protein> The RGMa-binding protein of the present invention is an isolated RGMa-binding protein that does not inhibit the binding between RGMa and Neogenin and neutralizes the neurite growth inhibitory activity of RGMa.

[0031] The RGMa protein is preferably an RGMa protein derived from a mammal, and examples of human RGMa proteins include the protein having the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing, mouse RGMa proteins include the protein having the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing, and rat RGMa proteins include the protein having the amino acid sequence of SEQ ID NO: 3 in the Sequence Listing. Furthermore, the RGMa protein may comprise an amino acid sequence in which one or several (preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5) amino acids have been substituted, deleted, inserted, and / or added within these sequences, and may be a polypeptide having substantially the same activity as an RGMa protein, or a polypeptide comprising an amino acid sequence having 90% or more (preferably 95% or more) identity to the amino acid sequence.

[0032] Here, "having substantially the same activity as the RGMa protein" includes any polypeptide as long as it has neurite outgrowth inhibitory activity.

[0033] The amino acid substitution is preferably a conservative substitution. Here, "conservative substitution" means replacing an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. For example, replacing a hydrophobic residue with another hydrophobic residue. Examples of functionally similar amino acids that can be substituted include substitutions of one polar residue with another polar residue of the same charge. Examples of functionally similar amino acids that can be substituted in this way include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged (basic) amino acids include arginine, histidine, and lysine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0034] The term "RGMa-binding protein of the present invention binds to RGMa" means that it specifically binds to RGMa. However, it is more preferable that the dissociation constant (Kd) for human RGMa is low, with the upper limit being, for example, 10 -8 M or less, preferably 10 -9 M or less, and even more preferably 10 -10 M or less The lower limit is not particularly limited, but for example, 10 -14 M or more, more preferably 10 -13 Examples of RGMa-binding proteins include those mentioned above.

[0035] The mature RGMa protein consists of an N-terminal domain and a C-terminal domain, but the C-terminal domain alone has neurite outgrowth inhibitory activity. The RGMa-binding protein of the present invention preferably binds to only the C-terminal domain of RGMa to neutralize the neurite outgrowth inhibitory activity. It is more preferable that the dissociation constant (Kd) for the C-terminal domain of human RGMa is low, with an upper limit of, for example, 10 -8 M or less, preferably 10 -9 M or less, and even more preferably 10 -10 M or less, and the lower limit is not particularly limited, but for example, 10 -14 M or more, more preferably 10 -13 Examples of RGMa-binding proteins include those mentioned above.

[0036] The RGMa-binding proteins of the present invention do not inhibit the binding of RGMa to neogenin. Here, "does not inhibit the binding of RGMa to neogenin" means that, in the RGMa-Neogenin binding system shown in the Examples below, increasing the concentration of the RGMa-binding protein does not substantially decrease the binding between RGMa and neogenin. For example, when the RGMa-binding protein is added to the RGMa-Neogenin binding system and the concentration is increased, if the RGMa-binding protein concentration showing the IC50 is 10 μg / mL or higher, more preferably 50 μg / mL or higher, and most preferably 100 μg / mL or higher, it can be said that the RGMa-binding protein does not inhibit the binding of RGMa to neogenin.

[0037] The neogenin used in the binding test with RGMa is preferably neogenin of the same species as RGMa. That is, it is preferable to use mouse neogenin for mouse RGMa, and human neogenin for human RGMa. An example of human neogenin is a protein having the amino acid sequence of SEQ ID NO: 10 in the Sequence Listing, but any protein that can bind to RGMa may have an amino acid sequence that is 90% or more (preferably 95% or more) identical to SEQ ID NO: 10 in the Sequence Listing.

[0038] The RGMa-binding proteins of the present invention neutralize the neurite outgrowth inhibitory activity of RGMa. The neurite outgrowth inhibitory activity can be evaluated by a neurite outgrowth assay, as shown in the Examples below. Addition of RGMa inhibits neurite outgrowth, whereas addition of an RGMa-binding protein abolishes the neurite outgrowth inhibition caused by RGMa. The RGMa-binding proteins of the present invention can neutralize the neurite outgrowth inhibition caused by addition of RGMa by 50% or more, more preferably 80% or more, and most preferably 90% or more.

[0039] The amino acid sequence of the RGMa protein varies depending on the animal species, and there are differences in the amino acid sequence between human RGMa shown in SEQ ID NO: 1, mouse RGMa shown in SEQ ID NO: 2, and rat RGMa shown in SEQ ID NO: 3. Generally, rodents such as mice and rats are used as experimental materials in pharmacological and safety tests of protein preparations such as antibody drugs. Therefore, the RGMa-binding protein of the present invention preferably binds to mouse or rat RGMa, and more preferably has a low Kd for mouse or rat RGMa. The upper limit of Kd is, for example, 5 x 10 -7 M or less, preferably 10 -8 M or less, and even more preferably 10 -9 M or less, and the lower limit is not particularly limited, but for example, 10 -12 M or more, more preferably 10 -11 Examples of RGMa-binding proteins include those mentioned above.

[0040] The RGMa-binding proteins of the present invention preferably have excellent thermal stability, which can be assessed by the decrease in binding to RGMa upon heat treatment, and are preferably stable upon heat treatment at 60°C or higher, more preferably stable upon heat treatment at 65°C or higher, and most preferably stable upon heat treatment at 70°C or higher.

[0041] The binding site of the RGMa-binding protein of the present invention when it binds to RGMa is not particularly limited, but examples thereof include, in human RGMa, EEVVNAVEDWDSQG (SEQ ID NO: 26 in the Sequence Listing) (amino acid numbers 298-311 of SEQ ID NO: 1 in the Sequence Listing), NQQIDFQAFHTNAE (SEQ ID NO: 27 in the Sequence Listing) (amino acid numbers 322-335 of SEQ ID NO: 1 in the Sequence Listing), and PTAPETFPYET (SEQ ID NO: 28 in the Sequence Listing). (amino acid numbers 349-359 of SEQ ID NO: 1 in the sequence listing), KLPVEDLYYQA (sequence number No. 29) (amino acid numbers 367-377 of SEQ ID NO: 1 in the Sequence Listing) It is more preferable to bind to SEQ ID NOs: 26 and 27 in the sequence listing, and it is even more preferable to bind to SEQ ID NOs: 26 and 27 in the sequence listing and SEQ ID NO: 28 or 29 in the sequence listing.

[0042] Specific examples of RGMa-binding proteins include anti-RGMa antibodies, RGMa-binding scaffold proteins, and fusion proteins thereof.

[0043] <Anti-RGMa antibody> The anti-RGMa antibodies of the present invention include polyclonal and monoclonal antibodies obtained by immunizing mammals such as mice with the RGMa protein or a partial fragment thereof (for example, a fragment comprising one or more of SEQ ID NOS: 26 to 29 in the above sequence listing), as well as 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.

[0044] Antigens can be used directly for immunization, or they can 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. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[0045] 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.

[0046] <Polyclonal antibody> Polyclonal antibodies can be obtained by a conventional method. For example, a mammal as described above is immunized with the antigen described above, optionally with Freund's adjuvant, and then the serum obtained from the immunized animal is extracted. It can be obtained from

[0047] <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.

[0048] 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)).

[0049] 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.

[0050] 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.

[0051] Examples of fusion promoters include polyethylene glycol, and cell fusion can usually be achieved by reacting polyethylene glycol (average molecular weight 1000 to 4000) at a concentration of about 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 usually about 1:1 to 10:1, for about 1 to 10 minutes.

[0052] 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.

[0053] In screening for antibody-producing hybridomas, in addition to binding assays with RGMa protein, we also evaluate whether the antibody inhibits the binding of RGMa protein to neogenin and whether the antibody neutralizes the function of RGMa protein (neurite outgrowth inhibitory activity). These screening methods allow us to select anti-RGMa antibodies of the present invention.

[0054] 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).

[0055] The production of monoclonal antibodies from hybridomas is carried out by culturing the hybridomas in vitro. The cells can be cultured in vitro or grown in vivo in the ascites of a mammal such as a mouse or rat, and then isolated from the resulting culture supernatant or from the ascites of the mammal.

[0056] When culturing in vitro, it is possible to use a nutrient medium suitable for growing, maintaining, and preserving hybridomas and producing monoclonal antibodies in the culture supernatant, depending on various conditions such as the characteristics of the cell type being cultured and the culture method.

[0057] 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.

[0058] Monoclonal antibodies can be isolated and purified by subjecting the culture supernatant or ascites fluid to saturated ammonium sulfate, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), or affinity column chromatography such as 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.

[0059] 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 determined sequence information. Monoclonal antibodies can then be produced by culturing a cell line transfected with such an expression vector. By using a human antibody library as the phage antibody library, human antibodies with the desired binding affinity can be generated.

[0060] Scaffold proteins such as the Kunitz domain of human serine protease inhibitors and the extracellular domain of human fibronectin are used, and by modifying the sequence of the target binding site on the scaffold, it is possible to generate scaffold proteins that bind to RGMa (Clifford Mintz et.al BioProcess International, 2013, Vol.11(2), pp40-48).

[0061] Fusion proteins include RGMa-binding proteins chemically or genetically engineered to functional molecules other than the RGMa-binding protein of the present application, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies.

[0062] 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 attached to the N-terminal amino group of an amino acid in the RGMa-binding protein, for example, by using an NHS-activated group.

[0063] When a radioactive substance is used as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 L u or 211 Radioactive materials are detected by RGM using the chloramine T method. a binding protein can be directly bound to the

[0064] 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.

[0065] When a low molecular weight compound is used as a functional molecule, examples thereof include daunomycin, doxorubicin, metrorexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0066] 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.

[0067] Linkers used to chemically attach toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2)nO (CR2)n- (R is an arbitrary substituent, n is a positive integer) Examples of suitable functional groups include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylaminos (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 this 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 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0068] A preferred embodiment of the RGMa-binding protein of the present invention is a chimeric antibody. Examples of "chimeric antibodies" include those whose variable regions are derived from immunoglobulins of non-human animals (such as mice, rats, hamsters, and chickens) and whose constant regions are derived from human immunoglobulins. For example, chimeric antibodies can be produced by immunizing a mouse with an antigen, excising the antigen-binding variable region from the mouse monoclonal antibody gene, and then 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, and IgG4), IgM, IgA (IgA1 and 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. Expression vectors can be constructed using the genes of chimeric antibodies produced in this manner. Host cells are transformed with the expression vector to obtain transformed cells that produce chimeric antibodies, and the transformed cells are cultured to obtain the desired chimeric antibodies from the culture supernatant.

[0069] Another preferred embodiment of the RGMa-binding protein 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, see JP-A-4-506458 and JP-A-2912618. They can be prepared by referring to the methods described in the manual, etc. Specifically, it refers to a humanized antibody characterized in that 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 the constant regions are derived from a human immunoglobulin.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Another preferred embodiment of the RGMa-binding protein 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.

[0074] For example, transgenic mice that produce human antibodies can be produced according to the methods described in Nature Genetics, Vol. 7, pp. 13-21, 1994; Nature Genetics, Vol. 15, pp. 146-156, 1997; Published Japanese Translation of PCT International Publication No. H4-504365; Published Japanese Translation of PCT International Publication No. H7-509137; International Publication WO94 / 25585; Nature, Vol. 368, pp. 856-859, 1994; and Published Japanese Translation of PCT International Publication No. H6-500233. More specific examples include HuMab® mice (Medarex, Princeton, NJ), KM™ mice (Kirin Pharma Company, Japan), and KM(FCγRIIb-KO) mice.

[0075] Specifically, the monoclonal antibody of the present invention comprises a heavy chain variable region CDR having the sequence number of the sequence listing. the light chain variable region comprising the amino acid sequences of 33 (HCDR1), 34 (HCDR2) and 35 (HCDR3); The CDRs of the region include those containing the amino acid sequences of SEQ ID NOs: 30 (LCDR1), 31 (LCDR2) and 32 (LCDR3) in the sequence listing. As long as the antibody of the present invention maintains its properties of having the ability to bind to RGMa, not inhibiting the binding of RGMa to Neogenin, and neutralizing the neurite outgrowth inhibitory activity of RGMa, any one of these CDRs may be used. In the above, one to several amino acids may be substituted. Here, one to several means, for example, one or two. It is preferable that the amino acid substitution is a conservative substitution in order to maintain the characteristics of the present invention. The term "maintaining the characteristics of the antibody" means that these characteristics are maintained by the amino acids in the CDR. This means that the amino acid sequence is maintained at the same level as before the modification, for example, 80% or more, preferably 90% or more, and more preferably 95% or more. Note that "maintained" also includes "improved."

[0076] There are no particular restrictions on the sequences of regions other than CDRs, as long as they maintain the structure of the antibody and can exert their functions. The constant regions may be any of mouse-derived sequences, human-derived sequences, sequences derived from other mammals, chimeric sequences thereof, and artificial sequences. When constant regions are included, examples of the amino acid sequences of the heavy and light chain constant regions include 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.

[0077] The mouse antibody having these CDRs has the amino acid sequence of SEQ ID NO: 4 in the light chain. An example of such an antibody is one having the amino acid sequence of SEQ ID NO: 5 in the heavy chain. These amino acid sequences may contain substitutions, deletions, additions, or insertions of one or several amino acids (1 to 20, 1 to 10, or 1 to 5) as long as they maintain the ability to bind to RGMa, do not inhibit the binding of RGMa to neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa. While such substitutions, deletions, or additions may be introduced into the CDRs, they are preferably introduced into regions other than the CDRs. Furthermore, the amino acid substitutions are preferably conservative substitutions in order to maintain the properties of the present invention.

[0078] Another example of the mouse antibody is a mouse / human chimeric antibody in which the constant region is derived from a human. An example of such a mouse / human chimeric antibody is an antibody having the amino acid sequence of SEQ ID NO: 8 in the Sequence Listing (variable region: 1-107) in the light chain and the amino acid sequence of SEQ ID NO: 9 in the heavy chain (variable region: 1-116). These amino acid sequences may contain substitutions, deletions, additions, or insertions of one or several amino acids (1-20, 1-10, or 1-5) as long as they maintain the ability to bind to RGMa, do not inhibit the binding of RGMa to neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa. While such substitutions, deletions, or additions may be introduced into the CDRs, they are preferably introduced into regions other than the CDRs. Furthermore, conservative substitutions are preferred to maintain the properties of the present invention.

[0079] Further examples include humanized antibodies in which the CDRs are derived from humans. Examples of such antibodies include those having an amino acid sequence in the heavy chain of any one of SEQ ID NOS: 11 to 18 (the variable region is up to 116 residues from the N-terminus) and an amino acid sequence in the light chain of any one of SEQ ID NOS: 19 to 25 (the variable region is up to 107 residues from the N-terminus). The amino acid sequences of the humanized antibodies (heavy chain: SEQ ID NOS: 11 to 18 in the Sequence Listing; light chain: SEQ ID NOS: 19 to 25 in the Sequence Listing) may contain substitutions, deletions, additions, or insertions of one or several amino acids (1 to 20, 1 to 10, or 1 to 5) as long as they maintain the ability to bind to RGMa, do not inhibit the binding of RGMa to Neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa. Such substitutions, deletions, or additions may be introduced into the CDRs, but are preferably introduced into regions other than the CDRs. Furthermore, the amino acid substitutions are preferably conservative substitutions in order to maintain the properties of the present invention.

[0080] The heavy chain amino acid sequence and light chain amino acid sequence may be any combination of these, but particularly preferred is an antibody having the amino acid sequence of SEQ ID NO: 15 in the heavy chain and the amino acid sequence of SEQ ID NO: 19 in the light chain. Of the amino acid sequence of SEQ ID NO: 15 in the Sequence Listing, the amino acid sequence corresponding to the heavy chain variable region is shown in SEQ ID NO: 41, and the amino acid sequence corresponding to the light chain variable region is shown in SEQ ID NO: 42. In other words, a particularly preferred antibody of the present invention is an antibody having the amino acid sequence of SEQ ID NO: 41 in the heavy chain variable region and the amino acid sequence of SEQ ID NO: 42 in the light chain variable region. These amino acid sequences have the ability to bind to RGMa and bind to Neogenin. As long as the property of neutralizing the neurite outgrowth inhibitory activity of RGMa without inhibiting its binding to the CDR is maintained, one or several amino acids (1 to 20, 1 to 10, or 1 to 5) may be substituted, deleted, added, or inserted. Such substitutions, deletions, and additions may be introduced into the CDR, but are preferably introduced into a region other than the CDR. Furthermore, the amino acid substitutions are preferably conservative substitutions in order to maintain the properties of the present invention. The amino acid sequences of the antibodies of the present invention, which contain substitutions, deletions, etc. in the amino acid sequences of SEQ ID NO: 41 and / or SEQ ID NO: 42 in the Sequence Listing, are amino acid sequences in which the heavy chain variable region has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) identity with SEQ ID NO: 41 in the Sequence Listing, and the light chain variable region has 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) identity with SEQ ID NO: 42 in the Sequence Listing.

[0081] As another specific example of the monoclonal antibody of the present invention, the CDR of the heavy chain variable region has the sequence shown in the sequence listing. Examples of such antibodies include those containing the amino acid sequences of SEQ ID NOS: 39 (HCDR1), 40 (HCDR2), and SFG (HCDR3), and those containing the amino acid sequences of SEQ ID NOS: 36 (LCDR1), 37 (LCDR2), and 38 (LCDR3) in the CDRs of the light chain variable region. One to several amino acids may be substituted in one or more of these CDRs, as long as the antibodies of the present invention maintain the properties of having the ability to bind to RGMa, not inhibiting the binding of RGMa to neogenin, and neutralizing the neurite outgrowth inhibitory activity of RGMa. Here, 1 to several means, for example, 1 or 2. The amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention. There are no particular restrictions on the sequences of regions other than CDRs, as long as they maintain the structure of the antibody and can exert their functions. The constant regions may be any of mouse-derived sequences, human-derived sequences, sequences derived from other mammals, chimeric sequences thereof, and artificial sequences. When constant regions are included, examples of the amino acid sequences of the heavy and light chain constant regions include 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.

[0082] The mouse antibody having these CDRs has the amino acid sequence of SEQ ID NO: 6 in the light chain. An example of such an antibody is one having the amino acid sequence of SEQ ID NO: 7 in the heavy chain. These amino acid sequences may contain substitutions, deletions, additions, or insertions of one or several amino acids (1 to 20, 1 to 10, or 1 to 5) as long as they maintain the ability to bind to RGMa, do not inhibit the binding of RGMa to neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa. While such substitutions, deletions, or additions may be introduced into the CDRs, they are preferably introduced into regions other than the CDRs. Furthermore, the amino acid substitutions are preferably conservative substitutions in order to maintain the properties of the present invention.

[0083] Other examples include chimeric antibodies in which the constant region of the above-mentioned mouse antibody is derived from a human, and humanized antibodies in which the regions other than the CDRs are derived from a human.

[0084] The anti-RGMa antibodies of the present invention include multispecific antibodies, functionally modified antibodies, and conjugated antibodies having CDRs consisting of specific amino acid sequences (e.g., the amino acid sequences of SEQ ID NO: 30 in the Sequence Listing for LCDR1, SEQ ID NO: 31 in the Sequence Listing for LCDR2, SEQ ID NO: 32 in the Sequence Listing for LCDR3, SEQ ID NO: 33 in the Sequence Listing for HCDR1, SEQ ID NO: 34 in the Sequence Listing for HCDR2, and SEQ ID NO: 35 in the Sequence Listing for HCDR3), or variable regions consisting of specific amino acid sequences (e.g., the amino acid sequence of SEQ ID NO: 41 in the Sequence Listing for the heavy chain variable region and SEQ ID NO: 42 for the light chain variable region).

[0085] The anti-RGMa antibody of the present invention can be used to generate multispecific antibodies such as bispecific antibodies by conjugating to an antibody having an antigen-binding specificity other than RGMa using genetic engineering techniques. Such genetic engineering techniques are well known in the art. For example, DVD-Ig (Wu et al., Nature Biotechnology 25(11), 1290(2007)), or by modifying the Fc region of an antibody, By using ART-Ig technology (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), which combines the heavy chains of two antibodies that bind to the same antigen, it is possible to create the desired bispecific antibody. Antigens other than RGMa include, but are not limited to, factors that inhibit neurite outgrowth, such as Nogo, MAG, Omgp, CSPG, Sema3A, and Lingo-1, as well as TNF-α, IL-6 receptor, CD3, CD20, α4 integrin, BLys, and Thymic These include immune-related molecules such as stromal lymphopoietin, IgE, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-23, and IL-25.

[0086] Modified molecules of the anti-RGMa antibodies of the present invention include functionally modified antibodies. Functionally modified antibodies refer to antibodies whose functions, such as cell killing function, complement activation function, and blood half-life extension function, have been modified primarily by modifying the Fc region (Shitara Kenya, Journal of Pharmacology, 2009, Vol. 129(1), p. 3; Ishii Akiko et al., Journal of Pharmacology, 2010, Vol. 136(5), p. 280; Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p. 710).

[0087] Functionally modified anti-RGMa antibodies can be prepared by the following methods. For example, when the present anti-RGMa 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 antibodies are produced using CHO cells into which the FUT8 gene has been introduced 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 No. 6,737,056, U.S. Patent No. 7,297,775, U.S. Patent No. 7,317,091). Furthermore, the blood half-life can be extended by using a mutant Fc region that enhances binding to FcRn, one of the Fc receptors (Shuhei Hashiguchi et al., Biochemistry, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering.

[0088] Conjugated antibodies are examples of modified molecules of the anti-RGMa 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, or other conjugated molecules. Examples include conjugated antibodies to which functional molecules other than antibodies are chemically or genetically bound.

[0089] When PEG is attached as a functional molecule, the PEG may have a molecular weight of, but not limited to, 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be linear or branched. PEG can be attached to the N-terminal amino group of an amino acid of an antibody, for example, by using an NHS active group.

[0090] 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 attached to antibodies using methods such as chloramine T. Direct binding can be achieved.

[0091] 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.

[0092] When using low molecular weight compounds as functional molecules, daunomycin, doxorubicin, methotrexate, Examples include Rexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0093] 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.

[0094] Linkers used to chemically attach toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2)nO (CR2)n- (R is an arbitrary substituent, n is a positive integer) Examples of suitable functional groups include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylaminos (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 this 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 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0095] In the present invention, the "antigen-binding fragment" of an antibody refers to a partial region of the antibody having antigen-binding ability, such as those described above, and specifically includes F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymerized forms thereof. Furthermore, the antigen-binding fragment may be a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular weight compound, a cytokine, a growth factor (TGF-β, NGF, Neurotrophin, etc.), albumin, an enzyme, or another antibody, which is an anti-RG antibody of the present invention. These include conjugated antigen-binding fragments to which functional molecules other than Ma antibodies are chemically or genetically engineered.

[0096] Here, "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulin with protease enzymes such as pepsin or papain, and digesting the fragments before and after 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 VL (light chain variable region) and CL (light chain constant region), and a heavy chain fragment consisting of VH (heavy chain variable region) and 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. When the 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. An antibody fragment can be produced, called F(ab')2.

[0097] Modified molecules of the antigen-binding fragment of the anti-RGMa antibody of the present invention include conjugated antigen-binding fragments, which are fragments of a functional molecule other than the anti-RGMa antibody of the present invention, such as a non-peptide polymer (e.g., polyethylene glycol (PEG)), radioactive material, toxin, low-molecular-weight compound, cytokine, growth factor (e.g., TGF-β, NGF, neurotrophin), albumin, enzyme, or other antibody, in a partial region of the anti-RGMa antibody that has antigen-binding activity. Examples of such antigen-binding fragments include conjugated antigen-binding fragments to which the antigen is chemically or genetically linked.

[0098] When PEG is attached as a functional molecule, the PEG may have a molecular weight of, but not limited to, 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be linear or branched. PEG can be attached to the N-terminal amino group of a portion of the anti-RGMa antibody that has antigen-binding ability, for example, by using an NHS-active group.

[0099] 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 in the detection of anti-RG antibodies using the chloramine T method. It can be directly linked to a region of the Ma antibody that has antigen-binding activity.

[0100] 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.

[0101] When a low molecular weight compound is used as a functional molecule, examples thereof include daunomycin, doxorubicin, metrorexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0102] 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.

[0103] Linkers used to chemically attach toxins, small molecules, or enzymes include divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2)nO (CR2)n- (R is an arbitrary substituent, n is a positive integer) Examples of suitable functional groups include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylaminos (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 this 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 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0104] In the anti-RGMa antibodies of the present invention comprising CDRs or variable regions having specific amino acid sequences, the constant region is preferably that of human IgG (IgG1, IgG2, IgG3, IgG4) in order to maintain a long half-life in blood.

[0105] The present invention provides an antibody having the above-described specific CDR amino acid sequence and an RGMa protein. Also included are anti-RGMa antibodies, and antigen-binding fragments thereof, that compete for binding with the antibody. Antibodies that compete with antibodies having the above-described specific CDR amino acid sequences for binding to RGMa Examples of such antibodies include antibodies having an epitope in a region selected from Glu298 to Gly311, Asn322 to Glu335, Lys367 to Ala377, and Pro349 to Thr359. The antibody is a binding system of an antibody having the above-described CDR sequence with an RGMa protein, By allowing them to coexist, they can be obtained (screened) and evaluated. For example, they can be obtained by screening using the following surface plasmon resonance (SPR) method. Cut.

[0106] Biotinylated human RGMa protein (4 μg / mL) is loaded as a ligand onto an avidin-immobilized sensor chip, immobilizing 1300 to 1600 RU of human RGMa protein. Next, an anti-RGMa antibody (15 μg / mL) is loaded as an analyte and allowed to bind to the human RGMa protein immobilized on the sensor chip. This process is repeated multiple times to create a state in which the anti-RGMa antibody binds to all of the human RGMa protein molecules on the sensor chip (saturation state), and the amount of binding at saturation (saturation binding amount 1) is determined. A similar experiment is also carried out with an anti-RGMa antibody of the present invention comprising a specific CDR amino acid sequence, and the amount of binding at saturation (saturation binding amount 2) is determined. Next, the human RGMa protein on the sensor chip is saturated with an anti-RGMa antibody containing the amino acid sequence of a specific CDR of the present invention, and then any anti-RGMa antibody (15 μg / mL) is loaded as an analyte to examine whether it additionally binds to the human RGMa protein saturated with the anti-RGMa antibody containing the amino acid sequence of a specific CDR of the present invention. Any anti-RGMa antibody is determined to be "non-competitive" if it can bind to a human RGMa protein saturated with an anti-RGMa antibody containing the amino acid sequence of a specific CDR of the present invention while exhibiting the saturation binding amount of the anti-RGMa antibody calculated above. On the other hand, any anti-RGMa antibody is determined to be "competitive" if it cannot bind to a human RGMa protein saturated with an anti-RGMa antibody containing the amino acid sequence of a specific CDR of the present invention. Furthermore, even if any anti-RGMa antibody can bind to a human RGMa protein saturated with an anti-RGMa antibody containing the amino acid sequence of a specific CDR of the present invention, if the additional binding amount does not reach the saturation binding amount of 1 with a significant difference, the antibody is determined to be "competitive." Significance is determined using a standard testing method (e.g., Student's t-test), with a significance level of 5% or less.

[0107] The anti-RGMa antibody that competes for binding to RGMa with the anti-RGMa antibody containing the amino acid sequence of the above-mentioned specific CDR may be an antibody derived from any animal, such as a mouse antibody, human antibody, rat antibody, rabbit antibody, goat antibody, or camel antibody, or may be a chimeric antibody or humanized antibody that is a combination of these antibodies, but is preferably a chimeric antibody, humanized antibody, or human antibody.

[0108] <Nucleic acid molecule of the present invention> The nucleic acid molecule of the present invention is a polynucleotide encoding the monoclonal antibody of the present invention. Examples of the nucleic acid molecule include a polynucleotide in which the region encoding the heavy chain variable region contains a base sequence encoding the amino acid sequences of SEQ ID NOs: 33, 34, and 35 in the Sequence Listing (one or several amino acids may be substituted, deleted, inserted, or added), respectively, and the region encoding the light chain variable region contains a base sequence encoding the amino acid sequences of SEQ ID NOs: 30, 31, and 32 in the Sequence Listing (one or several amino acids may be substituted, deleted, inserted, or added), respectively; and a polynucleotide in which the region encoding the heavy chain variable region contains a base sequence encoding the amino acid sequences of SEQ ID NOs: 39, 40, and SFG in the Sequence Listing (one or several amino acids may be substituted, deleted, inserted, or added). and a polynucleotide in which the region encoding the light chain variable region contains a base sequence encoding the amino acid sequence of SEQ ID NO: 36, 37, and 38 in the Sequence Listing (in which one or several amino acids may be substituted, deleted, inserted, or added).

[0109] Another example of the nucleic acid molecule of the present invention is a nucleic acid molecule in which the heavy chain coding region comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 in the Sequence Listing, and the light chain coding region comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: Examples of suitable polynucleotides include a polynucleotide having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, and a polynucleotide having a heavy chain-encoding region that includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 7 in the Sequence Listing and a light chain-encoding region that includes a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 in the Sequence Listing.

[0110] Another example of the nucleic acid molecule of the present invention is a polynucleotide in which the region encoding the heavy chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 in the Sequence Listing, and the region encoding the light chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:8 in the Sequence Listing.

[0111] Other examples of nucleic acid molecules of the present invention include polynucleotides in which the region encoding the heavy chain comprises a nucleotide sequence encoding any one of the amino acid sequences of SEQ ID NOs: 11 to 18 in the Sequence Listing, and the region encoding the light chain comprises a nucleotide sequence encoding any one of the amino acid sequences of SEQ ID NOs: 19 to 25 in the Sequence Listing.

[0112] A particularly preferred example of the nucleic acid molecule of the present invention is a polynucleotide in which the region encoding the heavy chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15 in the Sequence Listing, and the region encoding the light chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 19 in the Sequence Listing.

[0113] Another example of the nucleic acid molecule of the present invention is a polynucleotide in which the region encoding the heavy chain variable region comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 41 in the Sequence Listing, and the region encoding the light chain variable region comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 42 in the Sequence Listing.

[0114] A specific example of the nucleic acid molecule of the present invention is a polynucleotide in which the region encoding the heavy chain variable region comprises the base sequence of SEQ ID NO: 43 in the Sequence Listing, and the region encoding the light chain variable region comprises the base sequence of SEQ ID NO: 44 in the Sequence Listing.

[0115] The nucleic acid molecule of the present invention can hybridize under stringent conditions with the complementary strand DNA of the base sequence of SEQ ID NO: 43 in the Sequence Listing, as long as it encodes a monoclonal antibody that has the ability to bind to RGMa, does not inhibit the binding of RGMa to Neogenin, and neutralizes the neurite outgrowth inhibitory activity of RGMa. The soybean polynucleotide and the complementary strand DNA of the base sequence of SEQ ID NO: 44 in the sequence listing The hybridization target may contain polynucleotides that hybridize under stringent conditions, such as those performed after Southern hybridization at 68°C, 0.1×SSC, and a salt concentration equivalent to 0.1% SDS.

[0116] The nucleic acid molecule of the present invention 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 nucleotide sequences of the heavy and light chain constant regions are as described in Nucleic Acids Research, vol. 14, p. 1779, 1986, The Journal of Biological Chemistry, vol. 257, p1516, 1982 and Cell vol. 22, p197, 1980 are preferred.

[0117] The nucleic acid molecules 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 encoding the antibody is amplified by PCR using oligonucleotides with sequences conserved in the variable regions of known human antibody heavy chain and light chain genes as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by PCR. The base sequence of the DNA 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.

[0118] The present invention also provides a recombinant vector containing the nucleic acid molecule of the present invention and a transformant (host cell) containing the recombinant vector. The vector may be a vector (e.g., pBR322, pUC119, or a derivative thereof) that can be expressed in prokaryotic cells such as pcDNA3.1 (manufactured by Invitrogen), but a vector that can be expressed in eukaryotic cells is preferred, and a vector that can be expressed in mammalian cells is more preferred. Examples of vectors that can be expressed in mammalian cells include plasmid vectors such as pcDNA3.1 (manufactured by Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, and pREP4, and viral vectors such as pDON-AI DNA (manufactured by Takara Bio). It may be one vector containing the heavy chain coding sequence and the light chain coding sequence, or two vectors, one containing the heavy chain coding sequence and the other containing the light chain coding sequence.

[0119] The transformant into which the recombinant vector of the present invention is introduced may be a prokaryotic cell such as Escherichia coli or Bacillus subtilis, but is preferably a eukaryotic cell, more preferably a cell derived from a mammal. Examples of mammalian cells include Chinese hamster ovary cells (CHO cells), COS, and mammalian cells. Examples include Eloma, BHK, HeLa, Vero, 293, NS0, Namalwa, and YB2 / 0.

[0120] The obtained anti-RGMa antibody or its antigen-binding fragment can be purified to homogeneity. Separation and purification of antibodies and the like can be performed using methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining methods such as affinity chromatography columns, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David L ane, Cold Spring Harbor Laboratory, 1988) Examples of columns used for affinity chromatography include, but are not limited to, Protein A columns, Protein G columns, anti-immunoglobulin antibody binding columns, and antigen binding columns. For example, Protein A columns include Hyper D, Examples include POROS and Sepharose FF (Amersham Biosciences).

[0121] <Preventive or therapeutic drugs for immunological and neurological diseases> The RGMa-binding proteins of the present invention, particularly anti-RGMa antibodies or antigen-binding fragments thereof, promote the restoration of neuronal function by neutralizing the neurite outgrowth inhibitory activity of RGMa, and can therefore be used as drugs for the prevention, treatment, or recurrence prevention of neurological diseases. The RGMa-binding proteins of the present invention, particularly anti-RGMa antibodies or antigen-binding fragments thereof, also neutralize T cell activation by RGMa and are therefore useful as drugs for preventing, treating, or preventing recurrence of immunological diseases. It can be used.

[0122] Neurological disorders include amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barré syndrome, and cerebral leukodystrophy. 、 Multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), neuromyelitis optica, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal tumors, transverse myelitis, dementia (including senile dementia, mild cognitive impairment, Alzheimer's disease, and Alzheimer's-related dementia), Huntington's chorea, tardive dyskinesia, mania, Parkinson's disease, Steele-Richard syndrome, Down's syndrome, myasthenia gravis, neurotrauma (including optic nerve trauma), vascular amyloidosis, cerebral hemorrhage with amyloidosis, cerebral infarction, encephalitis, acute confusional disorder, glaucoma, schizophrenia, and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, and X-linked Examples of the optic neuropathy include retinoschisis, drug-induced optic neuropathy, retinal dystrophy, age-related macular degeneration, eye diseases characterized by optic disc drusen, eye diseases characterized by genetic determinants of photoreceptor degeneration, autosomal recessive cone-rod dystrophy, and mitochondrial disorders associated with optic neuropathy. Preferred are spinal cord injury and nerve trauma (including optic nerve trauma).

[0123] Immunological diseases include multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis, and psoriatic arthritis), Guillain-Barré syndrome, neuro-Behçet's disease, pernicious anemia, type 1 (insulin-dependent) diabetes mellitus, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjögren's syndrome, Good-Basture's syndrome, Graves' disease, and autoimmune hemolysis. Examples of the disease include chronic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis. Preferably, it is multiple sclerosis.

[0124] The RGMa-binding proteins of the present invention, particularly anti-RGMa antibodies or antigen-binding fragments thereof, can be used as agents for the prevention, treatment, or recurrence prevention of neurological diseases / immunological diseases, and preferred neurological diseases / immunological diseases include spinal cord injury, neurotrauma (including optic nerve trauma), and multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, and secondary progressive multiple sclerosis).

[0125] As used herein, "treatment" includes any treatment of a disease in a mammal, particularly a human, and includes inhibiting disease symptoms, i.e., arresting its progression or eliminating the disease or condition, and alleviating disease symptoms, i.e., causing regression of the disease or condition or slowing the progression of the condition.

[0126] Furthermore, "prevention" includes preventing the onset of the above-mentioned diseases in mammals, particularly humans.

[0127] Furthermore, "prevention of recurrence" includes preventing the recurrence of the above-mentioned diseases that repeat remission and relapse in mammals, particularly humans.

[0128] The RGMa binding proteins (anti-RGMa antibodies or antigen-binding fragments thereof) of the present invention can be formulated into pharmaceutical compositions for the prevention or treatment of neurological or immunological diseases.

[0129] The administration route of the RGMa-binding proteins (anti-RGMa antibodies or antigen-binding fragments thereof) of the present invention is not particularly limited, and they can be administered to mammals, including humans, by any of the following routes: oral administration, parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, intracerebral administration, intraspinal administration, and other local administration).

[0130] Dosage forms for oral and parenteral administration and methods for preparing them are well known to those skilled in the art, and pharmaceutical compositions can be produced by combining the antibody of the present invention with a pharmaceutically acceptable carrier, etc. Dosage forms for parenteral administration include injectable preparations (e.g., drip infusion preparations, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral administration preparations, and intraspinal administration preparations), topical preparations (e.g., ointments, poultices, and lotions), suppository inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes. In particular, when direct action on central nervous tissue is desired, continuous injection can be performed using a medical micropump, which is an osmotic pump, or the preparation can be mixed with fibrin glue or the like to form a sustained-release preparation and then placed in the affected tissue.

[0131] For example, an injectable formulation is usually prepared by dissolving an antibody in distilled water for injection. In addition, solubilizing agents, buffering agents, pH adjusting agents, isotonicity adjusting agents, soothing agents, preservatives, stabilizers, etc. may be added. Also, it may be made into a freeze-dried preparation for immediate preparation.

[0132] Dosage forms for oral administration include solid or liquid dosage forms, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, injections, troches, etc.

[0133] The pharmaceutical composition of the present invention may further contain other therapeutically effective drugs, and may also contain ingredients such as bactericides, anti-inflammatory agents, vitamins, and amino acids, as needed.

[0134] Examples of pharmacologically acceptable carriers include excipients, lubricants, binders, and disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents in liquid preparations. Furthermore, conventional additives such as preservatives, antioxidants, coloring agents, sweeteners, adsorbents, and wetting agents can also be used appropriately in appropriate amounts, if necessary.

[0135] The dosage of the antibody of the present invention will be determined by a physician based on various factors such as the route of administration, type of disease, severity of symptoms, age, sex, and body weight of the patient, severity of the disease, pharmacological knowledge such as pharmacokinetics and toxicological characteristics, whether or not a drug delivery system is used, and whether the antibody is administered as part of a combination of other drugs. Typically, for an adult (body weight 60 kg), the oral dosage is 1 to 5,000 μg / day, preferably 10 to 2,000 μg / day, and more preferably 50 to 2,000 μg / day, and the injection dosage is 1 to 5,000 μg / day, preferably 5 to 2,000 μg / day, and more preferably 50 to 2,000 μg / day, administered in a single dose or in divided doses. For systemic parenteral administration, the dosage can be 10 to 100,000 μg / kg of body weight, more preferably 100 to 50,000 μg / kg, and even more preferably 500 to 20,000 μg / kg, administered once a day, once a week, or once a month, or 1 to 7 times a year. For local administration using an osmotic pump or the like, the dosage can be continuously infused at a rate of 10 to 100,000 μg / day, more preferably 100 to 10,000 μg / day, and even more preferably 500 to 5,000 μg / day per adult (body weight 60 kg). [Example]

[0136] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the embodiments of the following examples.

[0137] Example 1: Preparation of human RGMa protein (C-terminal domain) Histidine at the C-terminus of Pro169 to Gly422 (referring to the 169th proline residue from the N-terminus to the 422nd glycine residue, hereinafter the same) of human RGMa protein (SEQ ID NO: 1 in the Sequence Listing) CHO cells expressing the tag-fused recombinant human RGMa protein were established. The C-terminal domain of human RGMa protein contained in the culture supernatant of CHO cells was adsorbed onto a nickel column (GE Healthcare, 17-5247-01) and then eluted with 100 mM imidazole solution. The imidazole-eluted fraction was replaced with phosphate buffered saline (PBS) by dialysis, and the immunoprecipitation was performed. It was used as a base.

[0138] Example 2: Generation of mouse anti-human RGMa monoclonal antibodies 10 μg of the recombinant human RGMa protein prepared in Example 1 was diluted with complete Freund's adjuvant ( The emulsion was mixed with the antibody (Sigma) to prepare an emulsion, which was then injected subcutaneously into the back of BALB / c mice (Charles River Japan, Inc.) at several locations. 10 μg of recombinant human RGMa protein was emulsified with Bant (Sigma) in the same manner. After several immunizations, blood samples were collected. Antibody titers were measured using the ELISA method described below, in which human or mouse RGMa protein was immobilized. Individuals showing an increase in antibody titer were tested for human RGMa protein. The mice were boosted with 10 μg of protein intravenously, and splenocytes were harvested 2-3 days later. Cell fusion was performed using the splenocytes and half of the mouse myeloma cells (SP2 / 0, Sumitomo Dainippon Pharma). The mixture was mixed and centrifuged to obtain a precipitate, which was then treated with polyethylene glycol (Roche Diagnostics). Cell fusion was then carried out by adding 10% fetal bovine serum (Invitrogen) and 1% BM conditioned medium (Russian). The cells were resuspended in GIT medium (Nihon Pharmaceutical) containing 1000kJ / well of 5 × 10 cells / well of Sigma-Aldrich Co., Ltd. (Gibberish, Germany) and HAT (Sigma-Aldrich Co., Ltd.). 4 Myeloma cells were seeded at 1000 cells / well in a 96-well plate. The culture supernatant was collected, and antibody-producing cells were identified by the human RGMa protein solid-phase ELISA method described in Example 3. Screening was carried out. The antibody-producing cells obtained through screening were cloned by limiting dilution and then used to generate two types of antibodies. Hybridoma cells producing monoclonal antibodies (B5.116A3 and B5.70E4) were selected. The isotypes of both monoclonal antibodies determined using an isotyping kit (Mouse MonoAB ID / SP KIT, ZYMED, 93-6550) were mouse IgG2b for the heavy chain and κ for the light chain. there were. Monoclonal antibodies are purified by immobilizing anti-mouse IgG antibodies from the culture supernatant of hybridomas. Affinity chromatography using agarose (Sigma-Aldrich Anti-Mouse IgG-Agarose) After the antibody was bound to the column, the column was washed with PBS and then eluted with 10 mM glycine hydrochloride (pH 2.7) and immediately neutralized. The eluted neutralizing solution was then replaced with PBS using an ultrafiltration membrane.

[0139] Example 3: ELISA with immobilized human or mouse RGMa protein Human RGMa protein (R&D Systems, 2459-RM) or mouse RGMa protein (R&D Systems, 2458-RG) prepared at 2 μg / mL in PBS was dispensed at 50 μL / well into a 96-well plate and allowed to stand at room temperature for 1 hour. After removing the liquid, ApplieBlock (Biochemical Biobusiness) diluted 5-fold with PBS was added. 200 μL / well of 200150 (manufactured by the company) was dispensed and left to stand at room temperature for 1 hour to block non-specific binding. After washing three times with PBST (PBS containing 0.05% Tween 20), the specimens (mouse serum, 50 μL / well of a hybridoma culture supernatant, a recombinant antibody expression culture supernatant, or a purified antibody (described later) was added and left to stand at room temperature for 1 hour. After that, the plate was washed three times with PBST and then diluted with PBS. Diluted peroxidase-labeled sheep anti-mouse IgG antibody (GE Healthcare, NA9310V) was added for 50 min. After washing three times, a peroxidase color development kit (Sumitomo Bakelite Co., Ltd., ML-1130O) was added to the wells and allowed to develop color for a certain period of time. The absorbance at 492 nm was then measured using a plate reader.

[0140] Example 4: Antibody epitope analysis The epitope to which the antibody binds was determined by peptide scanning. The epitope is a sequence of 11 consecutive residues, shifted by three residues, contained in Arg172 to Ala424 of the human RGMa protein (SEQ ID NO: 1 in the Sequence Listing). A total of 83 peptides were synthesized, each consisting of an amino acid sequence consisting of the following: fused to the N-terminus of a biotinylated spacer sequence (SGSG) (SEQ ID NO: 46 in the Sequence Listing). The peptides were immobilized on an avidin plate and then reacted with the test antibodies (B5.116A3, B5.70E4). The mixture was reacted with a dextrose-labeled rabbit anti-mouse Ig antibody (Dako, P026002), and then a substrate solution was added. After allowing the color to develop for a certain period of time, the absorbance was measured using a plate reader. As a result, B5.116A3 has two types of Glu298-Gly311 (Glu298-Asp308 and Val301-Gly311). B5.70E4 bound to the peptides Glu298-Gly311 (two peptides Glu298-Asp308 and Val301-Gly311), Asn322-Glu335 (two peptides Asn322-Thr332 and Ile325-Glu335), and Pro349-Thr359 derived from human RGMa.

[0141] Example 5: Sequence analysis and cloning of mouse antibody genes Total RNA was extracted from hybridoma cells producing mouse monoclonal antibodies (B5.116A3, B5.70E4). cDNA was synthesized by reverse transcription using the total RNA as a template. Using the above as a template, genes for the light chain variable and constant regions, and the heavy chain variable and constant regions were amplified by PCR, and the DNA sequences were determined. Next, based on the determined variable and constant region sequences, full-length antibody genes were amplified by PCR and cloned. These antibody genes were The encoded amino acid sequence was as follows:

[0142] (1) B5.116A3 light chain amino acid sequence (SEQ ID NO: 4 in the Sequence Listing) DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQLNTLPWTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0143] (2) B5.116A3 heavy chain amino acid sequence (SEQ ID NO: 5 in the Sequence Listing) EVKLEESGGGLVQPGGSMKLSCAASGTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRSVYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVT VSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINCPP PCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIER TISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0144] (3) B5.70E4 light chain amino acid sequence (SEQ ID NO: 6 in the sequence listing) DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKL EIKRADAAPTVSIFPPSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0145] (4) B5.70E4 heavy chain amino acid sequence (SEQ ID NO: 7 in the sequence listing) DVKLQESGPGLVKPSQSLSLTCSVTGYSITTSYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKNQFFLRLNSVTTEDTATYYCAGSFGYSQGTLVTVSA AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINCPPC KECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERT ISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0146] Example 6: Preparation of recombinant mouse antibodies and recombinant rat-mouse chimeric antibodies Two types of hybridoma-derived recombinant mouse anti-RGMa antibodies, B5.116A3 and B5.70E4, were prepared (hereinafter referred to as "r116A3" and "r70E4," respectively). As a comparative example, a combination of the variable region of rat antibody 5F9 and the constant region of mouse antibody (IgG2bκ) (the light chain constant region is Arg108 to Cys214 of SEQ ID NO: 4 in the sequence listing) was used based on Patent Document 1 (WO2009 / 106356). The heavy chain constant region was fused to Ala117 to Lys452 of SEQ ID NO: 5 in the Sequence Listing, to prepare a recombinant rat-mouse chimeric antibody (hereinafter referred to as "r5F9"). DNA encoding the light and heavy chains of each antibody was cloned into pcDNA3.3 (Life Technologies) The expression vector was then introduced into HEK293F cells (Life Technologies) using Neofection 293 (Astec). The cells were incubated at 37°C in 8% CO₂ atmosphere. After culturing for 6 days under these conditions, the culture supernatant was collected. The recombinant antibody was purified by applying the culture supernatant to an affinity column (GE Healthcare) on which Protein A or Protein G was immobilized. The antibody bound to the column was eluted with 10 mM glycine hydrochloride (pH 2.8) and immediately neutralized. Then, the medium was replaced with PBS. If higher purity is required depending on the intended use, use antibody purified using Protein A column. The antibody was purified using a Ceramic Hydroxyapatite Type 1 (CHT) column (BIORAD). The antibody bound to the CHT column was washed with 10 mM KH2PO4 (pH 6.5) and then eluted with 20 mM KH2PO4 (pH 6.5), 0.5 M NaCl. The eluted fraction was collected and replaced with PBS.

[0147] Example 7: Binding test to RGMa protein-expressing cells Full-length human RGMa protein (Met1 to Cys450 of SEQ ID NO: 1 in the Sequence Listing), human RGMa protein C the C-terminal domain (Pro169 to Cys450 of SEQ ID NO: 1 in the Sequence Listing), the full-length mouse RGMa protein (Met1 to Trp454 of SEQ ID NO: 2 in the Sequence Listing), or the C-terminal domain of rat RGMa protein (SEQ ID NO: A vector expressing Pro170 to Trp449 (column 3) was introduced into CHO cells or HEK293 cells. Antigen-expressing cells were generated. During the GPI-anchor attachment reaction, the C-terminal peptide of the RGMa protein is processed. Both mouse and rat RGMa proteins are cleaved at Ala427, resulting in removal of the C-terminal peptide. Therefore, the amino acid sequences of the full-length protein and the C-terminal domain expressed on cells via the GPI anchor are identical between mouse and rat. The test antibodies (r116A3 and r70E4) and r5F9 (comparison) at a final concentration of 10 μg / mL were added to the above antigen After incubation with the antigen-expressing cells, the cells were washed with PBS containing 0.1% bovine serum albumin and 0.05% NaN3. FITC-labeled anti-mouse immunoglobulin antibody (DAKO) was added, and the cells were washed again. Fluorescence was measured using a flow cytometer (Becton Dickinson, FaxCalibur) to evaluate the binding of the test antibody to the antigen-expressing cells (Table 1). As a result, r116A3 and r70E4, unlike r5F9, showed a significant difference in the expression of human and rat RGMa proteins. The C-terminal domain of the RGMa protein alone was found to have an inhibitory effect on neurite outgrowth, and r116A3 and r70E4 were found to bind to both the full-length RGMa protein and the C-terminal domain. It inhibits both the cytosolic and C-terminal domains.

[0148] [Table 1]

[0149] Example 8: Dissociation constant measurement for RGMa protein The affinity of the test antibodies (r116A3, r70E4) and r5F9 (comparative example) for the RGMa protein was measured by surface plasmon resonance (SPR) using Proteon XPR36 (Bio-Rad). Human RGMa protein (R&D Systems, 2459-RM), human RGMa protein C-terminal domain (prepared in Example 1), or mouse RGMa protein (R&D Systems, 2458-RG) diluted to 10 μg / mL with 10 mM acetate buffer (pH 4.5) was coupled to the GLC sensor by the amine coupling method. A serially diluted test antibody was applied as an analyte at a flow rate of 100 μL / min for 60 seconds to measure the dissociation constant (Kd value). As shown in Table 2, r116A3 and r70E4, unlike r5F9, also bound to the C-terminal domain of human RGMa protein. r116A3 bound 32-fold more strongly to human RGMa protein and 44-fold more strongly to mouse RGMa protein than r5F9.

[0150] [Table 2]

[0151] Example 9: RGMa-Neogenin binding inhibition test The extracellular domain (Ala34 to Leu1105) of recombinant human neogenin protein (SEQ ID NO: 10) was purified. A CHO cell line expressing the extracellular domain of human neogenin protein was established. A histidine tag was fused to the C-terminus. The antibody was adsorbed onto a nickel column (GE Healthcare, 17-5247-01) from the culture supernatant of CHO cells and then eluted with 100 mM imidazole solution. The imidazole-eluted fraction was replaced with PBS by dialysis. Human RGMa protein (R&D Systems, 2459-RM) was biotinylated using the ChromaLink Biotin Labeling Kit (Solulink). Biotin-labeled human RGMa protein (prepared at 2 μg / mL) was used. The protein was mixed with an equal volume of the test antibody (r116A3, r70E4) diluted two-fold serially and incubated at room temperature for 2 hours. The mixture was reacted to prepare a mixed solution. At the same time, 50 μL / well of the extracellular domain of human neogenin protein prepared at 2 μg / mL in PBS was added to a 96-well plate and left to stand at room temperature for 1 hour to prepare a neogenin solid-phase plate. After removal, 2.5% bovine serum albumin solution was added and left to stand for 1 hour to block non-specific binding. 50 μL / well of the above mixed solution was added to this Neogenin solid-phase plate and left to stand at room temperature for 1 hour. After washing, peroxidase-labeled Avidin (VECTASTAIN ABC system, Vector Laboratories) was added and left to stand at room temperature for 1 hour. After washing, a substrate solution was added to allow color development for a certain period of time, and the absorbance was measured using a plate reader. The absorbance ratio in the absence of antibody was set to 1 and plotted to show the concentration-dependent RGMa binding by the antibody. -Neogenin binding inhibition was evaluated (Figure 1). As a result, anti-human RGMa polyclonal antibody (R&D Systems, AF2459) and r5F9 In contrast to r116A3 and r70E4, r116A3 and r70E4 did not inhibit RGMa-Neogenin binding.

[0152] Example 10: RGMa-BMP2 binding inhibition test Human RGMa protein (R&D Systems, 2459-RM) prepared at 2 μg / mL in PBS was added to a 96-well plate at 50 μL / well and left to stand at room temperature for 1 hour. 2.5% bovine serum albumin solution was added. The plate is then left to stand for 1 hour to block non-specific binding and prepare a plate with RGMa protein immobilized on it. Test antibodies (B5.116A3, B5.70E4) serially diluted to 0.01-10 μg / mL were added to the RGMa protein solid-phase plate and allowed to stand at room temperature for 1 hour. After washing, the plate was diluted to 0.5 μg / mL. Diluted human BMP2 protein (R&D Systems, 355-BM) was added and the mixture was left to stand at room temperature for 1 hour. Biotin-labeled anti-BMP2 antibody was reacted, and then peroxidase-labeled Avidin (VECTASTAIN ABC system, Vector Laboratories) and a substrate solution were added to allow color development for a certain period of time, and the absorbance was measured using a plate reader (Figure 2). As a result, in particular, the anti-RGMa antibody (B5.116A3) weakly inhibited RGMa-BMP2 binding in a concentration-dependent manner (absorbance 0.45 at 0.01 μg / mL, 0.4 at 0.1 μg / mL, 0.32 at 1 μg / mL, and 0.1 at 10 μg / mL).

[0153] Example 11: Design of humanized antibodies The humanization of the mouse monoclonal antibody B5.116A3 was performed by Winter et al., as described in Japanese Patent Publication No. 2912618. This was carried out by complementarity determining region (CDR) grafting according to the method of [SEQ ID NO: 1]. First, we created a 3D homology model of the light and heavy chain variable regions of the mouse monoclonal antibody B5.116A3, and identified amino acid residues located near the CDRs in the framework (FW) region. We selected human antibody FWs that preserved these amino acids as much as possible, and then grafted the CDRs of the mouse antibody onto them. A humanized antibody sequence was designed. The designed humanized antibody sequence is shown as HA for the heavy chain (SEQ ID NO: 11 in the Sequence Listing) and KA for the light chain (SEQ ID NO: 19 in the Sequence Listing). Furthermore, additional mutations were introduced into the amino acids in the FW involved in the structural stability of the variable region, and multiple humanized antibody sequences were designed (the heavy chain is shown as HA). There are a total of eight types of HB to HH, and seven types of light chains, including KA, from KB to KG.

[0154] [Table 3]

[0155] Example 12: Preparation of recombinant mouse-human chimeric and recombinant humanized antibodies (1) In accordance with Example 6, a recombinant mouse-human chimeric anti-RGMa antibody 116A3 (r116A3C) having the following amino acid sequence was prepared.

[0156] Light chain (SEQ ID NO: 8 in the sequence listing) DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQLNTLPWTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0157] Heavy chain (SEQ ID NO: 9 in the sequence listing) EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRS VYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0158] (2) In accordance with Example 6, a total of 20 types of humanized anti-RGMa antibodies consisting of the following combinations of heavy chains and light chains were prepared.

[0159] heavy chain / light chain combination; HA / KA, HA / KB, HA / KC, HA / KG, HB / KC, HC / KA, HC / KB, HD / KA, HD / KB, HD / KC, HD / KD, HE / KA, HF / KA, HF / KF, HF / KG, HG / KD, HG / KH, HH / KA, HH / KD, HH / KF

[0160] (3) A recombinant humanized anti-RGMa antibody (hereinafter referred to as rH5F9) was prepared according to Example 6 (comparison). example). The variable region of the humanized anti-RGMa monoclonal antibody h5F9 described in Patent Document 1 (WO2009 / 106356) The light chain sequence was sequence ID_53 of Patent Document 1, and the heavy chain sequence was sequence ID_50 of Patent Document 1) were compared with the human antibody constant region. The light chain was linked to a region ranging from Arg108 to Cys214 of SEQ ID NO: 26 in the Sequence Listing, and the heavy chain was linked to a region ranging from Ala117 to Lys446 of SEQ ID NO: 27 in the Sequence Listing).

[0161] Example 13: Antibody thermostability test The culture supernatant expressing the recombinant antibody described in Example 12 was taken in 20 μL aliquots and thermally cycled. The samples were heat-treated for 10 minutes at eight temperatures: 40, 45, 50, 55, 60, 65, 70, and 75°C using a hopper (Takara Bio, TP600). The culture supernatant was diluted with PBS to a final antibody concentration of 125 ng / mL. The antibody was then subjected to ELISA using the human RGMa protein immobilized as described in Example 3 to evaluate the antigen-binding ability of the antibody (Figure 3). As a result, a humanized antibody consisting of a combination of a heavy chain HE and a light chain KA (hereinafter referred to as "rH116A3") was obtained. The mouse-human chimeric antibody (r116A3C) and the humanized antibody (rH5F9) showed superior thermal stability. Hereafter, this humanized antibody consisting of the HE / KA combination will be referred to as rH116A3. Furthermore, when not heat-treated, the mouse-human chimeric antibody (r116A3C) and the humanized antibody (rH116A3) exhibited equivalent antigen-binding activity, and there was no decrease in antigen-binding activity due to humanization.

[0162] Example 14: Establishment of a stable CHO cell line producing humanized anti-RGMa antibody (rH116A3) A stable CHO cell line producing the humanized anti-RGMa antibody (rH116A3) was established using the Lonza GS Xceed system (Lonza). A pXC double gene vector containing the light chain coding sequence (SEQ ID NO: 44) and heavy chain coding sequence (SEQ ID NO: 43) of the humanized anti-RGMa antibody (rH116A3) was inserted into the vector. , and then transfected into the CHOK1SV GS knockout parent cell line and incubated under methionine sulphoximine (MSX) selection. A pool of transformed cells was obtained. After separating them into single cells by flow cytometry, the amount of antibody produced in the culture supernatant and cell proliferation were evaluated, and a stable CHO cell line was obtained.

[0163] Example 15: Neurite outgrowth assay The cerebellum was removed from newborn rats (P7) and placed in a trypsin solution (0.25% trypsin containing 0.2% DNase). The cells were suspended in a PBS solution and digested at 37°C for 10-15 minutes. The cells were resuspended in the same medium and centrifuged, and the same procedure was repeated twice to separate the cells. The cells were washed. The cell suspension was then filtered through a 70 μm cell strainer and centrifuged. The precipitated fraction was resuspended in the same medium. B27 supplement (GIBCO) was added to the cell suspension, and the Cerebellar granule cells were prepared from neonatal rats. Next, rat neonatal cerebellar granule cells were seeded on the cell plate and cultured at 37°C for 1 day. Recombinant RGMa protein (R&D Systems, 2459-RM) was added at a final concentration of 2 μg / mL, and the cells were cultured at 37°C for 2 days. The neurite length was measured by microscopic observation, and as shown in Figure 4, Addition of RGMa inhibited neurite growth, reducing neurite length from 37 μm to 26 μm in the experiment shown on the left, and from 38 μm to 27 μm in the experiment shown on the right. Neurite length remained unchanged when test antibodies (B5.116A3, B5.70E4) were added alone at a final concentration of 10 μg / mL. However, when test antibodies were added simultaneously with recombinant RGMa protein, neurite length increased to the same extent as in the control (no RGMa addition). Transepithelial growth was observed, demonstrating the neutralization of RGMa protein by the antibody.

[0164] Example 16: Efficacy test using spinal cord injury model rats Wistar rats (female, 8 weeks old, weighing approximately 200 g) were anesthetized with halothane (Takeda Pharmaceutical Co., Ltd.) by inhalation and underwent laminectomy of one vertebra (T8-T10) anterior and posterior to the spinal level T9. The spinal cord was exposed. When evaluating the spinal cord crush injury model, an IH impactor (manufactured by Precision System) was used. A pressure of 200 kdyn was applied to the exposed spinal cord using a . Immediately after spinal cord injury as described above, an osmotic minipump (200 μL volume, 0.5 μL / hour delivery, 14 days) (Alzet, model 2002) filled with 400 μg / mL of the test antibody (r116A3, r70E4) or control mouse antibody (mo-IgG2bκ) was placed subcutaneously on the back of the rat. The tip of a silicone tube connected to the outlet of the osmotic minipump was placed under the dura mater at the spinal cord injury site. The tube was sewn to the spinous process on the side of the limb immediately below the laminectomy site, and the muscle and skin layers were sutured and the rat was then placed in a stable position. The motor function of spinal cord injury model rats was evaluated using the Basso-Beattie-Bresnahan (BBB) ​​score (Basso, D. M., Beattie, MS, & Bresnahan, JC, A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12, 1-21 (1995)) The effects were evaluated on days 0, 1, 3, and 7 after wounding, and then every week for up to 8 weeks. As a result, as shown in Figure 5(A), r116A3 and r70E4 showed a significant improvement in the efficacy of the control antibody (mo-IgG2bκ) compared to the control antibody (mo-IgG2bκ) at 4 days after administration. The BBB score was significantly improved after 3 weeks (p<0.05, Student's t-test). When evaluating the spinal cord hemisection model, the exposed dorsal part of the spinal cord was incised to a depth of 1.8 to 2.0 mm. As described above, the test antibody (B5.116A3) or the control mouse antibody (mo-IgG2bκ) was administered using an osmotic minipump on days 0, 1, 3, and 7 after injury, and then every week for up to 10 days. As shown in Figure 5(B), B5.116A3 significantly improved the BBB score compared to the control antibody (mo-IgG2bκ) from 4 weeks after administration (p<0.01, Student's t-test).

[0165] Example 17: Efficacy test using multiple sclerosis model mice PLP 139-151 The peptide (HSLGKWLGHPDKF: SEQ ID NO: 45 in the Sequence Listing, manufactured by Peptide Institute) was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Factory) and mixed with incomplete Freund's adjuvant (manufactured by Sigma) containing killed Mycobacterium tuberculosis H37 Ra (manufactured by Difco Laboratories) to prepare an emulsion. 139-151SJL / JorllcoCrj (SJL / J) mice (Charles River Japan) were immunized subcutaneously in the dorsal region with 50 μg of peptide per head, and the EAE score was evaluated. (H. Kataoka, K. Sugahara, K. Shimano, K. Teshima, M. Koyama, A. Fukunari and K. Chiba. FTY720, a sphingosine 1-phosphate receptor modulator, ameliorates experimental autoimmune encephalomyelitis by inhibition of T cell infiltration. Cellular & Molecular Immunology 6, 439-448, 2005.) and changes in body weight were evaluated (Fig. 6). The test antibody (B5.116A3) diluted in saline was added to the PLP 139-151 The peptide was administered intraperitoneally at 20 mg / kg on days 7 and 10 or days 18 and 21 after immunization. As a result, as shown in Figure 6, the anti-RGMa mouse monoclonal antibody (B5.116A3) inhibited the RGMa expression level more than the control. Compared to the antibody (mo-IgG2bκ), administration before the onset of EAE suppressed the worsening of EAE scores (Fig. 6, upper panel), and administration after the onset of EAE showed a preventive effect on recurrence (Fig. 6, lower panel).

[0166] Example 18: Antibody immunogenicity testing Undifferentiated dendritic cells contained in peripheral blood from 51 healthy donors were matured by stimulation with granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin-4. The test antibody (rH116A3) was added to the mature dendritic cells at a final concentration of 50 μg / mL, and the cells were cultured for 4 to 5 days, allowing the antibody to be incorporated into the dendritic cells. The cells were then mixed with peripheral blood CD4+ T cells (helper T cells) from the same donor and incubated for another week. After co-culture, T cell proliferation was measured by flow cytometry. The immunogenicity risk in humans was assessed using the T cell proliferation activity of the test antibodies as an index. As a result, T cell proliferation was observed in 4 of 51 donors (7.8%), indicating a low immunogenicity risk.

Claims

1. the light chain comprises the amino acid sequence of SEQ ID NO:6; An isolated anti-RGMa antibody, or antigen-binding fragment thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

7.

2. A nucleic acid molecule encoding a protein portion of the anti-RGMa antibody or antigen-binding fragment thereof of claim 1.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A host cell comprising the recombinant vector of claim 3.

5. A method for producing an anti-RGMa antibody or antigen-binding fragment thereof described in claim 1, comprising the step of culturing a host cell described in claim 4.

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