RGMa BINDING PROTEIN AND USE THEREOF

Abstract

To provide an anti-repulsive guidance molecule a (RGMa) antibody having a high binding activity and few side effects which can be used as a medicine for preventing, treating, or preventing the relapse of neurological or immunological diseases.SOLUTION: The problem is solved by providing an isolated RGMa binding protein which does not inhibit binding between RGMa and neogenin but neutralizes the neurite outgrowth inhibiting activity of RGMa, preferably by providing an anti-RGMa antibody which has complementarity determining regions having amino acid sequences of SEQ ID NOS: 30-35 or SEQ ID NOS: 36-40 in Sequence Listing, 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 includes 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 after central nervous system injury in adult humans and rats in addition to during development, and because 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. These functions increase the adhesion of T cells to ICAM-1 and fibronectin, and also promote the release of cytokines In multiple sclerosis model mice, administration of anti-RGMa antibodies suppresses symptoms of encephalomyelitis and also suppresses onset and recurrence. Anti-RGMa antibodies bind to RGMa expressed in dendritic cells, thereby suppressing T cell activation 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 nerve cells and T cells. RGMa binds to Neogenin on the cell membrane, inducing the activation of RhoA and the inactivation of Ras in cells. By binding to RGMa, it has an inhibitory effect on neurite outgrowth. On the other hand, it is known that in the developing chick brain, Neogenin induces apoptosis in the absence of RGMa binding (Matsunaga et al., Dev. Growth Differ. 46, 481, 2004). It is believed to have two opposing effects: a favorable effect for nerve regeneration that promotes the survival of nerve cells, and a negative effect that inhibits neurite outgrowth.

[0005] As a medicine 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 using an anti-RGM antibody that regulates the binding of RGM to the neogenin receptor. Patent Document 4 discloses the medicinal use of an anti-RGM antibody for multiple sclerosis, etc. In addition, Patent Document 5 discloses the use 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 therapeutic applications of RGM modulators such as anti-RGM antibodies for spinal cord injury and multiple sclerosis, and Non-Patent Document 3 discloses therapeutic applications 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, may impair the inherent functions of RGMa, and have problems with side effects, etc. In particular, conventional antibodies may inhibit the binding between RGMa and Neogenin, thereby inhibiting the desirable effects of Neogenin bound to RGMa, such as the inhibition of apoptosis. Therefore, the present invention provides a method for the inhibition of neurite outgrowth by RGMa without inhibiting the interaction between RGMa and Neogenin. 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 have conducted extensive research to achieve the above object, and as a result have 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 have found that this protein can be used as a medicine for neurological and immunological diseases, thereby completing 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 described in [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 a peptide of 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 described in any one of [1] to [3] that binds to the peptides of sequence numbers 26 and 27 in the sequence listing. [5] An RGMa-binding protein described in any of [1] to [4] that 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 described in any of [1] to [4] that 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 of any 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 an 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 described in any one of [1] to [7], comprising a step of culturing the host cell described in

[10] .

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

[13] The pharmaceutical composition according to

[12] for use in the prevention, treatment, or prevention of recurrence 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, 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 patient 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 with amyloidosis, cerebral infarction, encephalitis, acute confusion disorder, glaucoma, schizophrenia, and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal dystrophies, 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 with optic neuropathy).

[15] Immunologic disorders 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 syndrome, Graves' disease, and autoimmune hemolysis. The pharmaceutical composition according to

[13] , 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.

[16] The pharmaceutical composition according to

[13] , 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).

[17] The amino acid sequences of light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3), heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3) are LCDR1: RASQDISSYLN (Sequence Listing SEQ ID NO: 30) 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 (Sequence Listing SEQ ID NO: 36) 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. Alternatively, the antibody may be an isolated anti-RGMa antibody, or an antigen-binding fragment thereof.

[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 amount of the amino acid sequence An anti-RGMa antibody or antigen-binding fragment thereof described in

[17] , comprising an amino acid sequence that has at least 90% identity.

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

[17] or

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

[20] Any of the following:

[17] to

[19] , where the anti-RGMa antibody has a human IgG constant region. A described 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 described in any one of

[17] to

[20] .

[23] A nucleic acid sequence encoding the amino acid sequence of VH and VL. VH:gaagtgcagctggtggaatctggcggcggactggtgcagcctggcagatccctgagactgtcctgtaccgcctccggcttcaccttctccgacgcctggatggatgggtgcgacaggctcctggcaagggcctggaatgggtggccgagatccggtccaaggccaacaaccacgccacc and The nucleic acid molecule according to

[22] , which is a nucleic acid sequence comprising VL: gacatccagatgacccagtccccctccgtgtctgcttccgtgggcgacagagtgaccatcacctgtcgggcctcccaggacatctcctcctacctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctactacacctcccggctgcactccggcgtgccctctagattttccggctctggctccggcaccgactttaccctgaccatctccagcctgcagcccgaggacttcgcctcctacttctgtcagcagctgaacaccctgccctggacctttggcggaggcaccaaggtggaaatggaa (sequence number 44 in the sequence listing).

[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 described in any one of

[17] to

[20] .

[28] The pharmaceutical composition according to

[27] for use in the prevention, treatment or prevention of recurrence 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 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-Richards syndrome, Dow Jones Inflammatory Bowel Syndrome, and Parkinson's Disease. glaucoma, myasthenia gravis, neurotrauma (including optic nerve trauma), vascular amyloidosis, cerebral hemorrhage with amyloidosis, cerebral infarction, encephalitis, acute confusion disorder, glaucoma, schizophrenia, and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal dystrophies, 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, mitochondrial disorders with optic neuropathy).

[30] Immunologic disorders 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

[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 the step of administering an effective amount of an anti-RGMa antibody or antigen-binding fragment thereof described in any of

[17] to

[20] to a subject in need thereof. Effect 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, etc. Therefore, the humanized anti-RGMa antibody of the present invention has a strong protective effect on nerve cells and is less likely to cause side effects associated with a decrease in nerve cells. 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 medicine for neurological and immunological diseases with excellent efficacy and few side effects. [Brief description 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). [Diagram 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). [Diagram 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)). [Diagram 5] 1 shows the results of efficacy tests using control mouse IgG (mo-IgG2bk) and the antibodies of the present invention (r70E4, r116A3) in spinal cord injury rat models, (A) showing the results of efficacy tests in a spinal cord crush model, and (B) showing the results of efficacy tests in a spinal cord hemisection model. [Figure 6] 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, in which the left side shows the EAE score and the right side shows the change in body weight, with the upper panel showing the results when the test antibody was administered 7 and 10 days later and the lower panel showing the results when the test antibody was administered 18 and 21 days later. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] To facilitate understanding of the present invention, the following describes terms used in the present invention.

[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 (which refers to the peptide from the first methionine residue from the N-terminus to the proline residue just before the 47th residue, hereafter the same) is present at the N-terminus. The peptide bond between Asp168 and Pro169 is cleaved, the C-terminal peptide Arg423-Cys450 is removed, and a GPI anchor is added to the C-terminal carboxyl group of the C-terminal 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-Asp168) and the C-terminal domain (Pro169-Ala424) are linked by a disulfide bond. The precursor protein of mouse RGMa consists of the amino acid sequence shown in SEQ ID NO:2 in the sequence listing, and the precursor protein of rat RGMa consists of the amino acid sequence shown in SEQ ID NO:3 in the sequence listing, but since the C-terminal peptide is removed, the mature proteins have the same amino acid sequence. In the present invention, RGMa may refer to either a precursor protein, a mature protein, or an active fragment thereof, or may be a derivative or mutant thereof, as long as it acts by binding to Neogenin, which will be described later. In addition, human RGMa or RGMa derived from another organism may be used, but 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 1461 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 any of a precursor protein, a mature protein, or an RGMa-binding fragment thereof, or a derivative or mutant thereof, so long as it binds to RGMa. In addition, human neogenin or neogenin derived from another organism may be used, but human neogenin is preferred.

[0016] [Neutralization] As used herein, "neutralization" refers to the ability to bind to a target of interest and inhibit any of the functions 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 test described herein.

[0017] [Isolated] "Isolated" in the context of isolated RGMa binding protein, etc., means identified and separated and / or recovered from components in their natural state. Impurities in the natural state are substances that may interfere with diagnostic or therapeutic uses of the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, to isolate an RGMa binding protein, etc., it is sufficient to purify it by at least one purification step, and an RGMa binding protein purified by at least one purification step can be referred to as an "isolated RGMa binding protein."

[0018] [RGMa binding protein] In the present application, the term "RGMa-binding protein" refers to a molecule containing a protein that binds to RGMa. Examples of RGMa-binding proteins include anti-RGMa antibodies and antigen-binding fragments thereof, RGMa-binding scaffold proteins, soluble RGMa receptor proteins such as the extracellular domain of neogenin, and fusion proteins thereof. The term RGMa-binding scaffold protein refers to a protein that achieves the binding function with RGMa by introducing a mutation into the Kunitz domain of a serine protease inhibitor, the extracellular domain of human fibronectin, ankyrin, lipocalin, or the like. The term fusion protein refers to a protein that combines the RGMa-binding protein with 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 that binds to the RGMa of the present application. This refers to 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 refers to an antibody in which both the light chain and the heavy chain are derived from human immunoglobulin. Depending on the difference in the constant region of the heavy chain, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) having a γ heavy chain, IgM having a μ heavy chain, IgA (including IgA1 and IgA2) having an α heavy chain, IgD having a δ heavy chain, or IgE having an ε heavy chain. In principle, the light chain includes either a κ chain or a λ chain.

[0020] [Humanized antibody] A humanized antibody refers to an antibody that is composed of 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 a variable region of non-human origin and a constant region 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 of a multispecific antibody include a bispecific antibody, which has two antigen specificities, and a trispecific antibody, which has 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 a cell-killing function, a complement activation function, or a function to extend the blood half-life, have been modified mainly by modifying the amino acids or glycans in the Fc region of the antibody.

[0025] [Conjugated antibodies] In the present 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. It refers to an antibody that has been chemically or genetically linked to a target antigen.

[0026] [Antigen-binding fragment] In the present application, an antigen-binding fragment refers to a protein that contains a portion of an antibody and can bind 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 linked.

[0027] [Complementarity determining region] Complementarity determining region (CDR) refers to the region of the variable region of an immunoglobulin molecule that forms an antigen binding site, also called hypervariable region, which refers to a portion in which there is particularly large variation in amino acid sequence for each immunoglobulin molecule. There are three CDRs (LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3) in each light chain and heavy chain. In this application, the CDRs of an immunoglobulin molecule are numbered according to the Kabat numbering system (Kab at et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and The time course of treatment is determined according to the National Institutes of Health (NIH, USA).

[0028] [Percent (%) identity of amino acid sequence] "Percent (%) identity" with respect to an identified reference polypeptide sequence, such as a variable region, is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the particular reference polypeptide sequence after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent identity can be performed by a variety of methods within the skill of the art, such as by using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. It can be achieved. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve the maximum alignment for the full length of the sequences being compared. However, for the purposes here, the % identity value is obtained by using the sequence comparison computer program BLAST in a pairwise alignment. In the situation where BLAST is used for amino acid sequence comparison, the % identity of a given amino acid sequence A 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 with a score that matches as identical by the program alignment of A and B by the sequence alignment program BLAST, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % identity of A to B will be different from the % identity of B to A. Unless otherwise specified, all % identity values here are obtained using the BLAST computer program as shown in the 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 causes a significant decrease in the binding of the anti-RGMa antibody of the present invention to 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 of 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 for example, an example of a human RGMa protein is a protein having the amino acid sequence of SEQ ID NO: 1 in the sequence listing, an example of a mouse RGMa protein is a protein having the amino acid sequence of SEQ ID NO: 2 in the sequence listing, and an example of a rat RGMa protein is a protein having the amino acid sequence of SEQ ID NO: 3 in the sequence listing. In addition, the RGMa protein may be a polypeptide comprising 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 which has 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 the effect of inhibiting neurite outgrowth.

[0033] The above amino acid substitution is preferably a conservative substitution. Here, the term "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, a hydrophobic residue is replaced with another hydrophobic residue. Examples of functionally similar amino acids that can be substituted in this way include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc.; polar (neutral) amino acids such as glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc.; positively charged (basic) amino acids such as arginine, histidine, lysine, etc.; and negatively charged (acidic) amino acids such as aspartic acid, glutamic acid, etc.

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

[0035] As a mature protein, RGMa protein consists of an N-terminal domain and a C-terminal domain, but the C-terminal domain alone has neurite outgrowth inhibitory activity. It is preferable that the RGMa-binding protein of the present invention binds only to 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 the upper limit being, for example, 10 -8 M or less, more 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 protein of the present invention does not inhibit the binding between RGMa and Neogenin. Here, "does not inhibit the binding between RGMa and Neogenin" means that in the binding system between RGMa and Neogenin shown in the Examples below, the binding between RGMa and Neogenin is not substantially reduced even when the concentration of the RGMa binding protein is increased. For example, when the RGMa binding protein is added to the binding system between RGMa and Neogenin and the concentration is increased, if the RGMa binding protein concentration showing IC50 is 10 μg / mL or more, more preferably 50 μg / mL or more, and most preferably 100 μg / mL or more, it can be said that the RGMa binding protein does not inhibit the binding between RGMa and Neogenin.

[0037] The neogenin used in the binding test with RGMa is preferably the same type of neogenin 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 having an amino acid sequence that is 90% or more (preferably 95% or more) identical to SEQ ID NO: 10 in the sequence listing may be used as long as it can bind to RGMa.

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

[0039] The amino acid sequence of the RGMa protein varies depending on the animal species, so there are also differences in the amino acid sequence between human RGMa shown in SEQ ID NO:1 in the sequence listing, mouse RGMa shown in SEQ ID NO:2 in the sequence listing, and rat RGMa shown in SEQ ID NO:3 in the sequence listing. 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. -7 M or less, more 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 protein of the invention preferably has excellent thermal stability, which can be evaluated based on the decrease in binding to RGMa due to heat treatment, and is preferably stable even when heat-treated at 60°C or higher, more preferably stable even when heat-treated at 65°C or higher, and most preferably stable even when heat-treated 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 for example, in human RGMa, it is 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), 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 preferable to bind to SEQ ID NO:26 and SEQ ID NO:27 in the sequence listing, and it is more preferable to bind to SEQ ID NO:26 and SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:29 in the sequence listing.

[0042] RGMa-binding proteins specifically 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 a mammal such as a mouse with an antigen, 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 recombinant gene technology, and human antibodies produced using transgenic animals producing human antibodies, etc. When the antibodies of the present invention are administered to humans as a pharmaceutical, humanized or human antibodies are preferable from the viewpoint of side effects.

[0044] The antigen may be used for immunization as it is, or may be used as a complex with a carrier protein. Condensing agents such as glutaraldehyde, carbodiimide, and maleimide active ester can be used to prepare the complex of the antigen and carrier protein. Examples of carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.

[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 Freund's adjuvant or incomplete Freund's adjuvant, and is usually administered once every 2 to 5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of an immunized animal 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 existing general production methods. That is, for example, a mammal as described above is immunized with the above-mentioned antigen together with Freund's adjuvant as necessary, and serum from the immunized animal is obtained. 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 hindpaw of the above-mentioned mammals, if necessary, together with Freund's adjuvant. Immunization is performed by intraperitoneal or intraperitoneal injection or transplantation once or several times. Usually, immunization is performed 1 to 4 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] A "hybridoma" secreting a monoclonal antibody can be prepared according to the method of Kohler and Milstein et al. (Nature, 256, 495, 1975) or a modified method similar thereto. That is, the hybridoma can be prepared by fusing antibody-producing cells contained in the spleen or the like obtained from an immunized mammal with myeloma cells not capable of producing autoantibodies derived from a mammal, preferably a mouse, rat or human.

[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 / O, 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 carried out by reacting the cells using 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, with the ratio of antibody-producing cells to myeloma cells usually being about 1:1 to 10:1, for about 1 to 10 minutes.

[0052] Hybridoma clones that produce monoclonal antibodies can be screened 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 a binding assay with the RGMa protein, an evaluation is also performed as to whether the antibody inhibits the binding of the RGMa protein to Neogenin and whether the antibody neutralizes the function of the RGMa protein (neurite outgrowth inhibitory activity). By these screening methods, the anti-RGMa antibody of the present invention can be selected.

[0054] Cloning can be performed by limiting dilution from wells containing hybridomas that produce the desired antibody to obtain clones. Selection and breeding of hybridomas is usually performed 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 a culture medium or grown in vivo in ascites fluid of a mammal such as a mouse or rat, and isolated from the resulting culture supernatant or from the ascites fluid 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'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. The basal media may contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances, depending on the purpose.

[0058] Monoclonal antibodies can be isolated and purified by subjecting the above-mentioned culture supernatant or ascites to saturated ammonium sulfate, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography such as an anti-immunoglobulin column or protein A column, etc. Specifically, purification of monoclonal antibodies may be performed using known methods for purifying immunoglobulins, and can be easily achieved by, for example, ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, the use of an anion exchanger, affinity chromatography using RGMa protein, etc.

[0059] Monoclonal antibodies can also be obtained by phage display. In the phage display method, phages selected from any phage antibody library are screened using the target immunogen to select phages having the desired binding affinity to the immunogen. Next, the antibody-corresponding sequence contained in the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding an antibody or an antigen-binding domain is constructed based on the isolated sequence or the sequence information determined. Then, a cell line transfected with such an expression vector is cultured to produce a monoclonal antibody. By using a human antibody library as the phage antibody library, a human antibody having 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 in which functional molecules other than the RGMa-binding protein of the present application are chemically or genetically linked to the RGMa-binding protein, 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, other antibodies, etc.

[0062] When PEG is attached as a functional molecule, the PEG may have a molecular weight of, but is 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 the RGMa-binding protein, for example, by using an NHS active 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.

[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 are used.

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

[0066] When an enzyme is used as a 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, etc.), 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 Linkers and alkoxy represented by (CR2)n- (R is an arbitrary substituent, n is a positive integer) These include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for binding functional molecules have already been established in the field (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 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. An example of a "chimeric antibody" is a chimeric antibody whose variable region is derived from immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.) and whose constant region is derived from human immunoglobulin. For example, a chimeric antibody can be produced by immunizing a mouse with an antigen, excising a variable region that binds to the antigen from the gene of the mouse monoclonal antibody, and combining it with an antibody constant region derived from human bone marrow. The constant region derived from human immunoglobulin has a unique amino acid sequence depending on the isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE, and the constant region of the recombinant chimeric antibody of the present invention may be the constant region of human immunoglobulin belonging to any isotype. Preferably, it is the constant region of human IgG. An expression vector can be produced using the gene of the chimeric antibody produced in this way. A host cell is transformed with the expression vector to obtain a transformed cell producing a chimeric antibody, and the transformed cell is cultured to obtain the desired chimeric antibody from the culture supernatant.

[0069] Another preferred embodiment of the RGMa-binding protein of the present invention is a humanized antibody. In the present invention, a "humanized antibody" is an antibody in which only the DNA sequence of the antigen-binding site (CDR; complementarity determining region) of a non-human animal antibody such as a mouse is grafted onto a human antibody gene (CDR grafting). For example, see JP-A-4-506458 and JP-A-2912618. It can be prepared by referring to the method described in the specification, etc. Specifically, it means a humanized antibody characterized in that a part or all of its CDRs are CDRs derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), the framework region of its variable region is a framework region of a variable region derived from human immunoglobulin, and the constant region is a constant region derived from 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 portion and DNA of the mouse light chain CDR portion are isolated from a hybridoma producing a mouse monoclonal antibody, and a human heavy chain gene covering the entire region other than the human heavy chain CDR and a human light chain gene covering the entire region other than 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. A host cell is transformed with the expression vector thus prepared to obtain a humanized antibody-producing transformed cell, and the transformed cell is cultured to obtain the desired humanized antibody from the culture supernatant.

[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 an immunoglobulin, including the heavy chain variable region, the heavy chain constant region, and the light chain variable region, and the light chain constant region, are immunoglobulins 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 gene locus of a mammal other than a human, such as a mouse, can be produced by immunizing the animal with an antigen in the same manner as in the production method of a polyclonal or monoclonal antibody described above.

[0074] For example, transgenic mice producing human antibodies can be prepared according to the methods described in Nature Genetics, Vol.7, p. 13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; 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, p.856-859, 1994; and Published Japanese Translation of PCT International Publication No. H6-500233. More specifically, HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice, etc. are included.

[0075] Specifically, the monoclonal antibody of the present invention has a heavy chain variable region CDR having the sequence shown in the sequence listing. The light chain variable region comprises 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 NO: 30 (LCDR1), 31 (LCDR2) and 32 (LCDR3) in the sequence listing. In addition, as long as the antibody of the present invention maintains the characteristics 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, 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. The amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention. The expression "the antibody characteristics are maintained" means that these characteristics are maintained by the amino acids in the CDR. It 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 "improvement."

[0076] There are no particular restrictions on the sequence of the regions other than the CDRs, so long as they maintain the structure of the antibody and can exert their functions. The constant region may be any of a mouse-derived sequence, a human-derived sequence, a sequence derived from another mammal, a chimeric sequence thereof, and an artificial sequence. In the case where the constant region is included, the amino acid sequences of the heavy and light chain constant regions are exemplified by those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[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 an antibody having the amino acid sequence of SEQ ID NO: 5 in the sequence listing in its heavy chain. These amino acid sequences may have 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 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. Such substitutions, deletions, or 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.

[0078] Further, among the above mouse antibodies, a mouse / human chimeric antibody in which the constant region is derived from human is also exemplified. As such a mouse / human chimeric antibody, an antibody having an amino acid sequence of SEQ ID NO: 8 in the sequence listing (variable region 1-107) in the light chain and an amino acid sequence of SEQ ID NO: 9 in the sequence listing (variable region 1-116) in the heavy chain is exemplified. In these amino acid sequences, one or several amino acids (1-20, 1-10, or 1-5) may be substituted, deleted, added, or inserted, as long as 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 are maintained. Such substitutions, deletions, and additions may be introduced into the CDR, but are preferably introduced into a region other than the CDR. Moreover, the amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0079] Further, humanized antibodies in which the CDRs are not derived from humans are also exemplified. Examples of such antibodies include antibodies having an amino acid sequence in the heavy chain of any one of SEQ ID NOs: 11 to 18 in the Sequence Listing (the variable region is up to 116 residues on the N-terminus side), and an amino acid sequence in the light chain of any one of SEQ ID NOs: 19 to 25 in the Sequence Listing (the variable region is up to 107 residues on the N-terminus side). In addition, 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 have substitutions, deletions, additions or insertions of one or several amino acids (1 to 20, 1 to 10 or 1 to 5), so long as they 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. 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 the 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 in the sequence listing, and the amino acid sequence corresponding to the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing. That is, 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 are involved in the binding of RGMa to Neogenin. As long as the property of not inhibiting the binding of RGMa and neutralizing the neurite outgrowth inhibitory activity of RGMa 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 CDRs, but are preferably introduced into a region other than the CDRs. Furthermore, the amino acid substitutions are preferably conservative substitutions in order to maintain the properties of the present invention. The amino acid sequence of the antibody of the present invention, which includes substitutions, deletions, etc. in the amino acid sequence of SEQ ID NO:41 and / or SEQ ID NO:42 in the sequence listing, is an amino acid sequence in which the heavy chain variable region has 90% or more identity (more preferably 95%, 96%, 97%, 98%, 99% or more) with SEQ ID NO:41 in the sequence listing, and the light chain variable region has 90% or more identity (more preferably 95%, 96%, 97%, 98%, 99% or more) 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 having the amino acid sequences of SEQ ID NOs: 39 (HCDR1), 40 (HCDR2) and SFG (HCDR3), and those having the amino acid sequences of SEQ ID NOs: 36 (LCDR1), 37 (LCDR2) and 38 (LCDR3) in the CDRs of the light chain variable region. Note that one or more amino acids may be substituted in one or more of these CDRs, as long as the characteristics of the antibody of the present invention, that is, having the ability to bind to RGMa, not inhibiting the binding of RGMa to Neogenin, and neutralizing the neurite outgrowth inhibitory activity of RGMa, are maintained. Here, 1 to several means, for example, 1 or 2. The amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention. There are no particular restrictions on the sequence of the regions other than the CDRs, so long as they maintain the structure of the antibody and can exert their functions. The constant region may be any of a mouse-derived sequence, a human-derived sequence, a sequence derived from another mammal, a chimeric sequence thereof, and an artificial sequence. In the case where the constant region is included, the amino acid sequences of the heavy and light chain constant regions are exemplified by those described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982, and Cell vol.22, p197, 1980.

[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 an antibody having, in its heavy chain, the amino acid sequence of SEQ ID NO: 7 in the sequence listing. In these amino acid sequences, one or several amino acids (1 to 20, 1 to 10, or 1 to 5) may be substituted, deleted, added, or inserted, so long as 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 are maintained. 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.

[0083] Other examples of the above-mentioned mouse antibodies include chimeric antibodies in which the constant region is derived from human, and humanized antibodies in which the regions other than the CDRs are derived from 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 as LCDR1, SEQ ID NO: 31 in the Sequence Listing as LCDR2, SEQ ID NO: 32 in the Sequence Listing as LCDR3, SEQ ID NO: 33 in the Sequence Listing as HCDR1, SEQ ID NO: 34 in the Sequence Listing as HCDR2, and SEQ ID NO: 35 in the Sequence Listing as 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 as a heavy chain variable region and SEQ ID NO: 42 as a light chain variable region).

[0085] The anti-RGMa antibody of the present invention can be used to prepare a multispecific antibody such as a bispecific antibody by binding to an antibody having an antigen-binding specificity other than RGMa by genetic engineering techniques. Such genetic engineering techniques are well known in the art. For example, DVD-Ig (Wu et al., Nature 2013) is a DNA fragment in which variable regions are linked in series. Biotechnology 25(11), 1290(2007)) and by modifying the Fc region of the antibody, The ART-Ig technology (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), which combines the heavy chains of two types of antibodies that bind to the same antigen, can be used to create the desired bispecific antibodies. 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] The modified molecules of the anti-RGMa antibody of the present invention include functionally modified antibodies. The 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 mainly by modifying the Fc region, etc. (Shitara Kenya, Yakugaku Zasshi, 2009, Vol. 129(1), p. 3; Ishii Akiko et al., Nihon Yakurigaku Zasshi, 2010, Vol. 136(5), p. 280; Hashiguchi Shuhei et al., Seikagaku, 2010, Vol. 82(8), p. 710).

[0087] Functionally modified anti-RGMa antibodies are prepared by the following methods. For example, when the present anti-RGMa antibody is produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as a host cell, an antibody with a reduced fucose content in the glycan and enhanced cell killing function is obtained, and when the antibody is produced using CHO cells in which the FUT8 gene has been introduced as a host cell, an antibody with reduced cell killing function is obtained (WO 2005 / 035586, WO 2002 / 31140, WO 00 / 61739). In addition, 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 half-life in blood can be extended by using a mutant Fc region that enhances binding to FcRn, one of the Fc receptors (Shuhei Hashiguchi et al., Seikagaku, 2010, Vol. 82(8), p. 710). These functionally modified antibodies can be produced by genetic engineering techniques.

[0088] Conjugated antibodies are examples of modified molecules of the anti-RGMa antibody of the present invention. Conjugated antibodies include anti-RGMa antibodies and the like that are combined with non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, other antibodies, and other compounds that are not part of the anti-RGMa antibody of the present invention. Examples of such antibodies include conjugated antibodies to which functional molecules other than antibodies are chemically or genetically bound.

[0089] When PEG is bound as a functional molecule, the PEG may have a molecular weight of, but not limited to, 2000 to 100000 Da, more preferably 10000 to 50000 Da, and may be either linear or branched. PEG can be bound 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 by the Chloramine T method, etc. It can be directly bonded.

[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 are used.

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

[0093] When an enzyme is used as a 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, etc.), 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 Linkers and alkoxy represented by (CR2)n- (R is an arbitrary substituent, n is a positive integer) These include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for binding functional molecules have already been established in the field (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 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 means a partial region of the antibody having antigen-binding ability as 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. Further, the antigen-binding fragments include non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, other antibodies, and the like, which are anti-RG antibodies of the present invention. These include conjugated antigen-binding fragments to which functional molecules other than Ma antibodies are chemically or genetically linked.

[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 antibody 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. Each of these two homologous antibody fragments is called Fab. When the IgG is treated with pepsin, the disulfide bond between the two heavy chains in the hinge region is cleaved downstream to produce an antibody that is slightly larger than the one in which the two Fabs are connected at the hinge region. It is possible to produce an antibody fragment 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. Conjugated antigen-binding fragments include functional molecules other than the anti-RGMa antibody of the present application, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, and other antibodies, in a partial region of the anti-RGMa antibody that has antigen-binding activity. and 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 is not limited to, 2000 to 100000 Da, more preferably 10000 to 50000 Da, and may be either linear or branched. PEG can be attached to the N-terminal amino group of a portion of the region 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 as anti-RG antibodies by 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 are used.

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

[0102] When an enzyme is used as a 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, etc.), 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 Linkers and alkoxy represented by (CR2)n- (R is an arbitrary substituent, n is a positive integer) These include repeating units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (e.g., polyethyleneamino, Jeffamine™), as well as diacid esters and amides (succinate, succinamide, diglycolate, malonate, caproamide, etc.). Chemical modification methods for binding functional molecules have already been established in the field (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

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

[0105] The present invention relates to an antibody having a specific CDR amino acid sequence as described above, and an RGMa protein. Also included are anti-RGMa antibodies, and antigen-binding fragments thereof, that compete for binding with An antibody having the above-mentioned specific CDR amino acid sequence and an antibody that competes 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 has the following structure in a binding system between an antibody having the above-described CDR sequence and an RGMa protein: By allowing them to coexist, they can be acquired (screened) and evaluated. For example, they can be acquired 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 the avidin-immobilized sensor chip, immobilizing 1300 to 1600 RU of human RGMa protein. Next, any 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 is repeated multiple times to create a state in which any anti-RGMa antibody binds to all of the human RGMa protein molecules on the sensor chip (saturation state), and the amount of binding at the saturation state (saturation binding amount 1) is determined. A similar experiment is also carried out with an anti-RGMa antibody containing a specific CDR amino acid sequence of the present invention, and the binding amount 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 a specific CDR amino acid sequence 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 a specific CDR amino acid sequence of the present invention. If any anti-RGMa antibody can bind to a human RGMa protein saturated with an anti-RGMa antibody containing an amino acid sequence of a specific CDR of the present invention while exhibiting the saturation binding amount 1 of the anti-RGMa antibody calculated above, the antibody is judged to be "non-competitive". On the other hand, if any anti-RGMa antibody cannot bind to a human RGMa protein saturated with an anti-RGMa antibody containing an amino acid sequence of a specific CDR of the present invention, the antibody is judged to be "competitive". Also, even if any anti-RGMa antibody can bind to a human RGMa protein saturated with an anti-RGMa antibody containing an amino acid sequence of a specific CDR of the present invention, if the additional binding amount does not reach the saturation binding amount 1 with a significant difference, the antibody is judged to be "competitive". The significant difference is examined by a general test method (e.g., Student's t-test), and the significance level is 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 specific CDR described above 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 which 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 having a base sequence encoding the amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38 in the sequence listing (in which one or several amino acids may be substituted, deleted, inserted, or added), respectively.

[0109] In another embodiment of the nucleic acid molecule of the present invention, the heavy chain coding region comprises a nucleotide sequence coding for the amino acid sequence of SEQ ID NO:5 in the sequence listing, and the light chain coding region comprises a nucleotide sequence coding for the amino acid sequence of SEQ ID NO: and a polynucleotide in which the region encoding the heavy chain contains a base sequence encoding the amino acid sequence of SEQ ID NO:7 in the sequence listing and the region encoding the light chain contains a base 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 the nucleic acid molecule 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 set forth in 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 set forth in 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 contains 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 contains 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 has the ability to bind to RGMa, does not inhibit the binding of RGMa to Neogenin, and encodes a monoclonal antibody that neutralizes the neurite outgrowth inhibitory activity of RGMa, and hybridizes under stringent conditions with the complementary strand DNA of the base sequence of SEQ ID NO: 43 in the sequence listing. The polynucleotide sequence of soybean and the complementary strand DNA of the base sequence of SEQ ID NO: 44 in the sequence listing are The hybridization target may include a polynucleotide that hybridizes under stringent conditions, for example, conditions in which Southern hybridization is followed by washing at 68° C. with a salt concentration equivalent to 0.1×SSC and 0.1% SDS.

[0116] The nucleic acid molecule of the present invention may encode all of the constant and variable regions of the heavy and light chains, or may encode only the variable regions of the heavy and light chains. When encoding all of the constant and variable regions, the base sequences of the heavy and light chain constant regions are as described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell vol. 22, p197, 1980 are preferred.

[0117] The nucleic acid molecule 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 by reverse transcriptase using random primers or the like. Next, cDNA encoding an antibody is amplified by PCR using oligonucleotides of sequences conserved in the variable regions of known human antibody heavy chain genes 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 a conventional method, such as by incorporating it into a plasmid for sequencing. Alternatively, DNA encoding the monoclonal antibody of the present invention can be obtained by chemically synthesizing the sequence of the variable region or a portion thereof and linking 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 expressible in a prokaryotic cell such as pBR322, pUC119, or a derivative thereof, but a vector expressible in a eukaryotic cell is preferred, and a vector expressible in a mammalian cell is more preferred. Examples of vectors expressible in a mammalian cell 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 it may be 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 cells derived from a mammal include Chinese hamster ovary cells (CHO cells), COS, mycobacterium tuberculosis, and the like. Examples include Eloma, BHK, HeLa, Vero, 293, NS0, Namalwa, and YB2 / 0.

[0120] The obtained anti-RGMa antibody or antigen-binding fragment thereof can be purified to homogeneity. Separation and purification of antibodies and the like can be performed using methods used for separation and purification of ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David L ane, Cold Spring Harbor Laboratory, 1988) Examples of columns used in 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 protein of the present invention, particularly an anti-RGMa antibody or an antigen-binding fragment thereof, promotes the restoration of neural function by neutralizing the neurite outgrowth inhibitory activity of RGMa and can therefore be used as a drug for preventing, treating or preventing the recurrence 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 can therefore be used as drugs for the prevention, treatment, or prevention of recurrence of immunological diseases. It can be used.

[0122] Neurological diseases include amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barre 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 confusion disorder, glaucoma, schizophrenia, and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked Examples of the ophthalmopathy include retinoschisis, drug-induced optic neuropathy, retinal dystrophy, age-related macular degeneration, eye disease characterized by optic disc drusen, eye disease 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] Immunologic 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-Behcet's disease, pernicious anemia, type I (insulin-dependent) diabetes mellitus, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjogren's syndrome, Good-Basture 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. Preferred 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 drugs for the prevention, treatment, or relapse 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 a symptom of the disease, i.e., arresting its progression or eliminating the disease or condition, and alleviating a symptom of the disease, i.e., causing regression of the disease or condition, or slowing the progression of the condition.

[0126] Moreover, the term "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 which repeat remission and relapse in mammals, particularly humans.

[0128] The RGMa binding protein of the present invention (anti-RGMa antibody or antigen-binding fragment thereof) can be made into a pharmaceutical composition for the prevention or treatment of a neurological or immunological disease.

[0129] The administration form of the RGMa binding protein of the present invention (anti-RGMa antibody or antigen-binding fragment thereof) is not particularly limited, and it can be administered to mammals, including humans, via any of the following administration 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 pharma- ceutically acceptable carrier, etc. Dosage forms for parenteral administration include injection preparations (e.g., drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral administration preparations, intraspinal administration preparations), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, etc. In particular, when it is desired to directly act on the central nervous tissue, it can be continuously injected using a medical micropump of an osmotic pump, or it 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 agents, soothing agents, preservatives, stabilizers, etc. may be added. Also, it may be made into a lyophilized 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, and the like.

[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, amino acids, etc., as necessary.

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

[0135] The dosage of the antibody according to the present invention is 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 pharmacokinetic 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. Generally, for an adult (body weight 60 kg), the dosage is 1 to 5,000 μg / day, preferably 10 to 2,000 μg / day, and more preferably 50 to 2,000 μg / day for oral administration, and 1 to 5,000 μg / day, preferably 5 to 2,000 μg / day, and more preferably 50 to 2,000 μg / day for injection, which can be administered once or in divided doses. In the case of systemic parenteral administration, 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 can be administered once a day, once a week, once a month, or once to seven times a year. In the case of local administration using an osmotic pump or the like, the drug 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). EXAMPLES

[0136] The present invention will be specifically described below by way of 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-Gly422 (from the N-terminus, the 169th proline residue to the 422nd glycine residue; hereinafter the same) of human RGMa protein (SEQ ID NO: 1 in the sequence listing) We established CHO cells expressing recombinant human RGMa protein fused with a tag. 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 elution fraction was replaced with phosphate buffered saline (PBS) by dialysis, and the immunoprecipitation was performed. It was used as the source.

[0138] Example 2: Generation of mouse anti-human RGMa monoclonal antibodies The recombinant human RGMa protein (10 μg) prepared in Example 1 was diluted with Freund's complete adjuvant ( The emulsion was mixed with Sigma (Sigma) and used to immunize BALB / c mice (Charles River Biosciences Japan) at several subcutaneous sites on the back. 10 μg of recombinant human RGMa protein emulsified with Bant (Sigma) was similarly After several immunizations, blood samples were taken. Antibody titers were measured using the ELISA method with immobilized human or mouse RGMa protein, as described below. Individuals showing an increase in antibody titer were examined for human RGMa protein. The mice were boosted with 10 μg of protein intravenously, and splenocytes were harvested 2 to 3 days later. The spleen cells were fused with half of the mouse myeloma cells (SP2 / 0, Sumitomo Dainippon Pharma). The precipitate was mixed and centrifuged, and polyethylene glycol (Roche Diagnostics) was added to the precipitate. The cells were then washed twice with D-MEM (Invitrogen). The cells were then resuspended in 10% fetal bovine serum (Invitrogen), 1% BM condimed (Roscosmos). The cells were then resuspended in GIT medium (Nihon Pharmaceutical) containing 5 × 10 4 Myeloma cells were seeded at 100 / well in a 96-well plate. The culture supernatant was collected, and antibody-producing cells were detected by the human RGMa protein immobilized ELISA method described in Example 3. Screening was carried out. The antibody-producing cells obtained by screening were cloned by limiting dilution and two types of 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. The purification of monoclonal antibodies is carried out by isolating anti-mouse IgG antibodies from the culture supernatant of hybridomas. Affinity chromatography using agarose (Sigma Anti-Mouse IgG-Agarose) was performed. 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 left to stand at room temperature for 1 hour. After removing the liquid, ApplieBlock (Biochemical Bio Business) diluted 5-fold with PBS was added. 200μL / well of 200150) was dispensed and left 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, Hybridoma culture supernatant, recombinant antibody expression culture supernatant, or purified antibody (described later) was added at 50 μL / well and allowed to stand at room temperature for 1 hour. After that, the plate was washed three times with PBST and then diluted with PBS. The diluted peroxidase-labeled sheep anti-mouse IgG antibody (GE Healthcare, NA9310V) was added for 50 min. The plate was washed three times, and a peroxidase coloring kit (ML-1130O, manufactured by Sumitomo Bakelite Co., Ltd.) was added to the plate for coloring for a certain period of time. The absorbance at 492 nm was measured using a plate reader.

[0140] Example 4: Epitope analysis of antibodies The epitope to which the antibody binds was determined by peptide scanning. The epitope is a set of 11 consecutive residues, shifted by 3 residues, contained in Arg172 to Ala424 of human RGMa protein (SEQ ID NO: 1 in the sequence listing). A total of 83 types of peptides were synthesized, in which a spacer sequence (SGSG) (SEQ ID NO: 46 in the sequence listing) with a biotinylated N-terminus was fused to the N-terminus of the amino acid sequence consisting of the above. After the peptides were immobilized on an avidin plate, they were reacted with the test antibodies (B5.116A3, B5.70E4). The mixture was reacted with ELISA-labeled rabbit anti-mouse Ig antibody (Dako, P026002), and then a substrate solution was added. After allowing 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 human RGMa-derived peptides Glu298-Gly311 (Glu298-Asp308 and Val301-Gly311), Asn322-Glu335 (Asn322-Thr332 and Ile325-Glu335), and Pro349-Thr359.

[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 of the light chain variable region and constant region, and the heavy chain variable region and constant region were amplified by PCR, and the DNA sequences were determined. Next, based on the determined variable region 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) EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRSVYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVT 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 AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINCPPC 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) (light chain constant region is Arg108 to Cys214 of SEQ ID NO: 4 in the sequence listing, 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 inserted into pcDNA3.3 (Life Technologies, The expression vector was introduced into HEK293F cells (Life Technologies) using Neofection 293 (Astec). The cells were incubated at 37°C in 8% carbon dioxide gas. After culturing for 6 days under these conditions, the culture supernatant was collected. The recombinant antibodies were purified by applying the culture supernatant to an affinity column (GE Healthcare) on which Protein A or Protein G was immobilized, and the antibodies bound to the column were eluted with 10 mM glycine hydrochloride (pH 2.8) and quickly neutralized. Then, the medium was replaced with PBS. If the purpose of use requires higher purification purity, use antibodies 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 for 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 of the rat RGMa protein (Pro169 to Cys450 in SEQ ID NO:1 in the Sequence Listing), the full-length mouse RGMa protein (Met1 to Trp454 in SEQ ID NO:2 in the Sequence Listing), or the C-terminal domain of the rat RGMa protein ( A vector expressing Pro170 to Trp449 (column 3) was introduced into CHO cells or HEK293 cells, Antigen-expressing cells were generated. In addition, the C-terminal peptide of the RGMa protein is processed during the GPI anchor addition reaction. Both mouse and rat RGMa proteins are cleaved at Ala427, and the C-terminal peptide is removed. 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 (comparative example) were added at a final concentration of 10 μg / mL to the above antigens. After reacting 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 reacted and the cells were washed. Fluorescence was measured by flow cytometry (Becton Dickinson, FaxCalibur) to evaluate the binding ability of the test antibody to the antigen-expressing cells (Table 1). As a result, r116A3 and r70E4, unlike r5F9, were able to express the human and rat RGMa proteins. The C-terminal domain alone of the RGMa protein 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 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 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 stronger to human RGMa protein and 44-fold stronger to mouse RGMa protein than r5F9.

[0150] [Table 2]

[0151] Example 9: RGMa-Neogenin binding inhibition test The extracellular domain (Ala34-Leu1105) of recombinant human Neogenin protein (SEQ ID NO: 10 in the sequence listing) 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 protein 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 labeled with biotin using the ChromaLink Biotin Labeling Kit (Solulink). Biotin-labeled human RGMa protein was prepared at 2 μg / mL. The protein was mixed with an equal volume of the test antibody (r116A3, r70E4) diluted 2-fold and incubated at room temperature for 2 hours. The mixture was reacted to prepare a mixed solution. At the same time, human neogenin protein extracellular domain adjusted to 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 to prepare a neogenin solid-phase plate. After removal, a 2.5% bovine serum albumin solution was added and left to stand for 1 hour to block non-specific binding. The above mixed solution was added to this Neogenin solid-phase plate at 50 μL / well 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. -The inhibition of neogenin binding was evaluated (Figure 1). As a result, anti-human RGMa polyclonal antibody (R&D Systems, AF2459) and r5F9 In contrast, 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) adjusted to 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. Then, leave the plate for 1 hour to block non-specific binding, and prepare a plate with RGMa protein immobilized on it. The test antibodies (B5.116A3, B5.70E4) serially diluted to 0.01-10 μg / mL were added to the RGMa protein solid-phase plate and left 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 allowed to stand at room temperature for 1 hour. The plate was reacted with biotin-labeled anti-BMP2 antibody, 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, after which the absorbance was measured using a plate reader (FIG. 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, absorbance 0.4 at 0.1 μg / mL, absorbance 0.32 at 1 μg / mL, and absorbance 0.1 at 10 μg / mL).

[0153] Example 11: Design of humanized antibodies The humanization of the mouse monoclonal antibody B5.116A3 was carried out 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 described by et al. 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 maintained these amino acids as much as possible, and transplanted the CDRs of the mouse antibody. A humanized antibody sequence was designed. The heavy chain of the designed humanized antibody sequence is shown as HA (SEQ ID NO: 11 in the sequence listing) and the light chain is shown as KA (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 antigens, from HB to HH, and seven types of light chains, from KB to KG, including KA.

[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 / 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 (Comparative example). The variable region of the humanized anti-RGMa monoclonal antibody h5F9 described in Patent Document 1 (WO2009 / 106356) The regions (light chain: seq ID_53 in Patent Document 1, heavy chain: seq ID_50 in Patent Document 1) were compared with human antibody constant regions. The light chain was linked to Arg108 to Cys214 of SEQ ID NO:26 in the sequence listing, and the heavy chain was linked to 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 portions and thermal cycled. The samples were heat-treated for 10 minutes at eight temperatures: 40, 45, 50, 55, 60, 65, 70, and 75°C using a heat-treatment machine (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 the ELISA described in Example 3 in which human RGMa protein was immobilized, and the antigen-binding ability of the antibody was evaluated ( FIG. 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 to the humanized antibody (rH5F9). Hereafter, this humanized antibody consisting of the HE / KA combination is referred to as rH116A3. Furthermore, when heat treatment was not performed, the mouse-human chimeric antibody (r116A3C) and the humanized antibody (rH116A3) showed 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 CHO stable expression line producing 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 in the sequence listing) and the heavy chain coding sequence (SEQ ID NO: 43 in the sequence listing) of the humanized anti-RGMa antibody (rH116A3) was used. , and then transfected into 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 expression line was obtained.

[0163] Example 15: Neurite outgrowth assay The cerebellum was removed from newborn rats (P7) and then inoculated with trypsin solution (0.25% trypsin containing 0.2% DNase). The cells were then suspended in PBS (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 to separate the cells. 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 one 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, The addition of RGMa inhibited neurite growth, reducing the neurite length from 37 μm to 26 μm in the experiment on the left and from 38 μm to 27 μm in the experiment on the right. When only the test antibodies (B5.116A3, B5.70E4) were added at a final concentration of 10 μg / mL, no change was observed in the neurite length. However, when the test antibodies were added simultaneously with recombinant RGMa protein, the neurite length increased to the same extent as in the control (no RGMa addition). Transepithelial growth was observed, and the neutralization of RGMa protein by the antibody was observed.

[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 then 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 (Precision System) was used. A pressure of 200 kdyn was applied to the exposed spinal cord using a . Immediately after the spinal cord was injured as described above, an osmotic minipump (200 μL liquid volume, 0.5 μL / hour, delivered for 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 silicon tube connected to the outlet of the osmotic minipump was placed under the dura at the site of spinal cord injury. The tube was sewn and fixed to the spinous process on the side of the leg directly below the laminectomy site, and the muscle and skin layers were sutured and the rat was kept. 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)) was used to measure the damage. The results 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 FIG. 5(A), r116A3 and r70E4 showed a significant improvement in the efficacy of the control antibody (mo-IgG2bκ) compared with that of the control antibody (mo-IgG2bκ) at 4 days after administration. Or, after 3 weeks, the BBB score was significantly improved (p<0.05, Student's t-test). When evaluating using a 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 thereafter every week for up to 10 days. As shown in Figure 5(B), B5.116A3 significantly improved the BBB score after 4 weeks of administration compared to the control antibody (mo-IgG2bκ) (p<0.01, Student's t-test).

[0165] Example 17: Efficacy test using multiple sclerosis model mice PLP 139-151 The peptide (HSLGKWLGHPDKF: sequence number 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 subcutaneously immunized with 50 μg of peptide per head on the back, and the EAE score was evaluated. (H. Kataoka, K Sugahara, K. Shimano, K. Teshima, M. Koyama, A. Fukunari and K. Chiba. FTY720, 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 (Figure 6). The test antibody (B5.116A3) diluted in physiological saline was 139-151 The peptides were administered intraperitoneally at 20 mg / kg 7 and 10 days or 18 and 21 days after immunization, respectively. As a result, as shown in FIG. 6, the anti-RGMa mouse monoclonal antibody (B5.116A3) inhibited the expression of RGMa in mice compared with the control. Compared with the antibody (mo-IgG2bκ), administration before the onset of EAE suppressed the worsening of EAE scores (FIG. 6, top), and administration after the onset of EAE showed a recurrence prevention effect (FIG. 6, bottom).

[0166] Example 18: Immunogenicity testing of antibodies 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 to allow the antibody to be taken up by 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 evaluated using the T cell proliferation activity of the test antibody 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 amino acid sequence of the light chain complementarity determining region 1 (LCDR1) comprises RASQDISSYLN (SEQ ID NO: 30 in the sequence listing); The amino acid sequence of the light chain complementarity determining region 2 (LCDR2) comprises YTSRLHS (SEQ ID NO: 31 in the sequence listing); The amino acid sequence of the light chain complementarity determining region 3 (LCDR3) comprises QQLNTLP (SEQ ID NO: 32 in the sequence listing); The amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) comprises DAWMD (SEQ ID NO: 33 in the sequence listing); The amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) comprises EIRSKANNHATYYAESVKG (SEQ ID NO: 34 in the sequence listing); and The amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) comprises RDGAY (SEQ ID NO: 35 in the sequence listing); An isolated anti-RGMa antibody, or antigen-binding fragment thereof, which is stable even when heat-treated at 60°C.

2. Dissociation constant (Kd) for human RGMa is 10 -8 The anti-RGMa antibody, or antigen-binding fragment thereof, of claim 1 , having a molecular weight of M or less.

3. The anti-RGMa antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the anti-RGMa antibody is a humanized antibody.

4. An anti-RGMa antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the anti-RGMa antibody has a human IgG constant region.

5. A nucleic acid molecule encoding a protein portion of an anti-RGMa antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A recombinant vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the recombinant vector of claim 6.

8. A method for producing an anti-RGMa antibody or an antigen-binding fragment thereof described in any one of claims 1 to 4, comprising a step of culturing a host cell described in claim 7.

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