RGMa binding proteins and their use

Abstract

The objective of this invention is to obtain an anti-Repulsive Guidance Molecule a (RGMa) antibody with high binding activity and few side effects that can be used as a medicine for the prevention, treatment, or relapse prevention of neurological or immunological diseases. This is achieved by providing a monosomy of the RGMa binding protein that does not hinder the binding of RGMa to Neogenin and neutralizes the neurite growth inhibitory activity of RGMa. Preferably, the amino acid sequence of the complementarity-determining region is sequence number 30-35 or sequence number 36-40 of the sequence listing and SFG.

Description

Technical Field

[0001] The present invention relates to RGMa binding proteins and their uses. Prior Art

[0002] RGM (repulsive guidance molecule) is a GPI-anchored membrane protein with a molecular weight of about 33 kDa. It was originally identified as a membrane protein as an axonal guidance molecule of the visual system (see non-patent document 1). The RGM family includes three members called RGMa, RGMb and RGMc. Among them, RGMa is particularly prominent because it is expressed in adult humans and rats after central nervous system damage in addition to the developmental stage, and RGMa inhibition in rats can promote neurite growth after spinal cord injury and promote functional recovery (see non-patent document 2). Therefore, RGMa is considered to be a neurite-blocking substance after central nervous system damage.

[0003] RGMa has also been reported to act on the immune system. RGMa is expressed on dendritic cells and acts on T cells to increase the adhesion of T cells to ICAM-1 and fibronectin, or to induce the production of cytokines (Patent Document 4). In multiple sclerosis model mice, when anti-RGMa antibodies are administered, the symptoms of encephalomyelitis are suppressed, and the effect of suppressing the onset and relapse is also shown. Anti-RGMa antibodies are believed to show effects on multiple sclerosis by binding to RGMa expressed on dendritic cells, inhibiting the activation of T cells.

[0004] The signal transduction mechanism of RGMa has been gradually clarified, and Neogenin protein has been reported as a receptor of RGMa (Patent Document 3). Neogenin is a single-pass transmembrane protein expressed on nerve cells or T cells. RGMa binds to Neogenin on the cell membrane, inducing the activation of RhoA and the inactivation of Ras in the cell, thus inhibiting the growth of neurites. On the other hand, it is known that in the developing chicken brain, when Neogenin is not bound to RGMa, it causes apoptosis (Matsunaga et al., Dev. Growth Differ. 46, 481, 2004). In other words, the RGMa / Neogenin pathway is considered to have two opposite effects: promoting the survival of nerve cells, which is a better effect for nerve regeneration, and inhibiting the growth of neurites, which is a negative effect.

[0005] As a medicine targeting RGM, an axon regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient is disclosed in Patent Document 1. Further, in Patent Documents 2 and 3, a therapeutic agent for mechanical injury to the brain and spinal cord of an anti-RGM antibody that regulates the binding of RGM to the Neogenin receptor is disclosed. Further, in Patent Document 4, a medical use of an anti-RGM antibody for multiple sclerosis and the like is disclosed. Further, in Patent Document 5, a therapeutic use of an anti-RGM antibody for diseases including multiple sclerosis, mammalian brain injury, spinal cord injury, stroke, neurodegenerative diseases, and schizophrenia is disclosed. Further, in Patent Document 6, a therapeutic use of an RGM regulator such as an anti-RGM antibody for spinal cord injury or multiple sclerosis is disclosed, and a therapeutic use for progressive multiple sclerosis is not disclosed in Non-Patent Document 3. [Prior Art Documents] [Patent Documents]

[0006] [Patent Document 1] International Publication WO2005 / 087268 Pamphlet [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-537655 [Patent Document 3] Japanese Patent Application Laid-Open No. 2009-510002 [Patent Document 4] International Publication WO2011 / 071059 Pamphlet [Patent Document 5] Japanese Patent Application Laid-Open No. 2011-512806 [Patent Document 6] Japanese Patent Application Laid-Open No. 2004-525875 [Non-Patent Documents]

[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 Problems to be Solved by the Invention

[0008] As described above, although the therapeutic use of anti-RGMa antibodies for neurological diseases or immunological diseases has been revealed, conventional antibodies have problems such as insufficient activity, the possibility of damaging the original function of RGMa, and side effects. In particular, conventional antibodies may also hinder the binding between RGMa and Neogenin, thereby possibly hindering the better effects of Neonigenin bound to RGMa, such as inhibiting apoptosis. Therefore, the object of the present invention is to provide an RGMa binding protein that does not inhibit the RGMa / Neogenin interaction and neutralizes the neurite outgrowth inhibitory activity of RGMa. The means to solve the problem

[0009] The inventors of the present invention have made great efforts to solve the above problems. As a result, they have successfully obtained an RGMa binding protein that does not inhibit the binding of RGMa to Neogenin and neutralizes the neurite growth inhibitory activity of RGMa. They have found that the protein can be used as a medicine for neurological diseases or immunological diseases, and have completed 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 of [1], which binds to human RGMa, rat RGMa and / or mouse RGMa. [3] The RGMa binding protein of [1] or [2], which binds to the peptides EEVVNAVEDWDSQG (sequence number 26 in the sequence listing), NQQIDFQAFHTNAE (sequence number 27 in the sequence listing), PTAPETFPYET (sequence number 28 in the sequence listing), and / or KLPVEDLYYQA (sequence number 29 in the sequence listing). [4] The RGMa binding protein of any one of [1] to [3], which is bound to the peptides of sequence number 26 and sequence number 27 in the sequence listing. [5] The RGMa binding protein of any one of [1] to [4], which is bound to the peptides of sequence number 26, sequence number 27 and sequence number 28 in the sequence listing. [6] The RGMa binding protein of any one of [1] to [4], which is bound to the peptides of sequence number 26, sequence number 27 and sequence number 29 in the sequence listing. [7] The RGMa binding protein of any one of [1] to [6], wherein the RGMa binding protein is a human antibody, a humanized antibody or a chimeric antibody, or an antigen-binding fragment thereof. [8] A nucleic acid molecule encoding a protein portion of the RGMa binding protein of any one of [1] to [7]. [9] A recombinant vector comprising the nucleic acid molecule of [8].

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

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

[10] .

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

[13] The pharmaceutical composition of

[12] , which is used for the prevention, treatment or recurrence prevention of neurological diseases or immunological diseases.

[14] The pharmaceutical composition of

[13] , wherein the neurological disease is selected from the group consisting of amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barre syndrome, cerebral leukodystrophy, multiple sclerosis (including relapsing-remitting multiple sclerosis, first-episode progressive multiple sclerosis, and second-episode 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 disease, tardive dyskinesia, mania, Parkinson's disease, Steele-Richard syndrome, and syndrome), Down syndrome, myasthenia gravis, nerve trauma (including optic nerve trauma), angioamyloid, cerebral hemorrhage associated with amyloid, cerebral infarction, encephalitis, acute confusional disorder, glaucoma, schizophrenia and retinal nerve fiber layer disease (including eye diseases characterized by diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal nutritional atrophy, age-related macular degeneration, optic nerve head drusen; eye diseases characterized by genetic determinants of photoreceptor disease, autosomal recessive cone-rod dystrophy, mitochondrial damage associated with optic neuropathy).

[15] The pharmaceutical composition of

[13] , wherein the immunological disease is selected from the group consisting of multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis, 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), Sjögren's syndrome, Goodpasture's syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis.

[16] The pharmaceutical composition of

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

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

[18] The anti-RGMa antibody or antigen-binding fragment thereof as described in

[17] , wherein the heavy chain variable region (VH) comprises VH: (SEQ ID NO. 41 in the sequence listing) or an amino acid sequence having at least 90% identity with the amino acid sequence, and The light chain variable region (VL) comprises VL: (SEQ ID NO: 42 in the sequence listing) or an amino acid sequence having at least 90% identity with the amino acid sequence.

[19] The anti-RGMa antibody or antigen-binding fragment thereof as described in

[17] or

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

[20] The anti-RGMa antibody or antigen-binding fragment thereof according to any one of

[17] to

[19] , wherein the anti-RGMa antibody has a constant region of human IgG.

[21] An RGMa binding protein, wherein the binding to RGMa is competed with the anti-RGMa antibody of

[17] or

[18] .

[22] A nucleic acid molecule encoding the protein portion of the anti-RGMa antibody or antigen-binding fragment thereof as described in any one of

[17] to

[20] .

[23] The nucleic acid molecule of

[22] , wherein the nucleic acid sequence encoding the amino acid sequence of VH and VL comprises VH: (SEQ ID NO. 43 in the sequence listing), and VL: (SEQ ID NO: 44 in the sequence listing).

[24] A recombinant vector comprising the nucleic acid molecule of

[22] or

[23] .

[25] A host cell comprising the recombinant vector of

[24] .

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

[17] to

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

[25] .

[27] A pharmaceutical composition comprising the anti-RGMa antibody or antigen-binding fragment thereof as described in any one of

[17] to

[20] .

[28] The pharmaceutical composition of

[27] , which is used for the prevention, treatment or recurrence prevention of neurological diseases or immunological diseases.

[29] The pharmaceutical composition of

[28] , wherein the neurological disease is selected from the group consisting of amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barré syndrome, cerebral leukodystrophy, multiple sclerosis (including relapsing-remitting multiple sclerosis, first-episode progressive multiple sclerosis, and second-episode 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 disease, tardive dyskinesia, mania, and Parkinson's disease. , Still-Richard syndrome, Down syndrome, myasthenia gravis, neurological trauma (including optic nerve trauma), angioamyloid, cerebral hemorrhage associated with amyloid, cerebral infarction, encephalitis, acute confusional disorder, glaucoma, schizophrenia and retinal nerve fiber layer disease (including eye diseases characterized by diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal nutritional atrophy, age-related macular degeneration, optic nerve head drusen; eye diseases characterized by genetic determinants of photoreceptor disease, autosomal recessive cone-rod dystrophy, mitochondrial damage associated with optic neuropathy).

[30] The pharmaceutical composition as described in

[28] , wherein the immunological disease is selected from the group consisting of multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Guillain-Barré syndrome, neuro-Behçet's disease, pernicious anemia, type I (insulin-dependent) diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjögren's syndrome, Goodpasture's syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis.

[31] The pharmaceutical composition as described in

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

[32] A method for preventing, treating, or preventing recurrence of a neurological disease or immunological disease, which comprises administering an RGMa-binding protein as described in any one of [1] to [7] in an effective amount to a subject in need thereof.

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

[17] to

[20] in an effective amount to a subject in need thereof. Advantages of the Invention

[0011] The RGMa-binding protein of the present invention can maintain the function of Neogenin bound to RGMa, such as the inhibition of apoptosis of nerve cells, etc., by not interfering with the interaction between RGMa and Neogenin. Therefore, the protective effect on nerve cells is strong, and there is less concern about side effects associated with the reduction of nerve cells. In addition, the humanized anti-RGMa antibody of the present invention has excellent properties such as binding affinity and thermal stability to human RGMa compared with conventional antibodies. Therefore, it can be used as a medicine for neurological diseases or immunological diseases with excellent drug efficacy and few side effects. Brief Description of the Drawings

[0012] [Figure 1] A diagram showing the results of an RGMa-Neogenin binding inhibition test using an RGMa polyclonal antibody (AF2459), a comparative antibody (r5F9), and the antibodies of the present invention (r70E4, 116A3). [ Fig. 2 ] is a graph showing the results of an RGMa-BMP2 binding inhibition test using control group mouse IgG and the antibodies of the present invention (B5.70E4, B5.116A3). [ Fig. 3 ] is a graph showing the results of thermal stability tests of antibodies using a comparative example antibody (rH5F9), a chimeric antibody of the present invention (r116A3C), and a humanized antibody of the present invention (HE / KA, HA / KC). [ Fig. 4 ] A diagram showing the results of a neurite outgrowth assay using the antibodies of the present invention (B5.70E4 (left), B5.116A3 (right)). [Fig. 5] is a graph showing the results of efficacy tests using control group mouse IgG (mo-IgG2bk) and the antibodies of the present invention (r70E4, r116A3) in rats with spinal cord injury models. The results of efficacy tests in (A) spinal cord crush model and (B) hemisection model are shown. [Fig. 6] is a graph showing the results of an efficacy test of the antibody of the present invention (B5.116A3) using a multiple sclerosis model mouse induced with the PLP139-151 peptide. The left side shows the EAE score, the right side shows the change in body weight, the upper part shows the results when the test antibody was administered 7 days and 10 days later, and the lower part shows the results when the test antibody was administered 18 days and 21 days later. Implementation

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

[0014] [RGMa] RGMa is a neurite outgrowth inhibitory protein in the central nervous system. Human RGMa protein is synthesized as a precursor protein consisting of 450 amino acids as shown in SEQ ID NO. 1 in the sequence list. The signal peptide Met1~Pro47 (which refers to the peptide from the 1st methionine residue to the 47th proline residue from the N-terminal side, and the same description is used hereinafter) existing at the N-terminal is removed, the peptide bond between Asp168 and Pro169 is cut, and the C-terminal peptide Arg423~Cys450 is removed, and a GPI anchor is added to the C-terminal carboxyl group which is the C-terminal Gly422. Human RGMa protein is expressed on the cell membrane as a mature protein in which the N-terminal domain (Cys48~Asp168) and the C-terminal domain (Pro169~Ala424) are linked by a disulfide bond. The precursor protein of mouse RGMa is composed of the amino acid sequence shown in sequence number 2 of the sequence list, and the precursor protein of rat RGMa is composed of the amino acid sequence shown in sequence number 3 of the sequence list, but because the C-terminal peptide is removed, the amino acid sequence becomes the same as the mature protein. In the present invention, RGMa can refer to any of the precursor protein, mature protein or active fragment thereof, and can also be a derivative or mutant thereof, as long as it binds to Neogenin described later and acts. In addition, it can be human RGMa or RGMa from other organisms, but preferably human RGMa.

[0015] [Neogenin] Neogenin is expressed in nerve cells of the central nervous system and functions as one of the receptors of RGMa. As shown in sequence number 10 of the sequence table, human Neogenin protein is composed of 1461 amino acids, and is expressed as a mature membrane protein after removing the signal peptide Met1~Ala33. In the present invention, Neogenin, as long as it binds to RGMa, can refer to any of the precursor protein, mature protein or its RGMa binding fragment, and can also be a derivative or mutant thereof. In addition, it can be human Neogenin or Neogenin from other organisms, but preferably human Neogenin.

[0016] [Neutralization] In this case, "neutralization" means the ability to bind to a target and inhibit any function of the target. That is, "neutralizing the neurite growth inhibitory activity of RGMa" means inhibiting the neurite growth inhibitory activity of RGMa by binding RGMa binding protein to RGMa. Neurite growth inhibitory activity can be evaluated by one or more of several in vitro or in vivo analyses known in the art, for example, by the neurite growth inhibitory test described in the specification of this case.

[0017] [Single separation] The term "isolated" in the isolated RGMa binding protein, etc., means that the protein has been identified, separated, and / or recovered from its natural state. Natural impurities are substances that may interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally speaking, to isolate the RGMa binding protein, etc., it is sufficient to purify it by at least one purification step. The RGMa binding protein purified by at least one purification step may be referred to as an "isolated RGMa binding protein".

[0018] [RGMa binding protein] In the present case, "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. RGMa binding scaffold proteins refer to proteins that achieve the function of binding to RGMa by introducing mutations into Kunitz domains of serine protease inhibitors or extracellular domains of human fibronectin, ankyrin, lipocalin, etc. Fusion proteins refer to RGMa binding proteins obtained by chemically or genetically binding to 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., other functional molecules other than the RGMa binding protein of the present case.

[0019] [Human Antibodies] Human antibodies refer to antibodies whose light and heavy chains are derived from human immunoglobulins. Depending on the heavy chain constant region, human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) with a γ chain heavy chain, IgM with a μ chain heavy chain, IgA (including IgA1 and IgA2) with an α chain heavy chain, IgD with a δ chain heavy chain, or IgE with an ε chain heavy chain. In principle, the light chain includes either a κ chain or a λ chain.

[0020] [Humanized Antibodies] Humanized antibodies refer to antibodies composed of: complementarity determining regions of antibodies from non-human animals, variable regions consisting of framework regions from human antibodies, and constant regions from human antibodies.

[0021] [Chimeric antibody] Chimeric antibodies are antibodies whose light chains, heavy chains, or both chains are composed of variable regions from non-human sources and constant regions from humans.

[0022] [Anti-RGMa antibody] In this case, the anti-RGMa antibody refers to an immunoglobulin molecule or a modified molecule thereof that binds to RGMa. The modified molecule includes multispecific antibodies, chimeric antibodies, humanized antibodies, function-modified antibodies, and conjugated antibodies.

[0023] [Multispecific Antibodies] Multispecific antibodies refer to asymmetric antibodies that have two or more independent antigen recognition sites with two or more different antigen specificities, and examples thereof include bispecific antibodies with two antigen specificities and trispecific antibodies with three antigen specificities.

[0024] [Function-altering antibodies] In this case, function-altered antibodies refer to antibodies whose functions other than antigen binding function are altered by mainly changing the amino acids or sugar chains in the Fc region of the antibody, such as antibodies that have altered cell killing function, complement activation function, or half-life extension function in the blood.

[0025] [Linked Antibody] In this case, linked antibodies refer to antibodies that are chemically or genetically engineered to bind to functional molecules other than antibodies, 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, etc.

[0026] [Antigen-binding fragment] In the present case, antigen binding fragment refers to a protein that contains a part 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 bond Fv, single chain antibody (scFv), and polymers thereof. Furthermore, antigen binding fragments include conjugated antigen binding fragments obtained by chemically or genetically combining 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., with functional molecules other than the anti-RGMa antibody in the present case.

[0027] [Complementarity determining region] Complementarity determining region (CDR) refers to the region in the variable region of the immunoglobulin molecule that forms the antigen binding site, also known as the hypervariable region, and refers to the part of the amino acid sequence of each immunoglobulin molecule that varies greatly. Among the CDRs, the light chain and heavy chain each have 3 CDRs (LCDR1, LCDR2, LCDR3, and HCDR1, HCDR2, HCDR3). In this case, the CDR of the immunoglobulin molecule is determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0028] [Percentage (%) identity of amino acid sequence] Here, the "percent (%) identity" of a variable region, etc., with respect to an identified reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are the same as those in a specific reference polypeptide sequence after aligning the sequences to obtain the maximum % identity, introducing gaps as needed, and setting any conservative substitutions to not be considered as part of the sequence identity. The alignment for determining % identity can be achieved by using various methods within the technical scope of those with ordinary knowledge in the art, such as computer software publicly available like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those with ordinary knowledge in the art can determine appropriate parameters for aligning sequences, including any necessary algorithms, to achieve the maximum alignment of the full length of the sequences being compared. However, for the purposes here, the % identity value can be obtained by using the sequence comparison computer program BLAST in a pairwise alignment. In the situation of using BLAST in amino acid sequence comparison, the % identity of a given amino acid sequence A with respect to 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 that match as the same score in 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. When the length of amino acid sequence A is different from the length of amino acid sequence B, the % identity of A to B should be understood to 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 previous paragraph.

[0029] [Competition] In this case, "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 presence of the anti-RGMa antibody or its antigen-binding fragment significantly reduces 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 to Neogenin and neutralizes the neurite growth inhibiting activity of RGMa.

[0031] As RGMa protein, preferably, it is RGMa protein from mammals, for example, as human RGMa protein, there can be mentioned a protein having the amino acid sequence of sequence number 1 in the sequence list, as mouse RGMa protein, there can be mentioned a protein having the amino acid sequence of sequence number 2 in the sequence list, and as rat RGMa protein, there can be mentioned a protein having the amino acid sequence of sequence number 3 in the sequence list. In addition, it can also be a polypeptide containing an amino acid sequence in which one or several (preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5) amino acids are substituted, deleted, inserted, and / or added in these sequences, and has substantially the same activity as RGMa protein, and has an amino acid sequence with an identity of 90% or more (preferably 95% or more) to the polypeptide or the aforementioned amino acid sequence.

[0032] Here, "having substantially the same activity as RGMa protein" includes any peptide as long as the peptide has a neurite growth inhibitory effect.

[0033] The above amino acid substitution is preferably a conservative substitution. Here, "conservative substitution" means replacing an amino acid residue with another chemically similar amino acid residue in a manner that does not substantially change the activity of the peptide. For example, a case where a hydrophobic residue is replaced with another hydrophobic residue, a case where a polar residue is replaced with another polar residue having the same charge, etc. Examples of functionally similar amino acids that can be substituted in this way include alanine, threonine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. for non-polar (hydrophobic) amino acids. Examples include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. for polar (neutral) amino acids. Examples include arginine, histidine, lysine, etc. for amino acids with a positive charge (basicity). In addition, as the amino acids having a negative charge (acidity), there are aspartic acid, glutamic acid, etc.

[0034] The RGMa binding protein of the present invention binds to RGMa, which means that it binds specifically to RGMa, and preferably has a low dissociation constant (Kd) for human RGMa, with an upper limit of, for example, 10-8M or less, preferably 10-9M or less, and even more preferably 10-10M or less; the lower limit is not particularly limited, for example, RGMa binding proteins of 10-14M or more, and preferably 10-13 or more can be listed.

[0035] RGMa protein, as a mature protein, is composed of an N-terminal domain and a C-terminal domain, but the C-terminal domain alone also has neurite growth inhibitory activity. The RGMa binding protein of the present invention preferably also binds only to the C-terminal domain of RGMa to neutralize the neurite growth inhibitory activity. More preferably, the dissociation constant (Kd) for the C-terminal domain of human RGMa is low, and the upper limit is, for example, 10-8M or less, more preferably 10-9M or less, and even more preferably 10-10M or less; the lower limit is not particularly limited, for example, RGMa binding proteins of 10-14M or more, and more preferably 10-13 or more can be cited.

[0036] The RGMa binding protein of the present invention does not inhibit the binding of RGMa to Neogenin. Here, "does not inhibit the binding of RGMa to Neogenin" means that in the binding system of RGMa and Neogenin shown in the examples described below, even if the concentration of the RGMa binding protein is increased, the binding of RGMa to Neogenin does not substantially decrease. For example, when the RGMa binding protein is added to the binding system of RGMa and Neogenin and its concentration is increased, if the concentration of the RGMa binding protein representing 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 of RGMa to Neogenin.

[0037] Neogenin used in the binding test with RGMa is preferably Neogenin of the same species as RGMa. That is, for mouse RGMa, mouse Neogenin is preferably used, and for human RGMa, human Neogenin is preferably used. An example of human Neogenin is a protein having the amino acid sequence of SEQ ID NO. 10 in the sequence list, but as long as it can bind to RGMa, it may also be a protein having an amino acid sequence having 90% or more (preferably 95% or more) identity with SEQ ID NO. 10 in the sequence list.

[0038] The RGMa binding protein of the present invention neutralizes the neurite growth inhibitory activity of RGMa. The neurite growth inhibitory activity can be evaluated by the neurite growth test shown in the examples described below. When RGMa is added, the growth of neurites is inhibited. By adding the RGMa binding protein thereto, the inhibition of neurite growth caused by RGMa is no longer caused. The RGMa binding protein of the present invention can neutralize the inhibition of neurite growth caused by the addition of RGMa by more than 50%, more preferably more than 80%, and most preferably more than 90%.

[0039] The amino acid sequence of RGMa protein varies depending on the animal species. Therefore, the amino acid sequence of human RGMa represented by sequence number 1 in the sequence list is different from that of mouse RGMa represented by sequence number 2 in the sequence list and that of rat RGMa represented by sequence number 3 in the sequence list. Generally speaking, in pharmacological tests or safety tests of protein preparations such as antibody drugs, rodents such as mice or rats are used as experimental materials. Therefore, the RGMa binding protein of the present invention is preferably one that binds to RGMa of mice or rats, and more preferably one that has a low Kd for RGMa of mice or rats. The upper limit of Kd is, for example, 5x10-7M or less, more preferably 10-8M or less, and even more preferably 10-9M or less. The lower limit is not particularly limited, and for example, RGMa binding proteins of 10-12M or more, and more preferably 10-11 or more, can be cited.

[0040] The RGMa binding protein of the present invention preferably has excellent thermal stability. Thermal stability can be evaluated by the reduction of binding to RGMa caused by heat treatment. Preferably, it is stable even after heat treatment at 60°C or above, more preferably, it is stable even after heat treatment at 65°C or above, and most preferably, it is stable even after heat treatment at 70°C or above.

[0041] The binding site of the RGMa binding protein of the present invention when binding to RGMa is not particularly limited. For example, when it is human RGMa, it is preferably a peptide that binds to one or more of EEVVNAVEDWDSQG (SEQ ID NO. 26 of the sequence listing) (amino acid numbers 298-311 of sequence number 1 of the sequence listing), NQQIDFQAFHTNAE (SEQ ID NO. 27 of the sequence listing) (amino acid numbers 322-335 of sequence number 1 of the sequence listing), PTAPETFPYET (SEQ ID NO. 28 of the sequence listing) (amino acid numbers 349-359 of sequence number 1 of the sequence listing), and KLPVEDLYYQA (SEQ ID NO. 29 of the sequence listing) (amino acid numbers 367-377 of sequence number 1 of the sequence listing). It is more preferably a peptide that binds to sequence numbers 26 and 27 of the sequence listing, and more preferably a peptide that binds to sequence numbers 26 and 27 of the sequence listing, and to sequence numbers 28 or 29 of the sequence listing.

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

[0043] <Anti-RGMa Antibody> The anti-RGMa antibody of the present invention includes polyclonal antibodies or monoclonal antibodies obtained by immunizing mammals such as mice with RGMa protein or its partial fragments (e.g., fragments containing one or more of sequence numbers 26 to 29 in the above sequence table) as antigens, chimeric antibodies and humanized antibodies produced using gene recombination technology, and human antibodies produced using transgenic animals using human antibody-producing genes. When the antibody of the present invention is administered to humans as a medicine, it is desirable to use a humanized antibody or a human antibody from the viewpoint of side effects.

[0044] Antigens can be used directly to induce immunity or as a complex with a carrier protein. The complex of antigen and carrier protein can be prepared using condensation agents such as glutaraldehyde, carbodiimide, maleimide active ester, etc. Carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, KLH, etc.

[0045] Mammals to be induced to be immune include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses or cattle, and the inoculation method includes subcutaneous, intramuscular or intraperitoneal administration. When administered, it can be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant for administration, and the administration is usually performed once every 2 to 5 weeks. Antibody-producing cell lines obtained from the spleen or lymph nodes of the induced immune animal are fused with myeloma cells and isolated as fusion tumors. Myeloma cell lines used are from mammals, such as mice, rats, humans, etc.

[0046] <Multiclonal Antibodies> Polyclonal antibodies can be obtained by existing general production methods. That is, for example, the above antigens are used together with Freund's adjuvant as needed to induce immunization in mammals as described above, and the antibodies can be obtained from the serum obtained from the immunized animals.

[0047] <Monoclonal Antibody> Monoclonal antibodies can be obtained specifically as follows. That is, the antigen as described above is used as an immunogen, and the immunogen is injected one to several times or transplanted into the mammal as described above, subcutaneously, intramuscularly, intravenously, intrapedicularly, or intraperitoneally, together with Freund's Adjuvant as needed, to perform immune sensitization. Usually, 1 to 4 immunizations are performed about every 1 to 14 days from the first immunization, and antibody-producing cells are obtained from the immune-sensitized mammal about 1 to 5 days after the final immunization.

[0048] Monoclonal antibodies can be obtained using methods known to those skilled in the art (e.g., "Current Protocols in Molecular Biology" (John Wiley & Sons (1987)), Antibodies: A Laboratory Manual, Ed. Harlow and David Lane, Cold Spring Harbor Laboratory (1988)).

[0049] The preparation of "hybridoma" secreting monoclonal antibodies can be carried out according to the method of Koehler and Milstein et al. (Nature, 256, 495, 1975) and the modified method thereof. That is, it is prepared by cell fusion of antibody-producing cells contained in the spleen or the like obtained from an immunosensitized mammal with myeloma cells from a mammal, preferably a mouse, rat or human, which have no ability to produce self-antibodies.

[0050] Myeloma cells used for cell fusion include, for example, mouse 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 myeloma 210RCY3-Ag.2.3., human 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 be performed by using polyethylene glycol (average molecular weight 1000-4000) at a concentration of about 20-50%, at a temperature of 20-40°C, preferably 30-37°C, so that the ratio of the number of antibody-producing cells to the number of myeloma cells is usually about 1:1-10:1, and reacting for about 1-10 minutes.

[0052] Screening of fusion tumor clones producing monoclonal antibodies can be performed by culturing the fusion tumors in, for example, microtiter plates and measuring the reactivity of the culture supernatant of the wells to the immunizing antigen using an immunochemical method such as ELISA.

[0053] When screening antibody-producing fusion tumors, in addition to the binding analysis with RGMa protein, the evaluation of whether the antibody does not inhibit the binding of RGMa protein to Neogenin and the evaluation of whether the antibody neutralizes the function of RGMa protein (neurite outgrowth inhibitory activity) are also performed. By these screening methods, the anti-RGMa antibody of the present invention can be selected.

[0054] From the wells containing the fusion tumors producing the target antibody, the clones can be further selected by limiting dilution method. The selection and breeding of fusion tumors are usually carried out in animal cell culture medium supplemented with HAT (hypoxanthine, aminopterin, thymidine) and containing 10-20% fetal bovine serum.

[0055] Monoclonal antibodies can be produced from fusion tumors by culturing the fusion tumors in vitro or by growing them in vivo in the ascites of mammals such as mice and rats, and isolating them from the obtained culture supernatant or the ascites of mammals.

[0056] During in vitro culture, the fusion tumor can be proliferated, maintained and preserved by combining various conditions such as the characteristics of the cultured cell type and the culture method, and using a nutrient medium suitable for producing monoclonal antibodies in the culture supernatant.

[0057] Examples of the basic culture medium include low calcium culture media such as Ham'F12 culture media, MCDB153 culture media or low calcium MEM culture media, and high calcium culture media such as MCDB104 culture media, MEM culture media, D-MEM culture media, RPMI1640 culture media, ASF104 culture media or RD culture media. The basic culture media may contain, for example, serum, hormones, cytokines and / or various inorganic or organic substances, depending on the purpose.

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

[0059] Monoclonal antibodies can also be obtained by phage display. In the phage display method, the target immunogen is used to screen phages selected from any phage antibody gene library to select phages with the desired binding properties to the immunogen. Then, the antibody corresponding sequence contained in the phage is isolated or sequenced, and based on the isolated sequence or sequence information, a display vector containing a nucleic acid molecule encoding the antibody or antigen binding domain is constructed. Then, by culturing the cell line transfected with the expression vector, monoclonal antibodies can be produced. By using a human antibody gene library as a phage antibody gene library, human antibodies with the desired binding properties can be generated.

[0060] As scaffold proteins, the Kunitz domain of human serine protease inhibitor or the extracellular domain of human fibronectin are used. If the sequence of the target binding site on the scaffold is changed, a scaffold protein that binds to RGMa can be generated (Clifford Mintz et al BioProcess International, 2013, Vol. 11 (2), pp40-48).

[0061] Examples of fusion proteins include RGMa-binding proteins in which 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., are chemically or genetically bonded to the RGMa-binding protein to form functional molecules other than the RGMa-binding protein of the present invention.

[0062] When PEG is used as a functional molecule, the PEG can be used without limitation, with a molecular weight of 2000 to 100000 Da, preferably 10000 to 50000 Da, and can be straight chain or branched. PEG, for example, can be bound to the N-terminal amino group of the amino acid of the RGMa binding protein by using the NHS active group.

[0063] When a radioactive substance is used as a functional molecule, 131I, 125I, 90Y, 64Cu, 99Tc, 77Lu or 211At can be used. The radioactive substance can be directly bound to the RGMa binding protein by the chloramine T method or the like.

[0064] When toxins are used as functional molecules, bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), low molecular weight toxins (such as geldanamycin), maytansinoids, and calicheamicin can be used.

[0065] When low molecular weight compounds are used as functional molecules, examples thereof include daunomycin, doxorubicin, methotrexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC.

[0066] When enzymes are used as functional molecules, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. can be used.

[0067] Examples of linkers used when chemically binding toxins, low molecular weight compounds or enzymes include divalent free radicals (e.g., alkylene, arylene, heteroarylene), linkers represented by -(CR2)nO(CR2)n- (R is an arbitrary substituent, n is a positive integer), repeating units of alkoxy groups (e.g., polyethylene oxide, PEG, polymethylene oxide, etc.), and alkylamine groups (e.g., polyethyleneamine, JeffamineTM), as well as diacids and amides (e.g., succinate, succinamide, oxoglutarate, malonic acid esters and hexamethyleneamide, etc.). Chemical modification methods for binding functional molecules have been established in the art (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681).

[0068] The preferred embodiment of the RGMa binding protein of the present invention can be chimeric antibodies. "Chimeric antibodies" can be exemplified by chimeric antibodies whose variable regions are variable regions of immunoglobulins from non-human animals (mouse, rat, hamster, chicken, etc.) and whose constant regions are constant regions of human immunoglobulins. For example, mice can be immunized with antigens, and the variable regions that bind to the antigens can be cut out from the gene of the mouse monoclonal antibody, and combined with the constant regions of antibodies from human bone marrow to produce them. The constant regions from human immunoglobulins have their own amino acid sequences according to the isotypes of IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD and IgE, etc. The constant regions of the recombinant chimeric antibodies of the present invention can be the constant regions of human immunoglobulins of any isotype. Preferably, they are the constant regions of human IgG. The genes of chimeric antibodies produced in this way can be used to produce expression vectors. The host cell is transformed with the expression vector to obtain a transformed cell producing a chimeric antibody, and the target chimeric antibody is obtained from the culture supernatant by culturing the transformed cell.

[0069] Other preferred aspects of the RGMa binding protein of the present invention include humanized antibodies. The "humanized antibody" in the present invention is an antibody in which only the DNA sequence of the antigen binding site (CDR; complementarity determining region) of a non-human animal antibody such as a mouse is transplanted into a human antibody gene (CDR transplantation). For example, it can be prepared by referring to the methods described in Japanese Patent Publication No. 4-506458 and Japanese Patent No. 2912618. Specifically, it refers to a humanized antibody characterized in that part or all of its CDRs are CDRs of a monoclonal antibody from a non-human mammal (mouse, rat, hamster, etc.), the framework region of its variable region is the framework region of the variable region from a human immunoglobulin, and its constant region is the constant region from a human immunoglobulin.

[0070] The humanized antibodies of the present invention can be produced, for example, by the following method. However, the production method is not limited to this method.

[0071] For example, recombinant humanized antibodies from mouse monoclonal antibodies can be prepared by genetic engineering with reference to Japanese Patent Application No. 4-506458 and Japanese Patent Application Laid-Open No. 62-296890, etc. That is, DNA of mouse heavy chain CDR and DNA of mouse light chain CDR are isolated from the hybridoma producing mouse monoclonal antibodies, and human heavy chain genes of all regions other than human heavy chain CDR and human light chain genes of all regions other than human light chain CDR are isolated from human immunoglobulin genes.

[0072] The human heavy chain gene after transplanting the DNA of the isolated mouse heavy chain CDR part is introduced into an appropriate expression vector to enable it to be expressed. Similarly, the human light chain gene after transplanting the DNA of the mouse light chain CDR part is introduced into another appropriate expression vector to enable it to be expressed. Alternatively, the human heavy chain and light chain genes after transplanting the mouse CDR can also be introduced into the same expression vector to enable them to be expressed. By transforming the host cell with the expression vector prepared in this way, a humanized antibody-producing transformed cell is obtained, and by culturing the transformed cell, the target humanized antibody is obtained from the culture supernatant.

[0073] Other preferred aspects of the RGMa binding protein of the present invention include human antibodies. Human antibodies refer to antibodies in which all regions of the heavy chain variable region and heavy chain constant region, as well as the light chain variable region and light chain constant region constituting immunoglobulins are from genes encoding human immunoglobulins, and human antibody genes can be introduced into mice to produce them. Specifically, for example, by inserting at least a human immunoglobulin gene into the gene locus of a mammal other than humans such as a mouse, and sensitizing the produced gene-transfected animal with an antigen, the production can be carried out in the same manner as the aforementioned method for producing polyclonal antibodies or monoclonal antibodies.

[0074] For example, transgenic mice that produce human antibodies can be prepared according to the methods described in Nature Genetics, Vol.7, p.13-21, 1994; Nature Genetics, Vol.15, p.146-156, 1997; Japanese Patent Publication No. 4-504365; Japanese Patent Publication No. 7-509137; International Publication No. WO94 / 25585; Nature, Vol.368, p.856-859, 1994; and Japanese Patent Publication No. 6-500233. More specifically, HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FCγRIIb-KO) mice, etc. can be cited.

[0075] Specifically, the monoclonal antibodies of the present invention include those whose CDRs of the heavy chain variable region comprise the amino acid sequences of sequence numbers 33 (HCDR1), 34 (HCDR2) and 35 (HCDR3) in the sequence listing, and whose CDRs of the light chain variable region comprise the amino acid sequences of sequence numbers 30 (LCDR1), 31 (LCDR2) and 32 (LCDR3) in the sequence listing. Furthermore, as long as the antibody of the present invention has the ability to bind to RGMa, does not inhibit the binding of RGMa to Neogenin, and neutralizes the neurite growth inhibitory activity of RGMa, 1 to several amino acids in one or more of these CDRs may be substituted. Here, 1 to several refers to, for example, 1 or 2. Furthermore, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution. Maintaining the characteristics of the antibody means that these characteristics are maintained to the same degree as before the amino acid sequence of the CDR is changed, for example, maintained at 80% or more, preferably maintained at 90% or more, and more preferably maintained at 95% or more. Furthermore, maintenance also includes improvement.

[0076] Regions other than CDR are not particularly limited as long as they maintain the structure of an antibody and can function, and may be any of sequences derived from mice, humans, other mammals, chimeras thereof, and artificial sequences. When constant regions are included, amino acid sequences of the constant regions of the heavy and light chains may be 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] Mouse antibodies having such CDRs may include antibodies having the amino acid sequence of sequence number 4 in the sequence list for the light chain and the amino acid sequence of sequence number 5 in the sequence list for the heavy chain. Furthermore, in such amino acid sequences, as long as the ability to bind to RGMa is maintained, the binding of RGMa to Neogenin is not inhibited, and the neurite growth inhibitory activity of RGMa is neutralized, substitution, deletion, addition or insertion of one or several amino acids (1 to 20, 1 to 10 or 1 to 5) may be carried out. Such substitution, deletion or addition may also be introduced into the CDR, but it is preferably introduced into the region outside the CDR. In addition, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution.

[0078] In addition, among the above-mentioned mouse antibodies, mouse / human chimeric antibodies whose constant regions are derived from humans can also be exemplified. Such mouse / human chimeric antibodies can be exemplified by antibodies whose light chains have the amino acid sequence of sequence number 8 in the sequence list (variable regions are 1 to 107) and whose heavy chains have the amino acid sequence of sequence number 9 in the sequence list (variable regions are 1 to 116). Furthermore, in these amino acid sequences, as long as they maintain the ability to bind to RGMa, do not hinder the binding of RGMa to Neogenin, and neutralize the neurite growth inhibitory activity of RGMa, they can also have substitution, deletion, addition or insertion of 1 or several amino acids (1 to 20, 1 to 10 or 1 to 5). Such substitution, deletion, and addition can also be introduced into CDR, but it is preferably introduced into a region outside CDR. In addition, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution.

[0079] Furthermore, humanized antibodies derived from humans other than CDRs can also be exemplified. Such humanized antibodies can be exemplified by antibodies having an amino acid sequence of any one of sequence numbers 11 to 18 in the sequence list (variable region up to 116 residues on the N-terminal side) in the heavy chain and an amino acid sequence of any one of sequence numbers 19 to 25 in the sequence list (variable region up to 1 to 107 residues on the N-terminal side) in the light chain. Furthermore, in the amino acid sequence of the humanized antibody (heavy chain: sequence numbers 11 to 18 in the sequence list, light chain: sequence numbers 19 to 25 in the sequence list), as long as the ability to bind to RGMa is maintained, the binding of RGMa to Neogenin is not inhibited, and the neurite growth inhibitory activity of RGMa is neutralized, 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 also be introduced into CDRs, but are preferably introduced into regions outside CDRs. In addition, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution.

[0080] The heavy chain amino acid sequence and the light chain amino acid sequence may be any combination of the above, but the most preferred antibody is one in which the heavy chain has the amino acid sequence of sequence number 15 in the sequence listing and the light chain has the amino acid sequence of sequence number 19 in the sequence listing. Among the amino acid sequences of sequence number 15 in the sequence listing, the amino acid sequence corresponding to the heavy chain variable region is shown in sequence number 41 in the sequence listing, and the amino acid sequence corresponding to the light chain variable region is shown in sequence number 42 in the sequence listing. That is, the most preferred antibody of the present invention is one in which the heavy chain variable region has the amino acid sequence of sequence number 41 in the sequence listing, and the light chain variable region has the amino acid sequence of sequence number 42 in the sequence listing. Furthermore, in these amino acid sequences, as long as the ability to bind to RGMa is maintained, the binding of RGMa to Neogenin is not inhibited, and the neurite growth inhibitory activity of RGMa is neutralized, 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 also be introduced into CDRs, but are preferably introduced into regions outside CDRs. In addition, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution. Thus, the amino acid sequence of the antibody of the present invention comprising substitution, deletion, etc. in the amino acid sequence of SEQ ID NO. 41 in the sequence listing and / or SEQ ID NO. 42 in the sequence listing is an amino acid sequence in which the heavy chain variable region has an amino acid sequence that is more than 90% (preferably 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO. 41 in the sequence listing, and the light chain variable region has an amino acid sequence that is more than 90% (preferably 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO. 42 in the sequence listing.

[0081] Other specific examples of the monoclonal antibody of the present invention include those in which the CDR of the heavy chain variable region comprises the amino acid sequences of sequence numbers 39 (HCDR1), 40 (HCDR2) and SFG (HCDR3) in the sequence list, and the CDR of the light chain variable region comprises the amino acid sequences of sequence numbers 36 (LCDR1), 37 (LCDR2) and 38 (LCDR3) in the sequence list. Furthermore, as long as the characteristics of the antibody of the present invention 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, one to several amino acids in one or more of these CDRs may be substituted. Here, 1 to several means, for example, 1 or 2. Furthermore, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution. Regions other than CDR are not particularly limited as long as they maintain the structure of an antibody and can function, and may be any of sequences derived from mice, humans, other mammals, chimeras thereof, and artificial sequences. When constant regions are included, amino acid sequences of the constant regions of the heavy and light chains may be 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] Mouse antibodies having such CDRs may include antibodies having the amino acid sequence of sequence number 6 in the sequence list for the light chain and the amino acid sequence of sequence number 7 in the sequence list for the heavy chain. Furthermore, in such amino acid sequences, as long as the ability to bind to RGMa is maintained, the binding of RGMa to Neogenin is not inhibited, and the neurite growth inhibitory activity of RGMa is neutralized, substitution, deletion, addition or insertion of one or several amino acids (1 to 20, 1 to 10 or 1 to 5) may be carried out. Such substitution, deletion or addition may also be introduced into the CDR, but it is preferably introduced into the region outside the CDR. In addition, in order to maintain the characteristics of the present invention, the amino acid substitution is preferably a conservative substitution.

[0083] In addition, among the above mouse antibodies, chimeric antibodies in which the constant regions are derived from humans can also be exemplified. Furthermore, humanized antibodies in which regions other than CDRs are derived from humans can also be exemplified.

[0084] The anti-RGMa antibody of the present invention comprises a multispecific antibody, a function-modified antibody, or a linked antibody having a CDR composed of a specific amino acid sequence (e.g., sequence number 30 in the sequence listing for LCDR1, sequence number 31 in the sequence listing for LCDR2, sequence number 32 in the sequence listing for LCDR3, sequence number 33 in the sequence listing for HCDR1, sequence number 34 in the sequence listing for HCDR2, and sequence number 35 in the sequence listing for HCDR3), or a variable region composed of a specific amino acid sequence (e.g., sequence number 41 in the sequence listing for the heavy chain variable region, and amino acid sequence number 42 in the sequence listing for the light chain variable region).

[0085] The anti-RGMa antibody of the present invention can be combined with an antibody having antigen binding specificity other than RGMa by genetic engineering to produce a multispecific antibody such as a bispecific antibody. This genetic engineering technique has been established in the art. For example, by changing the DVD-Ig (Wu et al., Nature Biotechnology 25(11), 1290(2007)) that links the variable regions in series, or the Fc region of the antibody, the desired bispecific antibody can be obtained by using the ART-Ig technology (Kitazawa et al., Nature Medicine 18(10), 1570(2012)) that combines the heavy chains of two antibodies that bind to different antigens. As other antigens besides RGMa, non-limiting examples include factors that inhibit neurite growth, such as Nogo, MAG, Omgp, CSPG, Sema3A, Lingo-1, etc., or immune-related molecules, such as TNF-α, IL-6 receptor, CD3, CD20, α4 integrin, BLys, Thymic Stromal Lymphopoietin, IgE, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-23, IL-25, etc.

[0086] The modified molecules of the anti-RGMa antibody of the present invention include function-modified antibodies. Function-modified antibodies refer to antibodies whose functions such as cell killing function, complement activation function, and half-life extension function in blood are modified mainly by modifying the Fc region (Shitara Kenya, Journal of Pharmacy, 2009, Vol. 129 (1), p3; Ishii Akiko et al., Japanese Journal of Pharmacology, 2010, Vol. 136 (5), p280; Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82 (8), p710).

[0087] The function-modified antibody of the anti-RGMa antibody is prepared by the following method. For example, when the anti-RGMa antibody of the present invention is produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene is disrupted as a host cell, an antibody with reduced fucose content in the sugar chain and improved cell-killing function can be obtained. When the antibody is produced using CHO cells into which the FUT8 gene is introduced as a host cell, an antibody with low cell-killing function can be obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). In addition, by changing the amino acid residues in the Fc region, the complement activation function can be regulated (U.S. Patent No. 6737056, U.S. Patent No. 7297775, U.S. Patent No. 7317091). Furthermore, by using Fc region mutants that enhance binding to FcRn, one of the Fc receptors, it is possible to extend the half-life in blood (Hashiguchi Shuhei et al., Biochemistry, 2010, Vol. 82(8), p710). Such functionally altered antibodies can be produced by genetic engineering.

[0088] The modified molecules of the anti-RGMa antibody of the present invention include conjugated antibodies. The conjugated antibodies include those obtained by chemically or genetically combining the anti-RGMa antibody 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, etc., which are functional molecules other than the anti-RGMa antibody of the present invention.

[0089] When PEG is used as a functional molecule, PEG can be used without limitation, with a molecular weight of 2000 to 100000 Da, preferably 10000 to 50000 Da, and can be straight chain or branched. PEG can be bound to the N-terminal amino group of the amino acid of the antibody by using the NHS active group.

[0090] When a radioactive substance is used as a functional molecule, 131I, 125I, 90Y, 64Cu, 99Tc, 77Lu or 211At can be used. The radioactive substance can be directly bound to the antibody by the chloramine T method or the like.

[0091] When toxins are used as functional molecules, bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), low molecular weight toxins (such as geldanamycin), maytansine, and calicheamicin can be used.

[0092] When low molecular weight compounds are used as functional molecules, examples thereof include daunomycin, doxorubicin, methotrexate, mitomycin, neostatin, vindesine, and fluorescent dyes such as FITC.

[0093] When enzymes are used as functional molecules, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. can be used.

[0094] Examples of linkers used when chemically combining toxins, low molecular weight compounds or enzymes include divalent free radicals (e.g., alkylene, arylene, heteroarylene), linkers represented by -(CR2)nO(CR2)n- (R is an arbitrary substituent, n is a positive integer), repeating units of alkoxy groups (e.g., polyethylene oxide, PEG, polymethylene oxide, etc.), and alkylamine groups (e.g., polyethylene oxide, JeffamineTM), as well as diacids and amides (e.g., succinate, succinamide, oxoglutarate, malonate, and hexamethyleneamide, etc.). Chemical modification methods for binding functional molecules have been established in the art (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681).

[0095] The "antigen-binding fragment" of the antibody in the present invention refers to a region of a part of the antibody with antigen binding properties as described above, and specifically includes F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide bond Fv, single-chain antibody (scFv), and polymers thereof. Furthermore, the antigen-binding fragment is an antigen-binding fragment obtained by chemically or genetically binding to 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., which are functional molecules other than the anti-RGMa antibody in this case.

[0096] Here, "F(ab')2" and "Fab" refer to antibody fragments produced by treating immunoglobulins with proteolytic enzymes such as pepsin or papain, where the disulfide bond between two heavy chains in the hinge region is digested before and after. For example, when IgG is treated with papain, the upstream of the disulfide bond between two heavy chains in the hinge region is cut, and two identical antibody fragments are produced, in which the light chain composed of VL (light chain variable region) and CL (light chain constant region), and the heavy chain fragment composed of VH (heavy chain variable region) and CHγ1 (γ1 region in the heavy chain constant region) are bonded by a disulfide bond at the C-terminal region. Each of these two identical antibody fragments is called Fab. In addition, when IgG is treated with pepsin, the downstream of the disulfide bond between two heavy chains in the hinge region is cut, and an antibody fragment slightly larger than the above two Fabs linked by the hinge region is produced. This antibody fragment is called F(ab')2.

[0097] The modified molecules of the antigen-binding fragment of the anti-RGMa antibody of the present invention include linked antigen-binding fragments. Linked antigen-binding fragments include linked antigen-binding fragments obtained by chemically or genetically binding to a part of the anti-RGMa antibody with antigen-binding properties such as polyethylene glycol (PEG), non-peptide polymers, radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF-β, NGF, Neurotrophin, etc.), albumin, enzymes, other antibodies, etc., functional molecules other than the anti-RGMa antibody of the present invention.

[0098] When PEG is used as a functional molecule, PEG can be used without limitation, with a molecular weight of 2000 to 100000 Da, preferably 10000 to 50000 Da, and can be linear or branched. PEG can be bound to the N-terminal amino group of a part of the region with antigen binding of the anti-RGMa antibody, for example, by using the NHS active group.

[0099] When a radioactive substance is used as a functional molecule, 131I, 125I, 90Y, 64Cu, 99Tc, 77Lu or 211At can be used. The radioactive substance can be directly bound to a part of the anti-RGMa antibody having antigen binding properties by the chloramine T method or the like.

[0100] When toxins are used as functional molecules, bacterial toxins (such as diphtheria toxin), plant toxins (such as ricin), low molecular weight toxins (such as geldanamycin), maytansine, and calicheamicin can be used.

[0101] When low molecular weight compounds are used as functional molecules, examples thereof include daunomycin, doxorubicin, methotrexate, mitomycin, neostatin, vindesine, and fluorescent dyes such as FITC.

[0102] When enzymes are used as functional molecules, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. can be used.

[0103] Examples of linkers used when chemically combining toxins, low molecular weight compounds or enzymes include divalent free radicals (e.g., alkylene, arylene, heteroarylene), linkers represented by -(CR2)nO(CR2)n- (R is an arbitrary substituent, n is a positive integer), repeating units of alkoxy groups (e.g., polyethylene oxide, PEG, polymethylene oxide, etc.), and alkylamine groups (e.g., polyethylene oxide, JeffamineTM), as well as diacids and amides (e.g., succinate, succinamide, oxoglutarate, malonate, and hexamethyleneamide, etc.). Chemical modification methods for binding functional molecules have been established in the art (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681).

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

[0105] The present invention also includes antibodies having the amino acid sequence of the specific CDR as described above, anti-RGMa antibodies that compete with the binding of RGMa protein, and antigen-binding fragments thereof. As an antibody that competes for the binding of an antibody having an amino acid sequence of the above-mentioned specific CDR to RGMa, an antibody having an antigenic determinant in a region selected from Glu298 to Gly311, Asn322 to Glu335, Lys367 to Ala377, and Pro349 to Thr359 can be exemplified. The antibody is obtained (screened) or evaluated by coexisting an antibody having the above-mentioned CDR sequence with the RGMa protein binding system. For example, it can be obtained by screening using the following surface plasmon resonance (SPR) method.

[0106] Biotinylated human RGMa protein (4 μg / mL) was added as a ligand to the sensor chip with avidin fixed thereon, thereby fixing 1300 to 1600 RU of human RGMa protein. Then, any anti-RGMa antibody (15 μg / mL) was added as an analyte to bind to the human RGMa protein fixed on the sensor chip. By repeating this process multiple times, a state (saturation state) in which all molecules of human RGMa protein on the sensor chip were bound by any anti-RGMa antibody was created, and the binding amount in the saturation state (saturation binding amount 1) was obtained. The same experiment was also performed on the anti-RGMa antibody containing the amino acid sequence of the specific CDR of the present invention to obtain the binding amount at the saturation state (saturation binding amount 2). Next, after the human RGMa protein on the sensing chip was saturated with an anti-RGMa antibody containing the amino acid sequence of the specific CDR of the present invention, an arbitrary anti-RGMa antibody (15 μg / mL) was added as an analyte to investigate whether the human RGMa protein saturated with the anti-RGMa antibody containing the amino acid sequence of the specific CDR of the present invention can be bound in an additional form. If any anti-RGMa antibody can bind to human RGMa protein saturated with anti-RGMa antibody containing amino acid sequence of specific CDR of the present invention in the form of additional binding, when the saturation binding amount 1 of any anti-RGMa antibody calculated above is shown, the antibody is judged to "not compete". On the other hand, if any anti-RGMa antibody cannot bind to human RGMa protein saturated with anti-RGMa antibody containing amino acid sequence of specific CDR of the present invention in the form of additional binding, the antibody is judged to "compete". Moreover, even if any anti-RGMa antibody can bind to human RGMa protein saturated with anti-RGMa antibody containing amino acid sequence of specific CDR of the present invention in the form of additional binding, if the additional binding amount does not reach the saturation binding amount 1 with a significant difference, the antibody is judged to "compete". The significant difference is investigated 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 with the binding of RGMa to the anti-RGMa antibody containing the amino acid sequence of the above-mentioned specific CDR can be an antibody from any animal, such as mouse antibody, human antibody, rat antibody, rabbit antibody, goat antibody, camel antibody, etc., or it can be a chimeric antibody or humanized antibody that is a combination of these antibodies, but preferably it is a chimeric antibody, a humanized antibody, or a 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. As examples, the regions encoding the heavy chain variable region include base sequences encoding the amino acid sequences of sequence numbers 33, 34, and 35 in the sequence list (one or several amino acids may be substituted, deleted, inserted, or added), and the regions encoding the light chain variable region include base sequences encoding the amino acid sequences of sequence numbers 30, 31, and 32 in the sequence list (one or several amino acids may be substituted, deleted, inserted, or added); or the regions encoding the heavy chain variable region include base sequences encoding the amino acid sequences of sequence numbers 39, 40, and SFG in the sequence list (one or several amino acids may be substituted, deleted, inserted, or added), and the regions encoding the light chain variable region include base sequences encoding the amino acid sequences of sequence numbers 36, 37, and 38 in the sequence list (one or several amino acids may be substituted, deleted, inserted, or added).

[0109] Other examples of the nucleic acid molecules of the present invention include a polynucleotide in which the region encoding the heavy chain includes a base sequence encoding the amino acid sequence of sequence number 5 in the sequence list, and the region encoding the light chain includes a base sequence encoding the amino acid sequence of sequence number 4 in the sequence list; or a polynucleotide in which the region encoding the heavy chain includes a base sequence encoding the amino acid sequence of sequence number 7 in the sequence list, and the region encoding the light chain includes a base sequence encoding the amino acid sequence of sequence number 6 in the sequence list.

[0110] Other examples of the nucleic acid molecules of the present invention include polynucleotides in which the region encoding the heavy chain includes a base sequence encoding the amino acid sequence of sequence number 9 in the sequence listing, and the region encoding the light chain includes a base sequence encoding the amino acid sequence of sequence number 8 in the sequence listing.

[0111] Other examples of the nucleic acid molecule of the present invention include a polynucleotide in which the region encoding the heavy chain comprises a base sequence encoding the amino acid sequence of any one of sequence numbers 11 to 18 in the sequence listing, and the region encoding the light chain comprises a base sequence encoding the amino acid sequence of any one of sequence numbers 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 includes a base sequence encoding the amino acid sequence of sequence number 15 in the sequence listing, and the region encoding the light chain includes a base sequence encoding the amino acid sequence of sequence number 19 in the sequence listing.

[0113] Other examples of the nucleic acid molecules of the present invention include polynucleotides in which the region encoding the heavy chain variable region includes a base sequence encoding the amino acid sequence of sequence number 41 in the sequence listing, and the region encoding the light chain variable region includes a base sequence encoding the amino acid sequence of sequence number 42 in the sequence listing.

[0114] Specific examples of the nucleic acid molecules of the present invention include polynucleotides in which the region encoding the heavy chain variable region includes the base sequence of sequence number 43 in the sequence listing, and the region encoding the light chain variable region includes the base sequence of sequence number 44 in the sequence listing.

[0115] Furthermore, the nucleic acid molecule of the present invention may include a polynucleotide that hybridizes with the complementary strand DNA of the base sequence of SEQ ID NO. 43 in the sequence list under stringent conditions and a polynucleotide that hybridizes with the complementary strand DNA of the base sequence of SEQ ID NO. 44 in the sequence list under stringent conditions, as long as it encodes a monoclonal antibody that has the ability to bind to RGMa, does not inhibit the binding of RGMa to Neogenin, and neutralizes the neurite outgrowth inhibitory activity of RGMa. Here, stringent conditions include, for example, conditions of washing at 68°C, 0.1×SSC, and a salt concentration equivalent to 0.1% SDS after Southern hybridization.

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

[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 of a fusion tumor or the like using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using random primers or the like. Next, the cDNA encoding the antibody is amplified by PCR using oligonucleotides of respective preserved sequences as primers in the variable regions of known human antibody heavy chain genes and light chain genes. Sequences encoding constant regions can be obtained by amplifying known sequences by PCR. The base sequence of DNA can be determined by inserting a plasmid for sequence determination or the like according to general methods. Alternatively, the 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 combining it with a sequence containing the constant region.

[0118] In addition, the present invention provides a recombinant vector comprising the nucleic acid molecule of the present invention and a transformant (host cell) comprising the recombinant vector. As a recombinant vector, it can be a vector that can be expressed in prokaryotic cells such as Escherichia coli (e.g., pBR322, pUC119, or derivatives thereof), but it is preferably a vector that can be expressed in eukaryotic cells, and more preferably a vector that can be expressed in cells from mammals. Vectors that can be expressed in cells from mammals include, for example, plasmid vectors such as pcDNA3.1 (manufactured by Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, pREP4, and viral vectors such as pDON-AI DNA (manufactured by TAKARA BIO). It can be a single vector comprising a heavy chain coding sequence and a light chain coding sequence, or it can be two vectors comprising a vector comprising a heavy chain coding sequence and a vector comprising a 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, and more preferably a cell from a mammal. Examples of cells from mammals include Chinese hamster ovary cells (CHO cells), COS, myeloma, BHK, HeLa, Vero, 293, NS0, Namalwa, YB2 / 0, and the like.

[0120] The anti-RGMa antibody or its antigen-binding fragment obtained can be purified to be uniform. The separation and purification of antibodies and the like can be carried out by using separation and purification methods commonly used for proteins. For example, if a chromatography column, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric point electrophoresis, etc. of affinity chromatography are appropriately selected and combined, antibodies can be separated and purified (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but it is not limited to these. Columns used for affinity chromatography include protein A columns, protein G columns, anti-immunoglobulin antibody binding columns, antigen binding columns, etc. For example, as protein A columns, Hyper D, POROS, Sepharose FF (Amersham Biosciences), etc. can be listed.

[0121] <Preventive or therapeutic drugs for immunological and neurological diseases> The RGMa binding protein of the present invention, especially the anti-RGMa antibody or its antigen binding fragment, promotes the restoration of nerve function by neutralizing the neurite growth inhibitory activity of RGMa, and can therefore be used as a preventive, therapeutic or relapse prevention drug for neurological diseases. Furthermore, the RGMa binding protein of the present invention, particularly the anti-RGMa antibody or its antigen-binding fragment, neutralizes T cell activation caused by RGMa and can therefore be used as a drug for the prevention, treatment or recurrence prevention of immunological diseases.

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

[0123] Examples of immunological diseases include multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Guillain-Barré syndrome, neuro-Behcet's disease, pernicious anemia, type I (insulin-dependent) diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjögren's syndrome, Goodpasture's syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis. Multiple sclerosis is preferred.

[0124] The RGMa binding protein of the present invention, especially the anti-RGMa antibody or its antigen binding fragment, can be used as a preventive, therapeutic or relapse prevention drug for neurological diseases / immunological diseases. Preferred neurological diseases / immunological diseases include spinal cord injury, nerve trauma (including optic nerve trauma), and multiple sclerosis (including relapsing-remitting multiple sclerosis, first-onset progressive multiple sclerosis, and secondary progressive multiple sclerosis).

[0125] Here, "treatment" includes any treatment of diseases in mammals, especially humans, including hindering the symptoms of the disease, that is, preventing the progression or eliminating the disease or symptoms, and alleviating the symptoms of the disease, that is, causing the regression of the disease or symptoms, or delaying the progression of the symptoms.

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

[0127] Furthermore, "relapse prevention" includes preventing the recurrence of the above-mentioned diseases that have repeatedly remitted and relapsed in mammals, especially humans.

[0128] The RGMa binding protein (anti-RGMa antibody or antigen binding fragment thereof) of the present invention can be used as a pharmaceutical composition for preventing or treating neurological diseases or immunological diseases.

[0129] The administration form of the RGMa binding protein (anti-RGMa antibody or its antigen-binding fragment) of the present invention is not particularly limited, and can be administered to mammals including humans by any administration route of 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 their preparation methods are well known to those skilled in the art. Pharmaceutical compositions can be prepared by mixing the antibodies of the present invention with pharmaceutically acceptable drug carriers. The dosage forms for parenteral administration include injection preparations (e.g., drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, preparations for brain administration, and preparations for spinal administration), external preparations (e.g., ointments, poultices, lotions), suppositories for inhalation, eye preparations, eye ointments, nasal drops, ear drops, liposome preparations, etc. In particular, when the central nervous system is to be acted directly, it can be continuously injected using a medical micropump such as an osmotic pressure pump, or it can be mixed with fibrin glue or the like to be a sustained-release preparation and then left in the affected tissue.

[0131] For example, injection preparations can usually be prepared by dissolving the antibody in distilled water for injection, but dissolution aids, buffers, pH adjusters, isotonic agents, analgesics, preservatives, tranquilizers, etc. can be added as needed. In addition, freeze-dried preparations can also be prepared for use.

[0132] The dosage form for oral administration may be a solid or liquid dosage form, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, injections, tablets, etc.

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

[0134] Pharmacologically acceptable drug carriers include, for example, excipients, lubricants, binders and disintegrants in solid preparations, or solvents, dissolution aids, suspending agents, isotonic agents, buffers and analgesics in liquid preparations. Conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and other additives may also be used appropriately and in appropriate amounts as needed.

[0135] The dosage of the antibody of the present invention is determined by a physician based on various factors such as the route of administration, the type of disease, the degree of symptoms, the age, sex, weight of the patient, the severity of the disease, the pharmacological characteristics of the drug dynamics and toxicology, whether a drug delivery system is used, and whether it is administered as part of a combination with other drugs. However, generally, for an adult (weight 60 kg), the oral dosage can be 1-5000 μg / day, preferably 10-2000 μg / day, and more preferably 50-2000 μg / day; the injection dosage can be 1-5000 μg / day, preferably 5-2000 μg / day, and more preferably 50-2000 μg / day, and can be administered once or divided into several doses. For systemic parenteral administration, 10 to 100,000 μg / kg, preferably 100 to 50,000 μg / kg, and more preferably 500 to 20,000 μg / kg per unit body weight can be administered once a day, once a week, once a month, or once to seven times a year. For local administration using an osmotic pump, etc., usually 10 to 100,000 μg / day, preferably 100 to 10,000 μg / day, and more preferably 500 to 5,000 μg / day can be continuously injected for each adult (weight 60 kg). [Example]

[0136] Hereinafter, embodiments are listed to specifically illustrate the present invention, but the present invention is not limited to the following embodiments.

[0137] Example 1: Preparation of human RGMa protein (C-terminal domain) CHO cells expressing recombinant human RGMa protein fused with a histidine tag at the C-terminus of Pro169~Gly422 (meaning the 169th proline residue to the 422nd glycine residue from the N-terminal side, and the same will be recorded hereinafter) of the human RGMa protein (SEQ ID NO. 1 in the sequence list) were established. The C-terminal domain of human RGMa protein contained in the culture supernatant of CHO cells was adsorbed on a nickel column (GE Healthcare, 17-5247-01) and then eluted with a 100 mM imidazole solution. The imidazole elution fraction was replaced with phosphate buffered saline (PBS) by dialysis and used as an immunogen.

[0138] Example 2: Preparation of mouse anti-human RGMa monoclonal antibody 10 μg of the recombinant human RGMa protein prepared in Example 1 was mixed with complete Freund's adjuvant (manufactured by Sigma) to prepare an emulsion, and immunization was induced at several sites under the back of BALB / c mice (Charles River Co., Ltd., Japan). After that, 10 μg of recombinant human RGMa protein prepared in an emulsion with incomplete Freund's adjuvant (manufactured by Sigma) was similarly induced every 1 to 2 weeks, and blood was collected after several immunizations. The antibody titer was measured by ELISA method using human or mouse RGMa protein immobilized on a solid phase as described later. For individuals in whom an increase in antibody titer was observed, 10 μg of human RGMa protein was intravenously administered for boosting, and spleen cells were collected 2 to 3 days later. Cell fusion was performed by mixing the spleen cells with half of the mouse myeloma cells (SP2 / 0, Dainippon Sumitomo Pharmaceuticals), and adding polyethylene glycol (Roche Diagnostics) to the precipitate obtained by centrifugation to make the cells fuse. Then, the centrifuged cells were washed twice with D-MEM (Invitrogen). The cells were resuspended in GIT medium (Nippon Pharmaceutical) containing 10% fetal bovine serum (Invitrogen), 1% BM condimed (Roche Diagnostics) and HAT (Sigma-Aldrich), and seeded in a 96-well plate at 5×104 myeloma cells / well per well. The culture supernatant was recovered and the antibody-producing cells were screened by the human RGMa protein solid-phase ELISA method of Example 3. The antibody-producing cells obtained by screening were cloned by limiting dilution method, and fusion tumor cells producing two monoclonal antibodies (B5.116A3 and B5.70E4) were selected. The isotypes of the two monoclonal antibodies determined using an isotype kit (Mouse MonoAB ID / SP KIT, manufactured by ZYMED, 93-6550) were mouse IgG2b for the heavy chain and κ for the light chain. Monoclonal antibodies were purified by affinity chromatography using agarose (Sigma) immobilized with anti-mouse IgG antibodies from the culture supernatant of the fusion tumor. After the antibody was bound to the column, the column was washed with PBS, then eluted with 10 mM glycine hydrochloride (pH 2.7) and rapidly neutralized. Afterwards, the eluted neutralization solution was replaced with PBS through an ultrafiltration membrane.

[0139] Example 3: ELISA with solid-phased 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 into a 96-well plate at 50 μL / well and allowed to stand at room temperature for 1 hour. After removing the liquid, ApplieBlock (Seikagaku Biobusiness, 200150) diluted 5 times with PBS was dispensed at 200 μL / well and allowed to stand at room temperature for 1 hour to block nonspecific binding. After washing three times with PBST (PBS containing 0.05% Tween20), the sample (mouse serum, fusion tumor culture supernatant, recombinant antibody expression culture supernatant described later, or purified antibody, etc.) diluted in stages with PBS was added at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing three times with PBST, 50 μL / well of peroxidase-labeled goat anti-mouse IgG antibody (GE Healthcare, NA9310V) diluted with PBS was dispensed and allowed to stand at room temperature for 1 hour. After washing three times, a peroxidase color development kit (Sumitomo Bakelite, ML-1130O) was added to develop the color for a certain period of time, and the absorbance at 492 nm was measured with a microplate reader.

[0140] Example 4: Antibody antigenic determinant analysis The antigenic determinant bound by the antibody was determined by peptide scanning. A total of 83 peptides were synthesized, with an N-terminal biotinylated gap sequence (SGSG) (sequence number 46 in the sequence list) fused to the N-terminal side of the amino acid sequence consisting of 11 consecutive residues each offset by 3 residues contained in Arg172~Ala424 of the human RGMa protein (sequence number 1 in the sequence list). After the peptides were fixed on the avidin microplate, the test antibodies (B5.116A3, B5.70E4) were reacted. Then, the peroxidase-labeled rabbit anti-mouse Ig antibody (Dako, P026002) was reacted, and after adding the matrix solution for color development for a certain period of time, the absorbance was measured by a microplate analyzer. As a result, B5.116A3 bound to Glu298~Gly311 (two types of peptides of Glu298~Asp308 and Val301~Gly311), Asn322~Glu335 (two types of peptides of Asn322~Thr332 and Ile325~Glu335), and Lys367~Ala377 of a peptide derived from human RGMa, and B5.70E4 bound to Glu298~Gly311 (two types of peptides of Glu298~Asp308 and Val301~Gly311), Asn322~Glu335 (two types of peptides of Asn322~Thr332 and Ile325~Glu335), and Pro349~Thr359 of a peptide derived from human RGMa.

[0141] Example 5: Sequence analysis and cloning of mouse antibody genes Total RNA was extracted from fusion tumor cells producing mouse monoclonal antibodies (B5.116A3, B5.70E4). Total RNA was used as a template to synthesize cDNA by reverse transcription. Using cDNA as a template, PCR was used to amplify the genes of the light chain variable region and constant region, as well as the heavy chain variable region and constant region, and the DNA sequence was determined. Then, based on the determined variable region sequence and constant region sequence, the full-length antibody gene was amplified by PCR and cloned. The amino acid sequences encoded by these antibody genes are as follows.

[0142] (1) B5.116A3 light chain amino acid sequence (SEQ ID NO. 4 in the sequence listing)

[0143] (2) B5.116A3 heavy chain amino acid sequence (SEQ ID NO. 5 in the sequence listing)

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

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

[0146] Example 6: Preparation of recombinant mouse antibodies and recombinant rat-mouse chimeric antibodies Recombinant mouse antibodies of two anti-RGMa antibodies B5.116A3 or B5.70E4 (hereinafter referred to as "r116A3" and "r70E4", respectively) derived from fusion tumors were prepared. In addition, as a comparative example, based on Patent Document 1 (WO2009 / 106356), the variable region of rat antibody 5F9 and the constant region of mouse antibody (IgG2bκ) (the light chain constant region is Arg108 to Cys214 of sequence number 4 in the sequence list, and the heavy chain constant region is Ala117 to Lys452 of sequence number 5 in the sequence list) were fused to prepare a recombinant rat-mouse chimeric antibody (hereinafter referred to as "r5F9"). The DNA encoding the light chain and heavy chain of each antibody was inserted into pcDNA3.3 (manufactured by Life Technologies) to prepare an expression vector. The expression vector was introduced into HEK293F cells (manufactured by Life Technologies) using Neofection 293 (manufactured by Astec). The cells were cultured for 6 days at 37°C and 8% carbon dioxide, and the culture supernatant was recovered. The recombinant antibody was purified by adding the culture supernatant to an affinity column immobilized with Protein A or Protein G (manufactured by GE Healthcare), and the antibody bound to the column was eluted with 10mM glycine hydrochloride (pH2.8), rapidly neutralized, and replaced with PBS. When the purity of purification needs to be improved according to the purpose of use, the antibody purified by Protein A column is purified by Ceramic Hydroxyapatite Type 1 (CHT) column (manufactured by BIORAD). The antibody bound to the CHT column is washed with 10mM KH2PO4 (pH6.5) and then eluted with 20mM KH2PO4 (pH6.5) and 0.5M NaCl. After the eluted solution is recovered and separated, it is replaced with PBS.

[0147] Example 7: Binding assay for RGMa protein expressing cells Vectors expressing full-length human RGMa protein (Met1-Cys450 of sequence number 1 in the sequence list), C-terminal domain of human RGMa protein (Pro169-Cys450 of sequence number 1 in the sequence list), full-length mouse RGMa protein (Met1-Trp454 of sequence number 2 in the sequence list), or C-terminal domain of rat RGMa protein (Pro170-Trp449 of sequence number 3 in the sequence list) are introduced into CHO cells or HEK293 cells to prepare antigen-expressing cells. Furthermore, the C-terminal peptide of RGMa protein is processed during the GPI anchoring reaction. Both mouse RGMa protein and rat RGMa protein are cleaved at Ala427 and the C-terminal peptide is removed. Therefore, the amino acid sequence of the full-length protein and C-terminal domain expressed on cells through GPI anchoring is the same in mouse and rat lines. After the test antibodies (r116A3 and r70E4) and r5F9 (comparative example) were reacted with the antigen-expressing cells at a concentration of 10 μg / mL, the cells were washed with PBS containing 0.1% bovine serum albumin and 0.05% NaN3. FITC-labeled anti-mouse immunoglobulin antibody (manufactured by DAKO) was reacted and the cells were washed. Fluorescence was measured by flow cytometry (manufactured by Becton Dickinson, FACSCalibur) to evaluate the binding of the test antibodies to the antigen-expressing cells (Table 1). The results showed that r116A3 and r70E4, unlike r5F9, also bind to the C-terminal domain of human and rat RGMa proteins. The neurite outgrowth inhibitory effect was observed only with the C-terminal domain of RGMa protein, and r116A3 and r70E4 inhibited both the full-length RGMa protein and the C-terminal domain.

[0148]

[0149] Example 8: Determination of the dissociation constant of RGMa protein The affinity of the test antibodies (r116A3, r70E4) and r5F9 (comparative example) for RGMa protein was measured by the surface plasmon resonance (SPR) method using Proteon XPR36 (manufactured by BIORAD). 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 in 10 mM acetic acid buffer (pH 4.5) was immobilized on the GLC sensor chip by amine coupling. The test antibody diluted in stages was added at a flow rate of 100 μL / min for 60 seconds as an analyte to measure the dissociation constant (Kd value). As shown in Table 2, r116A3 and r70E4, unlike r5F9, also bind to the C-terminal domain of human RGMa protein. r116A3 binds 32 times more strongly to human RGMa protein and 44 times more strongly to mouse RGMa protein than r5F9.

[0150]

[0151] Example 9: RGMa-Neogenin binding inhibition test Purify the extracellular domain (Ala34~Leu1105) of recombinant human Neogenin protein (SEQ ID NO. 10 in the sequence list). Establish a CHO cell line that expresses the extracellular domain of human Neogenin protein. Fusion of histidine tag at the C-terminus. Adsorb the culture supernatant of CHO cells onto a nickel column (GE Healthcare, 17-5247-01) and then elute with 100mM imidazole solution. Replace the imidazole eluate with PBS by dialysis. Human RGMa protein (R&D systems, 2459-RM) was labeled with biotin using ChromaLink Biotin Labeling Kit (Solulink). Biotin-labeled human RGMa protein prepared at 2 μg / mL was mixed with equal amounts of test antibodies (r116A3, r70E4) diluted in two-fold stages and reacted at room temperature for 2 hours to prepare a mixed solution. At the same time, 50 μL / well of the extracellular domain of human Neogenin protein prepared at 2 μg / mL in PBS was added to a 96-well plate and allowed to stand at room temperature for 1 hour to prepare a Neogenin solid phase microplate. After the liquid was removed, a 2.5% bovine serum albumin solution was added and allowed to stand for 1 hour to block nonspecific binding. To the Neogenin solid phase microplate, 50 μL / well of the above mixed solution was added and allowed to stand at room temperature for 1 hour. After that, a washing operation was performed, and peroxidase-labeled Avidin (VECTASTAIN ABC system, manufactured by Vector Laboratories) was added and allowed to stand at room temperature for 1 hour. After that, a washing operation was performed, and a substrate solution was added to develop color for a certain period of time, and the absorbance was measured with a microplate analyzer. The absorbance ratio in the absence of the antibody was set as 1 to plot the concentration-dependent RGMa-Neogenin binding inhibition caused by the antibody (Figure 1). The results showed that, unlike the anti-human RGMa polyclonal antibody (manufactured by R&D Systems, AF2459) and r5F9, r116A3 and r70E4 did not inhibit the binding of RGMa-Neogenin.

[0152] Example 10: RGMa-BMP2 binding inhibition test Human RGMa protein (R&D systems, 2459-RM) prepared at 2 μg / mL in PBS was added to a 96-well plate at 50 μL / well and allowed to stand at room temperature for 1 hour. A 2.5% bovine serum albumin solution was added and allowed to stand for 1 hour to block nonspecific binding, and an RGMa protein solid-phase microplate was prepared. The test antibody (B5.116A3, B5.70E4) was added to the RGMa protein solid-phase microplate at a stepwise dilution of 0.01-10 μg / mL and allowed to stand at room temperature for 1 hour. After washing, human BMP2 protein (R&D systems, 355-BM) diluted to 0.5 μg / mL was added and allowed to stand at room temperature for 1 hour. The biotin-labeled anti-BMP2 antibody was allowed to react, and further, peroxidase-labeled Avidin (VECTASTAIN ABC system, manufactured by Vector Laboratories) and a substrate solution were added, and color was developed for a certain period of time, and the absorbance was measured with a microplate reader ( FIG. 2 ). The results showed that the anti-RGMa antibody (B5.116A3) in particular weakly inhibited the binding of RGMa-BMP2 in a concentration-dependent manner (absorbance was 0.45 at 0.01 μg / mL, 0.4 at 0.1 μg / mL, 0.32 at 1 μg / mL, and 0.1 at 10 μg / mL).

[0153] Example 11: Design of humanized antibodies The humanization of mouse monoclonal antibody B5.116A3 was performed by the complementarity determining region (CDR) transplantation method according to the method of Winter et al. described in Japanese Patent No. 2912618. First, a 3D homology model of the variable regions of the light and heavy chains of the mouse monoclonal antibody B5.116A3 was created, and the amino acid residues located near the CDR in the framework (FW) region were specified. The human antibody FW in which these amino acids were maintained as much as possible was selected, and the CDR of the mouse antibody was transplanted to design the humanized antibody sequence. The designed humanized antibody sequence is recorded as HA for the heavy chain (sequence number 11 in the sequence listing) and KA for the light chain (sequence number 19 in the sequence listing). Furthermore, additional mutations were introduced into the amino acids in the FW related to the structural stability of the variable region, and multiple humanized antibody sequences were designed (the heavy chain includes HA, HB~HH, a total of 8 types, and the light chain includes KA, KB~KG, a total of 7 types).

[0154]

[0155] Example 12: Preparation of recombinant mouse human chimeras and recombinant humanized antibodies (1) According to 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)

[0157] Heavy chain (SEQ ID NO. 9 in the sequence listing)

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

[0159] Heavy chain / light chain combinations: 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) According to Example 6, a recombinant humanized anti-RGMa antibody (hereinafter referred to as rH5F9) was prepared (Comparative Example). The variable region of the humanized anti-RGMa monoclonal antibody h5F9 described in Patent Document 1 (WO2009 / 106356) (the light chain is seq ID_53 of Patent Document 1, and the heavy chain is seq ID_50 of Patent Document 1) is linked to the human antibody constant region (the light chain is Arg108~Cys214 of sequence number 26 in the sequence list, and the heavy chain is Ala117~Lys446 of sequence number 27 in the sequence list).

[0161] Example 13: Thermal stability test of antibodies 20 μL of the culture supernatant expressing the recombinant antibody described in Example 12 was taken and heat treated at 8 temperatures of 40, 45, 50, 55, 60, 65, 70, and 75° C. for 10 minutes each using a thermal cycler (manufactured by Takara Bio, TP600). The culture supernatant was diluted with PBS to a final antibody concentration of 125 ng / mL. Then, the human RGMa protein described in Example 3 was immobilized on a solid phase and ELISA was performed to evaluate the antigen binding of the antibody ( FIG. 3 ). As a result, the humanized antibody composed of the combination of heavy chain HE and light chain KA (hereinafter referred to as "rH116A3") and the mouse human chimeric antibody (r116A3C) showed better thermal stability than the humanized antibody (rH5F9). Hereinafter, the humanized antibody composed of the combination of HE / KA will be described as rH116A3. Furthermore, when not subjected to heat treatment, the mouse human chimeric antibody (r116A3C) and the humanized antibody (rH116A3) showed equivalent antigen binding properties, and there was no decrease in antigen binding properties associated with humanization.

[0162] Example 14: Establishment of a stable CHO expression strain producing humanized anti-RGMa antibody (rH116A3) The stable CHO expression strain for producing humanized anti-RGMa antibody (rH116A3) was established using the Lonza GS Xceed system (Lonza). The pXC double-gene vector containing the light chain coding sequence (SEQ ID NO. 44 in the sequence list) and the heavy chain coding sequence (SEQ ID NO. 43 in the sequence list) of the humanized anti-RGMa antibody (rH116A3) was introduced into the CHOK1SV GS knock-out parent cell line, and the transformed cell pool was obtained under methionine sulphoximine (MSX) selection. After separation into single cells by flow cytometry, the antibody production amount in the culture supernatant, cell proliferation, etc. were evaluated to obtain a stable CHO expression strain.

[0163] Example 15: Neurite Growth Assay Cerebellum was removed from rat neonates (P7), suspended in trypsin solution (0.25% trypsin PBS solution containing 0.2% DNase), and digested at 37°C for 10-15 minutes. Then, DMEM medium containing 10% fetal bovine serum was added and centrifuged. Resuspended in the same medium and centrifuged, the same operation was repeated twice to wash the cells. Further, the cell suspension was filtered through a 70μm cell strainer and centrifuged, and the precipitate was resuspended in the same medium. B27 supplement (GIBCO) was added to the cell suspension to prepare rat neonatal cerebellar granule cells. Next, rat neonatal cerebellar granule cells were seeded into cell microtiter plates and cultured at 37°C for 1 day. Recombinant RGMa protein (R&D systems, 2459-RM) was added at a final concentration of 2 μg / mL and cultured at 37°C for 2 days. After measuring the length of neurites by microscopic observation, as shown in Figure 4, the addition of RGMa changed 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, and neurite growth was inhibited. 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 the recombinant RGMa protein, the neurites grew to the same extent as the control (RGMa not added), and the neutralization effect of the RGMa protein by the antibody was observed.

[0164] Example 16: Efficacy test using a spinal cord injury model rat Wistar rats (female, 8 weeks old, weighing about 200 g) were anesthetized with ethyl halide (Takeda Pharmaceutical Co.), and a laparotomy was performed on the anterior and posterior vertebrae (T8-T10) centered at the spinal segment T9 to expose the spinal cord. When the spinal cord contusion model was used for evaluation, an IH impactor (Precision System Co., Ltd.) was used to apply a pressure of 200 kdyn to the exposed spinal cord. Immediately after the spinal cord was injured as described above, an osmotic pressure mini pump (200 μL volume, 0.5 μL / hour, 14-day delivery) (Alzet, model 2002) filled with 400 μg / mL of the test antibody (r116A3, r70E4) or the control mouse antibody (mo-IgG2bκ) was placed under the skin of the back of the rat. The tip of the silicon tube connected to the outlet of the osmotic pressure mini pump was placed under the dura mater at the site of spinal cord injury. The tube was sutured to the spinous process on the lower limb side of the laminectomy site and fixed, and the muscle and skin layers were sutured and fed. The motor function of rats with spinal cord injury was evaluated using the Basso-Beattie-Bresnahan (BBB) ​​score (Basso, DM, Beattie, MS, & Bresnahan, JC, A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12, 1-21 (1995)) at 0, 1, 3, 7 days after injury, and every week thereafter for up to 8 weeks. As shown in Figure 5 (A), r116A3 or r70E4 significantly improved the BBB score 4 or 3 weeks after administration, respectively, compared with the control antibody (mo-IgG2bκ) (p<0.05, Student's t-test). When the hemisection model was used for evaluation, the exposed dorsal part of the spinal cord was cut at a depth of 1.8 mm to 2.0 mm. The test antibody (B5.116A3) or the control mouse antibody (mo-IgG2bκ) was administered using an osmotic pressure mini-pump in the same manner as above, and the BBB score was evaluated at 0, 1, 3, and 7 days after injury, and every week thereafter, up to 10 weeks. As shown in Figure 5 (B), B5.116A3 significantly improved the BBB score compared with the control antibody (mo-IgG2bκ) 4 weeks after administration (p<0.01, Student's t-test).

[0165] Example 17: Efficacy test using multiple sclerosis model mice The PLP139-151 peptide (HSLGKWLGHPDKF: sequence number 45 in the sequence list, manufactured by Peptide Research Institute) was dissolved in physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) and mixed with incomplete Freund's adjuvant (manufactured by Sigma) to which killed tuberculosis bacteria H37 Ra (manufactured by Difco Laboratories) were added to prepare an emulsion. As PLP139-151 peptide, 50 μg / head was used to induce subcutaneous immunization on the back of SJL / JorllcoCrj (SJL / J) mice (Charles River, Japan), and the EAE score (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 body weight changes (Figure 6) were evaluated. The test antibody (B5.116A3) diluted with physiological saline was intraperitoneally administered at 20 mg / kg 7 and 10 days or 18 and 21 days after immunization with PLP139-151 peptide. As shown in FIG6 , the anti-RGMa mouse monoclonal antibody (B5.116A3) inhibited the deterioration of EAE scores when administered before the onset of the disease ( FIG6 upper part), and showed a relapse prevention effect when administered after the onset of the disease ( FIG6 lower part), compared with the control antibody (mo-IgG2bκ).

[0166] Example 18: Antibody immunogenicity test Undifferentiated dendritic cells contained in peripheral blood from 51 healthy donors were stimulated to mature 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 antibody was taken up by dendritic cells by culturing for 4 and 5 days. The peripheral blood CD4+T cells (helper T cells) from the same donor were mixed with the test antibody, and after further co-culture for 1 week, 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 indicator. As a result, T cell proliferation was observed in 4 of the 51 donors (7.8%), indicating a low immunogenicity risk.

[0167]

Claims

1. An isolated anti-RGMa antibody or its antigen-binding fragment, wherein the heavy chain comprises the following amino acid sequence: (Sequence No. 15), and the light chain comprises the following amino acid sequence: (Sequence No. 19).

2. The anti-RGMa antibody or its antigen-binding fragment as requested in claim 1, wherein the anti-RGMa antibody is a humanized antibody.

3. A nucleic acid molecule that is a protein portion encoding an anti-RGMa antibody or an antigen-binding fragment thereof as claimed in claim 1 or 2.

4. A recombinant vector comprising the nucleic acid molecule as claimed in claim 3.

5. A host cell comprising the recombinant vector as claimed in claim 4.

6. A pharmaceutical composition comprising an anti-RGMa antibody or an antigen-binding fragment thereof as claimed in claim 1 or 2.

7. The pharmaceutical composition described in claim 6 is for the prevention, treatment or relapse prevention of neurological or immunological diseases.

8. The pharmaceutical composition as described in claim 7, including neurological disorders, is selected from the following: amyotrophic lateral sclerosis (ALS), brachial plexus injury, brain injury, cerebral palsy, Guillain-Barré syndrome, leukoencephalopathy, multiple sclerosis, post-poliomyelitis syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal tumor, transverse myelitis, dementia, Huntington's disease, tardive dyskinesia, mania, Parkinson's disease, Steele-Richard syndrome, Down syndrome, myasthenia gravis, nerve injury, angioamylindrica, cerebral hemorrhage associated with amylindrica, cerebral infarction, encephalitis. A group of acute confusional disorders, glaucoma, schizophrenia, or immunological diseases, including multiple sclerosis, psoriasis, arthritis, Guillain-Barré syndrome, neurobesette syndrome, pernicious anemia, type I (insulin-dependent) diabetes mellitus, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Hugh Grant's syndrome, Goobard's syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis.

9. The pharmaceutical composition of the claim 7, wherein the neurological or immunological disease is selected from the group consisting of spinal cord injury, nerve injury and multiple sclerosis.

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