WO2016175236A1 - RGMa結合タンパク質及びその使用 - Google Patents
RGMa結合タンパク質及びその使用 Download PDFInfo
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- WO2016175236A1 WO2016175236A1 PCT/JP2016/063166 JP2016063166W WO2016175236A1 WO 2016175236 A1 WO2016175236 A1 WO 2016175236A1 JP 2016063166 W JP2016063166 W JP 2016063166W WO 2016175236 A1 WO2016175236 A1 WO 2016175236A1
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Definitions
- the present invention relates to RGMa binding proteins and uses thereof.
- RGM (repulsive guidance molecule) is a GPI-anchored membrane protein having a molecular weight of about 33 kDa, and is a membrane protein initially identified as an axon-inducing molecule in the visual system (see Non-Patent Document 1).
- the RGM family includes three members called RGMa, RGMb and RGMc, among which RGMa is re-expressed after central nervous system injury in adult humans and rats, in addition to developmental stage, RGMa in rats. Since inhibition enhances neurite growth after spinal cord injury and promotes functional recovery (see Non-Patent Document 2), RGMa is considered to be a neurite inhibitor after central nervous system injury.
- RGMa has also been reported to act in the immune system.
- RGMa is expressed on dendritic cells and acts on T cells to enhance the adhesion of T cells to ICAM-1 / fibronectin and induce cytokine production (Patent Document 4).
- Patent Document 4 In multiple sclerosis model mice, when anti-RGMa antibody is administered, symptoms due to encephalomyelitis are suppressed, and the onset and recurrence are also suppressed.
- the anti-RGMa antibody is considered to inhibit T cell activation by binding to RGMa expressed in dendritic cells and to have an effect on multiple sclerosis.
- Neogenin protein has been reported as a receptor for RGMa (Patent Document 3).
- Neogenin is a single-transmembrane protein that is expressed on neurons and T cells.
- RGMa binds to Neogenin on the cell membrane and induces intracellular activation of RhoA and inactivation of Ras, thereby producing a neurite growth inhibitory effect.
- Neogenin is known to cause apoptosis when RGMa is not bound in the developing chicken brain (Matsunaga et al., Dev. Growth Differ. 46, 481, 2004). That is, it is considered that the RGMa / Neogenin pathway has two contradictory effects of promoting the survival of nerve cells, favorable for nerve regeneration, and negative for inhibiting neurite growth.
- Patent Document 1 discloses an axonal regeneration promoter containing an anti-RGM neutralizing antibody as an active ingredient.
- Patent Documents 2 and 3 disclose therapeutic agents for mechanical damage to the brain and spinal cord of an anti-RGM antibody that regulates the binding of RGM to the Neogenin receptor.
- Patent Document 4 discloses an anti-RGM antibody pharmaceutical use such as multiple sclerosis.
- Patent Document 5 discloses therapeutic use of anti-RGM antibodies for diseases including multiple sclerosis, mammalian brain trauma, spinal cord injury, stroke, neurodegenerative diseases and schizophrenia.
- Patent Document 6 discloses a therapeutic use for spinal cord injury and multiple sclerosis of an RGM modulator such as an anti-RGM antibody
- Non-Patent Document 3 discloses a therapeutic use for progressive multiple sclerosis. It is disclosed.
- an object of the present invention is to provide an RGMa-binding protein that does not inhibit the RGMa / Neogenin interaction and neutralizes the neurite growth inhibitory activity of RGMa.
- the present inventors have intensively studied to solve the above problems. As a result, we succeeded in obtaining an RGMa binding protein that does not inhibit the binding of RGMa and Neogenin and neutralizes the neurite growth inhibitory activity of RGMa, which is used as a medicine for neurological diseases and immunological diseases. As a result, the present invention has been completed.
- the present invention is as follows. [1] An isolated RGMa binding protein that does not inhibit the binding of RGMa and Neogenin and neutralizes the neurite growth inhibitory activity of RGMa. [2] The RGMa binding protein according to [1], which binds to human RGMa, rat RGMa and / or mouse RGMa. [3] Binds to a peptide of EEVVNAVEDWDSQG (SEQ ID NO: 26 in the sequence listing), NQQIDFQAFHTNAE (SEQ ID NO: 27 in the sequence listing), PTAPETFPYET (SEQ ID NO: 28 in the sequence listing), and / or KLPVEDLYYQA (SEQ ID NO: 29 in the sequence listing).
- RGMa-binding protein according to [1] or [2].
- [6] The RGMa binding protein according to any one of [1] to [4], which binds to the peptide of SEQ ID NO: 26 in the sequence listing, SEQ ID NO: 27 in the sequence listing, and SEQ ID NO: 29 in the sequence listing.
- [7] The RGMa binding protein according to any one of [1] to [6], wherein the RGMa binding protein is a human antibody, a humanized antibody or a chimeric antibody, or an antigen-binding fragment thereof.
- [8] A nucleic acid molecule encoding the protein portion of the RGMa binding protein according to any one of [1] to [7].
- [9] A recombinant vector comprising the nucleic acid molecule according to [8].
- a host cell comprising the recombinant vector according to [9].
- a pharmaceutical composition comprising the RGMa binding protein according to any one of [1] to [7].
- Neurological diseases include amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain Valley, cerebral white matter atrophy, multiple sclerosis (recurrence) Remission-type multiple sclerosis, primary progressive multiple sclerosis, including secondary progressive multiple sclerosis), optic neuritis, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spine Tumor, transverse myelitis, dementia (including senile dementia, mild cognitive impairment, Alzheimer's disease, Alzheimer-related dementia), Huntington's chorea, late-onset dyskinesia, mania, Parkinson's disease, Steel-Richard syndrome Down syndrome, myasthenia gravis, neurotrauma (including optic nerve trauma), vascular amyloidosis, cerebral hemorrhage with amyloidosis, cerebral infarction, encephalitis, acute confusion disorder, glaucoma, schizophrenia and retinal nerve fiber Degeneration (diabe), brain
- the immunological disease is multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), optic neuromyelitis, psoriasis, Arthritis (including rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Guillain-Barre syndrome, neurobehcet's disease, pernicious anemia, type I (insulin-dependent) diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) , Sjogren's syndrome, Goodbascher syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, And the pharmaceutical composition according to [13], which is selected from the group comprising sarcoidosis.
- Neurological or immunological diseases are spinal cord injury, nerve trauma (including optic nerve trauma) and multiple sclerosis (relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary [13]
- Light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3), heavy chain complementarity determining region 1 (HCDR1)
- the amino acid sequences of the heavy chain complementarity determining region 2 (HCDR2) and the heavy chain complementarity determining region 3 (HCDR3) are: LCDR1: RASQDISSYLN (SEQ ID NO: 30 in the sequence listing) LCDR2: YTSRLHS (SEQ ID NO: 31 in the sequence listing) LCDR3: QQLNTLP (SEQ ID NO: 32 in the sequence listing)
- HCDR1 DAWMD (SEQ ID NO: 33 in the sequence listing)
- HCDR2 EIRSKANNHATYYAESVKG (SEQ ID NO: 34 in the sequence listing)
- HCDR3 including RDGAY (SEQ ID NO: 35 of the Sequence Listing),
- LCDR1 RSSQSLVHSNGNTYLH (SEQ ID NO: 36
- Heavy chain variable region (VH) VH EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS or an amino acid sequence having at least 90% identity to the amino acid sequence
- Light chain variable region (VL) VL DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 42 in the sequence listing) or an anti-RG according to [17], wherein the amino acid sequence has at least 90% identity.
- nucleic acid sequence encoding the amino acid sequence of VH and VL VH: gaagtgcagctggtggaatctggcggactggtgcagcctggcagatccctgagactgtcctgtaccgcctccggcttcaccttctccgacgcctggatggattgggtgcgacaggctcctggcaagggcctggaatgggtggccgagatccggtccaaggccaacaaccacgccacctactacgccgagtctgtgaagggccggttcaccatctcccgggacgactccaagtccatcgtgtacctgcagatgaactccctgcggaccgaggacaccgccctgtactactggggaccgaggacaccgccctgtactactggggaccgagg
- a recombinant vector comprising the nucleic acid molecule according to [22] or [23].
- a host cell comprising the recombinant vector according to [24].
- a pharmaceutical composition comprising the anti-RGMa antibody or antigen-binding fragment thereof according to any one of [17] to [20].
- Neurological diseases include amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain Valley, cerebral white matter atrophy, multiple sclerosis (relapse) Remission-type multiple sclerosis, primary progressive multiple sclerosis, including secondary progressive multiple sclerosis), optic neuritis, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spine Tumor, transverse myelitis, dementia (including senile dementia, mild cognitive impairment, Alzheimer's disease, Alzheimer-related dementia), Huntington's chorea, late-onset dyskinesia, mania, Parkinson's disease, Steel-Richard syndrome Down syndrome, myasthenia gravis, neurotrauma (including optic nerve trauma), vascular amyloidosis, cerebral hemorrhage with amyloidosis, cerebral infarction, encephalitis, acute confusion disorder, glaucoma, schizophrenia and retinal nerve fiber Degeneration (diabetic ,
- the immunological disease is multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), optic neuromyelitis, psoriasis, Arthritis (including rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Guillain-Barre syndrome, neurobehcet's disease, pernicious anemia, type I (insulin-dependent) diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD) , Sjogren's syndrome, Goodbascher syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, And the pharmaceutical composition according to [28], which is selected from the group comprising sarcoidosis.
- Neurological or immunological diseases are spinal cord injury, neurotrauma (including optic nerve trauma) and multiple sclerosis (relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary [28].
- Prevention or treatment of a neurological or immunological disease comprising a step of administering an effective amount of the RGMa binding protein according to any one of [1] to [7] to a subject in need thereof Or recurrence prevention method.
- a neurological disease or immunological method comprising the step of administering an effective amount of the anti-RGMa antibody or antigen-binding fragment thereof according to any one of [17] to [20] to a subject in need thereof Disease prevention, treatment or recurrence prevention methods.
- the RGMa binding protein of the present invention does not inhibit the interaction between RGMa and Neogenin, it can maintain the action such as inhibition of Apoptosis on nerve cells etc. possessed by Neogenin bound to RGMa, and thus has a strong protective effect on nerve cells. Less worry about side effects associated with neuronal loss.
- the humanized anti-RGMa antibody of the present invention is superior in properties such as binding to human RGMa and thermal stability to conventional antibodies. Therefore, it can be used as a medicament for neurological diseases and immunological diseases having excellent medicinal effects and few side effects.
- the results of the efficacy test in (A) spinal cord crush model and (B) spinal cord hemisection model are shown. It shows the results of efficacy testing of the antibody (B5.116A3) of the present invention using a multiple sclerosis model mice induced with PLP 139-151 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 after 7 and 10 days
- RGMa is a neurite growth inhibitory protein in the central nervous system, and human RGMa protein is biosynthesized as a precursor protein consisting of 450 amino acids as shown in SEQ ID NO: 1 in the Sequence Listing.
- the signal peptide Met1 to Pro47 (referring to the peptide from the 1st methionine residue to the 47th proline residue from the N-terminal side, which will be described in the same manner) present at the N-terminal is removed, and between Asp168 and Pro169 The peptide bond is cleaved to remove the C-terminal peptides Arg423 to Cys450, and a GPI anchor is added to the C-terminal carboxyl group of Gly422 that has become the C-terminal.
- the human RGMa protein is expressed on the cell membrane via a GPI anchor as a mature protein in which the N-terminal domain (Cys48-Asp168) and the C-terminal domain (Pro169-Ala424) are connected by a disulfide bond.
- the mouse RGMa precursor protein consists of the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing
- the rat RGMa precursor protein consists of the amino acid sequence shown in SEQ ID NO: 3 of the sequence listing, but the C-terminal peptide is removed, so that the mature protein As the same amino acid sequence.
- RGMa may refer to either a precursor protein, a mature protein, or an active fragment thereof, as long as it functions by binding to Neogenin described later, or a derivative or mutant thereof. Good. Moreover, human RGMa or RGMa derived from other organisms may be used, but human RGMa is preferable.
- Neogenin is expressed in neurons of the central nervous system and functions as one of the receptors for RGMa. As shown in SEQ ID NO: 10 in the sequence listing, human Neogenin protein is composed of 1461 amino acids, and is expressed as a mature membrane protein by removing signal peptides Met1 to Ala33. In the present invention, Neogenin may refer to either a precursor protein, a mature protein or an RGMa-binding fragment thereof as long as it binds to RGMa, or may be a derivative or variant thereof. Moreover, although human Neogenin or Neogenin derived from other organisms may be used, human Neogenin is preferable.
- Neutralization refers to an action capable of binding to a target of interest and inhibiting any function of the target. That is, “to neutralize RGMa's neurite growth inhibitory activity” refers to inhibiting RGMa's neurite growth inhibitory activity by binding to RGMa. Neurite outgrowth inhibitory activity can be assessed by one or more of several in vitro or in vivo analyzes known in the art, for example by the neurite outgrowth inhibition test described herein. Can be evaluated.
- Isolated such as an isolated RGMa binding protein, means that it has been identified and separated and / or recovered from a component in its natural state. Impurities in the natural state are substances that can interfere with the diagnostic or therapeutic use of the antibody and include enzymes, hormones and other proteinaceous or non-proteinaceous solutes.
- an RGMa-binding protein in order to isolate an RGMa-binding protein or the like, it may be purified by at least one purification step, and an RGMa-binding protein purified by at least one purification step is referred to as an “isolated RGMa-binding protein”. Can do.
- RGMa binding protein refers to a molecule containing a protein that binds to RGMa.
- RGMa-binding proteins include anti-RGMa antibodies and antigen-binding fragments thereof, RGMa-binding scaffold proteins, soluble RGMa receptor proteins such as Neogenin's extracellular domain, and fusion proteins thereof.
- the RGMa-binding scaffold protein refers to a protein that realizes a function of binding to RGMa by introducing a mutation into the Kunitz domain of a serine protease inhibitor, the extracellular domain of human fibronectin, ankyrin, lipocalin, or the like.
- Fusion proteins include RGMa binding proteins, non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF- ⁇ , NGF, Neurotrophin, etc.), albumins, enzymes, etc.
- RGMa binding proteins non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF- ⁇ , NGF, Neurotrophin, etc.), albumins, enzymes, etc.
- PEG polyethylene glycol
- An RGMa-binding protein obtained by chemically or genetically binding a functional molecule other than the RGMa-binding protein of the present application, such as the above-mentioned antibody.
- human antibody refers to an antibody derived from human immunoglobulin for both light chain and heavy chain. Due to the difference in the constant region of the heavy chain, human antibodies include IgG having IgG heavy chain (including IgG1, IgG2, IgG3 and IgG4), IgM having ⁇ heavy chain, IgA having ⁇ heavy chain (Including IgA1 and IgA2), IgD having a heavy chain of ⁇ chain, or IgE having a heavy chain of ⁇ chain.
- the light chain includes either a kappa chain or a lambda chain.
- the humanized antibody refers to an antibody consisting of a variable region composed of a complementarity determining region of a non-human animal-derived antibody and a framework region derived from a human antibody, and a constant region derived from a human antibody.
- a chimeric antibody refers to an antibody in which the light chain, the heavy chain, or both are composed of a variable region derived from non-human and a constant region derived from human.
- an anti-RGMa antibody refers to an immunoglobulin molecule that binds to RGMa or a modified molecule thereof. Modified molecules are meant to include multispecific antibodies, chimeric antibodies, humanized antibodies, functionally modified antibodies, and conjugated antibodies.
- a multispecific antibody is an asymmetric antibody having two or more independent antigen recognition sites having two or more different antigen specificities, a bispecific antibody having two antigen specificities, and three antigen specificities. And the like.
- a function-modified antibody refers to a function other than the antigen-binding function of an antibody, for example, a cell killing function, a complement activation function or a blood half-life, mainly by modifying amino acids and sugar chains in the Fc region of the antibody An antibody with altered extension function.
- the conjugated antibody is a non-peptidic polymer such as polyethylene glycol (PEG), radioactive substance, toxin, low molecular weight compound, cytokine, growth factor (TGF- ⁇ , NGF, Neurotrophin etc.), albumin, enzyme, etc.
- PEG polyethylene glycol
- radioactive substance such as radioactive substance, toxin, low molecular weight compound, cytokine, growth factor (TGF- ⁇ , NGF, Neurotrophin etc.), albumin, enzyme, etc.
- an antigen-binding fragment refers to a protein containing a part of an antibody and capable of binding to an antigen.
- the antigen-binding fragment include F (ab ′) 2 , Fab ′, Fab, Fv (variable fragment of antibody), disulfide bond Fv, single chain antibody (scFv), and polymers thereof.
- antigen-binding fragments include non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular compounds, cytokines, growth factors (TGF- ⁇ , NGF, Neurotrophin, etc.), albumins, enzymes, other antibodies and a conjugate antigen-binding fragment to which a functional molecule other than the anti-RGMa antibody of the present application is chemically or genetically bound.
- PEG polyethylene glycol
- Complementarity determining region refers to a region that forms an antigen-binding site among variable regions of immunoglobulin molecules, and is also referred to as a hypervariable region, and refers to a portion that has a particularly large change in amino acid sequence for each immunoglobulin molecule. .
- CDR Complementarity determining region
- CDRs of immunoglobulin molecules are determined according to the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interstitut, US Department of Health and Human Services, NIH, USA).
- Percent (%) identity with respect to an identified reference polypeptide sequence, such as a variable region, refers to any conservative, introducing gaps if necessary to align the sequences and obtain maximum% identity. Substitution is also defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of a particular reference polypeptide sequence after not considering it as part of the sequence identity. Alignments for the purpose of measuring% identity can be obtained by various methods within the skill of the art, such as BLAST, BLAST-2, ALIGN, or publicly available computers such as Megalign (DNASTAR) software. This can be achieved by using software.
- % identity values are obtained by using the sequence comparison computer program BLAST in pairwise alignments.
- sequence comparison computer program BLAST in pairwise alignments.
- percent identity of a given amino acid sequence A with a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues with a score that is identical by the program alignment of A and B of the sequence alignment program BLAST, and Y is the total number of amino acid residues of B It is.
- “competing” with the anti-RGMa antibody of the present invention means the presence of the anti-RGMa antibody or an antigen-binding fragment thereof, as measured by the surface plasmon resonance (SPR) method described herein, It means that the binding between the anti-RGMa antibody of the present invention and RGMa decreases with a significant difference.
- SPR surface plasmon resonance
- the RGMa binding protein of the present invention is an isolated RGMa binding protein that does not inhibit the binding of RGMa and Neogenin and neutralizes the neurite growth inhibitory activity of RGMa.
- the RGMa protein is preferably a mammal-derived RGMa protein.
- a human RGMa protein includes a protein having the amino acid sequence of SEQ ID NO: 1 in the sequence listing
- a mouse RGMa protein includes a sequence of SEQ ID NO: 2 in the sequence listing.
- Examples include a protein having an amino acid sequence
- examples of the rat RGMa protein include a protein having the amino acid sequence of SEQ ID NO: 3 in the Sequence Listing.
- one or several (preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5) amino acids are substituted, deleted, inserted, and / or added.
- RGMa protein having substantially the same activity as the RGMa protein
- RGMa protein includes any polypeptide having a neurite growth inhibitory action.
- the amino acid substitution is preferably a conservative substitution.
- “conservative substitution” means substitution of an amino acid residue with another chemically similar amino acid residue so as not to substantially alter the activity of the peptide. For example, when a certain hydrophobic residue is substituted with another hydrophobic residue, a certain polar residue is substituted with another polar residue having the same charge, and the like.
- Examples of functionally similar amino acids that can make such substitutions include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine and the like as nonpolar (hydrophobic) amino acids.
- Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine and the like.
- Examples of positively charged (basic) amino acids include arginine, histidine, and lysine.
- Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- the binding of the RGMa binding protein of the present invention to RGMa means binding specific to RGMa, but preferably has a low dissociation constant (Kd) for human RGMa, and the upper limit is, for example, 10 ⁇ 8 M or less, more preferably Is 10 ⁇ 9 M or less, and even more preferably 10 ⁇ 10 M or less, and the lower limit is not particularly limited, and examples thereof include RGMa-binding proteins of 10 ⁇ 14 M or more, more preferably 10 ⁇ 13 or more. .
- Kd dissociation constant
- the RGMa protein consists of an N-terminal domain and a C-terminal domain as a mature protein, but the C-terminal domain alone has neurite growth inhibitory activity.
- the RGMa binding protein of the present invention preferably binds only to the C-terminal domain of RGMa to neutralize neurite growth inhibitory activity.
- Those having a low dissociation constant (Kd) for the C-terminal domain of human RGMa are more preferred, and the upper limit is, for example, 10 ⁇ 8 M or less, more preferably 10 ⁇ 9 M or less, and even more preferably 10 ⁇ 10 M or less.
- the lower limit is not particularly limited, and examples thereof include an RGMa-binding protein having 10 ⁇ 14 M or more, more preferably 10 ⁇ 13 or more.
- the RGMa binding protein of the present invention does not inhibit the binding between RGMa and Neogenin.
- “does not inhibit the binding between RGMa and Neogenin” means that in the binding system of RGMa and Neogenin, which will be described later, the binding between RGMa and Neogenin even if the concentration of RGMa binding protein is increased. Does not substantially decrease.
- the concentration of RGMa binding protein showing IC50 is 10 ⁇ g / mL or more, more preferably 50 ⁇ g / mL or more, most preferably If it is 100 ⁇ g / mL or more, it can be said that the RGMa-binding protein does not inhibit the binding between RGMa and Neogenin.
- the Neogenin used for the binding test with RGMa is preferably the same type of Neogenin as RGMa. That is, it is preferable to use mouse Neogenin for mouse RGMa and human Neogenin for human RGMa.
- An example of human Neogenin is a protein having the amino acid sequence of SEQ ID NO: 10 in the sequence listing, and 90% or more (preferably 95% or more preferably) of SEQ ID NO: 10 in the sequence listing as long as it can bind to RGMa. It may have an amino acid sequence having the identity of
- 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 a neurite growth test shown in Examples described later. When RGMa is added, neurite growth is suppressed. By adding RGMa-binding protein, neurite growth inhibition by RGMa does not occur.
- the RGMa-binding protein of the present invention can neutralize neurite growth inhibition by addition of RGMa by 50% or more, more preferably 80% or more, and most preferably 90% or more.
- the amino acid sequence of the RGMa protein varies depending on the animal species, the amino acid sequence of human RGMa represented by SEQ ID NO: 1 in the sequence listing, mouse RGMa represented by SEQ ID NO: 2 in the sequence listing, and rat RGMa represented by SEQ ID NO: 3 in the sequence listing There is a difference.
- rodents such as mice and rats are used as experimental materials in pharmacological tests and safety tests of protein preparations such as antibody drugs. Therefore, the RGMa-binding protein of the present invention should bind to RGMa of mice and rats. Preferred are those having a low Kd for RGMa in mice and rats.
- the upper limit value of Kd is, for example, 5 ⁇ 10 ⁇ 7 M or less, more preferably 10 ⁇ 8 M or less, even more preferably 10 ⁇ 9 M or less, and the lower limit value is not particularly limited, but for example, 10 ⁇ 12 M or more
- An RGMa-binding protein that is preferably 10 ⁇ 11 or more can be mentioned.
- the RGMa binding protein of the present invention is preferably excellent in thermal stability.
- Thermal stability can be evaluated by a decrease in binding with RGMa by heat treatment, but it is preferably stable even by heat treatment at 60 ° C. or higher, more preferably stable by heat treatment at 65 ° C. or higher, Most preferably, it is stable even by heat treatment at 70 ° C. or higher.
- the binding site when the RGMa binding protein of the present invention binds to RGMa is not particularly limited.
- EEVVNAVEDWDSQG (SEQ ID NO: 26 in the sequence listing) (amino acid number 298- of SEQ ID NO: 1 in the sequence listing) 311)
- NQQIDFQAFHTNAE (SEQ ID NO: 27 of the Sequence Listing) (amino acids 322 to 335 of SEQ ID NO: 1 of the Sequence Listing)
- PTAPETFPYET SEQ ID NO: 28 of Sequence Listing
- It is preferable to bind to one or more peptides of KLPVEDLYYQA (SEQ ID NO: 29 of the Sequence Listing) (amino acids 367-377 of SEQ ID NO: 1 of the Sequence Listing). More preferably, it binds to SEQ ID NO: 26 and 27 in the sequence listing, and more preferably
- RGMa binding protein examples include anti-RGMa antibody, RGMa binding scaffold protein, and fusion proteins thereof.
- the anti-RGMa antibody of the present invention immunizes a mammal such as a mouse with the RGMa protein or a partial fragment thereof (for example, a fragment containing one or more of SEQ ID NOs: 26 to 29 in the above sequence listing) as an antigen.
- a mammal such as a mouse
- the RGMa protein or a partial fragment thereof for example, a fragment containing one or more of SEQ ID NOs: 26 to 29 in the above sequence listing
- examples include polyclonal antibodies and monoclonal antibodies obtained, chimeric antibodies and humanized antibodies produced using gene recombination techniques, and human antibodies produced using human antibody-producing transgenic animals.
- a humanized antibody or a human antibody is desirable from the viewpoint of side effects.
- the antigen may be used for immunization as it is, or may be used as a complex with a carrier protein.
- Condensation agents such as glutaraldehyde, carbodiimide, and maleimide active ester can be used for the preparation of the complex of antigen and carrier protein.
- carrier proteins include bovine serum albumin, thyroglobulin, hemocyanin, and KLH.
- mammals to be immunized examples include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses or cows, and inoculation methods include subcutaneous, intramuscular or intraperitoneal administration. In administration, it may be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant, and is usually administered once every 2 to 5 weeks.
- Antibody-producing cells obtained from the spleen or lymph nodes of the immunized animal are fused with myeloma cells and isolated as hybridomas. As the myeloma cells, those derived from mammals such as mice, rats, humans and the like are used.
- the polyclonal antibody can be obtained by an existing general production method. That is, for example, the antigen as described above can be obtained from the serum obtained from the immunized animal by immunizing the mammal as described above together with Freund's Adjuvant as necessary.
- the monoclonal antibody can be obtained as follows. That is, an antigen as described above is used as an immunogen, and the immunogen is optionally combined with Freund's Adjuvant, as described above, in the subcutaneous, intramuscular, intravenous, food pad, or intraperitoneal of mammals as described above. Immunization is performed by injecting 1 to several times or transplanting. Usually, immunization is carried out 1 to 4 times about every 1 to 14 days from the initial immunization, and antibody producing cells are obtained from the immunized mammal about 1 to 5 days after the final immunization.
- Monoclonal antibodies can be obtained using methods well known to those skilled in the art (for example, “Current Protocols in Molecular Molecular Biology” (John Wiley and Sons (1987)), Antibodies: A Laboratory and Manual, Ed. Harlow, and David David, Cold. Spring Harbor Laboratory (1988)).
- a “hybridoma” that secretes a monoclonal antibody can be prepared according to the method of Köhler and Milstein et al. (Nature, 256,495, 1975) and a modification method according thereto. That is, it is prepared by cell fusion of an antibody-producing cell contained in a spleen obtained from an immunized mammal and a myeloma cell having no autoantibody-producing ability derived from a mammal, preferably a mouse, rat or human. Is done.
- myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8.
- Human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, CEM-T15, and the like can be used.
- the fusion promoter examples include polyethylene glycol and the like.
- polyethylene glycol average molecular weight 1000 to 4000 having a concentration of about 20 to 50% is used, and the temperature is 20 to 40 ° C., preferably 30 to 37 ° C.
- the ratio of the number of antibody-producing cells to the number of myeloma cells is usually about 1: 1 to 10: 1, and cell fusion can be carried out by reacting for about 1 to 10 minutes.
- Hybridoma clones that produce monoclonal antibodies can be screened by culturing the hybridomas in, for example, a microtiter plate, and measuring the reactivity of the culture supernatant of the wells with the immunoantigen by an immunochemical method such as ELISA. it can.
- the anti-RGMa antibody of the present invention in addition to the binding assay with RGMa protein, evaluation of whether the antibody does not inhibit the binding of RGMa protein to Neogenin and the function of RGMa protein (neurite growth inhibitory activity) The neutralization is also evaluated.
- the anti-RGMa antibody of the present invention can be selected.
- a clone can be obtained by further cloning from a well containing a hybridoma producing the target antibody by a limiting dilution method. Selection and breeding of hybridomas are usually carried out in an animal cell medium containing 10-20% fetal calf serum with the addition of HAT (hypoxanthine, aminopterin, thymidine).
- HAT hypoxanthine, aminopterin, thymidine
- Production of monoclonal antibodies from hybridomas can be accomplished by culturing the hybridomas in vitro or growing in vivo in the ascites of mammals such as mice, rats, etc., and isolating the resulting culture supernatant or the ascites of mammals. This can be done.
- a nutrient medium suitable for growing, maintaining and storing hybridomas and producing monoclonal antibodies in the culture supernatant in accordance with various conditions such as the characteristics of the cell type to be cultured and the culture method Can be used.
- Examples of the basic medium include a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium, and a high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium.
- the basic medium can contain, for example, serum, hormones, cytokines and / or various inorganic or organic substances depending on the purpose.
- Isolation and purification of the monoclonal antibody can be carried out by using the above-mentioned culture supernatant or ascites, saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52 etc.), anti-immunoglobulin column or It can be performed by subjecting to affinity column chromatography such as a protein A column.
- monoclonal antibodies may be purified by known methods for immunoglobulin purification, such as ammonium sulfate fractionation, PEG fractionation, ethanol fractionation, use of anion exchanger, and RGMa. This can be easily achieved by means such as affinity chromatography using a protein.
- Monoclonal antibodies can also be obtained by the phage display method.
- a phage selected from an arbitrary phage antibody library is screened using a target immunogen, and a phage having a desired binding property to the immunogen is selected.
- an antibody corresponding sequence contained in the phage is isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding an antibody or an antigen binding domain is constructed based on the isolated sequence or the determined sequence information.
- a monoclonal antibody can be produced by culturing a cell line transfected with such an expression vector.
- a human antibody library as the phage antibody library, a human antibody having a desired binding property can be generated.
- the Kunitz domain of human serine protease inhibitor and the extracellular domain of human fibronectin are used.
- a scaffold protein that binds to RGMa is generated. (Clifford Mintz et.al BioProcess International, 2013, Vol.11 (2), pp40-48).
- Fusion proteins include RGMa binding proteins, non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF- ⁇ , NGF, Neurotrophin, etc.), albumins, enzymes, etc. And RGMa-binding protein obtained by chemically or genetically binding a functional molecule other than the RGMa-binding protein of the present application.
- PEG polyethylene glycol
- RGMa-binding protein obtained by chemically or genetically binding a functional molecule other than the RGMa-binding protein of the present application.
- PEG When PEG is bonded as a functional molecule, PEG can be used with a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be linear or branched. PEG can be bound to the N-terminal amino group of the amino acid of the RGMa binding protein, for example, by using an NHS active group.
- radioactive substance When a radioactive substance is used as the functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, 211 At, or the like is used.
- the radioactive substance can be directly bound to the RGMa-binding protein, such as by the Kuguchi Lamin T method.
- bacterial toxins eg, diphtheria toxin
- plant toxins eg, ricin
- low molecular toxins eg, geldanamycin
- maytansinoids calicheamicins, and the like
- fluorescent dyes such as daunomycin, doxorubicin, metrolexate, mitomycin, neocalcinostatin, vindesine, and FITC are exemplified.
- luciferase eg, firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456
- malate dehydrogenase urease, peroxidase (eg, horseradish peroxidase (HRPO)), alkaline phosphatase, ⁇ -galactosidase , Glucoamylase, lysozyme, saccharide oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg, uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
- HRPO horseradish peroxidase
- Glucoamylase eg, Glucoamylase
- saccharide oxidase eg, glucose oxidase, galactos
- Linkers used when chemically linking toxins, low molecular compounds or enzymes include divalent radicals (eg, alkylene, arylene, heteroarylene),-(CR 2 ) nO (CR 2 ) n- (R is optional And a repeating unit of alkoxy (eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (eg, polyethyleneamino, Jeffamine TM), and diacid esters And amides (including succinate, succinamide, diglycolate, malonate and caproamide).
- divalent radicals eg, alkylene, arylene, heteroarylene
- R is optional And a repeating unit of alkoxy (eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (eg, polyethyleneamino, Jeffamine TM)
- alkoxy eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.
- alkylamino eg, polyethylene
- a preferred embodiment of the RGMa binding protein of the present invention is a chimeric antibody.
- “Chimeric antibody” is a chimeric antibody in which the variable region is a variable region derived from an immunoglobulin of a non-human animal (mouse, rat, hamster, chicken, etc.), and the constant region is a constant region derived from a human immunoglobulin. Illustrated. For example, it can be prepared by immunizing a mouse with an antigen, cutting out a variable region that binds to the antigen from the gene of the mouse monoclonal antibody, and binding to an antibody constant region derived from human bone marrow.
- the constant regions derived from human immunoglobulin have unique amino acid sequences depending on isotypes such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE.
- the constant region of the chimeric antibody may be a constant region of human immunoglobulin belonging to any isotype. Preferably, it is a constant region of human IgG.
- An expression vector can be prepared using the gene of the chimeric antibody thus prepared.
- a host cell is transformed with the expression vector to obtain a chimeric antibody-producing transformed cell. By culturing the transformed cell, the desired chimerized antibody is obtained from the culture supernatant.
- the “humanized antibody” in the present invention is an antibody obtained by transplanting only the DNA sequence of the antigen binding site (CDR; complementarity determining region) of a non-human animal antibody such as a mouse (CDR grafting). For example, it can be produced with reference to the methods described in JP-T-4-506458 and JP-A-2912618. Specifically, a part or all of the CDR is a CDR derived from a monoclonal antibody of a non-human mammal (mouse, rat, hamster, etc.), and the framework region of the variable region is a variable region derived from human immunoglobulin.
- a humanized antibody characterized in that the constant region is a constant region derived from human immunoglobulin.
- the humanized antibody in the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
- a recombinant humanized antibody derived from a mouse monoclonal antibody can be prepared by genetic engineering with reference to JP-T-4-506458 and JP-A-62-296890. That is, from the hybridoma producing the mouse monoclonal antibody, the DNA of the mouse heavy chain CDR portion and the DNA of the mouse light chain CDR portion are isolated, and the human heavy chain gene of the entire region other than the human heavy chain CDR from the human immunoglobulin gene; The human light chain gene of the entire region other than the human light chain CDR is isolated.
- human heavy chain gene grafted with the isolated mouse heavy chain CDR portion DNA can be expressed
- human light chain gene grafted with the mouse light chain CDR portion DNA can be expressed
- it is introduced into another appropriate expression vector.
- human heavy and light chain genes grafted with mouse CDRs can be introduced into the same expression vector so that they can be expressed.
- a human antibody is an immunoglobulin in which all regions including the variable region of the heavy chain and the constant region of the heavy chain, and the variable region of the light chain and the constant region of the light chain that are part of the immunoglobulin are derived from a gene encoding human immunoglobulin.
- An antibody that is a globulin which can be prepared by introducing a human antibody gene into a mouse.
- the polyclonal antibody described above can be obtained by immunizing with a antigen a transgenic animal produced by incorporating at least a human immunoglobulin gene into the locus of a mammal such as a mouse. Alternatively, it can be produced in the same manner as the monoclonal antibody production method.
- transgenic mice that produce human antibodies include Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 15, p. 146-156, 1997; Japanese Patent Publication No. 7-509137; International Publication WO94 / 25585 pamphlet; Nature, Vol.368, p.856-859, 1994; Can do. More specifically, HuMab (registered trademark) mice (Medarex, Princeton NJ), KMTM mice (Kirin Pharma Company, Japan), KM (FC ⁇ RIIb-KO) mice and the like can be mentioned.
- the monoclonal antibody of the present invention includes the amino acid sequence of SEQ ID NO: 33 (HCDR1), 34 (HCDR2) and 35 (HCDR3) in the CDR of the heavy chain variable region, and the CDR of the light chain variable region.
- Examples include those containing the amino acid sequences of SEQ ID NO: 30 (LCDR1), 31 (LCDR2) and 32 (LCDR3) in the Sequence Listing.
- the antibody 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, one of these CDRs is maintained.
- one to several amino acids may be substituted.
- one to several is, for example, one or two.
- the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention. Maintaining the characteristics of an antibody means that these characteristics are maintained at the same level as before CDR amino acid sequence modification, for example, 80% or more, preferably 90% or more, more preferably 95% or more. Say. Maintenance includes improvement.
- the region other than CDR is not particularly limited as long as it maintains the structure as an antibody and can exert its function, and is derived from a mouse-derived sequence, a human-derived sequence, a sequence derived from another mammal, a chimeric sequence thereof, an artificial sequence Any of the sequences may be used.
- the amino acid sequences of the heavy and light chain constant regions are Nucleic Acids Research14vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell vol.22, Examples are described in p197, 1980.
- mice antibody having these CDRs include an antibody having the amino acid sequence of SEQ ID NO: 4 in the light chain and the amino acid sequence of SEQ ID NO: 5 in the heavy chain.
- amino acid sequences one or several amino acid sequences can be used as long as they have the ability to bind to RGMa, do not inhibit the binding between RGMa and Neogenin, and maintain the properties of neutralizing the neurite growth inhibitory activity of RGMa.
- the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention.
- mouse antibody examples include mouse / human chimeric antibodies whose constant region is derived from human.
- the amino acid sequence of SEQ ID NO: 8 of the Sequence Listing (variable region is 1 to 107) in the light chain and the amino acid sequence of SEQ ID NO: 9 of the Sequence Listing in the heavy chain (the variable region is 1 to 10).
- one or several of the amino acid sequences have the ability to bind RGMa, do not inhibit the binding of RGMa and Neogenin, and maintain the properties of neutralizing the neurite growth inhibitory activity of RGMa.
- substitutions, deletions, additions or insertions of amino acids (1-20, 1-10 or 1-5). Such substitutions, deletions and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
- the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention.
- humanized antibodies derived from humans other than CDRs are also exemplified.
- the heavy chain has any amino acid sequence of SEQ ID NO: 11 to 18 (variable region up to 116 residues on the N-terminal side), and the light chain has SEQ ID NO:
- An antibody having any amino acid sequence of 19 to 25 (variable region is 1 to 107 residues on the N-terminal side) is exemplified.
- amino acid sequences of humanized antibodies have binding ability to RGMa, and binding between RGMa and Neogenin Substitution of one or several amino acids (1-20, 1-10 or 1-5), lacking, as long as the properties of neutralizing RGMa's neurite outgrowth inhibitory activity are maintained. There may be loss, addition or insertion. Such substitutions, deletions and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
- the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention.
- the heavy chain amino acid sequence and the light chain amino acid sequence may be any combination thereof, but particularly preferred is that the heavy chain has the amino acid sequence of SEQ ID NO: 15 in the sequence listing and the light chain has the amino acid sequence of SEQ ID NO: 19 in the sequence listing Antibody.
- the amino acid sequence of SEQ ID NO: 15 in the sequence listing the amino acid sequence corresponding to the heavy chain variable region is shown in SEQ ID NO: 41, and the amino acid sequence corresponding to the light chain variable region is shown in SEQ ID NO: 42 in the sequence listing.
- a particularly preferred antibody of the present invention is an antibody having the amino acid sequence of SEQ ID NO: 41 in the heavy chain variable region and the amino acid sequence of SEQ ID NO: 42 in the light chain variable region.
- amino acid sequences one or several of these amino acid sequences can be used as long as they have the ability to bind to RGMa, do not inhibit the binding of RGMa to Neogenin, and neutralize the neurite growth inhibitory activity of RGMa.
- substitutions, deletions, additions or insertions of amino acids (1-20, 1-10 or 1-5). Such substitutions, deletions and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
- the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention.
- the amino acid sequence of the antibody of the present invention containing substitution, deletion, etc.
- the heavy chain variable region is SEQ ID NO: 41 and 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) of the amino acid sequence
- the light chain variable region is 90% of SEQ ID NO: 42 in the sequence listing
- An amino acid sequence having the above identity (more preferably 95%, 96%, 97%, 98%, 99% or more).
- the CDR of the heavy chain variable region contains the amino acid sequences of SEQ ID NO: 39 (HCDR1), 40 (HCDR2) and SFG (HCDR3) in the sequence listing, and the CDR of the light chain variable region Include those containing the amino acid sequences of SEQ ID NO: 36 (LCDR1), 37 (LCDR2) and 38 (LCDR3) in the Sequence Listing.
- the antibody 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, one of these CDRs is maintained.
- one to several amino acids may be substituted.
- one to several is, for example, one or two.
- the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention.
- the region other than CDR is not particularly limited as long as it maintains the structure as an antibody and can exert its function, and is derived from a mouse-derived sequence, a human-derived sequence, a sequence derived from another mammal, a chimeric sequence thereof, an artificial sequence Any of the sequences may be used.
- the amino acid sequences of the heavy and light chain constant regions are Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell vol.22, p197, 1980 is exemplified.
- mouse antibodies having these CDRs include antibodies having the amino acid sequence of SEQ ID NO: 6 in the sequence list in the light chain and the amino acid sequence of SEQ ID NO: 7 in the heavy chain.
- amino acid sequences one or several of the amino acid sequences have the ability to bind RGMa, do not inhibit the binding of RGMa and Neogenin, and maintain the properties of neutralizing the neurite growth inhibitory activity of RGMa.
- substitutions, deletions, additions or insertions of amino acids (1-20, 1-10 or 1-5). Such substitutions, deletions and additions may be introduced into the CDR, but are preferably introduced into regions other than the CDR.
- the amino acid substitution is preferably a conservative substitution in order to maintain the characteristics of the present invention.
- a chimeric antibody whose constant region is derived from human is also exemplified.
- humanized antibodies derived from humans other than CDRs are also exemplified.
- the anti-RGMa antibody of the present invention comprises a CDR comprising a specific amino acid sequence (for example, SEQ ID NO: 30 in the Sequence Listing as LCDR1, SEQ ID NO: 31 in the Sequence Listing as LCDR2, SEQ ID NO: 32 in the Sequence Listing as LCDR3, and Sequence Listing as HCDR1). SEQ ID NO: 33, SEQ ID NO: 34 of the Sequence Listing as HCDR2, and SEQ ID NO: 35 of the Sequence Listing as HCDR3), or a variable region comprising a specific amino acid sequence (for example, SEQ ID NO: 41 of the Sequence Listing as the heavy chain variable region) Multispecific antibody having 42 amino acid sequences as a light chain variable region), a functionally modified antibody, and a conjugated antibody.
- a CDR comprising a specific amino acid sequence
- SEQ ID NO: 30 in the Sequence Listing as LCDR1 for example, SEQ ID NO: 31 in the Sequence Listing as LCDR2, SEQ ID NO: 32 in the Sequence Listing as LCDR3, and
- the anti-RGMa antibody of the present invention can produce a multispecific antibody such as a bispecific antibody by binding an antibody having another antigen binding specificity other than RGMa to itself by a genetic engineering technique.
- the genetic engineering technique has already been established in this field. For example, DVD-Ig (Wu et al., Nature Biotechnology 25 (11), 1290 (2007)) in which variable regions are connected in series, or two types of antibodies that bind to different antigens by modifying the Fc region of an antibody.
- the desired bispecific antibody can be obtained by using the ART-Ig technology (Kitazawa et al., Nature Medicine 18 (10), 1570 (2012)) in which the heavy chains are combined.
- antigens other than RGMa include, but are not limited to, factors that inhibit neurite growth such as Nogo, MAG, Omgp, CSPG, Sema3A, Lingo-1, TNF- ⁇ , IL-6 receptor, CD3, CD20 , ⁇ 4 integrin, BLys, Thymic Strom lypopoietin, IgE, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-23, IL-25, etc.
- factors that inhibit neurite growth such as Nogo, MAG, Omgp, CSPG, Sema3A, Lingo-1, TNF- ⁇ , IL-6 receptor, CD3, CD20 , ⁇ 4 integrin, BLys, Thymic Strom lypopoietin, IgE, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-23, IL-25, etc.
- Examples of the modified molecule of the anti-RGMa antibody of the present invention include a functionally modified antibody.
- the functionally modified antibody means an antibody whose functions such as cell killing function, complement activation function, blood half-life extension function, etc. are modified mainly by modifying the Fc region or the like. 2009, Vol.129 (1), p3; Akiko Ishii et al., Journal of Japanese Pharmacology, 2010, Vol.136 (5), p280; Shuhei Hashiguchi, Biochemistry, 2010, Vol.82 (8), p710).
- the function-modified antibody of the anti-RGMa antibody is prepared by the following method.
- the anti-RGMa antibody of the present application is produced using a CHO cell in which the ⁇ 1,6-fucose transferase (FUT8) gene is disrupted as a host cell, an antibody having a reduced cell chain fucose content and an increased cell killing function can be obtained.
- FUT8 ⁇ 1,6-fucose transferase
- an antibody having a reduced cell chain fucose content and an increased cell killing function can be obtained.
- a CHO cell into which the FUT8 gene is introduced is produced as a host cell, an antibody having a low cell killing function can be obtained (WO 2005/035586, WO 2002/31140, WO 00/61739).
- the complement activation function can be regulated by modifying amino acid residues in the Fc region (US Pat. No.
- conjugate antibodies examples include conjugate antibodies.
- Conjugate antibodies include anti-RGMa antibodies, non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low molecular weight compounds, cytokines, growth factors (TGF- ⁇ , NGF, Neurotrophin, etc.), albumins, enzymes, Examples thereof include conjugate antibodies in which functional molecules other than the anti-RGMa antibody of the present application such as other antibodies are chemically or genetically bound.
- PEG When PEG is bonded as a functional molecule, PEG can be used with a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be linear or branched. PEG can be bound to the N-terminal amino group of an amino acid of an antibody by using, for example, an NHS active group.
- radioactive substance When a radioactive substance is used as the functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, 211 At, or the like is used.
- the radioactive substance can be directly bound to the antibody by the Kuguchi Lamin T method or the like.
- bacterial toxins eg, diphtheria toxin
- plant toxins eg, ricin
- low molecular toxins eg, geldanamycin
- maytansinoids calicheamicins, and the like
- fluorescent dyes such as daunomycin, doxorubicin, metrolexate, mitomycin, neocalcinostatin, vindesine, and FITC are exemplified.
- luciferase eg, firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456
- malate dehydrogenase urease, peroxidase (eg, horseradish peroxidase (HRPO)), alkaline phosphatase, ⁇ -galactosidase , Glucoamylase, lysozyme, saccharide oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg, uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
- HRPO horseradish peroxidase
- Glucoamylase eg, Glucoamylase
- saccharide oxidase eg, glucose oxidase, galactos
- Linkers used when chemically linking toxins, low molecular compounds or enzymes include divalent radicals (eg, alkylene, arylene, heteroarylene),-(CR 2 ) nO (CR 2 ) n- (R is optional And a repeating unit of alkoxy (eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (eg, polyethyleneamino, Jeffamine TM), and diacid esters And amides (including succinate, succinamide, diglycolate, malonate and caproamide).
- divalent radicals eg, alkylene, arylene, heteroarylene
- R is optional And a repeating unit of alkoxy (eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (eg, polyethyleneamino, Jeffamine TM)
- alkoxy eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.
- alkylamino eg, polyethylene
- the “antigen-binding fragment” of an antibody in the present invention means a partial region having the antigen-binding property of the antibody as described above, specifically, F (ab ′) 2 , Fab ′, Fab, Fv ( variable fragment of antibody), disulfide bond Fv, single chain antibody (scFv), and polymers thereof, and further, antigen-binding fragments include non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, By chemically or genetically linking functional molecules other than the anti-RGMa antibody of the present application, such as toxins, low molecular weight compounds, cytokines, growth factors (TGF- ⁇ , NGF, Neurotrophin, etc.), albumins, enzymes, and other antibodies. Conjugated antigen binding fragments are included.
- F (ab ′) 2 and “Fab” are produced by treating immunoglobulin with a proteolytic enzyme such as pepsin or papain, and between two heavy chains in the hinge region. It means an antibody fragment produced by digestion before and after an existing disulfide bond.
- a light chain consisting of VL (light chain variable region) and CL (light chain constant region) is cleaved upstream of a disulfide bond existing between two heavy chains in the hinge region
- two homologous antibody fragments in which a heavy chain fragment consisting of VH (heavy chain variable region) and CH ⁇ 1 ( ⁇ 1 region in the heavy chain constant region) is linked by a disulfide bond in the C-terminal region can be produced.
- Fab fragment fragments in which a heavy chain fragment consisting of VH (heavy chain variable region) and CH ⁇ 1 ( ⁇ 1 region in the heavy chain constant region) is linked by a disulfide bond in the C-terminal region.
- an antibody fragment that is cleaved downstream of a disulfide bond existing between two heavy chains in the hinge region to produce a slightly larger antibody fragment than the two Fabs connected in the hinge region can be produced. It can.
- This antibody fragment is called F (ab ') 2 .
- Examples of the modified molecule of the antigen-binding fragment of the anti-RGMa antibody of the present invention include a conjugated antigen-binding fragment.
- Conjugated antigen-binding fragments include non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular compounds, cytokines, growth factors (TGF- ⁇ ) in a partial region having antigen-binding properties of anti-RGMa antibodies. , NGF, Neurotrophin, etc.), albumin, enzyme, and other antibodies other than the anti-RGMa antibody of the present application, such as conjugate antigen-binding fragments chemically or genetically bound.
- PEG When PEG is bonded as a functional molecule, PEG can be used with a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, and may be linear or branched. PEG can be bound to the N-terminal amino group or the like of a partial region having the antigen-binding property of the anti-RGMa antibody by using, for example, an NHS active group.
- radioactive substance When a radioactive substance is used as the functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, 211 At, or the like is used.
- the radioactive substance can be directly bound to a partial region having the antigen-binding property of the anti-RGMa antibody by the Kuguchi Lamin T method or the like.
- bacterial toxins eg, diphtheria toxin
- plant toxins eg, ricin
- low molecular toxins eg, geldanamycin
- maytansinoids calicheamicins, and the like
- fluorescent dyes such as daunomycin, doxorubicin, metrolexate, mitomycin, neocalcinostatin, vindesine, and FITC are exemplified.
- luciferase eg, firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456
- malate dehydrogenase urease, peroxidase (eg, horseradish peroxidase (HRPO)), alkaline phosphatase, ⁇ -galactosidase , Glucoamylase, lysozyme, saccharide oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg, uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
- HRPO horseradish peroxidase
- Glucoamylase eg, Glucoamylase
- saccharide oxidase eg, glucose oxidase, galactos
- Linkers used when chemically linking toxins, low molecular compounds or enzymes include divalent radicals (eg, alkylene, arylene, heteroarylene),-(CR 2 ) nO (CR 2 ) n- (R is optional And a repeating unit of alkoxy (eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (eg, polyethyleneamino, Jeffamine TM), and diacid esters And amides (including succinate, succinamide, diglycolate, malonate and caproamide).
- divalent radicals eg, alkylene, arylene, heteroarylene
- R is optional And a repeating unit of alkoxy (eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylamino (eg, polyethyleneamino, Jeffamine TM)
- alkoxy eg, polyethyleneoxy, PEG, polymethyleneoxy, etc.
- alkylamino eg, polyethylene
- the anti-RGMa antibody comprising a CDR having a specific amino acid sequence or a variable region of the present invention has a constant region of human IgG (IgG1, IgG2, IgG3, IgG4) because it wants to maintain a long half-life in blood. Preferably there is.
- the present invention also includes an anti-RGMa antibody that competes for binding to an RGMa protein and an antibody having a specific CDR amino acid sequence as described above, and an antigen-binding fragment thereof.
- an epitope is a region selected from Glu298 to Gly311, Asn322 to Glu335, Lys367 to Ala377, and Pro349 to Thr359.
- the antibody which has is illustrated.
- the antibody can be obtained (screened) or evaluated by coexistence in the binding system of the antibody having the CDR sequence as described above and the RGMa protein. For example, it can be obtained by screening by the following surface plasmon resonance (SPR) method.
- SPR surface plasmon resonance
- a human RGMa protein equivalent to 1300 to 1600 RU is immobilized by loading biotinylated human RGMa protein (4 ⁇ g / mL) as a ligand onto a sensor chip on which avidin is immobilized.
- an optional anti-RGMa antibody (15 ⁇ g / mL) is loaded as an analyte and allowed to bind to the human RGMa protein immobilized on the sensor chip.
- a state (saturated state) in which any anti-RGMa antibody is bound to all molecules of the human RGMa protein on the sensor chip is created, and the binding amount in the saturated state (saturated binding amount 1) Ask for.
- a similar experiment is also carried out with an anti-RGMa antibody containing the specific CDR amino acid sequence of the present invention to determine the amount of binding in a saturated state (saturated binding amount 2).
- an anti-RGMa antibody containing the specific CDR amino acid sequence of the present invention is loaded as an analyte. It is examined whether it binds in an added manner to a human RGMa protein saturated with an anti-RGMa antibody comprising the specific CDR amino acid sequence.
- any anti-RGMa antibody calculated above in the form that any anti-RGMa antibody is added to the human RGMa protein saturated with the anti-RGMa antibody containing the amino acid sequence of the specific CDR of the present invention.
- the antibody is determined to be “non-competing”.
- any anti-RGMa antibody is unable to bind in an added form to a human RGMa protein saturated with an anti-RGMa antibody comprising the amino acid sequence of a particular CDR of the present invention, the antibody is said to “compete”. To be judged.
- the added amount of binding has a significant difference. If the saturation binding amount of 1 is not reached, the antibody is determined to “compete”. The significant difference is examined by a general test method (for example, Student's t test), and the significance level is set to 5% or less.
- the anti-RGMa antibody that competes with RGMa for binding to the anti-RGMa antibody including the amino acid sequence of the above-mentioned specific CDR may be any antibody such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody.
- An antibody derived from an animal may be used, and a chimeric antibody or a humanized antibody that is a combination of these antibodies may be used, but a chimeric antibody, a humanized antibody, or a human antibody is preferable.
- the nucleic acid molecule of the present invention is a polynucleotide encoding the monoclonal antibody of the present invention.
- the region encoding the heavy chain variable region is the amino acid sequence of SEQ ID NOs: 33, 34, and 35 (1 or number).
- each of which includes a nucleotide sequence that may be substituted, deleted, inserted or added), and the region encoding the light chain variable region is the amino acid sequence of SEQ ID NOs: 30, 31, and 32 in the sequence listing Polynucleotides each including a base sequence encoding (one or several amino acids may be substituted, deleted, inserted or added), or a region encoding a heavy chain variable region is SEQ ID NO: 39, 40, And a base sequence encoding the amino acid sequence of SFG (one or several amino acids may be substituted, deleted, inserted or added), and
- the region encoding the chain variable region includes the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36, 37, and 38 in the sequence listing (one or several amino acids may be substituted, deleted, inserted or added), respectively.
- a polynucleotide is mentioned.
- the region encoding the heavy chain includes a base sequence encoding the amino acid sequence of SEQ ID NO: 5 in the sequence listing, and the region encoding the light chain is SEQ ID NO: 4 of the sequence listing.
- a polynucleotide comprising the base sequence encoding the amino acid sequence of SEQ ID NO: 6 is exemplified.
- the region encoding the heavy chain includes a base sequence encoding the amino acid sequence of SEQ ID NO: 9 of the sequence listing, and the region encoding the light chain is SEQ ID NO: 8 of the sequence listing.
- the region encoding the heavy chain includes a base sequence encoding any one of the amino acid sequences of SEQ ID NOs: 11 to 18 in the sequence listing, and the region encoding the light chain is the sequence listing.
- Examples thereof include a polynucleotide comprising a base sequence encoding any amino acid sequence of SEQ ID NOs: 19 to 25.
- the region encoding the heavy chain comprises a base sequence encoding the amino acid sequence of SEQ ID NO: 15 of the sequence listing
- the region encoding the light chain is the amino acid of SEQ ID NO: 19 of the sequence listing
- Examples of the polynucleotide include a base sequence encoding the sequence.
- the region encoding the heavy chain variable region includes a base sequence encoding the amino acid sequence of SEQ ID NO: 41 in the sequence listing, and the region encoding the light chain variable region is a sequence listing sequence A polynucleotide containing the base sequence encoding the amino acid sequence of No. 42 is exemplified.
- the region encoding the heavy chain variable region includes the base sequence of SEQ ID NO: 43 in the sequence listing, and the region encoding the light chain variable region includes the base sequence of SEQ ID NO: 44 of the sequence listing.
- the polynucleotide include.
- nucleic acid molecule of the present invention encodes a monoclonal antibody that has a binding ability to RGMa, does not inhibit the binding of RGMa and Neogenin, and neutralizes the neurite growth inhibitory activity of RGMa
- stringent conditions include, for example, conditions of washing at a salt concentration corresponding to 68 ° C., 0.1 ⁇ SSC, 0.1% SDS after Southern hybridization.
- the nucleic acid molecule of the present invention may encode all of the heavy chain and light chain constant regions and variable regions, or may encode only the heavy chain and light chain variable regions.
- the base sequences of the heavy and light chain constant regions when coding all of the constant region and the variable region are Nucleic ⁇ Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell Those described in vol.22, p197, 1980 are preferable.
- the nucleic acid molecule of the present invention can be obtained, for example, by the following method. First, total RNA is prepared from a cell such as a hybridoma using a commercially available RNA extraction kit, and cDNA is synthesized by reverse transcriptase using a random primer or the like. Next, in the variable regions of known human antibody heavy chain genes and light chain genes, cDNAs encoding the antibodies are amplified by PCR using oligonucleotides having conserved sequences as primers. The sequence encoding the constant region can be obtained by amplifying a known sequence by the PCR method. The base sequence of DNA can be determined by a conventional method by incorporating it into a sequencing plasmid. Alternatively, the DNA encoding the monoclonal antibody of the present invention can also be obtained by chemically synthesizing a variable region or a partial sequence thereof and binding it to a sequence containing a constant region.
- the present invention also provides a recombinant vector containing the nucleic acid molecule of the present invention and a transformant (host cell) containing the recombinant vector.
- the recombinant vector may be a vector that can be expressed in prokaryotic cells such as Echerichia coli (for example, pBR322, pUC119, or a derivative thereof), but a vector that can be expressed in eukaryotic cells is preferred. More preferred are vectors that can be expressed in mammalian cells.
- vectors that can be expressed in mammalian cells include plasmid vectors such as pcDNA3.1 (manufactured by Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, and pREP4, and pDON-AI DNA (treasure).
- Virus vectors such as those manufactured by Bio Inc.). There may be one vector containing a heavy chain coding sequence and a light chain coding sequence, or two vectors, a vector containing a heavy chain coding sequence and a vector containing a light chain coding sequence.
- the transformant into which the recombinant vector of the present invention is introduced may be a prokaryotic cell such as E. coli or Bacillus subtilis, but is preferably a eukaryotic cell, more preferably a mammal-derived cell.
- mammalian cells include Chinese hamster ovary cells (CHO cells), COS, myeloma, BHK, HeLa, Vero, 293, NS0, Namalwa, YB2 / 0, and the like.
- the obtained anti-RGMa antibody and its antigen-binding fragment can be purified to homogeneity. Separation and purification of antibodies and the like may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited thereto. Examples of the column used for affinity chromatography include a protein A column, a protein G column, an anti-immunoglobulin antibody binding column, and an antigen binding column. For example, as a protein A column, Hyper D, POROS, Sepharose F. F. (Amersham Biosciences) and the like.
- the RGMa-binding protein of the present invention particularly the anti-RGMa antibody or antigen-binding fragment thereof, promotes the repair of nerve function by neutralizing the neurite growth inhibitory activity of RGMa, it prevents, treats or relapses of neurological diseases. Can be used as a prophylactic agent. Since the RGMa-binding protein of the present invention, particularly anti-RGMa antibody or antigen-binding fragment thereof, also neutralizes T cell activation by RGMa, it can be used as a preventive, therapeutic or relapse preventive for immunological diseases. .
- Neurological disorders include amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain Valley, cerebral white matter atrophy , multiple sclerosis (relapsing-remitting type) Multiple sclerosis, primary progressive multiple sclerosis, including secondary progressive multiple sclerosis), optic neuritis, 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-related dementia), Huntington's disease, late-onset dyskinesia, epidemic, Parkinson's disease, Steel-Richard syndrome, Down's syndrome , Myasthenia gravis, nerve trauma (including optic nerve trauma), vascular amyloidosis, cerebral hemorrhage with amyloidosis, cerebral infarction, encephalitis, acute confusion disorder, glaucoma, schizophrenia and retinal nerve fiber layer Gender (diabe
- Immunological disorders include multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), optic neuromyelitis, psoriasis, arthritis ( Rheumatoid arthritis, osteoarthritis, including psoriatic arthritis), Guillain-Barre syndrome, neurobehcet's disease, pernicious anemia, type I (insulin-dependent) diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjogren Syndrome, Goodbascher syndrome, Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease, and sarcoidosis Is exemplified.
- multiple sclerosis including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple
- the RGMa-binding protein of the present invention can be used as a preventive, therapeutic or relapse preventive agent for neurological / immunological diseases.
- Diseases include spinal cord injury, nerve trauma (including optic nerve trauma), multiple sclerosis (relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis) ).
- treatment includes any treatment of disease in mammals, particularly humans, that inhibits disease symptoms, ie, prevents its progression or eliminates disease or symptoms, and reduces disease symptoms. I.e. causing the disease or symptom to retreat or delaying the progression of the symptom.
- prevention includes preventing the onset of the above diseases in mammals, particularly humans.
- recurrence prevention includes preventing recurrence of the above-mentioned disease that repeatedly remits and recurs in mammals, particularly humans.
- the RGMa binding protein (anti-RGMa antibody or antigen-binding fragment thereof) of the present invention can be used as a pharmaceutical composition for prevention or treatment of neurological diseases or immunological diseases.
- the administration form of the RGMa-binding protein (anti-RGMa antibody or antigen-binding fragment thereof) of the present invention is not particularly limited, and is orally or parenterally administered (for example, intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, brain) It can be administered to mammals including humans by any route of administration (internal administration, intraspinal administration, other local administration).
- the dosage form for oral administration and parenteral administration and the preparation method thereof are well known to those skilled in the art, and the pharmaceutical composition is produced by blending the antibody according to the present invention with a pharmaceutically acceptable carrier or the like.
- the dosage form for parenteral administration includes injectable preparations (for example, intravenous injection, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracerebral administration, intraspinal preparation), external preparation (For example, ointments, poultices, lotions), suppository inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes and the like.
- the central nervous tissue when it is desired to directly act on the central nervous tissue, it can be continuously infused by using a medical micropump of an osmotic pump, or mixed with fibrin glue or the like to form a sustained release preparation and then applied to the affected tissue. It can also be detained.
- an injectable preparation is usually prepared by dissolving an antibody in distilled water for injection. If necessary, a solubilizing agent, buffer, pH adjuster, isotonic agent, soothing agent, preservative, Stabilizers and the like can be added. Moreover, it can also be set as the freeze-dried formulation for business preparation.
- the dosage forms for oral administration are solid or liquid dosage forms, specifically tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, injections. Agents, lozenges and the like.
- the pharmaceutical composition of the present invention may further contain other therapeutically effective drugs, and if necessary, components such as bactericides, anti-inflammatory agents, vitamins, amino acids and the like can be blended. .
- pharmacologically acceptable carriers examples include excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers in liquid formulations. And soothing agents. If necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.
- the dosage of the antibody according to the present invention includes, for example, pharmacological findings such as administration route, disease type, symptom severity, patient age, sex, body weight, disease severity, pharmacokinetics and toxicological characteristics, It is determined by a doctor based on various factors such as whether or not a drug delivery system is used and whether it is administered as a part of a combination of other drugs.
- pharmacological findings such as administration route, disease type, symptom severity, patient age, sex, body weight, disease severity, pharmacokinetics and toxicological characteristics. It is determined by a doctor based on various factors such as whether or not a drug delivery system is used and whether it is administered as a part of a combination of other drugs.
- per adult (60 kg body weight) 1 to 5000 ⁇ g / day, preferably 10 to 2000 ⁇ g / day, more preferably 50 to 2000 ⁇ g / day, for injection administration 1 to 5000 ⁇ g / day, preferably 5 to 2000 ⁇ g / day, more preferably
- 10 to 100000 ⁇ g / kg per body weight more preferably 100 to 50000 ⁇ g / kg, even more preferably 500 to 20000 ⁇ g / kg once a day, once a week, once a month, or once a year
- 10 to 100000 ⁇ g / kg per body weight more preferably 100 to 50000 ⁇ g / kg, even more preferably 500 to 20000 ⁇ g / kg once a day, once a week, once a month, or once a year
- continuous infusion is usually performed at a rate of 10 to 100,000 ⁇ g / day, more preferably 100 to 10,000 ⁇ g / day, even more preferably 500 to 5000 ⁇ g / day per adult (body weight 60 kg).
- body weight 60 kg body weight
- Example 1 Preparation of human RGMa protein (C-terminal domain) Pro169 to Gly422 of human RGMa protein (SEQ ID NO: 1 in the sequence listing) (the 169th proline residue from the N-terminal side to the 422th glycine)
- a CHO cell expressing a recombinant human RGMa protein was established in which a histidine tag was fused to the C-terminus of the residue (referred to hereinafter).
- the human RGMa protein C terminal domain 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.
- PBS Phosphate buffered saline
- Example 2 Preparation of mouse anti-human RGMa monoclonal antibody 10 ⁇ g 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 BALB / c The mice were immunized at several sites under the back of a mouse (Charles River Japan Co., Ltd.). Thereafter, 10 ⁇ g of recombinant human RGMa protein prepared with an incomplete Freund's adjuvant (manufactured by Sigma) was immunized every 1-2 weeks in the same manner, and blood was collected after several immunizations.
- complete Freund's adjuvant manufactured by Sigma
- the antibody titer was measured by an ELISA method in which a human or mouse RGMa protein described below was immobilized. Individuals with increased antibody titers were boosted by intravenous administration of 10 ⁇ g of human RGMa protein, and spleen cells were collected 2-3 days later. Cell fusion is performed by mixing the spleen cells and half of the mouse myeloma cells (SP2 / 0, Dainippon Sumitomo Pharma Co., Ltd.), adding polyethylene glycol (Roche Diagnostics) to the precipitate fraction obtained by centrifugation. Fused. The centrifuged cells were then washed twice with D-MEM (Invitrogen).
- the isotypes of both monoclonal antibodies determined using an isotyping kit were mouse IgG2b for the heavy chain and ⁇ for the light chain.
- Purification of the monoclonal antibody was performed by affinity chromatography using agarose (anti-mouse IgG-Agarose manufactured by Sigma) to which anti-mouse IgG antibody was immobilized from the culture supernatant of the hybridoma. After binding the antibody to the column, the column was washed with PBS, then eluted with 10 mM glycine hydrochloride (pH 2.7) and neutralized immediately. Thereafter, the elution neutralized solution was replaced with PBS by an ultrafiltration membrane.
- Example 3 ELISA on which human or mouse RGMa protein is immobilized Human RGMa protein (R & D systems, 2459-RM) or mouse RGMa protein (R & D systems, 2458-RG) prepared at 2 ⁇ g / mL with PBS is dispensed into a 96-well plate at 50 ⁇ L / well at room temperature. Let stand for 1 hour. After removing the solution, ApplieBlock (Seikagaku Biobusiness, Inc., 200150) diluted 5-fold with PBS was dispensed at 200 ⁇ L / well and allowed to stand at room temperature for 1 hour to block nonspecific binding.
- ApplieBlock Seikagaku Biobusiness, Inc., 200150
- Samples (mouse serum, hybridoma culture supernatant, recombinant antibody expression culture supernatant, or purified antibody, etc.) serially diluted with PBST after washing 3 times with PBST (PBS containing 0.05% Tween20) at 50 ⁇ L / well Each was added and allowed to stand at room temperature for 1 hour. Thereafter, the plate was washed with PBST three times, and then peroxidase-labeled sheep anti-mouse IgG antibody (GE Healthcare, NA9310V) diluted with PBS was dispensed at 50 ⁇ L / well and allowed to stand at room temperature for 1 hour.
- PBST PBS containing 0.05% Tween20
- a peroxidase coloring kit manufactured by Sumitomo Bakelite Co., Ltd., ML-1130O was added for color development for a certain period of time, and the absorbance at 492 nm was measured with a plate reader.
- Example 4 Epitope analysis of antibody The epitope to which the antibody binds was determined by the peptide scanning method. A spacer sequence in which the N-terminus is biotinylated on the N-terminal side of an amino acid sequence consisting of 11 consecutive residues shifted by 3 residues, contained in Arg172 to Ala424 of human RGMa protein (SEQ ID NO: 1 in the sequence listing) SGSG) (SEQ ID NO: 46 in the Sequence Listing) was fused to synthesize 83 types of peptides. After the peptide was immobilized on the avidin plate, the test antibody (B5.116A3, B5.70E4) was reacted.
- B5.116A3 is a human of Glu298 to Gly311 (Glu298 to Asp308, Val301 to Gly311), Asn322 to Glu335 (Asn322 to Thr332, two peptides of Ile325 to Glu335), and Lys367 to Ala377.
- B5.70E4 binds to RGMa-derived peptides, Glu298 to Gly311 (two types of peptides Glu298 to Asp308, Val301 to Gly311), Asn322 to Glu335 (two types of peptides Asn322 to Thr332, Ile325 to Glu335), and Pro349 ⁇ Thr359 human RGMa-derived peptide bound.
- Example 5 Sequence analysis and cloning of mouse antibody gene
- Total RNA was extracted from hybridoma cells producing mouse monoclonal antibodies (B5.116A3, B5.70E4).
- CDNA was synthesized by reverse transcription using Total RNA as a template.
- the light chain variable region and constant region gene, and the heavy chain variable region and constant region genes were PCR amplified to determine the DNA sequence.
- the antibody full-length gene was amplified by PCR and cloned.
- the amino acid sequences encoded by these antibody genes were as follows.
- B5.116A3 light chain amino acid sequence (SEQ ID NO: 4 in the sequence listing) DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQLNTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINKKWKIDGSERQNGVETWTSTSTSTST
- B5.116A3 heavy chain amino acid sequence (SEQ ID NO: 5 in the sequence listing) EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRSVYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIY
- B5.70E4 light chain amino acid sequence (SEQ ID NO: 6 in the sequence listing) DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWTCSTDSGA
- Example 6 Preparation of recombinant mouse antibody and recombinant rat mouse chimeric antibody
- Two types of anti-RGMa antibodies B5.116A3 or B5.70E4 derived from hybridoma were prepared (hereinafter referred to as " r116A3 "and” r70E4 ").
- r116A3 anti-RGMa antibodies B5.116A3 or B5.70E4 derived from hybridoma
- the chain constant region was fused with Ala117 to Lys452 (SEQ ID NO: 5 in the sequence listing) to prepare a recombinant rat mouse chimeric antibody (hereinafter referred to as “r5F9”).
- DNAs encoding the light chain and heavy chain of each antibody were inserted into pcDNA3.3 (manufactured by Life Technologies) to prepare an expression vector.
- the expression vector was introduced into HEK293F cells (Life Technologies) using Neofection 293 (Astech). The cells were cultured for 6 days at 37 ° C. and 8% carbon dioxide gas, and then the culture supernatant was collected.
- the culture supernatant was applied to an affinity column (GE healthcare) immobilized with Protein A or Protein G, and the antibody bound to the column was eluted with 10 mM glycine hydrochloride (pH 2.8). After neutralization immediately, it was replaced with PBS.
- affinity column GE healthcare
- 10 mM glycine hydrochloride pH 2.8
- PBS PBS
- the antibody after protein A column purification was purified with a Ceramic Hydroxyapatite Type 1 (CHT) column (manufactured by BIORAD).
- CHT Ceramic Hydroxyapatite Type 1
- the antibody bound to the CHT column was washed with 10 mM KH 2 PO 4 (pH 6.5) and then eluted with 20 mM KH 2 PO 4 (pH 6.5) and 0.5 M NaCl. The elution fraction was collected and replaced with PBS.
- Example 7 Binding test on cells expressing RGMa protein Full-length human RGMa protein (Met1 to Cys450 of SEQ ID NO: 1 in the sequence listing), human RGMa protein C-terminal domain (Pro169 to Cys450 of SEQ ID NO: 1 of the sequence listing) ), A full-length mouse RGMa protein (Met1 to Trp454 of SEQ ID NO: 2 in the sequence listing), or a rat RGMa protein C-terminal domain (Pro170 to Trp449 of SEQ ID NO: 3 of the sequence listing) is expressed in CHO cells or HEK293 cells To produce antigen-expressing cells. The RGMa protein is processed at the C-terminal peptide during the GPI anchor addition reaction.
- Both mouse RGMa protein and rat RGMa protein are cleaved with Ala427, and the C-terminal peptide is removed. Therefore, the amino acid sequences of the full-length protein and C-terminal domain expressed on the cell via the GPI anchor are the same in mice and rats.
- the test antibodies (r116A3 and r70E4) and r5F9 (comparative example) with final concentrations of 10 ⁇ g / mL were reacted with the above antigen-expressing cells, and then washed with PBS containing 0.1% bovine serum albumin and 0.05% NaN 3 did.
- FITC-labeled anti-mouse immunoglobulin antibody manufactured by DAKO was reacted and the cells were washed.
- Example 8 Measurement of dissociation constant for RGMa protein
- the affinity of test antibodies (r116A3, r70E4) and r5F9 (comparative example) to RGMa protein was determined by surface plasmon resonance using Proteon XPR36 (manufactured by Bio-Rad) ( SPR).
- Human RGMa protein (R & D Systems, 2459-RM) diluted with 10 mM acetate buffer (pH 4.5) to 10 ⁇ g / mL, human RGMa protein C-terminal domain (prepared in Example 1), or mouse RGMa protein ( R & D Systems 2458-RG) was fixed to the GLC sensor chip by the amine coupling method.
- test antibody serially diluted as an analyte was applied at a flow rate of 100 ⁇ L / min for 60 seconds, and the dissociation constant (Kd value) was measured.
- Kd value dissociation constant
- Example 9 RGMa-Neogenin binding inhibition test
- the extracellular domain (Ala34 to Leu1105) of a recombinant human Neogenin protein (SEQ ID NO: 10 in the sequence listing) was purified.
- a CHO cell line expressing human Neogenin protein extracellular domain was established.
- a histidine tag was fused to the C-terminus.
- the CHO cell culture supernatant was adsorbed onto a nickel column (GE Healthcare, 17-5247-01) and then eluted with a 100 mM imidazole solution. The imidazole elution fraction was replaced with PBS by dialysis.
- Human RGMa protein (R & D systems, 2459-RM) was labeled with biotin using ChromaLink Biotin Labeling Kit (Solulink).
- a biotin-labeled human RGMa protein prepared to 2 ⁇ g / mL was mixed with an equal amount of a test antibody (r116A3, r70E4) diluted 2-fold and reacted at room temperature for 2 hours to prepare a mixed solution.
- the human Neogenin protein extracellular domain prepared at 2 ⁇ g / mL with PBS was added to each 96-well plate at 50 ⁇ L / well and allowed to stand at room temperature for 1 hour to prepare a Neogenin solid phase plate.
- Example 10 RGMa-BMP2 binding inhibition test Human RGMa protein (R & D systems, 2459-RM) prepared to 2 ⁇ g / mL with PBS was added to a 96-well plate at 50 ⁇ L / well and allowed to stand at room temperature for 1 hour. Nonspecific binding was blocked by adding a 2.5% bovine serum albumin solution and allowing to stand for 1 hour to prepare an RGMa protein-immobilized plate. Test antibodies (B5.116A3, B5.70E4) serially diluted to 0.01 to 10 ⁇ g / mL were added to this RGMa protein solid phase plate and allowed to stand at room temperature for 1 hour.
- Test antibodies (B5.116A3, B5.70E4) serially diluted to 0.01 to 10 ⁇ g / mL were added to this RGMa protein solid phase plate and allowed to stand at room temperature for 1 hour.
- the anti-RGMa antibody (B5.116A3) weakly inhibited the binding of RGMa-BMP2 in a concentration-dependent manner (absorbance 0.45 at 0.01 ⁇ g / mL, absorbance 0.4 at 0.1 ⁇ g / mL, absorbance at 1 ⁇ g / mL). 0.32, absorbance at 10 ⁇ g / mL 0.1).
- Example 11 Design of humanized antibody Human monoclonal antibody B5.116A3 was humanized by complementation determining region (CDR) transplantation according to the method of Winter et al. Described in Japanese Patent No. 2912618. It was. First, a 3D homology model of the light chain and heavy chain variable regions of mouse monoclonal antibody B5.116A3 was created, and amino acid residues located in the vicinity of the CDRs were identified in the framework (FW) region. A human antibody FW in which these amino acids were maintained as much as possible was selected, a mouse antibody CDR was transplanted, and a humanized antibody sequence was designed.
- CDR complementation determining region
- the heavy chain is described as HA (SEQ ID NO: 11 in the sequence listing), and the light chain is described as KA (SEQ ID NO: 19 in the sequence listing).
- additional mutations were introduced into amino acids in FW involved in the structural stability of the variable region, and multiple humanized antibody sequences were designed (heavy chain including HB to HB to HH, 8 types in total, light chain 7 types from KB to KG including KA).
- Example 12 Preparation of recombinant mouse human chimera and recombinant humanized antibody (1) A recombinant mouse human chimeric anti-RGMa antibody 116A3 (r116A3C) having the following amino acid sequence was prepared according to Example 6.
- Example 6 In accordance with Example 6, a total of 20 humanized anti-RGMa antibodies comprising the following heavy and light chain combinations were prepared.
- Heavy / light chain combination HA / KA, HA / KB, HA / KC, HA / KG, HB / KC, HC / KA, HC / KB, HD / KA, HD / KB, HD / KC, HD / KD, HE / KA, HF / KA, HF / KF, HF / KG, HG / KD, HG / KH, HH / KA, HH / KD, HH / KF
- Example 6 A recombinant humanized anti-RGMa antibody (hereinafter referred to as rH5F9) was prepared according to Example 6 (Comparative Example).
- the variable region of humanized anti-RGMa monoclonal antibody h5F9 described in Patent Document 1 (WO2009 / 106356) (the light chain is seq ID_53 of Patent Document 1, the heavy chain is seq ID_50 of Patent Document 1), the human antibody constant region ( The light chain was linked to Arg108 to Cys214 in SEQ ID NO: 26 in the sequence listing, and the heavy chain was linked to Ala117 to Lys446 in SEQ ID NO: 27 in the sequence listing.
- Example 13 Thermal stability test of antibody 20 ⁇ L each of the culture supernatant expressing the recombinant antibody described in Example 12 was collected and using a thermal cycler (Takara Bio, TP600), Heat treatment was performed for 10 minutes at eight temperatures of 40, 45, 50, 55, 60, 65, 70, and 75 ° C., respectively.
- the culture supernatant was diluted with PBS so that the final antibody concentration was 125 ng / mL.
- the human RGMa protein described in Example 3 was subjected to ELISA on which the solid phase was immobilized, and the antigen binding property of the antibody was evaluated (FIG. 3).
- the humanized antibody (hereinafter referred to as “rH116A3”) composed of a combination of heavy chain HE and light chain KA, and mouse human chimeric antibody (r116A3C) have better thermal stability than humanized antibody (rH5F9).
- the humanized antibody comprising this HE / KA combination is referred to as rH116A3.
- the mouse human chimeric antibody (r116A3C) and the humanized antibody (rH116A3) showed equivalent antigen binding, and there was no decrease in antigen binding due to humanization.
- Example 14 Establishment of a humanized anti-RGMa antibody (rH116A3) -producing CHO stable expression strain
- a CHO stable expression strain producing a humanized anti-RGMa antibody (rH116A3) was obtained using the Lonza GS Xceed system (Lonza).
- Lonza Lonza GS Xceed system
- a pXC double gene vector containing the light chain coding sequence (SEQ ID NO: 44 of the sequence listing) and heavy chain coding sequence (SEQ ID NO: 43 of the sequence listing) of the humanized anti-RGMa antibody (rH116A3) is introduced into the CHOK1SV GS knock out parent cell line.
- a transformed cell pool was obtained under the selection of methionine sulphoximine (MSX). After separation into single cells by flow cytometry, the amount of antibody production in the culture supernatant, cell proliferation, etc. were evaluated, and a CHO stable expression strain was obtained.
- MSX methionine sulphoximine
- Example 15 Neurite growth test The cerebellum was removed from a rat newborn (P7), suspended in a trypsin solution (0.25% trypsin in PBS containing 0.2% DNase), and digested at 37 ° C for 10-15 minutes. did. Subsequently, DMEM medium containing 10% fetal bovine serum was added and centrifuged. The cells were resuspended in the same medium and centrifuged, and the operation was repeated twice to wash the cells. Further, this cell suspension was filtered with a 70 ⁇ m cell strainer and then centrifuged to resuspend the precipitate fraction in the same medium. B27 supplement (GIBCO) was added to the cell suspension to prepare rat neonatal cerebellar granule cells.
- trypsin solution 0.25% trypsin in PBS containing 0.2% DNase
- rat neonatal cerebellar granule cells were seeded on a cell plate and cultured at 37 ° C. for 1 day.
- Recombinant RGMa protein R & D systems, 2459-RM
- RGMa Recombinant RGMa protein
- FIG. 4 the addition of RGMa changed the neurite length from 37 ⁇ m to 26 ⁇ m in the experiment on the left and from 38 ⁇ m to 27 ⁇ m in the right, which inhibited neurite growth. It was done.
- test antibodies B5.116A3, B5.70E4
- a final concentration of 10 ⁇ g / mL there was no change in neurite length, but when the test antibody was added simultaneously with the recombinant RGMa protein Neurites grew to the same extent as the control (no RGMa added), and the neutralizing action of the RGMa protein by the antibody was observed.
- Example 16 Efficacy test using spinal cord injury model rats Wistar rats (female, 8 weeks old, approximately 200 g body weight) anesthetized with halothane (manufactured by Takeda Pharmaceutical Company Limited) were given spinal level T9. At the center, a laminectomy was performed on one anteroposterior vertebrae (T8-T10) to expose the spinal cord. When evaluating using a spinal cord crushing model, a pressure of 200 kdyn was applied to the exposed spinal cord using an IH impactor (Precision System).
- IH impactor Precision System
- an osmotic minipump 200 ⁇ L volume, 0.5 ⁇ L / hour, 14 days filled with 400 ⁇ g / mL test antibody (r116A3, r70E4) or control mouse antibody (mo-IgG2b ⁇ ) Delivery) (Alzet, model 2002) was placed subcutaneously on the rat's back.
- the tip of a silicone tube connected to the outlet of the osmotic minipump was placed under the dura mater at the site of spinal cord injury. The tube was sewed and fixed to the spinous process on the lower limb side of the laminectomy site, and the muscle and skin layer were sutured and reared.
- BBB Basso-Beattie-Bresnahan
- Test antibody (B5.116A3) or control mouse antibody (mo-IgG2b ⁇ ) is administered using an osmotic minipump in the same manner as above, and 0, 1, 3, 7 days after injury, and thereafter up to 10 Evaluation was made using the BBB score over the course of the week.
- B5.116A3 significantly improved the BBB score after 4 weeks after administration compared to the control antibody (mo-IgG2b ⁇ ) (p ⁇ 0.01, Student's t-test).
- Example 17 Efficacy test using multiple sclerosis model mice PLP 139-151 peptide (HSLGKWLGHPDKF: SEQ ID NO: 45 in the sequence listing, manufactured by Peptide Institute) was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Factory) and tuberculosis killed H37 Ra (manufactured by Difco Laboratories) was added. It was mixed with Freund's adjuvant (manufactured by Sigma) to prepare an emulsion.
- physiological saline manufactured by Otsuka Pharmaceutical Factory
- tuberculosis killed H37 Ra manufactured by Difco Laboratories
- PLP 139-151 peptide was immunized subcutaneously in the back of SJL / JorllcoCrj (SJL / J) mice (Nippon Charles River) at 50 ⁇ g / head and 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.) Evaluation was made (FIG. 6).
- test antibody (B5.116A3) diluted in physiological saline was intraperitoneally administered at 20 mg / kg 7 days and 10 days or 18 days and 21 days after immunization with the PLP 139-151 peptide.
- the anti-RGMa mouse monoclonal antibody (B5.116A3) suppressed the worsening of the EAE score when administered before the onset compared to the control antibody (mo-IgG2b ⁇ ) (the upper panel of FIG. 6).
- the administration after the onset showed recurrence prevention effect (lower part of FIG. 6).
- Example 18 Antibody immunogenicity test Undifferentiated dendritic cells contained in peripheral blood from 51 healthy donors were matured by granulocyte-monocyte colony-stimulating factor (GM-CSF) and Interleukin-4 stimulation. A test antibody (rH116A3) having a final concentration of 50 ⁇ g / mL was added to mature dendritic cells, and the cells were cultured for 4 to 5 days, whereby the antibodies were taken up into dendritic cells.
- peripheral blood CD4 + T cells (helper T cells) derived from the same donor were mixed and further co-cultured for 1 week, and then the proliferation of T cells was measured by flow cytometry.
- the immunogenicity risk in humans was evaluated using the T cell proliferation activity of the test antibody as an index. As a result, T cells proliferated in 4 out of 51 donors (7.8%), and the immunogenicity risk was low.
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Abstract
Description
RGMaは、細胞膜上のNeogeninに結合して、細胞内のRhoAの活性化、Rasの不活性化を誘導することで、神経突起成長阻害効果をもたらす。一方で、発生途上のニワトリの脳において、RGMaが結合していない場合、Neogeninはアポトーシスを引き起こすことが知られている(Matsunagaら、Dev.Growth Differ.46, 481, 2004)。すなわち、RGMa/Neogenin経路は、神経細胞の生存を促進する、神経再生にとって好ましい作用、および神経突起成長を阻害する負の作用の二つの相反する作用を有していると考えられる。
そこで、本発明は、RGMa/Neogeninの相互作用を阻害せず、かつRGMaの神経突起成長阻害活性を中和するRGMa結合タンパク質を提供することを課題とする。
[1]RGMaとNeogeninの結合を阻害せず、かつRGMaの神経突起成長阻害活性を中和する、単離されたRGMa結合タンパク質。
[2]ヒトRGMa、ラットRGMa及び/又はマウスRGMaと結合する、[1]に記載のRGMa結合タンパク質。
[3]EEVVNAVEDWDSQG(配列表の配列番号26)、NQQIDFQAFHTNAE(配列表の配列番号27)、PTAPETFPYET(配列表の配列番号28)、及び/又はKLPVEDLYYQA(配列表の配列番号29)のペプチドに結合する、[1]または[2]に記載のRGMa結合タンパク質。
[4]配列表の配列番号26及び配列番号27のペプチドに結合する、[1]から[3]のいずれかに記載のRGMa結合タンパク質。
[5]配列表の配列番号26、配列表の配列番号27及び配列表の配列番号28のペプチドに結合する、[1]から[4]のいずれかに記載のRGMa結合タンパク質。
[6]配列表の配列番号26、配列表の配列番号27及び配列表の配列番号29のペプチドに結合する、[1]から[4]のいずれかに記載のRGMa結合タンパク質。
[7]RGMa結合タンパク質がヒト抗体、ヒト化抗体またはキメラ抗体、またはこれらの抗原結合断片である、[1]から[6]のいずれかに記載のRGMa結合タンパク質。
[8][1]から[7]のいずれかに記載のRGMa結合タンパク質のタンパク質部分をコードする核酸分子。
[9][8]に記載の核酸分子を含む組み換えベクター。
[10][9]に記載の組み換えベクターを含む宿主細胞。
[11][1]から[7]のいずれかに記載のRGMa結合タンパク質の製造方法であって、[10]に記載の宿主細胞を培養する工程を含む方法。
[12][1]から[7]のいずれかに記載のRGMa結合タンパク質を含む、医薬組成物。
[13]神経学的疾患または免疫学的疾患の予防、治療または再発予防用の[12]に記載の医薬組成物。
[14]神経学的疾患が、筋萎縮性側索硬化症、上腕神経叢損傷、脳損傷(外傷性脳損傷を含む)、脳性麻痺、ギランバレー、大脳白質萎縮症、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、ポリオ後症候群、二分脊椎、脊髄損傷、脊髄性筋萎縮症、脊椎腫瘍、横断性脊髄炎、認知症(老年性認知症、軽度認知機能障害、アルツハイマー病、アルツハイマー関連認知症を含む)、ハンチントン舞踏病、遅発性ジスキネジー、そう病、パーキンソン病、スティール-リチャード症候群、ダウン症、重症筋無力症、神経外傷(視神経外傷を含む)、血管アミロイド症、アミロイド症を伴う大脳出血、脳梗塞、脳炎、急性混乱障害、緑内障、統合失調症及び網膜神経線維層変性(糖尿病性網膜症、虚血性視神経症、X染色体連鎖性網膜分離症、薬物誘発性視神経症、網膜ジストロフィー、加齢黄斑変性、視神経乳頭ドルーゼンにより特徴づけられる眼病、光受容器変性の遺伝的決定因子により特徴づけられる眼病、常染色体劣性錐体-桿性ジストロフィー、視神経症を伴うミトコンドリア障害を含む)を含む群から選択される、[13]に記載の医薬組成物。
[15]免疫学的疾患が、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、乾癬、関節炎(関節リウマチ、変形性関節症、乾癬性関節炎を含む)、ギランバレー症候群、神経ベーチエツト病、悪性貧血、I型(インスリン依存型)糖尿病、全身エリテマトーデス(SLE) 、炎症性腸疾患(IBD) 、シェーグレン症候群、グッドバスチャー症候群、グレーブス病、自己免疫性溶血性貧血、自己免疫性血小板減少性紫斑病、喘息、花粉症、アトピー性皮膚炎、糸球体腎炎、重症筋無力症、橋本病、及びサルコイドーシスを含む群から選択される、[13]に記載の医薬組成物。
[16]神経学的疾患または免疫学的疾患が、脊髄損傷、神経外傷(視神経外傷を含む)及び多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)を含む群から選択される[13]に記載の医薬組成物。
[17]軽鎖の相補性決定領域1(LCDR1)、軽鎖の相補性決定領域2(LCDR2)、軽鎖の相補性決定領域3(LCDR3)、重鎖の相補性決定領域1(HCDR1)、重鎖の相補性決定領域2(HCDR2)及び重鎖の相補性決定領域3(HCDR3)のそれぞれのアミノ酸配列が、
LCDR1:RASQDISSYLN(配列表の配列番号30)
LCDR2:YTSRLHS(配列表の配列番号31)
LCDR3:QQLNTLP(配列表の配列番号32)
HCDR1:DAWMD(配列表の配列番号33)
HCDR2:EIRSKANNHATYYAESVKG(配列表の配列番号34)及び
HCDR3:RDGAY(配列表の配列番号35)を含む、
または、
LCDR1:RSSQSLVHSNGNTYLH(配列表の配列番号36)
LCDR2:KVSNRFS(配列表の配列番号37)
LCDR3:SQSTHVP(配列表の配列番号38)
HCDR1:TSYYWN(配列表の配列番号39)
HCDR2:YISYDGTNNYNPSLKN(配列表の配列番号40)及び
HCDR3:SFGを含み、
各CDR配列においては、1または数個のアミノ酸が置換、欠失、及び/または付加されていてもよい、単離された抗RGMa抗体、またはその抗原結合断片。
[18]重鎖可変領域(VH)が
VH:EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS(配列表の配列番号41)または該アミノ酸配列に少なくとも90%の同一性のあるアミノ酸配列を含み、
軽鎖可変領域(VL)が
VL:DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME(配列表の配列番号42)または該アミノ酸配列に少なくとも90%の同一性のあるアミノ酸配列を含む、[17]に記載の抗RGMa抗体またはその抗原結合断片。
[19]抗RGMa抗体がヒト化抗体である、[17]または[18]に記載の抗RGMa抗体またはその抗原結合断片。
[20]抗RGMa抗体がヒトIgGの定常領域を有する、[17]から[19]のいずれかに記載の抗RGMa抗体またはその抗原結合断片。
[21]RGMaとの結合が[17]または[18]に記載の抗RGMa抗体と競合する、RGMa結合タンパク質。
[22][17]から[20]のいずれかに記載の抗RGMa抗体またはその抗原結合断片のタンパク質部分をコードする核酸分子。
[23]VH及びVLのアミノ酸配列をコードする核酸配列が
VH:gaagtgcagctggtggaatctggcggcggactggtgcagcctggcagatccctgagactgtcctgtaccgcctccggcttcaccttctccgacgcctggatggattgggtgcgacaggctcctggcaagggcctggaatgggtggccgagatccggtccaaggccaacaaccacgccacctactacgccgagtctgtgaagggccggttcaccatctcccgggacgactccaagtccatcgtgtacctgcagatgaactccctgcggaccgaggacaccgccctgtactactgcaccagaagggacggcgcctactggggcaagggcaccacagtgacagtgtcctcc(配列表の配列番号43)、及び
VL:gacatccagatgacccagtccccctcctccgtgtctgcttccgtgggcgacagagtgaccatcacctgtcgggcctcccaggacatctcctcctacctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctactacacctcccggctgcactccggcgtgccctctagattttccggctctggctccggcaccgactttaccctgaccatctccagcctgcagcccgaggacttcgcctcctacttctgtcagcagctgaacaccctgccctggacctttggcggaggcaccaaggtggaaatggaa(配列表の配列番号44)を含む核酸配列である、[22]に記載の核酸分子。
[24][22]または[23]に記載の核酸分子を含む組み換えベクター。
[25][24]に記載の組み換えベクターを含む宿主細胞。
[26][17]から[20]のいずれかに記載の抗RGMa抗体またはその抗原結合断片の製造方法であって、[25]に記載の宿主細胞を培養する工程を含む方法。
[27][17]から[20]のいずれかに記載の抗RGMa抗体またはその抗原結合断片を含む、医薬組成物。
[28]神経学的疾患または免疫学的疾患の予防、治療または再発予防用の[27]に記載の医薬組成物。
[29]神経学的疾患が、筋萎縮性側索硬化症、上腕神経叢損傷、脳損傷(外傷性脳損傷を含む)、脳性麻痺、ギランバレー、大脳白質萎縮症、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、ポリオ後症候群、二分脊椎、脊髄損傷、脊髄性筋萎縮症、脊椎腫瘍、横断性脊髄炎、認知症(老年性認知症、軽度認知機能障害、アルツハイマー病、アルツハイマー関連認知症を含む)、ハンチントン舞踏病、遅発性ジスキネジー、そう病、パーキンソン病、スティール-リチャード症候群、ダウン症、重症筋無力症、神経外傷(視神経外傷を含む)、血管アミロイド症、アミロイド症を伴う大脳出血、脳梗塞、脳炎、急性混乱障害、緑内障、統合失調症及び網膜神経線維層変性(糖尿病性網膜症、虚血性視神経症、X染色体連鎖性網膜分離症、薬物誘発性視神経症、網膜ジストロフィー、加齢黄斑変性、視神経乳頭ドルーゼンにより特徴づけられる眼病、光受容器変性の遺伝的決定因子により特徴づけられる眼病、常染色体劣性錐体-桿性ジストロフィー、視神経症を伴うミトコンドリア障害を含む)を含む群から選択される、[28]に記載の医薬組成物。
[30]免疫学的疾患が、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、乾癬、関節炎(関節リウマチ、変形性関節症、乾癬性関節炎を含む)、ギランバレー症候群、神経ベーチエツト病、悪性貧血、I型(インスリン依存型)糖尿病、全身エリテマトーデス(SLE) 、炎症性腸疾患(IBD) 、シェーグレン症候群、グッドバスチャー症候群、グレーブス病、自己免疫性溶血性貧血、自己免疫性血小板減少性紫斑病、喘息、花粉症、アトピー性皮膚炎、糸球体腎炎、重症筋無力症、橋本病、及びサルコイドーシスを含む群から選択される、[28]に記載の医薬組成物。
[31]神経学的疾患または免疫学的疾患が、脊髄損傷、神経外傷(視神経外傷を含む)及び多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)を含む群から選択される[28]に記載の医薬組成物。
[32][1]から[7]のいずれかに記載のRGMa結合タンパク質を、それを必要とする対象に、有効量投与する工程を含む、神経学的疾患または免疫学的疾患の予防、治療または再発予防方法。
[33][17]から[20]のいずれかに記載の抗RGMa抗体またはその抗原結合断片を、それを必要とする対象に、有効量投与する工程を含む、神経学的疾患または免疫学的疾患の予防、治療または再発予防方法。
RGMaは中枢神経系における神経突起成長阻害タンパク質であり、ヒトRGMaタンパク質は配列表の配列番号1に示すように450アミノ酸からなる前駆タンパク質として生合成される。N末端に存在するシグナルペプチドMet1~Pro47(N末端側から1番目のメチオニン残基から47番前のプロリン残基までのペプチドを指す、以後同様に記載)が除去され、Asp168とPro169の間のペプチド結合が切断され、C末端ペプチドArg423~Cys450が除去されるとともに、C末端となったGly422のC末端カルボキシル基にGPIアンカーが付加される。ヒトRGMaタンパク質は、N末側ドメイン(Cys48~Asp168)とC末側ドメイン(Pro169~Ala424)がジスルフィド結合により繋がった成熟タンパク質として、GPIアンカーを介して細胞膜上に発現する。マウスのRGMaの前駆タンパク質は配列表の配列番号2に示すアミノ酸配列、ラットのRGMaの前駆タンパク質は配列表の配列番号3に示すアミノ酸配列からなるが、C末端ペプチドが除去されるため、成熟タンパク質としては同一のアミノ酸配列となる。本発明では、RGMaは後述するNeogeninに結合して作用するものであれば、前駆タンパク質、成熟タンパク質またはその活性型断片のいずれかを指してもよいし、それらの誘導体若しくは変異体であってもよい。また、ヒトRGMaでも他の生物由来のRGMaでもよいが、ヒトRGMaが好ましい。
Neogeninは中枢神経の神経細胞等に発現し、RGMaの受容体の1つとして機能している。ヒトNeogeninタンパク質は配列表の配列番号10に示すように、1461アミノ酸からなり、シグナルペプチドMet1~Ala33が除かれ成熟した膜タンパク質として発現する。本発明では、NeogeninはRGMaに結合するものであれば、前駆タンパク質、成熟タンパク質またはそのRGMa結合断片のいずれかを指してもよいし、それらの誘導体若しくは変異体であってもよい。また、ヒトNeogeninでも他の生物由来のNeogeninでもよいが、ヒトNeogeninが好ましい。
本願において「中和」とは目的の標的に結合し、かつ、その標的のいずれかの機能を阻害することができる作用のことをいう。すなわち、「RGMaの神経突起成長阻害活性を中和する」とは、RGMa結合タンパク質がRGMaに結合することにより、RGMaの神経突起成長阻害活性を阻害することをいう。神経突起成長阻害活性は、当分野において知られたいくつかのin vitroまたはin vivo分析の1つまたはそれ以上によって評価することができるが、例えば、本願明細書に記載の神経突起成長阻害試験により評価することができる。
単離されたRGMa結合タンパク質等の「単離された」とは、同定され、かつ、分離された、及び/または、自然状態での成分から回収された、という意味である。自然状態での不純物は、その抗体の診断的または治療的使用を妨害し得る物質であり、酵素、ホルモン及びその他の蛋白質性のまたは非蛋白質性の溶質が挙げられる。一般的に、RGMa結合タンパク質等を単離するには、少なくとも1つの精製工程によって精製すればよく、少なくとも1つの精製工程により精製されたRGMa結合タンパク質を「単離されたRGMa結合タンパク質」ということができる。
本願において「RGMa結合タンパク質」とはRGMaに結合するタンパク質を含む分子をいう。RGMa結合タンパク質としては、抗RGMa抗体及びその抗原結合断片、並びにRGMa結合スキャフォールドタンパク質、並びにNeogeninの細胞外ドメインなどの可溶性RGMa受容体タンパク質、並びにこれらの融合タンパク質があげられる。RGMa結合スキャフォールドタンパク質とは、セリンプロテアーゼ阻害剤のKunitzドメインやヒトのファイブロネクチンの細胞外ドメイン、アンキリン、リポカリンなどに変異を導入することにより、RGMaとの結合機能を実現するタンパク質をいう。融合タンパク質とは、RGMa結合タンパク質にポリエチレングリコール(PEG)等の非ペプチド性ポリマー、放射性物質、毒素、低分子化合物、サイトカイン、成長因子(TGF-β、NGF、Neurotrophinなど)、アルブミン、酵素、他の抗体などの本願のRGMa結合タンパク質以外の機能分子を化学的または遺伝子工学的に結合したRGMa結合タンパク質をいう。
ヒト抗体とは、軽鎖、重鎖ともにヒト免疫グロブリン由来の抗体をいう。ヒト抗体は、重鎖の定常領域の違いにより、γ鎖の重鎖を有するIgG(IgG1、IgG2、IgG3及びIgG4を含む)、μ鎖の重鎖を有するIgM、α鎖の重鎖を有するIgA(IgA1,IgA2を含む)、δ鎖の重鎖を有するIgD、またはε鎖の重鎖を有するIgEを含む。また原則として軽鎖は、κ鎖とλ鎖のどちらか一方を含む。
ヒト化抗体は、非ヒト動物由来抗体の相補性決定領域と、ヒト抗体由来のフレームワーク領域とからなる可変領域、及びヒト抗体由来の定常領域からなる抗体をいう。
キメラ抗体とは、軽鎖、重鎖、またはその両方が、非ヒト由来の可変領域と、ヒト由来の定常領域からなる抗体をいう。
本願において抗RGMa抗体は、RGMaに結合する免疫グロブリン分子またはその改変分子のことをいう。改変分子には、マルチスペシフィック抗体、キメラ抗体、ヒト化抗体、機能改変抗体、及びコンジュゲート抗体を含むものとする。
マルチスペシフィック抗体とは、2つ以上の異なる抗原特異性を有する2つ以上の独立した抗原認識部位を持ち合わせた非対称の抗体であり、2つの抗原特異性を有するバイスペシフィック抗体、3つの抗原特異性を有するトリスペシフィック抗体などが挙げられる。
本願において機能改変抗体とは、主に抗体のFc領域のアミノ酸や糖鎖を改変することにより、抗体の有する抗原結合機能以外の機能、例えば細胞殺傷機能、補体活性化機能や血中半減期延長機能を改変した抗体をいう。
本願においてコンジュゲート抗体とは、抗体にポリエチレングリコール(PEG)等の非ペプチド性ポリマー、放射性物質、毒素、低分子化合物、サイトカイン、成長因子(TGF-β、NGF、Neurotrophinなど)、アルブミン、酵素などの抗体以外の機能分子を化学的または遺伝子工学的に結合した抗体をいう。
本願において抗原結合断片とは、抗体の一部分を含む蛋白質であり、抗原に結合できるものをいう。抗原結合断片の例としては、F(ab')2 、Fab'、Fab 、Fv(variable fragment of antibody)、ジスルフィド結合Fv、一本鎖抗体(scFv)、およびこれらの重合体等が挙げられる。さらに、抗原結合断片は、ポリエチレングリコール(PEG)等の非ペプチド性ポリマー、放射性物質、毒素、低分子化合物、サイトカイン、成長因子(TGF-β、NGF、Neurotrophinなど)、アルブミン、酵素、他の抗体などの本願の抗RGMa抗体以外の機能分子を化学的または遺伝子工学的に結合しているコンジュゲート抗原結合断片を含むものとする。
相補性決定領域(CDR)とは免疫グロブリン分子の可変領域のうち、抗原結合部位を形成する領域をいい、超可変領域とも呼ばれ、免疫グロブリン分子ごとに特にアミノ酸配列の変化が大きい部分をいう。CDRには軽鎖及び重鎖それぞれに3つのCDR(LCDR1、LCDR2、LCDR3、及びHCDR1、HCDR2、HCDR3)がある。本願では、免疫グロブリン分子のCDRはカバット(Kabat)の番号付けシステム(Kabatら、1987、Sequences of Proteins of Immunological Interest、US Department of Health and Human Services、NIH、USA)に従って決定される。
ここで可変領域等の、同定した参照ポリペプチド配列に関する「パーセント(%)同一性」とは、配列を整列させ、最大の%同一性を得るために必要ならば間隙を導入し、如何なる保存的置換も配列同一性の一部と考えないとした後の、特定の参照ポリペプチド配列のアミノ酸残基と同一である候補配列中のアミノ酸残基のパーセントとして定義される。%同一性を測定する目的のためのアラインメントは、当業者の技量の範囲にある種々の方法、例えばBLAST、BLAST-2、ALIGN、又はMegalign(DNASTAR)ソフトウエアのような公に入手可能なコンピュータソフトウエアを使用することにより達成可能である。当業者であれば、比較される配列の完全長に対して最大のアラインメントを達成するために必要な任意のアルゴリズムを含む、配列をアラインメントするための適切なパラメータを決定することができる。しかし、ここでの目的のためには、%同一性値は、ペアワイズアラインメントにおいて、配列比較コンピュータプログラムBLASTを使用することによって得られる。
アミノ酸配列比較にBLASTが用いられる状況では、与えられたアミノ酸配列Aの、与えられたアミノ酸配列Bとの%同一性は次のように計算される:
分率X/Yの100倍
ここで、Xは配列アラインメントプログラムBLASTのA及びBのプログラムアラインメントによって同一であると一致したスコアのアミノ酸残基の数であり、YはBの全アミノ酸残基数である。アミノ酸配列Aの長さがアミノ酸配列Bの長さと異なる場合、AのBに対する%同一性は、BのAに対する%同一性とは異なることは理解されるであろう。特に断らない限りは、ここでの全ての%同一性値は、直ぐ上のパラグラフに示したようにBLASTコンピュータプログラムを用いて得られる。
本願において、本発明の抗RGMa抗体と「競合する」とは、本明細書に記載された表面プラズモン共鳴(SPR)法によって測定した場合に、該抗RGMa抗体またはその抗原結合断片の存在により、有意差をもって本発明の抗RGMa抗体とRGMaとの結合が低下することをいう。
<RGMa結合タンパク質>
本発明のRGMa結合タンパク質は、RGMaとNeogeninの結合を阻害せず、かつRGMaの神経突起成長阻害活性を中和する、単離されたRGMa結合タンパク質である。
本発明の抗RGMa抗体は、RGMaタンパク質またはその部分断片(例えば、上記配列表の配列番号26~29の一つ以上を含む断片)を抗原として、該抗原をマウス等の哺乳動物に免疫して得られるポリクローナル抗体やモノクローナル抗体、遺伝子組換え技術を用いて製造されるキメラ抗体およびヒト化抗体、並びにヒト抗体産生トランスジェニック動物等を用いて製造されるヒト抗体などが含まれる。本発明の抗体を医薬としてヒトに投与する場合は、副作用の観点から、ヒト化抗体またはヒト抗体が望ましい。
ポリクローナル抗体は、既存の一般的な製造方法によって取得することができる。即ち、例えば、前述のような抗原を、必要に応じてフロイントアジュバント(Freund's Adjuvant)とともに、上記のような哺乳動物に免疫することで該免疫感作動物から得た血清から取得することができる。
モノクローナル抗体は、具体的には下記のようにして取得することができる。即ち、前述のような抗原を免疫原とし、該免疫原を、必要に応じてフロイントアジュバント(Freund's Adjuvant)とともに、上記のような哺乳動物の皮下、筋肉内、静脈内、フッドパッド内あるいは腹腔内に1~数回注射するかあるいは移植することにより免疫感作を施す。通常、初回免疫から約1~14日毎に1~4回免疫を行って、最終免疫より約1~5日後に免疫感作された該哺乳動物から抗体産生細胞が取得される。
なお、RGMa との結合能を有し、RGMa とNeogeninの結合を阻害せず、RGMaの神経突起成長阻害活性を中和するという本発明の抗体の特性が維持される限り、これらのCDRの1つ以上において、1~数個のアミノ酸が置換されてもよい。ここで、1~数個とは、例えば、1個または2個である。なお、該アミノ酸置換は本発明の特性を維持するために保存的置換であることが好ましい。抗体の特性が維持されるとは、これらの特性がCDRのアミノ酸配列改変前と比較して同程度、例えば、80%以上、好ましくは90%以上、さらに好ましくは95%以上に維持されることをいう。なお、維持は向上も含む。
なお、ヒト化抗体のアミノ酸配列(重鎖:配列表の配列番号11~18、軽鎖:配列表の配列番号19~25)においては、RGMa との結合能を有し、RGMa とNeogeninの結合を阻害せず、RGMaの神経突起成長阻害活性を中和するという特性が維持される限り、1または数個のアミノ酸(1~20個、1~10個または1~5個)の置換、欠失、付加又は挿入があってもよい。このような置換、欠失、付加はCDRに導入されてもよいが、CDR以外の領域に導入されることが好ましい。また、該アミノ酸置換は本発明の特性を維持するために保存的置換であることが好ましい。
なお、これらのアミノ酸配列において、RGMa との結合能を有し、RGMa とNeogeninの結合を阻害せず、RGMaの神経突起成長阻害活性を中和するという特性が維持される限り、1または数個のアミノ酸(1~20個、1~10個または1~5個)の置換、欠失、付加又は挿入があってもよい。このような置換、欠失、付加はCDRに導入されてもよいが、CDR以外の領域に導入されることが好ましい。また、該アミノ酸置換は本発明の特性を維持するために保存的置換であることが好ましい。
このような配列表の配列番号41および/または配列表の配列番号42のアミノ酸配列において置換、欠失等が含まれた本発明の抗体のアミノ酸配列は、重鎖可変領域が配列表の配列番号41と90%以上(より好ましくは95%、96%、97%、98%、99%以上、)の同一性を有するアミノ酸配列であり,軽鎖可変領域が配列表の配列番号42と90%以上(より好ましくは95%、96%、97%、98%、99%以上)の同一性を有するアミノ酸配列である。
ここで、1~数個とは、例えば、1個または2個である。なお、該アミノ酸置換は本発明の特性を維持するために保存的置換であることが好ましい。
CDR以外の領域は抗体としての構造を維持し、機能を発揮できる配列であれば特に制限されず、マウス由来の配列、ヒト由来の配列、他の哺乳動物由来の配列、それらのキメラ配列、人工配列のいずれであってもよい。定常領域を含む場合においては、重鎖および軽鎖の定常領域のアミノ酸配列は、Nucleic Acids Research vol.14, p1779, 1986、The Journal of Biological Chemistry vol.257, p1516, 1982 およびCell vol.22, p197, 1980 に記載のものが例示される。
上記のような特定のCDRのアミノ酸配列を有する抗体とRGMaとの結合において競合する抗体としては、エピトープとして、Glu298~Gly311、Asn322~Glu335、Lys367~Ala377、およびPro349~Thr359から選ばれる領域にエピトープを有する抗体が例示される。
該抗体は、上記のようなCDR配列を有する抗体とRGMaタンパク質との結合系において、共存させることにより、取得(スクリーニング)したり、評価したりすることができる。例えば、以下の表面プラズモン共鳴(SPR)法によりスクリーニングすることにより取得できる。
同様の実験を本発明の特定のCDRのアミノ酸配列を含む抗RGMa抗体でも実施し、飽和状態での結合量(飽和結合量2)を求める。
続いて、センサーチップ上のヒトRGMa蛋白質を本発明の特定のCDRのアミノ酸配列を含む抗RGMa抗体で飽和させた後、任意の抗RGMa抗体(15μg/mL)をアナライトとしてロードし、本発明の特定のCDRのアミノ酸配列を含む抗RGMa抗体で飽和されたヒトRGMa蛋白質に追加される形で結合するかどうかを調べる。
任意の抗RGMa抗体が、本発明の特定のCDRのアミノ酸配列を含む、抗RGMa抗体で飽和されたヒトRGMa蛋白質に追加される形で、上記で算出した任意の抗RGMa抗体の飽和結合量1を示しながら結合できる場合は、その抗体は「競合しない」と判断される。一方、任意の抗RGMa抗体が、本発明の特定のCDRのアミノ酸配列を含む、抗RGMa抗体で飽和されたヒトRGMa蛋白質に追加される形で結合できない場合は、その抗体は「競合する」と判断される。また任意の抗RGMa抗体が、本発明の特定のCDRのアミノ酸配列を含む、抗RGMa抗体で飽和されたヒトRGMa蛋白質に追加される形で結合できる場合でも、追加される結合量が有意差をもって飽和結合量1に達しない場合は、その抗体は「競合する」と判断される。有意差は一般的な検定方法(例えば、スチューデントのt検定)で調べ、有意水準は5%以下とする。
本発明の核酸分子は本発明のモノクローナル抗体をコードするポリヌクレオチドであるが、例として、重鎖可変領域をコードする領域が配列表の配列番号33、34、および35のアミノ酸配列(1または数個のアミノ酸が置換、欠失、挿入または付加されてよい)をコードする塩基配列をそれぞれ含み、かつ、軽鎖可変領域をコードする領域が配列表の配列番号30、31、および32のアミノ酸配列(1または数個のアミノ酸が置換、欠失、挿入または付加されてよい)をコードする塩基配列をそれぞれ含むポリヌクレオチドや、重鎖可変領域をコードする領域が配列表の配列番号39、40、およびSFGのアミノ酸配列(1または数個のアミノ酸が置換、欠失、挿入または付加されてよい)をコードする塩基配列をそれぞれ含み、かつ、軽鎖可変領域をコードする領域が配列表の配列番号36、37、および38のアミノ酸配列(1または数個のアミノ酸が置換、欠失、挿入または付加されてよい)をコードする塩基配列をそれぞれ含むポリヌクレオチドが挙げられる。
あるいは、可変領域又はその一部の配列を化学合成し、定常領域を含む配列に結合することによっても本発明のモノクローナル抗体をコードするDNAを得ることができる。
本発明のRGMa結合タンパク質、特に抗RGMa抗体またはその抗原結合断片は、RGMaの神経突起成長阻害活性を中和することで神経機能の修復を促すことから、神経学的疾患の予防、治療または再発予防薬として使用することができる。
本発明のRGMa結合タンパク質、特に抗RGMa抗体またはその抗原結合断片はまた、RGMaによるT細胞活性化を中和することから、免疫学的疾患の予防、治療または再発予防薬として使用することができる。
非経口投与のための剤型は、注射用製剤(例えば、点滴注射剤、静脈注射剤、筋肉注射剤、皮下注射剤、皮内注射剤、脳内投与製剤、脊髄内投与製剤)、外用剤(例えば、軟膏剤、パップ剤、ローション剤)、坐剤吸入剤、眼剤、眼軟膏剤、点鼻剤、点耳剤、リポソーム剤等が挙げられる。特に、中枢神経組織に直接作用させたい場合は浸透圧ポンプの医療用マイクロポンプを利用して持続的に注入することもできるし、フィブリン糊などと混合し徐放製剤としたうえで患部組織に留置することもできる。
ヒトRGMaタンパク質(配列表の配列番号1)のPro169~Gly422(N末端側から169番目のプロリン残基から422番目のグリシン残基までを指す、以後同様に記載)のC末端にヒスチジンタグを融合した組換えヒトRGMaタンパク質発現するCHO細胞を樹立した。
CHO細胞の培養上清中に含まれるヒトRGMaタンパク質C末側ドメインを、ニッケルカラム(GE Healthcare 社製、17-5247-01)に吸着させた後、100mMイミダゾール溶液で溶出した。透析により、イミダゾール溶出画分からPhosphate bufferd saline(PBS)に置換し、免疫原として用いた。
実施例1で調製した組換えヒトRGMaタンパク質10μgを、完全フロイントアジュバント(シグマ社製)と混合してエマルジョンを作製し、BALB/cマウス(日本チャールス・リバー株式会社)の背部皮下の数か所に免疫した。その後1~2週間おきに不完全フロイントアジュバント(シグマ社製)でエマルジョンを作製した組換えヒトRGMaタンパク質10μgを同様に免疫し、数回免疫後に採血した。後述するヒトまたはマウスRGMaタンパク質を固相化したELISA法にて抗体価を測定した。抗体価の上昇が認められた個体について、ヒトRGMaタンパク質10μgを静脈内投与してブーストし、2~3日後に脾細胞を回収した。
細胞融合は前記脾細胞とその半数のマウスミエローマ細胞(SP2/0、大日本住友製薬)を混合し、遠心して得た沈殿画分にポリエチレングリコール(ロシュ・ダイアグノスティック社製)を加えて細胞融合させた。次いで遠心した細胞をD-MEM(インビトロジェン社製)で2回洗浄した。細胞を10%ウシ胎児血清(インビトロジェン社製)、1% BM condimed(ロシュ・ダイアグノスティック社製)およびHAT(シグマアルドリッチ社製)を含むGIT培地(日本製薬製)に再懸濁し、各ウェル5 ×104ミエローマ細胞/wellで96穴プレートに播種した。培養上清を回収し、実施例3のヒトRGMaタンパク質固相化ELISA法により、抗体産生細胞のスクリーニングを行った。
スクリーニングで得られた抗体産生細胞を限界希釈法によりクローニングし、2種類のモノクローナル抗体(B5.116A3およびB5.70E4)を産生するハイブリドーマ細胞を選定した。
アイソタイピングキット(Mouse MonoAB ID/SP KIT、ZYMED社製、93-6550)を用いて判定した両モノクローナル抗体のアイソタイプは、いずれも重鎖はマウスIgG2b、軽鎖はκであった。
モノクローナル抗体の精製は、ハイブリドーマの培養上清から、抗マウスIgG抗体を固定したアガロース(Sigma社製 Anti-Mouse IgG -Agarose)を用いたアフィティークロマトグラフィーにより行った。抗体をカラムに結合させた後、PBSでカラムを洗浄し、次いで10mMグリシン塩酸(pH2.7)で溶出し、すみやかに中和した。その後、溶出中和液を限外濾過膜によりPBSに置換した。
PBSで2μg/mLに調製したヒトRGMaタンパク質(R&D systems社製、2459-RM)またはマウスRGMaタンパク質(R&D systems社製、2458-RG)を96穴プレートに50μL/wellずつ分注し、室温で1時間静置した。液を除去後、PBSで5倍希釈したApplieBlock(生化学バイオビジネス社製、200150)を200μL/wellずつ分注し室温で1時間静置し、非特異結合をブロックした。PBST (0.05% Tween20を含むPBS)で3回洗浄後、PBSで段階希釈した検体(マウス血清、ハイブリドーマ培養上清、後述する組換え抗体発現培養上清、または精製抗体など)を、50μL/wellずつ添加し、室温で1時間静置した。その後PBSTで3回洗浄し、次いでPBSで希釈したペルオキシダーゼ標識ヒツジ抗マウスIgG抗体(GE Healthcare社製、NA9310V)を50μL/wellずつ分注し、室温で1時間静置した。3回洗浄した後、ペルオキシダーゼ発色キット(住友ベークライト社製、ML-1130O)を添加して一定時間発色させ、プレートリーダーにて492nmの吸光度を測定した。
抗体が結合するエピトープは、ペプチドスキャン法にて決定した。ヒトRGMaタンパク質(配列表の配列番号1)のArg172~Ala424に含まれる、3残基ずつずらした連続する11残基から成るアミノ酸配列のN末端側に、N末端をビオチン化したスペーサ―配列(SGSG) (配列表の配列番号46)を融合した計83種類のペプチドを合成した。ペプチドをアビジンプレートに固定した後、被験抗体(B5.116A3、B5.70E4)を反応させた。次いで、ペルオキシダーゼ標識ウサギ抗マウスIg抗体(Dako社、P026002)を反応させ、基質溶液を添加して一定時間発色させた後、プレートリーダーにて吸光度を測定した。
その結果、B5.116A3は、Glu298~Gly311(Glu298~Asp308、Val301~Gly311の2種類のペプチド)、Asn322~Glu335(Asn322~Thr332、Ile325~Glu335の2種類のペプチド)、およびLys367~Ala377のヒトRGMa由来のペプチドに結合し、B5.70E4はGlu298~Gly311(Glu298~Asp308、Val301~Gly311の2種類のペプチド)、Asn322~Glu335(Asn322~Thr332、Ile325~Glu335の2種類のペプチド)、およびPro349~Thr359のヒトRGMa由来のペプチドに結合した。
マウスモノクローナル抗体(B5.116A3、B5.70E4)を産生するハイブリドーマ細胞から、total RNAを抽出した。Total RNAを鋳型にして、逆転写反応によりcDNAを合成した。cDNAを鋳型に、軽鎖可変領域および定常領域、並びに重鎖可変領域および定常領域の遺伝子をPCR増幅し、DNA配列を決定した。次いで、決定した可変領域遺配列および定常領域配列に基づき、抗体全長遺伝子をPCRにより増幅し、クローニングした。これらの抗体遺伝子がコードするアミノ酸配列は、以下の通りであった。
DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQLNTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRSVYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
DVKLQESGPGLVKPSQSLSLTCSVTGYSITTSYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKNQFFLRLNSVTTEDTATYYCAGSFGYSQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK
ハイブリドーマ由来の2種類の抗RGMa抗体B5.116A3またはB5.70E4の組換えマウス抗体を調製した(それぞれ以後、「r116A3」、「r70E4」と記載する。)。
また、比較例として、特許文献1(WO2009/106356)に基づき、ラット抗体5F9の可変領域とマウス抗体(IgG2bκ)の定常領域(軽鎖定常領域は配列表の配列番号4のArg108~Cys214、重鎖定常領域は配列表の配列番号5のAla117~Lys452)を融合し組換えラットマウスキメラ抗体(以後、「r5F9」と記載する。)を調製した。
それぞれの抗体の軽鎖および重鎖をコードするDNAを、pcDNA3.3(Life Technologies社製)に挿入し、発現ベクターを作製した。発現ベクターを、HEK293F細胞(Life Technologies社製)にNeofection 293(アステック社製)を用いて導入した。細胞を37℃、8%炭酸ガス条件下で6日間培養した後、培養上清を回収した。組換え抗体の精製は、培養上清を、Protein AまたはProtein Gを固定化したアフィニティーカラム(GE healthcare社製)にアプライし、カラムに結合した抗体を10mMグリシン塩酸(pH2.8)で溶出し、すみやかに中和した後
、PBSに置換した。
使用目的により、精製純度を高める必要がある場合は、Protein Aカラム精製後の抗体を、Ceramic Hydroxyapatite Type1(CHT)カラム(BIORAD社製)にて精製した。CHTカラムに結合した抗体を10 mM KH2PO4(pH6.5)で洗浄後、20 mM KH2PO4(pH6.5),0.5 M NaClで溶出した。溶出画分を回収後、PBSに置換した。
全長ヒトRGMaタンパク質(配列表の配列番号1のMet1~Cys450)、ヒトRGMaタンパク質C末側ドメイン(配列表の配列番号1のPro169~Cys450)、全長マウスRGMaタンパク質(配列表の配列番号2のMet1~Trp454)、またはラットRGMaタンパク質C末側ドメイン(配列表の配列番号3のPro170~Trp449)を発現するベクターを、CHO細胞またはHEK293細胞に導入し、抗原発現細胞を作製した。
尚、RGMaタンパク質は、GPIアンカー付加反応の際にC末端ペプチドがプロセッシングを受ける。マウスRGMaタンパク質およびラットRGMaタンパク質は、いずれもAla427で切断され、C末端側のペプチドは除去される。従って、GPIアンカーを介して細胞上に発現する全長タンパク質およびC末側ドメインのアミノ酸配列は、マウスとラットでは同一である。
終濃度10μg/mLの被験抗体(r116A3およびr70E4)及びr5F9(比較例)を、上記の抗原発現細胞と反応させた後、0.1%ウシ血清アルブミン、0.05%NaN3を含むPBSにて細胞を洗浄した。FITC標識抗マウスイムノグロブリン抗体(DAKO社製)を反応させ、細胞を洗浄した。フローサイトメトリー(ベクトン・ディッキンソン社製、ファックスキャリバー)にて蛍光を測定し、被験抗体の抗原発現細胞への結合性を評価した(表1)。
その結果、r116A3およびr70E4は、r5F9と異なり、ヒトおよびラットRGMaタンパク質のC末側ドメインにも結合することが分かった。RGMaタンパク質はC末側ドメインのみでも神経突起成長阻害作用が認められており、r116A3およびr70E4は、全長RGMaタンパク質およびC末側ドメインの両者を阻害する。
被験抗体(r116A3, r70E4)及びr5F9(比較例)のRGMaタンパク質に対する親和性は、Proteon XPR36(バイオラッド社製)を用いた表面プラズモン共鳴(SPR)法により測定した。
10mM 酢酸バッファー(pH4.5)で10μg/mLに希釈したヒトRGMaタンパク質(R&D Systems社製、2459-RM)、ヒトRGMaタンパク質C末端側ドメイン(実施例1にて調製)、またはマウスRGMaタンパク質(R&D Systems社製、2458-RG)を、アミンカップリング法によりGLCセンサーチップに固定した。アナライトとして段階希釈した被験抗体を流速100 μL/minで60 秒間アプライし、解離定数(Kd値)を測定した。
表2に示すように、r116A3およびr70E4は、r5F9と異なり、ヒトRGMaタンパク質のC末側ドメインにも結合した。r116A3はr5F9よりも、ヒトRGMaタンパク質に対して32倍、マウスRGMaタンパク質に対しては44倍強く結合した。
組換えヒトNeogeninタンパク質(配列表の配列番号10)の細胞外ドメイン(Ala34~Leu1105)を精製した。ヒトNeogeninタンパク質細胞外ドメインを発現するCHO細胞株を樹立した。C末端にヒスチジンタグを融合した。CHO細胞の培養上清から、ニッケルカラム(GE Healthcare 社製、17-5247-01) に吸着させた後、100mMイミダゾール溶液で溶出した。透析により、イミダゾール溶出画分からPBSに置換した。
ChromaLink Biotin Labeling Kit(Solulink社製)を用いて、ヒトRGMaタンパク質(R&D systems社製、2459-RM)をビオチン標識した。2μg/mLに調製したビオチン標識ヒトRGMaタンパク質を、2倍段階希釈した被験抗体(r116A3、r70E4)と等量混和し、室温で2時間反応させ、混合溶液を調製した。
同時に、PBSで2μg/mLに調製したヒトNeogeninタンパク質細胞外ドメインを96穴プレートに50μL/wellずつ加えて室温で1時間静置し、Neogenin固相プレートを作製した。液を除去後、2.5%ウシ血清アルブミン溶液を添加し、1時間静置することで非特異結合をブロックした。このNeogenin固相プレートに、上記の混合溶液を50μL/wellで添加し、室温で1時間静置した。その後洗浄操作を行い、ペルオキシダーゼ標識Avidin(VECTASTAIN ABC システム、ベクターラボラトリーズ社製)を添加し、室温で1時間静置した。その後洗浄操作を行い、基質溶液を添加して一定時間発色させ、プレートリーダーにて吸光度を測定した。抗体が存在しない場合の吸光度比を1としてプロットし、抗体による濃度依存的なRGMa-Neogeninの結合阻害を評価した(図1)。
その結果、抗ヒトRGMaポリクローナル抗体(R&D Systems社製、AF2459)およびr5F9とは異なり、r116A3およびr70E4は、RGMa-Neogeninの結合を阻害しなかった。
PBSで2μg/mLに調製したヒトRGMaタンパク質(R&D systems社製、2459-RM)を96穴プレートに50μL/wellずつ加えて室温で1時間静置した。2.5%ウシ血清アルブミン溶液を添加し、1時間静置することで非特異結合をブロックし、RGMaタンパク質固相化プレートを調製した。このRGMaタンパク質固相プレートに0.01~10μg/mLに段階希釈した被験抗体(B5.116A3、B5.70E4)を加えて室温で1時間静置した。その後洗浄操作を行い、0.5μg/mLに希釈したヒトBMP2タンパク質(R&D systems社製、355-BM)を加え室温で1時間静置した。
ビオチン標識抗BMP2抗体を反応させ、更にペルオキシダーゼ標識Avidin(VECTASTAIN ABC システム、ベクターラボラトリーズ社製)、基質溶液を添加して一定時間発色させ、プレートリーダーにて吸光度を測定した(図2)。
その結果、特に、抗RGMa抗体(B5.116A3)は、濃度依存的にRGMa-BMP2の結合を弱く阻害した(0.01μg/mL で吸光度0.45、0.1μg/mL で吸光度0.4、1μg/mL で吸光度0.32、10μg/mLで吸光度0.1)。
マウスモノクローナル抗体B5.116A3のヒト化は、特許第2912618号公報に記載のWinterらの方法に従い、相補性決定領域(CDR)移植法にて行った。
まず、マウスモノクローナル抗体B5.116A3の軽鎖および重鎖可変領域の3D homology modelを作成し、フレームワーク(FW)領域で、CDR近傍に位置するアミノ酸残基を特定した。これらアミノ酸が可能な限り維持されるヒト抗体FWを選択し、マウス抗体のCDRを移植し、ヒト化抗体配列を設計した。設計したヒト化抗体配列は、重鎖はHA(配列表の配列番号11)、軽鎖はKA(配列表の配列番号19)と記載する。更に、可変領域の構造安定性に関与するFW内のアミノ酸に追加の変異導入を行い、複数のヒト化抗体配列を設計した(重鎖はHAを含
めHB~HHまで計8種、軽鎖はKAを含めKB~KGの計7種)。
(1)実施例6に準じて、以下のアミノ酸配列を有する組換えマウスヒトキメラ抗RGMa抗体116A3(r116A3C)を調製した。
DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQLNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRSVYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
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
特許文献1(WO2009/106356)に記載されたヒト化抗RGMaモノクローナル抗体h5F9の可変領域(軽鎖は特許文献1のseq ID_53、重鎖は特許文献1のseq ID_50)を、ヒト抗体定常領域(軽鎖は配列表の配列番号26のArg108~Cys214、重鎖は配列表の配列番号27のAla117~Lys446)と連結した。
実施例12に記載の組換え抗体を発現する培養上清を20μLずつ分取し,サーマルサイクラー(Takara bio社製、TP600)を用いて、40、45、50、55、60、65、70、75℃の8点の温度でそれぞれ10分間熱処理した。
抗体終濃度が125ng/mLになるよう培養上清をPBSで希釈した。その後、実施例3に記載したヒトRGMaタンパク質を固相化したELISAに供し、抗体の抗原結合性を評価した(図3)。
その結果、重鎖HE、軽鎖KAの組み合わせから成るヒト化抗体(以下、「rH116A3」と称する。)、およびマウスヒトキメラ抗体(r116A3C)は、ヒト化抗体(rH5F9)より優れた熱安定性を示した。以後、このHE/KAの組み合わせから成るヒト化抗体をrH116A3と記載する。
尚、熱処理をしなかった場合、マウスヒトキメラ抗体(r116A3C)とヒト化抗体(rH116A3)は同等の抗原結合性を示し、ヒト化に伴う抗原結合性の低下は無かった。
ヒト化抗RGMa抗体(rH116A3)を生産するCHO安定発現株は、Lonza GS Xceedシステム(Lonza社)にて樹立した。ヒト化抗RGMa抗体(rH116A3)の軽鎖コード配列(配列表の配列番号44)および重鎖コード配列(配列表の配列番号43)を含むpXCダブルジーンベクターを、CHOK1SV GS knock out親細胞株に導入し、methionine sulphoximine (MSX)選択下にて形質転換細胞プールを取得した。フローサイトメトリーにて単一細胞に分離した後、培養上清中への抗体産生量、細胞増殖性などを評価し、CHO安定発現株を取得した。
ラット新生仔(P7)より小脳を摘出し、トリプシン溶液(0.2%DNaseを含む0.25%トリプシンPBS溶液)に懸濁し、37℃で10~15分間消化した。続いて10%ウシ胎児血清含有DMEM培地を加えて遠心分離した。同培地にて再懸濁して遠心分離し、同様に操作を2回繰り返し細胞を洗浄した。更に、この細胞懸濁液を70μmセルストレーナーで濾過後、遠心分離して沈殿画分を同培地にて再懸濁した。細胞浮遊液にB27 supplement(GIBCO社製)を加えてラット新生仔小脳顆粒細胞を調製した。
次に、細胞プレートにラット新生仔小脳顆粒細胞を播種し、37℃で1日間培養を行った。
終濃度2μg/mLの組換えRGMaタンパク質(R&D systems社製、2459-RM)を添加して37℃、2日間培養した。神経突起長を顕微鏡観察により測定したところ、図4に示すように、RGMaの添加により、左図の実験では神経突起長が37μmから26μmとなり、右図では38μmから27μmとなり、神経突起成長が阻害された。被験抗体(B5.116A3、B5.70E4)のみを終濃度10μg/mLで添加しても、神経突起長に変化は見られなかったが、組換えRGMaタンパク質と同時に被験抗体を添加した場合には、対照(RGMa非添加)と同程度に神経突起が成長し、抗体によるRGMaタンパク質の中和作用が認められた。
ハロタン(武田薬品工業社製)にて吸引麻酔したWistar系ラット(雌、8週齢、体重約200g)に、脊椎レベルT9を中心に前後1椎骨分(T8~T10)の椎弓切除術を施し、脊髄を露出させた。脊髄圧挫モデルを用いて評価する場合には、IH impactor(Precision System社製)を用いて、露出した脊髄に200 kdynの圧力をかけた。
前記のように脊髄を損傷させた直後に、400μg/mLの被験抗体(r116A3, r70E4)または対照マウス抗体(mo-IgG2bκ)を満たした浸透圧ミニポンプ(200μL液量、0.5μL/時間、14日間送達)(Alzet社製、model 2002)をラットの背中の皮下に置いた。浸透圧ミニポンプの出口に接続されたシリコン製チューブの先端は、脊髄損傷部位の硬膜下に置いた。該チューブは、椎弓切除術部位の直下肢側の棘突起に縫いつけて固定し、筋肉及び皮膚層を縫合して、飼育した。
脊髄損傷モデルラットの運動機能は、Basso-Beattie-Bresnahan(BBB)スコア(Basso, D.M., Beattie, M.S., & Bresnahan, J.C., A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 12, 1-21 (1995))を用い、損傷後0、1、3、7日目、以降1週間毎に最長8週間に亘り、評価した。その結果、図5(A)に示すように、r116A3またはr70E4は、対照抗体(mo-IgG2bκ)と比較してそれぞれ投与後4または3週間以後に有意にBBBスコアを改善した(p<0.05, Student's t-検定)。
脊髄半側切断モデル用いて評価する場合には、露出させた脊髄の背側部を1.8mm~2.0mmの深さに切断した。上記と同様に被検抗体(B5.116A3)または対照マウス抗体(mo-IgG2bκ)を浸透圧ミニポンプを用いて投与し、損傷後0、1、3、7日目、以降1週間毎に最長10週間に亘り、BBBスコアを用いて評価した。図5(B)に示すように、B5.116A3は対照抗体(mo-IgG2bκ)と比較して投与後4週間以後に有意にBBBスコアを改善した(p<0.01, Student's t-検定)。
PLP139-151ペプチド(HSLGKWLGHPDKF:配列表の配列番号45、ペプチド研究所製) を生理食塩液(大塚製薬工場製)で溶解し、結核死菌H37 Ra (Difco Laboratories社製) を加えた不完全フロイントアジュバント (シグマ社製) と混合し、エマルジョンを作製した。PLP139-151ペプチドとして50 μg/headでSJL/JorllcoCrj (SJL/J) マウス (日本チャールス・リバー) の背部皮下に免疫し、EAEスコア (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.) および体重変化を評価した(図6)。
生理食塩液に希釈した被験抗体 (B5.116A3)を、PLP139-151ペプチドで免疫した7日及び10日後 または18日及び21日後にそれぞれ20 mg/kgで腹腔内に投与した。
その結果、図6に示すように、抗RGMaマウスモノクローナル抗体 (B5.116A3) は、対照抗体(mo-IgG2bκ)と比較して、発症前の投与でEAEスコアの悪化を抑制し(図6上段)、発症後の投与で再発予防効果を示した(図6下段)。
51名の健常人ドナー由来の末梢血中に含まれる未分化樹状細胞を顆粒球単球コロニー刺激因子(GM-CSF)およびInterleukin-4刺激により成熟させた。成熟樹状細胞に終濃度50μg/mLの被験抗体(rH116A3)を添加し、4、5日間培養することで、抗体を樹状細胞に取り込ませた。ここへ、同一ドナー由来の末梢血CD4+T細胞(ヘルパーT細胞)を混合し、更に1週間共培養した後、T細胞の増殖をフローサイトメトリーにて測定した。被験抗体のT細胞増殖活性を指標として、ヒトにおける免疫原性リスクを評価した。その結果、51名のドナーのうち、4名(7.8%)についてT細胞の増殖が認められ、免疫原性リスクは低かった。
Claims (31)
- Repulsive Guidance Molecule a(RGMa)とNeogeninの結合を阻害せず、かつRGMaの神経突起成長阻害活性を中和する、単離されたRGMa結合タンパク質。
- ヒトRGMa、ラットRGMa及び/又はマウスRGMaと結合する、請求項1に記載のRGMa結合タンパク質。
- EEVVNAVEDWDSQG(配列表の配列番号26)、NQQIDFQAFHTNAE(配列表の配列番号27)、PTAPETFPYET(配列表の配列番号28)、及び/又はKLPVEDLYYQA(配列表の配列番号29)のペプチドに結合する、請求項1または2に記載のRGMa結合タンパク質。
- 配列表の配列番号26及び配列表の配列番号27のペプチドに結合する、請求項1から3のいずれか1項に記載のRGMa結合タンパク質。
- 配列表の配列番号26、配列表の配列番号27及び配列表の配列番号28のペプチドに結合する、請求項1から4のいずれか1項に記載のRGMa結合タンパク質。
- 配列表の配列番号26、配列表の配列番号27及び配列表の配列番号29のペプチドに結合する、請求項1から4のいずれか1項に記載のRGMa結合タンパク質。
- RGMa結合タンパク質がヒト抗体、ヒト化抗体またはキメラ抗体、またはこれらの抗原結合断片である、請求項1から6のいずれか1項に記載のRGMa結合タンパク質。
- 請求項1から7のいずれか1項に記載のRGMa結合タンパク質のタンパク質部分をコードする核酸分子。
- 請求項8に記載の核酸分子を含む組み換えベクター。
- 請求項9に記載の組み換えベクターを含む宿主細胞。
- 請求項1から7のいずれか1項に記載のRGMa結合タンパク質の製造方法であって、請求項10に記載の宿主細胞を培養する工程を含む方法。
- 請求項1から7のいずれか1項に記載のRGMa結合タンパク質を含む、医薬組成物。
- 神経学的疾患または免疫学的疾患の予防、治療または再発予防用の請求項12に記載の医薬組成物。
- 神経学的疾患が、筋萎縮性側索硬化症、上腕神経叢損傷、脳損傷(外傷性脳損傷を含む)、脳性麻痺、ギランバレー、大脳白質萎縮症、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、ポリオ後症候群、二分脊椎、脊髄損傷、脊髄性筋萎縮症、脊椎腫瘍、横断性脊髄炎、認知症(老年性認知症、軽度認知機能障害、アルツハイマー病、アルツハイマー関連認知症を含む)、ハンチントン舞踏病、遅発性ジスキネジー、そう病、パーキンソン病、スティール-リチャード症候群、ダウン症、重症筋無力症、神経外傷(視神経外傷を含む)、血管アミロイド症、アミロイド症を伴う大脳出血、脳梗塞、脳炎、急性混乱障害、緑内障、統合失調症及び網膜神経線維層変性(糖尿病性網膜症、虚血性視神経症、X染色体連鎖性網膜分離症、薬物誘発性視神経症、網膜ジストロフィー、加齢黄斑変性、視神経乳頭ドルーゼンにより特徴づけられる眼病、光受容器変性の遺伝的決定因子により特徴づけられる眼病、常染色体劣性錐体-桿性ジストロフィー、視神経症を伴うミトコンドリア障害を含む)を含む群から選択される、請求項13に記載の医薬組成物。
- 免疫学的疾患が、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、乾癬、関節炎(関節リウマチ、変形性関節症、乾癬性関節炎を含む)、ギランバレー症候群、神経ベーチエツト病、悪性貧血、I型(インスリン依存型)糖尿病、全身エリテマトーデス(SLE) 、炎症性腸疾患(IBD) 、シェーグレン症候群、グッドバスチャー症候群、グレーブス病、自己免疫性溶血性貧血、自己免疫性血小板減少性紫斑病、喘息、花粉症、アトピー性皮膚炎、糸球体腎炎、重症筋無力症、橋本病、及びサルコイドーシスを含む群から選択される、請求項13に記載の医薬組成物。
- 神経学的疾患または免疫学的疾患が、脊髄損傷、神経外傷(視神経外傷を含む)及び多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)を含む群から選択される請求項13に記載の医薬組成物。
- 軽鎖の相補性決定領域1(LCDR1)、軽鎖の相補性決定領域2(LCDR2)、軽鎖の相補性決定領域3(LCDR3)、重鎖の相補性決定領域1(HCDR1)、重鎖の相補性決定領域2(HCDR2)及び重鎖の相補性決定領域3(HCDR3)のそれぞれのアミノ酸配列が、
LCDR1:RASQDISSYLN(配列表の配列番号30)
LCDR2:YTSRLHS(配列表の配列番号31)
LCDR3:QQLNTLP(配列表の配列番号32)
HCDR1:DAWMD(配列表の配列番号33)
HCDR2:EIRSKANNHATYYAESVKG(配列表の配列番号34)及び
HCDR3:RDGAY(配列表の配列番号35)を含む、
または、
LCDR1:RSSQSLVHSNGNTYLH(配列表の配列番号36)
LCDR2:KVSNRFS(配列表の配列番号37)
LCDR3:SQSTHVP(配列表の配列番号38)
HCDR1:TSYYWN(配列表の配列番号39)
HCDR2:YISYDGTNNYNPSLKN(配列表の配列番号40)及び
HCDR3:SFGを含み、
各CDR配列においては、1または数個のアミノ酸が置換、欠失、及び/または付加されていてもよい、単離された抗RGMa抗体、またはその抗原結合断片。 - 重鎖可変領域(VH)が
VH:EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS(配列表の配列番号41)または該アミノ酸配列に少なくとも90%の同一性のあるアミノ酸配列を含み、
軽鎖可変領域(VL)が
VL:DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME(配列表の配列番号42)または該アミノ酸配列に少なくとも90%の同一性のあるアミノ酸配列を含む、請求項17に記載の抗RGMa抗体またはその抗原結合断片。 - 抗RGMa抗体がヒト化抗体である、請求項17または18に記載の抗RGMa抗体またはその抗原結合断片。
- 抗RGMa抗体がヒトIgGの定常領域を有する、請求項17から19のいずれか1項に記載の抗RGMa抗体またはその抗原結合断片。
- RGMaとの結合が請求項17または18に記載の抗RGMa抗体と競合する、RGMa結合タンパク質。
- 請求項17から20のいずれか1項に記載の抗RGMa抗体またはその抗原結合断片のタンパク質部分をコードする核酸分子。
- VH及びVLのアミノ酸配列をコードする核酸配列が
VH:gaagtgcagctggtggaatctggcggcggactggtgcagcctggcagatccctgagactgtcctgtaccgcctccggcttcaccttctccgacgcctggatggattgggtgcgacaggctcctggcaagggcctggaatgggtggccgagatccggtccaaggccaacaaccacgccacctactacgccgagtctgtgaagggccggttcaccatctcccgggacgactccaagtccatcgtgtacctgcagatgaactccctgcggaccgaggacaccgccctgtactactgcaccagaagggacggcgcctactggggcaagggcaccacagtgacagtgtcctcc(配列表の配列番号43)、及び
VL:gacatccagatgacccagtccccctcctccgtgtctgcttccgtgggcgacagagtgaccatcacctgtcgggcctcccaggacatctcctcctacctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctactacacctcccggctgcactccggcgtgccctctagattttccggctctggctccggcaccgactttaccctgaccatctccagcctgcagcccgaggacttcgcctcctacttctgtcagcagctgaacaccctgccctggacctttggcggaggcaccaaggtggaaatggaa(配列表の配列番号44)
を含む核酸配列である、請求項22に記載の核酸分子。 - 請求項22または23に記載の核酸分子を含む組み換えベクター。
- 請求項24に記載の組み換えベクターを含む宿主細胞。
- 請求項17から20のいずれか1項に記載の抗RGMa抗体またはその抗原結合断片の製造方法であって、請求項25に記載の宿主細胞を培養する工程を含む方法。
- 請求項17から20のいずれか1項に記載の抗RGMa抗体またはその抗原結合断片を含む、医薬組成物。
- 神経学的疾患または免疫学的疾患の予防、治療または再発予防用の請求項27に記載の医薬組成物。
- 神経学的疾患が、筋萎縮性側索硬化症、上腕神経叢損傷、脳損傷(外傷性脳損傷を含む)、脳性麻痺、ギランバレー、大脳白質萎縮症、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、ポリオ後症候群、二分脊椎、脊髄損傷、脊髄性筋萎縮症、脊椎腫瘍、横断性脊髄炎、認知症(老年性認知症、軽度認知機能障害、アルツハイマー病、アルツハイマー関連認知症を含む)、ハンチントン舞踏病、遅発性ジスキネジー、そう病、パーキンソン病、スティール-リチャード症候群、ダウン症、重症筋無力症、神経外傷(視神経外傷を含む)、血管アミロイド症、アミロイド症を伴う大脳出血、脳梗塞、脳炎、急性混乱障害、緑内障、統合失調症及び網膜神経線維層変性(糖尿病性網膜症、虚血性視神経症、X染色体連鎖性網膜分離症、薬物誘発性視神経症、網膜ジストロフィー、加齢黄斑変性、視神経乳頭ドルーゼンにより特徴づけられる眼病、光受容器変性の遺伝的決定因子により特徴づけられる眼病、常染色体劣性錐体-桿性ジストロフィー、視神経症を伴うミトコンドリア障害を含む)を含む群から選択される、請求項28に記載の医薬組成物。
- 免疫学的疾患が、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)、視神経脊髄炎、乾癬、関節炎(関節リウマチ、変形性関節症、乾癬性関節炎を含む)、ギランバレー症候群、神経ベーチエツト病、悪性貧血、I型(インスリン依存型)糖尿病、全身エリテマトーデス(SLE) 、炎症性腸疾患(IBD) 、シェーグレン症候群、グッドバスチャー症候群、グレーブス病、自己免疫性溶血性貧血、自己免疫性血小板減少性紫斑病、喘息、花粉症、アトピー性皮膚炎、糸球体腎炎、重症筋無力症、橋本病、及びサルコイドーシスを含む群から選択される、請求項28に記載の医薬組成物。
- 神経学的疾患または免疫学的疾患が、脊髄損傷、神経外傷(視神経外傷を含む)、多発性硬化症(再発寛解型多発性硬化症、一次性進行型多発性硬化症、二次性進行型多発性硬化症を含む)を含む群から選択される、請求項28に記載の医薬組成物。
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EP16786512.0A EP3290441B1 (en) | 2015-04-28 | 2016-04-27 | Rgma binding protein and use thereof |
LTEP16786512.0T LT3290441T (lt) | 2015-04-28 | 2016-04-27 | Rgma surišantys baltymai ir jų panaudojimas |
DK16786512.0T DK3290441T3 (da) | 2015-04-28 | 2016-04-27 | Rgma-bindingsprotein og anvendelse deraf |
CA2983898A CA2983898A1 (en) | 2015-04-28 | 2016-04-27 | Rgma binding protein and use thereof |
US15/569,382 US10287346B2 (en) | 2015-04-28 | 2016-04-27 | RGMa binding protein and use thereof |
JP2017515573A JP6497760B2 (ja) | 2015-04-28 | 2016-04-27 | RGMa結合タンパク質及びその使用 |
MX2017013894A MX2017013894A (es) | 2015-04-28 | 2016-04-27 | Proteina de union a la molecula de guia repelente a (rgma) y su uso. |
EP19179190.4A EP3584260A1 (en) | 2015-04-28 | 2016-04-27 | Rgma binding protein and use thereof |
KR1020237042670A KR20230173734A (ko) | 2015-04-28 | 2016-04-27 | RGMa 결합 단백질 및 그 사용 |
PL16786512T PL3290441T3 (pl) | 2015-04-28 | 2016-04-27 | Białko wiążące RGMa i jego zastosowanie |
RU2017135353A RU2705304C2 (ru) | 2015-04-28 | 2016-04-27 | Связывающий rgma белок и его использование |
IL287291A IL287291B (en) | 2015-04-28 | 2016-04-27 | A protein that binds to rgma and its use |
CR20170539A CR20170539A (es) | 2015-04-28 | 2016-04-27 | PROTEINA DE UNIÓN A RGma Y SU USO |
KR1020197032016A KR102451786B1 (ko) | 2015-04-28 | 2016-04-27 | RGMa 결합 단백질 및 그 사용 |
CN201680023980.4A CN107531791B (zh) | 2015-04-28 | 2016-04-27 | RGMa结合蛋白质及其使用 |
SG11201708848TA SG11201708848TA (en) | 2015-04-28 | 2016-04-27 | RGMa BINDING PROTEIN AND USE THEREOF |
KR1020227034319A KR102613528B1 (ko) | 2015-04-28 | 2016-04-27 | RGMa 결합 단백질 및 그 사용 |
BR112017022994-3A BR112017022994A2 (pt) | 2015-04-28 | 2016-04-27 | proteína de ligação de rgma e uso da mesma |
RS20191502A RS59847B1 (sr) | 2015-04-28 | 2016-04-27 | Protein koji vezuje rgma i njegova upotreba |
AU2016253886A AU2016253886B2 (en) | 2015-04-28 | 2016-04-27 | RGMa binding protein and use thereof |
KR1020177034449A KR102040235B1 (ko) | 2015-04-28 | 2016-04-27 | RGMa 결합 단백질 및 그 사용 |
ES16786512T ES2758480T3 (es) | 2015-04-28 | 2016-04-27 | Proteína de unión a RGMa y uso de la misma |
SI201630515T SI3290441T1 (sl) | 2015-04-28 | 2016-04-27 | RGMA-vezavni protein in njegova uporaba |
PE2022001997A PE20230381A1 (es) | 2015-04-28 | 2016-04-27 | Proteina de union a rgma |
SA517390230A SA517390230B1 (ar) | 2015-04-28 | 2017-10-25 | Rgma بــروتــيــن رابــط لــ واســـتــخـدامـــه |
PH12017501941A PH12017501941A1 (en) | 2015-04-28 | 2017-10-25 | RGMa BINDING PROTEIN AND USE THEREOF |
IL255293A IL255293B (en) | 2015-04-28 | 2017-10-26 | A protein that binds to rgma and its use |
CONC2017/0011975A CO2017011975A2 (es) | 2015-04-28 | 2017-11-24 | Proteína de unión a rgma |
ZA2017/08047A ZA201708047B (en) | 2015-04-28 | 2017-11-27 | Rgma binding protein and use thereof |
HK18105139.1A HK1245808A1 (zh) | 2015-04-28 | 2018-04-19 | RGMa 結合蛋白質及其使用 |
US16/358,657 US11008388B2 (en) | 2015-04-28 | 2019-03-19 | RGMa binding protein and use thereof |
AU2019210655A AU2019210655A1 (en) | 2015-04-28 | 2019-08-02 | RGMa binding protein and use thereof |
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HRP20192099TT HRP20192099T1 (hr) | 2015-04-28 | 2019-11-22 | Protein koji veže rgma i njegova uporaba |
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