RU2809500C2 - RGMa BINDING PROTEIN AND ITS USE - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention is related to an isolated antibody to RGMa or its antigen-binding fragment. A nucleic acid molecule encoding said antibody; an expression vector containing said nucleic acid molecule; a host cell containing said vector; a composition containing said antibody are also disclosed. A method for producing said antibody is disclosed.

EFFECT: invention makes it possible to effectively treat diseases in which the activity of RGMa is involved.

14 cl, 18 ex, 3 tbl, 6 dwg

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0001] The present invention relates to an RGMa binding protein and its use.

BACKGROUND ART

[0002] RGM (repulsive guidance molecule), which is a GPI-anchored membrane protein with a molecular weight of approximately 33 kDa, was initially identified as an axon guidance molecule in the visual system (see Non-Patent Document 1). The RGM family includes three members, named RGMa, RGMb and RGMc. Among them, RGMa is re-expressed after central nervous system injury in adult humans and rats, as well as during the developmental stage, and inhibition of RGMa in rat accelerates neurite outgrowth after spinal cord injury and promotes functional recovery (see Non-Patent Document 2). Thus, RGMa is believed to be an inhibitor of neurites after injury to the central nervous system.

[0003] RGMa has also been reported to have effects on the immune system. RGMa is expressed in dendritic cells and acts on T cells, thereby increasing T cell adhesion to ICAM-1 and fibronectin and inducing cytokine production (Patent Document 4). In a mouse model of multiple sclerosis, administration of an anti-RGMa antibody suppressed symptoms associated with encephalomyelitis and also suppressed disease onset and relapse. Anti-RGMa antibody is believed to bind to RGMa expressed by dendritic cells, inhibiting T cell activation, thereby affecting multiple sclerosis.

[0004] The RGMa signal transduction mechanism is also being studied, and it has been published that the neogenin protein is a receptor for RGMa (Patent Document 3). Neogenin is the only transmembrane protein expressed by neurons and T cells.

RGMa binds to neogenin on the cell membrane, inducing intracellular activation of RhoA and inactivation of Ras, thereby providing an inhibitory effect on neurite outgrowth. However, neogenin is known to cause apoptosis in the absence of RGMa binding during chicken brain development (Matsunaga et al., Dev. Growth Differ. 46, 481, 2004). Thus, the RGMa/neogenin pathway is believed to have two opposing actions, promoting neuronal survival, which is a beneficial effect for nerve regeneration, and inhibiting neurite outgrowth, a negative effect.

[0005] As a pharmaceutical agent targeting RGM, Patent Document 1 describes an axon regeneration promoting agent containing an anti-RGM neutralizing antibody as an active ingredient. Patent Documents 2 and 3 describe a therapeutic agent for mechanical injury to the brain and spinal cord, an anti-RGM antibody that regulates the binding of RGM to its receptor neogenin. Patent Document 4 describes medical uses of an anti-RGM antibody, such as for multiple sclerosis. Patent Document 5 describes therapeutic uses of an anti-RGM antibody for diseases including multiple sclerosis, mammalian brain injury, spinal cord injury, apoplexy, neurodegenerative disease, and schizophrenia. In addition, Patent Document 6 describes therapeutic uses of RGM modulators such as anti-RGM antibody for spinal cord injury and multiple sclerosis, and Non-Patent Document 3 describes therapeutic uses for progressive multiple sclerosis.

BACKGROUND DOCUMENTS

PATENT DOCUMENTS

[0006] Patent Document 1: WO 2005/087268

Patent Document 2: Published Translated Japanese PCT Patent Application No. 2010-537655

Patent Document 3: Published Translated Japanese PCT Patent Application No. 2009-510002

Patent Document 4: WO 2011/071059

Patent Document 5: Published Translated Japanese PCT Patent Application No. 2011-512806

Patent Document 6: Published Translated Japanese PCT Patent Application No. 2004-525875

NON-PATENT DOCUMENTS

[0007] Non-patent document 1: Neuron 5, 735-743 (1990)

Non-Patent Document 2: J. Cell Biol. 173, 47-58 (2006)

Non-patent document 3: Cell Reports 10, 1-12 (2015)

SUMMARY OF THE INVENTION

TECHNICAL PROBLEM

[0008] As stated above, therapeutic uses of anti-RGMa antibodies have been described in neurological and immunological diseases, but conventional antibodies have problems such as lack of activity, the potential to interfere with the intrinsic functions of RGMa, and side effects. In particular, conventional antibodies can inhibit the binding of RGMa and neogenin, thereby also inhibiting beneficial effects, such as inhibition of apoptosis, resulting from the binding of neogenin to RGMa.

Thus, it is an object of the present invention to provide an RGMa binding protein that does not inhibit the RGMa/neogenin interaction but neutralizes the neurite outgrowth inhibitory activity of RGMa.

SOLUTION

[0009] The inventors have conducted extensive research to solve the above problems. As a result, the present inventors were able to obtain an RGMa binding protein that does not inhibit the binding of RGMa and neogenin, but neutralizes the neurite outgrowth inhibitory activity of RGMa, and found that the RGMa binding protein can be used as a drug for neurological or immunological diseases, thus achieving real invention.

[0010] The present invention is as follows.

[1] An isolated RGMa binding protein that does not inhibit RGMa and neogenin binding but neutralizes the neurite outgrowth inhibitory activity of RGMa.

[2] The RGMa binding protein described in [1], which binds to human, rat and/or mouse RGMa.

[3] The RGMa binding protein described in paragraph [1] or [2], which binds to the peptides EEVVNAVEDWDSQG (SEQ ID NO: 26 in the sequence listing), NQQIDFQAFHTNAE (SEQ ID NO: 27 in the sequence listing), PTAPETFPYET (SEQ ID NO: 28 in the sequence list) and/or KLPVEDLYYQA (SEQ ID NO: 29 in the sequence list).

[4] An RGMa binding protein described in any of [1] to [3] that binds to peptides SEQ ID NOs: 26 and 27 in the sequence listing.

[5] An RGMa binding protein described in any of [1] to [4] that binds to peptides SEQ ID NOs: 26, 27 and 28 in the sequence listing.

[6] An RGMa binding protein described in any of [1] to [4] that binds to peptides SEQ ID NOs: 26, 27 and 29 in the sequence listing.

[7] The RGMa binding protein described in any one of [1] to [6], wherein the RGMa binding protein is a human antibody, a humanized antibody or a chimeric antibody or an antigen binding fragment thereof.

[8] A nucleic acid molecule encoding a protein region of the RGMa binding protein described in any of paragraphs [1] to [7].

[9] A recombinant vector containing the nucleic acid molecule described in [8].

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

[11] The method for producing RGMa binding protein described in paragraphs [1]-[7], where the method includes the step of culturing a host cell described in [10].

[12] A pharmaceutical composition containing an RGMa binding protein described in any of paragraphs [1] to [7].

[13] The pharmaceutical composition described in paragraph [12] for use in the prevention, treatment or prevention of relapse of neurological or immunological diseases.

[14] The pharmaceutical composition described in paragraph [13], wherein the neurological diseases are selected from the group consisting of amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barre syndrome, cerebral leukodystrophy, multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), neuromyelitis optica, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal neoplasia, transverse myelitis, dementia (including senile dementia, mild cognitive impairment, Alzheimer's disease, dementia associated with Alzheimer's disease), Huntington's disease, tardive dyskinesia, mania, Parkinson's disease, Steele-Richardson syndrome, Down syndrome, myasthenia gravis, nervous system trauma (including optic nerve injury), amyloidosis vascular disease, intracerebral hemorrhage associated with amyloidosis, cerebral infarction, encephalitis, acute confusional states, glaucoma, schizophrenia and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal degeneration associated with age-related macular degeneration, eye diseases characterized by optic disc drusen, eye diseases characterized by genetically determined photoreceptor degeneration, autosomal recessive rod-cone dystrophy, mitochondrial disorder associated with optic neuropathy).

[15] The pharmaceutical composition described in paragraph [13], wherein the immunological diseases are selected from the group consisting of multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Guillain-Barré syndrome, Behçet's disease, pernicious anemia, (insulin-dependent) diabetes mellitus type I, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjögren's syndrome, Goodpasture's syndrome, Graves' disease , autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease and sarcoidosis.

[16] The pharmaceutical composition described in paragraph [13], wherein the neurological or immunological diseases are selected from the group consisting of spinal cord injury, nervous system injury (including optic nerve injury) and multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis , secondary progressive multiple sclerosis).

[17] An isolated anti-RGMa antibody or antigen binding fragment thereof, wherein the amino acid sequence of each of the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), the complementarity determining region heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3) contains the following sequences:

LCDR1:RASQDISSYLN (SEQ ID NO: 30 in sequence list)

LCDR2:YTSRLHS (SEQ ID NO: 31 in sequence listing)

LCDR3:QQLNTLP (SEQ ID NO: 32 in sequence list)

HCDR1:DAWMD (SEQ ID NO: 33 in sequence listing)

HCDR2:EIRSKANNHATYYAESVKG (SEQ ID NO: 34 in the sequence list) and

HCDR3:RDGAY (SEQ ID NO: 35 in sequence listing);

or

LCDR1:RSSQSLVHSNGNTYLH (SEQ ID NO: 36 in sequence list)

LCDR2:KVSNRFS (SEQ ID NO: 37 in sequence listing)

LCDR3:SQSTHVP (SEQ ID NO: 38 in sequence list)

HCDR1:TSYYWN (SEQ ID NO: 39 in sequence list)

HCDR2:YISYDGTNNYNPSLKN (SEQ ID NO: 40 in sequence listing) and

HCDR3:SFG, and

wherein in each of the CDR sequences one or more amino acids can be substituted, deleted and/or added.

[18] Antibody to RGMa or its antigen-binding fragment described in paragraph [17],

wherein the heavy chain variable region (VH) contains the following:

VH: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS (SEQ ID NO: 41 in the sequence listing) or an amino acid sequence having at least 90% identity with the specified amino acid sequence; And

wherein the light chain variable region (VL) contains the following:

VL: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 42 in the sequence listing) or an amino acid sequence having at least 90% identity with the specified amino acid sequence.

[19] An anti-RGMa antibody or an antigen-binding fragment thereof as described in [17] or [18], wherein the anti-RGMa antibody is a humanized antibody.

[20] An anti-RGMa antibody or an antigen-binding fragment thereof as described in any of [17]-[19], wherein the anti-RGMa antibody comprises a human IgG constant region.

[21] An RGMa binding protein that competes with the anti-RGMa antibody described in [17] or [18] for binding to RGMa.

[22] A nucleic acid molecule encoding an anti-RGMa antibody protein region or an antigen-binding fragment thereof described in any of paragraphs [17]-[20].

[23] The nucleic acid molecule described in paragraph [22], wherein each of the nucleotide sequences encoding the amino acid sequences VH and VL is a nucleotide sequence containing:

VH: gaagtgcagctggtggaatctggcggcggactggtgcagcctggcagatccctgagactgtcctgtaccgcctccggcttcaccttctccgacgcctggatggattgggtgcgacaggctcctggcaagggcctggaatgggtggccgagatccggtccaaggccaacaaccacgccacctactacgccgagtctgtgaaggg ccggttcaccatctcccgggacgactccaagtccatcgtgtacctgcagatgaactccctgcggaccgaggacaccgccctgtactactgcaccagaagggacggcgcctactggggcaagggcaccacagtgacagtgtcctcc (SEQ ID NO: 43 in sequence listing), and

VL: gacatccagatgacccagtccccctcctccgtgtctgcttccgtgggcgacagagtgaccatcacctgtcgggcctcccaggacatctcctcctacctgaactggtatcagcagaagcccggcaaggcccccaagctgctgatctactacacctcccggctgcactccggcgtgccctctagattttccggctctggctccggcacc gactttaccctgaccatctccagcctgcagcccgaggacttcgcctcctacttctgtcagcagctgaacaccctgccctggacctttggcggaggcaccaaggtggaaatggaa (SEQ ID NO: 44 in sequence listing).

[24] A recombinant vector containing the nucleic acid molecule described in paragraphs [22] or [23].

[25] A host cell containing the recombinant vector described in paragraph [24].

[26] A method for producing an antibody to RGMa or an antigen-binding fragment thereof described in any one of paragraphs [17]-[20], where the method includes the step of culturing a host cell described in paragraph [25].

[27] A pharmaceutical composition containing an antibody to RGMa or an antigen-binding fragment thereof described in any of paragraphs [17]-[20].

[28] The pharmaceutical composition described in paragraph [27], for use in the prevention, treatment or prevention of relapse of neurological or immunological diseases.

[29] The pharmaceutical composition described in paragraph [28], wherein the neurological diseases are selected from the group consisting of amyotrophic sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barre syndrome, cerebral leukodystrophy, multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), neuromyelitis optica, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal neoplasia, transverse myelitis, dementia (including senile dementia, mild cognitive impairment, Alzheimer's disease, dementia associated with Alzheimer's disease), Huntington's disease, tardive dyskinesia, mania, Parkinson's disease, Steele-Richardson syndrome, Down syndrome, myasthenia gravis, nervous system trauma (including optic nerve injury), amyloidosis vascular disease, intracerebral hemorrhage associated with amyloidosis, cerebral infarction, encephalitis, acute confusional states, glaucoma, schizophrenia and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal degeneration, age-related macular degeneration, eye diseases characterized by optic disc drusen, eye diseases characterized by genetically determined photoreceptor degeneration, autosomal recessive rod-cone dystrophy, mitochondrial disorder associated with optic neuropathy).

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

[31] The pharmaceutical composition described in paragraph [28], wherein the neurological or immunological diseases are selected from the group consisting of spinal cord injury, nervous system injury (including optic nerve injury) and multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis , secondary progressive multiple sclerosis).

[32] A method for preventing, treating, or preventing relapse of neurological or immunological diseases, wherein the method includes the step of administering an effective dose of the RGMa binding protein described in any of paragraphs [1] to [7] to an individual in need thereof.

[33] A method for preventing, treating, or preventing the recurrence of neurological or immunological diseases, wherein the method includes the step of administering an effective dose of an anti-RGMa antibody or an antigen-binding fragment thereof described in any of paragraphs [17]-[20] to an individual in need thereof.

USEFUL EFFECT OF THE INVENTION

[0011] The RGMa binding protein of the present invention does not inhibit the interaction of RGMa and neogenin, thus is capable of maintaining effects such as inhibition of neuronal apoptosis and the like caused by the binding of neogenin to RGMa, thus having a strong protective effect against neurons and little risk of side effects associated with neuronal depletion. In addition, the humanized anti-RGMa antibody of the present invention has superior properties compared with conventional antibodies, such as binding to human RGMa and heat stability. Thus, the antibody can be used as a drug for neurological or immunological diseases, which is highly effective and has few side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In FIG. 1 shows the result of an RGMa-neogenin binding inhibition assay using a polyclonal anti-RGMa antibody (AF2459), a comparative example antibody (r5F9) and an antibody of the present invention (r70E4 and 116A3).

In fig. 2 shows the result of an RGMa-BMP2 binding inhibition assay using control mouse IgG and antibodies of the present invention (B5.70E4 and B5.116A3).

In fig. 3 shows the result of a heat stability test for antibodies using the comparative example antibody (rH5F9), the chimeric antibody of the present invention (r116A3C) and the humanized antibodies of the present invention (HE/KA and HA/KC).

In fig. 4 shows the result of a neurite outgrowth assay using antibodies of the present invention (B5.70E4 (left) and B5.116A3 (right)).

In fig. 5 shows the result of an efficacy test using mouse control IgG (mo-IgG2bk) and antibodies of the present invention (r70E4 and r116A3) and using a rat spinal cord injury model. Results from performance tests in (A) a spinal cord crush model and (B) a spinal cord hemisection model are demonstrated.

In fig. 6 shows the result of an efficacy test of the antibody of the present invention (B5.116A3) using a PLP _139-151γ peptide-induced multiple sclerosis model in mice. The left side shows the EAE scores, the right side shows the changes in body weight, and the top and bottom sides show the results when the antibody test was administered after 7 and 10 days and after 18 and 21 days, respectively.

DESCRIPTION OF IMPLEMENTATION OPTIONS

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

[0014] RGMa

RGMa is a neurite outgrowth inhibitory protein in the central nervous system, and human RGMa protein is biosynthesized as a precursor protein containing 450 amino acids, as demonstrated in SEQ ID NO: 1 in the sequence listing. The signal peptide Met 1-Pro 47 contained at the N-terminus (which represents the peptide from the first methionine residue to the 47th proline residue on the N-terminal side, similarly described below) is removed, the peptide bond between Asp 168 and Pro 169 is cleaved, and C- the terminal peptide Arg 423-Cys 450 is removed and a GPI anchor is simultaneously added to the carboxyl group of Gly 422 at the C terminus, which becomes the C terminus. The human RGMa protein is expressed on the cell membrane via a GPI anchor as a mature protein in which the N-terminal domain (Cys 48-Asp 168) and the C-terminal domain (Pro 169-Ala 424) are linked to each other by a disulfide bond. The mouse RGMa precursor protein contains the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and the rat RGMa precursor protein contains the amino acid sequence shown in SEQID NO: 3 in the sequence listing. Due to the fact that the peptides at the C-terminus are removed, the mature proteins from them have the same amino acid sequences. In the present invention, RGMa may refer to a precursor protein, a mature protein or an active fragment thereof, or may be a derivative or variant thereof, provided that it acts by binding to neogenin, as described below. RGMa may be human RGMa or RGMa derived from other organisms, but human RGMa is preferred.

[0015] Neogenin

Neogenin is expressed, for example, in neurons of the central nervous system and functions as one of the RGMa receptors. As demonstrated in SEQ ID NO: 10 in the sequence listing, the human neogenin protein contains 1461 amino acids and is expressed as a mature membrane protein after removal of the Met 1-Ala 33 signal peptide. In the present invention, neogenin may refer to a precursor protein, a mature protein, or a binding protein. RGMa fragment, or may be a derivative or variant thereof, provided that it binds to RGMa. The neogenin may be human neogenin or neogenin derived from other organisms, but human neogenin is preferred.

[0016] Neutralization

The term "neutralization" as used herein refers to the action by which binding to a target of interest and inhibition of any function of the target occurs. In other words, the phrase “neutralizing the neurite outgrowth inhibitory activity of RGMa” means that the RGMa binding protein binds to RGMa, thereby inhibiting the neurite outgrowth inhibitory activity of RGMa. Neurite outgrowth inhibitory activity can be assessed by one or more of a number of in vitro or in vivo assays known in the art and can be assessed, for example, by the neurite outgrowth inhibition assay described herein.

[0017] Dedicated

The term “isolated,” such as an isolated RGMa binding protein, means identified and separated and/or purified from its components in its natural state. Naturally occurring impurities are substances that may interfere with the diagnostic or therapeutic use of an antibody, including enzymes, hormones, and other protein or non-protein solutes. Typically, isolation of RGMa binding protein or the like. can be carried out through at least one purification step. An RGMa binding protein purified by at least one purification step may be referred to as an "isolated RGMa binding protein."

[0018] RGMa binding protein

As used herein, the term “RGMa binding protein” refers to a molecule containing a protein that binds to RGMa. Examples of RGMa binding proteins include anti-RGMa antibodies and antigen binding fragments thereof; RGMa binding scaffold proteins; solubilized RGMa receptor proteins, such as the extracellular domain of neogenin, and fusion proteins thereof. The term “RGMa-binding scaffold protein” refers to a protein that performs the function of binding RGMa by introducing mutations in the Kunitz domain of serine protease inhibitor, the extracellular domain of fibronectin, ankyrin, human lipocalin, or the like. The term "fusion protein" refers to RGMa binding proteins linked chemically or genetically to functional molecules other than the RGMa binding protein of the present invention, such as non-protein polymers such as polyethylene glycol (PEG), radioactive substances, toxins, small molecules, cytokines, growth factors (eg, TGF-β, NGF, neurotrophin), albumin, enzymes and other antibodies.

[0019] Human antibody

The term "human antibody" refers to an antibody in which the light and heavy chains are derived from human immunoglobulin. Depending on the difference in heavy chain constant regions, human antibodies include IgG containing heavy γ chains (including IgG1, IgG2, IgG3 and IgG4), IgM containing heavy μ chains, IgA containing heavy α chains (including IgA1 and IgA2 ), IgD containing heavy δ chains, or IgE containing heavy ε chains. Light chains mainly contain κ chains or λ chains.

[0020] Humanized antibody

The term “humanized antibody” refers to an antibody comprising variable regions containing the complementarity determining regions of an antibody derived from a non-human animal, and framework regions derived from a human antibody, and constant regions derived from a human antibody.

[0021] Chimeric antibody

The term "chimeric antibody" refers to an antibody in which the light chain, the heavy chain, or both chains contain a non-human variable region and a human constant region.

[0022] Antibody to RGMa

As used herein, the term “anti-RGMa antibody” refers to immunoglobulin molecules that bind to RGMa, or modified molecules thereof. Modified molecules include multispecific antibodies, chimeric antibodies, humanized antibodies, functionally modified antibodies and conjugated antibodies.

[0023] Polyspecific antibody

The term "multispecific antibody" refers to an asymmetric antibody containing two or more independent antigen recognition regions having two or more different antigen specificities, including a bispecific antibody having two antigen specificities and a trispecific antibody having three antigen specificities.

[0024] Functionally modified antibody

As used herein, the term "functionally modified antibody" refers to an antibody in which functions other than the antigen-binding function of the antibody, including a cell cytolysis function, a complement activation function, and an extended serum half-life function, are modified primarily by amino acid or sugar modification. chains of the Fc region of an antibody.

[0025] Conjugated antibody

As used herein, the term "conjugated antibody" refers to an antibody linked chemically or genetically to functional molecules other than the antibody, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, small molecules, cytokines, factors growth (eg, TGF-β, NGF, neurotrophin), albumin and enzymes.

[0026] Antigen binding fragment

As used herein, the term “antigen-binding fragment” refers to a protein that contains a portion of an antibody and can bind to an antigen. Examples of the antigen binding fragment include F(ab') ₂ , Fab', Fab, Fv (antibody variable fragment), disulfide-linked Fv, single chain antibody (scFv) and polymers thereof. In addition, the antigen binding fragment includes conjugated antigen binding fragments chemically or genetically linked to functional molecules other than the anti-RGMa antibody herein, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, small molecules, cytokines, factors growth (eg, TGF-β, NGF, neurotrophin), albumin, enzymes and other antibodies.

[0027] Complementarity determining region

The term “complementarity determining region (CDR)” refers to the region forming the antigen-binding region in the variable region of an immunoglobulin molecule, which is also called the hypervariable region, and in particular refers to the region in which the amino acid sequence undergoes significant changes in each immunoglobulin molecule. Regarding CDRs, their light and heavy chains each contain three CDRs (LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3). In this application, CDR immunoglobulin molecules are determined in accordance with the Kabat numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0028] Percentage (%) of amino acid sequence identity

"Percentage (%) identity" with respect to an identified reference polypeptide sequence, such as a variable region, is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular reference polypeptide sequence after sequence alignment and the introduction of gaps as necessary to obtain maximum % identity and provided that conservative substitutions do not constitute a significant portion of the sequence identity. Alignment to determine % identity can be carried out in various ways known to one skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to obtain maximum alignment throughout the full-length sequences that are being compared. However, for the purposes of the present invention, % identity values are obtained using the BLAST sequence comparison computer program in pairwise alignments.

In cases where BLAST is used for amino acid sequence comparisons, the % identity of a given amino acid sequence A with a given amino acid sequence B is calculated as follows:

100 multiplied by fraction X/Y

where X is the number of amino acid residues scored as identical matches by the Blast sequence alignment program when aligned by A and B, and where Y is the total number of amino acid residues in B. It should be understood that if the length of the amino acid sequence A is different from the length of the amino acid sequence B , the % identity of A with B will be different from the % identity of B with A. Unless otherwise noted, all % identity values herein are obtained using the BLAST computer program as shown in the preceding paragraph.

[0029] Competition

As used herein, the term “competition” with the anti-RGMa antibody of the present invention means that, as measured by surface plasmon resonance (SPR) described herein, the binding of the anti-RGMa antibody of the present invention to RGMa is reduced by a significant difference due to the presence of said anti-RGMa antibody or antigen-binding fragment thereof.

[0030] The present invention is described in more detail below.

RGMa binding protein

The RGMa binding protein of the present invention is an isolated RGMa binding protein that does not inhibit the binding of RGMa and neogenin, but neutralizes the neurite outgrowth inhibitory activity of RGMa.

[0031] Preferably, the RGMa proteins are mammalian derived RGMa proteins. For example, human RGMa proteins include a protein containing the amino acid sequence SEQ ID NO: 1 in the sequence listing, mouse RGMa proteins include a protein containing the amino acid sequence SEQ ID NO: 2 in the sequence listing, and rat RGMa proteins include a protein containing the amino acid sequence SEQ ID NO: 3 in sequence list. Preferred may also be a polypeptide containing an amino acid sequence where one or more (preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5) amino acids in these sequences are replaced, deleted, introduced and/or added, and having substantially the same activity as the RGMa protein; or a polypeptide comprising an amino acid sequence having 90% or more (preferably 95% or more) amino acid sequence identity.

[0032] As used herein, a polypeptide "having substantially the same activity as the RGMa protein" includes any polypeptide so long as the polypeptide has a neurite outgrowth inhibitory effect.

[0033] The amino acid substitution is preferably a conservative substitution. As used herein, a “conservative substitution” means the replacement of an amino acid residue with another chemically similar amino acid residue such that the activity of the peptide is not substantially altered. For example, they include replacing one hydrophobic residue with another hydrophobic residue, replacing one polar residue with another polar residue having the same electrical charge, and the like. Examples of functionally similar amino acids with which such a substitution is possible include the non-polar (hydrophobic) amino acids alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine and cysteine. 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.

[0034] Binding of the RGMa binding protein of the present invention to RGMa means specific binding of RGMa. More preferably, an RGMa binding protein has a low dissociation constant (Kd) with human RGMa, for example, 10 ^-8 M or less, more preferably 10 ^-9 M or less, even more preferably 10 ^-10 M or less as an upper limit value , and for example, without limitation, 10 ^-14 M or more, more preferably 10 ^-13 or more as the lower limit value.

[0035] The RGMa protein as a mature protein contains an N-terminal domain and a C-terminal domain, but has neurite outgrowth inhibitory activity with only one C-terminal domain. The RGMa binding protein of the present invention preferentially binds only to the C-terminal domain of RGMa, neutralizing the neurite outgrowth inhibitory activity. More preferably, an RGMa binding protein has a low dissociation constant (Kd) with the human RGMa C-terminal domain, e.g., 10 ^-8 M or less, more preferably 10 ^-9 M or less, even more preferably 10 ^-10 M or less in as the upper limit value, and for example, without limitation, 10 ^-14 M or more, more preferably 10 ^-13 or more as the lower limit value.

[0036] The RGMa binding protein of the present invention does not inhibit the binding of RGMa and neogenin. As used herein, the phrase “does not inhibit the binding of RGMa and neogenin” means that in the RGMa and neogenin binding system shown in the examples described below, even when the concentration of the RGMa binding protein is increased, the binding of RGMa and neogenin is not substantially decreased. For example, in the case where the RGMa binding protein is added to the RGMa-neogenin binding system and its concentration is increased, if the concentration of the RGMa binding protein having an _IC50 is not less than 10 μg/ml, more preferably not less than 50 μg/ml, most preferably at least 100 μg/ml, it can be said that the RGMa binding protein does not inhibit the binding of RGMa and neogenin.

[0037] The neogenin used for the RGMa binding assay is preferably the same type of neogenin as RGMa. In other words, it is preferable to use mouse or human neogenin for mouse or human RGMa, respectively. An example of human neogenin includes a protein containing the amino acid sequence of SEQ ID NO: 10 in the sequence listing. However, provided that it is capable of binding to RGMa, a protein containing an amino acid sequence with 90% or more (preferably 95% or more) identity to SEQ ID NO: 10 in the sequence listing may be preferred.

[0038] The RGMa binding protein of the present invention neutralizes the neurite outgrowth inhibitory activity of RGMa. The neurite outgrowth inhibitory activity can be assessed by the neurite outgrowth assay shown in the examples described below. The addition of RGMa inhibits neurite outgrowth, but the addition of RGMa binding protein prevents the inhibition of RGMa neurite outgrowth. The RGMa binding protein of the present invention can counteract the inhibition of neurite outgrowth resulting from the addition of RGMa by 50% or more, more preferably by 80% or more, and most preferably by 90% or more.

[0039] Due to the fact that the amino acid sequences of RGMa proteins vary depending on the animal species, there are also differences in the amino acid sequence of human RGMa provided by SEQ ID NO: 1 in the sequence listing, mouse RGMa provided by SEQ ID NO: 2 in the sequence listing, and rat RGMa provided by SEQ ID NO: 3 in the sequence listing. Due to the fact that rodents such as mice and rats are mainly used as experimental material in pharmacological and safety tests of protein drugs such as antibody drugs, the RGMa binding protein of the present invention preferably binds to mouse and/or rat RGMa , and more preferred are RGMa binding proteins having a low Kd to mouse and/or rat RGMa. Includes RGMa binding proteins whose Kd has an upper limit, for example, 5×10 ^-7 M or less, more preferably 10 ^-8 M or less, even more preferably 10 ^-9 M or less, and a lower limit value, for example, without limitation, 10 ^-12 M or more, more preferably 10 ^-11 or more.

[0040] The RGMa binding protein of the present invention preferably has high thermostability. Thermal stability can be assessed by the decrease in binding to RGMa as a result of heat treatment, and the RGMa binding protein of the present invention is preferably stable even when cooked at 60°C or higher, more preferably even when cooked at 65°C or higher, most preferably even when cooked at 70°C or higher.

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

[0042] Specific examples of the RGMa binding protein include an anti-RGMa antibody, an RGMa binding scaffold protein, and fusion proteins thereof.

[0043] Antibody to RGMa

The anti-RGMa antibody of the present invention refers to polyclonal or monoclonal antibodies produced by using the RGMa protein or a partial fragment thereof (e.g., a fragment containing one or more of SEQ ID NOs: 26 to 29 in the above sequence listing) as an antigen and immunizing a mammal , such as mouse antigens; chimeric antibodies and humanized antibodies obtained using gene recombination technology; antibodies and humans, obtained using, for example, transgenic animals that produce human antibodies, etc. When the antibody of the present invention is administered as a pharmaceutical to humans, a humanized or human antibody is preferred from the viewpoint of side effects.

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

[0045] Examples of mammals that can be immunized include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, horses and cattle, and methods of inoculation include subcutaneous, intramuscular or intraperitoneal administration. When administered, the antigens can be administered in mixtures with complete Freund's adjuvant or incomplete Freund's adjuvant, and administration is typically done once every 2-5 weeks. Antibody-producing cells obtained from the spleen or lymph nodes of immunized animals are fused with myeloma cells and isolated as hybridomas. As myeloma cells, myeloma cells obtained from a mammal such as a mouse, rat or human are used.

[0046] Polyclonal antibody

Polyclonal antibodies can be produced by known production methods. In other words, polyclonal antibodies can be obtained from serum obtained from animals that have been immunized, for example, by immunizing a mammal as described above with an antigen as described above, together with Freund's adjuvant as needed.

[0047] Monoclonal antibody

In particular, monoclonal antibodies can be produced as follows. In other words, the antigen as described above is used as an immunogen, and the immunogen is injected or transplanted one or more times in combination with Freund's adjuvant, if necessary, into the mammal as described above, subcutaneously, intramuscularly, intravenously, into the ball of the foot, or intraperitoneally for immunization. Typically, immunizations are administered 1 to 4 times every 1 to 14 days from the primary immunization, and antibody-producing cells are obtained from the immunized mammal approximately 1 to 5 days after the final immunization.

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

[0049] The production of "hybridomas" secreting monoclonal antibodies can be carried out by the method and Milstein et al. (Nature, 256, 495, 1975) and similar methods. Thus, hybridomas are produced by fusing an antibody-producing cell contained in a spleen or the like obtained from an immunized mammal with a myeloma cell lacking the ability to produce an autoantibody obtained from a mammal, preferably a mouse, rat or human.

[0050] Examples of myeloma cells that can be used for cell fusion include mouse-derived myeloma P3/X63-AG8,653 (653), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8 .U1 (P3U1), SP2/0-Ag14 (Sp2/O, Sp2), PAI, F0 and BW5147, rat myeloma 210RCY3-Ag.2.3 and human myeloma U-266AR1, GM1500-6TG-A1-2 , UC729-6, CEM-AGR, D1R11 and CEM-T15.

[0051] Examples of fusion accelerators include polyethylene glycol and the like. In general, the interaction of antibody-producing cells and myeloma cells in a ratio of typically about 1:1 to 10:1 is achieved by using polyethylene glycol (average molecular weight: 1000 to 4000) at a concentration of about 20 to 50%, at a temperature 20 to 40°C, preferably 30 to 37°C for about 1 to 10 minutes, so that cell fusion can be carried out.

[0052] Screening for hybridoma clones producing monoclonal antibodies can be accomplished by culturing the hybridomas, for example, in microtiter plates, and measuring the reactivity of the culture supernatants in the wells to the immunogen by immunochemical methods such as ELISA.

[0053] When screening antibody-producing hybridomas, in addition to the RGMa protein binding assay, the antibody also evaluates whether the antibody inhibits the binding of RGMa protein and neogenin, and whether the antibody neutralizes the function of the RGMa protein (neurite outgrowth inhibitory activity). Such screening methods select for the anti-RGMa antibody of the present invention.

[0054] Clones can further be obtained from wells containing hybridomas producing the desired antibodies by cloning using limiting dilution. Selection and propagation of hybridomas is typically carried out in animal cell culture medium containing 10 to 20% fetal bovine serum supplemented with HAT (hypoxanthine, aminopterin and thymidine).

[0055] Monoclonal antibodies from hybridomas can be obtained by culturing the hybridoma in vitro or growing them in vivo , for example, in the ascitic fluid of mammals such as mice and rats, and isolating the monoclonal antibodies from the resulting culture supernatant or ascitic fluid of the mammal.

[0056] In in vitro culture, hybridomas are grown, maintained and stored according to various conditions such as the characteristics and culture method of the cell species to be cultured, and a culture medium suitable for the production of monoclonal antibodies in the culture supernatant can be used.

[0057] Examples of basic media include low calcium media such as Ham's F12 medium, MCDB 153 medium, or low calcium MEM medium, and high calcium media such as MCDB 104 medium, MEM medium, D-MEM medium, RPMI 1640 environment, ASF 104 environment or RD environment. Basic media may contain, for example, serum, hormones, cytokines and/or various inorganic or organic substances according to the purpose.

[0058] Monoclonal antibodies can be isolated and purified, for example, by subjecting the above culture supernatant or ascitic fluid to saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (such as DEAE or DE52) or affinity column chromatography such as anti-immunoglobulin antibody column or protein A column chromatography. In particular, the purification of a monoclonal antibody can be carried out by any methods known as an immunoglobulin purification method, and can be easily obtained by methods such as ammonium sulfate fractionation, PEG fractionation , ethanol fractionation and affinity chromatography using anion exchange agent and additional use of RGMa proteins.

[0059] Monoclonal antibodies can also be produced by phage display. In a phage display method, phages selected from an optional phage antibody library are screened using a desired immunogen and phages having the desired binding ability to the immunogen are selected. The antibody-corresponding sequence contained in the phage is then isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding the antibody or antigen-binding domain is constructed based on the isolated sequence or determined sequence information. Finally, monoclonal antibodies can be produced by culturing a cell line transfected with such expression vectors. The human antibody library can be used as a phage antibody library to produce human antibodies having the desired binding properties.

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

[0061] Fusion proteins include RGMa binding proteins chemically or genetically linked to functional molecules other than the RGMa binding protein of the present invention, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, small molecules, cytokines, growth factors (eg TGF-β, NGF, neurotrophin), albumin, enzyme and other antibodies.

[0062] When PEG is linked as a functional molecule, a PEG with a molecular weight without limitation of 2000 to 100,000 Da, more preferably 10,000 to 50,000 Da, which may be linear or branched, can be used. Using, for example, an NHS active group, PEG can be linked, for example, to the N-terminal amino acid groups of the RGMa binding protein.

[0063] In the case of using a radioactive substance as a functional molecule, for example, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ⁶⁴ Cu, ⁹⁹ Tc, ⁷⁷ Lu or ²¹¹ At are used. Radioactive substances can be directly coupled to the RGMa binding protein, for example by the chloramine T method.

[0064] When using a toxin as the functional molecule, for example, bacterial toxins (eg, diphtheria toxin), phytotoxins (eg, ricin), low molecular weight toxins (eg, geldanamycin), maytansinoids, and calicheamycin are used.

[0065] When using low molecular weight compounds as the functional molecule, for example, daunomycin, doxorubicin, methotrexate, mitomycin, neocarzinostatin, vindesine and fluorescent dyes such as FITC are used.

[0066] When using an enzyme, the functional molecule is, for example, luciferase (eg, firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (eg, horseradish peroxidase (HRPO)), alkaline phosphatase, β- galactosidase, glucoamylase, lysozyme, saccharide oxidase (eg glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg uricase and xanthine oxidase), lactoperoxidase and microperoxidase.

[0067] Examples of linkers used to chemically link a toxin, small molecule compound, or enzyme include divalent radicals (eg, alkylene, arylene, heteroarylene), a repeating linker unit, or an alkoxy represented by -(CR ₂ ) _n O(CR ₂ ) _n - (where R is an optional substituent and n is a positive integer) (for example, polyethyleneoxy, PEG and polymethyleneoxy) and alkylamino (for example, polyethyleneamino, Jeffamine™) and diacid esters and amide (for example, succinate, succinamide, diglycolate, malonate and caproamide). Chemical modification methods for binding functional molecules have already been established in this area (DJ King., Applications and Engineering of Monoclonal antibodies., 1998, TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998, Marcel Dekker Inc; Chari et al. , Cancer Res., 1992 Vol 152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0068] A preferred embodiment of the RGMa binding protein of the present invention is a chimeric antibody. As a “chimeric antibody,” an exemplary chimeric antibody is one in which the variable region is a variable region derived from a non-human animal immunoglobulin (such as a mouse, rat, hamster, or chicken) and the constant region is a human immunoglobulin constant region. A chimeric antibody can be produced, for example, by immunizing a mouse with an antigen, excising the variable region that binds the antigen from a gene encoding a mouse monoclonal antibody, and combining the variable region with the constant region of an antibody derived from human bone marrow. The constant region derived from human immunoglobulin contains a unique amino acid sequence depending on the isotype, such as IgG (IgG1, IgG2, IgG3 and IgG4), IgM, IgA (IgA1 and IgA2), IgD and IgE, and the constant region of a recombinant chimeric antibody according to of the present invention may be a human immunoglobulin constant region belonging to any isotype. The constant region is preferably a human IgG constant region. An expression vector can be produced using the chimeric antibody gene thus produced. Host cells are transformed with an expression vector to produce transformant cells producing the chimeric antibody, and then the transformed cells are cultured to produce the desired chimeric antibody from the culture supernatant.

[0069] Another preferred embodiment of the RGMa binding protein of the present invention is a humanized antibody. A “humanized antibody” in the present invention is an antibody produced by transplanting only the DNA sequence of the antigen binding region (CDR; complementarity determining region) of an antibody from a non-human animal such as a mouse into a human antibody gene (CDR graft). Humanized antibodies can be produced based on methods described, for example, in Japanese PCT Patent Application Published Translation No. Hei 4-506458 and Japanese Patent No. 2912618. In particular, a humanized antibody is provided, characterized in that some or all of the CDRs are CDRs derived from monoclonal antibodies from non-human mammals (such as mouse, rat and hamster), that the variable region framework regions are variable region framework regions, derived from human immunoglobulin, and that the constant regions are constant regions derived from human immunoglobulin.

[0070] The humanized antibody of the present invention can be produced, for example, as indicated, but it goes without saying that the production method is not limited thereto.

[0071] For example, a recombinant humanized antibody derived from a mouse monoclonal antibody can be produced by genetic engineering based on published translated Japanese PCT patent applications No. Hei 4-506458 and Sho 62-296890. Thus, DNA encoding a mouse heavy chain CDR region and DNA encoding a mouse light chain CDR region are isolated from hybridomas produced by a mouse monoclonal antibody and a human heavy chain gene encoding a complete region other than the human heavy chain CDR, and a human light chain gene encoding a complete region other than the human light chain CDR is isolated from the human immunoglobulin gene.

[0072] The human heavy chain gene into which the isolated DNA encoding the mouse heavy chain CDR region is transplanted is introduced into a corresponding expression vector so that its expression is possible. Likewise, a human light chain gene into which the DNA encoding the mouse light chain CDR region has been transplanted is introduced into another suitable expression vector such that its expression is possible. Alternatively, the human heavy and light chain genes into which the mouse CDRs are transplanted can be introduced into the same expression vector such that their expression is possible. The host cells are transformed with the thus obtained expression vector to obtain transformant cells producing the humanized antibody, and then the transformed cells are cultured to obtain the desired humanized antibody from the culture supernatant.

[0073] Another preferred embodiment of the RGMa binding protein of the present invention is a human antibody. Human antibody refers to an antibody in which all regions, including heavy chain variable regions and heavy chain constant regions, and light chain variable regions and light chain constant regions, included in the immunoglobulin are derived from genes encoding human immunoglobulin. Human antibodies can be produced by introducing human antibody genes into mice. Human antibodies can be produced in a similar manner as in the above method for producing polyclonal antibodies or monoclonal antibodies, in particular, for example, by immunizing a transgenic animal obtained by integrating at least human immunoglobulin genes into the gene locus of a non-human mammal, such as a mouse.

[0074] For example, transgenic mice that produce human antibodies can be generated by methods described, for example, in Nature Genetics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol.15, p. 146-156, 1997; published translated Japanese PCT patent applications No. Hei 4-504365 and Hei 7-509137; WO 94/25585; Nature, Vol. 368, p. 856-859, 1994 and published translated Japanese PCT Patent Application No. Hei 6-500233. Specific examples of transgenic mice include HuMab™ mouse (Medarex, Princeton NJ), KMTM mouse (Kirin Pharma Company, Japan) and KM mouse (FCγRIIb-KO).

[0075] Specific examples of the monoclonal antibody of the present invention include monoclonal antibodies in which the CDRs in the heavy chain variable region contain the amino acid sequences of SEQ ID NO: 33 (HCDR 1), 34 (HCDR 2) and 35 (HCDR 3) in the sequence listing, and wherein the CDRs in the light chain variable region comprise the amino acid sequences of SEQ ID NO: 30 (LCDR 1), 31 (LCDR 2) and 32 (LCDR 3) in the sequence listing. One to more amino acids in one or more CDRs may be replaced so long as the antibody of the present invention retains the properties of binding to RGMa, not inhibiting the binding of RGMa and neogenin, and neutralizing the neurite outgrowth inhibitory activity of RGMa. One to several means, for example, one or two. The amino acid substitution is preferably a conservative substitution to maintain the properties of the present invention. Retaining the properties of an antibody means that those properties are retained to the same extent, for example, 80% or more, preferably 90% or more, more preferably 95% or more, compared to the properties before modifications to the CDR amino acid sequence. Conservation also includes improvement.

[0076] The region other than the CDR is not particularly limited as long as it is a sequence that can retain the structure of an antibody and perform its function, and can be any of the sequences obtained from mouse, human and other mammals, their chimeric sequences and artificial sequences. In the case of a constant region, the amino acid sequences of the constant regions in the heavy chain and light chain are illustrated by the amino acid sequences described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell vol.22 , p197, 1980.

[0077] Examples of mouse antibodies containing such CDRs include antibodies in which the light chain contains the amino acid sequence SEQ ID NO: 4 in the sequence listing, and in which the heavy chain contains the amino acid sequence SEQ ID NO: 5 in the sequence listing. These amino acid sequences may include substitution, deletion, addition, or insertion of one or more (1 to 20, 1 to 10, or 1 to 5) amino acids, provided that the ability of the antibody to bind to RGMa is not to inhibit binding. RGMa and neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa. Such a substitution, deletion or addition can be introduced into a CDR, but preferably it is introduced into a region other than the CDR. The amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0078] Also included are mouse/human chimeric antibodies in which the constant regions in the above mouse antibody are derived from a human. An example of such mouse/human chimeric antibodies is an antibody wherein the light chain contains the amino acid sequence SEQ ID NO: 8 in the sequence listing (variable region extends from 1 to 107), and wherein the heavy chain contains the amino acid sequence SEQ ID NO: 9 in sequence list (variable region extends from 1 to 116). These amino acid sequences may include substitution, deletion, addition or insertion of one or more (1 to 20, 1 to 10, or 1 to 5) amino acids, provided that the antibody retains the ability to bind to RGMa without inhibiting RGMa binding and neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa. Such a substitution, deletion or addition can be introduced into CDRs, but is preferably introduced into a region other than the CDR. The amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0079] Also exemplary are humanized antibodies in which the region other than the CDR is derived from a human. An example of such humanized antibodies is an antibody in which the heavy chain contains the amino acid sequence of any SEQ ID NO: 11-18 (the variable region is located up to the 116th residue from the N-terminus), and in which the light chain contains the amino acid sequence of any SEQ ID NO: : 19-25 in the sequence list (variable region extends from residues 1 to 107 from the N-terminus).

The amino acid sequences of the humanized antibody (heavy chain: SEQ ID NO: 11-18 in the sequence listing, light chain: SEQ ID NO: 19-25 in the sequence listing) may contain a substitution, deletion, addition or insertion of one or more (1 to 20, from 1 to 10 or from 1 to 5) amino acids, provided that the properties of the antibody are retained to bind to RGMa, not to inhibit the binding of RGMa and neogenin, and to neutralize the neurite outgrowth inhibitory activity of RGMa. Such a substitution, deletion or addition may conveniently be introduced into a CDR, but is preferably introduced into a region other than the CDR. The amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0080] The heavy chain amino acid sequence and the light chain amino acid sequence may be any combination thereof, but particularly preferred is an antibody comprising heavy chains containing the amino acid sequence SEQ ID NO: 15 in the sequence listing and light chains containing the amino acid sequence SEQ ID NO: 19 in the sequence list. In the amino acid sequence of SEQ ID NO: 15 in the sequence listing, the amino acid sequence corresponding to the heavy chain variable region is shown in SEQ ID NO: 41 in the sequence listing, and the amino acid sequence corresponding to the light chain variable region is shown in SEQ ID NO: 42 in the listing sequences. Thus, a particularly preferred antibody of the present invention is an antibody in which the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 41 in the sequence listing, and in which the light chain variable region contains the amino acid sequence of SEQ ID NO: 42 in the sequence listing.

These amino acid sequences may include substitution, deletion, addition or insertion of one or more (1 to 20, 1 to 10, or 1 to 5) amino acids, provided that the antibody retains the ability to bind to RGMa without inhibiting RGMa binding and neogenin and neutralize the neurite outgrowth inhibitory activity of RGMa. Such a substitution, deletion or addition can be introduced into the CDR, but is preferably introduced into a region other than the CDR. The amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

In the amino acid sequence of the antibody of the present invention containing a substitution, deletion or the like. in the amino acid sequence SEQ ID NO: 41 and/or 42 in the sequence listing as described above, the heavy chain variable region is an amino acid sequence having 90% or more (more preferably 95%, 96%, 97%, 98%, 99 % or more) identity to SEQ ID NO: 41 of the sequence listing, and the light chain variable region is an amino acid sequence having 90% or more (more preferably 95%, 96%, 97%, 98%, 99% or more) identity with SEQ ID NO: 42 sequence listings.

[0081] Other specific examples of the monoclonal antibody of the present invention include monoclonal antibodies in which the CDRs in the heavy chain variable region contain the amino acid sequences of SEQ ID NO: 39 (HCDR 1), 40 (HCDR 2) and SFG (HCDR 3) in the sequence listing and wherein the CDRs in the light chain variable region comprise the amino acid sequences of SEQ ID NOs: 36 (LCDR 1), 37 (LCDR 2) and 38 (LCDR 3) in the sequence listing. One to more amino acids in one or more CDRs can be replaced so long as the antibody of the present invention retains its ability to bind to RGMa, not inhibit the binding of RGMa and neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa.

One to several means, for example, one or two. The amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

The region other than the CDR is not particularly limited as long as it is a sequence that can retain the structure of an antibody and perform its function, and can be any of the sequences derived from mouse, human and other mammals, chimeric sequences thereof and artificial sequences. In the case of a constant region, exemplary amino acid sequences of the constant regions in the heavy chain and light chain are described in Nucleic Acids Research vol.14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell vol. 22, p197, 1980.

[0082] Examples of mouse antibodies containing such CDRs include antibodies in which the light chain contains the amino acid sequence SEQ ID NO: 6 in the sequence listing, and in which the heavy chain contains the amino acid sequence SEQ ID NO: 7 in the sequence listing. Such amino acid sequences may contain substitution, deletion, addition or insertion of one or more (1 to 20, 1 to 10 or 1 to 5) amino acids, provided that the antibody retains the ability to bind to RGMa and does not inhibit the binding of RGMa and neogenin, and neutralize the neurite outgrowth inhibitory activity of RGMa. Such a substitution, deletion or addition can be introduced into the CDR, but is preferably introduced into a region other than the CDR. The amino acid substitution is preferably a conservative substitution in order to maintain the properties of the present invention.

[0083] As stated above, also included are mouse antibodies, chimeric antibodies whose constant regions are derived from humans. Additionally included are humanized antibodies in which the region other than the CDR is derived from a human.

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

[0085] The anti-RGMa antibody of the present invention can itself be coupled to an antibody having a different antigen binding specificity than that of the anti-RGMa antibody by genetic engineering methods to produce multispecific antibodies, such as bispecific antibodies. Genetic engineering methods have already been established in this area. For example, using the DVD-Ig method in which the variable regions are connected in series (Wu et al., Nature Biotechnology 25(11), 1290 (2007)) or the ART-Ig method in which the heavy chains of two types of antibodies bind different antigens are combined by modifying the Fc region of the antibody (Kitazawa et al., Nature Medicine, 18(10), 1570(2012), the desired bispecific antibodies can be obtained. Other antigens other than RGMa include, but are not limited to, factors , inhibiting neurite outgrowth such as Nogo, MAG, Omgp, CSPG, Sema 3A and Lingo-1, and immune-dependent molecules such as TNF-α, IL-6 receptor, CD3, CD20, α4 integrin, BLys, thymic stromal lymphopoietin, IgE, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-23 and IL-25.

[0086] Examples of functionally modified antibodies are the modified anti-RGMa antibody molecules of the present invention. A functionally modified antibody means an antibody in which functions such as cell cytolysis-inducing function, complement activating function and blood half-life increasing function are modified mainly by modifying the Fc region or the like. (Shitara, Journal of the Pharmaceutical Society of Japan, 2009, Vol. 129(1), p3; Ishii et al., Nippon Yakubutsugaku Zasshi (Folia Pharmacologica Japonica), 2010, Vol. 136(5), p280; Hashiguchi et al ., The Journal of Japanese Biochemical Society, 2010, Vol. 82(8), p710).

[0087] Functionally modified antibodies from anti-RGMa antibodies are produced by the following method. For example, when the anti-RGMa antibody of the present invention is produced using CHO cells in which the α1,6-fucosyltransferase (FUT 8) gene has been disrupted as host cells, antibodies with reduced content of fucose sugar chains and increased cell cytolysis-inducing function are obtained, and when the anti-RGMa antibody of the present invention is produced using CHO cells into which the FUT8 gene has been introduced as a host cell, antibodies with low cell cytolysis-inducing function are obtained (WO 2005/035586, WO 2002/31140 and WO 00/61739). The function of complement activation can be regulated by modification of amino acid residues in the Fc region (US patent No. 6737056, 7297775 and 7317091). Increased blood half-life can be obtained by using Fc region variants with increased binding to FcRn, which is one of the Fc receptors (Hashiguchi et al., The Journal of Japanese Biochemical Society, 2010, Vol. 82 (8), p710 ). These functionally modified antibodies can be produced by genetic engineering methods.

[0088] Exemplary conjugated antibodies are modified anti-RGMa antibody molecules of the present invention. Examples of the conjugated antibody include conjugated antibodies in which the anti-RGMa antibodies are chemically or genetically linked to functional molecules other than the anti-RGMa antibody of the present invention, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, small molecules, cytokines, growth factors (eg, TGF-β, NGF, neurotrophin), albumin, enzymes and other antibodies.

[0089] When PEG is linked as a functional molecule, a PEG with a molecular weight without limitation of 2000 to 100,000 Da, more preferably 10,000 to 50,000 Da, which may be linear or branched, can be used. Using, for example, an NHS active group, PEG can be linked, for example, to the N-terminal amino acid group of antibodies.

[0090] In the case of using a radioactive substance as the functional molecule, for example, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ⁶⁴ Cu, ⁹⁹ Tc, ⁷⁷ Lu or ²¹¹ At are used. Radioactive substances can be directly coupled to antibodies, for example using the chloramine T method.

[0091] When using a toxin as the functional molecule, for example, bacterial toxins (eg, diphtheria toxin), phytotoxins (eg, ricin), low molecular weight toxins (eg, geldanamycin), maytansinoids, and calicheamycin are used.

[0092] When using a low molecular weight compound, daunomycin, doxorubicin, methotrexate, mitomycin, neocarzinostatin, vindesine and fluorescent dyes such as FITC are used as the functional molecule, for example.

[0093] When using an enzyme, the functional molecule is, for example, luciferase (eg, firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (eg, horseradish peroxidase (HRPO)), alkaline phosphatase, β- galactosidase, glucoamylase, lysozyme, saccharide oxidase (eg glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg uricase and xanthine oxidase), lactor peroxidase and microperoxidase.

[0094] Examples of linkers used to chemically link a toxin, small molecule compound, or enzyme include divalent radicals (e.g., alkylene, arylene, heteroarylene), a repeating linker unit, or alkoxy, provided by -(CR ₂ ) _n O(CR ₂ )n - (where R is an optional substituent and n is a positive integer) (for example, polyethyleneoxy, PEG and polymethyleneoxy) and alkylamino (for example, polyethyleneamino, Jeffamine™) and diacid ester and amide (for example, succinate, succinamide, diglycolate, malonate and caproamide). Methods for chemically modifying the binding of functional molecules have already been established in this area (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res ., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0095] "Antigen binding fragments" of antibodies in the present invention means a partial region having the antigen binding property of the antibodies described above, including, in particular, F(ab')2, Fab', Fab, Fv (antibody variable fragment) linked by Fv disulfide bonds , single chain antibody (scFv) and polymers thereof. Antigen binding fragments further include conjugated antigen binding fragments chemically or genetically linked to functional molecules other than the anti-RGMa antibody of the present invention, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, small molecules, cytokines, growth factors (eg , TGF-β, NGF, neurotrophin), albumin, enzymes and other antibodies.

[0096] As used herein, "F(ab') ₂ " and "Fab" mean antibody fragments that are produced by treating immunoglobulins with the protease pepsin or papain and are produced by cleavage upstream and downstream of the disulfide bond located between the two heavy chains in the hinge region . For example, papain treatment of IgG can cause cleavage upstream of the disulfide bond located between the two heavy chains in the hinge region, producing two homologous antibody fragments, each containing a light chain containing a VL (light chain variable region) and a CL (light chain constant region). ), and a heavy chain fragment containing VH (heavy chain variable region) and CHγ1 (γ1 region in the heavy chain constant region), in which the light chain and heavy chain fragments are linked to each other through a disulfide bond in the C-terminal domain. Each of the two homologous antibody fragments is designated Fab. Pepsin treatment of IgG can cause cleavage downstream of the disulfide bond located between the two heavy chains at the hinge region, yielding an antibody fragment that is not significantly lighter than a fragment in which two Fabs are linked to each other at the hinge region. This antibody fragment is designated F(ab') ₂ .

[0097] Exemplary conjugated antigen binding fragments are modified anti-RGMa antibody antigen binding fragment molecules of the present invention. Examples of a conjugated antigen binding fragment include fragments in which the partial region having the antigen binding property of an anti-RGMa antibody is chemically or genetically linked to a functional molecule other than the anti-RGMa antibody of the present invention, such as non-peptide polymers such as polyethylene glycol (PEG), radioactive substances, toxins, small molecules, cytokines, growth factors (eg, TGF-β, NGF, neurotrophin), albumin, enzymes and other antibodies.

[0098] When PEG is linked as a functional molecule, a PEG with a molecular weight without limitation of 2000 to 100,000 Da, more preferably 10,000 to 50,000 Da, which may be linear or branched, can be used. PEG can be linked, for example, to the N-terminal amino group of the partial region of the antigen-binding properties of the anti-RGMa antibody, using, for example, an NHS active group.

[0099] In the case of using a radioactive substance as the functional molecule, for example, ¹³¹ I, ¹²⁵ I, ⁹⁰ Y, ⁶⁴ Cu, ⁹⁹ Tc, ⁷⁷ Lu or ²¹¹ At are used. Radioactive substances can be directly coupled to the partial region having the antigen-binding property of the RGMa antibody, for example, using the chloramine T method.

[0100] When using a toxin as the functional molecule, for example, bacterial toxins (eg, diphtheria toxin), phytotoxins (eg, ricin), low molecular weight toxins (eg, geldanamycin), maytansinoids, and calicheamycin are used.

[0101] When using a small molecule compound, daunomycin, doxorubicin, methotrexate, mitomycin, neocarzinostatin, vindesine, and fluorescent dyes such as FITC are used as the functional molecule, for example.

[0102] When using enzymes, the functional molecule is, for example, luciferase (eg, firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), malate dehydrogenase, urease, peroxidase (eg, horseradish peroxidase (HRPO)), alkaline phosphatase, β- galactosidase, glucoamylase, lysozyme, saccharide oxidase (eg glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (eg uricase and xanthine oxidase), lactor peroxidase and microperoxidase.

[0103] Examples of linkers used to chemically link a toxin, small molecule compound, or enzyme include divalent radicals (eg, alkylene, arylene, heteroarylene), a repeating linker unit, or an alkoxy represented by -(CR ₂ ) _n O(CR ₂ ) _n - (where R is an optional substituent and n is a positive integer) (for example, polyethyleneoxy, PEG and polymethyleneoxy) and alkylamino (for example, polyethyleneamino, Jeffamine™) and diacid ester and amide (for example, succinate, succinamide, diglycolate, malonate and caproamide). Methods for chemically modifying the binding of functional molecules have already been established in this area (DJKing., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res. , 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Volatl Acad Sci USA, 1996 Vol 93:8681).

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

[0105] The present invention also provides an anti-RGMa antibody that competes with an antibody containing the amino acid sequences of specific CDRs, as described above, for binding to RGMa proteins and antigen-binding fragments thereof. An antibody that competes with an antibody containing the amino acid sequences of specific CDRs as described above for binding to RGMa proteins contains epitopes in blasts selected from Glu 298-Gly 311, Asn 322-Glu 335, Lys 367-Ala 377 and Pro 349 -Thr 359.

The antibody can be produced (screened) or evaluated by allowing the antibody to be present in a binding system of an antibody containing CDR sequences as described above and RGMa proteins. For example, an antibody can be produced by screening using the following surface plasmon resonance (SPR) method.

[0106] Biotinylated human RGMa protein (4 μg/ml) is liganded onto an avidin-immobilized sensor chip to immobilize human RGMa proteins equivalent to 1300 to 1600 RU. An optional anti-RGMa antibody (15 μg/ml) is then applied as an analyte and binds to the human RGMa protein immobilized on the sensor chip. By repeating this procedure many times, a state is created in which the optional anti-RGMa antibody is bound to all human RGMa protein molecules on the sensor chip (saturated state) and the binding amount in the saturated state is determined (saturated binding value 1).

A similar experiment is performed with an anti-RGMa antibody containing the amino acid sequences of the specific CDRs of the present invention to determine the saturated binding value (saturated binding value 2).

The human RGMa proteins on the sensor chip are then saturated with an anti-RGMa antibody containing the amino acid sequences of the specific CDRs of the present invention, and then an optional anti-RGMa antibody (15 μg/ml) is applied as an analyte to examine whether the optional anti-RGMa antibody additionally binds to the RGMa protein human, saturated with an antibody to RGMa containing the amino acid sequences of specific CDRs according to the invention.

If the optional anti-RGMa antibody can further bind to a human RGMa protein saturated with an anti-RGMa antibody containing the amino acid sequences of the specific CDRs of the invention, exhibiting a saturated binding value of 1 of the optional anti-RGMa antibody calculated above, then the antibody is judged to be “non-competitive.” On the other hand, if the optional anti-RGMa antibody cannot further bind to a human RGMa protein saturated with an anti-RGMa antibody containing the amino acid sequences of the specific CDRs of the invention, then the antibody is considered to be “competing.” Even if an optional anti-RGMa antibody can further bind to a human RGMa protein saturated with an anti-RGMa antibody containing the amino acid sequences of specific CDRs of the invention, the additional amount of binding does not reach the amount of saturated binding 1 with a significant difference, then the antibody is considered "competing". Significant differences are illustrated by conventional statistical techniques (eg, Student's t test), and the significance level is set at 5% or less.

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

[0108] Nucleic acid molecules of the present invention

Examples of the nucleic acid molecules of the present invention include polynucleotides in which the heavy chain variable region encoding region contains a core sequence encoding the amino acid sequence of SEQ ID NO: 33, 34 and 35 in the sequence listing, respectively (in which one or more amino acids may be substituted , delete, introduce or add), and the light chain variable region encoding region contains a basic sequence encoding the amino acid sequence of SEQ ID NO: 30, 31 and 32 in the sequence listing, respectively (in which one or more amino acids can be replaced, deleted, enter or add); and polynucleotides wherein the heavy chain variable region encoding region contains a core sequence encoding the amino acid sequence of SEQ ID NO: 39, 40, and SFG in the sequence listing, respectively (in which one or more amino acids may be replaced, deleted, introduced, or added ), and the light chain variable region encoding region contains a basic sequence encoding the amino acid sequence of SEQ ID NO: 36, 37 and 38 in the sequence listing, respectively (in which one or more amino acids may be substituted, deleted, introduced or added).

[0109] Other examples of the nucleic acid molecule of the present invention include polynucleotides in which the heavy chain encoding region contains a core sequence encoding the amino acid sequence of SEQ ID NO: 5 in the sequence listing, and the light chain encoding region contains a core sequence encoding amino acid sequence SEQ ID NO: 4 in sequence listing; and polynucleotides, wherein the heavy chain coding region contains a basic sequence encoding the amino acid sequence of SEQ ID NO: 7 in the sequence listing, and the light chain coding region contains a basic sequence encoding the amino acid sequence of SEQ ID NO: 6 in the sequence listing.

[0110] Other examples of the nucleic acid molecule of the present invention include polynucleotides in which the heavy chain encoding region contains a core sequence encoding the amino acid sequence of SEQ ID NO: 9 in the sequence listing, and the light chain encoding region contains a core sequence encoding amino acid sequence SEQ ID NO: 8 in the sequence listing.

[0111] Other examples of the nucleic acid molecule of the present invention include polynucleotides in which the heavy chain coding region contains a core sequence encoding the amino acid sequence of any SEQ ID NO: 11 to 18 in the sequence listing, and the light chain coding region contains a core sequence a sequence encoding the amino acid sequence of any SEQ ID NO: 19 to 25 in the sequence listing.

[0112] Particularly preferred examples of the nucleic acid molecule of the present invention include polynucleotides in which the heavy chain encoding region contains a core sequence encoding the amino acid sequence of SEQ ID NO: 15 in the sequence listing, and the light chain encoding region contains a core sequence coding amino acid sequence SEQ ID NO: 19 in the sequence listing.

[0113] Other examples of the nucleic acid molecule of the present invention include polynucleotides in which the heavy chain variable region encoding region contains a basic sequence encoding the amino acid sequence of SEQ ID NO: 41 in the sequence listing, and the light chain variable region encoding region contains a basic sequence. sequence encoding the amino acid sequence SEQ ID NO: 42 in the sequence listing.

[0114] Specific examples of the nucleic acid molecule of the present invention include polynucleotides in which the heavy chain variable region encoding region contains the basic sequence of SEQ ID NO: 43 in the sequence listing and the light chain variable region encoding region contains the basic sequence of SEQ ID NO: 43 : 44 in sequence list.

[0115] The nucleic acid molecule of the present invention may be polynucleotides that hybridize under stringent conditions to a complementary DNA strand containing the basic sequence of SEQ ID NO: 43 in the sequence listing, and polynucleotides that hybridize under stringent conditions to a complementary DNA strand containing the basic sequence SEQ ID NO: 44 in the sequence listing, provided that the nucleic acid molecule of the present invention encodes a monoclonal antibody that has the ability to bind to RGMa, does not inhibit the binding of RGMa and neogenin, and neutralizes the neurite outgrowth inhibitory activity of RGMa. Examples of stringent conditions include a Southern hybridization condition and a salt concentration wash corresponding to 0.1× SSC, 0.1% SDS at 68°C.

[0116] The nucleic acid molecule of the present invention can encode all the constant regions and variable regions of the heavy and light chains, and can only encode the variable regions of the heavy and light chains. The basic sequence of the constant region of the heavy and light chains, in the case of encoding all of the constant region and the variable region, is preferably the basic sequences described in Nucleic Acids Research vol. 14, p1779, 1986, The Journal of Biological Chemistry vol.257, p1516, 1982 and Cell, vol.22, p197, 1980.

[0117] The nucleic acid molecule of the present invention can be obtained, for example, by the following method. First, total RNA is obtained from cells such as hybridomas using a commercial RNA extraction kit, and cDNA is synthesized by reverse transcriptase using random primers or the like. The cDNA encoding the antibody is then amplified by PCR using oligonucleotides as primers containing sequences conserved in the variable regions of the known human antibody heavy and light chain genes, respectively. Regarding the constant region coding sequence, it can be obtained by amplifying a known sequence by PCR method. The basic DNA sequence can be determined in a conventional manner, for example by insertion into a sequencing plasmid.

Alternatively, DNA encoding a monoclonal antibody of the present invention can also be obtained by chemically synthesizing a variable region sequence or a portion thereof and linking it to a sequence containing a constant region.

[0118] The present invention also relates to a recombinant vector containing a nucleic acid molecule of the present invention, and a transformant (host cell) containing the recombinant vector. The recombinant vector may be vectors that can be expressed in prokaryotic cells, such as E. coli ( Escherichia coli ) (for example, pBR322, pUC119, or a derivative thereof), and is preferably vectors that can be expressed in eukaryotic cells, and more preferably are vectors that can be expressed in mammalian-derived cells. Examples of vectors that can be expressed in mammalian-derived cells include plasmid vectors such as pcDNA 3.1 (Invitrogen), pConPlus, pcDM8, pcDNA I/Amp, pcDNA 3.1, pREP4, and viral vectors such as pDON-AI DNA (Takara Bio). The vector may be one vector containing a heavy chain coding sequence and a light chain coding sequence, or may be two vectors: a vector containing a heavy chain coding sequence and a vector containing a light chain coding sequence.

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

[0120] The resulting anti-RGMa antibody or antigen-binding fragment thereof can be purified to a homogeneous state. To separate and purify antibodies, separation and purification methods used for conventional proteins and the like can be used. Separation and purification of antibodies can be carried out appropriately by screening and combining, for example, but not limited to, column chromatography such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis or isoelectric focusing (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). Examples of columns used for affinity chromatography include a protein A column, a protein G column, an anti-immunoglobulin antibody-conjugated column, and an antigen-conjugated column. Examples of Protein A column include Hyper D, POROS and Sepharose F.F. (Amersham Biosciences).

[0121] Agents for the prevention or treatment of immunological and neurological diseases

The RGMa binding protein, in particular the anti-RGMa antibody or antigen-binding fragment thereof of the present invention neutralizes the neurite outgrowth inhibitory activity of RGMa, promoting the restoration of neuronal function, and thus can be used as an agent for the prevention, treatment or prevention of relapse of neurological diseases.

An RGMa binding protein, in particular an anti-RGMa antibody or an antigen-binding fragment thereof of the present invention, also neutralizes T cell activation caused by RGMa and thus can be used as an agent for the prevention, treatment or prevention of relapse of immunological diseases.

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

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

[0124] An RGMa binding protein, in particular an anti-RGMa antibody or an antigen binding fragment thereof of the present invention, can be used as an agent for the prevention, treatment or prevention of relapse of neurological/immunological diseases, which preferably include spinal cord injury, nervous system trauma (including trauma optic nerve) and multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis).

[0125] As used herein, the term “treatment” includes any treatment of diseases in a mammal, particularly a human, and includes inhibition of symptoms of a disease, thereby inhibiting the progression thereof or eliminating the disease or symptoms, and alleviation of symptoms of the disease, thereby causing regression of diseases or symptoms or slowing of progression of symptoms.

[0126] "Prevention" includes preventing the onset of the diseases described above in a mammal, particularly a human.

[0127] “Relapse prevention” includes prevention of relapse of remission and recurrence of the diseases described above in a mammal, particularly a human.

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

[0129] The administration form of the RGMa binding protein (RGMa antibody or antigen binding fragment thereof) of the present invention is not particularly limited and can be administered to mammals, including humans, by any route: oral or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, rectal, transdermal, intracerebral, intraspinal or other local administration).

[0130] Dosage forms for oral and parenteral administration and methods for their preparation are well known to those skilled in the art, and a pharmaceutical composition can be prepared by combining the antibody of the invention with a pharmaceutically acceptable carrier or the like.

Parenteral dosage forms include injectables (eg, drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracerebral injections and intraspinal preparations), topical preparations (eg, ointments, compresses and lotions) , suppository, inhalation dosage forms, eye drops, ophthalmic ointments, nasal drops, ear drops and liposomes. Particularly, when the antibody of the present invention is to directly act on tissue of the central nervous system, it can be continuously infused using a medical micropump, which is an osmotic pump, or mixed with fibrin glue or the like. to produce a sustained release drug and then applied to the affected tissue.

[0131] For example, injectables are typically prepared by dissolving the antibody in distilled water for injection, and a solubilizer, buffer, pH adjuster, isotonicity agent, emollient, preservative, and stabilizer may be added as needed. Injectable preparations may also be lyophilized preparations prepared prior to use.

[0132] Oral dosage forms include solid or liquid dosage forms, such as tablets, coated tablets, pills, fine granules, granules, powders, capsules, syrups, emulsions, suspensions, injections, and lozenges.

[0133] The pharmaceutical composition of the present invention may further contain other therapeutically effective drugs, and components such as bactericidal agents, anti-inflammatory agents, vitamins and amino acids may be mixed as necessary.

[0134] Pharmacologically acceptable carriers include, for example, excipients, lubricants, binders and disintegrants for solid preparations and solvents, solubilizers, suspending agents, isotonicity agents, buffers and emollients for liquid preparations. If necessary, additives such as conventional antiseptics, antioxidants, colorants, sweeteners, adsorbents and wetting agents may be appropriately used in appropriate quantities.

[0135] The dose of the antibody of the present invention can be determined based on various factors such as route of administration, type of disease, severity of symptoms, age, gender, body weight, disease severity of the patients, based on pharmacological results such as pharmacokinetic and toxicological characteristics, whether or not a drug delivery system is used, and whether or not it is administered as part of a combination with other drugs. Typically, 1 to 5000 mcg/day, preferably 10 to 2000 mcg/day, more preferably 50 to 2000 mcg/day for oral administration, or 1 to 5000 mcg/day, preferably 5 to 2000 mcg/day, may be administered. day, more preferably 50 to 2000 mcg/day for injection in an adult (60 kg body weight) in one or more doses. For parenteral administration, 10 to 100,000 μg/kg, more preferably 100 to 50,000 μg/kg, and even more preferably 500 to 20,000 μg/kg body weight may be administered for the whole body at intervals of once daily, once weekly, once a month or from 1 to 7 times a year. For local administration using an osmotic pump or the like. In general, continuous infusion at a rate of 10 to 100,000 mcg/day, more preferably 100 to 10,000 mcg/day, even more preferably 500 to 5000 mcg/day for an adult (60 kg body weight) is possible.

EXAMPLES

[0136] The present invention is described in more detail below with reference to examples, but the present invention is not limited to the aspects of the following examples.

[0137] Example 1: Preparation of Human RGMa Protein (C-Terminal Domain)

A CHO cell was generated expressing a recombinant human RGMa protein in which a histidine tag was fused to the C-terminus of Pro 169-Gly 422 (which refers to the proline residue at position 169 - the glycine residue at position 422 on the N-terminal side, hereinafter described similarly) human RGMa protein (SEQ ID NO: 1 in sequence listing).

The C-terminal domain of human RGMa protein contained in the culture supernatant of CHO cells was adsorbed onto a nickel chelate chromatography column (GE Healthcare, 17-5247-01) and eluted with 100 mM imidazole solution. The imidazole elution fraction was replaced by dialysis with phosphate-buffered saline (PBS) and used as an immunogen.

[0138] Example 2: Production of Mouse Anti-Human RGMa Monoclonal Antibody

Ten micrograms of the recombinant human RGMa protein prepared in Example 1 was mixed with Freund's complete adjuvant (Sigma) to form an emulsion and immunized a BALB/c mouse (Charles River Japan) at several subcutaneous sites on the back. Immunizations were then carried out similarly at intervals of 1 to 2 weeks with 10 μg of recombinant human RGMa protein emulsified with Freund's incomplete adjuvant (Sigma), and blood was collected after several immunizations. Antibody titer was measured by ELISA, described below, in which human or mouse RGMa proteins were immobilized. Individuals who demonstrated elevated antibody titers were given 10 μg of human RGMa protein intravenously for booster immunization, and splenocytes were isolated 2 or 3 days later.

For cell fusion, spleen cells and mouse myeloma cells (SP2/0, Dainippon Sumitomo Pharma), where the number of spleen cells was half as many, were mixed and centrifuged, and polyethylene glycol (Roche Diagnostics) was added to the resulting precipitated fraction to obtain cell fusions. Cells were then centrifuged and washed twice with D-MEM (Invitrogen). Cells were resuspended in GIT medium (Nippon Pharmaceutical) containing 10% fetal bovine serum (Invitrogen), 1% BM-Condimed (Roche Diagnostics), and HAT (Sigma-Aldrich) and seeded into each well of a 96-well plate at 5 × 10 ⁴ myeloma cells/well. The culture supernatant was isolated and screened for antibody-producing cells by human RGMa protein-immobilized ELISA according to Example 3.

The antibody-producing cells obtained from the screen were cloned by limiting dilution, and hybridoma cells producing two types of monoclonal antibodies (B5.116A3 and B5.70E4) were selected.

The isotypes of both monoclonal antibodies determined using an isotyping kit (Mouse MonoAB ID/SP KIT, ZYMED 93-6550) were mouse IgG2b for the heavy chain and kappa for the light chain.

Hybridoma culture supernatants were subjected to affinity chromatography using agarose immobilized with anti-mouse IgG antibodies (Anti-Mouse IgG-Agarose, manufactured by Sigma) to purify the monoclonal antibodies. After the antibody was bound to the column, the column was washed with PBS. The antibody was then eluted with 10 mM glycine hydrochloride (pH 2.7) and the eluate was immediately neutralized. The neutralized eluate was then replaced with PBS through an ultrafilter.

[0139] Example 3: ELISA in which human or mouse RGMa proteins are immobilized

Human RGMa protein (R&D systems, 2459-RM) or mouse RGMa protein (R&D systems, 2458-RG), obtained at a concentration of 2 μg/ml with PBS, was dosed into a 96-well plate at 50 μl/well, and the plate was allowed to settle. at room temperature for 1 hour. After removing the solution, ApplieBlock (Seikagaku Bio-Business, 200150), diluted 5 times with PBS, was dispensed at 200 μl/well and the plate was allowed to sit at room temperature for 1 hour to block nonspecific binding. After three washes with PBST (PBS containing 0.05% Tween 20), serially diluted PBS samples (e.g., mouse serum, hybridoma culture supernatant, supernatant from cultures expressing the recombinant antibody described below, or purified antibody) were added to each well in 50 μl. /well and left the plate to settle at room temperature for 1 hour. The plate was then washed three times with PBST, and then diluted PBS, peroxidase-labeled sheep anti-mouse IgG antibody (GE Healthcare, NA9310V) was dosed into each well at 50 μl/well and the plate was allowed to sit at room temperature for 1 hour. After three washes, Peroxidase-coloring kit (Sumitomo Bakelite, ML-1130O) was added and the color was allowed to develop for a period of time, and the absorbance was measured at 492 nm using a plate spectrophotometer.

[0140] Example 4: Antibody Epitope Analysis

The epitopes to which the antibodies bind were determined by peptide scanning. A total of 83 peptide species were synthesized by fusion of amino acid sequences from the N-terminus, consisting of 11 consecutive residues, shifted by 3 residues, contained in Arg 172-Ala 424 of the human RGMa protein (SEQ ID NO: 1 in the sequence list), with a spacer sequence (SGSG) with a biotinylated N-terminus (SEQ ID NO: 46 in the sequence listing). After immobilization of the peptides on avidin-coated plates, the test antibodies (B5.116A3 and B5.70E4) were allowed to react. Next, peroxidase-labeled rabbit anti-mouse Ig (Dako, P026002) was allowed to react. After the substrate solution was added and the color developed for a certain time, the absorbance was measured using a plate spectrophotometer.

As a result, B5.116A3 bound to human RGMa-derived peptides Glu 298-Gly 311 (two types of peptides: Glu 298-Asp 308 and Val 301-Gly 311), Asn 322-Glu 335 (two types of peptides: Asn 322- Thr 332 and Ile 325-Glu 335) and Lys 367-Ala 377; and B5.70E4 bound to human RGMa-derived peptides, Glu 298-Gly 311 (two types of peptides: Glu 298-Asp 308 and Val 301-Gly 311), Asn 322-Glu 335 (two types of peptides: Asn 322-Thr 332 and Ile 325-Glu 335) and Pro 349-Thr 359.

[0141] Example 5: Mouse Antibody Gene Sequence Analysis and Cloning

Total RNA was extracted from hybridoma cells producing a mouse monoclonal antibody (B5.116A3 or B5.70E4). Using total RNA as a template, cDNA was synthesized by reverse transcription reaction. Using cDNA as a template, the light chain variable and constant region and heavy chain variable and constant region genes were amplified by PCR and the DNA sequences were determined. Then, based on the determined sequences of the variable and constant regions, PCR amplification was performed and the full-length antibody genes were cloned. The amino acid sequences encoded by such antibody genes are as follows.

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

DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQLNTLPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEAT HKTSTSPIVKSFNRNEC

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

EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRSVYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASS TTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSD GSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

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

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSTHVPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEY ERHNSYTCEATHKTSSTSPIVKSFNRNEC

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

DVKLQESGPGLVKPSQSLSLTCSVTGYSITTSYYWNWIRQFPGNKLEWMGYISYDGTNNYNPSLKNRISITRDTSKNQFFLRLNSVTTEDTATYYCAGSFGYSQGTLVTVSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASST TVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDG SYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK

[0146] Example 6: Preparation of Mouse Recombinant Antibody and Rat-Mouse Chimeric Recombinant Antibody

Recombinant mouse antibodies of two types of RGMa antibodies B5.116A3 and B5.70E4 obtained from hybrids (hereinafter referred to as “r116A3” and “r70E4”, respectively) were obtained.

In addition, as a comparative example, a recombinant rat-mouse chimeric antibody (hereinafter referred to as "r5F9") was prepared by fusing the variable regions of the rat antibody 5F9 with the constant regions of the mouse antibody (IgG2bκ) (the light chain constant region is Arg 108-Cys 214 SEQ ID NO: 4 in the sequence listing, and the heavy chain constant region is Ala 117-Lys 452 SEQ ID NO: 5 in the sequence listing) according to Patent Document 1 (WO 2009/106356).

DNA encoding the light and heavy chains of each antibody was introduced into pcDNA 3.3 (Life Technologies) to generate an expression vector. Expression vectors were introduced into HEK293F cells (Life Technologies) using Neofection 293 (ASTEC). The cells were cultured at 37°C in an 8% carbon dioxide gas atmosphere for 6 days, and then the culture supernatant was isolated. To purify the recombinant antibody, the culture supernatant was applied to a Protein A or Protein G affinity column (GE Healthcare) and the antibodies bound to the column were eluted with 10 mM glycine hydrochloride (pH2.8). The eluate was immediately neutralized and then replaced with PBS.

Where increased purity requirements are necessary depending on the intended use, antibodies purified using a protein A column were purified using a type 1 ceramic hydroxyapatite (CHT) column (BIORAD). Antibodies bound to the CHT column were washed with 10 mM KH ₂ PO ₄ (pH6.5) and then eluted with 20 mM KH ₂ PO ₄ (pH6.5), 0.5 M NaCl. The eluted fractions were collected and then replaced with PBS.

[0147] Example 7: Binding Assay on Cells Expressing RGMa Protein

Vector expressing full-length human RGMa protein (Met 1-Cys 450 SEQ ID NO: 1 in sequence listing), C-terminal domain of human RGMa protein (Pro 169-Cys 450 SEQ ID NO: 1 in sequence listing), full-length mouse RGMa protein (Met 1-Trp 454 SEQ ID NO: 2 in the sequence listing) or the C-terminal domain of the rat RGMa protein (Pro 170-Trp 449 SEQ ID NO: 3 in the sequence listing) were introduced into CHO or HEK 293 cells to produce antigen-expressing cells.

In RGMa proteins, the C-terminal peptide is processed during the GPI anchor addition reaction. Mouse and rat RGMa proteins are cleaved at Ala 427 and the C-terminal peptide is removed. Thus, the amino acid sequences of the full-length protein and the C-terminal domain expressed by the cell through the GPI anchor are the same in mouse and rat.

Test antibodies (r116A3 and r70E4) and r5F9 (comparative example) at a final concentration of 10 μg/ml were reacted with the antigen-expressing cells described above, and then the cells were washed with PBS containing 0.1% bovine serum albumin and 0.05% NaN ₃ . FITC-labeled anti-mouse immunoglobulin antibody (DAKO) was allowed to react and the cells were washed. Fluorescence was measured by flow cytometry (FACSCalibur, manufactured by Beckton Dickinson), and the binding properties of the test antibodies were assessed using antigen-expressing cells (Table 1).

As a result, it was revealed that r116A3 and r70E4 bind to the C-terminal domain of human and rat RGMa proteins, in contrast to r5F9. The RGMa protein has neurite outgrowth inhibitory effects even in the C-terminal domain alone, and r116A3 and r70E4 inhibit the full-length RGMa protein and the C-terminal domain.

[0148] Table 1: Evaluation of binding of various recombinant anti-RGMa antibodies to antigen-expressing cells*

Full size human RGMa C-terminal domain of human RGMa Full size RGMa mouse C-terminal domain of rat RGMa r116A3 ++ ++ ++ ++ r70E4 ++ ++ + ++ r5F9 ++ - ++ -

*; ++ strong binding

+ weak binding

- no binding

[0149] Example 8: Determination of the dissociation constant for the RGMa protein

The affinity of the test antibodies (r116A3 and r70E4) and r5F9 (comparative example) for the RGMa protein was measured by surface plasmon resonance (SPR) using Proteon XPR36 (Bio-Rad).

Human RGMa protein (R&D Systems, 2459-RM), C-terminal domain of human RGMa protein (prepared in Example 1) or mouse RGMa protein (R&D Systems, 2458-RG), diluted to 10 μg/ml in 10 mM acetate buffer (pH4 ,5) immobilized on the GLC sensor chip by amine coupling method. Serially diluted test antibodies were added as analytes at a flow rate of 100 μL/min for 60 seconds to determine the dissociation constant (Kd value).

As demonstrated in Table 2, r116A3 and r70E4 also bound to the C-terminal domain of the human RGMa protein, but r5F9 did not. R116A3 bound to human RGMa protein 32 times more strongly than r5F9, and to mouse RGMa protein 44 times more strongly than r5F9.

[0150] Table 2: Monoclonal Antibody Affinity for RGMa

Antibody Dissociation constant (Kd value, nM) C-terminal domain of human RGMa RGMa mouse

r116A3 0.0487 0.0568 0.201 r70E4 2.43 1.12 174 r5F9 (Comparative example) 1.59 no binding 8.98

[0151] Example 9: RGMa-Neogenin Binding Inhibition Assay

The extracellular domain (Ala 34-Leu 1105) of recombinant human neogenin protein (SEQ ID NO: 10 in the sequence listing) was isolated. A CHO cell line expressing the extracellular domain of the human neogenin protein was established. The histidine tag was fused to the C-terminus. From the culture supernatant of CHO cells, the extracellular domain was adsorbed onto a nickel chelate chromatography column (GE Healthcare, 17-5247-01) and then eluted with 100 mM imidazole solution. The imidazole elution fractions were replaced by dialysis with PBS.

Human RGMa protein (R&D systems, 2459-RM) was labeled with biotin using the ChromaLink Biotin Labeling kit (Solulink). Equivalent amounts of biotin-labeled human RGMa protein, adjusted to 2 μg/ml, and test antibodies (r116A3 and r70E4), subjected to 2-fold serial dilution, were mixed and allowed to react at room temperature for 2 hours to obtain a mixed solution.

At the same time, 50 μl/well of the extracellular domain of human neogenin protein, adjusted to 2 μg/ml with PBS, was added to a 96-well plate, and the plate was allowed to settle at room temperature for 1 hour to obtain an immobilized neogenin plate. After removing the solution, 2.5% bovine serum albumin was added and the plate was allowed to sit for 1 hour to block nonspecific binding. The mixed solution described above (50 μl/well) was added to the plate with immobilized neogenin and the plate was left to settle at room temperature for 1 hour. Washing was then performed and peroxidase-labeled avidin (VECTASTAIN ABC system, available from Vector Laboratories) was added and the plate was allowed to sit at room temperature for 1 hour. Washing was then carried out, a substrate solution was added, the color was developed for a certain period of time, and the absorbance was measured using a plate spectrophotometer. the absorbance ratio in the absence of antibody was plotted as 1, and the concentration-dependent inhibition of RGMa-neogenin binding by antibody was assessed (Fig. 1).

As a result, in contrast to the polyclonal anti-human RGMa antibody (R&D Systems, AF2459), r5F9, r116A3, and r70E4 did not inhibit RGMa-neogenin binding.

[0152] Example 10: RGMa-BMP2 Binding Inhibition Assay

Human RGMa protein (R&D systems, 2459-RM) adjusted to 2 μg/ml PBS was added to each well of a 96-well plate at 50 μl/well and the plate was allowed to settle at room temperature for 1 hour. A 2.5% bovine serum albumin solution was added and the mixture was allowed to sit for 1 hour to block nonspecific binding, thereby obtaining an immobilized RGMa protein plate. Test antibodies (B5.116A3 and B5.70E4), serially diluted from 0.01 to 10 μg/ml, were added to the plate with immobilized RGMa protein, and the plate was left to settle at room temperature for 1 hour. The plate was then washed and human BMP2 protein (R&D systems, 355-BM) diluted to 0.5 μg/ml was added and the plate was allowed to sit at room temperature for 1 hour.

Biotin-labeled anti-BMP2 antibody was allowed to react and additional peroxidase-labeled avidin (VECTASTAIN ABC system, available from Vector Laboratories) was added and substrate solution was added, color developed over a period of time, and absorbance measured using a plate spectrophotometer (Fig. 2). ).

As a result, in particular, the anti-RGMa antibody (B5.116A3) weakly inhibited RGMa-BMP2 binding in a concentration-dependent manner (optical density: 0.45 at 0.01 μg/ml, 0.4 at 0.1 μg/ml, 0.32 at 1 µg/ml and 0.1 at 10 µg/ml).

[0153] Example 11: Construction of a Humanized Antibody

Humanization of the mouse monoclonal antibody B5.116A3 was carried out by the complementarity determining region (CDR) transfer method of Winter et al., described in patent No. 2912618.

First, 3D homology models of the light and heavy chain variable regions of the mouse monoclonal antibody B5.116A3 were obtained and amino acid residues in the framework (FW) region located near the CDR were identified. FWs of human antibodies were selected, in which these amino acids are preserved to the greatest extent possible, CDRs of mouse antibodies were transplanted into them, and thus a humanized antibody was constructed. In the engineered humanized antibody sequence, 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). In addition, a set of humanized antibody sequences was constructed by introducing additional mutations into amino acids in FW involved in the stability of the variable region structure (a total of 8 heavy chains ranging from HB to HH, including HA, and a total of 7 light chains ranging from KB to KG, including KA).

[0154] Table 3: Designed Humanized Antibody Sequences

Heavy chain SEQ ID NO in sequence list H.A. Sequence in which the CDR of mouse B5.116A3 antibodies is transplanted into human FW antibodies eleven HB HA sequence with substitution Ala 81 Val and Lys 89 Arg 12 H.C. HA sequence with Ala 81 Val substitution 13 HD HA sequence with Lys 89 Arg substitution 14 HE HA sequence with substitution Ala 81 Val, Lys 89 Arg and Phe 37 Val 15 HF HA sequence with Leu 95 Val substitution 16

H.G. HA sequence with Phe 37 Val substitution 17 HH HA sequence with Phe 37 Val and Leu 95 Val substitution 18 Light chain K.A. Sequence in which the CDR of mouse B5.116A3 antibodies is transplanted into human FW antibodies 19 K.B. KA sequence with Phe 71 Tyr substitution 20 KC KA sequence with Phe 71 Tyr and Phe 44 Val substitution 21 KD KA sequence with Ser 85 Thr substitution 22 KE KA sequence with Pro 44 Val replacement 23 KF KA sequence with replacement of Pro 44 Val and Ser 85 Thr 24 KG KA sequence with Phe 71 Tyr, Pro 44 Val and Ser 85 Thr substitution 25

[0155] Example 12: Production of recombinant mouse-human chimeric antibody and recombinant humanized antibody

(1) Recombinant chimeric anti-mouse-human RGMa antibody 116A3 (r116A3C) containing the following amino acid sequences was prepared in accordance with Example 6.

[0156] Light chain (SEQ ID NO: 8 in sequence list)

DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQLNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

[0157] Heavy Chain (SEQ ID NO: 9 in sequence listing)

EVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKRSVYLQMNNLRAEDTGIYYCTRRDGAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0158] (2) A total of 20 humanized anti-RGMa antibodies containing the following combinations of heavy and light chains were prepared according to Example 6.

[0159] Heavy chain/light chain combinations;

HA/KA, HA/KB, HA/KC, HA/KG, HB/KC, HC/KA, HC/KB, HD/KA, HD/KB, HD/KC, HD/KD, HE/KA, HF/ KA, HF/KF, HF/KG, HG/KD, HG/KH, HH/KA, HH/KD, HH/KF

[0160] (3) Recombinant humanized anti-RGMa antibody (hereinafter referred to as rH5F9) was prepared in accordance with Example 6 (Comparative Example).

The variable region of the humanized anti-RGMa monoclonal antibody h5F9 described in Patent Document 1 (WO2009/106356) (the light chain is seq ID_53 of Patent Document 1 and the heavy chain is seq ID_50 of Patent Document 1) was ligated with the constant region of a human antibody (the light chain is Arg 108-Cys 214 SEQ ID NO: 26 in the sequence listing, and the heavy chain is Ala 117-Lys 446 SEQ ID NO: 27 in the sequence listing).

[0161] Example 13: Antibody Thermal Stability Test

Twenty μl aliquots of the culture supernatant in which the recombinant antibody described in Example 12 was expressed were collected and treated with heat for 10 minutes at 8 temperatures of 40, 45, 50, 55, 60, 65, 70 and 75°C, respectively. using a thermal cycler (Takara bio, TP 600). The culture supernatant was diluted with PBS such that the final antibody concentration was 125 ng/ml. The culture supernatant was then subjected to the ELISA in which human RGMa proteins were immobilized, described in Example 3, to evaluate the antigen-binding properties of the antibody (Fig. 3).

As a result, the humanized antibody containing a combination of heavy chain HE and light chain KA (hereinafter referred to as "rH116A3") and the mouse-human chimeric antibody (r116A3C) had better thermostability compared with the humanized antibody (rH5F9). Hereinafter, a humanized antibody containing such a HE/KA combination is referred to as rH116A3.

When heat treatment was not performed, the mouse-human chimeric antibody (r116A3C) and the humanized antibody (rH116A3) had equivalent antigen-binding properties, and no attenuation of antigen-binding property associated with humanization was observed.

[0162] Example 14: Establishment of a stable CHO cell line producing a humanized anti-RGMa antibody (rH116A3)

A stable line of CHO cells producing a humanized anti-RGMa antibody (rH116A3) was established using the Lonza GS Xceed system (Lonza). The pXC dual gene vector containing the light chain coding sequence (SEQ ID NO: 44 in the sequence listing) and the heavy chain coding sequence (SEQ ID NO: 43 in the sequence listing) of the humanized anti-RGMa antibody (rH116A3) was introduced into the parental cell line CHOK1SV with GS knockout and obtained a set of transformed cells by selection on methionine sulfoxyimine (MSX). After separation into single cells by flow cytometry, the amount of antibody produced in the culture supernatant, cell proliferation, etc. were assessed, and a stable CHO cell line was obtained.

[0163] Example 15: Neurite Outgrowth Assay

The cerebellum of a neonatal (P7) rat was excised, suspended in trypsin solution (0.25% trypsin in PBS containing 0.2% DNase) and digested at 37°C for 10-15 minutes. DMEM containing 10% fetal calf serum was then added and the mixture was centrifuged. The cells were resuspended in the same medium and centrifuged, and the same procedure was repeated twice to wash the cells. Additionally, this cell suspension was filtered through a 70 μm cell filter and centrifuged, and the pelleted fraction was resuspended in the same medium. Supplement B27 (GIBCO) was added to the cell suspension to obtain neonatal rat cerebellar granulocytes.

Then neonatal rat cerebellar granulocytes were seeded onto a cell culture plate and cultured at 37°C for 1 day.

Recombinant RGMa protein (R&D systems, 2459-RM) was added at a final concentration of 2 μg/ml, and cells were cultured at 37°C for 2 days. Neurite length was measured by microscopy. As shown in FIG. 4, the addition of RGMa resulted in changes in neurite length from 37 μm to 26 μm in the experiment on the left figure and from 38 μm to 27 μm in the right figure, thus inhibiting neurite outgrowth. Although addition of the test antibody alone (B5.116A3 or B5.70E4) at a final concentration of 10 μg/ml did not change neurite length, simultaneous addition of recombinant RGMa protein and test antibody induced neurite outgrowth to the same extent as the control (without RGMa addition), which indicates a neutralizing effect of the antibody against the RGMa protein.

[0164] Example 16: Efficacy Testing Using a Rat Spinal Cord Injury Model

A Wistar rat (female, 8 weeks old, weighing approximately 200 g), anesthetized by inhalation of halothane (Takeda Pharmaceutical), underwent laminectomy of the anterior and posterior vertebrae around the T9 level of the spine (T8-T10) and the spinal cord was exposed. For evaluation using the spinal cord crush model, 200 kdyn of pressure was applied to the exposed spinal cord using an IH impactor (Precision System).

Immediately after spinal cord injury as described above, an osmotic minipump filled with 400 μg/ml test antibody (r116A3 or r70E4) or mouse control antibody (mo-IgG2bκ) was placed under the skin on the rat's back (volume 200 μl, 0.5 μl/ hour, delivery within 14 days) (Alzet, model 2002). The tip of the silicone tube connected to the outlet of the osmotic minipump was placed into the dura mater at the site of spinal cord injury. The tube was sutured and fixed to the spinous process on the right side of the lower limb of the laminectomy site, sutures were placed on the muscle and skin layers, and the rat was raised.

Motor function in a rat model of spinal cord injury was assessed using the Basso-Beattie-Bresnahan (BBB) scale (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)) 0, 1, 3 and 7 days after injury, and every week thereafter for 8 weeks. As a result, as shown in FIG. 5(A), r116A3 or r70E4 significantly improved the BBB score at 4 or 3 weeks post-administration, respectively, compared with the control antibody (mo-IgG2bκ) (p<0.05, Student's t test).

In the case of evaluation using the spinal cord hemisection model, the dorsal side of the exposed spinal cord was incised to a depth of 1.8 mm–2.0 mm. Similarly, as above, the test antibody (B5.116A3) or mouse control antibody (mo-IgG2bκ) was administered using an osmotic minipump and scored using the BBB scale at 0, 1, 3 and 7 days after injury and every week thereafter. for a period of up to 10 weeks. As shown in FIG. 5(B), B5.116A3 significantly improved BBB score 4 weeks post-administration compared with control antibody (mo-IgG2bκ) (p<0.01, Student's t test).

[0165] Example 17: Efficacy Testing Using a Mouse Model of Multiple Sclerosis

Peptide PLP _139-151 (HSLGKWLGHPDKF: SEQ ID NO: 45 in the sequence list, Peptide Institute) was dissolved in saline (Otsuka Pharmaceutical Factory) and mixed with Freund's incomplete adjuvant (Sigma) to which was added dead tubercle bacillus H37Ra (Difco Laboratories) , to obtain an emulsion. An SJL/JorllcoCrj (SJL/J) mouse (Charles River Japan) was immunized subcutaneously in the back with PLP139-151 peptide at 50 μg/head and assessed for EAE and body weight change (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. (Fig. .6).

The test antibody (B5.116A3), diluted in saline, was administered intraperitoneally at 20 mg/kg on days 7 and 10 or days 18 and 21 after immunization with the PLP139-151 peptide.

As a result, as shown in FIG. 6, compared with the control antibody (mo-IgG2bκ), the mouse monoclonal antibody to RGMa (B5.116A3) inhibited the worsening of EAE scores as a result of administration before initiation (top of Fig. 6) and exhibited a preventive effect against relapse as a result of administration after initiation (bottom of Fig. 6).

[0166] Example 18: Antibody Immunogenicity Testing

Undifferentiated dendritic cells contained in peripheral blood obtained from 51 healthy donors were subjected to maturation by stimulations of granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin 4. The test antibody (rH116A3) at a final concentration of 50 μg/ml was added to the mature dendritic cells and the cells were cultured for 4.5 days, ensuring the capture of the antibody by dendritic cells. Peripheral blood CD4+ T cells (helper T cells) obtained from the same donor were mixed with dendritic cells and cocultured for an additional week, and then T cell proliferation was measured by flow cytometry. Using the T cell proliferation activity of the test antibody as an indicator, the risk of immunogenicity in humans was assessed. As a result, T cell proliferation was observed in 4 (7.8%) of 51 donors and thus the risk of immunogenicity is low.

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LIST OF SEQUENCES

<110> Mitsubishi Tanabe Pharma Corporation

Osaka University

National University Corporation Chiba University

<120> RGMa BINDING PROTEIN AND ITS USE

<130> A16005-16140

<140> PCT/JP2016/063166

<141> 2016-04-27

<150> JP2015-091095

<151> 2015-04-28

<160> 46

<170> Patent In version 3.5

<210> 1

<211> 450

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<213> Homo sapiens

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Phe Pro Leu Ala Pro Gln Ile Leu Leu Gly Thr Ile Pro Leu Leu Val

435 440 445

Leu Leu Pro Val Leu Trp

450

<210> 3

<211> 449

<212> Protein

<213> Rattus norvegicus

<400> 3

Met Gln Pro Pro Arg Glu Arg Leu Val Val Thr Gly Arg Ala Gly Trp

1 5 10 15

Met Gly Met Gly Arg Gly Ala Gly Arg Ser Ala Leu Gly Leu Trp Pro

20 25 30

Thr Leu Ala Phe Leu Leu Cys Ser Phe Pro Ala Ala Ile Ser Pro Cys

35 40 45

Lys Ile Leu Lys Cys Asn Ser Glu Phe Trp Ser Ala Thr Ser Ser Gly

50 55 60

Ser His Ala Pro Ala Ser Asp Asp Val Pro Glu Phe Cys Ala Ala Leu

65 70 75 80

Arg Thr Tyr Ala Leu Cys Thr Arg Arg Thr Ala Arg Thr Cys Arg Gly

85 90 95

Asp Leu Ala Tyr His Ser Ala Val His Gly Ile Glu Asp Leu Met Ser

100 105 110

Gln His Asn Cys Ser Lys Asp Gly Pro Thr Ser Gln Pro Arg Val Arg

115 120 125

Thr Leu Pro Pro Ala Gly Asp Ser Gln Glu Arg Ser Asp Ser Pro Glu

130 135 140

Ile Cys His Tyr Glu Lys Ser Phe His Lys His Ser Ala Ala Pro Asn

145 150 155 160

Tyr Thr His Cys Gly Leu Phe Gly Asp Pro His Leu Arg Thr Phe Thr

165 170 175

Asp His Phe Gln Thr Cys Lys Val Gln Gly Ala Trp Pro Leu Ile Asp

180 185 190

Asn Asn Tyr Leu Asn Val Gln Val Thr Asn Thr Pro Val Leu Pro Gly

195 200 205

Ser Ala Ala Thr Ala Thr Ser Lys Leu Thr Ile Ile Phe Lys Asn Phe

210 215 220

Gln Glu Cys Val Asp Gln Lys Val Tyr Gln Ala Glu Met Asp Glu Leu

225 230 235 240

Pro Ser Ala Phe Ala Asp Gly Ser Lys Asn Gly Gly Asp Lys His Gly

245 250 255

Ala Asn Ser Leu Lys Ile Thr Glu Lys Val Ser Gly Gln His Val Glu

260 265 270

Ile Gln Ala Lys Tyr Ile Gly Thr Thr Ile Val Val Arg Gln Val Gly

275 280 285

Arg Tyr Leu Thr Phe Ala Val Arg Met Pro Glu Glu Val Val Asn Ala

290 295 300

Val Glu Asp Arg Asp Ser Gln Gly Leu Tyr Leu Cys Leu Arg Gly Cys

305 310 315 320

Pro Leu Asn Gln Gln Ile Asp Phe Gln Ala Phe Arg Ala Asn Ala Glu

325 330 335

Ser Pro Arg Arg Pro Ala Ala Ala Ser Pro Ser Pro Val Val Pro Glu

340 345 350

Thr Phe Pro Tyr Glu Thr Ala Val Ala Lys Cys Lys Glu Lys Leu Pro

355 360 365

Val Glu Asp Leu Tyr Tyr Gln Ala Cys Val Phe Asp Leu Leu Thr Thr

370 375 380

Gly Asp Val Asn Phe Thr Leu Ala Ala Tyr Tyr Ala Leu Glu Asp Gly

385 390 395 400

Lys Met Leu His Ser Asn Lys Asp Lys Leu His Leu Phe Glu Arg Thr

405 410 415

Arg Glu Leu Pro Gly Ala Val Ala Ala Ala Ala Phe Pro Leu Ala Pro

420 425 430

Glu Met Leu Pro Gly Thr Val Thr Leu Leu Val Leu Leu Pro Leu Phe

435 440 445

Trp

<210> 4

<211> 234

<212> Protein

<213> Mus musculus

<400> 4

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 15

Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp

35 40 45

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val

50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser

85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Leu Asn

100 105 110

Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

115 120 125

Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

130 135 140

Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr

145 150 155 160

Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

165 170 175

Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

195 200 205

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

210 215 220

Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230

<210> 5

<211> 471

<212> Protein

<213> Mus musculus

<400> 5

Met Tyr Leu Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly

1 5 10 15

Val Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr

65 70 75 80

Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser

85 90 95

Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr

100 105 110

Gly Ile Tyr Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Gln Gly

115 120 125

Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr

130 135 140

Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu

145 150 155 160

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp

165 170 175

Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu

180 185 190

Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser

195 200 205

Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser

210 215 220

Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr

225 230 235 240

Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro

245 250 255

Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys

260 265 270

Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val

275 280 285

Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn

290 295 300

Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr

305 310 315 320

Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln His Gln Asp

325 330 335

Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu

340 345 350

Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg

355 360 365

Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg

370 375 380

Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp

385 390 395 400

Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys

405 410 415

Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser

420 425 430

Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser

435 440 445

Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr

450 455 460

Ile Ser Arg Ser Pro Gly Lys

465 470

<210> 6

<211> 238

<212> Protein

<213> Mus musculus

<400> 6

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Arg Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Phe Cys

100 105 110

Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro

130 135 140

Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu

145 150 155 160

Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly

165 170 175

Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp

195 200 205

Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr

210 215 220

Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 7

<211> 466

<212> Protein

<213> Mus musculus

<400> 7

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 15

Met Ser Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45

Thr Ser Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Thr Asn Asn Tyr Asn Pro

65 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95

Phe Phe Leu Arg Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110

Tyr Cys Ala Gly Ser Phe Gly Tyr Ser Gln Gly Thr Leu Val Thr Val

115 120 125

Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly

130 135 140

Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys

145 150 155 160

Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu

165 170 175

Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr

180 185 190

Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln

195 200 205

Thr Val Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp

210 215 220

Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro

225 230 235 240

Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly

245 250 255

Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile

260 265 270

Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp

275 280 285

Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His

290 295 300

Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg

305 310 315 320

Val Val Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys

325 330 335

Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu

340 345 350

Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr

355 360 365

Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu

370 375 380

Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp

385 390 395 400

Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys

420 425 430

Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His

435 440 445

Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro

450 455 460

Gly Lys

465

<210> 8

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric antibody light chain

<400> 8

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> chimeric antibody heavy chain

<400> 9

Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Arg Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 10

<211> 1461

<212> Protein

<213> Homo sapiens

<400> 10

Met Ala Ala Glu Arg Gly Ala Arg Arg Leu Leu Ser Thr Pro Ser Phe

1 5 10 15

Trp Leu Tyr Cys Leu Leu Leu Leu Gly Arg Arg Ala Pro Gly Ala Ala

20 25 30

Ala Ala Arg Ser Gly Ser Ala Pro Gln Ser Pro Gly Ala Ser Ile Arg

35 40 45

Thr Phe Thr Pro Phe Tyr Phe Leu Val Glu Pro Val Asp Thr Leu Ser

50 55 60

Val Arg Gly Ser Ser Val Ile Leu Asn Cys Ser Ala Tyr Ser Glu Pro

65 70 75 80

Ser Pro Lys Ile Glu Trp Lys Lys Asp Gly Thr Phe Leu Asn Leu Val

85 90 95

Ser Asp Asp Arg Arg Gln Leu Leu Pro Asp Gly Ser Leu Phe Ile Ser

100 105 110

Asn Val Val His Ser Lys His Asn Lys Pro Asp Glu Gly Tyr Tyr Gln

115 120 125

Cys Val Ala Thr Val Glu Ser Leu Gly Thr Ile Ile Ser Arg Thr Ala

130 135 140

Lys Leu Ile Val Ala Gly Leu Pro Arg Phe Thr Ser Gln Pro Glu Pro

145 150 155 160

Ser Ser Val Tyr Ala Gly Asn Asn Ala Ile Leu Asn Cys Glu Val Asn

165 170 175

Ala Asp Leu Val Pro Phe Val Arg Trp Glu Gln Asn Arg Gln Pro Leu

180 185 190

Leu Leu Asp Asp Arg Val Ile Lys Leu Pro Ser Gly Met Leu Val Ile

195 200 205

Ser Asn Ala Thr Glu Gly Asp Gly Gly Leu Tyr Arg Cys Val Val Glu

210 215 220

Ser Gly Gly Pro Pro Lys Tyr Ser Asp Glu Val Glu Leu Lys Val Leu

225 230 235 240

Pro Asp Pro Glu Val Ile Ser Asp Leu Val Phe Leu Lys Gln Pro Ser

245 250 255

Pro Leu Val Arg Val Ile Gly Gln Asp Val Val Leu Pro Cys Val Ala

260 265 270

Ser Gly Leu Pro Thr Pro Thr Ile Lys Trp Met Lys Asn Glu Glu Ala

275 280 285

Leu Asp Thr Glu Ser Ser Glu Arg Leu Val Leu Leu Ala Gly Gly Ser

290 295 300

Leu Glu Ile Ser Asp Val Thr Glu Asp Asp Ala Gly Thr Tyr Phe Cys

305 310 315 320

Ile Ala Asp Asn Gly Asn Glu Thr Ile Glu Ala Gln Ala Glu Leu Thr

325 330 335

Val Gln Ala Gln Pro Glu Phe Leu Lys Gln Pro Thr Asn Ile Tyr Ala

340 345 350

His Glu Ser Met Asp Ile Val Phe Glu Cys Glu Val Thr Gly Lys Pro

355 360 365

Thr Pro Thr Val Lys Trp Val Lys Asn Gly Asp Met Val Ile Pro Ser

370 375 380

Asp Tyr Phe Lys Ile Val Lys Glu His Asn Leu Gln Val Leu Gly Leu

385 390 395 400

Val Lys Ser Asp Glu Gly Phe Tyr Gln Cys Ile Ala Glu Asn Asp Val

405 410 415

Gly Asn Ala Gln Ala Gly Ala Gln Leu Ile Ile Leu Glu His Ala Pro

420 425 430

Ala Thr Thr Gly Pro Leu Pro Ser Ala Pro Arg Asp Val Val Ala Ser

435 440 445

Leu Val Ser Thr Arg Phe Ile Lys Leu Thr Trp Arg Thr Pro Ala Ser

450 455 460

Asp Pro His Gly Asp Asn Leu Thr Tyr Ser Val Phe Tyr Thr Lys Glu

465 470 475 480

Gly Ile Ala Arg Glu Arg Val Glu Asn Thr Ser His Pro Gly Glu Met

485 490 495

Gln Val Thr Ile Gln Asn Leu Met Pro Ala Thr Val Tyr Ile Phe Arg

500 505 510

Val Met Ala Gln Asn Lys His Gly Ser Gly Glu Ser Ser Ala Pro Leu

515 520 525

Arg Val Glu Thr Gln Pro Glu Val Gln Leu Pro Gly Pro Ala Pro Asn

530 535 540

Leu Arg Ala Tyr Ala Ala Ser Pro Thr Ser Ile Thr Val Thr Trp Glu

545 550 555 560

Thr Pro Val Ser Gly Asn Gly Glu Ile Gln Asn Tyr Lys Leu Tyr Tyr

565 570 575

Met Glu Lys Gly Thr Asp Lys Glu Gln Asp Val Asp Val Ser Ser His

580 585 590

Ser Tyr Thr Ile Asn Gly Leu Lys Lys Tyr Thr Glu Tyr Ser Phe Arg

595 600 605

Val Val Ala Tyr Asn Lys His Gly Pro Gly Val Ser Thr Pro Asp Val

610 615 620

Ala Val Arg Thr Leu Ser Asp Val Pro Ser Ala Ala Pro Gln Asn Leu

625 630 635 640

Ser Leu Glu Val Arg Asn Ser Lys Ser Ile Met Ile His Trp Gln Pro

645 650 655

Pro Ala Pro Ala Thr Gln Asn Gly Gln Ile Thr Gly Tyr Lys Ile Arg

660 665 670

Tyr Arg Lys Ala Ser Arg Lys Ser Asp Val Thr Glu Thr Leu Val Ser

675 680 685

Gly Thr Gln Leu Ser Gln Leu Ile Glu Gly Leu Asp Arg Gly Thr Glu

690 695 700

Tyr Asn Phe Arg Val Ala Ala Leu Thr Ile Asn Gly Thr Gly Pro Ala

705 710 715 720

Thr Asp Trp Leu Ser Ala Glu Thr Phe Glu Ser Asp Leu Asp Glu Thr

725 730 735

Arg Val Pro Glu Val Pro Ser Ser Leu His Val Arg Pro Leu Val Thr

740 745 750

Ser Ile Val Val Ser Trp Thr Pro Pro Glu Asn Gln Asn Ile Val Val

755 760 765

Arg Gly Tyr Ala Ile Gly Tyr Gly Ile Gly Ser Pro His Ala Gln Thr

770 775 780

Ile Lys Val Asp Tyr Lys Gln Arg Tyr Tyr Thr Ile Glu Asn Leu Asp

785 790 795 800

Pro Ser Ser His Tyr Val Ile Thr Leu Lys Ala Phe Asn Asn Val Gly

805 810 815

Glu Gly Ile Pro Leu Tyr Glu Ser Ala Val Thr Arg Pro His Thr Asp

820 825 830

Thr Ser Glu Val Asp Leu Phe Val Ile Asn Ala Pro Tyr Thr Pro Val

835 840 845

Pro Asp Pro Thr Pro Met Met Pro Pro Val Gly Val Gln Ala Ser Ile

850 855 860

Leu Ser His Asp Thr Ile Arg Ile Thr Trp Ala Asp Asn Ser Leu Pro

865 870 875 880

Lys His Gln Lys Ile Thr Asp Ser Arg Tyr Tyr Thr Val Arg Trp Lys

885 890 895

Thr Asn Ile Pro Ala Asn Thr Lys Tyr Lys Asn Ala Asn Ala Thr Thr

900 905 910

Leu Ser Tyr Leu Val Thr Gly Leu Lys Pro Asn Thr Leu Tyr Glu Phe

915 920 925

Ser Val Met Val Thr Lys Gly Arg Arg Ser Ser Thr Trp Ser Met Thr

930 935 940

Ala His Gly Thr Thr Phe Glu Leu Val Pro Thr Ser Pro Pro Lys Asp

945 950 955 960

Val Thr Val Val Ser Lys Glu Gly Lys Pro Lys Thr Ile Ile Val Asn

965 970 975

Trp Gln Pro Pro Ser Glu Ala Asn Gly Lys Ile Thr Gly Tyr Ile Ile

980 985 990

Tyr Tyr Ser Thr Asp Val Asn Ala Glu Ile His Asp Trp Val Ile Glu

995 1000 1005

Pro Val Val Gly Asn Arg Leu Thr His Gln Ile Gln Glu Leu Thr

1010 1015 1020

Leu Asp Thr Pro Tyr Tyr Phe Lys Ile Gln Ala Arg Asn Ser Lys

1025 1030 1035

Gly Met Gly Pro Met Ser Glu Ala Val Gln Phe Arg Thr Pro Lys

1040 1045 1050

Ala Asp Ser Ser Asp Lys Met Pro Asn Asp Gln Ala Ser Gly Ser

1055 1060 1065

Gly Gly Lys Gly Ser Arg Leu Pro Asp Leu Gly Ser Asp Tyr Lys

1070 1075 1080

Pro Pro Met Ser Gly Ser Asn Ser Pro His Gly Ser Pro Thr Ser

1085 1090 1095

Pro Leu Asp Ser Asn Met Leu Leu Val Ile Ile Val Ser Val Gly

1100 1105 1110

Val Ile Thr Ile Val Val Val Val Ile Ile Ala Val Phe Cys Thr

1115 1120 1125

Arg Arg Thr Thr Ser His Gln Lys Lys Lys Arg Ala Ala Cys Lys

1130 1135 1140

Ser Val Asn Gly Ser His Lys Tyr Lys Gly Asn Ser Lys Asp Val

1145 1150 1155

Lys Pro Pro Asp Leu Trp Ile His His Glu Arg Leu Glu Leu Lys

1160 1165 1170

Pro Ile Asp Lys Ser Pro Asp Pro Asn Pro Ile Met Thr Asp Thr

1175 1180 1185

Pro Ile Pro Arg Asn Ser Gln Asp Ile Thr Pro Val Asp Asn Ser

1190 1195 1200

Met Asp Ser Asn Ile His Gln Arg Arg Asn Ser Tyr Arg Gly His

1205 1210 1215

Glu Ser Glu Asp Ser Met Ser Thr Leu Ala Gly Arg Arg Gly Met

1220 1225 1230

Arg Pro Lys Met Met Met Pro Phe Asp Ser Gln Pro Pro Gln Pro

1235 1240 1245

Val Ile Ser Ala His Pro Ile His Ser Leu Asp Asn Pro His His

1250 1255 1260

His Phe His Ser Ser Ser Leu Ala Ser Pro Ala Arg Ser His Leu

1265 1270 1275

Tyr His Pro Gly Ser Pro Trp Pro Ile Gly Thr Ser Met Ser Leu

1280 1285 1290

Ser Asp Arg Ala Asn Ser Thr Glu Ser Val Arg Asn Thr Pro Ser

1295 1300 1305

Thr Asp Thr Met Pro Ala Ser Ser Ser Gln Thr Cys Cys Thr Asp

1310 1315 1320

His Gln Asp Pro Glu Gly Ala Thr Ser Ser Ser Tyr Leu Ala Ser

1325 1330 1335

Ser Gln Glu Glu Asp Ser Gly Gln Ser Leu Pro Thr Ala His Val

1340 1345 1350

Arg Pro Ser His Pro Leu Lys Ser Phe Ala Val Pro Ala Ile Pro

1355 1360 1365

Pro Pro Gly Pro Pro Thr Tyr Asp Pro Ala Leu Pro Ser Thr Pro

1370 1375 1380

Leu Leu Ser Gln Gln Ala Leu Asn His His Ile His Ser Val Lys

1385 1390 1395

Thr Ala Ser Ile Gly Thr Leu Gly Arg Ser Arg Pro Pro Met Pro

1400 1405 1410

Val Val Val Pro Ser Ala Pro Glu Val Gln Glu Thr Thr Arg Met

1415 1420 1425

Leu Glu Asp Ser Glu Ser Ser Tyr Glu Pro Asp Glu Leu Thr Lys

1430 1435 1440

Glu Met Ala His Leu Glu Gly Leu Met Lys Asp Leu Asn Ala Ile

1445 1450 1455

Thr Thr Ala

1460

<210> 11

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized Antibody Heavy Chain HA

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Leu Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 12

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> HB heavy chain humanized antibody

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 13

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> Humanized Antibody Heavy Chain HC

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Leu Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 14

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> HD heavy chain humanized antibody

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 15

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223>HE heavy chain humanized antibody

<400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 16

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> HF heavy chain humanized antibody

<400> 16

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 17

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> HG humanized antibody heavy chain

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Leu Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 18

<211> 446

<212> Protein

<213> Artificial sequence

<220>

<223> HH humanized antibody heavy chain

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 19

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> KA humanized antibody light chain

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 20

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> KB humanized antibody light chain

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 21

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> KC light chain of humanized antibody

<400> 21

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 22

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> KD of humanized antibody light chain

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 23

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> KE of humanized antibody light chain

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 24

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223>KF humanized antibody light chain

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 25

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> humanized antibody light chain KG

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 26

<211> 14

<212> Protein

<213> Mus musculus

<400> 26

Glu Glu Val Val Asn Ala Val Glu Asp Trp Asp Ser Gln Gly

1 5 10

<210> 27

<211> 14

<212> Protein

<213> Mus musculus

<400> 27

Asn Gln Gln Ile Asp Phe Gln Ala Phe His Thr Asn Ala Glu

1 5 10

<210> 28

<211> 11

<212> Protein

<213> Mus musculus

<400> 28

Pro Thr Ala Pro Glu Thr Phe Pro Tyr Glu Thr

1 5 10

<210> 29

<211> 11

<212> Protein

<213> Mus musculus

<400> 29

Lys Leu Pro Val Glu Asp Leu Tyr Tyr Gln Ala

1 5 10

<210> 30

<211> 11

<212> Protein

<213> Mus musculus

<400> 30

Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 31

<211> 7

<212> Protein

<213> Mus musculus

<400> 31

Tyr Thr Ser Arg Leu His Ser

15

<210> 32

<211> 7

<212> Protein

<213> Mus musculus

<400> 32

Gln Gln Leu Asn Thr Leu Pro

15

<210> 33

<211> 5

<212> Protein

<213> Mus musculus

<400> 33

Asp Ala Trp Met Asp

15

<210> 34

<211> 19

<212> Protein

<213> Mus musculus

<400> 34

Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu Ser

1 5 10 15

Val Lys Gly

<210> 35

<211> 5

<212> Protein

<213> Mus musculus

<400> 35

Arg Asp Gly Ala Tyr

15

<210> 36

<211> 16

<212> Protein

<213> Mus musculus

<400> 36

Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His

1 5 10 15

<210> 37

<211> 7

<212> Protein

<213> Mus musculus

<400> 37

Lys Val Ser Asn Arg Phe Ser

15

<210> 38

<211> 7

<212> Protein

<213> Mus musculus

<400> 38

Ser Gln Ser Thr His Val Pro

15

<210> 39

<211> 6

<212> Protein

<213> Mus musculus

<400> 39

Thr Ser Tyr Tyr Trp Asn

15

<210> 40

<211> 16

<212> Protein

<213> Mus musculus

<400> 40

Tyr Ile Ser Tyr Asp Gly Thr Asn Asn Tyr Asn Pro Ser Leu Lys Asn

1 5 10 15

<210> 41

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> humanized antibody heavy chain

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asp Ala

20 25 30

Trp Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr Tyr Ala Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Leu Tyr

85 90 95

Tyr Cys Thr Arg Arg Asp Gly Ala Tyr Trp Gly Lys Gly Thr Thr Val

100 105 110

Thr Val Ser Ser

115

<210> 42

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> humanized antibody light chain

<400> 42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Leu Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Met Glu

100 105

<210> 43

<211> 348

<212> DNA

<213> Artificial sequence

<220>

<223> gene encoding the heavy chain of a humanized antibody

<400> 43

gaagtgcagc tggtggaatc tggcggcgga ctggtgcagc ctggcagatc cctgagactg 60

tcctgtaccg cctccggctt caccttctcc gacgcctgga tggattgggt gcgacaggct 120

cctggcaagg gcctggaatg ggtggccgag atccggtcca aggccaacaa ccacgccacc 180

tactacgccg agtctgtgaa gggccggttc accatctccc gggacgactc caagtccatc 240

gtgtacctgc agatgaactc cctgcggacc gaggacaccg ccctgtacta ctgcaccaga 300

agggacggcg cctactgggg caagggcacc acagtgacag tgtcctcc 348

<210> 44

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> gene encoding the light chain of a humanized antibody

<400> 44

gacatccaga tgacccagtc cccctcctcc gtgtctgctt ccgtgggcga cagagtgacc 60

atcacctgtc gggcctccca ggacatctcc tcctacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctactac acctcccggc tgcactccgg cgtgccctct 180

agattttccg gctctggctc cggcaccgac tttaccctga ccatctccag cctgcagccc 240

gaggacttcg cctcctactt ctgtcagcag ctgaacaccc tgccctggac ctttggcgga 300

ggcaccaagg tggaaatgga a 321

<210> 45

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223>PLP139-151

<400> 45

His Ser Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe

1 5 10

<210> 46

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> spacer

<400> 46

Ser Gly Ser Gly

1

<---

Claims (25)

1. Isolated antibody to RGMa or its antigen-binding fragment, wherein the amino acid sequence of each of heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3) comprises the following sequences: HCDR1: DAWMD (SEQ ID NO: 33 in sequence listing), HCDR2: EIRSKANNHATYYAESVKG (SEQ ID NO: 34 in the sequence list) and HCDR3: RDGAY (SEQ ID NO: 35 in sequence listing); And wherein the light chain variable region (VL) contains the following amino acid sequence: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 42 in sequence listing). 2. An anti-RGMa antibody or an antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region (VH) contains the following amino acid sequence: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS (SEQ ID NO: 41 in sequence listing). 3. Isolated antibody to RGMa or its antigen-binding fragment, wherein the heavy chain variable region (VH) contains the following amino acid sequence: EVQLVESGGGLVQPGRSLRLSCTASGFTFSDAWMDWVRQAPGKGLEWVAEIRSKANNHATYYAESVKGRFTISRDDSKSIVYLQMNSLRTEDTALYYCTRRDGAYWGKGTTVTVSS (SEQ ID NO: 41 in sequence listing); And wherein the light chain variable region (VL) contains the following amino acid sequence: DIQMTQSPSSVSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFASYFCQQLNTLPWTFGGGTKVEME (SEQ ID NO: 42 in sequence listing). 4. An antibody to RGMa or an antigen-binding fragment thereof according to any one of paragraphs. 1-3, wherein the anti-RGMa antibody is a humanized antibody. 5. An antibody to RGMa or an antigen-binding fragment thereof according to any one of paragraphs. 1-3, wherein the anti-RGMa antibody contains human IgG constant regions. 6. An antibody to RGMa or an antigen-binding fragment thereof according to any one of paragraphs. 1-3, wherein a substitution, deletion, addition or insertion of 1-20 amino acids may be introduced into a region other than the CDR. 7. A nucleic acid molecule encoding the protein of an antibody to RGMa or an antigen-binding fragment thereof according to any one of claims. 1-6. 8. An expression vector containing a nucleic acid molecule according to claim 7. 9. A host cell for producing an antibody, containing the expression vector according to claim 8. 10. A method for producing an antibody to RGMa or an antigen-binding fragment thereof according to any one of claims. 1-6, where the method includes the step of culturing the host cell according to claim 9. 11. A pharmaceutical composition for the prevention, treatment or prevention of relapse of neurological or immunological diseases in which the activity of RGMa is involved, containing an effective amount of an antibody to RGMa or an antigen-binding fragment thereof according to any one of claims. 1-6. 12. The pharmaceutical composition according to claim 11, where the neurological diseases are selected from the group consisting of amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain-Barré syndrome, cerebral leukodystrophy, multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), optic neuromyelitis, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal neoplasia, transverse myelitis, dementia (including senile dementia, mild cognitive disorder, Alzheimer's disease, dementia associated with Alzheimer's disease), Huntington's disease, tardive dyskinesia, mania, Parkinson's disease, Steele-Richardson syndrome, Down syndrome, myasthenia gravis, nervous system trauma (including optic nerve injury), vascular amyloidosis, intracerebral hemorrhage amyloidosis, cerebral infarction, encephalitis, acute confusional states, glaucoma, schizophrenia, and retinal nerve fiber layer degeneration (including diabetic retinopathy, ischemic optic neuropathy, X-linked retinoschisis, drug-induced optic neuropathy, retinal dystrophy associated age-related macular degeneration, eye diseases characterized by optic disc drusen, eye diseases characterized by genetically determined photoreceptor degeneration, autosomal recessive rod-cone dystrophy, mitochondrial disorder associated with optic neuropathy). 13. The pharmaceutical composition according to claim 11, where the immunological diseases are selected from the group consisting of multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis), neuromyelitis optica, psoriasis, arthritis (including rheumatoid arthritis, osteoarthritis , psoriatic arthritis), Guillain-Barré syndrome, Behçet's disease, pernicious anemia, (insulin-dependent) diabetes mellitus type I, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), Sjögren's syndrome, Goodpasture's syndrome, Graves' disease, autoimmune hemolytic anemia , autoimmune thrombocytopenic purpura, asthma, hay fever, atopic dermatitis, glomerulonephritis, myasthenia gravis, Hashimoto's disease and sarcoidosis. 14. The pharmaceutical composition according to claim 11, where the neurological or immunological diseases are selected from the group consisting of spinal cord injury, nervous system injury (including optic nerve injury), multiple sclerosis (including relapsing-remitting multiple sclerosis, primary progressive multiple sclerosis, secondary progressive multiple sclerosis sclerosis).