WO2010126169A1 - Pharmaceutical composition for preventing vascular disorders which comprises alk1 inhibitor as active ingredient - Google Patents

Pharmaceutical composition for preventing vascular disorders which comprises alk1 inhibitor as active ingredient Download PDF

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WO2010126169A1
WO2010126169A1 PCT/JP2010/057924 JP2010057924W WO2010126169A1 WO 2010126169 A1 WO2010126169 A1 WO 2010126169A1 JP 2010057924 W JP2010057924 W JP 2010057924W WO 2010126169 A1 WO2010126169 A1 WO 2010126169A1
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bmp9
alk1
antibody
pharmaceutical composition
protein
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French (fr)
Japanese (ja)
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冨塚一磨
清水清
柿谷誠
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協和発酵キリン株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a pharmaceutical composition containing an activin receptor-like kinase 1 (Activin receptor-like kinase 1; hereinafter referred to as ALK1) inhibitor as an active ingredient, and a vascular disorder treatment using the same. It is about the method.
  • ALK1 activin receptor-like kinase 1
  • a vascular endothelial cell refers to a flat and thin layer of cells constituting the inner surface of a blood vessel.
  • Vascular endothelial cells produce and secrete various physiologically active substances, and are involved in antithrombotic action, regulation of vascular tonus, and inflammatory reaction (white blood cell adhesion, migration).
  • Healthy endothelial cells express various antithrombotic molecules, function in platelet aggregation inhibition, blood coagulation inhibition, fibrinolytic activity, and vascular relaxation, and function in antithrombotic properties (Non-patent Document 1).
  • vascular endothelial cells constantly express extracellular nucleotide-degrading enzymes on the cell membrane, degrade ADP released from platelets, and suppress platelet aggregation.
  • Non-patent Document 1 vascular endothelial cells are damaged, specifically, a state in which vasodilatability to a specific stimulating substance such as acetylcholine and bradykinin is reduced, an inflammatory state, a thrombotic state (thrombus tends to occur) (Non-patent documents 2 and 3).
  • vascular endothelial cells refers to inflammatory substances such as oxidized LDL, glycated protein, inflammatory cytokines (IL-1, TNF- ⁇ , etc.), endotoxin, etc. acting on vascular endothelial cells, and E-selectin, VCAM- 1, refers to the state in which ICAM-1 and NF-kB are expressed (Non-patent Document 2).
  • E-selectin and VCAM-1 are molecules that are specifically expressed in vascular endothelial cells, and are regarded as vascular disorder markers because they are induced during inflammation.
  • the E-selectin, VCAM-1 is considered to be involved in inflammation through the following mechanism.
  • E-selectin and VCAM-1 which are adhesion molecules, are expressed on vascular endothelial cells.
  • E- selectin on vascular endothelial cells express, leukocytes such as monocytes and neutrophils, via the selectin ligand (sugar (such as sialyl Le x antigens) expressed on the cell surface, weak vascular endothelial cells
  • leukocytes are integrin receptors such as LFA-1 or VLA4 expressed on the surface of leukocytes and VCAM-1 or ICAM-I expressed on the surface of vascular endothelial cells by inflammatory substances.
  • Non-patent Document 4 vascular endothelial injury and thrombotic state
  • normal vascular endothelial cells are in an antithrombotic state, but once inflammation is caused by injury or infection, antithrombosis in endothelial cells is part of the physiological defense reaction.
  • VWF von Willebrand factor
  • TF tissue factor
  • PAI- plasminogen activator inhibitor-1
  • thrombus formation is likely to be induced (Non-patent Document 1).
  • arteriosclerosis, hypertension, chronic kidney disease (CKD), diabetes, heart disease, lipid which are known to be caused by vascular endothelial disorder
  • lifestyle-related diseases such as abnormalities, acute renal failure, and thrombosis is particularly well known.
  • vascular endothelial damage is considered the first stage of atherosclerosis progression, and recent studies have demonstrated that vascular endothelial damage is a useful prognostic indicator of atherosclerotic disease, etc.
  • the importance of vascular endothelial injury in the development of atherosclerosis is unquestionable (Non-Patent Documents 3 and 5).
  • vascular endothelial damage is observed not only in hemodialysis patients but also in patients in the preservation period, and it has been reported that vascular endothelial damage is related to pathological progression (Non-Patent Documents 6-8).
  • Vascular endothelial injury due to renal disease is said to occur because the imported renal arterioles fail to contract due to abnormalities in the paraglomerular device caused by renal disease, and systemic blood pressure is transmitted directly to the glomeruli (non-patented). Reference 8).
  • pathological progression in hypertension, type I diabetes, type II diabetes, heart disease, dyslipidemia, etc., and high cardiovascular disease (CVD) risk in these diseases are also regarded as vascular endothelial disorder.
  • Non-Patent Documents 9-15 In addition, in type II diabetes and hypertension, the presence of vascular endothelial damage has been recognized before the onset of the symptom, and a relationship with the onset of the disease has also been suggested (Non-patent Documents 16 and 17).
  • Non-patent Document 18 vascular endothelial injury in the local region of the kidney is involved in pathological progression such as acute renal failure.
  • Vascular endothelial injury is also known to be deeply related to insulin resistance, which is considered to be the background of metabolic syndrome.
  • Vascular endothelial cells receive the action of insulin and produce nitric oxide (NO) via phosphatidylinositol 3-kinase (PI3K) to control vasodilation, but are considered to act on vascular endothelial damage, high blood sugar, lipid
  • NO nitric oxide
  • PI3K phosphatidylinositol 3-kinase
  • vascular endothelial disorders are closely related to insulin resistance, and lifestyle habits such as arteriosclerotic disease, hypertension, chronic kidney disease, acute renal failure, diabetes, heart disease, and thrombosis are observed. Perceived as the cause of the disease.
  • lifestyle habits such as arteriosclerotic disease, hypertension, chronic kidney disease, acute renal failure, diabetes, heart disease, and thrombosis.
  • Perceived as the cause of the disease.
  • many factors aging, smoking, inflammation, trauma, dyslipidemia, hyperglycemia, etc.
  • the true mechanism is not elucidated (Non-patent Document 22).
  • ALK1 belongs to the type I receptor group of the TGF-beta superfamily and is specifically expressed on the vascular endothelium, and is considered to be a causative gene of hereditary hemorrhagic telangiectasia (HHT) ( Non-patent document 23).
  • ALK1 phosphorylates intracellular signaling molecules Smad1 and Smad5 and transmits signals into the cell. From the analysis using ALK1-deficient mice, it is also known that ALK1 is an important molecule for blood vessel construction (Non-patent Documents 24 and 25).
  • BMP9 belonging to the bone morphogenetic protein (hereinafter referred to as BMP) family is known (Non-patent Documents 26 and 27).
  • BMP belongs to the TGF-beta superfamily and has been identified as a molecule having the ability to induce ectopic bone growth and cartilage formation (Non-patent Documents 28 and 29). Recently, BMP family molecules have generally been found to be involved in the growth, differentiation, and apoptosis of various cells and are important for tissue and organ morphogenesis (Non-patent Documents 30 and 31). BMP9 belonging to the BMP family molecule has been reported to promote the formation of hypertrophic chondrocytes and differentiation from mesenchymal cells to cartilage (Non-patent Document 32), as well as other BMP family molecules.
  • Non-patent Document 33 hepcidin expression regulation
  • Non-Patent Document 34 sugar metabolism involved in maintaining the homeostasis of the drug
  • the expression organ it has been reported that it is mainly expressed in the spinal cord and inter-segmental membrane in the fetal period and in the liver in the adult period (Non-patent Documents 34 to 36).
  • BMP9 is a circulating factor in blood (Non-patent Document 37).
  • the protein structure of BMP9 as with other BMP families, after synthesis as a single-chain precursor protein (pre-pro body), the signal peptide region is cleaved, and the C-terminal side is cleaved in the cell.
  • the cysteine residue present in the dimer forms a dimer (prodimer) via a disulfide bond. Then, it is cleaved by furin-like protease into the C-terminal side (matter dimer), which is the active body, and the N-terminal propeptide region (pro-region) having no disulfide bond. Two molecules of the cleaved N-terminal propeptide region form a complex with one molecule of the C-terminal nature dimer via a non-covalent bond, and are secreted from the cell in the form of the complex. Is known (Non-patent Document 38).
  • BMP signaling generally requires two receptors of type I and type II belonging to the TGFbeta superfamily having a serine / threonine kinase domain in the intracellular region.
  • ALK1 is known as a type I receptor for BMP9, and BMP type II receptor (BMPRII), activin type IIa receptor (ActRIIa), and activin type IIb receptor (ActRIIb) are known as type II receptors (Non-patent Document 27). .
  • ALK1 extracellular region polypeptide-Fc fusion protein or ALK1 extracellular region polypeptide antibody as a therapeutic agent for angiogenesis-related diseases such as cancer and rheumatoid arthritis
  • BMP9 protein The possibility as a therapeutic agent in breast cancer or prostate cancer (Patent Document 3) has been reported.
  • an ALK1 inhibitor comprising a compound having a pyrazolopyrimidine skeleton as an active ingredient as a therapeutic agent in angiogenesis-related diseases has been reported (Patent Document 4).
  • an ALK1 inhibitor has a vascular disorder inhibitory activity. That is, the present inventors have found that when BMP9, which is a ligand of ALK1, is allowed to act on vascular endothelial cells, E-selectin and VCAM-1 that are vascular endothelial injury markers are remarkably induced. Furthermore, when a mouse overexpressing BMP9 was produced, it was found that blood was accumulated in the abdominal cavity / thoracic cavity due to the excessive action of BMP9, and redness due to blood leakage was caused in lymph nodes. We have succeeded in confirming the action in vivo.
  • anti-BMP9 therapy that inhibits has anti-inflammatory effects including suppression of vascular injury.
  • anti-BMP9 therapy is effective for diseases such as heart disease, arteriosclerosis, renal disease, and dyslipidemia. The possibility that it could be an effective treatment was confirmed.
  • the present inventor considered that the present invention can be provided as a pharmaceutical composition for suppressing vascular injury using an ALK1 inhibitor such as a BMP9 neutralizing antibody as an active ingredient, and has completed the present invention. That is, the present invention is as follows. [1] A pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient. [2] The pharmaceutical composition of [1], wherein the ALK1 inhibitor is selected from any of the following.
  • the antibody that binds to BMP9 or the antibody fragment thereof (2) The antibody that binds to the ALK1 extracellular region polypeptide or the antibody fragment is a polyclonal antibody, a peptide antibody, a monoclonal antibody or a fragment thereof.
  • the pharmaceutical composition is a polyclonal antibody, a peptide antibody, a monoclonal antibody or a fragment thereof.
  • [5] Peptide in which the antibody fragment contains Fab, Fab ′, F (ab ′) 2, single chain antibody (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) and CDR The pharmaceutical composition of [3] or [4], which is an antibody fragment selected from [6]
  • [8] Diseases associated with vascular disorders include renal diseases, arteriosclerotic diseases, hypertension, pulmonary hypertension, heart disease, diabetes, diabetic complications, thrombosis, dyslipidemia and other lifestyle-related diseases, vasculitis
  • the pharmaceutical composition according to [7] which is selected from a disease accompanied by the disease or a disease shown to be possibly involved in BMP9.
  • the pharmaceutical composition of [8], wherein the renal disease is chronic glomerulonephritis including IgA nephropathy, diabetic nephropathy, lupus nephritis, nephrosclerosis or rapidly progressive glomerulonephritis.
  • Arteriosclerotic diseases include cerebrovascular disorders (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc.), ischemic heart disease (myocardial infarction, angina pectoris, etc.), aortic aneurysm, [8] The pharmaceutical composition according to [8], which is aortic dissection, nephrosclerosis or obstructive arteriosclerosis. [11] The pharmaceutical composition according to [8], wherein the heart disease is valvular heart disease, myocardial infarction, angina pectoris, cardiomyopathy.
  • the pharmaceutical composition according to [8], wherein the diabetic complication is arteriosclerosis, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic gangrene, chronic infection or cataract.
  • the thrombosis is pulmonary thromboembolism, cerebral infarction, myocardial infarction, acute limb thrombosis, intestinal necrosis (upper mesenteric artery thrombosis).
  • vasculitis Diseases associated with vasculitis are Takayasu's arteritis, giant cell arteritis (temporal arteritis), polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, Kawasaki disease, Henoch-Schönlein purpura
  • the pharmaceutical composition according to [8] which is hypersensitivity vasculitis, systemic lupus erythematosus, or rheumatoid arthritis.
  • liver disease acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, etc.
  • cancer with cancerous ascites / pleural effusion chronic Pancreatitis
  • allergic diseases allergic rhinitis, asthma, airway hypersensitivity, atopic dermatitis, etc.
  • inflammatory diseases delayed allergy, rheumatoid arthritis, arthritis, lung disease, hepatitis, ulcerative colitis, etc.
  • Alzheimer The pharmaceutical composition according to [8], which is multiple sclerosis, diabetic retinopathy, Raynaud's syndrome, Crohn's disease or cancer.
  • a method for treating a disease associated with a vascular disorder using the pharmaceutical composition according to any one of [1] to [15].
  • FIG. 1 is a diagram showing the neutralizing action of purified hsALK1Fc protein on BMP9.
  • FIG. 2 shows gene expression analysis using normal human vascular endothelial cells supplemented with BMP9.
  • a vascular endothelial disorder marker gene and lifestyle diseases hypertension, various renal diseases, diabetes, diabetic complications, (Dyslipidemia, arteriosclerotic disease, thrombosis) related gene is a diagram showing that the expression changes.
  • FIG. 3 is a diagram showing the structure of an N-terminal His-tagged mBMP9 complex recombinant expression vector.
  • FIG. 4 is a diagram showing an SDS-PAGE electrophoretic image of a purified N-terminal His-type mBMP9 complex purified sample by CBB staining.
  • FIG. 4 is a diagram showing an SDS-PAGE electrophoretic image of a purified N-terminal His-type mBMP9 complex purified sample by CBB staining.
  • FIG. 5 is a diagram showing analysis results of systolic blood pressure, diastolic blood pressure, mean blood pressure, and heart rate in rats administered with N-terminal His-type mBMP9 complex protein in a rat telemetry test.
  • FIG. 6 shows the results of blood biochemical analysis using sera of 6 control chimeric mice and 12 UShBMP9 KI chimeric mice reaching 5 weeks of age.
  • FIG. 7 is a diagram showing the structure of an N-terminal His-tagged hBMP9 complex recombinant expression vector.
  • FIG. 8 is a diagram showing an SDS-PAGE electrophoretic image of the N-terminal His-type hBMP9 complex purified preparation by silver staining.
  • FIG. 8 is a diagram showing an SDS-PAGE electrophoretic image of the N-terminal His-type hBMP9 complex purified preparation by silver staining.
  • FIG. 9 shows serum biochemical data of individual mice administered with hBMP9 complex at 100 ⁇ g / head or 360 ⁇ g / head.
  • FIG. 10 shows the results of conducting a biomarker survey (RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen)) using mouse serum administered with hBMP9 complex. Each value (times) at 100 ⁇ g / head or 360 ⁇ g / head indicates a variation ratio with respect to each control group.
  • FIG. 11 is a diagram showing the platelet number lowering action of the BMP9 neutralizing antibody.
  • FIG. 12 shows data obtained when a biomarker survey (RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen)) was performed using mouse serum administered with a BMP9 neutralizing antibody. Each value (times) is calculated by setting the variation ratio in the neutralizing antibody administration group to 1 in the control group.
  • FIG. 13 is a diagram showing an SDS-PAGE electrophoretic image of the purified hsALK1mFc preparation by CBB staining.
  • FIG. 14 is a diagram showing the lowering effect on neutral fat (triglycerol) and total cholesterol of the BMP9 neutralizing antibody found in an in vivo test using SHR-ND rats.
  • FIG. 15 is a diagram showing an inhibitory action on cardiac hypertrophy possessed by a BMP9 neutralizing antibody found from an in vivo test using SHR-ND rats. The heart weight value was corrected using the body weight value of each individual.
  • FIG. 16 is a view showing an inhibitory action of an ALK1 antagonist containing a BMP9 neutralizing antibody on plaque formation, which is a main lesion of arteriosclerosis. The figure shows the plaque area ratio in the aorta of APOE-deficient mice.
  • FIG. 17 shows the antihypertensive action of ALK1 extracellular region polypeptide-Fc fusion protein. The figure shows the systolic blood pressure of SHR rats implanted with telemetry equipment.
  • FIG. 18 is a diagram showing the inhibitory effect of BMP9 neutralizing antibody on the progression of nephropathy found from an in vivo test using a WKY anti-GBM nephritis model. The figure shows the change in serum BUN over time.
  • the present invention provides a pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient.
  • the ALK1 inhibitor contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is a substance that can suppress ALK1 activation, that is, activation of ALK1 kinase activity and suppress ALK1 signal.
  • the ALK1 inhibitor used in the present invention is not particularly limited, and examples thereof include the following (1) to (10): (1) an antibody that binds to BMP9 or an antibody fragment thereof; (2) ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof; (3) A nucleic acid encoding an ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof; (4) an expression vector comprising a nucleic acid encoding an ALK1 extracellular region polypeptide or a variant thereof, or a protein comprising the ALK1 extracellular region polypeptide or a variant thereof; (5) an antibody or an antibody fragment thereof that binds to an ALK1 extracellular region polypeptide; (6) an aptamer for BMP9 or an aptamer for ALK1 extracellular region polypeptide; (7) RNAi molecule targeting ALK1 gene or RNAi molecule targeting
  • the ALK1 inhibitor used in the present invention is particularly preferably an antibody that binds to BMP9 or an antibody fragment thereof that has neutralizing activity.
  • the ALK1 inhibitor that can be included in the pharmaceutical composition of the present invention is described below.
  • BMP9 antibody The antibody that binds to BMP9 is preferably a neutralizing antibody and can be prepared by the following method.
  • BMP9 is known and isolated from humans, mice, Japanese zelkova, etc., and sequence information is disclosed in databases such as GenBank.
  • human BMP9 is registered with GenBank as accession numbers Q9UK05, AAD56960, etc.
  • mouse BMP9 is registered with GenBank as accession numbers Q9WV56, NP_062379, etc., and these can be used.
  • the BMP9 protein a BMP9 protein having a known amino acid sequence registered in GenBank or the like can be used.
  • the BMP9 protein has an amino acid sequence represented by SEQ ID NO: 10 (mouse BMP9) or SEQ ID NO: 24 (human BMP9).
  • BMP9 protein particularly preferably BMP9 protein having the amino acid sequence shown in SEQ ID NO: 24 is used.
  • sequences represented by SEQ ID NO: 10 and SEQ ID NO: 24 include a signal sequence and pro-region.
  • SEQ ID NO: 10 the 319th to 428th sequences correspond to mature parts, and in SEQ ID NO: 24, The 320th to 429th arrays correspond to mature parts.
  • BMP9 includes a variant of BMP9. Such mutants include both natural mutants and artificial mutants.
  • amino acid sequence of BMP9 at least 319 to 428 of SEQ ID NO: 10, or at least 320 to 429 of SEQ ID NO: 24 1 to several amino acid deletions, substitutions, additions or insertions are included in the amino acid sequence having the sequence shown in the second sequence, or 80% or more, preferably 85% or more, more preferably It has an amino acid sequence having 90% or more, for example, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity.
  • the range of “1 to several” is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 1. Three, or one or two.
  • Antigen epitopes for obtaining the target antibody include regions having high antigenicity in the amino acid sequence of BMP9, regions having superficiality, regions that may not have secondary structure, homology with other proteins, or A low region can be selected.
  • the region having high antigenicity can be estimated by the method of Parker et al. [Biochemistry, 25, 5425-5432 (1986)].
  • the superficial region can be estimated by calculating and plotting a hydropathic index, for example. Regions that may not have secondary structure are described, for example, by the method of Chou and Fasman [Adv Enzymol Relat Areas Mol Biol. 47, 45-148 (1978)].
  • a peptide comprising the amino acid sequence can be synthesized by using a peptide synthesis method.
  • peptides of interest include R.I. B. Synthesized using a commercially available peptide synthesizer based on solid phase peptide synthesis developed by Merrifield [Science, 232, 341-347 (1986)], and after removing the protecting group, ion exchange chromatography , Purification by gel filtration chromatography, reverse phase chromatography, etc., alone or in combination.
  • the obtained purified peptide can be used as an immunogen by binding to a carrier protein such as keyhole limpet hemocyanin (KLH) or albumin.
  • KLH keyhole limpet hemocyanin
  • a polyclonal antibody or a monoclonal antibody that binds to BMP9 using BMP9 as an immunogen can also be prepared by a known technique.
  • the term “recombinant” used for BMP9 or a monoclonal antibody, a polyclonal antibody, or other proteins means that these proteins are produced by recombinant DNA in a host cell. Means.
  • a host cell any of prokaryotes (for example, bacteria such as E. coli) and eukaryotes (for example, yeast, CHO cells, insect cells, etc.) can be used.
  • the “antibody” of the present invention may be a peptide antibody, a polyclonal antibody, or a monoclonal antibody.
  • Antibodies produce antibodies that will specifically bind to proteins used for immunization of mice or other suitable host animals (eg, rabbits, cows, horses, sheep, pigs, rodents, etc.). Alternatively, it can be obtained by immunizing an antigen or antigen-expressing cell by subcutaneous, intraperitoneal, or intramuscular route to extract lymphocytes that would be produced. Further, as a host animal, a desired humanized antibody may be obtained by administering an antigen or an antigen-expressing cell to a transgenic animal having a repertoire of human antibody genes [Proc. Natl. Acad. Sci.
  • antibodies can be obtained by immunizing lymphocytes in vitro.
  • a polyclonal antibody can be obtained by collecting and purifying fractions that bind to the antigen from serum obtained from the host animal.
  • monoclonal antibodies can be prepared by fusing lymphocytes with myeloma cells using a suitable fusion reagent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principals. and Practice, 59-103, Academic press, (1986)].
  • the monoclonal antibody of the present invention can be prepared using the hybridoma method [Nature, 256, 495 (1975)] or using the recombinant DNA method (Cabilly et al., US Pat. No. 4,816,567). it can.
  • the antigen protein can be prepared by expressing DNA encoding all or a partial sequence of the BMP9 protein in E. coli, yeast, insect cells, animal cells and the like.
  • Recombinant BMP9 is purified by a method such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography or the like alone or in combination, and this purified preparation is used as an immunogen.
  • the antibody of the present invention may be an intact antibody or an antibody fragment such as (Fab ′) 2 or Fab.
  • chimeric antibodies or mosaic antibodies in which the constant region is replaced with a human constant region eg, mouse-human chimeric antibody; Cabilly et al., US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 89, 6851 (1984)
  • Fully human antibodies are also included in the antibodies of the present invention.
  • the use of chimeric antibodies, mosaic antibodies or humanized antibodies is preferred in order to prevent the appearance of antibodies against heterologous antigens, such as human anti-mouse antibodies (HAMA), Most preferred is a fully human antibody.
  • BMP9 antibody fragment In the present invention, examples of the antibody fragment include Fab, F (ab ′) 2, Fab ′, scFv, diabody, dsFv, and a peptide containing CDR.
  • Fab is a fragment obtained by treating IgG with papain, a proteolytic enzyme (cleaved at the 224th amino acid residue of the H chain), about half of the N chain side of the H chain and the entire L chain are disulfides. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000 bound by binding.
  • the Fab of the present invention can be obtained by treating a monoclonal antibody that binds to BMP9 with papain.
  • a Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. it can.
  • F (ab ′) 2 was obtained by decomposing the lower part of two disulfide bonds in the hinge region of IgG with pepsin, a proteolytic enzyme, and was constructed by binding two Fab regions at the hinge portion. It is a fragment having an antigen binding activity with a molecular weight of about 100,000.
  • F (ab ′) 2 of the present invention can be obtained by treating a monoclonal antibody that binds to BMP9 with pepsin.
  • Fab ′ described below can be produced by thioether bond or disulfide bond.
  • Fab ′ is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2.
  • the Fab ′ of the present invention can be obtained by treating F (ab ′) 2 binding to BMP9 with a reducing agent such as dithiothreitol.
  • DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the vector is introduced into prokaryotic or eukaryotic cells to express Fab ′.
  • VH-P-VL or VL-P-VH polypeptide which is an antibody fragment having antigen-binding activity.
  • the scFv of the present invention obtains cDNAs encoding monoclonal antibodies VH and VL that bind to BMP9, constructs a DNA encoding scFv, and inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector.
  • the expression vector can be expressed and produced by introducing the expression vector into a prokaryotic or eukaryotic organism.
  • Diabody is an antibody fragment in which scFv is dimerized and is an antibody fragment having a bivalent antigen-binding activity.
  • the bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.
  • the diabody of the present invention obtains cDNA encoding VH and VL of the monoclonal antibody that binds to BMP9, constructs the DNA encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less,
  • the DNA can be expressed and produced by inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are bonded via a disulfide bond between the cysteine residues.
  • the amino acid residue to be substituted with the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method [Protein Engineering, 7, 697 (1994)].
  • the dsFv of the present invention obtains cDNAs encoding the monoclonal antibodies VH and VL that bind to the BMP9 of the present invention, constructs a DNA encoding the dsFv, and the DNA is expressed in a prokaryotic expression vector or eukaryotic expression. It can be expressed and produced by inserting into a vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR is composed of at least one region of CDR of VH or VL.
  • Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • the peptide containing the CDR of the present invention constructs DNA encoding the VH and VL CDRs of the monoclonal antibody that binds to BMP9 of the present invention, and inserts the DNA into a prokaryotic expression vector or a eukaryotic expression vector. It can be expressed and produced by introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc t method.
  • ALK1 is a type I cell surface receptor for transforming growth factor ⁇ receptor type 1 (TGF- ⁇ -1).
  • TGF- ⁇ -1 transforming growth factor ⁇ receptor type 1
  • Human ALK1 is a polypeptide of 503 amino acids, which includes a signal sequence (amino acids: 1 to 21), an N-terminal extracellular TGF- ⁇ -1 ligand binding domain, or an extracellular domain (amino acids 22 to 22).
  • Attisano et al. Cell, 1993, vol. 75, pp. 671-680 includes human ALK1 amino acid sequence containing serine at position 172 (Genbank accession number L17075), and US Pat. No. 6,316,217 discloses human ALK1 amino acid sequence having threonine at position 172. (Genbank accession number NM — 000020) is disclosed.
  • the “ALK1 extracellular region polypeptide” is a protein consisting of all or part of the amino acid sequence of the ALK1 extracellular region polypeptide derived from a mammal.
  • the amino acid sequence and cDNA sequence of human ALK1 extracellular region polypeptide are as follows. Amino acid sequence of human ALK1 extracellular region polypeptide (SEQ ID NO: 29): (Genbank ACCESSION No.
  • the underline indicates the signal sequence, and the encircled line indicates the extracellular region polypeptide after cleavage of the signal sequence.
  • CDNA sequence of human ALK1 extracellular region polypeptide SEQ ID NO: 30: In the present invention, the ALK1 extracellular region polypeptide also encompasses variants of the above ALK1 extracellular region polypeptide.
  • Such mutants include both natural mutants and artificial mutants, and substitution of one or more (preferably one or several) amino acids in the amino acid sequence of the ALK1 extracellular region polypeptide, Deletion or addition, or 80% or more, preferably 85% or more, more preferably 90% or more, such as 93% or more, 95% or more, 97% or more, 98% or more, or 99% with the amino acid sequence It contains an amino acid sequence having the above identity and has vascular disorder inhibitory activity.
  • the variant includes one or more (preferably one or several) amino acid substitutions, deletions or additions in the amino acid sequence of SEQ ID NO: 29, or 80% or more of the amino acid sequence.
  • vascular disorder inhibitory activity Preferably 85% or more, more preferably 90% or more, for example, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, and an vascular disorder inhibitory activity. It is what has.
  • the term “several” as used herein generally refers to any integer from 2 to 10. Preferably it is any integer from 2 to 5.
  • the term “identity” refers to the alignment of two amino acid sequences (or nucleotide sequences) so that the number of identical amino acid residues (or nucleotides) is maximized.
  • a protein having a sequence identity of 80% or more, preferably 85% or more accesses a sequence database such as NCBI (US) or EMBL (Europe) and uses a sequence homology search program such as BLAST or FASTA. (Altschul, SF et al. (1990) J. Mol. Biol.
  • BLAST breaks the sequence into fixed-length words, searches for similar fragments in word units, stretches them in both directions until the degree of similarity is maximized, performs local alignment, and finally combines them into the final It is a method of performing a general alignment.
  • FASTA searches for fragments of sequences that match continuously at high speed, performs local alignment by focusing on those fragments that have high similarity, and finally considers these gaps. Is an alignment method.
  • a site-directed mutagenesis method using a PCR method using a primer (including a complementary mutant sequence) synthesized based on the sequence of the ALK1 extracellular region polypeptide is preferable (Kunkel et al., Proc Natl.Acad.Sci.USA, 1985, 82: 488-492; F.M. Ausubel et al., Short Protocols in Molecular Biology, 1995, John Wiley & Clones, J. Sambrook et al., Molecular A: Mol. ed., 1989, Cold Spring Harbor Laboratory Press).
  • Mutation introduction kits for example, manufactured by Takara Shuzo Co., Ltd. are also commercially available, and mutations can be introduced according to the instructions.
  • the template is annealed to the template, DNA synthesis is performed, and the ends are ligated with T4 DNA ligase to purify the DNA containing the target mutation.
  • the mutation includes substitution, deletion, addition, insertion, or a combination thereof.
  • substitution may be either a conservative substitution or a non-conservative substitution, but a conservative substitution is preferred so as not to substantially change the conformation of the ALK1 extracellular region polypeptide.
  • Conservative substitutions can be made between amino acids with similar chemical and physical properties such as structural (eg, branched, aromatic, etc.), electrical (eg, acidic, basic, etc.), polar or hydrophobic, etc. Refers to replacement.
  • Branched amino acids include valine, leucine and isoleucine.
  • Aromatic amino acids include tyrosine, tryptophan, phenylalanine, histidine.
  • Acidic amino acids include glutamic acid and aspartic acid.
  • Basic amino acids include lysine, arginine, and histidine.
  • Polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, cysteine, glycine, proline and the like.
  • Hydrophobic amino acids include alanine, valine, leucine, isoleucine, methionine and the like.
  • Deletion is the loss of one or more amino acid residues.
  • Addition is the attachment of one or more amino acid residues to the N-terminus or C-terminus of the protein.
  • Insertion is the joining of one or more amino acid residues inside a protein.
  • deletion and insertion can be performed on the assumption that the conformation of the ALK1 extracellular region polypeptide is not substantially changed. Therefore, it is preferably limited to deletion or insertion of about 1 to 5 amino acid residues.
  • the protein containing the ALK1 extracellular region polypeptide or a variant thereof is a protein containing the above ALK1 extracellular region polypeptide or a variant thereof, and is not limited to the ALK1 extracellular region polypeptide or a variant thereof.
  • a fusion protein of a mammal and a mammal-derived immunoglobulin Fc protein or a variant thereof for example, a fusion protein described in WO 2008/057461.
  • the expression “comprising” is necessary for the ALK1 extracellular region polypeptide or variant thereof and the heterologous peptide, polypeptide or protein on the N-terminal or C-terminal side of the domain or variant thereof.
  • a heterologous protein is a mammal-derived immunoglobulin Fc protein or a variant thereof.
  • a preferred Fc protein is a human immunoglobulin Fc protein for use in humans.
  • the class and subclass of the immunoglobulin are not limited to the following, but any of IgG, IgD, IgE, IgM, IgA, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgA1, IgA2, etc. Can be used. However, for use in humans, it is desirable to use human immunoglobulin classes and subclasses.
  • the Fc protein can improve the in vivo stability of the ALK1 extracellular domain or a variant thereof. In this case, however, the Fc protein exhibits biological activities such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities in order to avoid in vivo effects due to its biological activity.
  • ADCC antibody-dependent cytotoxicity
  • CDC complement-dependent cytotoxicity
  • a preferred example of the Fc protein is a human IgG1 Fc variant represented by the amino acid sequence of SEQ ID NO: 31 below.
  • SEQ ID NO: 31 The binding position of the Fc protein may be either the N-terminal side or the C-terminal side of the ALK1 extracellular region polypeptide or a variant thereof, but the C-terminal side is preferred.
  • a specific example of the fusion protein of the ALK1 extracellular region polypeptide and the Fc protein is a protein represented by the amino acid sequence of SEQ ID NO: 32 below, for example.
  • the underlined portion indicates the ALK1 extracellular region polypeptide (SEQ ID NO: 29), and the non-underlined portion indicates the human IgG1 Fc variant protein (SEQ ID NO: 31).
  • a protein containing an ALK1 extracellular region polypeptide or a variant thereof does not necessarily have to be bound or fused with a heterologous peptide, polypeptide or protein.
  • the protein containing the ALK1 extracellular region polypeptide or a variant thereof in the present invention can be prepared by a gene recombination technique commonly used in this industry.
  • the protein is prepared by preparing a DNA encoding the protein of the present invention, constructing an expression vector containing the DNA, transforming or transfecting prokaryotic or eukaryotic cells with the vector, Recovering the desired recombinant protein from the cultured cells.
  • Protein purification is performed by appropriately combining conventional protein purification methods such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and HPLC. Is possible.
  • ⁇ ALK1 extracellular region polypeptide or variant thereof or nucleic acid encoding ALK1 extracellular region polypeptide or protein containing the variant> and ⁇ ALK1 extracellular region polypeptide or its Since it is described in the section, Examples, etc. of the expression vector> containing the nucleic acid encoding the mutant or the ALK1 extracellular region polypeptide or the protein containing the mutant, it can be referred to.
  • gene recombination techniques are described in Ausubel et al. (Above), Sambrook et al. (Above), and can be used for the present invention.
  • the protein containing the ALK1 extracellular region polypeptide or variant thereof in the present invention may be chemically modified.
  • Chemical modifications include, but are not limited to, for example, glycosylation, PEGylation (PEG), acetylation, amidation, phosphorylation and the like. Particularly preferred chemical modifications that can be utilized are glycosylation and pegylation.
  • Pegylation is the attachment of one or more polyethylene glycol (PEG) molecules to amino acid residues such as the N-terminal amino group of proteins and the ⁇ -amino group of lysine (Lys).
  • PEG polyethylene glycol
  • Lys ⁇ -amino group of lysine
  • a PEG molecule is attached to the free amino group of an amino acid.
  • the average molecular weight of PEG is not limited to the following, but can be used in the range of about 3,000 to about 50,000.
  • the PEG end can be, for example, carboxylated, formylated, succinimidylated, or maleimidylated and reacted with the free amino group of the protein.
  • Glycosylation is the attachment of a carbohydrate chain (ie, sugar chain) to an asparagine, serine or threonine residue of a protein.
  • sugar chain binding occurs by recognizing the sequence of Asn-X-Thr / Ser (where X is any amino acid residue other than Pro).
  • a sugar chain can be introduced at a position different from the natural type.
  • a recombinant protein can be glycosylated by expressing a nucleic acid encoding the recombinant protein in a eukaryotic cell (yeast cell, animal cell, plant cell, etc.) by a genetic recombination technique.
  • a eukaryotic cell yeast cell, animal cell, plant cell, etc.
  • the sugar chain structure is not particularly limited, and it is considered that the sugar chain structure varies depending on the cell type selected for expression.
  • human-derived cells, yeast cells capable of synthesizing human sugar chains, Chinese hamster ovary (CHO) cells, and the like can be used. Acetylation and amidation are preferably performed mainly at the N-terminus or C-terminus of the protein.
  • nucleic acid includes both DNA and RNA.
  • DNA includes genomic DNA and cDNA
  • RNA includes mRNA.
  • the nucleic acid in the present invention includes the nucleic acid encoding the ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof, as explained and specifically exemplified above. .
  • the nucleic acid includes a nucleic acid encoding an amino acid sequence containing at least the 22nd to 118th amino acids in the amino acid sequence of the extracellular region polypeptide (SEQ ID NO: 29) of human ALK1.
  • the nucleic acid may further include a nucleotide sequence encoding a signal sequence. Examples of signal sequences are BMP9 signal sequence, Ig ⁇ and the like.
  • the nucleotide sequence encoding the precursor of the human-derived ALK1 extracellular region polypeptide is exemplified below.
  • the underlined site indicates a nucleotide sequence encoding a signal sequence
  • the non-underlined site indicates a nucleotide sequence encoding a mature sequence of an extracellular domain protein.
  • the nucleic acid in the present invention also includes a nucleic acid encoding a fusion protein of a protein containing an ALK1 extracellular region polypeptide or a variant thereof and a heterologous protein as defined above.
  • heterologous protein is an immunoglobulin Fc protein derived from a mammal, and a human Fc protein is particularly preferable, but it is desirable to introduce a mutation so as to reduce or lose its biological activity (particularly ADCC and CDC).
  • the nucleotide sequence encoding the mutant human IgG1-derived Fc protein is shown in SEQ ID NO: 33.
  • SEQ ID NO: 34 a nucleotide sequence (SEQ ID NO: 34) encoding a fusion protein of this mutant human IgG1-derived Fc protein (underlined portion) and a protein containing a human-derived ALK1 extracellular region polypeptide (non-underlined portion) is as follows: This is illustrated in SEQ ID NO: 34:
  • the nucleotide sequence encoding the fusion protein can further include a nucleotide sequence encoding a signal sequence. Examples of signal sequences are human protein-derived signal sequences such as BMP9 and Ig ⁇ .
  • Nucleic acid homologs encoding the above proteins can be obtained by using well-known techniques using primers and probes prepared based on cDNA synthesized from mRNA encoding ALK1 extracellular region polypeptide gene derived from human or mouse. It can be obtained from cDNA libraries prepared from other mammals and prepared from cells or tissues known to express the gene. Such techniques include PCR methods, hybridization methods (Southern method, Northern method, etc.) and the like. The PCR method is a polymerase chain reaction, which is a denaturing step (about 94 to 96 ° C., about 30 seconds to 1 minute) for dissociating double-stranded DNA into single strands, using a primer as a template.
  • Annealing step for binding to single-stranded DNA (about 55 to 68 ° C., about 30 seconds to 1 minute), extension step for extending DNA strand (about 72 ° C., about 30 seconds to about 30 seconds to 1 minute)
  • a cycle consisting of 1 minute) is regarded as one cycle, and about 25 to 40 cycles are carried out.
  • a preheating treatment is performed at about 94 to 95 ° C. for about 5 to 12 minutes, and after the final cycle of the extension step, an extension reaction can be further performed at 72 ° C. for about 7 to 15 minutes. it can.
  • PCR is performed with a commercially available thermal cycler using a thermostable DNA polymerase (for example, AmpliTaq Gold (registered trademark) (Applied Biosystems)), MgCl 2 , In a PCR buffer containing dNTP (dATP, dGTP, dCTP, dTTP), etc., in the presence of sense and antisense primers (size: about 17-30b, preferably 20-25b) and template DNA.
  • dNTP dATP, dGTP, dCTP, dTTP
  • sense and antisense primers size: about 17-30b, preferably 20-25b
  • template DNA size: about 17-30b, preferably 20-25b
  • the amplified DNA can be separated and purified (ethidium bromide staining) by agarose gel electrophoresis.
  • Hybridization is a technique for detecting a target nucleic acid by forming a double strand with a labeled probe having a length of about 20 to 100 b or more.
  • hybridization can generally be performed under stringent conditions.
  • the stringent conditions are, for example, about 1 to 5 ⁇ SSC, hybridization at room temperature to about 40 ° C., and then about 0.1 to 1 ⁇ SSC, 0.1% SDS, about 45 to 65 ° C. Consists of washing.
  • 1 ⁇ SSC refers to a solution of 150 mM NaCl, 15 mM Na-citric acid, pH 7.0.
  • nucleic acids with a sequence identity of about 80% or more, preferably 85% or more.
  • expression vector containing nucleic acid encoding ALK1 extracellular region polypeptide or variant thereof, or protein containing ALK1 extracellular region polypeptide or variant thereof The nucleic acid can be inserted into a vector and used to produce a protein that is an active ingredient of the pharmaceutical composition of the present invention, or the vector itself can be formulated and used as a pharmaceutical composition.
  • Vectors include, for example, plasmids, phages, viruses and the like. Examples of plasmids include, but are not limited to, E.
  • coli-derived plasmids eg, pRSET, pTZ19R, pBR322, pBR325, pUC118, pUC119, etc.
  • Bacillus subtilis-derived plasmids eg, pUB110, pTP5, etc.
  • yeast-derived plasmids eg, YEp13, YEp24, YCp50, etc.
  • Ti plasmids etc.
  • examples of phages include ⁇ phages
  • viral vectors include animal virus vectors such as retroviruses, vaccinia viruses, lentiviruses, adenoviruses, adeno-associated viruses, etc.
  • the vector may contain a polylinker or multicloning site for integrating the DNA of interest, and may contain several control elements to express the DNA.
  • the control element includes, for example, a promoter, an enhancer, a poly A addition signal, a replication origin, a selection marker, a ribosome binding sequence, a terminator and the like.
  • selectable markers include drug resistance genes (eg, neomycin resistance gene, ampicillin resistance gene, kanamycin resistance gene, puromycin resistance gene, etc.), auxotrophic complementary genes (eg, dihydrofolate reductase (DHFR) gene, HIS3 gene, LEU2 gene) URA3 gene, etc.).
  • the promoter may vary depending on the host cell.
  • host cells include, but are not limited to, bacteria such as Escherichia such as E. coli, Bacillus such as Bacillus subtilis, Pseudomonas such as Pseudomonas putida, Saccharomyces cerevisiae, Saccharomyces such as Schizosaccharomyces pombe And yeast such as Candida and Pichia, animal cells such as CHO, COS, HEK293, and NIH3T3, insect cells such as Sf9 and Sf21, and plant cells.
  • examples of the promoter include trp promoter, lac promoter, PL or PR promoter.
  • examples of the promoter include gal1 promoter, gal10 promoter, heat shock protein promoter, MF ⁇ 1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like.
  • examples of promoters include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV promoter, human CMV early gene promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, metallothionein promoter, polyhedron promoter, etc. Is done.
  • examples of promoters include CaMV promoter and TMV promoter.
  • Examples of transformation or transfection of host cells using expression vectors include electroporation, spheroplast, lithium acetate, calcium phosphate, Agrobacterium, virus infection, liposome, microinjection, Examples include gene gun method and lipofection method.
  • the transformed host is cultured under culture conditions according to the types of bacteria, yeast, animal cells, and plant cells, and the target protein is recovered from the cells or from the culture solution.
  • a medium containing a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by microorganisms is used.
  • carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, as nitrogen sources, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc.
  • Inorganic acids, ammonium salts of organic acids, peptone, meat extract, corn steep liquor, etc., as inorganic substances, potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, Manganese sulfate, copper sulfate, calcium carbonate and the like are used.
  • a medium in which DMEM medium, RPMI 1640 medium, or the like is used as a basic medium and fetal calf serum (FCS) or the like is added thereto is used.
  • FCS fetal calf serum
  • the target protein can be recovered by conventional methods for protein purification, such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, It can be carried out by HPLC or the like.
  • the vector is used for therapy, it is preferably a vector that is not integrated into the subject's genome and that infects cells but is unable to replicate, such as a non-viral vector.
  • Such vectors include, for example, adeno-associated virus vectors, adenovirus vectors and the like. These vectors can include promoters, enhancers, polyadenylation sites, selectable markers, reporter genes, and the like. Examples of viral vectors are described in J. Virol. 67: 5911-5921 (1993), Human Gene Therapy 5: 717-729 (1994), Gene Therapy 1: 51-58 (1994), Human Gene Therapy 5: 793-801 (1994), Gene Therapy 1: 165. 169 (1994) and the like, or improved vectors thereof.
  • an example of a non-viral vector is a human artificial chromosome vector, which is a vector composed of chromosome fragments containing human chromosome-derived centromeres and telomeres.
  • the human chromosome fragment is not particularly limited, but includes, for example, human chromosome 14 fragment, human chromosome 21 fragment (re-listed 2004/031385, Japanese Patent Application Laid-Open No. 2007-295860, etc.).
  • the nucleic acid as defined above is inserted into the vector and administered to the subject, or the vector is administered to the subject by introducing the vector into a tissue or cell collected from the subject and then returning to the subject. can do.
  • the antibody that binds to the ALK1 extracellular region polypeptide in the present invention is an antibody that can bind to the ALK1 extracellular region polypeptide, and is preferably a neutralizing antibody.
  • An antibody that inhibits the binding between ALK1 and ALK1 is preferred.
  • the antibody that binds to the ALK1 extracellular region polypeptide in the present invention includes, for example, an antibody described in WO 2007/40912.
  • An antibody that binds to the ALK1 extracellular region polypeptide or the antibody fragment can be prepared by using the ALK1 extracellular region polypeptide as an antigen protein by the technique specifically shown in the above section (BMP9 antibody). it can.
  • Aptamers are nucleic acid ligands that bind to specific molecules of proteins.
  • the aptamer in the present invention binds to the BMP9 protein or ALK1 extracellular region and can inhibit the function of the BMP9 protein or ALK1.
  • Aptamers can be obtained by preparing a library composed of various nucleic acid chains and selecting a nucleic acid chain that can bind to the target protein from the library. Suitable methods for identifying aptamers include, for example, Systematic Evolution of Ligands by Exponential Enrichment (SELEX). TM ) Method (US Pat. No. 5,270,163).
  • RNAi molecule targeting ALK1 gene or RNAi molecule targeting BMP9 gene An RNAi molecule is a single-stranded or double-stranded nucleic acid molecule comprising an antisense strand having a base sequence complementary to mRNA encoding a target protein. In the RNAi molecule, the antisense strand specifically binds to mRNA encoding the target protein and inhibits expression of the target protein (protein synthesis).
  • target means that the antisense strand contained in the RNAi molecule hybridizes with the target mRNA under stringent conditions.
  • the stringent conditions are, for example, about 1 to 5 ⁇ SSC, hybridization at room temperature to about 40 ° C., and then about 0.1 to 1 ⁇ SSC, 0.1% SDS, about 45 to 65 ° C. Consists of washing.
  • 1 ⁇ SSC refers to a solution of 150 mM NaCl, 15 mM Na-citric acid, pH 7.0.
  • the RNAi molecule in the present invention specifically binds to mRNA encoding BMP9 or ALK1, and inhibits its protein synthesis.
  • the RNAi molecule in the present invention includes antisense RNA, antisense DNA, siRNA, and shRNA.
  • siRNA is a low molecular double-stranded RNA obtained by hybridizing an antisense strand having a base sequence complementary to mRNA encoding a target protein and a sense strand complementary to the antisense strand.
  • shRNA is a single-stranded RNA in which the antisense strand and the sense strand are linked via a linker portion, and the linker portion is folded by forming a loop, and the antisense strand and the sense strand are Hybridizes to form a double stranded portion.
  • the RNAi molecule of the present invention can be designed based on the above-described BMP9 and ALK1 gene sequences, for example, using a siRNA Design Support System (Takara Bio Inc.).
  • a siRNA Design Support System TiRNA Design Support System (Takara Bio Inc.).
  • the low molecular weight compound having an inhibitory activity on the kinase activity of ALK1 in the present invention is not particularly limited as long as it is a compound known to inhibit ALK1 kinase activity.
  • WO 2007 / 147647 N- (2-dimethylamino-ethyl) -3- [5- (4-isopropyl-phenylamino) -pyrazolo [1,5-a] pyrimidine-3-yl] -benzamide Phenyl- [3- (3,4,5-trimethoxy-phenyl) -pyrazolo [1,5-a] pyrimidine-5-yl] -amine; (4-fluoro-phenyl)-[3- (3,4 , 5-trimethoxy-phenyl) -pyrazolo [1,5-a] pyrimidin-5-yl] -amine; N ′-[3- (3-chloro-phenyl) -pyrazolo [1 5-a] pyrimidin -5-yl] -N, N- diethyl - propane -e-1,3-diamine, but not limited to) can be mentioned.
  • the low molecular weight compound having an activity of inhibiting the binding between BMP9 and ALK1 or the binding between BMP9 and type II receptor is the inhibition of the binding between BMP9 and ALK1 or between BMP9 and type II receptor. It refers to a compound that can be produced and is not particularly limited.
  • Type II receptor includes BMP type II receptor (BMPRII), activin type IIa receptor (ActRIIa), and activin type IIb receptor (ActRIIb).
  • the low molecular weight compound included in the present invention may have an activity of inhibiting any one of the binding between BMP9 and BMPRII, the binding between BMP9 and ActRIIa, and the binding between BMP9 and ActRIIb. In addition, it may have an activity of inhibiting any two bonds, or may have an activity of inhibiting these three bonds. Preferable examples include low molecular weight compounds having an activity of inhibiting the binding between BMP9 and BMPRII.
  • ⁇ BMP9 antagonist protein having BMP9 inhibitory activity> Although no BMP9 antagonist protein having BMP9 inhibitory activity has been reported so far, various biologically derived BMP antagonists having BMP inhibitory activity have been reported for BMP, and such BMP9 antagonist is also present in BMP9. May exist.
  • Such a BMP9 antagonist can be used as an active ingredient of the pharmaceutical composition of the present invention.
  • the pharmaceutical composition of the present invention can be used for diseases associated with vascular disorders, such as renal diseases, arteriosclerotic diseases, hypertension, heart diseases, diabetes, diabetic complications, thrombosis, dyslipidemia, etc. Lifestyle-related diseases, diseases associated with vasculitis, and diseases in which BMP9 has been shown to be involved.
  • the renal disease is not particularly limited, and includes chronic glomerulonephritis including IgA nephropathy, diabetic nephropathy, lupus nephritis, nephrosclerosis or rapidly progressive glomerulonephritis.
  • Arteriosclerotic disease is not particularly limited, but ischemic heart such as cerebrovascular disorders (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc.), myocardial infarction and angina Diseases, aortic aneurysm, aortic dissection, nephrosclerosis, obstructive arteriosclerosis and the like can be mentioned.
  • the heart disease is not particularly limited, and examples thereof include valvular heart disease, myocardial infarction, angina pectoris, and cardiomyopathy.
  • Diabetic complications include arteriosclerosis, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic gangrene, chronic infection, cataract and the like.
  • the thrombosis is not particularly limited, and examples thereof include pulmonary thromboembolism, cerebral infarction, myocardial infarction, lower limb acute arterial thrombosis, intestinal necrosis (upper mesenteric artery thrombosis) and the like.
  • vasculitis Diseases associated with vasculitis are not particularly limited, but Takayasu arteritis, giant cell arteritis (temporal arteritis), polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, Kawasaki disease, Examples include Henoch-Schönlein purpura, hypersensitivity vasculitis, systemic lupus erythematosus, and rheumatoid arthritis.
  • Diseases that may be involved in BMP9 are liver disease, cancer with cancerous ascites / pleural effusion, chronic pancreatitis, allergic disease, inflammatory disease, Alzheimer, multiple sclerosis, diabetic retinopathy, Raynaud Syndrome, Crohn's disease, cancer, etc.
  • the allergic disease is not particularly limited, and examples include allergic rhinitis, asthma, airway hypersensitivity, and atopic dermatitis.
  • the inflammatory disease is not particularly limited, and examples thereof include delayed allergy, rheumatoid arthritis, arthritis, pulmonary disease, hepatitis, ulcerative colitis and the like.
  • the liver disease is not particularly limited, and examples include acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, and metastatic liver cancer.
  • the pharmaceutical composition of the present invention is preferably a renal disease, arteriosclerotic disease, hypertension, heart disease, diabetes, diabetic complications, thrombosis, dyslipidemia and other lifestyle diseases, particularly preferably renal disease, arteriosclerosis It can be applied to sex diseases, hypertension and thrombosis.
  • Chronic kidney disease (CKD) is considered to be the largest independent risk factor for cardiovascular complications (CVD), but the presence of arteriosclerosis is also recognized in the early stages of CKD.
  • CVD cardiovascular complications
  • the pharmaceutical composition of the present invention contains one or a combination of the above active ingredients.
  • the pharmaceutical composition of the present invention contains an antibody that binds to BMP9 or the antibody fragment as an active ingredient.
  • the amount of the active ingredient contained in the pharmaceutical composition of the present invention should be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the patient to be administered, and is not limited to the following, It can be appropriately determined within a range of about 0.1 ⁇ g / kg to 100 mg / kg, preferably within a range of about 1 ⁇ g / kg to 10 mg / kg.
  • the form (namely, preparation) of the pharmaceutical composition of the present invention is not limited, and includes both oral preparations and parenteral preparations.
  • a preferred form is a parenteral preparation, which includes, but is not limited to, an intravenous preparation, an intramuscular preparation, an intraperitoneal preparation, a subcutaneous preparation, a topical preparation and the like.
  • the parenteral preparation includes, for example, an injection, an instillation, a suppository, a transdermal absorption agent, a liposome or a nanoparticle-encapsulated preparation.
  • Oral preparations include, for example, tablets, pills, granules, capsules, powders, solutions, suspensions, delayed release preparations, enteric preparations and the like.
  • the pharmaceutical composition of the present invention can contain pharmaceutically acceptable excipients, carriers such as diluents, and additives.
  • Carriers include, for example, saline, glycerol, ethanol, almond oil, vegetable oil, sucrose, starch, lactose and the like.
  • Additives include, for example, binders (eg pregelatinized corn starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone etc.), lubricants (eg magnesium stearate, talc, silica etc.), dispersants (eg polyvinylpyrrolidone, corn starch etc.) ), Suspension (eg talc, gum arabic, etc.), emulsifier (eg lecithin, gum arabic, etc.), disintegrant (potato starch, sodium starch glycolate, crospovidone, etc.), buffer (eg phosphate, acetate) Citrate, tris salt, etc.), antioxidants (eg, ascorbic acid, tocopherol, etc.), preservatives (eg, sorbic acid, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate), isotonic agents (eg Eg sodium chloride) , And the like agents (eg, gly
  • enteric preparations for example, polymers such as hydroxypropyl methylcellulose phthalate, methacrylic acid-methyl methacrylate copolymer, methacrylic acid-ethyl acrylate copolymer, hydroxypropyl acetate succinate are used.
  • the dosage of the pharmaceutical composition of the present invention should be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the patient, and is not limited to the following, but for example, about 0.1 ⁇ g per adult day / Kg to 100 mg / kg, preferably about 1 ⁇ g / kg to 10 mg / kg.
  • Administration of the formulation may be administered daily during treatment or at intervals such as several days, two weeks or one month.
  • the pharmaceutical composition of the present invention can be administered to a patient by an administration method suitable for the type and dosage form of the active ingredient contained therein.
  • the pharmaceutical composition of the present invention is administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intranasally, intravaginally, intrarectally, topically, intramuscularly, orally.
  • Injection, infusion, cell transplantation of transformed cells expressing ALK1 extracellular region polypeptide or the like prepared using the above vector, or any combination of the above.
  • the pharmaceutical composition of the present invention contains any of the nucleic acids, expression vectors, aptamers or RNAi molecules described in detail above or a combination thereof as an active ingredient
  • the pharmaceutical composition is administered by using the active ingredient as a target tissue.
  • it can be performed using a technique or technique generally used in gene therapy that can be introduced into cells.
  • Gene delivery methods that can be used to administer the pharmaceutical composition of the present invention include colloidal dispersion systems, liposome-derived systems, artificial virus envelopes, and the like.
  • the delivery system should use macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, liposomes, calcium phosphate method, DEAE dextran method, electroporation method, lipofection method, etc. Can do.
  • ⁇ Evaluation of therapeutic agents> The effect of the pharmaceutical composition of the present invention can be evaluated by alleviating or eliminating the symptoms of the disease in a patient suffering from the disease that has received administration of the pharmaceutical composition.
  • the effect of the pharmaceutical composition of the present invention can be evaluated by genetic or biochemical analysis of genes, biomarkers and serum biochemical parameters that are known to be related to the above-mentioned diseases.
  • genes, biomarkers and serum biochemical parameters include (but are not limited to) the following: gene Biomarker Serum biochemistry parameters EXAMPLES
  • the present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
  • this application is a contract research and industrial technology related to “Development of biological system control infrastructure technology using compounds, etc.” in 2007, New Energy and Industrial Technology Development Organization. This is a patent application subject to Article 19 of the Strengthening Law.
  • hsALK1Fc expression vector is a PEAK8 expression vector obtained by enzymatic digestion of the extracellular region cDNA fragment of human ALK1 and the hIgG1 Fc region cDNA fragment with EcoRI and NotI (both from Roche Diagnostics, Japan). It was prepared by incorporating into (Edge Biosystems).
  • the cDNA sequence of the incorporated hsALK1Fc is as follows (SEQ ID NO: 1).
  • SEQ ID NO: 1 346 amino acids, SEQ ID NO: 2) is shown below.
  • SEQ ID NO: 2 Each fragment was obtained as follows.
  • the extracellular region cDNA of human ALK1 is hsALK1 FW: agaattccccaccatgaccttgtccccccag (SEQ ID NO: 3), hsALK1 RV: aacttagctgggcccatctgtttccccggctg (SEQ ID NO: 4) was cloned from a human lung cDNA library using the PCR method. The obtained fragment was enzymatically digested with EcoR1 and SpeI, and then electrophoresed on an agarose gel to cut out the band. For extraction of DNA from agarose, QIAquick Gel Extraction Kit (Qiagen, Japan) was used.
  • the FIgG region cDNA of hIgG1 is hIgG1 Fc FW: actagtgacaaaactcacacatgcc (SEQ ID NO: 5)
  • hIgG1 Fc RV gcggccgctcatcatttaccc (SEQ ID NO: 6)
  • the obtained fragment was enzymatically digested with SpeI and NotI (both Roche Diagnostics, Japan), and then electrophoresed on an agarose gel to cut out the band.
  • QIAquick Gel Extraction Kit Qiagen, Japan
  • the hsALK1Fc high expression strain was prepared by linearizing the expression vector prepared above using Sfi-I restriction enzyme and then converting the linearized DNA to Lipofectamine TM LTX. Using a reagent (Invitrogen, Japan), the gene was introduced into CHO Ras clone I cells according to the package insert. Twenty-four hours after gene transfer, drug-selected cells were selected using alpha MEM containing 6 ⁇ g / ml puromycin and 10% serum (FCS), and single clones were isolated by limiting dilution. It was.
  • Human IgG ELISA kit purchased from Japan Cosmo Bio Co., Ltd. was used for selection of high expression strains. Next, the obtained high expression strain was inoculated into a large flask of 225 cm 2 and cultured at 37 ° C. until it became confluent. After confirming that the cells were confluent, the cells were washed with PBS (Dulecco's Phosphate Buffered Saline; SIGMA), and then replaced with EX-Cell 302 (SAFC Biosciences) medium diluted to 25% in D / F medium. did.
  • PBS Dulecco's Phosphate Buffered Saline
  • EX-Cell 302 SAFC Biosciences
  • the pretreated culture supernatant was applied to a column equilibrated with PBS, washed with a PBS solution containing PBS and 1.85 M NaCl, and then eluted with 20 mM sodium citrate, 50 mM NaCl, pH 2.
  • the target protein was eluted using 7).
  • AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used.
  • the eluate was subjected to sample concentration while substituting the solvent with PBS using an ultrafiltration membrane VIVASPIN20 10,000 MWCO PES (Sartorius Stedim Japan, Japan). After concentration, sterilization was performed using a 0.22 ⁇ m filter (Millex GV; Japan Millipore, Japan).
  • hsALK1Fc activity measurement of purified hsALK1Fc protein in vitro For evaluation of hsALK1Fc activity, a cell line into which a reporter plasmid capable of detecting a BMP signal was stably introduced was used. Specifically, a reporter plasmid (p (GCCG) 12-Luc / neo) that can detect a BMP signal is introduced into a human liver cancer cell line HepG2 (available from ATCC), and the reporter plasmid is introduced. A stably transfected strain HepG2 (p (GCCG) 12-Luc / HepG2 (38-5)) was prepared.
  • p (GCCG) 12-Luc / HepG2 available from ATCC
  • the BMP signal detection reporter plasmid (p (GCCG) 12-Luc / neo) was linked in tandem with 12 binding sequences of Smad1 / 5/8, which is a BMP signaling factor, upstream of the luciferase gene.
  • Plasmid (p (GCCG) 12-Luc / neo) was obtained from Mol. Biol. Cell. 2000, 11 (2): 555-65. Were prepared according to the method described by Kusanagi et al. The neutralization activity of purified hsALK1Fc against BMP9 was measured as follows.
  • EBM endothelial basic medium-2
  • EBM containing 10 ng / ml BMP9 protein purchased from R & D Systems
  • registered registered medium containing only EBM (registered trademark) -2
  • n 2.
  • the supernatant was removed, washed carefully twice with PBS, and RNA was obtained according to the package insert of RNeasy mini kit (Qiagen, Japan). The obtained RNA was sent to Moritex Co., Ltd.
  • FIG. 2 The values in FIG. 2 are obtained by calculating the gene expression change (fold) due to the addition of BMP9 with the value of the BMP9 unstimulated sample as 1.
  • FIG. 2 it was revealed that the addition of BMP9 to vascular endothelial cells strongly induced expression of E-selectin and VCAM-1 which are markers for vascular endothelial injury (FIG. 2).
  • E-selectin and VCAM-1 which are markers for vascular endothelial injury
  • FIG. 2 strong expression induction of genes such as IL-8 and COX-2, which are considered to be involved in vascular disorders and inflammation, was also observed (FIG. 2).
  • SEQ ID NO: 8 pUSmBMP9 KI vector
  • the polynucleotide sequence from the start codon to the stop codon of the mBMP9 expression unit (the mouse BMP9 signal sequence was replaced with the mouse Ig ⁇ signal sequence [underlined portion] containing the intron region, and the mouse BMP9 pro body sequence was downstream of it.
  • Including 1522 bp, SEQ ID NO: 9) and the amino acid sequence encoded by the cDNA (426 amino acids, the boxed portion indicates the mouse Ig ⁇ signal sequence, SEQ ID NO: 10) are shown below.
  • Mouse Ig ⁇ signal sequence information including an intron region was obtained from the UCSC mouse genome database based on MUSIGKVR1 (accession number K02159) obtained from GenBank.
  • SEQ ID NO: 9 SEQ ID NO: 10
  • a USmBMP9 KI chimeric mouse that expresses mouse BMP9 in a B cell specific manner was prepared according to the examples of the pamphlet of International Publication No. WO 2006/78072. Further, mouse control individuals (control chimeric mice) used in Examples 5 and 6 below were prepared according to the method described in Example 11 of International Publication No. WO 2006/78072.
  • red coloration of mesenteric lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age. 5-1-4.
  • Reddish submandibular lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied.
  • reddening of the submandibular lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age. 5-1-5.
  • Reddish elbow lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied.
  • pancreas reddening 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, 2 individuals at 3 weeks of age, 5 individuals at 4 weeks of age, and 4 individuals at 5 weeks of age, redness was observed on a part of the pancreas surface. From the above results, it is clarified that the excessive action of BMP9 causes reddening of ascites, pleural effusion, lymphoid tissue, small intestine and pancreas, which is thought to be caused by hemorrhagic changes. It was confirmed not only in vitro but also in vivo. 5-2.
  • Vascular permeability test of USmBMP9 KI chimeric mouse Evans Blue (L.S., Inc., Japan) was prepared according to the method described in Example 4 and the control chimeric mouse and USmBMP9 KI chimeric mouse that reached 5 weeks of age. I. At a dose of 30 mg / kg. v. Administered. Sixty minutes later, the administered individuals were necropsied and vascular permeability was evaluated using ascites, pleural effusion and organ properties / changes as indicators. As a result, blue-colored ascites and pleural effusion were observed only in the USmBMP9 KI chimeric mice administered with Evans Blue.
  • N-terminal His-type mBMP9 complex recombinant 7-1 Construction of N-terminal His-type mBMP9 complex recombinant expression vector 7-1-1. Construction of pLN1V5 vector Sense oligo DNA having a BamHI / NheI / SalI site at the 5 ′ end and an XhoI site at the 3 ′ end (V5 tag + Stop codon) and its antisense oligo DNA: V5S and V5AS (Hokkaido System Science Co., Ltd., Japan) Company).
  • V5S GATCCGCTAGCGTCGACGGGTAAGCCCTCCTCAACCCTCTCCTGCGTCTCGATCTCGTGGAC (SEQ ID NO: 11)
  • V5AS TCGAGTCACGTAGAGATCGAGACCGAGGAGAGGGTTTAGGATAGGGCTTACCGTCGACGCTAGCG (SEQ ID NO: 12)
  • the synthetic oligo DNA was introduced into the BamHI-XhoI site on the pLN1 vector described in a report by Kakeda et al. (Gene Ther. 12: 852-856, 2005) to construct a pLN1V5 vector. 7-1-2.
  • Synthesis of mBMP9 DNA fragment having His tag sequence at N terminus PCR primer for adding His tag to N terminus of mouse full length sequence of BMP9 (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16)
  • a reaction solution was prepared according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan), SEQ ID NOS: 13 and 14, 10 pmol of each of two primers in a 50 ⁇ l reaction solution, mouse BMP9-cDNA (as a template) SEQ ID NO: 7) was added and incubated at 94 ° C. for 5 minutes, followed by 25 cycles of amplification at 98 ° C.
  • the resulting 101 bp amplified fragment was amplified Separated and recovered with 0.8% gel.
  • An amplified fragment (NheI His mBMP9) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • a reaction solution was prepared according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan), SEQ ID NOS: 15 and 16, 10 pmol of each of the two primers in a 50 ⁇ l reaction solution, mouse BMP9-cDNA (as a template) SEQ ID NO: 7) was added and incubated at 94 ° C. for 5 minutes, followed by 25 cycles of amplification at 98 ° C. for 10 seconds, 57 ° C. for 5 seconds, and 72 ° C. for 1 minute 20 seconds. The resulting 1249 bp amplified fragment was amplified Separated and recovered with 0.8% gel.
  • the amplified fragment (His mBMP9 SalI) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the DNA amplified fragments (NheI His mBMP9) and (His mBMP9 SalI) obtained by the above two PCRs were added to PrimeSTAR buffer to a total volume of 100 ⁇ l, heated to 100 ° C. for 10 minutes, returned to room temperature, and the His tag region was removed. Annealed. Thereafter, SEQ ID NOs: 13 and 16, 2 ps of each primer, 10 pmol each, Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan) were added, an extension reaction was performed at 72 ° C.
  • the enzyme-treated fragment was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the obtained enzyme-treated fragment was introduced into the NheI / SalI site of the pLN1V5 vector prepared in Example 7-1-1 to construct an N-terminal His-type mBMP9 complex recombinant expression vector (FIG. 3).
  • SEQ ID NO: 17 The following is a polynucleotide sequence (1305 bp, SEQ ID NO: 17) from the start codon to the stop codon of the N-terminal His-type mBMP9 complex recombinant cDNA, and an amino acid sequence (434 amino acids, including the signal sequence of mBMP9 encoded by the cDNA) SEQ ID NO: 18) is shown.
  • the underlined portion represents the signal sequence portion of mouse BMP9
  • the boxed portion represents the histidine tag portion
  • italic represents the mouse BMP9 pro body portion.
  • SEQ ID NO: 17 SEQ ID NO: 18 (including mBMP9 signal sequence (underlined portion)) 7-2.
  • N-terminal His type mBMP9 complex using N-terminal His type mBMP9 complex recombinant expression vector 7-2-1.
  • the N-terminal His-type mBMP9 complex recombinant expression vector obtained in 1) was introduced into E. coli DH5 ⁇ , and DNA was prepared from the resulting transformant using a plasmid purification kit (Qiagen plasmid Maxi kit; Qiagen, Japan). . 7-2-2.
  • Free style 293F cells (Invitrogen, Japan) were used with Free style 293 Expression Medium (Invitrogen, Japan) at 37 ° C, 5% CO 2 , 125 rpm, cell density Were cultured in the range of 1 ⁇ 10 5 to 3 ⁇ 10 6 cells / ml.
  • 35 ml of Opti-MEM I Reduced Serum Medium (Invitrogen, Japan) was added to 1 mg of the expression vector, and 33 ml of 1.3 ml of 293fectin Transfection Reagent (Invitrogen, Japan) was added.
  • Opti-MEM I Reduced Serum Medium 7 ml of Opti-MEM I Reduced Serum Medium was added and incubated at room temperature for 5 minutes each. After incubation, the two solutions were mixed and incubated for another 20-30 minutes at room temperature. Thereafter, the expression vector treated by the above method was added to a medium containing 1 ⁇ 10 9 cells / L Free style 293F cells and cultured for 3 days. 7-3. Purification and preparation of N-terminal His-type mBMP9 complex 7-3-1. Pretreatment of culture supernatant After culture, the supernatant was collected, filtered through a 0.22 ⁇ m filter (0.22 ⁇ m GP Express Membrane 500 ml; Japan Millipore, Japan), and then cooled at 4 ° C. (cold room).
  • Example 7-3 The operation was performed in a clean bench as much as possible. All the processes performed in Example 7-3 were performed in a low temperature room (+ 4 ° C.) or on ice, except for work on a clean bench. SDS-PAGE (CBB staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and a small amount under reducing conditions. Pro monomer was detected (FIG. 4). From this result, it was considered that the purified preparation prepared by the above operation mainly contains a complex (complex body) in which two molecules of pro-region and one molecule of molecule dimer are bound.
  • CBB staining SDS-PAGE (CBB staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and
  • Endotoxin-containing PBS was intravenously administered as a control solution to two rats implanted with a transmitter and reached 11 weeks of age, and the telemetry system Dataquest A.
  • R. T. T. was used to measure systolic blood pressure, diastolic blood pressure, mean blood pressure and heart rate, and it was confirmed that there was no blood pressure fluctuation by administration of endotoxin-containing PBS.
  • the N-terminal His-type mBMP9 complex body prepared by the method of Example 7 was intravenously administered so as to have a dosage of 0.5 mg / kg. Minimum blood pressure, mean blood pressure and heart rate were measured.
  • SEQ ID NO: 20 pUShBMP9 KI vector
  • the polynucleotide sequence from the start codon to the stop codon of the hBMP9 expression unit (the human BMP9 signal sequence was replaced with the mouse Ig ⁇ signal sequence [underlined portion] containing the intron region, and the human BMP9 pro body sequence was downstream of it.
  • Including 1525 bp, SEQ ID NO: 21) and the amino acid sequence encoded by the cDNA (427 amino acids, the boxed portion indicates the mouse Ig ⁇ signal sequence, SEQ ID NO: 22) are shown below.
  • Mouse Ig ⁇ signal sequence information including an intron region was obtained from the UCSC mouse genome database based on MUSIGKVR1 (accession number K02159) obtained from GenBank.
  • Serum biochemical test in UShBMP9 KI chimeric mice Using the serum collected from the posterior vena cava of control chimera mice and UShBMP9 KI chimeric mice, which were prepared according to the method described in Example 9 above, Blood biochemistry analysis was performed with an analyzer 7180 (manufactured by HITACHI). As a result, it was found that the serum LDH, BUN, LDL cholesterol, GOT, and sodium values of the UShBMP9 KI chimeric mice were significantly higher than those of the control chimeric mice (FIG. 6). In addition, a significant decrease in serum albumin and serum total protein was also observed (FIG. 6).
  • BUN is a diagnostic marker for kidney disease and is associated with chronic nephritis, edema, obstructive urinary tract disease, LDL cholesterol is dyslipidemia and arteriosclerotic disease (angina, acute coronary syndrome, myocardial infarction, Serum LDH and GOT are associated with acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, acute myocardial infarction, heart failure, serum sodium It is known that an increase is associated with hypertension, and a decrease in serum albumin and serum total protein is known to be associated with liver dysfunction and nephrotic syndrome. It was suggested.
  • anti-BMP9 therapy is chronic nephritis, edema, obstructive urinary tract disease, dyslipidemia, arteriosclerotic disease, acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, acute myocardial infarction, heart failure It has been shown that it may be an effective treatment for diseases such as nephrotic syndrome and hypertension.
  • N-terminal His-type hBMP9 complex recombinant 12-1 Construction of N-terminal His-type hBMP9 complex recombinant expression vector 12-1-1. Synthesis of hBMP9 DNA fragment having His tag sequence at N-terminus PCR primer for adding His tag to N-terminus of full-length human BMP9 sequence (SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26) Prepare a reaction solution according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan).
  • An amplified fragment (His hBMP9 XhoI) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the DNA amplified fragments (NheI His hBMP9) and (HisBMP9 XhoI) obtained by the above two PCRs were added to PrimeSTAR buffer to a total volume of 100 ⁇ l, heated to 100 ° C. for 10 minutes, returned to room temperature, and the His tag region was removed. Annealed. Thereafter, SEQ ID NOs: 23 and 26, 10 pmol each of two kinds of primers, Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan) were added, an extension reaction was performed at 72 ° C.
  • the enzyme-treated fragment was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
  • the obtained enzyme-treated fragment was introduced into the NheI / XhoI site of the pLN1V5 vector to construct an N-terminal His-type hBMP9 complex recombinant expression vector (FIG. 7).
  • SEQ ID NO: 27 The following is a polynucleotide sequence (1308 bp, SEQ ID NO: 27) from the start codon to the stop codon of the N-terminal His-type hBMP9 complex recombinant cDNA, and an amino acid sequence (435 amino acids, including the signal sequence of hBMP9 encoded by the cDNA) SEQ ID NO: 28).
  • the underlined portion indicates the signal sequence portion of human BMP9
  • the surrounded line indicates the histidine tag portion
  • the italic type indicates the human BMP9 pro body portion.
  • SEQ ID NO: 27 SEQ ID NO: 28 (including hBMP9 signal sequence [ underlined portion ]): 12-2.
  • N-terminal His-type hBMP9 complex using N-terminal His-type hBMP9 complex recombinant expression vector 12-2-1.
  • the N-terminal His-type hBMP9 complex recombinant expression vector obtained in (1) was introduced into Escherichia coli DH5 ⁇ , and DNA was prepared from the obtained transformant using a plasmid purification kit (Qiagen plasmid Maxi kit; Qiagen, Japan). . 12-2-2.
  • Free style 293F cells (Invitrogen, Japan) were used with Free style 293 Expression Medium (Invitrogen, Japan) at 37 ° C, 5% CO 2 , 125 rpm, cell density Is cultured in the range of 1 ⁇ 10 5 to 3 ⁇ 10 6 cells / ml.
  • Opti-MEM I Reduced Serum Medium (Invitrogen, Japan) was added to 1 mg of the expression vector, and 33.7 ml in 1.3 ml of 293fectin Transfection Reagent (Invitrogen, Japan). Of Opti-MEM I Reduced Serum Medium was added and incubated at room temperature for 5 minutes each.
  • PBS Dulecco's Phosphate Buffered Saline; SIGMA
  • a buffer PBS containing 0.5 M Imidazole
  • B buffer PBS containing 0.5 M Imidazole
  • the pretreated 1 L culture supernatant was applied to a Ni Sepharose column (His Trap HP 5 ml; GE Healthcare Biosciences, Japan) equilibrated with PBS. Thereafter, the column was washed in the order of 25 ml of 0% B buffer, 25 ml of buffer solution in which NaCl was added to PBS to adjust the NaCl concentration to 1.85 M, 25 ml of 0% B buffer, 30 ml of 9% B buffer, and 40 ml of 11% B buffer.
  • Example 12-3-2 Ion Exchange Chromatography A buffer was prepared by adding NaCl to PBS as A buffer and adjusting NaCl concentration to 1.85 M by adding NaCl to PBS as B buffer. The purpose obtained in Example 12-3-2 on a strong anion exchange column equilibrated with PBS (Hi Trap Q HP 1 mL; GE Healthcare Biosciences, Japan) under a flow rate of 1 mL / min Protein was added.
  • the column was washed sequentially with 20 ml of 0% B buffer and 10 ml of 2% B buffer. After the washing operation, 20 ml of 7% B buffer was applied to the column, and the target protein was recovered.
  • AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used. Endotoxin removal treatment was performed before use. 12-3-4.
  • Purified sample preparation After replacing the solvent in the purified sample obtained in Example 12-3-3 with PBS using an ultrafiltration membrane VIVASPIN20 10,000 MWCO PES (Sartorius Stedim Japan, Japan), The sample was concentrated. After the concentration operation, filtration was performed with a 0.22 ⁇ m filter (Millex GV; Japan Millipore Corporation, Japan).
  • Example 12-3 The operation was performed in a clean bench as much as possible. All steps performed in Example 12-3 were performed at 4 ° C. (cold room or on ice) except for work on a clean bench. SDS-PAGE (silver staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and a small amount under reducing conditions. Pro monomer was detected (FIG. 8). From this result, it was considered that the purified preparation prepared by the above operation mainly contains a complex (complex body) in which two molecules of pro-region and one molecule of molecule dimer are bound.
  • SDS-PAGE silver staining
  • BMP9 activity was evaluated according to the method described in Example 2.
  • the purified hBMP9 complex body increased Luciferase activity in a dose-dependent manner, and was confirmed to have BMP ligand activity.
  • mice N-terminal His-type hBMP9 complex administration test to mice (in vivo test)
  • Each group composition is as follows.
  • each administration solution was administered into the tail vein at Day 1, Day 3, Day 5, and Day 8, and whole blood was collected from the posterior vena cava at Day 9 and prepared as serum.
  • blood biochemistry analysis was performed using the obtained serum with an automatic analyzer 7180 (manufactured by HITACHI), and a biomarker survey was conducted by entrusting it to Charles River Japan Co., Ltd.
  • RODENTS MULTI-ANALYTE PROFILES Rost MAP V2.0 Plasma Antigen
  • LMP cholesterol increased, pancreatic lipase (LIP) decreased, and serum albumin decreased by BMP9 administration in the 100 ⁇ g / head administered group (3), and all of these fluctuations were controlled by (5) It was significant compared to the group (FIG. 9).
  • Increased LDL cholesterol was associated with dyslipidemia and arteriosclerosis, and decreased serum albumin was associated with nephrotic syndrome, severe liver disease, malnutrition, protein-losing gastroenteropathy, and inflammation.
  • LIP Reduced lipase
  • the biomarkers are known to be associated with the following diseases.
  • anti-BMP9 therapy includes thrombosis, arteriosclerotic disease (acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.), various renal diseases (chronic kidney disease, acute kidney injury, glomerulus).
  • arteriosclerotic disease acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.
  • various renal diseases chronic kidney disease, acute kidney injury, glomerulus.
  • Nephritis hypertension
  • vascular disorder diseases autoimmune diseases such as systemic lupus erythematosus (SLE), Kawasaki disease, vasculitis syndrome, sepsis, etc.
  • allergic diseases allergic rhinitis, asthma, atopic dermatitis, etc.
  • Various inflammatory diseases delayed allergy, rheumatoid arthritis, arthritis, lung disease, hepatitis, acute neutrophilic inflammation, etc.
  • heart diseases hypertensive heart hypertrophy, cardiomyopathy, viral cardiomyopathy, etc.
  • Potential for treatment of cancer Alzheimer's, multiple sclerosis, abnormal angiogenesis in Retinopathy of diabetics, Raynaud's syndrome, and Crohn's disease.
  • the dose was administered intraperitoneally to achieve an amount.
  • administration was carried out 6 times in total including Day1, Day3, Day5, Day8, Day10, and Day13.
  • the blood collected from the orbit was subjected to hematological examination using Bayer ADVIA (registered trademark) 120 Hematology System Analyzer and biomarker investigation was performed using serum collected from the posterior vena cava.
  • biomarker survey RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen) was commissioned to Charles River Japan Co., Ltd. and conducted.
  • hematological examination revealed that the administration of BMP9 neutralizing antibody significantly decreased the number of platelets related to thrombus formation and inflammation by about 25% (FIG. 11).
  • CD40 ligand, CRP, EGF, Endothelin-1, Factor VII, Haptoglobin, IL-1alpha, IL-5, MCP-1, MCP-3, MCP-5, and BMP9 neutralizing antibody were administered.
  • Biomarkers such as MIP-1 beta, MIP-1 gamma, MMP-9, Tissue Factor, TNF-a, TIMP-1, VEGF, RANTES, Lymphoactin, Eotaxin, GCP-2 (Granulocyte Chemical Protein-2) (Fig. 12).
  • biomarkers shown in Example 14 that were confirmed to be increased by administration of recombinant BMP9 were decreased by anti-BMP9 therapy using a BMP9 neutralizing antibody.
  • the biomarkers are known to be associated with the following diseases.
  • anti-BMP9 therapy includes various thrombosis, arteriosclerotic diseases (acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.), various renal diseases (chronic kidney disease, acute kidney injury, thread) Sphere nephritis), hypertension, vascular disorder diseases (autoimmune diseases such as systemic lupus erythematosus (SLE), Kawasaki disease, vasculitis syndrome, sepsis), allergic diseases (allergic rhinitis, airway hypersensitivity, asthma, atopy) Dermatitis, etc.), various inflammatory diseases (delayed allergy, rheumatoid arthritis, arthropathy, lung disease, hepatitis, acute neutrophilic inflammation, ulcerative colitis, etc.), heart disease (hypertensive cardiac hypertrophy, cardiomyopathy) , Chronic heart failure such as viral cardiomyopathy), cancer, diabetic retinopathy, Raynaud's syndrome, and the
  • hsALK1mFc expression vector is a PEAK8 expression vector obtained by enzymatic digestion of the extracellular region cDNA fragment of human ALK1 and the Fc region cDNA fragment of mIgG1 with EcoRI and NotI (both from Roche Diagnostics, Japan). It was prepared by incorporating into (Edge Biosystems).
  • the cDNA sequence of the incorporated hsALK1mFc is as follows (SEQ ID NO: 35).
  • the amino acid sequence encoded by SEQ ID NO: 35 (346 amino acids, SEQ ID NO: 36) is shown below.
  • SEQ ID NO: 36 Each fragment was obtained as follows.
  • the extracellular region cDNA of human ALK1 is hsALK1 FW: agaattccccaccatgaccttgtccccccag (SEQ ID NO: 3), hsALK1 RV: aacttagctgggcccatctgtttccccggctg (SEQ ID NO: 4) was cloned from a human lung cDNA library using the PCR method. The obtained fragment was enzymatically digested with EcoRI and SpeI, and then electrophoresed on an agarose gel to cut out the band.
  • the Fc region cDNA of mIgG1 is mIgG1 FP: acttagcctaaggtcacgtgtgtgt (SEQ ID NO: 37)
  • mIgG1 RP gcggccgcttattattattaccagaggaggggg (SEQ ID NO: 38)
  • the obtained fragment was enzymatically digested with SpeI and NotI (both Roche Diagnostics, Japan), and then electrophoresed on an agarose gel to cut out the band.
  • hsALK1mFc high expression strain was prepared by linearizing the expression vector prepared above using Sfi-I restriction enzyme and then converting the linearized DNA to Lipofectamine TM LTX. Using a reagent (Invitrogen, Japan), the gene was introduced into CHO Ras clone I cells according to the package insert.
  • the cells were washed with PBS (Dulecco's Phosphate Buffered Saline; SIGMA), and then the EX-Cell 302 (SAFC Biosciences) medium was diluted to 25% with the DMEM / F-12 mixed medium. The medium was replaced. After culturing for 5 to 7 days, the culture supernatant was collected, filtered through a 0.22 ⁇ m filter (0.22 ⁇ m GP Express Membrane 500 ml; Japan Millipore, Japan), and then at 4 ° C. (cold room). Cooled down. When stored frozen, it was re-filtered through a 0.22 ⁇ m filter after thawing. 16-3.
  • PBS Dulecco's Phosphate Buffered Saline
  • SIGMA Dulecco's Phosphate Buffered Saline
  • SAFC Biosciences SAFC Biosciences
  • a column of RESOURCE S 6 ml (manufactured by GE Healthcare) is connected to AKTA Explorer, 20 mM MES (pH 5.8) is used as the A1 solution, and 20 mM MES (pH 5.8) containing 1 M NaCl is used as the B1 solution.
  • the ProSep eluate diluted 4 to 10 times with the A1 solution was filtered through a 0.22 ⁇ m filter and applied to the column. After the application, A1 was fed, and after confirming that OD280nm returned to the baseline, the elution program was started.
  • 0.1M sodium phosphate (pH 7.2) containing 2M (NH 4 ) 2 SO 4 was used for the A2 solution
  • 0.1M sodium phosphate (pH 7.2) was used for the B2 solution.
  • the B2 concentration was increased from 50 to 90% and eluted in 20 columns. Each eluted fraction was subjected to SDS-PAGE under non-reducing conditions and then subjected to silver staining, and the fraction containing only dimer was selected and mixed.
  • the ammonium sulfate concentration in the mixture of the 3rd chromatographic fraction was estimated on the same column (RESOURCE PHE) from the electrical conductivity, and the final concentration of ammonium sulfate exceeded 1M.
  • A2 was added and then applied to the column. After the sample was applied, the ammonium sulfate concentration was lowered to 100 to 200 mM at once, stepwise elution was performed, and the sample was collected at 0.5 mL / tube.
  • the fraction containing the purified protein was selected and mixed based on the absorbance at OD 280 nM, and then the buffer was replaced with PBS using NAP-25 (manufactured by GE Healthcare), followed by filter sterilization.
  • the purified hsALK1mFc protein showed a mobility of slightly over 50 kDa under reduction and 90-95 kDa under non-reduction, and was confirmed to be a highly purified protein (FIG. 13).
  • This SHR / NDmc-cp (cp / cp) is known as a spontaneously hypertensive rat with obesity, and is also known as an animal that causes cardiac hypertrophy with hypertension (Metabolism 47, 1199-1204 (1998)).
  • the breeding feed was changed from FR-2 to a special feed (Nippon Nosan Kogyo Co., Ltd.) based on Lab H Standard containing 20% casein, 20% shoecloth, and 8% lard.
  • urine was collected in a metabolic cage for about 24 hours, and then blood was collected from the tail vein.
  • the group composition includes PBS administration group (vehicle administration group), BMP9 neutralizing antibody 0.04 mg / kg administration group, BMP9 neutralizing antibody 0.2 mg / kg administration group, and SHR / NDmc ⁇ as a negative control.
  • PBS administration group vehicle administration group
  • BMP9 neutralizing antibody 0.04 mg / kg administration group
  • BMP9 neutralizing antibody 0.2 mg / kg administration group
  • SHR / NDmc ⁇ as a negative control.
  • One group of cp (+ / +) was set.
  • human BMP9 neutralizing antibody (R & D System, Inc, Clone NO .: 360107) diluted with PBS to 0.10 mg / mL and 0.020 mg / mL was prepared at a dose of 2 mL / kg.
  • the weight was calculated based on the body weight before administration and administered intraperitoneally.
  • PBS was intraperitoneally administered to SHR / NDmc-cp (+ / +) which is control. Thereafter, the test substance was administered once a week. The body weight was measured once a week or twice a week, and blood was collected from the tail vein every other week from one week after administration of the test substance.
  • BMP9 neutralizing antibody had no effect on kidney weight, but suppressed heart hypertrophy in a dose-dependent and significant manner (test by Kruskal-Wallis test + Steel test) for heart weight.
  • the neutralizing antibody for BMP9 is effective for cardiac hypertrophy, which is a problem in heart disease, that is, it may be a therapeutic agent for heart disease.
  • a physiological saline administration group (vehicle administration group), a BMP9 neutralizing antibody administration group, and an hsALK1mFc administration group
  • one group of C57BL / 6N was set as a negative control.
  • test substances saline, BMP9 neutralizing antibody, hsALK1mFc
  • physiological saline was intraperitoneally administered to C57BL / 6 mice as controls.
  • the doses of the BMP9 neutralizing antibody and hsALK1mFc were 1 mg / kg and 0.1 mg / kg, respectively.
  • the breeding feed was changed to a Western diet (F2WTD) (produced by Oriental Yeast Co., Ltd.), which is known to easily induce arteriosclerosis after grouping.
  • F2WTD Western diet
  • BMP9 neutralizing antibody a product purchased from R & D (Clone: 360107) was used, and both the BMP9 neutralizing antibody and hsALK1mFc were prepared in a normal saline solution. Thereafter, test substances (physiological saline, BMP9 neutralizing antibody, hsALK1mFc) were administered intraperitoneally at a frequency of once a week. Blood was collected over time every 2 weeks or 4 weeks after the start of test substance administration.
  • Blood biochemical analysis was performed with an automatic analyzer (Hitachi 7170) using reagents sold by Kyowa Medex Co., Ltd.
  • the whole blood was collected from the abdominal vena cava under isoflurane anesthesia, and then the aorta in the region from directly under the heart to the lower descending aorta was removed.
  • the aorta is cut longitudinally from the anterior and posterior sides with scissors and then cut into 10% formalin solution. Soaked and fixed overnight.
  • the fixed aorta was washed twice with PBS and once with 60% isopropanol, and then immersed in 1.8 mg / mL oil red O staining solution overnight. The next day, after washing once with 60% isopropanol and twice with PBS, excess fat adhering to the outside of the aorta was removed using tweezers and scissors under an optical microscope. Used for image analysis. In the image analysis, the total area of the extracted aorta and the area stained with Oil Red O were calculated, and the plaque area ratio in each sample was calculated by dividing the area stained with Oil Red O by the total area of the aorta.
  • the plaque formation inhibition rate (%) in the hsALKmFc and neutralizing antibody administration group was calculated by the following formula. ((Plaque area rate in the APOE-deficient mouse physiological saline administration group) ⁇ (Plaque area rate in the APOE-deficient mouse test substance administration group)) ⁇ ((Plaque area rate in the APOE-deficient mouse physiological saline administration group)) — (C57BL / 6 plaque area ratio in saline administration group)) X100
  • plaque formation in APOE-deficient mice was suppressed by about 32% by hsALKmFc administration and by about 15% by BMP9 neutralizing antibody administration (FIG. 16).
  • ALK1 antagonists containing BMP9 neutralizing antibody The possibility of being effective was found.
  • the diastolic blood pressure, systolic blood pressure, and heart rate of a rat implanted with a transmitter and reaching the age of 33 weeks are measured by the telemetry system Dataquest A. R. T. T.
  • the groups were divided into 4 groups so that their values were equal to each group.
  • a BMP9 neutralizing antibody purchased from R & D (Clone: 360107) was used.
  • the antibody dilution medium PBS
  • the test substance such as BMP9 neutralizing antibody and hsALK1mFc was administered intraperitoneally the next day. Blood pressure, systolic blood pressure, and heart rate were measured.
  • BMP9 neutralizing antibody was not observed to have an effect on blood pressure
  • a decrease in diastolic blood pressure and systolic blood pressure was observed from about 14 hours after administration, and the decrease was observed on the 4th day. (FIG. 17). From the above results, it was revealed that hsALK1mFc has a hypotensive action.
  • test substance PBS, BMP9 neutralizing antibody
  • PBS was administered to negative control rats.
  • test substance was administered once a week.
  • a BMP9 neutralizing antibody purchased from R & D (Clone: 360107) was used.
  • the body weight was measured once or twice a week, and blood was collected from the tail vein at Day 15, Day 22, Day 29, Day 35 after administration of the anti-GBM antibody.
  • whole blood was collected from the abdominal aorta under isoflurane anesthesia, and then the kidney was removed and the kidney weight was measured.

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Abstract

Disclosed is use/usage of an activin receptor-like kinase 1 (ALK1) inhibitor in a pharmaceutical composition for preventing vascular disorders. Specifically disclosed are: a pharmaceutical composition for preventing vascular disorders, which contains an ALK1 inhibitor as an active ingredient; and a method for treating vascular disorders using the pharmaceutical composition.

Description

ＡＬＫ１阻害剤を有効成分とする血管障害を抑制するための医薬組成物Pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient
　本発明は、アクチビン受容体様キナーゼ１（Ａｃｔｉｖｉｎ　ｒｅｃｅｐｔｏｒ−ｌｉｋｅ　ｋｉｎａｓｅ　１；以下、ＡＬＫ１と表記）阻害剤を有効成分として含む血管障害を抑制するための医薬組成物およびそれを用いた血管障害の治療方法に関するものである。 The present invention relates to a pharmaceutical composition containing an activin receptor-like kinase 1 (Activin receptor-like kinase 1; hereinafter referred to as ALK1) inhibitor as an active ingredient, and a vascular disorder treatment using the same. It is about the method.
　血管内皮細胞とは、血管の内表面を構成する扁平で薄い細胞の層をいう。血管内皮細胞は、様々な生理活性物質を産生・分泌し、抗血栓作用、血管トーヌスの調節、炎症反応（白血球の接着、遊走）に関与している。健常な内皮細胞は様々な抗血栓分子を発現し、血小板凝集抑制、血液凝固抑制、線溶活性、さらには血管弛緩に働き、抗血栓性に機能している（非特許文献１）。
　例えば、血管内皮細胞は、細胞膜上に細胞外ヌクレオチド分解酵素を恒常的に発現し、血小板から放出されるＡＤＰを分解し、血小板凝集を抑制する。また、刺激により、抗血小板作用、血管拡張作用を有する一酸化窒素やプロスタグランジンＩ２を分泌し、抗血栓性を発揮する（非特許文献１）。
　血管障害とは血管内皮細胞が障害を受けた状態で、具体的には、アセチルコリンやブラジキニンなどの特定の刺激物質に対する血管拡張性が低下した状態、炎症状態、易血栓状態（血栓が生じやすくなった状態）などを指す（非特許文献２，３）。
　また血管内皮細胞の炎症状態とは、酸化ＬＤＬ、糖化蛋白、炎症性サイトカイン（ＩＬ−１、ＴＮＦ−α等）、エンドトキシンなどの炎症性物質が血管内皮細胞に作用し、Ｅ−セレクチン、ＶＣＡＭ−１、ＩＣＡＭ−１、ＮＦ−ｋＢが発現した状態を指す（非特許文献２）。Ｅ−セレクチン、ＶＣＡＭ−１は血管内皮細胞に特異的に発現する分子で、炎症時に発現誘導されることより、血管障害マーカーとして捉えられている。そのＥ−セレクチン、ＶＣＡＭ−１は次のような機序を介し炎症に関与するとされる。炎症性サイトカインなどの炎症性物質が血管内皮細胞に作用すると、血管内皮細胞上に接着分子であるＥ−セレクチン、ＶＣＡＭ−１が発現するようになる。血管内皮細胞上にＥ−セレクチンが発現すると、単球や好中球などの白血球は、その細胞表面上に発現するセレクチンリガンド（糖鎖（シアリルＬｅｘ抗原など）を介し、血管内皮細胞と弱く接触できるようになる。さらに、白血球は、その白血球表面上に発現するＬＦＡ−１もしくはＶＬＡ４というインテグリンと、炎症性物質により血管内皮細胞表面上に発現されるＶＣＡＭ−１もしくはＩＣＡＭ−Ｉといったインテグリンレセプターとの結合を介し、より強固に血管内皮細胞に結合できるようになり、炎症反応が開始される（非特許文献４）。
　血管内皮障害と易血栓状態については、正常時の血管内皮細胞は抗血栓性の状態にあるが、一旦、障害や感染などにより炎症をきたすと、生理防御反応の一環として、内皮細胞における抗血栓性因子の産生は低下し、血小板凝集惹起因子のｖｏｎ　Ｗｉｌｌｅｂｒａｎｄ因子（ＶＷＦ）、凝固促進因子の組織因子（ＴＦ）や第Ｖ因子、線溶阻害因子のプラスミノーゲンアクチベーターインヒビター−１（ＰＡＩ−１）などの産生増加が起こり、血栓形成が誘発されやすくなることが知られている（非特許文献１）。
　血管内皮障害と疾患との関係については、血管内皮障害を成因とすることが知られている、動脈硬化症、高血圧症、慢性腎疾患（Ｃｈｒｏｎｉｃ　ｋｉｄｎｅｙ　ｄｉｓｅａｓｅ（ＣＫＤ））、糖尿病、心疾患、脂質異常症、急性腎不全、血栓症などの生活習慣病との関連が特によく知られている。
　動脈硬化との関連では、血管内皮障害はアテローム性動脈硬化進行の第１段階とされ、最近の研究では、血管内皮障害は動脈硬化性疾患の有用な予後指標になることが証明されるなど、血管内皮障害の、動脈硬化症の発症進展における重要性は疑いようのない事実となっている（非特許文献３，５）。
　慢性腎疾患においても、血管内皮障害は血液透析患者だけでなく、保存期の患者においても認められ、血管内皮障害は病態進行に関わることが報告されている（非特許文献６−８）。腎疾患での血管内皮障害は、腎疾患で生じる傍糸球体装置の異常により輸入腎細動脈の収縮がうまくいかないため、全身血圧がそのまま糸球体内に伝わるようになり、生じるとされる（非特許文献８）。
　その他、高血圧症、Ｉ型糖尿病、ＩＩ型糖尿病、心疾患、脂質異常症などでの病態進行や、それら疾患での高い心血管病（ＣＶＤ）リスクも、血管内皮障害によるものとして捉えられている（非特許文献９−１５）。また、ＩＩ型糖尿病や高血圧症などでは、血管内皮障害の存在は、当該症状発症前より認められており、疾患発症との関連も示唆されている（非特許文献１６，１７）。また腎局所における血管内皮障害が、急性腎不全などの病態進行にかかわるとの報告もなされている（非特許文献１８）。
　また血管内皮障害は、メタボリックシンドロームの背景にあるとされるインシュリン抵抗性とも深く関わることが知られている。血管内皮細胞はインシュリンの作用を受けて、Ｐｈｏｓｐｈａｔｉｄｙｌｉｎｏｓｉｔｏｌ　３−ｋｉｎａｓｅ（ＰＩ３Ｋ）を介して一酸化窒素（ＮＯ）を産生し、血管拡張を制御するが、血管内皮障害に働くとされる高血糖、脂質異常、炎症性物質は、ＰＩ３Ｋ依存性のインシュリンシグナル伝達に対し抑制的に働き、インシュリン抵抗性を引き起こすことが報告されている（非特許文献１９−２１）。
　このように、血管内皮障害は、インシュリン抵抗性と密接に関わることからも明らかなように、動脈硬化性疾患、高血圧症、慢性腎疾患、急性腎不全、糖尿病、心疾患、血栓症といった生活習慣病の成因として捉えられている。
　しかしながら、血管内皮障害の発症機構については、多くの要因（老化、喫煙、炎症、外傷、脂質異常症、高血糖など）の関与が示唆されているが、実際に、どの要因が深く血管障害と結びつくのか、真の機構については解明されていない（非特許文献２２）。
　ＡＬＫ１は、ＴＧＦ−ｂｅｔａスーパーファミリーのＩ型レセプター群に属する、血管内皮に特異的に発現する遺伝子であり、遺伝性出血性毛細血管拡張症（ｈｅｒｅｄｉｔａｒｙ　ｈｅｍｏｒｒｈａｇｉｃ　ｔｅｌａｎｇｉｅｃｔａｓｉａ：ＨＨＴ）の原因遺伝子とされる（非特許文献２３）。ＡＬＫ１は、細胞内情報伝達分子であるＳｍａｄ１とＳｍａｄ５をリン酸化し、細胞内にシグナルを伝える。ＡＬＫ１欠損マウスを用いた解析から、ＡＬＫ１は血管構築に重要な分子であることも分かっている（非特許文献２４，２５）。
　ＡＬＫ１のリガンドとしては、骨形成タンパク質（Ｂｏｎｅ　ｍｏｒｐｈｏｇｅｎｅｔｉｃ　ｐｒｏｔｅｉｎ；以下、ＢＭＰと表記）ファミリーに属するＢＭＰ９が知られている（非特許文献２６，２７）。
　ＢＭＰは、ＴＧＦ−ｂｅｔａスーパーファミリーに属し、異所性の骨成長や軟骨形成に対する誘導能を持つ分子として同定されてきた（非特許文献２８，２９）。また最近では、ＢＭＰファミリー分子は、一般に種々の細胞の増殖、分化、アポトーシスに関わり、組織、臓器の形態生成に重要であることが分かってきている（非特許文献３０，３１）。
　ＢＭＰファミリー分子に属するＢＭＰ９は、他のＢＭＰファミリー分子と同様に、肥大軟骨細胞の形成や間葉細胞からの軟骨への分化に対する促進作用（非特許文献３２）が報告されている他、鉄イオンの恒常性維持に関わるｈｅｐｃｉｄｉｎの発現調節（非特許文献３３）や糖代謝に関わる可能性も報告されている（非特許文献３４）。その発現臓器に関しては、胎児期では脊髄や体節間膜で、成体期では肝臓で主に発現していることが報告されている（非特許文献３４−３６）。更には、ＢＭＰ９が血中循環因子であることも報告されている（非特許文献３７）。
　また、ＢＭＰ９のタンパク質構造に関して言えば、他のＢＭＰファミリーと同様に、一本鎖の前駆体タンパク質（ｐｒｅ−ｐｒｏ体）として合成された後、シグナルペプチド領域が切断され、細胞内でＣ末端側に存在するシステイン残基がジスルフィド結合を介し２量体（ｐｒｏ２量体）を形成する。その後、ｆｕｒｉｎ様プロテアーゼにより、活性本体であるＣ末側（ｍａｔｕｒｅ２量体）とジスルフィド結合を持たないＮ末プロペプチド領域（ｐｒｏ−ｒｅｇｉｏｎ）に切断される。切断されたＮ末側のプロペプチド領域２分子はＣ末側のｍａｔｕｒｅ２量体１分子と非共有結合を介し複合体（ｃｏｍｐｌｅｘ体）を形成し、その複合体の形で細胞から分泌されることが知られている（非特許文献３８）。また、ｍａｔｕｒｅ２量体同様、Ｎ末プロペプチド領域が結合した複合体（ｃｏｍｐｌｅｘ体）も、シグナル伝達能を有することが知られている（非特許文献３８）。
　また、ＢＭＰのシグナル伝達には、一般にセリン・スレオニンキナーゼドメインを細胞内領域に有するＴＧＦｂｅｔａスーパーファミリーに属するタイプＩ、タイプＩＩの２つのレセプターを必要とする。ＢＭＰ９のタイプＩレセプターとしては、ＡＬＫ１が、タイプＩＩレセプターとしてはＢＭＰタイプＩＩレセプター（ＢＭＰＲＩＩ）、ａｃｔｉｖｉｎタイプＩＩａレセプター（ＡｃｔＲＩＩａ）、ａｃｔｉｖｉｎタイプＩＩｂレセプター（ＡｃｔＲＩＩｂ）が知られている（非特許文献２７）。
　ＡＬＫ１および／またはＢＭＰ９と所定の疾患との関連についての報告は、幾つかなされている。例えば、ＡＬＫ１細胞外領域ポリペプチド−Ｆｃ融合蛋白質またはＡＬＫ１細胞外領域ポリペプチド抗体の癌、関節リウマチ等の血管新生関連疾患での治療剤としての可能性（特許文献１、２）、ＢＭＰ９蛋白質の乳癌または前立腺癌における治療剤としての可能性（特許文献３）が報告されている。また、低分子化合物としては、ピラゾロピリミジン骨格を有する化合物を有効成分とするＡＬＫ１阻害剤の血管新生関連疾患での治療剤としての可能性（特許文献４）についても報告されている。
　しかしながら、ＡＬＫ１および／またはＢＭＰ９と血管内皮細胞の機能低下がもたらす血管障害抑制活性との関連、すなわち、ＡＬＫ１阻害剤の血管障害を起因とする疾患における治療剤としての可能性については見出されていない。
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For example, vascular endothelial cells constantly express extracellular nucleotide-degrading enzymes on the cell membrane, degrade ADP released from platelets, and suppress platelet aggregation. Further, upon stimulation, nitric oxide and prostaglandin I2 having antiplatelet action and vasodilatory action are secreted, and antithrombotic properties are exhibited (Non-patent Document 1).
Vascular injury is a state in which vascular endothelial cells are damaged, specifically, a state in which vasodilatability to a specific stimulating substance such as acetylcholine and bradykinin is reduced, an inflammatory state, a thrombotic state (thrombus tends to occur) (Non-patent documents 2 and 3).
Further, the inflammatory state of vascular endothelial cells refers to inflammatory substances such as oxidized LDL, glycated protein, inflammatory cytokines (IL-1, TNF-α, etc.), endotoxin, etc. acting on vascular endothelial cells, and E-selectin, VCAM- 1, refers to the state in which ICAM-1 and NF-kB are expressed (Non-patent Document 2). E-selectin and VCAM-1 are molecules that are specifically expressed in vascular endothelial cells, and are regarded as vascular disorder markers because they are induced during inflammation. The E-selectin, VCAM-1, is considered to be involved in inflammation through the following mechanism. When inflammatory substances such as inflammatory cytokines act on vascular endothelial cells, E-selectin and VCAM-1, which are adhesion molecules, are expressed on vascular endothelial cells. If E- selectin on vascular endothelial cells express, leukocytes such as monocytes and neutrophils, via the selectin ligand (sugar (such as sialyl Le x antigens) expressed on the cell surface, weak vascular endothelial cells Furthermore, leukocytes are integrin receptors such as LFA-1 or VLA4 expressed on the surface of leukocytes and VCAM-1 or ICAM-I expressed on the surface of vascular endothelial cells by inflammatory substances. It becomes possible to bind to vascular endothelial cells more firmly through the binding to inflammatory reaction (Non-patent Document 4).
Regarding vascular endothelial injury and thrombotic state, normal vascular endothelial cells are in an antithrombotic state, but once inflammation is caused by injury or infection, antithrombosis in endothelial cells is part of the physiological defense reaction. Production of sex factors is reduced, and von Willebrand factor (VWF), a platelet aggregation inducer, tissue factor (TF), procoagulant factor, factor V, and plasminogen activator inhibitor-1 (PAI-), a fibrinolysis inhibitor It is known that production increases such as 1) occur and thrombus formation is likely to be induced (Non-patent Document 1).
Regarding the relationship between vascular endothelial disorder and disease, arteriosclerosis, hypertension, chronic kidney disease (CKD), diabetes, heart disease, lipid, which are known to be caused by vascular endothelial disorder The relationship with lifestyle-related diseases such as abnormalities, acute renal failure, and thrombosis is particularly well known.
In the context of arteriosclerosis, vascular endothelial damage is considered the first stage of atherosclerosis progression, and recent studies have demonstrated that vascular endothelial damage is a useful prognostic indicator of atherosclerotic disease, etc. The importance of vascular endothelial injury in the development of atherosclerosis is unquestionable (Non-Patent Documents 3 and 5).
Even in chronic kidney disease, vascular endothelial damage is observed not only in hemodialysis patients but also in patients in the preservation period, and it has been reported that vascular endothelial damage is related to pathological progression (Non-Patent Documents 6-8). Vascular endothelial injury due to renal disease is said to occur because the imported renal arterioles fail to contract due to abnormalities in the paraglomerular device caused by renal disease, and systemic blood pressure is transmitted directly to the glomeruli (non-patented). Reference 8).
In addition, pathological progression in hypertension, type I diabetes, type II diabetes, heart disease, dyslipidemia, etc., and high cardiovascular disease (CVD) risk in these diseases are also regarded as vascular endothelial disorder. (Non-Patent Documents 9-15). In addition, in type II diabetes and hypertension, the presence of vascular endothelial damage has been recognized before the onset of the symptom, and a relationship with the onset of the disease has also been suggested (Non-patent Documents 16 and 17). In addition, it has been reported that vascular endothelial injury in the local region of the kidney is involved in pathological progression such as acute renal failure (Non-patent Document 18).
Vascular endothelial injury is also known to be deeply related to insulin resistance, which is considered to be the background of metabolic syndrome. Vascular endothelial cells receive the action of insulin and produce nitric oxide (NO) via phosphatidylinositol 3-kinase (PI3K) to control vasodilation, but are considered to act on vascular endothelial damage, high blood sugar, lipid It has been reported that abnormal and inflammatory substances act in a suppressive manner on PI3K-dependent insulin signaling and cause insulin resistance (Non-patent Documents 19-21).
As described above, vascular endothelial disorders are closely related to insulin resistance, and lifestyle habits such as arteriosclerotic disease, hypertension, chronic kidney disease, acute renal failure, diabetes, heart disease, and thrombosis are observed. Perceived as the cause of the disease.
However, regarding the onset mechanism of vascular endothelial damage, many factors (aging, smoking, inflammation, trauma, dyslipidemia, hyperglycemia, etc.) have been suggested to be involved. The true mechanism is not elucidated (Non-patent Document 22).
ALK1 belongs to the type I receptor group of the TGF-beta superfamily and is specifically expressed on the vascular endothelium, and is considered to be a causative gene of hereditary hemorrhagic telangiectasia (HHT) ( Non-patent document 23). ALK1 phosphorylates intracellular signaling molecules Smad1 and Smad5 and transmits signals into the cell. From the analysis using ALK1-deficient mice, it is also known that ALK1 is an important molecule for blood vessel construction (Non-patent Documents 24 and 25).
As a ligand of ALK1, BMP9 belonging to the bone morphogenetic protein (hereinafter referred to as BMP) family is known (Non-patent Documents 26 and 27).
BMP belongs to the TGF-beta superfamily and has been identified as a molecule having the ability to induce ectopic bone growth and cartilage formation (Non-patent Documents 28 and 29). Recently, BMP family molecules have generally been found to be involved in the growth, differentiation, and apoptosis of various cells and are important for tissue and organ morphogenesis (Non-patent Documents 30 and 31).
BMP9 belonging to the BMP family molecule has been reported to promote the formation of hypertrophic chondrocytes and differentiation from mesenchymal cells to cartilage (Non-patent Document 32), as well as other BMP family molecules. It has also been reported that hepcidin expression regulation (Non-patent Document 33) and sugar metabolism involved in maintaining the homeostasis of the drug (Non-Patent Document 34). As for the expression organ, it has been reported that it is mainly expressed in the spinal cord and inter-segmental membrane in the fetal period and in the liver in the adult period (Non-patent Documents 34 to 36). Furthermore, it has been reported that BMP9 is a circulating factor in blood (Non-patent Document 37).
As for the protein structure of BMP9, as with other BMP families, after synthesis as a single-chain precursor protein (pre-pro body), the signal peptide region is cleaved, and the C-terminal side is cleaved in the cell. The cysteine residue present in the dimer forms a dimer (prodimer) via a disulfide bond. Then, it is cleaved by furin-like protease into the C-terminal side (matter dimer), which is the active body, and the N-terminal propeptide region (pro-region) having no disulfide bond. Two molecules of the cleaved N-terminal propeptide region form a complex with one molecule of the C-terminal nature dimer via a non-covalent bond, and are secreted from the cell in the form of the complex. Is known (Non-patent Document 38). Moreover, it is known that the complex (complex body) which the N terminal propeptide area | region couple | bonded with a signal dimer has signal transduction ability similarly to a nature dimer (nonpatent literature 38).
In addition, BMP signaling generally requires two receptors of type I and type II belonging to the TGFbeta superfamily having a serine / threonine kinase domain in the intracellular region. ALK1 is known as a type I receptor for BMP9, and BMP type II receptor (BMPRII), activin type IIa receptor (ActRIIa), and activin type IIb receptor (ActRIIb) are known as type II receptors (Non-patent Document 27). .
There have been several reports on the association between ALK1 and / or BMP9 and certain diseases. For example, ALK1 extracellular region polypeptide-Fc fusion protein or ALK1 extracellular region polypeptide antibody as a therapeutic agent for angiogenesis-related diseases such as cancer and rheumatoid arthritis (Patent Documents 1 and 2), BMP9 protein The possibility as a therapeutic agent in breast cancer or prostate cancer (Patent Document 3) has been reported. In addition, as a low molecular weight compound, the possibility that an ALK1 inhibitor comprising a compound having a pyrazolopyrimidine skeleton as an active ingredient as a therapeutic agent in angiogenesis-related diseases has been reported (Patent Document 4).
However, the relationship between ALK1 and / or BMP9 and the vascular injury-suppressing activity caused by the decreased function of vascular endothelial cells, that is, the potential of ALK1 inhibitors as therapeutic agents in diseases caused by vascular injury has been found. Absent.
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　本発明者は、哺乳動物においてＢＭＰ９遺伝子産物の生理的作用について鋭意研究を行った結果、ＡＬＫ１阻害剤が血管障害抑制活性を有することを見出した。すなわち、本発明者は、ＡＬＫ１のリガンドであるＢＭＰ９を血管内皮細胞に作用させたところ、血管内皮障害マーカーであるＥ−ｓｅｌｅｃｔｉｎやＶＣＡＭ−１が顕著に発現誘導されることを見出した。更に、ＢＭＰ９を過剰発現するマウスを作製したところ、ＢＭＰ９の過剰作用により、腹腔・胸腔内に血液が貯留することや、リンパ節で血液漏出による赤色化が引き起こされることを見出し、ＢＭＰ９の血管障害作用をｉｎ　ｖｉｖｏにおいても確認することに成功した。更に、ＢＭＰ９のマウスへの過剰作用は、血管障害マーカーを含め、各種炎症マーカーを増加させる一方、正常マウスへのＢＭＰ９中和抗体投与は、反対に炎症マーカーの発現を低下させ、ＢＭＰ９の機能を阻害するような抗ＢＭＰ９療法は、血管障害抑制を含めた、抗炎症作用を有することを見出した。また、ＢＭＰ９中和抗体やＡＬＫ１細胞外領域ポリペプチド−Ｆｃ融合蛋白質を用いた各種疾患モデルに対するインビボ試験からは、抗ＢＭＰ９療法が、心疾患、動脈硬化症、腎疾患、脂質異常症といった疾患の有効な治療法となりうる可能性が確認された。本発明者はこれらの知見に基づき、ＢＭＰ９中和抗体等のＡＬＫ１阻害剤を有効成分とする血管障害を抑制するための医薬組成物として提供できると考え、本発明を完成させるに至った。
　すなわち、本発明は、以下のとおりである。
［１］　ＡＬＫ１阻害剤を有効成分とする血管障害を抑制するための医薬組成物。
［２］　ＡＬＫ１阻害剤が以下のいずれかから選択される、［１］の医薬組成物。
（１）ＢＭＰ９に結合する抗体または該抗体断片
（２）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質
（３）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸
（４）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸を含む発現ベクター
（５）ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片
（６）ＢＭＰ９に対するアプタマーまたはＡＬＫ１細胞外領域ポリペプチドに対するアプタマー
（７）ＡＬＫ１遺伝子を標的とするＲＮＡｉ分子、またはＢＭＰ９遺伝子を標的とするＲＮＡｉ分子
（８）ＡＬＫ１のキナーゼ活性に対して阻害活性を有する低分子化合物
（９）ＢＭＰ９とＡＬＫ１との結合、もしくはＢＭＰ９とタイプＩＩレセプターとの結合を阻害する活性を有する低分子化合物
（１０）ＢＭＰ９阻害活性を有するＢＭＰ９アンタゴニスト蛋白質
［３］　（１）ＢＭＰ９に結合する抗体または該抗体断片、（２）ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片が、ヒト抗体、ヒト化抗体、またはその断片である、［２］の医薬組成物。
［４］　（１）ＢＭＰ９に結合する抗体または該抗体断片、（２）ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片が、ポリクローナル抗体、ペプチド抗体、モノクローナル抗体またはその断片である、［３］の医薬組成物。
［５］　抗体断片が、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、一本鎖抗体（ｓｃＦｖ）、二量体化Ｖ領域（Ｄｉａｂｏｄｙ）、ジスルフィド安定化Ｖ領域（ｄｓＦｖ）およびＣＤＲを含むペプチドから選ばれる抗体断片である、［３］または［４］の医薬組成物。
［６］　ＡＬＫ１細胞外領域ポリペプチドまたはその変異体が、哺乳動物由来免疫グロブリンＦｃ蛋白質又はその変異体との融合蛋白質である、［２］の医薬組成物。
［７］　血管障害を伴う疾患に対して用いられる、［１］~［６］のいずれかの医薬組成物。
［８］　血管障害を伴う疾患が、腎疾患、動脈硬化性疾患、高血圧症、肺性高血圧症、心疾患、糖尿病、糖尿病合併症、血栓症、脂質異常症などの生活習慣病、血管炎を伴う疾患またはＢＭＰ９の関与の可能性が示された疾患から選択される、［７］の医薬組成物。
［９］　腎疾患が、ＩｇＡ腎症を含む慢性糸球体腎炎、糖尿病性腎症、ループス腎炎、腎硬化症または急速進行性糸球体腎炎である、［８］の医薬組成物。
［１０］　動脈硬化性疾患が、脳血管障害（脳卒中、ラクナ梗塞も含む脳梗塞、脳血栓、脳出血、クモ膜下出血など）、虚血性心疾患（心筋梗塞や狭心症など）、大動脈瘤、大動脈解離、腎硬化症または閉塞性動脈硬化症である、［８］の医薬組成物。
［１１］　心疾患が、心臓弁膜症、心筋梗塞、狭心症、心筋症である、［８］の医薬組成物。
［１２］　糖尿病合併症が、動脈硬化症、糖尿病性網膜症、糖尿病性腎症、糖尿病性神経障害、糖尿病性壊疽、慢性感染症または白内障である、［８］の医薬組成物。
［１３］　血栓症が、肺血栓塞栓症、脳梗塞、心筋梗塞、下肢急性動脈血栓症、腸壊死（上腸間膜動脈血栓症）である、［８］の医薬組成物。
［１４］　血管炎を伴う疾患が、高安動脈炎、巨細胞動脈炎（側頭動脈炎）、結節性多発動脈炎、ウェゲナー肉芽腫症、チャーグ・ストラウス症候群、川崎病、ヘノッホ・シェーンライン紫斑病、過敏性血管炎、全身性エリテマトーデスまたは関節リウマチである、［８］の医薬組成物。
［１５］　ＢＭＰ９の関与の可能性が示された疾患が、肝疾患（急性肝炎、慢性肝炎、肝臓がん、肝硬変、転移性肝臓がんなど）、癌性腹水・胸水を伴うがん、慢性膵炎、アレルギー性疾患（アレルギー性鼻炎、喘息、気道過敏症、アトピー性皮膚炎など）、炎症性疾患（遅延型アレルギー、関節リウマチ、関節炎、肺疾患、肝炎、潰瘍性大腸炎など）、アルツハイマー、多発性硬化症、糖尿病性網膜症、レイノー症候群、クローン病またはがんである、［８］の医薬組成物。
［１６］　［１］~［１５］のいずれかの医薬組成物を用いた血管障害を伴う疾患の治療法。 As a result of intensive studies on the physiological action of the BMP9 gene product in mammals, the present inventor has found that an ALK1 inhibitor has a vascular disorder inhibitory activity. That is, the present inventors have found that when BMP9, which is a ligand of ALK1, is allowed to act on vascular endothelial cells, E-selectin and VCAM-1 that are vascular endothelial injury markers are remarkably induced. Furthermore, when a mouse overexpressing BMP9 was produced, it was found that blood was accumulated in the abdominal cavity / thoracic cavity due to the excessive action of BMP9, and redness due to blood leakage was caused in lymph nodes. We have succeeded in confirming the action in vivo. Furthermore, excessive action of BMP9 on mice increases various inflammatory markers, including vascular injury markers, while administration of BMP9 neutralizing antibody to normal mice, conversely, decreases the expression of inflammatory markers and increases the function of BMP9. It has been found that anti-BMP9 therapy that inhibits has anti-inflammatory effects including suppression of vascular injury. In addition, from in vivo studies on various disease models using BMP9 neutralizing antibody and ALK1 extracellular domain polypeptide-Fc fusion protein, anti-BMP9 therapy is effective for diseases such as heart disease, arteriosclerosis, renal disease, and dyslipidemia. The possibility that it could be an effective treatment was confirmed. Based on these findings, the present inventor considered that the present invention can be provided as a pharmaceutical composition for suppressing vascular injury using an ALK1 inhibitor such as a BMP9 neutralizing antibody as an active ingredient, and has completed the present invention.
That is, the present invention is as follows.
[1] A pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient.
[2] The pharmaceutical composition of [1], wherein the ALK1 inhibitor is selected from any of the following.
(1) an antibody that binds to BMP9 or the antibody fragment (2) ALK1 extracellular region polypeptide or variant thereof, or ALK1 extracellular region polypeptide or protein containing the variant (3) ALK1 extracellular region polypeptide or Nucleic acid encoding a variant thereof, or an ALK1 extracellular region polypeptide or a protein comprising the variant (4) ALK1 extracellular region polypeptide or a variant thereof, or an ALK1 extracellular region polypeptide or a protein comprising the variant (5) An antibody that binds to an ALK1 extracellular region polypeptide or an antibody fragment thereof (6) An aptamer to BMP9 or an aptamer to an ALK1 extracellular region polypeptide (7) RNAi targeting the ALK1 gene Molecule, or BMP9 RNAi molecule targeting a gene (8) Low molecular weight compound having inhibitory activity against the kinase activity of ALK1 (9) It has an activity of inhibiting the binding of BMP9 and ALK1, or the binding of BMP9 and type II receptor Low molecular compound (10) BMP9 antagonist protein having BMP9 inhibitory activity [3] (1) An antibody or antibody fragment that binds to BMP9, (2) An antibody or antibody fragment that binds to an ALK1 extracellular region polypeptide [2] The pharmaceutical composition of [2], which is an antibody, a humanized antibody, or a fragment thereof.
[4] (1) The antibody that binds to BMP9 or the antibody fragment thereof, (2) The antibody that binds to the ALK1 extracellular region polypeptide or the antibody fragment is a polyclonal antibody, a peptide antibody, a monoclonal antibody or a fragment thereof. 3] The pharmaceutical composition.
[5] Peptide in which the antibody fragment contains Fab, Fab ′, F (ab ′) 2, single chain antibody (scFv), dimerization V region (Diabody), disulfide stabilized V region (dsFv) and CDR The pharmaceutical composition of [3] or [4], which is an antibody fragment selected from
[6] The pharmaceutical composition according to [2], wherein the ALK1 extracellular region polypeptide or a variant thereof is a fusion protein with a mammal-derived immunoglobulin Fc protein or a variant thereof.
[7] The pharmaceutical composition according to any one of [1] to [6], which is used for diseases associated with vascular disorders.
[8] Diseases associated with vascular disorders include renal diseases, arteriosclerotic diseases, hypertension, pulmonary hypertension, heart disease, diabetes, diabetic complications, thrombosis, dyslipidemia and other lifestyle-related diseases, vasculitis The pharmaceutical composition according to [7], which is selected from a disease accompanied by the disease or a disease shown to be possibly involved in BMP9.
[9] The pharmaceutical composition of [8], wherein the renal disease is chronic glomerulonephritis including IgA nephropathy, diabetic nephropathy, lupus nephritis, nephrosclerosis or rapidly progressive glomerulonephritis.
[10] Arteriosclerotic diseases include cerebrovascular disorders (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc.), ischemic heart disease (myocardial infarction, angina pectoris, etc.), aortic aneurysm, [8] The pharmaceutical composition according to [8], which is aortic dissection, nephrosclerosis or obstructive arteriosclerosis.
[11] The pharmaceutical composition according to [8], wherein the heart disease is valvular heart disease, myocardial infarction, angina pectoris, cardiomyopathy.
[12] The pharmaceutical composition according to [8], wherein the diabetic complication is arteriosclerosis, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic gangrene, chronic infection or cataract.
[13] The pharmaceutical composition according to [8], wherein the thrombosis is pulmonary thromboembolism, cerebral infarction, myocardial infarction, acute limb thrombosis, intestinal necrosis (upper mesenteric artery thrombosis).
[14] Diseases associated with vasculitis are Takayasu's arteritis, giant cell arteritis (temporal arteritis), polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, Kawasaki disease, Henoch-Schönlein purpura The pharmaceutical composition according to [8], which is hypersensitivity vasculitis, systemic lupus erythematosus, or rheumatoid arthritis.
[15] Diseases that may be involved in BMP9 include liver disease (acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, etc.), cancer with cancerous ascites / pleural effusion, chronic Pancreatitis, allergic diseases (allergic rhinitis, asthma, airway hypersensitivity, atopic dermatitis, etc.), inflammatory diseases (delayed allergy, rheumatoid arthritis, arthritis, lung disease, hepatitis, ulcerative colitis, etc.), Alzheimer, The pharmaceutical composition according to [8], which is multiple sclerosis, diabetic retinopathy, Raynaud's syndrome, Crohn's disease or cancer.
[16] A method for treating a disease associated with a vascular disorder using the pharmaceutical composition according to any one of [1] to [15].
　図１は、精製したｈｓＡＬＫ１Ｆｃタンパク質のＢＭＰ９に対する中和作用を示す図である。
　図２は、ＢＭＰ９を添加した正常ヒト血管内皮細胞を用いた遺伝子発現解析を行い、ＢＭＰ９刺激により、血管内皮障害マーカー遺伝子、および生活習慣病（高血圧症、各種腎疾患、糖尿病、糖尿病合併症、脂質異常症、動脈硬化性疾患、血栓症）関連遺伝子が発現変動することを示した図である。
　図３は、Ｎ末端Ｈｉｓタグ付加ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターの構造を示す図である。Ｋｏｚａｋ：コザック配列、ＳＰ：マウスＢＭＰ９固有のシグナル配列、Ｈｉｓ−ｔａｇ：Ｈｉｓ６タグ配列、ｍＢＭＰ（−ＳＰ）：固有のシグナルペプチドコード領域を持たないマウスＢＭＰ９遺伝子、ＳＶ４０　ｐｏｌｙＡ：ＳＶ４０由来ｐｏｌｙＡシグナル領域、５’ｎｅｏ：ｎｅｏ耐性遺伝子の５’側領域、ｐＬＮ１Ｖ５：ｐＬＮ１ベクターにＶ５タグ配列が挿入されたベクター。
　図４は、Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ精製標品のＣＢＢ染色によるＳＤＳ−ＰＡＧＥ泳動像を示す図である。
　図５は、ラットテレメトリー試験によるＮ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘタンパク質を投与したラットにおける最高血圧、最低血圧、平均血圧及び心拍数の解析結果を示す図である。
　図６は、５週齢に達したコントロールキメラマウス６個体およびＵＳｈＢＭＰ９　ＫＩキメラマウス１２個体の血清を用いた血液生化学解析の結果を示す。
　図７は、Ｎ末端Ｈｉｓタグ付加ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターの構造を示す図である。Ｋｏｚａｋ：コザック配列、ＳＰ：マウスＢＭＰ９固有のシグナル配列、Ｈｉｓ−ｔａｇ：Ｈｉｓ６タグ配列、ｈＢＭＰ（−ＳＰ）：固有のシグナルペプチドコード領域を持たないヒトＢＭＰ９遺伝子、ＳＶ４０　ｐｏｌｙＡ：ＳＶ４０由来ｐｏｌｙＡシグナル領域、５’ｎｅｏ：ｎｅｏ耐性遺伝子の５’側領域、ｐＬＮ１Ｖ５：ｐＬＮ１ベクターにＶ５タグ配列が挿入されたベクター。
　図８は、Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ精製標品の銀染色によるＳＤＳ−ＰＡＧＥ泳動像を示す図である。
　図９は、ｈＢＭＰ９　ｃｏｍｐｌｅｘ体を１００μｇ／ｈｅａｄもしくは３６０μｇ／ｈｅａｄで投与したマウス個体の血清生化学検査データを示す。
　図１０は、ｈＢＭＰ９　ｃｏｍｐｌｅｘ体を投与したマウス血清を用いてバイオマーカー調査（ＲＯＤＥＮＴＳ　ＭＵＬＴＩ−ＡＮＡＬＹＴＥ　ＰＲＯＦＩＬＥＳ（Ｒｏｄｅｎｔ　ＭＡＰ　Ｖ２．０　Ｐｌａｓｍａ　Ａｎｔｉｇｅｎ））を実施した時の結果を示す。各々の１００μｇ／ｈｅａｄ、もしくは３６０μｇ／ｈｅａｄでの値（倍）は、それぞれのコントール群に対する変動割合を示す。
　図１１は、ＢＭＰ９中和抗体の持つ血小板数低下作用を示す図である。
　図１２は、ＢＭＰ９中和抗体を投与したマウス血清を用いてバイオマーカー調査（ＲＯＤＥＮＴＳ　ＭＵＬＴＩ−ＡＮＡＬＹＴＥ　ＰＲＯＦＩＬＥＳ（Ｒｏｄｅｎｔ　ＭＡＰ　Ｖ２．０　Ｐｌａｓｍａ　Ａｎｔｉｇｅｎ））を実施した時のデータを示す。各々の値（倍）は、中和抗体投与群での変動割合をコントール群を１とし算出したものである。
　図１３は、ｈｓＡＬＫ１ｍＦｃ精製標品のＣＢＢ染色によるＳＤＳ−ＰＡＧＥ泳動像を示す図である。
　図１４は、ＳＨＲ−ＮＤラットを用いたインビボ試験より見出されたＢＭＰ９中和抗体の持つ中性脂肪（トリグリセロール）、総コレステロールに対する低下作用を示す図である。
　図１５は、ＳＨＲ−ＮＤラットを用いたインビボ試験より見出されたＢＭＰ９中和抗体の持つ心肥大に対する抑制作用を示す図である。心臓重量の値は各個体の体重の値を用いて補正した。
　図１６は、ＢＭＰ９中和抗体を含むＡＬＫ１拮抗薬の持つ、動脈硬化の主要病変であるプラーク形成に対する抑制作用を示した図である。図はＡＰＯＥ欠損マウスの大動脈におけるプラーク面積率を示す。
　図１７、ＡＬＫ１細胞外領域ポリペプチド−Ｆｃ融合蛋白質の降圧作用を示した図である。図はテレメトリー機器を移植したＳＨＲラットの収縮期血圧を示し、左のカラムは被験物質投与後０−２４時間までの経時的な推移、右のカラムは投与後２４~４８時間までの血圧の推移を示す。
　図１８は、ＷＫＹ抗ＧＢＭ腎炎モデルを用いたインビボ試験より見出されたＢＭＰ９中和抗体の腎症進展に対する抑制作用を示した図である。図は経時的な血清ＢＵＮの推移を示す。 FIG. 1 is a diagram showing the neutralizing action of purified hsALK1Fc protein on BMP9.
FIG. 2 shows gene expression analysis using normal human vascular endothelial cells supplemented with BMP9. By stimulation with BMP9, a vascular endothelial disorder marker gene and lifestyle diseases (hypertension, various renal diseases, diabetes, diabetic complications, (Dyslipidemia, arteriosclerotic disease, thrombosis) related gene is a diagram showing that the expression changes.
FIG. 3 is a diagram showing the structure of an N-terminal His-tagged mBMP9 complex recombinant expression vector. Kozak: Kozak sequence, SP: signal sequence specific to mouse BMP9, His-tag: His6 tag sequence, mBMP (-SP): mouse BMP9 gene without a specific signal peptide coding region, SV40 polyA: SV40-derived polyA signal region, 5′neo: a 5 ′ region of a neo resistance gene, pLN1V5: a vector in which a V5 tag sequence is inserted into a pLN1 vector.
FIG. 4 is a diagram showing an SDS-PAGE electrophoretic image of a purified N-terminal His-type mBMP9 complex purified sample by CBB staining.
FIG. 5 is a diagram showing analysis results of systolic blood pressure, diastolic blood pressure, mean blood pressure, and heart rate in rats administered with N-terminal His-type mBMP9 complex protein in a rat telemetry test.
FIG. 6 shows the results of blood biochemical analysis using sera of 6 control chimeric mice and 12 UShBMP9 KI chimeric mice reaching 5 weeks of age.
FIG. 7 is a diagram showing the structure of an N-terminal His-tagged hBMP9 complex recombinant expression vector. Kozak: Kozak sequence, SP: signal sequence unique to mouse BMP9, His-tag: His6 tag sequence, hBMP (-SP): human BMP9 gene without a unique signal peptide coding region, SV40 polyA: SV40-derived polyA signal region, 5′neo: a 5 ′ region of a neo resistance gene, pLN1V5: a vector in which a V5 tag sequence is inserted into a pLN1 vector.
FIG. 8 is a diagram showing an SDS-PAGE electrophoretic image of the N-terminal His-type hBMP9 complex purified preparation by silver staining.
FIG. 9 shows serum biochemical data of individual mice administered with hBMP9 complex at 100 μg / head or 360 μg / head.
FIG. 10 shows the results of conducting a biomarker survey (RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen)) using mouse serum administered with hBMP9 complex. Each value (times) at 100 μg / head or 360 μg / head indicates a variation ratio with respect to each control group.
FIG. 11 is a diagram showing the platelet number lowering action of the BMP9 neutralizing antibody.
FIG. 12 shows data obtained when a biomarker survey (RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen)) was performed using mouse serum administered with a BMP9 neutralizing antibody. Each value (times) is calculated by setting the variation ratio in the neutralizing antibody administration group to 1 in the control group.
FIG. 13 is a diagram showing an SDS-PAGE electrophoretic image of the purified hsALK1mFc preparation by CBB staining.
FIG. 14 is a diagram showing the lowering effect on neutral fat (triglycerol) and total cholesterol of the BMP9 neutralizing antibody found in an in vivo test using SHR-ND rats.
FIG. 15 is a diagram showing an inhibitory action on cardiac hypertrophy possessed by a BMP9 neutralizing antibody found from an in vivo test using SHR-ND rats. The heart weight value was corrected using the body weight value of each individual.
FIG. 16 is a view showing an inhibitory action of an ALK1 antagonist containing a BMP9 neutralizing antibody on plaque formation, which is a main lesion of arteriosclerosis. The figure shows the plaque area ratio in the aorta of APOE-deficient mice.
FIG. 17 shows the antihypertensive action of ALK1 extracellular region polypeptide-Fc fusion protein. The figure shows the systolic blood pressure of SHR rats implanted with telemetry equipment. The left column shows the change over time from 0 to 24 hours after administration of the test substance, and the right column shows the change in blood pressure from 24 to 48 hours after administration. Indicates.
FIG. 18 is a diagram showing the inhibitory effect of BMP9 neutralizing antibody on the progression of nephropathy found from an in vivo test using a WKY anti-GBM nephritis model. The figure shows the change in serum BUN over time.
　以下、本発明を詳細に説明する。
　本発明は、ＡＬＫ１阻害剤を有効成分とする血管障害を抑制するための医薬組成物を提供する。
　本発明の医薬組成物に含まれるＡＬＫ１阻害剤としては、ＡＬＫ１の活性化、すなわちＡＬＫ１のキナーゼ活性の活性化を抑制し、ＡＬＫ１シグナルを抑制しうる物質であれば特に限定されない。本発明に用いられるＡＬＫ１阻害剤として、特に限定されるものではないが、例えば、以下の（１）~（１０）が挙げられる：
（１）ＢＭＰ９に結合する抗体または該抗体断片；
（２）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質；
（３）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸；
（４）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸を含む発現ベクター；
（５）ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片；
（６）ＢＭＰ９に対するアプタマーまたはＡＬＫ１細胞外領域ポリペプチドに対するアプタマー；
（７）ＡＬＫ１遺伝子を標的とするＲＮＡｉ分子、またはＢＭＰ９遺伝子を標的とするＲＮＡｉ分子；
（８）ＡＬＫ１のキナーゼ活性に対して阻害活性を有する低分子化合物；
（９）ＢＭＰ９とＡＬＫ１との結合、もしくはＢＭＰ９とタイプＩＩレセプターとの結合を阻害する活性を有する低分子化合物；または
（１０）ＢＭＰ９阻害活性を有するＢＭＰ９アンタゴニスト蛋白質。
　本発明に用いられるＡＬＫ１阻害剤として特に好ましくは、ＢＭＰ９に結合する抗体または該抗体断片であって中和活性を有するものが挙げられる。
　以下に、本発明の医薬組成物に含めることができるＡＬＫ１阻害剤について説明する。
＜ＢＭＰ９に結合する抗体または該抗体断片＞
（ＢＭＰ９抗体）
　ＢＭＰ９に結合する抗体は、中和抗体が望ましく、下記の方法により作製することができる
　ＢＭＰ９は公知であり、ヒト、マウス、セキショクヤケイなどから単離され、配列情報がＧｅｎＢａｎｋなどのデータベースに公開されている。ヒト又はマウス由来ＢＭＰ９の配列情報は、例えばヒトＢＭＰ９はアクセッション番号Ｑ９ＵＫ０５，ＡＡＤ５６９６０等として、マウスＢＭＰ９はアクセッション番号Ｑ９ＷＶ５６，ＮＰ＿０６２３７９等として、ＧｅｎＢａｎｋに登録されておりこれらを利用することが可能である。
　ＢＭＰ９蛋白質は、ＧｅｎＢａｎｋなどに登録された公知のアミノ酸配列を有するＢＭＰ９蛋白質を用いることができるが、好ましくは配列番号１０（マウスＢＭＰ９）または配列番号２４（ヒトＢＭＰ９）で示されたアミノ酸配列を有するＢＭＰ９蛋白質、特に好ましくは配列番号２４で示されたアミノ酸配列を有するＢＭＰ９蛋白質を用いる。また、配列番号１０および配列番号２４で示される配列は、シグナル配列、ｐｒｏ−ｒｅｇｉｏｎを含み、配列番号１０においては、３１９番目から４２８番目の配列がマチュア部分に相当し、配列番号２４においては、３２０番目から４２９番目の配列がマチュア部分に相当する。本明細書におけるＢＭＰ９には、ＢＭＰ９の変異体も包含される。このような変異体は、天然の突然変異体及び人工変異体のいずれも含まれ、上記ＢＭＰ９のアミノ酸配列において、配列番号１０の少なくとも３１９番目から４２８番目、または配列番号２４の少なくとも３２０番目から４２９番目の配列に示される配列を有するアミノ酸配列に１~数個のアミノ酸の欠失、置換、付加又は挿入を含むか、或いは、該アミノ酸配列と８０％以上、好ましくは８５％以上、より好ましくは９０％以上、例えば９３％以上、９５％以上、９７％以上、９８％以上又は９９％以上の同一性を有するアミノ酸配列を有するものである。「１から数個」の範囲は特には限定されないが、例えば、１から２０個、好ましくは１から１０個、より好ましくは１から７個、さらに好ましくは１から５個、特に好ましくは１から３個、あるいは１個又は２個である。
　目的の抗体を得るための抗原エピトープは、ＢＭＰ９のアミノ酸配列において抗原性の高い領域、表在性がある領域、二次構造をとらない可能性のある領域、他の蛋白質とホモロジーがないか又は低い領域から選択され得る。ここで抗原性の高い領域は、Ｐａｒｋｅｒらの方法［Ｂｉｏｃｈｅｍｉｓｔｒｙ、２５巻、５４２５−５４３２頁（１９８６年）］によって推定可能である。表在性がある領域は、例えばハイドロパシーインデックスを計算しプロットすることによって推定可能である。二次構造をとらない可能性のある領域は、例えば、ＣｈｏｕとＦａｓｍａｎの方法［Ａｄｖ　Ｅｎｚｙｍｏｌ　Ｒｅｌａｔ　Ａｒｅａｓ　Ｍｏｌ　Ｂｉｏｌ．、４７巻、４５−１４８頁（１９７８年）］によって推定可能である。
　上記の手法で推定されたＢＭＰ９の部分アミノ酸配列を基に、ペプチド合成法を利用することによって該アミノ酸配列からなるペプチドを合成することができる。目的のペプチドは、例えば、Ｒ．Ｂ．Ｍｅｒｒｉｆｉｅｌｄ［Ｓｃｉｅｎｃｅ、２３２巻、３４１−３４７頁（１９８６年）］によって開発された固相ペプチド合成に基づいた市販のペプチド合成機を使用して合成し、保護基を脱離後、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、逆相クロマトグラフィー等を単独もしくは組合わせた方法により精製する。得られた精製ペプチドはキーホールリンペットヘモシアニン（ＫＬＨ）やアルブミンなどのキャリヤ蛋白質と結合し免疫原として使用することができる。
　さらに、ＢＭＰ９を免疫原としてＢＭＰ９と結合するポリクローナル抗体あるいはモノクローナル抗体を公知の手法により作製することもできる。なお、本明細書中において、ＢＭＰ９若しくはモノクローナル抗体、ポリクローナル抗体、又は他の蛋白質に関して用いられる「組換え」という用語は、これらの蛋白質が宿主細胞内で組換えＤＮＡによって生産されたものであることを意味する。宿主細胞としては、原核生物（例えば大腸菌のような細菌）および真核生物（例えば酵母、ＣＨＯ細胞、昆虫細胞等）のいずれも使用され得る。
　本発明の「抗体」はペプチド抗体、ポリクローナル抗体、モノクローナル抗体のいずれでもよい。「抗体」は、マウスまたは他の適した宿主動物（たとえばウサギ、ウシ、ウマ、ヒツジ、ブタ、げっ歯類など）を免疫に用いられた蛋白質に特異的に結合するであろう抗体を産生するか、産生するであろうリンパ球を引き出すために、皮下、腹腔内、または筋肉内の経路によって、抗原あるいは抗原発現細胞を免疫することによって得られる。さらに宿主動物としてはヒト抗体遺伝子のレパートリーを有するトランスジェニック動物に抗原または抗原発現細胞を投与し、所望のヒト化抗体を取得してもよい［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、９７巻、７２２−７２７頁（２０００年）、国際公開ＷＯ９６／３３７３５、ＷＯ９７／０７６７１、ＷＯ９７／１３８５２、ＷＯ９８／３７７５７参照］。上記の手法に代えて、リンパ球をインビトロで免疫化することによって抗体を得ることができる。宿主動物から得られた血清から、抗原に結合する画分を集め、精製することにより、ポリクローナル抗体を取得することができる。また、ハイブリドーマ細胞を形成するために、ポリエチレングリコールのような適当な融合試薬を用いて、リンパ球を骨髄腫細胞と融合させることにより、モノクローナル抗体を作製することができる［Ｇｏｄｉｎｇ，Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ：Ｐｒｉｎｃｉｐａｌｓ　ａｎｄ　Ｐｒａｃｔｉｃｅ、５９−１０３頁、Ａｃａｄｅｍｉｃ　ｐｒｅｓｓ、（１９８６年）］。例えば本発明のモノクローナル抗体は、ハイブリドーマ法［Ｎａｔｕｒｅ、２５６巻、４９５頁（１９７５年）］を用いても、組換えＤＮＡ法（Ｃａｂｉｌｌｙら、米国特許第４８１６５６７号）を用いても作製することができる。
　抗原蛋白質は、ＢＭＰ９の蛋白質の全てまたは部分配列をコードするＤＮＡを、大腸菌や酵母、昆虫細胞、動物細胞などで発現させることにより調製することができる。遺伝子組換えＢＭＰ９は、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、逆相クロマトグラフィー等を単独もしくは組合わせた方法により精製し、この精製標品を免疫原として用いる。
　また、本発明の抗体は、無傷の抗体であってもよいし、あるいは（Ｆａｂ’）２やＦａｂなどの抗体断片であってもよい。
　また、定常領域をヒトの定常領域に置き換えたキメラ抗体やモザイク抗体［例えばマウス−ヒトキメラ抗体；Ｃａｂｉｌｌｙら、米国特許４８１６５６７およびＭｏｒｒｉｓｏｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、８９巻、６８５１頁（１９８４年）］、定常領域および超可変領域（または、Ｃｏｍｐｌｅｍｅｎｔａｒｙ−ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ；以下、ＣＤＲと表記）を除く全ての可変領域をヒトの配列に置き換えたヒト化抗体［Ｃａｒｔｅｒら，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、８９巻、４２８５頁（１９９２年）およびＣａｒｔｅｒら、ＢｉｏＴｅｃｈｎｏｌｏｇｙ、１０巻、１６３頁、（１９９２年）］、完全ヒト抗体（国際公開ＷＯ０２／４３４７８号公報）も本発明の抗体に含まれる。ヒトに使用した場合、異種抗原に対する抗体、たとえばヒト抗マウス抗体（ｈｕｍａｎ　ａｎｔｉｍｏｕｓｅ　ｍｏｎｏｃｌｏｎａｌ　ａｎｔｉｂｏｄｙ；ＨＡＭＡ）などの出現が起こらないようにするために、キメラ抗体やモザイク抗体もしくはヒト化抗体の使用が好ましく、最も好ましくは完全ヒト抗体である。
（ＢＭＰ９抗体断片）
　本発明において、抗体断片としては、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、ｓｃＦｖ、ｄｉａｂｏｄｙ、ｄｓＦｖおよびＣＤＲを含むペプチドなどがあげられる。
　Ｆａｂは、ＩｇＧを蛋白質分解酵素であるパパインで処理して得られる断片のうち（Ｈ鎖の２２４番目のアミノ酸残基で切断される）、Ｈ鎖のＮ末端側約半分とＬ鎖全体がジスルフィド結合で結合した分子量約５万の抗原結合活性を有する抗体断片である。本発明のＦａｂは、ＢＭＰ９に結合するモノクローナル抗体をパパインで処理して得ることができる。また、該抗体のＦａｂをコードするＤＮＡを原核生物用発現ベクターあるいは真核生物用発現ベクターに挿入し、該ベクターを原核生物あるいは真核生物へ導入することにより発現させ、Ｆａｂを製造することもできる。
　Ｆ（ａｂ’）２は、ＩｇＧのヒンジ領域の２個のジスルフィド結合の下部を蛋白質分解酵素であるペプシンで分解して得られた、２つのＦａｂ領域がヒンジ部分で結合して構成された、分子量約１０万の抗原結合活性を有する断片である。本発明のＦ（ａｂ’）２は、ＢＭＰ９に結合するモノクローナル抗体をペプシンで処理して得ることができる。また、下記のＦａｂ’をチオエーテル結合あるいはジスルフィド結合させ、作製することもできる。
　Ｆａｂ’は、上記Ｆ（ａｂ’）２のヒンジ領域のジスルフィド結合を切断した分子量約５万の抗原結合活性を有する抗体断片である。本発明のＦａｂ’は、ＢＭＰ９に結合するＦ（ａｂ’）２をジチオスレイトールなどの還元剤で処理して得ることができる。また、該抗体のＦａｂ’断片をコードするＤＮＡを原核生物用発現ベクターあるいは真核生物用発現ベクターに挿入し、該ベクターを原核生物あるいは真核生物へ導入することにより発現させ、Ｆａｂ’を製造することもできる。
　ｓｃＦｖは、１本の重鎖可変領域（以下、ＶＨと表記する）と１本の軽鎖可変領域（以下、ＶＬと表記する）とを適当なペプチドリンカー（以下、Ｐと表記する）を用いて連結した、ＶＨ−Ｐ−ＶＬないしはＶＬ−Ｐ−ＶＨポリペプチドで、抗原結合活性を有する抗体断片である。本発明のｓｃＦｖは、ＢＭＰ９に結合するモノクローナル抗体のＶＨおよびＶＬをコードするｃＤＮＡを取得し、ｓｃＦｖをコードするＤＮＡを構築し、該ＤＮＡを原核生物用発現ベクターあるいは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物あるいは真核生物へ導入することにより発現させ、製造することができる。
　ｄｉａｂｏｄｙは、ｓｃＦｖが二量体化した抗体断片で、二価の抗原結合活性を有する抗体断片である。二価の抗原結合活性は、同一であることもできるし、一方を異なる抗原結合活性とすることもできる。本発明のｄｉａｂｏｄｙは、ＢＭＰ９に結合するモノクローナル抗体のＶＨおよびＶＬをコードするｃＤＮＡを取得し、ｓｃＦｖをコードするＤＮＡをペプチドリンカーのアミノ酸配列の長さが８残基以下となるように構築し、該ＤＮＡを原核生物用発現ベクターあるいは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物あるいは真核生物へ導入することにより発現させ、製造することができる。
　ｄｓＦｖは、ＶＨおよびＶＬ中のそれぞれ１アミノ酸残基をシステイン残基に置換したポリペプチドを該システイン残基間のジスルフィド結合を介して結合させたものをいう。システイン残基に置換するアミノ酸残基は既知の方法［Ｐｒｏｔｅｉｎ　Ｅｎｇｉｎｅｅｒｉｎｇ，７，６９７（１９９４）］に従って、抗体の立体構造予測に基づいて選択することができる。本発明のｄｓＦｖは、本発明のＢＭＰ９に結合するモノクローナル抗体のＶＨおよびＶＬをコードするｃＤＮＡを取得し、ｄｓＦｖをコードするＤＮＡを構築し、該ＤＮＡを原核生物用発現ベクターあるいは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物あるいは真核生物へ導入することにより発現させ、製造することができる。
　ＣＤＲを含むペプチドは、ＶＨまたはＶＬのＣＤＲの少なくとも１領域以上を含んで構成される。複数のＣＤＲを含むペプチドは、直接または適当なペプチドリンカーを介して結合させることができる。本発明のＣＤＲを含むペプチドは、本発明のＢＭＰ９に結合するモノクローナル抗体のＶＨおよびＶＬのＣＤＲをコードするＤＮＡを構築し、該ＤＮＡを原核生物用発現ベクターあるいは真核生物用発現ベクターに挿入し、該発現ベクターを原核生物あるいは真核生物へ導入することにより発現させ、製造することができる。また、ＣＤＲを含むペプチドは、Ｆｍｏｃ法、またはｔＢｏｃ　ｔ法などの化学合成法によって製造することもできる。
＜ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質＞
（ＡＬＫ１細胞外領域ポリペプチドまたはその変異体）
　ＡＬＫ１は、トランスフォーミング増殖因子β受容体１型（ＴＧＦ−β−１）のＩ型細胞表面受容体である。ヒトＡＬＫ１は、アミノ酸５０３個のポリペプチドであり、これには、シグナル配列（アミノ酸：１~２１位）、Ｎ−末端細胞外ＴＧＦ−β−１リガンド結合ドメイン、または細胞外ドメイン（アミノ酸２２~１１８位）、１回膜貫通ドメイン（アミノ酸１１９~１４１位）、調節グリシン／セリンリッチ（ＧＳ）ドメイン（アミノ酸：１４２~２０２位）、およびＣ−末端セリン−トレオニンキナーゼドメイン（２０２~４９２位）が含まれる。Ａｔｔｉｓａｎｏ　ｅｔ　ａｌ．Ｃｅｌｌ，１９９３，ｖｏｌ．７５，ｐｐ．６７１−６８０には、１７２位にセリンが含まれるヒトＡＬＫ１のアミノ酸配列（Ｇｅｎｂａｎｋアクセッション番号Ｌ１７０７５）が、米国特許第６，３１６，２１７号には、１７２位にトレオニンを有するヒトＡＬＫ１のアミノ酸配列（Ｇｅｎｂａｎｋアクセッション番号ＮＭ＿００００２０）が開示されている。Ａｔｔｉｓａｎｏらにおいて開示されている完全長のヒトＡＬＫ１をコードするＡＣＶＲＬ１遺伝子は、Ｉｎｖｉｔｒｏｇｅｎ　Ｉｎｃ．からクローンＩＤ　ＩＯＨ２１０４８として市販されている。
　本発明において、「ＡＬＫ１細胞外領域ポリペプチド」とは、哺乳動物由来のＡＬＫ１の細胞外領域ポリペプチドのアミノ酸配列の全体または一部からなる蛋白質である。
　ヒトＡＬＫ１細胞外領域ポリペプチドのアミノ酸配列、ｃＤＮＡ配列は、以下のとおりである。
　ヒトＡＬＫ１細胞外領域ポリペプチドのアミノ酸配列（配列番号２９）：
Figure JPOXMLDOC01-appb-I000001
（Ｇｅｎｂａｎｋ　ＡＣＣＥＳＳＩＯＮ　Ｎｏ．Ｌ１７０７５）下線はシグナル配列を示し、囲み線はシグナル配列切断後の細胞外領域ポリペプチドを示す。
　ヒトＡＬＫ１細胞外領域ポリペプチドのｃＤＮＡ配列（配列番号３０）：
Figure JPOXMLDOC01-appb-I000002
　本発明においてＡＬＫ１細胞外領域ポリペプチドには、上記のＡＬＫ１細胞外領域ポリペプチドの変異体も包含される。このような変異体は、天然の突然変異体及び人工変異体のいずれも含まれ、上記ＡＬＫ１細胞外領域ポリペプチドのアミノ酸配列において、１もしくは複数（好ましくは１もしくは数個）のアミノ酸の置換、欠失又は付加を含むか、或いは、該アミノ酸配列と８０％以上、好ましくは８５％以上、より好ましくは９０％以上、例えば９３％以上、９５％以上、９７％以上、９８％以上又は９９％以上の同一性を有するアミノ酸配列を含み、かつ、血管障害抑制活性を有するものである。
　例示的に、上記変異体は、配列番号２９のアミノ酸配列において、１もしくは複数（好ましくは１もしくは数個）のアミノ酸の置換、欠失又は付加を含むか、或いは、該アミノ酸配列と８０％以上、好ましくは８５％以上、より好ましくは９０％以上、例えば９３％以上、９５％以上、９７％以上、９８％以上又は９９％以上の同一性を有するアミノ酸配列を含み、かつ、血管障害抑制活性を有するものである。
　本明細書で使用される「数個」なる用語は、通常、２から１０までの任意の整数を指す。好ましくは、それは２~５の任意の整数である。
　本明細書で使用される「同一性」なる用語は、２つのアミノ酸配列（又はヌクレオチド配列）のアラインメントにおいて、同一のアミノ酸残基数（又はヌクレオチド数）が最大となるように該２つの配列を整列させたときの配列間の一致度を意味し、具体的には、総アミノ酸残基数（又は総ヌクレオチド数）に対する同一アミノ酸残基数（又は同一ヌクレオチド数）のパーセンテージ（％）で表わされる。ＦＡＳＴＡのようにギャップを導入する場合、ギャップの数も総アミノ酸残基数（又は総ヌクレオチド数）に加算する。
　８０％以上、好ましくは８５％以上の配列同一性を有する蛋白質は、例えばＮＣＢＩ（米国）、ＥＭＢＬ（欧州）などの配列データベースにアクセスし、例えばＢＬＡＳＴ、ＦＡＳＴＡなどの配列相同性検索プログラムを利用して検索することが可能である（Ａｌｔｓｃｈｕｌ，Ｓ．Ｆ．ら（１９９０）Ｊ．Ｍｏｌ．Ｂｉｏｌ．１５：４０３−４１０；Ｋａｒｌｉｎ，Ｓ．とＡｌｔｓｃｈｕｌ　Ｓ．Ｆ．（１９９０）Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８７：２２６４−２２６８など）。ＢＬＡＳＴは、配列を固定長のワードに区切り、ワード単位で類似する断片を検索し、これらを類似度が最大になるまで両方向に伸ばして局所的なアラインメントを行い、最後にこれらを結合して最終的なアラインメントを行う方法である。また、ＦＡＳＴＡは、連続して一致する配列の断片を高速に検索し、それらの断片の中で類似度の高いものに着目して局所的なアラインメントを行い、最後にギャップを考慮した上でこれらを結合しアラインメントを行う方法である。
　変異導入法としては、ＡＬＫ１細胞外領域ポリペプチドの配列に基づいて合成した（相補的変異配列を含む）プライマーを使用するＰＣＲ法を利用した部位特異的突然変異誘発法が好ましい（Ｋｕｎｋｅｌら，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，１９８５，８２：４８８−４９２；Ｆ．Ｍ．Ａｕｓｕｂｅｌら，Ｓｈｏｒｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，１９９５，Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ；Ｊ．Ｓａｍｂｒｏｏｋら，Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ，２ｎｄ　ｅｄ．，１９８９，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓなど）。変異導入用キット（例えば、宝酒造製）も市販されているので、指示書に従って変異を導入することもできる。
　簡単に説明すると、Ｋｕｎｋｅｌらの方法によると、ＡＬＫ１細胞外領域ポリペプチドをコードするＤＮＡを含むプラスミドを鋳型にし、Ｔ４　ＤＮＡポリヌクレオチドキナーゼで予め５’末端をリン酸化したプライマー（相補的変異配列を含む）を該鋳型にアニーリングし、ＤＮＡ合成を行ったのち、Ｔ４　ＤＮＡリガーゼで末端同士を連結して、目的の変異を含むＤＮＡを精製することを含む。
　本発明において、変異は、置換、欠失、付加、挿入、又はそれの組み合わせを含む。
　置換は、保存的置換又は非保存的置換のいずれでもよいが、ＡＬＫ１細胞外領域ポリペプチドのコンホメーションを実質的に変化させないためには保存的置換が好ましい。保存的置換は、構造的（例えば、分岐状、芳香族性など）、電気的（例えば、酸性、塩基性など）、極性又は疎水性、などの化学的・物理的性質の類似したアミノ酸間での置換をいう。分岐状アミノ酸には、バリン、ロイシン、イソロイシンが含まれる。芳香族アミノ酸には、チロシン、トリプトファン、フェニルアラニン、ヒスチジンが含まれる。酸性アミノ酸には、グルタミン酸、アスパラギン酸が含まれる。塩基性アミノ酸には、リシン、アルギニン、ヒスチジンが含まれる。極性アミノ酸には、セリン、トレオニン、グルタミン、アスパラギン、チロシン、システイン、グリシン、プロリンなどが含まれる。疎水性アミノ酸には、アラニン、バリン、ロイシン、イソロイシン、メチオニンなどが含まれる。
　欠失は、１もしくは複数のアミノ酸残基を失うことである。付加は、１もしくは複数のアミノ酸残基を蛋白質のＮ末端又はＣ末端に結合することである。挿入は、蛋白質の内部に１もしくは複数のアミノ酸残基を結合することである。このうち、欠失と挿入は、ＡＬＫ１細胞外領域ポリペプチドのコンホメーションを実質的に変化させないことを前提として行うことができる。そのために、好ましくは約１~５個程度のアミノ酸残基の欠失又は挿入に制限される。
（ＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質）
　ＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質とは、上記ＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質であり、限定されるものではないが、ＡＬＫ１細胞外領域ポリペプチド又はその変異体と哺乳動物由来免疫グロブリンＦｃ蛋白質又はその変異体との融合蛋白質（例えばＷＯ　２００８／０５７４６１に記載された融合蛋白質）が挙げられる。
　ここで、「含む」なる表現は、上記ＡＬＫ１細胞外領域ポリペプチド又はその変異体に、異種ペプチド、ポリペプチド又は蛋白質を、該ドメイン又はその変異体のＮ末端又はＣ末端側に、必要であれば適当なペプチドリンカー（例えば、アミノ酸数１~２０）を介して、結合又は融合してもよいことを意味する。例えば、そのような異種蛋白質として好ましい例は、哺乳動物由来免疫グロブリンＦｃ蛋白質又はその変異体などが挙げられる。しかし、このような異種蛋白質が生体内に投与されると、拒絶反応を引き起こす可能性があるため、それをできる限り回避するためにも、投与する哺乳動物が本来もつ蛋白質を該異種蛋白質として使用することが望ましい。
　好ましいＦｃ蛋白質は、ヒトへの使用を考慮すると、ヒト免疫グロブリンのＦｃ蛋白質である。また、免疫グロブリンのクラス及びサブクラスは、以下のものに制限されないが、例えばＩｇＧ，ＩｇＤ，ＩｇＥ，ＩｇＭ，ＩｇＡ，ＩｇＧ１，ＩｇＧ２，ＩｇＧ２ａ，ＩｇＧ２ｂ，ＩｇＧ２ｃ，ＩｇＧ３，ＩｇＧ４，ＩｇＡ１，ＩｇＡ２などのいずれも使用することができる。ただし、ヒトに使用するのであれば、ヒト免疫グロブリンのクラス及びサブクラスを使用することが望ましい。Ｆｃ蛋白質は、ＡＬＫ１細胞外ドメイン又はその変異体の生体内（ｉｎ　ｖｉｖｏ）での安定性を向上させることができる。ただし、この場合、Ｆｃ蛋白質は、その生物活性による生体内に及ぼす影響を避けるために、例えば抗体依存性細胞障害活性（ＡＤＣＣ）及び補体依存性細胞障害活性（ＣＤＣ）活性などの生物活性を予め低下させることが望ましく、そのために、前記のような生物活性を抑制、低下又は喪失させるための変異を導入することが好ましい。
　Ｆｃ蛋白質の好適例は、以下の配列番号３１のアミノ酸配列で表されるヒトＩｇＧ１Ｆｃ変異体である。
配列番号３１
Figure JPOXMLDOC01-appb-I000003
Ｆｃ蛋白質の結合位置は、ＡＬＫ１細胞外領域ポリペプチド又はその変異体のＮ末端側、Ｃ末端側のいずれでもよいが、Ｃ末端側が好ましい。
　上記ＡＬＫ１細胞外領域ポリペプチドとＦｃ蛋白質との融合蛋白質の具体例は、例えば下記の配列番号３２のアミノ酸配列で表される蛋白質である。ここで、下線部分は、ＡＬＫ１細胞外領域ポリペプチド（配列番号２９）を、非下線部分は、ヒトＩｇＧ１Ｆｃ変異体蛋白質（配列番号３１）をそれぞれ示す。
配列番号３２：
Figure JPOXMLDOC01-appb-I000004
　本発明においては、ＡＬＫ１細胞外領域ポリペプチド又はその変異体を含む蛋白質は、必ずしも異種ペプチド、ポリペプチド又は蛋白質と結合又は融合する必要はない。
　本発明におけるＡＬＫ１細胞外領域ポリペプチド又はその変異体を含む蛋白質は、この業界で慣用の遺伝子組換え技術によって作製しうる。簡単に説明すると、該蛋白質の作製は、本発明の蛋白質をコードするＤＮＡを用意し、このＤＮＡを含む発現ベクターを構築し、該ベクターで原核又は真核細胞を形質転換又はトランスフェクションし、得られた細胞の培養から目的の組換え蛋白質を回収することを含む。蛋白質の精製は、蛋白質の慣用の精製法、例えば硫酸アンモニウム沈殿、有機溶媒沈殿、透析、電気泳動、クロマトフォーカシング、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、ＨＰＬＣなどを適宜組み合わせることによって実施可能である。
　上記ＤＮＡやベクターに関しては、後述の＜ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸＞ならびに＜ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸を含む発現ベクター＞の節、実施例等に記載しているので、それらを参照することができる。また、遺伝子組換え技術は、Ａｕｓｕｂｅｌら（上記）、Ｓａｍｂｒｏｏｋら（上記）などに記載されており、本発明のために利用できる。
　本発明におけるＡＬＫ１細胞外領域ポリペプチド又はその変異体を含む蛋白質は、化学修飾されていてもよい。
　化学修飾には、非限定的に、例えばグリコシル化、ペグ（ＰＥＧ）化、アセチル化、アミド化、リン酸化などが含まれる。特に好ましく利用できる化学修飾は、グリコシル化及びペグ化である。
　ペグ化は、例えば蛋白質のＮ末端アミノ基、リシン（Ｌｙｓ）のεアミノ基などのアミノ酸残基に１又は複数のポリエチレングリコール（ＰＥＧ）分子を結合させることである。一般的には、アミノ酸の遊離アミノ基にＰＥＧ分子が結合される。ＰＥＧの平均分子量は、以下に限定されないが、約３，０００~約５０，０００の範囲で使用可能である。ＰＥＧを蛋白質に結合させるには、ＰＥＧの末端を、例えばカルボキシル化、ホルミル化、スクシンイミジル化、又はマレイイミジル化し、蛋白質の遊離アミノ基と反応させることができる。
　グリコシル化は、蛋白質のアスパラギン、セリン又はトレオニン残基に炭水化物鎖（即ち、糖鎖）が結合することである。一般に、Ａｓｎ−Ｘ−Ｔｈｒ／Ｓｅｒ（ここで、ＸはＰｒｏ以外の任意のアミノ酸残基である。）の配列を認識して糖鎖の結合が起こる。このような配列をもつように該蛋白質のアミノ酸配列を改変するときは、天然型と異なる位置に糖鎖を導入することもできる。通常、遺伝子組換え技術によって、組換え蛋白質をコードする核酸を、真核細胞（酵母細胞、動物細胞、植物細胞など）中で発現することによって、組換え蛋白質のグリコシル化を起こすことができる。本発明では、糖鎖の構造は特に制限されないものとし、発現のために選択された細胞の種類によって糖鎖構造が異なると考えられる。ヒトにおける使用の場合、ヒト由来細胞、ヒト糖鎖を合成可能な酵母細胞、チャイニーズハムスター卵巣（ＣＨＯ）細胞などを利用しうる。
　アセチル化やアミド化は、主に、蛋白質のＮ末端又はＣ末端で行うことが望ましい。これらの反応は、例えば脂肪族アルコールや脂肪酸などのアルコール類やカルボン酸類を用いて行うことができる。アルキル部分の炭素数は、例えば約１~２０程度であるが、水溶性を損なわない、無毒性であるなどの条件を満たす必要がある。
＜ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸＞
　本明細書で使用する「核酸」なる用語には、ＤＮＡ及びＲＮＡの両方を含むものとし、ＤＮＡにはゲノムＤＮＡやｃＤＮＡを含み、ＲＮＡにはｍＲＮＡを含む。
　ＡＬＫ１細胞外領域ポリペプチド、その変異体及びそれらを含む蛋白質については、Ｆｃ蛋白質との融合蛋白質を含めて、上記＜ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質＞の各節で説明したとおりであり、それらの節で説明した全ての記載をここでも引用する。したがって、本発明における核酸は、上で説明し具体的に例示した、ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸を包含する。
　具体的には、該核酸は、ヒトＡＬＫ１の細胞外領域ポリペプチド（配列番号２９）のアミノ酸配列において、少なくとも、２２番目~１１８番目のアミノ酸を含むアミノ酸配列をコードする核酸を包含する。
　真核細胞での該核酸の発現及び発現産物の細胞外への分泌を考慮するならば、シグナル配列をコードするヌクレオチド配列をさらに含むとよい。シグナル配列の例は、ＢＭＰ９シグナル配列、Ｉｇκなどである。
　以下にヒト由来ＡＬＫ１細胞外領域ポリペプチドの前駆体をコードするヌクレオチド配列を例示する。下線部位はシグナル配列をコードするヌクレオチド配列、非下線部位は、細胞外ドメイン蛋白質の成熟配列をコードするヌクレオチド配列を示す。
ヒトＡＬＫ１細胞外領域ポリペプチドをコードするＤＮＡ（配列番号３０）：
Figure JPOXMLDOC01-appb-I000005
　本発明における核酸は、ＡＬＫ１細胞外領域ポリペプチドを含む蛋白質又はその変異体と、上記定義の異種蛋白質との融合蛋白質をコードする核酸も含まれる。異種蛋白質として好ましい例は、哺乳動物由来の免疫グロブリンＦｃ蛋白質であり、特にヒトＦｃ蛋白質が好ましいが、その生物活性（特にＡＤＣＣ及びＣＤＣ）を低下又は喪失させるように変異を導入することが望ましい。例えば、変異型ヒトＩｇＧ１由来Ｆｃ蛋白質をコードするヌクレオチド配列を配列番号３３に示す。
配列番号３３
Figure JPOXMLDOC01-appb-I000006
　さらにまた、この変異型ヒトＩｇＧ１由来Ｆｃ蛋白質（下線部）と、ヒト由来ＡＬＫ１細胞外領域ポリペプチドを含む蛋白質（非下線部）との融合蛋白質をコードするヌクレオチド配列（配列番号３４）を、以下に例示する。
配列番号３４：
Figure JPOXMLDOC01-appb-I000007
　上記融合蛋白質をコードするヌクレオチド配列には、シグナル配列をコードするヌクレオチド配列をさらに含むことができる。シグナル配列の例は、ヒト蛋白質由来のシグナル配列、例えばＢＭＰ９、Ｉｇκなどである。
　上記蛋白質類をコードする核酸ホモログは、ヒト又はマウス由来のＡＬＫ１細胞外領域ポリペプチド遺伝子をコードするｍＲＮＡから合成したｃＤＮＡに基づいて作製したプライマーやプローブを使用する周知の技術によって、ヒト及びマウス以外の他の哺乳動物由来の、該遺伝子を発現することが公知の細胞や組織から調製したｃＤＮＡライブラリーから取得することができる。そのような技術には、ＰＣＲ法、ハイブリダイゼーション法（サザン法、ノーザン法など）などが含まれる。
　ＰＣＲ法は、ポリメラーゼ連鎖反応であり、これは、二本鎖ＤＮＡを一本鎖に解離するための変性（ｄｅｎａｔｕｒｉｎｇ）工程（約９４~９６℃、約３０秒~１分）、プライマーを鋳型の一本鎖ＤＮＡに結合するためのアニーリング（ａｎｎｅａｌｉｎｇ）工程（約５５~６８℃、約３０秒~１分）、ＤＮＡ鎖を伸長するための伸長（ｅｘｔｅｎｓｉｏｎ）工程（約７２℃、約３０秒~１分）からなるサイクルを１サイクルとして約２５~４０サイクルを実施する。また、変性工程の前に、約９４~９５℃で約５~１２分の前加熱処理を行い、伸長工程の最終サイクル後に、さらに７２℃で約７~１５分の伸長反応を実施することができる。ＰＣＲは、市販のサーマルサイクラーにて、耐熱性ＤＮＡポリメラーゼ（例えば、ＡｍｐｌｉＴａｑ　Ｇｏｌｄ（登録商標）（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）など）、ＭｇＣｌ２、ｄＮＴＰ（ｄＡＴＰ，ｄＧＴＰ，ｄＣＴＰ，ｄＴＴＰ）などを含有するＰＣＲバッファー中で、センス及びアンチセンスプライマー（サイズ：約１７~３０ｂ、好ましくは２０~２５ｂ）と鋳型ＤＮＡの存在下で行う。増幅されたＤＮＡは、アガロースゲル電気泳動で分離・精製（臭化エチジウム染色）することができる。
　ハイブリダイゼーションは、約２０~１００ｂ又はそれ以上の長さの標識プローブと二本鎖を形成して目的核酸を検出する技法である。選択性を高めるために、一般にストリンジェントな条件でハイブリダイゼーションを行うことができる。ストリンジェントな条件は、例えば約１~５×ＳＳＣ、室温~約４０℃でのハイブリダイゼーション、及びその後の、約０．１~１×ＳＳＣ、０．１％　ＳＤＳ、約４５~６５℃での洗浄からなる。ここで、１×ＳＳＣは、１５０ｍＭ　ＮａＣｌ、１５ｍＭ　Ｎａ−クエン酸、ｐＨ７．０の溶液を指す。このような条件は、配列同一性が約８０％以上、好ましくは８５％以上の核酸を検出することを可能にするであろう。
＜ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸を含む発現ベクター＞
　上記核酸はベクターに挿入され、本発明の医薬組成物の有効成分である蛋白質の製造のために使用されるか、或いは、ベクター自体を製剤化して医薬組成物として使用できる。
　ベクターは、例えばプラスミド、ファージ、ウイルスなどを含む。プラスミドの例は、非限定的に、大腸菌由来プラスミド（例えばｐＲＳＥＴ、ｐＴＺ１９Ｒ、ｐＢＲ３２２、ｐＢＲ３２５、ｐＵＣ１１８、ｐＵＣ１１９など）、枯草菌由来プラスミド（例えばｐＵＢ１１０、ｐＴＰ５など）、酵母由来プラスミド（例えばＹＥｐ１３、ＹＥｐ２４、ＹＣｐ５０など）、Ｔｉプラスミドなどが挙げられ、ファージの例はλファージなどが挙げられ、さらに、ウイルスベクターの例は、レトロウイルス、ワクシニアウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルスなどの動物ウイルスベクター、バキュロウイルスなどの昆虫ウイルスベクターなどが挙げられる。
　ベクターは、目的ＤＮＡを組み込むためのポリリンカーもしくはマルチクローニングサイトを含んでもよく、また、該ＤＮＡを発現するためにいくつかの制御エレメントを含むことができる。制御エレメントには、例えばプロモーター、エンハンサー、ポリＡ付加シグナル、複製開始点、選択マーカー、リボソーム結合配列、ターミネーターなどが含まれる。
　選択マーカーの例は、薬剤耐性遺伝子（例えばネオマイシン耐性遺伝子、アンピシリン耐性遺伝子、カナマイシン耐性遺伝子、ピューロマイシン耐性遺伝子など）、栄養要求性相補遺伝子（例えばジヒドロ葉酸レダクターゼ（ＤＨＦＲ）遺伝子、ＨＩＳ３遺伝子、ＬＥＵ２遺伝子、ＵＲＡ３遺伝子など）などである。
　プロモーターは、宿主細胞に応じて異なる場合がある。
　宿主細胞の例としては、非限定的に、大腸菌などのエシェリヒア属、バチルス・ズブチリスなどのバチルス属、シュードモナス・プチダなどのシュードモナス属などの細菌、サッカロミセス・セレビシエ、シゾサッカロミセス・ポンベなどのサッカロミセス属、カンジダ属、ピキア属などの酵母、ＣＨＯ、ＣＯＳ、ＨＥＫ２９３、ＮＩＨ３Ｔ３などの動物細胞、Ｓｆ９、Ｓｆ２１などの昆虫細胞、植物細胞などが挙げられる。
　大腸菌等の細菌を宿主とする場合、プロモーターとして、例えばｔｒｐプロモーター、ｌａｃプロモーター、ＰＬ又はＰＲプロモーターなどが例示される。
　酵母を宿主とする場合、プロモーターとして、例えばｇａｌ１プロモーター、ｇａｌ１０プロモーター、ヒートショックタンパク質プロモーター、ＭＦα１プロモーター、ＰＨＯ５プロモーター、ＰＧＫプロモーター、ＧＡＰプロモーター、ＡＤＨプロモーター、ＡＯＸ１プロモーターなどが例示される。
　動物細胞を宿主とする場合、プロモーターとして、ＳＲαプロモーター、ＳＶ４０プロモーター、ＬＴＲプロモーター、ＣＭＶプロモーター、ヒトＣＭＶ初期遺伝子プロモーター、アデノウイルス後期プロモーター、ワクシニアウイルス７．５Ｋプロモーター、メタロチオネインプロモーター、多角体プロモーターなどが例示される。
　植物細胞を宿主とする場合、プロモーターとして、例えばＣａＭＶプロモーター、ＴＭＶプロモーターなどが例示される。
　発現ベクターを用いた宿主細胞の形質転換又はトランスフェクションとしては、例えばエレクトロポレーション法、スフェロプラスト法、酢酸リチウム法、リン酸カルシウム法、アグロバクテリウム法、ウイルス感染法、リポソーム法、マイクロインジェクション法、遺伝子銃法、リポフェクション法などが挙げられる。
　形質転換宿主は、細菌、酵母、動物細胞、植物細胞の種類に応じた培養条件で培養され、細胞内又は培養液から目的蛋白質を回収する。
　微生物の培養では、微生物が資化しうる炭素源、窒素源、無機塩類等を含有する培地を使用する。炭素源として、グルコース、フラクトース、スクロース、デンプン等の炭水化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノール等のアルコール類、窒素源として、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム、リン酸アンモニウム等の無機酸や有機酸のアンモニウム塩、ペプトン、肉エキス、コーンスティープリカー等、無機物として、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウム等が用いられる。
　動物細胞の培養では、例えばＤＭＥＭ培地、ＲＰＭＩ１６４０培地などを基本培地とし、これに牛胎児血清（ＦＣＳ）等を添加した培地が用いられる。
　目的蛋白質の回収は、上で説明したとおり、蛋白質精製のための慣用手法、例えば硫酸アンモニウム沈殿、有機溶媒沈殿、透析、電気泳動、クロマトフォーカシング、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、アフィニティークロマトグラフィー、ＨＰＬＣなどにて実施しうる。
　ベクターを治療用に使用する場合には、被験体のゲノムに組み込まれないベクターであって、細胞に感染するが複製が不能にされたウイルスベクター、非ウイルスベクターなどが望ましい。このようなベクターには、例えばアデノ随伴ウイルスベクター、アデノウイルスベクターなどが含まれる。これらのベクターは、プロモーター、エンハンサー、ポリアデニル化部位、選択マーカー、レポーター遺伝子などを含みうる。ウイルスベクターの例は、Ｊ．Ｖｉｒｏｌ．６７：５９１１−５９２１（１９９３），Ｈｕｍａｎ　Ｇｅｎｅ　Ｔｈｅｒａｐｙ　５：７１７−７２９（１９９４），Ｇｅｎｅ　Ｔｈｅｒａｐｙ　１：５１−５８（１９９４），Ｈｕｍａｎ　Ｇｅｎｅ　Ｔｈｅｒａｐｙ　５：７９３−８０１（１９９４），Ｇｅｎｅ　Ｔｈｅｒａｐｙ　１：１６５−１６９（１９９４）などに記載されているベクター、或いは、それらの改良ベクターである。更に、非ウイルスベクターの例は、ヒト人工染色体ベクターであり、これは、ヒト染色体由来のセントロメア及びテロメアを含む染色体断片から構成されるベクターである。ヒト染色体断片は特に制限されないが、例えばヒト１４番染色体断片、ヒト２１番染色体断片などが含まれる（再表２００４／０３１３８５号、特開２００７−２９５８６０など）。上記ベクターに、上記定義の核酸を挿入し、被験体に投与するか、或いは、被験体から採取した組織若しくは細胞にベクターを導入したのち該被験体に戻す方法などにより、ベクターを被験体に投与することができる。
＜ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片＞
　本発明におけるＡＬＫ１細胞外領域ポリペプチドに結合する抗体は、上記ＡＬＫ１細胞外領域ポリペプチドに結合することができる抗体であって、中和抗体が望ましく、限定されるものではないが、例えば、ＢＭＰ９とＡＬＫ１との結合を阻害する抗体が好ましい。本発明におけるＡＬＫ１細胞外領域ポリペプチドに結合する抗体には、例えばＷＯ　２００７／４０９１２に記載された抗体が含まれる。
　ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片は、上記ＡＬＫ１細胞外領域ポリペプチドを抗原蛋白質として用いて、上記（ＢＭＰ９抗体）の節にて具体的に示した手法により作製することができる。
＜ＢＭＰ９に対するアプタマーまたはＡＬＫ１細胞外領域ポリペプチドに対するアプタマー＞
　アプタマーとは、蛋白質の特定の分子に結合する核酸リガンドである。本発明におけるアプタマーは、ＢＭＰ９蛋白質またはＡＬＫ１細胞外領域に結合し、当該ＢＭＰ９蛋白質またはＡＬＫ１の機能を阻害することができる。
　アプタマーは、多種の核酸鎖からなるライブラリーを製造し、その中から標的蛋白質に結合しうる核酸鎖を選択することによって取得することができる。アプタマーを同定するための好適な方法としては、例えば、Ｓｙｓｔｅｍａｔｉｃ　Ｅｖｏｌｕｔｉｏｎ　ｏｆ　Ｌｉｇａｎｄｓ　ｂｙ　Ｅｘｐｏｎｅｎｔｉａｌ　Ｅｎｒｉｃｈｍｅｎｔ（ＳＥＬＥＸＴＭ）法（米国特許５２７０１６３）が挙げられる。本発明においては、上記ＢＭＰ９蛋白質または上記ＡＬＫ１細胞外領域ポリペプチドとライブラリー核酸とを混合した後、当該ＢＭＰ９蛋白質またはＡＬＫ１細胞外領域ポリペプチドと結合している核酸を同定することによって、目的のアプタマーを得ることができる。
＜ＡＬＫ１遺伝子を標的とするＲＮＡｉ分子またはＢＭＰ９遺伝子を標的とするＲＮＡｉ分子＞
　ＲＮＡｉ分子は、標的とする蛋白質をコードするｍＲＮＡに相補的な塩基配列を有するアンチセンス鎖を含む、一本鎖または二本鎖核酸分子である。ＲＮＡｉ分子は、当該アンチセンス鎖が標的蛋白質をコードするｍＲＮＡと特異的に結合してその標的蛋白質の発現（蛋白質合成）を阻害する。
　「標的とする」なる表現は、ＲＮＡｉ分子に含まれる上記アンチセンス鎖が、標的ｍＲＮＡとストリンジェントな条件下でハイブリダイズすることをいう。ストリンジェントな条件は、例えば約１~５×ＳＳＣ、室温~約４０℃でのハイブリダイゼーション、及びその後の、約０．１~１×ＳＳＣ、０．１％　ＳＤＳ、約４５~６５℃での洗浄からなる。ここで、１×ＳＳＣは、１５０ｍＭ　ＮａＣｌ、１５ｍＭ　Ｎａ−クエン酸、ｐＨ７．０の溶液を指す。
　本発明におけるＲＮＡｉ分子は、ＢＭＰ９またはＡＬＫ１をコードするｍＲＮＡと特異的に結合して、その蛋白質合成を阻害する。本発明におけるＲＮＡｉ分子には、アンチセンスＲＮＡ、アンチセンスＤＮＡ、ｓｉＲＮＡ、ｓｈＲＮＡが含まれる。ｓｉＲＮＡとは、標的蛋白質をコードするｍＲＮＡに相補的な塩基配列を有するアンチセンス鎖と当該アンチセンス鎖に相補的なセンス鎖とがハイブリダイズしてなる低分子二本鎖ＲＮＡである。ｓｈＲＮＡとは、上記アンチセンス鎖とセンス鎖とがリンカー部分を介して連結された一本鎖ＲＮＡであり、当該リンカー部分がループを形成することにより折りたたまれ、当該アンチセンス鎖と当該センス鎖がハイブリダイズして、二本鎖部分を形成する。
　本発明のＲＮＡｉ分子は、上記のＢＭＰ９およびＡＬＫ１の遺伝子配列を基に公知の手法、例えば、ｓｉＲＮＡ　Ｄｅｓｉｇｎ　Ｓｕｐｐｏｒｔ　Ｓｙｓｔｅｍ（タカラバイオ株式会社）を用いて設計することができる。
＜ＡＬＫ１のキナーゼ活性に対して阻害活性を有する低分子化合物＞
　本発明におけるＡＬＫ１のキナーゼ活性に対して阻害活性を有する低分子化合物としては、ＡＬＫ１キナーゼ活性を阻害することが知られている化合物であれば、特に限定されるものではないが、例えばＷＯ　２００７／１４７６４７に記載された低分子化合物（Ｎ−（２−ジメチルアミノ−エチル）−３−［５−（４−イソプロピル−フェニルアミノ）−ピラゾロ［１，５−ａ］ピリミジン−３−ｙｌ］−ベンズアミド；フェニル−［３−（３，４，５−トリメトキシ−フェニル）−ピラゾロ［１，５−ａ］ピリミジン−５−ｙｌ］−アミン；（４−フルオロ−フェニル）−［３−（３，４，５−トリメトキシ−フェニル）−ピラゾロ［１，５−ａ］ピリミジン−５−ｙｌ］−アミン；Ｎ’−［３−（３−クロロ−フェニル）−ピラゾロ［１，５−ａ］ピリミジン−５−ｙｌ］−Ｎ，Ｎ−ジエチル−プロパン−ｅ−１，３−ジアミンなど、これらに限定されない）が挙げられる。
＜ＢＭＰ９とＡＬＫ１の結合、またはＢＭＰ９とタイプＩＩレセプターとの結合を阻害する活性を有する低分子化合物＞
　本発明においてＢＭＰ９とＡＬＫ１の結合、またはＢＭＰ９とタイプＩＩレセプターとの結合を阻害する活性を有する低分子化合物とは、ＢＭＰ９とＡＬＫ１の結合、もしくはＢＭＰ９とタイプＩＩレセプターとの結合を阻害することができる化合物を指し、特に限定されるものではない。「タイプＩＩレセプター」には、ＢＭＰタイプＩＩレセプター（ＢＭＰＲＩＩ）、ａｃｔｉｖｉｎタイプＩＩａレセプター（ＡｃｔＲＩＩａ）、ａｃｔｉｖｉｎタイプＩＩｂレセプター（ＡｃｔＲＩＩｂ）が含まれる。本発明に含まれる低分子化合物は、ＢＭＰ９とＢＭＰＲＩＩとの結合、ＢＭＰ９とＡｃｔＲＩＩａとの結合、ＢＭＰ９とＡｃｔＲＩＩｂとの結合のうち、いずれか一つの結合を阻害する活性を有するものであってもよいし、いずれか二つの結合を阻害する活性を有するものであってもよいし、また、これら三つの結合を阻害する活性を有するものであってもよい。好ましいものとして、ＢＭＰ９とＢＭＰＲＩＩとの結合を阻害する活性を有する低分子化合物が挙げられる。
＜ＢＭＰ９阻害活性を有するＢＭＰ９アンタゴニスト蛋白質＞
　ＢＭＰ９阻害活性を有するＢＭＰ９アンタゴニスト蛋白質は今のところ報告はないが、ＢＭＰには、ＢＭＰ阻害活性を有する、種々の生体由来のＢＭＰアンタゴニストが報告されており、ＢＭＰ９にも、そのようなＢＭＰ９アンタゴニストが存在する可能性がある。そのようなＢＭＰ９アンタゴニストを本発明の医薬組成物の有効成分として用いることができる。
＜疾患＞
　本発明の医薬組成物は、血管障害を伴う疾患に対して用いることができ、例えば、腎疾患、動脈硬化性疾患、高血圧症、心疾患、糖尿病、糖尿病合併症、血栓症、脂質異常症などの生活習慣病、血管炎を伴う疾患およびＢＭＰ９の関与が示された疾患が挙げられる。
　腎疾患は、特に限定されるものではないが、ＩｇＡ腎症を含む慢性糸球体腎炎、糖尿病性腎症、ループス腎炎、腎硬化症または急速進行性糸球体腎炎などが挙げられる。動脈硬化性疾患は、特に限定されるものではないが、脳血管障害（脳卒中、ラクナ梗塞も含む脳梗塞、脳血栓、脳出血、クモ膜下出血など）、心筋梗塞や狭心症などの虚血性心疾患、大動脈瘤、大動脈解離、腎硬化症、閉塞性動脈硬化症などが挙げられる。心疾患は、特に限定されるものではないが、心臓弁膜症、心筋梗塞、狭心症、心筋症などが挙げられる。糖尿病合併症は、動脈硬化症、糖尿病性網膜症、糖尿病性腎症、糖尿病性神経障害、糖尿病性壊疽、慢性感染症、白内障などが挙げられる。血栓症は、特に限定されるものではないが、肺血栓塞栓症、脳梗塞、心筋梗塞、下肢急性動脈血栓症、腸壊死（上腸間膜動脈血栓症）などが挙げられる。血管炎を伴う疾患は、特に限定されるものではないが、高安動脈炎、巨細胞動脈炎（側頭動脈炎）、結節性多発動脈炎、ウェゲナー肉芽腫症、チャーグ・ストラウス症候群、川崎病、ヘノッホ・シェーンライン紫斑病、過敏性血管炎、全身性エリテマトーデス、関節リウマチなどが挙げられる。ＢＭＰ９の関与の可能性が示された疾患は、肝疾患、癌性腹水・胸水を伴うがん、慢性膵炎、アレルギー性疾患、炎症性疾患、アルツハイマー、多発性硬化症、糖尿病性網膜症、レイノー症候群、クローン病、がんなどが挙げられる。アレルギー性疾患は、特に限定されるものではないが、アレルギー性鼻炎、喘息、気道過敏症、アトピー性皮膚炎などが挙げられる。炎症性疾患は、特に限定されるものではないが、遅延型アレルギー、関節リウマチ、関節炎、肺疾患、肝炎、潰瘍性大腸炎などが挙げられる。肝疾患は、特に限定されるものではないが、急性肝炎、慢性肝炎、肝臓がん、肝硬変、転移性肝臓がんなどが挙げられる。
　本発明の医薬組成物は、好ましくは腎疾患、動脈硬化性疾患、高血圧症、心疾患、糖尿病、糖尿病合併症、血栓症、脂質異常症などの生活習慣病、特に好ましくは腎疾患、動脈硬化性疾患、高血圧症、血栓症において適用が可能である。
　慢性腎臓病（ＣＫＤ）は、心血管合併症（ＣＶＤ）の独立した最大のリスクファクターとされているが、ＣＫＤの初期段階において動脈硬化の存在も認められていることから、腎疾患治療では、ＣＫＤ治療のみならずＣＶＤ治療も求められている。本発明の医薬組成物は、これまでに得られた結果より、ＣＫＤだけでなくＣＶＤに対しても有効である可能性が示唆されていることから、本発明の医薬組成物が特に腎疾患において臨床上有用な治療剤なり得る可能性が高いことが期待される。
＜製剤・投与法＞
　本発明の医薬組成物は、上記有効成分を１種または複数種組み合わせて含有する。好ましくは、本発明の医薬組成物は、ＢＭＰ９に結合する抗体または該抗体断片を有効成分として含有する。本発明の医薬組成物に含まれる有効成分の量は、投与される患者の年齢、性別、体重、症状及び投与経路などに応じて適宜決定されるべきであり、以下に限定されないが、一投与あたり約０．１μｇ／ｋｇ~１００ｍｇ／ｋｇの範囲、好ましくは約１μｇ／ｋｇ~１０ｍｇ／ｋｇの範囲内で適宜決定できる。
　本発明の医薬組成物の形態（即ち、製剤）は、制限されないものとし、経口製剤、非経口製剤のいずれも包含する。
　好ましい形態は、非経口製剤であり、非限定的に、静脈内投与製剤、筋肉内投与製剤、腹腔内投与製剤、皮下投与製剤、局所投与製剤などが含まれる。非経口投与製剤には、例えば、注射剤、点滴剤、坐剤、経皮吸収剤、リポソーム又はナノ粒子封入製剤などが含まれる。
　経口製剤には、例えば、錠剤、丸剤、顆粒剤、カプセル剤、散剤、溶液剤、懸濁剤、遅延放出製剤、腸溶性製剤などが含まれる。
　本発明の医薬組成物は、製薬上許容される賦形剤、希釈剤などの担体、及び添加剤を含むことができる。
　担体は、例えば生理食塩水、グリセロール、エタノール、アーモンド油、植物油、スクロース、デンプン、ラクトースなどを含む。
　添加剤は、例えば、結合剤（例えばα化トウモロコシデンプン、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドンなど）、滑沢剤（例えば、ステアリン酸マグネシウム、タルク、シリカなど）、分散剤（例えばポリビニルピロリドン、トウモロコシデンプンなど）、懸濁剤（例えばタルク、アラビアゴムなど）、乳化剤（例えばレシチン、アラビアゴムなど）、崩壊剤（ジャガイモデンプン、グリコール酸ナトリウムデンプン、クロスポピドンなど）、緩衝剤（例えばリン酸塩、酢酸塩、クエン酸塩、トリス塩など）、抗酸化剤（例えばアスコルビン酸、トコフェロールなど）、保存剤（例えばソルビン酸、ｐ−ヒドロキシ安息香酸メチル、ｐ−ヒドロキシ安息香酸プロピルなど）、等張化剤（例えば塩化ナトリウムなど）、安定化剤（例えばグリセロールなど）などを含むことができる。
　腸溶性製剤には、例えば、ヒドロキシプロピルメチルセルロースフタレート、メタクリル酸−メタクリル酸メチルコポリマー、メタクリル酸−アクリル酸エチルコポリマー、ヒドロキシプロピルアセテートサクシネートなどのポリマーが使用される。
　本発明の医薬組成物の投与量は、患者の年齢、性別、体重、症状及び投与経路などに応じて適宜決定されるべきであり、以下に限定されないが、例えば成人一日あたり約０．１μｇ／ｋｇ~１００ｍｇ／ｋｇの範囲内であり、好ましくは約１μｇ／ｋｇ~１０ｍｇ／ｋｇの範囲内である。製剤の投与は、治療中毎日投与してもよいし、数日間隔、２週間間隔又は１ヶ月間隔など時間間隔をとって投与してもよい。
　本発明の医薬組成物は、含まれる有効成分の種類および剤形に適した投与法により患者に投与することができる。本発明の医薬組成物は、静脈内投与、皮内投与、動脈内投与、腹腔内投与、病巣内投与、鼻腔内投与、膣内投与、直腸内投与、局所的投与、筋肉内投与、経口投与、注射、点滴、上記ベクターを用いて作製されたＡＬＫ１細胞外領域ポリペプチド等を発現する形質転換細胞の細胞移植等または上記いずれかの組み合わせにより投与することができる。
　本発明の医薬組成物が、上に詳述した核酸、発現ベクター、アプタマーまたはＲＮＡｉ分子のいずれか又はそれらの組合せを有効成分とする場合、当該医薬組成物の投与は上記有効成分を目的の組織又は細胞に導入することが可能な遺伝子治療で一般的に行われる技術又は手法を用いて実施することができる。
　本発明の医薬組成物を投与するために使用し得る遺伝子送達法には、コロイド分散系、リポソーム誘導系、人工ウイルスエンベロープなどが含まれる。例えば、送達系は巨大分子複合体、ナノカプセル、ミクロスフェア、ビーズ、水中油型乳剤、ミセル、混合ミセル、リポソーム、リン酸カルシウム法、ＤＥＡＥデキストラン法、エレクトロポレーション法、、リポフェクション法等を使用することができる。
＜治療剤の評価＞
　本発明の医薬組成物の効果は、当該医薬組成物の投与を受けた上記疾患に罹患する患者において、上記疾患の症状が緩和または消失することによって評価することができる。
　また、本発明の医薬組成物の効果は、上記疾患に関連することが知られている遺伝子、バイオマーカー及び血清生化学パラメーターの遺伝子学的または生化学的解析により評価することができる。本発明の医薬組成物の投与を受けた上記疾患に罹患する患者（好ましくは、患者由来の血液サンプル）において、当該遺伝子の発現量、バイオマーカー値、もしくは血清生化学パラメーター値の変化を指標にして評価することができる。このような遺伝子、バイオマーカー及び血清生化学パラメーターとしては、以下のものが挙げられる（ただし、これらに限定されない）：
遺伝子
Figure JPOXMLDOC01-appb-I000008
Figure JPOXMLDOC01-appb-I000009
バイオマーカー
Figure JPOXMLDOC01-appb-I000010
Figure JPOXMLDOC01-appb-I000011
血清生化学パラメーター
Figure JPOXMLDOC01-appb-I000012
　以下に実施例を示して本発明を具体的に説明するが、本発明はこれらの実施例に限定されるものでない。
　なお、本願は、日本国においては、平成１９年（２００７年）度、独立行政法人新エネルギー・産業技術総合開発機構「化合物等を活用した生物システム制御基盤技術開発」に係る委託研究、産業技術力強化法第１９条の適用を受ける特許出願である。 Hereinafter, the present invention will be described in detail.
The present invention provides a pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient.
The ALK1 inhibitor contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is a substance that can suppress ALK1 activation, that is, activation of ALK1 kinase activity and suppress ALK1 signal. The ALK1 inhibitor used in the present invention is not particularly limited, and examples thereof include the following (1) to (10):
(1) an antibody that binds to BMP9 or an antibody fragment thereof;
(2) ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof;
(3) A nucleic acid encoding an ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof;
(4) an expression vector comprising a nucleic acid encoding an ALK1 extracellular region polypeptide or a variant thereof, or a protein comprising the ALK1 extracellular region polypeptide or a variant thereof;
(5) an antibody or an antibody fragment thereof that binds to an ALK1 extracellular region polypeptide;
(6) an aptamer for BMP9 or an aptamer for ALK1 extracellular region polypeptide;
(7) RNAi molecule targeting ALK1 gene or RNAi molecule targeting BMP9 gene;
(8) a low molecular weight compound having an inhibitory activity against the kinase activity of ALK1;
(9) a low molecular weight compound having an activity of inhibiting the binding between BMP9 and ALK1, or the binding between BMP9 and a type II receptor; or
(10) A BMP9 antagonist protein having BMP9 inhibitory activity.
The ALK1 inhibitor used in the present invention is particularly preferably an antibody that binds to BMP9 or an antibody fragment thereof that has neutralizing activity.
The ALK1 inhibitor that can be included in the pharmaceutical composition of the present invention is described below.
<Antibodies that bind to BMP9 or antibody fragments thereof>
(BMP9 antibody)
The antibody that binds to BMP9 is preferably a neutralizing antibody and can be prepared by the following method.
BMP9 is known and isolated from humans, mice, Japanese zelkova, etc., and sequence information is disclosed in databases such as GenBank. Regarding the sequence information of human or mouse-derived BMP9, for example, human BMP9 is registered with GenBank as accession numbers Q9UK05, AAD56960, etc., and mouse BMP9 is registered with GenBank as accession numbers Q9WV56, NP_062379, etc., and these can be used. .
As the BMP9 protein, a BMP9 protein having a known amino acid sequence registered in GenBank or the like can be used. Preferably, the BMP9 protein has an amino acid sequence represented by SEQ ID NO: 10 (mouse BMP9) or SEQ ID NO: 24 (human BMP9). BMP9 protein, particularly preferably BMP9 protein having the amino acid sequence shown in SEQ ID NO: 24 is used. The sequences represented by SEQ ID NO: 10 and SEQ ID NO: 24 include a signal sequence and pro-region. In SEQ ID NO: 10, the 319th to 428th sequences correspond to mature parts, and in SEQ ID NO: 24, The 320th to 429th arrays correspond to mature parts. In the present specification, BMP9 includes a variant of BMP9. Such mutants include both natural mutants and artificial mutants. In the amino acid sequence of BMP9, at least 319 to 428 of SEQ ID NO: 10, or at least 320 to 429 of SEQ ID NO: 24 1 to several amino acid deletions, substitutions, additions or insertions are included in the amino acid sequence having the sequence shown in the second sequence, or 80% or more, preferably 85% or more, more preferably It has an amino acid sequence having 90% or more, for example, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity. The range of “1 to several” is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 1. Three, or one or two.
Antigen epitopes for obtaining the target antibody include regions having high antigenicity in the amino acid sequence of BMP9, regions having superficiality, regions that may not have secondary structure, homology with other proteins, or A low region can be selected. Here, the region having high antigenicity can be estimated by the method of Parker et al. [Biochemistry, 25, 5425-5432 (1986)]. The superficial region can be estimated by calculating and plotting a hydropathic index, for example. Regions that may not have secondary structure are described, for example, by the method of Chou and Fasman [Adv Enzymol Relat Areas Mol Biol. 47, 45-148 (1978)].
Based on the partial amino acid sequence of BMP9 estimated by the above method, a peptide comprising the amino acid sequence can be synthesized by using a peptide synthesis method. Examples of peptides of interest include R.I. B. Synthesized using a commercially available peptide synthesizer based on solid phase peptide synthesis developed by Merrifield [Science, 232, 341-347 (1986)], and after removing the protecting group, ion exchange chromatography , Purification by gel filtration chromatography, reverse phase chromatography, etc., alone or in combination. The obtained purified peptide can be used as an immunogen by binding to a carrier protein such as keyhole limpet hemocyanin (KLH) or albumin.
Furthermore, a polyclonal antibody or a monoclonal antibody that binds to BMP9 using BMP9 as an immunogen can also be prepared by a known technique. In the present specification, the term “recombinant” used for BMP9 or a monoclonal antibody, a polyclonal antibody, or other proteins means that these proteins are produced by recombinant DNA in a host cell. Means. As a host cell, any of prokaryotes (for example, bacteria such as E. coli) and eukaryotes (for example, yeast, CHO cells, insect cells, etc.) can be used.
The “antibody” of the present invention may be a peptide antibody, a polyclonal antibody, or a monoclonal antibody. “Antibodies” produce antibodies that will specifically bind to proteins used for immunization of mice or other suitable host animals (eg, rabbits, cows, horses, sheep, pigs, rodents, etc.). Alternatively, it can be obtained by immunizing an antigen or antigen-expressing cell by subcutaneous, intraperitoneal, or intramuscular route to extract lymphocytes that would be produced. Further, as a host animal, a desired humanized antibody may be obtained by administering an antigen or an antigen-expressing cell to a transgenic animal having a repertoire of human antibody genes [Proc. Natl. Acad. Sci. USA, 97, 722-727 (2000), International Publication WO96 / 33735, WO97 / 07671, WO97 / 13852, WO98 / 37757]. As an alternative to the above approach, antibodies can be obtained by immunizing lymphocytes in vitro. A polyclonal antibody can be obtained by collecting and purifying fractions that bind to the antigen from serum obtained from the host animal. In addition, monoclonal antibodies can be prepared by fusing lymphocytes with myeloma cells using a suitable fusion reagent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principals. and Practice, 59-103, Academic press, (1986)]. For example, the monoclonal antibody of the present invention can be prepared using the hybridoma method [Nature, 256, 495 (1975)] or using the recombinant DNA method (Cabilly et al., US Pat. No. 4,816,567). it can.
The antigen protein can be prepared by expressing DNA encoding all or a partial sequence of the BMP9 protein in E. coli, yeast, insect cells, animal cells and the like. Recombinant BMP9 is purified by a method such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, reverse phase chromatography or the like alone or in combination, and this purified preparation is used as an immunogen.
The antibody of the present invention may be an intact antibody or an antibody fragment such as (Fab ′) 2 or Fab.
Also, chimeric antibodies or mosaic antibodies in which the constant region is replaced with a human constant region [eg, mouse-human chimeric antibody; Cabilly et al., US Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 89, 6851 (1984)], humanized antibodies in which all variable regions except the constant region and the hypervariable region (or complementary-determining region; hereinafter referred to as CDR) are replaced with human sequences [ Carter et al., Proc. Natl. Acad. Sci. USA, 89, 4285 (1992) and Carter et al., BioTechnology, 10, 163, (1992)], fully human antibodies (International Publication WO02 / 43478) are also included in the antibodies of the present invention. When used in humans, the use of chimeric antibodies, mosaic antibodies or humanized antibodies is preferred in order to prevent the appearance of antibodies against heterologous antigens, such as human anti-mouse antibodies (HAMA), Most preferred is a fully human antibody.
(BMP9 antibody fragment)
In the present invention, examples of the antibody fragment include Fab, F (ab ′) 2, Fab ′, scFv, diabody, dsFv, and a peptide containing CDR.
Fab is a fragment obtained by treating IgG with papain, a proteolytic enzyme (cleaved at the 224th amino acid residue of the H chain), about half of the N chain side of the H chain and the entire L chain are disulfides. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000 bound by binding. The Fab of the present invention can be obtained by treating a monoclonal antibody that binds to BMP9 with papain. Alternatively, a Fab may be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. it can.
F (ab ′) 2 was obtained by decomposing the lower part of two disulfide bonds in the hinge region of IgG with pepsin, a proteolytic enzyme, and was constructed by binding two Fab regions at the hinge portion. It is a fragment having an antigen binding activity with a molecular weight of about 100,000. F (ab ′) 2 of the present invention can be obtained by treating a monoclonal antibody that binds to BMP9 with pepsin. Alternatively, Fab ′ described below can be produced by thioether bond or disulfide bond.
Fab ′ is an antibody fragment having a molecular weight of about 50,000 and having an antigen binding activity obtained by cleaving the disulfide bond in the hinge region of F (ab ′) 2. The Fab ′ of the present invention can be obtained by treating F (ab ′) 2 binding to BMP9 with a reducing agent such as dithiothreitol. In addition, DNA encoding the Fab ′ fragment of the antibody is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the vector is introduced into prokaryotic or eukaryotic cells to express Fab ′. You can also
scFv uses one heavy chain variable region (hereinafter referred to as VH) and one light chain variable region (hereinafter referred to as VL) using an appropriate peptide linker (hereinafter referred to as P). VH-P-VL or VL-P-VH polypeptide, which is an antibody fragment having antigen-binding activity. The scFv of the present invention obtains cDNAs encoding monoclonal antibodies VH and VL that bind to BMP9, constructs a DNA encoding scFv, and inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector. The expression vector can be expressed and produced by introducing the expression vector into a prokaryotic or eukaryotic organism.
Diabody is an antibody fragment in which scFv is dimerized and is an antibody fragment having a bivalent antigen-binding activity. The bivalent antigen binding activity can be the same, or one can be a different antigen binding activity. The diabody of the present invention obtains cDNA encoding VH and VL of the monoclonal antibody that binds to BMP9, constructs the DNA encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less, The DNA can be expressed and produced by inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the expression vector into a prokaryotic or eukaryotic organism.
dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are bonded via a disulfide bond between the cysteine residues. The amino acid residue to be substituted with the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to a known method [Protein Engineering, 7, 697 (1994)]. The dsFv of the present invention obtains cDNAs encoding the monoclonal antibodies VH and VL that bind to the BMP9 of the present invention, constructs a DNA encoding the dsFv, and the DNA is expressed in a prokaryotic expression vector or eukaryotic expression. It can be expressed and produced by inserting into a vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
The peptide containing CDR is composed of at least one region of CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker. The peptide containing the CDR of the present invention constructs DNA encoding the VH and VL CDRs of the monoclonal antibody that binds to BMP9 of the present invention, and inserts the DNA into a prokaryotic expression vector or a eukaryotic expression vector. It can be expressed and produced by introducing the expression vector into a prokaryotic or eukaryotic organism. Moreover, the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc t method.
<ALK1 extracellular region polypeptide or variant thereof, or protein containing ALK1 extracellular region polypeptide or variant thereof>
(ALK1 extracellular region polypeptide or variant thereof)
ALK1 is a type I cell surface receptor for transforming growth factor β receptor type 1 (TGF-β-1). Human ALK1 is a polypeptide of 503 amino acids, which includes a signal sequence (amino acids: 1 to 21), an N-terminal extracellular TGF-β-1 ligand binding domain, or an extracellular domain (amino acids 22 to 22). 118) single transmembrane domain (amino acids 119-141), regulatory glycine / serine rich (GS) domain (amino acids: 142-202), and C-terminal serine-threonine kinase domain (202-492) Is included. Attisano et al. Cell, 1993, vol. 75, pp. 671-680 includes human ALK1 amino acid sequence containing serine at position 172 (Genbank accession number L17075), and US Pat. No. 6,316,217 discloses human ALK1 amino acid sequence having threonine at position 172. (Genbank accession number NM — 000020) is disclosed. The ACVRL1 gene encoding full-length human ALK1 disclosed in Attisano et al. Is described in Invitrogen Inc. Commercially available as clone ID IOH21048.
In the present invention, the “ALK1 extracellular region polypeptide” is a protein consisting of all or part of the amino acid sequence of the ALK1 extracellular region polypeptide derived from a mammal.
The amino acid sequence and cDNA sequence of human ALK1 extracellular region polypeptide are as follows.
Amino acid sequence of human ALK1 extracellular region polypeptide (SEQ ID NO: 29):
Figure JPOXMLDOC01-appb-I000001
(Genbank ACCESSION No. L17075) The underline indicates the signal sequence, and the encircled line indicates the extracellular region polypeptide after cleavage of the signal sequence.
CDNA sequence of human ALK1 extracellular region polypeptide (SEQ ID NO: 30):
Figure JPOXMLDOC01-appb-I000002
In the present invention, the ALK1 extracellular region polypeptide also encompasses variants of the above ALK1 extracellular region polypeptide. Such mutants include both natural mutants and artificial mutants, and substitution of one or more (preferably one or several) amino acids in the amino acid sequence of the ALK1 extracellular region polypeptide, Deletion or addition, or 80% or more, preferably 85% or more, more preferably 90% or more, such as 93% or more, 95% or more, 97% or more, 98% or more, or 99% with the amino acid sequence It contains an amino acid sequence having the above identity and has vascular disorder inhibitory activity.
Illustratively, the variant includes one or more (preferably one or several) amino acid substitutions, deletions or additions in the amino acid sequence of SEQ ID NO: 29, or 80% or more of the amino acid sequence. , Preferably 85% or more, more preferably 90% or more, for example, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more, and an vascular disorder inhibitory activity. It is what has.
The term “several” as used herein generally refers to any integer from 2 to 10. Preferably it is any integer from 2 to 5.
As used herein, the term “identity” refers to the alignment of two amino acid sequences (or nucleotide sequences) so that the number of identical amino acid residues (or nucleotides) is maximized. This means the degree of coincidence between sequences when aligned, and is specifically expressed as a percentage (%) of the same number of amino acid residues (or the same number of nucleotides) to the total number of amino acid residues (or the total number of nucleotides). . When gaps are introduced as in FASTA, the number of gaps is also added to the total number of amino acid residues (or the total number of nucleotides).
A protein having a sequence identity of 80% or more, preferably 85% or more accesses a sequence database such as NCBI (US) or EMBL (Europe) and uses a sequence homology search program such as BLAST or FASTA. (Altschul, SF et al. (1990) J. Mol. Biol. 15: 403-410; Karlin, S. and Altschul SF (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268). BLAST breaks the sequence into fixed-length words, searches for similar fragments in word units, stretches them in both directions until the degree of similarity is maximized, performs local alignment, and finally combines them into the final It is a method of performing a general alignment. In addition, FASTA searches for fragments of sequences that match continuously at high speed, performs local alignment by focusing on those fragments that have high similarity, and finally considers these gaps. Is an alignment method.
As a mutagenesis method, a site-directed mutagenesis method using a PCR method using a primer (including a complementary mutant sequence) synthesized based on the sequence of the ALK1 extracellular region polypeptide is preferable (Kunkel et al., Proc Natl.Acad.Sci.USA, 1985, 82: 488-492; F.M. Ausubel et al., Short Protocols in Molecular Biology, 1995, John Wiley & Clones, J. Sambrook et al., Molecular A: Mol. ed., 1989, Cold Spring Harbor Laboratory Press). Mutation introduction kits (for example, manufactured by Takara Shuzo Co., Ltd.) are also commercially available, and mutations can be introduced according to the instructions.
Briefly, according to the method of Kunkel et al., A primer containing a DNA encoding the ALK1 extracellular region polypeptide as a template, and a primer (complementary mutant sequence that has been phosphorylated at the 5 ′ end with T4 DNA polynucleotide kinase in advance). The template is annealed to the template, DNA synthesis is performed, and the ends are ligated with T4 DNA ligase to purify the DNA containing the target mutation.
In the present invention, the mutation includes substitution, deletion, addition, insertion, or a combination thereof.
The substitution may be either a conservative substitution or a non-conservative substitution, but a conservative substitution is preferred so as not to substantially change the conformation of the ALK1 extracellular region polypeptide. Conservative substitutions can be made between amino acids with similar chemical and physical properties such as structural (eg, branched, aromatic, etc.), electrical (eg, acidic, basic, etc.), polar or hydrophobic, etc. Refers to replacement. Branched amino acids include valine, leucine and isoleucine. Aromatic amino acids include tyrosine, tryptophan, phenylalanine, histidine. Acidic amino acids include glutamic acid and aspartic acid. Basic amino acids include lysine, arginine, and histidine. Polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, cysteine, glycine, proline and the like. Hydrophobic amino acids include alanine, valine, leucine, isoleucine, methionine and the like.
Deletion is the loss of one or more amino acid residues. Addition is the attachment of one or more amino acid residues to the N-terminus or C-terminus of the protein. Insertion is the joining of one or more amino acid residues inside a protein. Among these, deletion and insertion can be performed on the assumption that the conformation of the ALK1 extracellular region polypeptide is not substantially changed. Therefore, it is preferably limited to deletion or insertion of about 1 to 5 amino acid residues.
(Protein containing ALK1 extracellular domain polypeptide or variant thereof)
The protein containing the ALK1 extracellular region polypeptide or a variant thereof is a protein containing the above ALK1 extracellular region polypeptide or a variant thereof, and is not limited to the ALK1 extracellular region polypeptide or a variant thereof. And a fusion protein of a mammal and a mammal-derived immunoglobulin Fc protein or a variant thereof (for example, a fusion protein described in WO 2008/057461).
Here, the expression “comprising” is necessary for the ALK1 extracellular region polypeptide or variant thereof and the heterologous peptide, polypeptide or protein on the N-terminal or C-terminal side of the domain or variant thereof. For example, it means that they may be bonded or fused via an appropriate peptide linker (for example, 1 to 20 amino acids). For example, a preferred example of such a heterologous protein is a mammal-derived immunoglobulin Fc protein or a variant thereof. However, if such a heterologous protein is administered in vivo, it may cause a rejection reaction. Therefore, in order to avoid it as much as possible, the protein originally possessed by the administered mammal is used as the heterologous protein. It is desirable to do.
A preferred Fc protein is a human immunoglobulin Fc protein for use in humans. The class and subclass of the immunoglobulin are not limited to the following, but any of IgG, IgD, IgE, IgM, IgA, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgA1, IgA2, etc. Can be used. However, for use in humans, it is desirable to use human immunoglobulin classes and subclasses. The Fc protein can improve the in vivo stability of the ALK1 extracellular domain or a variant thereof. In this case, however, the Fc protein exhibits biological activities such as antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities in order to avoid in vivo effects due to its biological activity. It is desirable to reduce it in advance, and therefore, it is preferable to introduce a mutation for suppressing, reducing or losing the biological activity as described above.
A preferred example of the Fc protein is a human IgG1 Fc variant represented by the amino acid sequence of SEQ ID NO: 31 below.
SEQ ID NO: 31
Figure JPOXMLDOC01-appb-I000003
The binding position of the Fc protein may be either the N-terminal side or the C-terminal side of the ALK1 extracellular region polypeptide or a variant thereof, but the C-terminal side is preferred.
A specific example of the fusion protein of the ALK1 extracellular region polypeptide and the Fc protein is a protein represented by the amino acid sequence of SEQ ID NO: 32 below, for example. Here, the underlined portion indicates the ALK1 extracellular region polypeptide (SEQ ID NO: 29), and the non-underlined portion indicates the human IgG1 Fc variant protein (SEQ ID NO: 31).
SEQ ID NO: 32:
Figure JPOXMLDOC01-appb-I000004
In the present invention, a protein containing an ALK1 extracellular region polypeptide or a variant thereof does not necessarily have to be bound or fused with a heterologous peptide, polypeptide or protein.
The protein containing the ALK1 extracellular region polypeptide or a variant thereof in the present invention can be prepared by a gene recombination technique commonly used in this industry. Briefly, the protein is prepared by preparing a DNA encoding the protein of the present invention, constructing an expression vector containing the DNA, transforming or transfecting prokaryotic or eukaryotic cells with the vector, Recovering the desired recombinant protein from the cultured cells. Protein purification is performed by appropriately combining conventional protein purification methods such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and HPLC. Is possible.
Regarding the above DNA and vector, <ALK1 extracellular region polypeptide or variant thereof, or nucleic acid encoding ALK1 extracellular region polypeptide or protein containing the variant> and <ALK1 extracellular region polypeptide or its Since it is described in the section, Examples, etc. of the expression vector> containing the nucleic acid encoding the mutant or the ALK1 extracellular region polypeptide or the protein containing the mutant, it can be referred to. In addition, gene recombination techniques are described in Ausubel et al. (Above), Sambrook et al. (Above), and can be used for the present invention.
The protein containing the ALK1 extracellular region polypeptide or variant thereof in the present invention may be chemically modified.
Chemical modifications include, but are not limited to, for example, glycosylation, PEGylation (PEG), acetylation, amidation, phosphorylation and the like. Particularly preferred chemical modifications that can be utilized are glycosylation and pegylation.
Pegylation is the attachment of one or more polyethylene glycol (PEG) molecules to amino acid residues such as the N-terminal amino group of proteins and the ε-amino group of lysine (Lys). Generally, a PEG molecule is attached to the free amino group of an amino acid. The average molecular weight of PEG is not limited to the following, but can be used in the range of about 3,000 to about 50,000. To attach PEG to a protein, the PEG end can be, for example, carboxylated, formylated, succinimidylated, or maleimidylated and reacted with the free amino group of the protein.
Glycosylation is the attachment of a carbohydrate chain (ie, sugar chain) to an asparagine, serine or threonine residue of a protein. In general, sugar chain binding occurs by recognizing the sequence of Asn-X-Thr / Ser (where X is any amino acid residue other than Pro). When the amino acid sequence of the protein is modified so as to have such a sequence, a sugar chain can be introduced at a position different from the natural type. Usually, a recombinant protein can be glycosylated by expressing a nucleic acid encoding the recombinant protein in a eukaryotic cell (yeast cell, animal cell, plant cell, etc.) by a genetic recombination technique. In the present invention, the sugar chain structure is not particularly limited, and it is considered that the sugar chain structure varies depending on the cell type selected for expression. For human use, human-derived cells, yeast cells capable of synthesizing human sugar chains, Chinese hamster ovary (CHO) cells, and the like can be used.
Acetylation and amidation are preferably performed mainly at the N-terminus or C-terminus of the protein. These reactions can be performed using, for example, alcohols such as aliphatic alcohols and fatty acids, and carboxylic acids. The number of carbon atoms in the alkyl portion is, for example, about 1 to 20, but it is necessary to satisfy the conditions such as not impairing water solubility and nontoxicity.
<ALK1 extracellular region polypeptide or a variant thereof, or a nucleic acid encoding a protein containing the ALK1 extracellular region polypeptide or a variant thereof>
As used herein, the term “nucleic acid” includes both DNA and RNA. DNA includes genomic DNA and cDNA, and RNA includes mRNA.
ALK1 extracellular region polypeptide, its variant and protein containing them, including fusion proteins with Fc protein, <ALK1 extracellular region polypeptide or variant thereof, or ALK1 extracellular region polypeptide or its Proteins containing mutants> as described in each section, and all descriptions described in those sections are cited here. Accordingly, the nucleic acid in the present invention includes the nucleic acid encoding the ALK1 extracellular region polypeptide or a variant thereof, or a protein containing the ALK1 extracellular region polypeptide or a variant thereof, as explained and specifically exemplified above. .
Specifically, the nucleic acid includes a nucleic acid encoding an amino acid sequence containing at least the 22nd to 118th amino acids in the amino acid sequence of the extracellular region polypeptide (SEQ ID NO: 29) of human ALK1.
In consideration of expression of the nucleic acid in a eukaryotic cell and secretion of the expression product outside the cell, it may further include a nucleotide sequence encoding a signal sequence. Examples of signal sequences are BMP9 signal sequence, Igκ and the like.
The nucleotide sequence encoding the precursor of the human-derived ALK1 extracellular region polypeptide is exemplified below. The underlined site indicates a nucleotide sequence encoding a signal sequence, and the non-underlined site indicates a nucleotide sequence encoding a mature sequence of an extracellular domain protein.
DNA encoding human ALK1 extracellular region polypeptide (SEQ ID NO: 30):
Figure JPOXMLDOC01-appb-I000005
The nucleic acid in the present invention also includes a nucleic acid encoding a fusion protein of a protein containing an ALK1 extracellular region polypeptide or a variant thereof and a heterologous protein as defined above. A preferable example of the heterologous protein is an immunoglobulin Fc protein derived from a mammal, and a human Fc protein is particularly preferable, but it is desirable to introduce a mutation so as to reduce or lose its biological activity (particularly ADCC and CDC). For example, the nucleotide sequence encoding the mutant human IgG1-derived Fc protein is shown in SEQ ID NO: 33.
SEQ ID NO: 33
Figure JPOXMLDOC01-appb-I000006
Furthermore, a nucleotide sequence (SEQ ID NO: 34) encoding a fusion protein of this mutant human IgG1-derived Fc protein (underlined portion) and a protein containing a human-derived ALK1 extracellular region polypeptide (non-underlined portion) is as follows: This is illustrated in
SEQ ID NO: 34:
Figure JPOXMLDOC01-appb-I000007
The nucleotide sequence encoding the fusion protein can further include a nucleotide sequence encoding a signal sequence. Examples of signal sequences are human protein-derived signal sequences such as BMP9 and Igκ.
Nucleic acid homologs encoding the above proteins can be obtained by using well-known techniques using primers and probes prepared based on cDNA synthesized from mRNA encoding ALK1 extracellular region polypeptide gene derived from human or mouse. It can be obtained from cDNA libraries prepared from other mammals and prepared from cells or tissues known to express the gene. Such techniques include PCR methods, hybridization methods (Southern method, Northern method, etc.) and the like.
The PCR method is a polymerase chain reaction, which is a denaturing step (about 94 to 96 ° C., about 30 seconds to 1 minute) for dissociating double-stranded DNA into single strands, using a primer as a template. Annealing step for binding to single-stranded DNA (about 55 to 68 ° C., about 30 seconds to 1 minute), extension step for extending DNA strand (about 72 ° C., about 30 seconds to about 30 seconds to 1 minute) A cycle consisting of 1 minute) is regarded as one cycle, and about 25 to 40 cycles are carried out. Further, before the denaturation step, a preheating treatment is performed at about 94 to 95 ° C. for about 5 to 12 minutes, and after the final cycle of the extension step, an extension reaction can be further performed at 72 ° C. for about 7 to 15 minutes. it can. PCR is performed with a commercially available thermal cycler using a thermostable DNA polymerase (for example, AmpliTaq Gold (registered trademark) (Applied Biosystems)), MgCl 2 , In a PCR buffer containing dNTP (dATP, dGTP, dCTP, dTTP), etc., in the presence of sense and antisense primers (size: about 17-30b, preferably 20-25b) and template DNA. The amplified DNA can be separated and purified (ethidium bromide staining) by agarose gel electrophoresis.
Hybridization is a technique for detecting a target nucleic acid by forming a double strand with a labeled probe having a length of about 20 to 100 b or more. In order to enhance selectivity, hybridization can generally be performed under stringent conditions. The stringent conditions are, for example, about 1 to 5 × SSC, hybridization at room temperature to about 40 ° C., and then about 0.1 to 1 × SSC, 0.1% SDS, about 45 to 65 ° C. Consists of washing. Here, 1 × SSC refers to a solution of 150 mM NaCl, 15 mM Na-citric acid, pH 7.0. Such conditions will make it possible to detect nucleic acids with a sequence identity of about 80% or more, preferably 85% or more.
<Expression vector containing nucleic acid encoding ALK1 extracellular region polypeptide or variant thereof, or protein containing ALK1 extracellular region polypeptide or variant thereof>
The nucleic acid can be inserted into a vector and used to produce a protein that is an active ingredient of the pharmaceutical composition of the present invention, or the vector itself can be formulated and used as a pharmaceutical composition.
Vectors include, for example, plasmids, phages, viruses and the like. Examples of plasmids include, but are not limited to, E. coli-derived plasmids (eg, pRSET, pTZ19R, pBR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.), Ti plasmids, etc., examples of phages include λ phages, and examples of viral vectors include animal virus vectors such as retroviruses, vaccinia viruses, lentiviruses, adenoviruses, adeno-associated viruses, etc. And insect virus vectors such as baculovirus.
The vector may contain a polylinker or multicloning site for integrating the DNA of interest, and may contain several control elements to express the DNA. The control element includes, for example, a promoter, an enhancer, a poly A addition signal, a replication origin, a selection marker, a ribosome binding sequence, a terminator and the like.
Examples of selectable markers include drug resistance genes (eg, neomycin resistance gene, ampicillin resistance gene, kanamycin resistance gene, puromycin resistance gene, etc.), auxotrophic complementary genes (eg, dihydrofolate reductase (DHFR) gene, HIS3 gene, LEU2 gene) URA3 gene, etc.).
The promoter may vary depending on the host cell.
Examples of host cells include, but are not limited to, bacteria such as Escherichia such as E. coli, Bacillus such as Bacillus subtilis, Pseudomonas such as Pseudomonas putida, Saccharomyces cerevisiae, Saccharomyces such as Schizosaccharomyces pombe And yeast such as Candida and Pichia, animal cells such as CHO, COS, HEK293, and NIH3T3, insect cells such as Sf9 and Sf21, and plant cells.
When a bacterium such as E. coli is used as a host, examples of the promoter include trp promoter, lac promoter, PL or PR promoter.
When yeast is used as a host, examples of the promoter include gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like.
When animal cells are used as hosts, examples of promoters include SRα promoter, SV40 promoter, LTR promoter, CMV promoter, human CMV early gene promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, metallothionein promoter, polyhedron promoter, etc. Is done.
When plant cells are used as hosts, examples of promoters include CaMV promoter and TMV promoter.
Examples of transformation or transfection of host cells using expression vectors include electroporation, spheroplast, lithium acetate, calcium phosphate, Agrobacterium, virus infection, liposome, microinjection, Examples include gene gun method and lipofection method.
The transformed host is cultured under culture conditions according to the types of bacteria, yeast, animal cells, and plant cells, and the target protein is recovered from the cells or from the culture solution.
In culturing microorganisms, a medium containing a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by microorganisms is used. As carbon sources, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, as nitrogen sources, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, etc. Inorganic acids, ammonium salts of organic acids, peptone, meat extract, corn steep liquor, etc., as inorganic substances, potassium phosphate, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, Manganese sulfate, copper sulfate, calcium carbonate and the like are used.
In culturing animal cells, for example, a medium in which DMEM medium, RPMI 1640 medium, or the like is used as a basic medium and fetal calf serum (FCS) or the like is added thereto is used.
As described above, the target protein can be recovered by conventional methods for protein purification, such as ammonium sulfate precipitation, organic solvent precipitation, dialysis, electrophoresis, chromatofocusing, gel filtration chromatography, ion exchange chromatography, affinity chromatography, It can be carried out by HPLC or the like.
When the vector is used for therapy, it is preferably a vector that is not integrated into the subject's genome and that infects cells but is unable to replicate, such as a non-viral vector. Such vectors include, for example, adeno-associated virus vectors, adenovirus vectors and the like. These vectors can include promoters, enhancers, polyadenylation sites, selectable markers, reporter genes, and the like. Examples of viral vectors are described in J. Virol. 67: 5911-5921 (1993), Human Gene Therapy 5: 717-729 (1994), Gene Therapy 1: 51-58 (1994), Human Gene Therapy 5: 793-801 (1994), Gene Therapy 1: 165. 169 (1994) and the like, or improved vectors thereof. Furthermore, an example of a non-viral vector is a human artificial chromosome vector, which is a vector composed of chromosome fragments containing human chromosome-derived centromeres and telomeres. The human chromosome fragment is not particularly limited, but includes, for example, human chromosome 14 fragment, human chromosome 21 fragment (re-listed 2004/031385, Japanese Patent Application Laid-Open No. 2007-295860, etc.). The nucleic acid as defined above is inserted into the vector and administered to the subject, or the vector is administered to the subject by introducing the vector into a tissue or cell collected from the subject and then returning to the subject. can do.
<Antibodies that bind to ALK1 extracellular region polypeptide or antibody fragments thereof>
The antibody that binds to the ALK1 extracellular region polypeptide in the present invention is an antibody that can bind to the ALK1 extracellular region polypeptide, and is preferably a neutralizing antibody. An antibody that inhibits the binding between ALK1 and ALK1 is preferred. The antibody that binds to the ALK1 extracellular region polypeptide in the present invention includes, for example, an antibody described in WO 2007/40912.
An antibody that binds to the ALK1 extracellular region polypeptide or the antibody fragment can be prepared by using the ALK1 extracellular region polypeptide as an antigen protein by the technique specifically shown in the above section (BMP9 antibody). it can.
<Aptamer for BMP9 or aptamer for ALK1 extracellular region polypeptide>
Aptamers are nucleic acid ligands that bind to specific molecules of proteins. The aptamer in the present invention binds to the BMP9 protein or ALK1 extracellular region and can inhibit the function of the BMP9 protein or ALK1.
Aptamers can be obtained by preparing a library composed of various nucleic acid chains and selecting a nucleic acid chain that can bind to the target protein from the library. Suitable methods for identifying aptamers include, for example, Systematic Evolution of Ligands by Exponential Enrichment (SELEX). TM ) Method (US Pat. No. 5,270,163). In the present invention, the BMP9 protein or the ALK1 extracellular region polypeptide and a library nucleic acid are mixed, and then the nucleic acid bound to the BMP9 protein or the ALK1 extracellular region polypeptide is identified, thereby Aptamers can be obtained.
<RNAi molecule targeting ALK1 gene or RNAi molecule targeting BMP9 gene>
An RNAi molecule is a single-stranded or double-stranded nucleic acid molecule comprising an antisense strand having a base sequence complementary to mRNA encoding a target protein. In the RNAi molecule, the antisense strand specifically binds to mRNA encoding the target protein and inhibits expression of the target protein (protein synthesis).
The expression “target” means that the antisense strand contained in the RNAi molecule hybridizes with the target mRNA under stringent conditions. The stringent conditions are, for example, about 1 to 5 × SSC, hybridization at room temperature to about 40 ° C., and then about 0.1 to 1 × SSC, 0.1% SDS, about 45 to 65 ° C. Consists of washing. Here, 1 × SSC refers to a solution of 150 mM NaCl, 15 mM Na-citric acid, pH 7.0.
The RNAi molecule in the present invention specifically binds to mRNA encoding BMP9 or ALK1, and inhibits its protein synthesis. The RNAi molecule in the present invention includes antisense RNA, antisense DNA, siRNA, and shRNA. siRNA is a low molecular double-stranded RNA obtained by hybridizing an antisense strand having a base sequence complementary to mRNA encoding a target protein and a sense strand complementary to the antisense strand. shRNA is a single-stranded RNA in which the antisense strand and the sense strand are linked via a linker portion, and the linker portion is folded by forming a loop, and the antisense strand and the sense strand are Hybridizes to form a double stranded portion.
The RNAi molecule of the present invention can be designed based on the above-described BMP9 and ALK1 gene sequences, for example, using a siRNA Design Support System (Takara Bio Inc.).
<Low molecular compound having inhibitory activity against ALK1 kinase activity>
The low molecular weight compound having an inhibitory activity on the kinase activity of ALK1 in the present invention is not particularly limited as long as it is a compound known to inhibit ALK1 kinase activity. For example, WO 2007 / 147647 (N- (2-dimethylamino-ethyl) -3- [5- (4-isopropyl-phenylamino) -pyrazolo [1,5-a] pyrimidine-3-yl] -benzamide Phenyl- [3- (3,4,5-trimethoxy-phenyl) -pyrazolo [1,5-a] pyrimidine-5-yl] -amine; (4-fluoro-phenyl)-[3- (3,4 , 5-trimethoxy-phenyl) -pyrazolo [1,5-a] pyrimidin-5-yl] -amine; N ′-[3- (3-chloro-phenyl) -pyrazolo [1 5-a] pyrimidin -5-yl] -N, N- diethyl - propane -e-1,3-diamine, but not limited to) can be mentioned.
<Low molecular weight compound having activity of inhibiting binding of BMP9 and ALK1, or binding of BMP9 and type II receptor>
In the present invention, the low molecular weight compound having an activity of inhibiting the binding between BMP9 and ALK1 or the binding between BMP9 and type II receptor is the inhibition of the binding between BMP9 and ALK1 or between BMP9 and type II receptor. It refers to a compound that can be produced and is not particularly limited. “Type II receptor” includes BMP type II receptor (BMPRII), activin type IIa receptor (ActRIIa), and activin type IIb receptor (ActRIIb). The low molecular weight compound included in the present invention may have an activity of inhibiting any one of the binding between BMP9 and BMPRII, the binding between BMP9 and ActRIIa, and the binding between BMP9 and ActRIIb. In addition, it may have an activity of inhibiting any two bonds, or may have an activity of inhibiting these three bonds. Preferable examples include low molecular weight compounds having an activity of inhibiting the binding between BMP9 and BMPRII.
<BMP9 antagonist protein having BMP9 inhibitory activity>
Although no BMP9 antagonist protein having BMP9 inhibitory activity has been reported so far, various biologically derived BMP antagonists having BMP inhibitory activity have been reported for BMP, and such BMP9 antagonist is also present in BMP9. May exist. Such a BMP9 antagonist can be used as an active ingredient of the pharmaceutical composition of the present invention.
<Disease>
The pharmaceutical composition of the present invention can be used for diseases associated with vascular disorders, such as renal diseases, arteriosclerotic diseases, hypertension, heart diseases, diabetes, diabetic complications, thrombosis, dyslipidemia, etc. Lifestyle-related diseases, diseases associated with vasculitis, and diseases in which BMP9 has been shown to be involved.
The renal disease is not particularly limited, and includes chronic glomerulonephritis including IgA nephropathy, diabetic nephropathy, lupus nephritis, nephrosclerosis or rapidly progressive glomerulonephritis. Arteriosclerotic disease is not particularly limited, but ischemic heart such as cerebrovascular disorders (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc.), myocardial infarction and angina Diseases, aortic aneurysm, aortic dissection, nephrosclerosis, obstructive arteriosclerosis and the like can be mentioned. The heart disease is not particularly limited, and examples thereof include valvular heart disease, myocardial infarction, angina pectoris, and cardiomyopathy. Diabetic complications include arteriosclerosis, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic gangrene, chronic infection, cataract and the like. The thrombosis is not particularly limited, and examples thereof include pulmonary thromboembolism, cerebral infarction, myocardial infarction, lower limb acute arterial thrombosis, intestinal necrosis (upper mesenteric artery thrombosis) and the like. Diseases associated with vasculitis are not particularly limited, but Takayasu arteritis, giant cell arteritis (temporal arteritis), polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, Kawasaki disease, Examples include Henoch-Schönlein purpura, hypersensitivity vasculitis, systemic lupus erythematosus, and rheumatoid arthritis. Diseases that may be involved in BMP9 are liver disease, cancer with cancerous ascites / pleural effusion, chronic pancreatitis, allergic disease, inflammatory disease, Alzheimer, multiple sclerosis, diabetic retinopathy, Raynaud Syndrome, Crohn's disease, cancer, etc. The allergic disease is not particularly limited, and examples include allergic rhinitis, asthma, airway hypersensitivity, and atopic dermatitis. The inflammatory disease is not particularly limited, and examples thereof include delayed allergy, rheumatoid arthritis, arthritis, pulmonary disease, hepatitis, ulcerative colitis and the like. The liver disease is not particularly limited, and examples include acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, and metastatic liver cancer.
The pharmaceutical composition of the present invention is preferably a renal disease, arteriosclerotic disease, hypertension, heart disease, diabetes, diabetic complications, thrombosis, dyslipidemia and other lifestyle diseases, particularly preferably renal disease, arteriosclerosis It can be applied to sex diseases, hypertension and thrombosis.
Chronic kidney disease (CKD) is considered to be the largest independent risk factor for cardiovascular complications (CVD), but the presence of arteriosclerosis is also recognized in the early stages of CKD. There is a need for CVD treatment as well as CKD treatment. The results obtained so far suggest that the pharmaceutical composition of the present invention may be effective not only for CKD but also for CVD. Therefore, the pharmaceutical composition of the present invention is particularly useful in renal diseases. It is expected that the possibility of being a clinically useful therapeutic agent is high.
<Formulation and administration method>
The pharmaceutical composition of the present invention contains one or a combination of the above active ingredients. Preferably, the pharmaceutical composition of the present invention contains an antibody that binds to BMP9 or the antibody fragment as an active ingredient. The amount of the active ingredient contained in the pharmaceutical composition of the present invention should be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the patient to be administered, and is not limited to the following, It can be appropriately determined within a range of about 0.1 μg / kg to 100 mg / kg, preferably within a range of about 1 μg / kg to 10 mg / kg.
The form (namely, preparation) of the pharmaceutical composition of the present invention is not limited, and includes both oral preparations and parenteral preparations.
A preferred form is a parenteral preparation, which includes, but is not limited to, an intravenous preparation, an intramuscular preparation, an intraperitoneal preparation, a subcutaneous preparation, a topical preparation and the like. The parenteral preparation includes, for example, an injection, an instillation, a suppository, a transdermal absorption agent, a liposome or a nanoparticle-encapsulated preparation.
Oral preparations include, for example, tablets, pills, granules, capsules, powders, solutions, suspensions, delayed release preparations, enteric preparations and the like.
The pharmaceutical composition of the present invention can contain pharmaceutically acceptable excipients, carriers such as diluents, and additives.
Carriers include, for example, saline, glycerol, ethanol, almond oil, vegetable oil, sucrose, starch, lactose and the like.
Additives include, for example, binders (eg pregelatinized corn starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone etc.), lubricants (eg magnesium stearate, talc, silica etc.), dispersants (eg polyvinylpyrrolidone, corn starch etc.) ), Suspension (eg talc, gum arabic, etc.), emulsifier (eg lecithin, gum arabic, etc.), disintegrant (potato starch, sodium starch glycolate, crospovidone, etc.), buffer (eg phosphate, acetate) Citrate, tris salt, etc.), antioxidants (eg, ascorbic acid, tocopherol, etc.), preservatives (eg, sorbic acid, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate), isotonic agents (eg Eg sodium chloride) , And the like agents (eg, glycerol, etc.).
For enteric preparations, for example, polymers such as hydroxypropyl methylcellulose phthalate, methacrylic acid-methyl methacrylate copolymer, methacrylic acid-ethyl acrylate copolymer, hydroxypropyl acetate succinate are used.
The dosage of the pharmaceutical composition of the present invention should be appropriately determined according to the age, sex, weight, symptom, route of administration, etc. of the patient, and is not limited to the following, but for example, about 0.1 μg per adult day / Kg to 100 mg / kg, preferably about 1 μg / kg to 10 mg / kg. Administration of the formulation may be administered daily during treatment or at intervals such as several days, two weeks or one month.
The pharmaceutical composition of the present invention can be administered to a patient by an administration method suitable for the type and dosage form of the active ingredient contained therein. The pharmaceutical composition of the present invention is administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intranasally, intravaginally, intrarectally, topically, intramuscularly, orally. , Injection, infusion, cell transplantation of transformed cells expressing ALK1 extracellular region polypeptide or the like prepared using the above vector, or any combination of the above.
When the pharmaceutical composition of the present invention contains any of the nucleic acids, expression vectors, aptamers or RNAi molecules described in detail above or a combination thereof as an active ingredient, the pharmaceutical composition is administered by using the active ingredient as a target tissue. Alternatively, it can be performed using a technique or technique generally used in gene therapy that can be introduced into cells.
Gene delivery methods that can be used to administer the pharmaceutical composition of the present invention include colloidal dispersion systems, liposome-derived systems, artificial virus envelopes, and the like. For example, the delivery system should use macromolecular complexes, nanocapsules, microspheres, beads, oil-in-water emulsions, micelles, mixed micelles, liposomes, calcium phosphate method, DEAE dextran method, electroporation method, lipofection method, etc. Can do.
<Evaluation of therapeutic agents>
The effect of the pharmaceutical composition of the present invention can be evaluated by alleviating or eliminating the symptoms of the disease in a patient suffering from the disease that has received administration of the pharmaceutical composition.
The effect of the pharmaceutical composition of the present invention can be evaluated by genetic or biochemical analysis of genes, biomarkers and serum biochemical parameters that are known to be related to the above-mentioned diseases. In patients suffering from the above-mentioned diseases (preferably blood samples derived from patients) who have received administration of the pharmaceutical composition of the present invention, changes in the expression level of the gene, biomarker values, or serum biochemical parameter values are used as indicators. Can be evaluated. Such genes, biomarkers and serum biochemical parameters include (but are not limited to) the following:
gene
Figure JPOXMLDOC01-appb-I000008
Figure JPOXMLDOC01-appb-I000009
Biomarker
Figure JPOXMLDOC01-appb-I000010
Figure JPOXMLDOC01-appb-I000011
Serum biochemistry parameters
Figure JPOXMLDOC01-appb-I000012
EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
In addition, in Japan, this application is a contract research and industrial technology related to “Development of biological system control infrastructure technology using compounds, etc.” in 2007, New Energy and Industrial Technology Development Organization. This is a patent application subject to Article 19 of the Strengthening Law.
ｈｓＡＬＫ１Ｆｃ組み換えタンパク質の取得・調整
１−１．ｈｓＡＬＫ１Ｆｃ発現ベクターの作製
　ｈｓＡＬＫ１Ｆｃ発現ベクターはヒトＡＬＫ１の細胞外領域ｃＤＮＡ断片、およびｈＩｇＧ１のＦｃ領域ｃＤＮＡ断片を、ＥｃｏＲＩ、ＮｏｔＩ（ともに日本国ロシュ・ダイアグノスティックス株式会社）で酵素消化したＰＥＡＫ８発現ベクター（Ｅｄｇｅ　Ｂｉｏｓｙｓｔｅｍｓ社）に組み込むことにより作製した。
組み込まれたｈｓＡＬＫ１ＦｃのｃＤＮＡ配列は下記の通り（配列番号１）。
Figure JPOXMLDOC01-appb-I000013
　以下に、配列番号１がコードするアミノ酸配列（３４６アミノ酸、配列番号２）を示す。
配列番号２：
Figure JPOXMLDOC01-appb-I000014
　それぞれの断片は以下のようにして取得した。ヒトＡＬＫ１の細胞外領域ｃＤＮＡは、
ｈｓＡＬＫ１　ＦＷ：ａｇａａｔｔｃｃｃａｃｃａｔｇａｃｃｔｔｇｔｃｃｃｃｃａｇ（配列番号３）、
ｈｓＡＬＫ１　ＲＶ：ａａｃｔａｇｔｃｔｇｇｃｃａｔｃｔｇｔｔｃｃｃｇｇｃｔｇ（配列番号４）
を用いて、ヒト肺ｃＤＮＡライブラリーよりＰＣＲ法を用いてクローニングした。得られた断片をＥｃｏＲ１、ＳｐｅＩで酵素消化した後、アガロースゲルにて電気泳動し、当該バンドを切り出した。アガロースからのＤＮＡの抽出にはＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用いた。
　一方、ｈＩｇＧ１のＦｃ領域ｃＤＮＡは、
ｈＩｇＧ１　Ｆｃ　ＦＷ：ａｃｔａｇｔｇａｃａａａａｃｔｃａｃａｃａｔｇｃｃ（配列番号５）
ｈＩｇＧ１　Ｆｃ　ＲＶ：ｇｃｇｇｃｃｇｃｔｃａｔｃａｔｔｔａｃｃｃ（配列番号６）
を用いてＰＣＲ法にてクローニングした。得られた断片をＳｐｅＩ、ＮｏｔＩ（ともに日本国ロシュ・ダイアグノスティックス株式会社）で酵素消化した後、アガロースゲルにて電気泳動し、当該バンドを切り出した。アガロースからのＤＮＡの抽出にはＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用いた。
１−２．ｈｓＡＬＫ１Ｆｃ高発現株の作製・培養上清取得
　ｈｓＡＬＫ１Ｆｃ高発現株の作製は、上記で作成した発現ベクターをＳｆｉ−Ｉ制限酵素を用いて線状化した後、その線状化したＤＮＡをＬｉｐｏｆｅｃｔａｍｉｎｅＴＭ　ＬＴＸ試薬（日本国インビトロジェン株式会社）を用いて添付文書に従いＣＨＯ　Ｒａｓ　ｃｌｏｎｅＩ細胞に遺伝子導入した。遺伝子導入２４時間後、６μｇ／ｍｌのｐｕｒｏｍｙｃｉｎｅおよび１０％血清（ＦＣＳ）を含むａｌｐｈａ　ＭＥＭを用いて遺伝子導入された細胞を薬剤選抜した後、限外希釈法にて、シングルクローンの単離を行った。高発現株の選別には、ヒトＩｇＧ　ＥＬＩＳＡ　ｋｉｔ（日本国コスモバイオ株式会社より購入）を用いた。
　次に、得られた高発現株を２２５ｃｍ２の大フラスコに播種し、３７℃にてコンフルエントになるまで培養した。コンフルエントになったことを確認した後、ＰＢＳ（Ｄｕｌｅｃｃｏ’ｓ　Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ；ＳＩＧＭＡ）を用いて洗浄した後、Ｄ／Ｆ培地にて２５％に希釈したＥＸ−Ｃｅｌｌ３０２（ＳＡＦＣ　Ｂｉｏｓｃｉｅｎｃｅｓ社）培地に置換した。５日間培養した後、培養上清を回収し、０．２２μｍフィルター（０．２２μｍ　ＧＰ　Ｅｘｐｒｅｓｓ　Ｍｅｍｂｒａｎｅ　５００ｍｌ；日本国日本ミリポア株式会社）で濾過処理を行った後、４℃（低温室）で冷却した。凍結保存した場合には融解後０．２２μｍフィルターで再濾過した。
１−３．ｈｓＡＬＫ１Ｆｃの精製・調製
　ｈｓＡＬＫ１Ｆｃの精製には、Ａｎｔｉｂｏｄｙ　Ａｆｆｉｎｉｔｙカラム（Ｈｉ　Ｔｒａｐ　ＰｒｏｔｅｉｎＧ　ＨＰ　５ｍｌ；日本国ＧＥヘルスケア　バイオサイエンス株式会社）を用いた。前処理した前記の培養上清をＰＢＳにて平衡化したカラムにアプライし、ＰＢＳおよび１．８５ＭのＮａＣｌを含むＰＢＳ溶液にて洗浄した後、溶出液（２０ｍＭ　クエン酸ナトリウム、５０ｍＭ　ＮａＣｌ、ｐＨ２．７）を用いて目的蛋白質の溶出を行った。上記分離精製操作にはＡＫＴＡｅｘｐｌｏｒｅｒ１０ｓ（日本国ＧＥヘルスケア　バイオサイエンス株式会社）を用いた。次に、その溶出物を限外濾過膜ＶＩＶＡＳＰＩＮ２０　１０，０００　ＭＷＣＯ　ＰＥＳ（日本国ザルトリウス・ステディム・ジャパン株式会社）を用いてＰＢＳへの溶媒置換を行いながら、サンプル濃縮を行った。濃縮後、０．２２μｍフィルター（Ｍｉｌｌｅｘ　ＧＶ；日本国日本ミリポア株式会社）を用いて滅菌処理を行った。蛋白質濃度はＡ２８０ｎｍを測定し、分子吸光係数（Ｅ１％，１ｃｍ＝１０）より算出した。 Acquisition and adjustment of hsALK1Fc recombinant protein 1-1. Preparation of hsALK1Fc Expression Vector The hsALK1Fc expression vector is a PEAK8 expression vector obtained by enzymatic digestion of the extracellular region cDNA fragment of human ALK1 and the hIgG1 Fc region cDNA fragment with EcoRI and NotI (both from Roche Diagnostics, Japan). It was prepared by incorporating into (Edge Biosystems).
The cDNA sequence of the incorporated hsALK1Fc is as follows (SEQ ID NO: 1).
Figure JPOXMLDOC01-appb-I000013
The amino acid sequence encoded by SEQ ID NO: 1 (346 amino acids, SEQ ID NO: 2) is shown below.
SEQ ID NO: 2:
Figure JPOXMLDOC01-appb-I000014
Each fragment was obtained as follows. The extracellular region cDNA of human ALK1 is
hsALK1 FW: agaattccccaccatgaccttgtccccccag (SEQ ID NO: 3),
hsALK1 RV: aacttagctgggcccatctgtttccccggctg (SEQ ID NO: 4)
Was cloned from a human lung cDNA library using the PCR method. The obtained fragment was enzymatically digested with EcoR1 and SpeI, and then electrophoresed on an agarose gel to cut out the band. For extraction of DNA from agarose, QIAquick Gel Extraction Kit (Qiagen, Japan) was used.
On the other hand, the FIgG region cDNA of hIgG1 is
hIgG1 Fc FW: actagtgacaaaaactcacacatgcc (SEQ ID NO: 5)
hIgG1 Fc RV: gcggccgctcatcatttaccc (SEQ ID NO: 6)
Was cloned by PCR method. The obtained fragment was enzymatically digested with SpeI and NotI (both Roche Diagnostics, Japan), and then electrophoresed on an agarose gel to cut out the band. For extraction of DNA from agarose, QIAquick Gel Extraction Kit (Qiagen, Japan) was used.
1-2. Preparation of hsALK1Fc high expression strain and acquisition of culture supernatant The hsALK1Fc high expression strain was prepared by linearizing the expression vector prepared above using Sfi-I restriction enzyme and then converting the linearized DNA to Lipofectamine ™ LTX. Using a reagent (Invitrogen, Japan), the gene was introduced into CHO Ras clone I cells according to the package insert. Twenty-four hours after gene transfer, drug-selected cells were selected using alpha MEM containing 6 μg / ml puromycin and 10% serum (FCS), and single clones were isolated by limiting dilution. It was. Human IgG ELISA kit (purchased from Japan Cosmo Bio Co., Ltd.) was used for selection of high expression strains.
Next, the obtained high expression strain was inoculated into a large flask of 225 cm 2 and cultured at 37 ° C. until it became confluent. After confirming that the cells were confluent, the cells were washed with PBS (Dulecco's Phosphate Buffered Saline; SIGMA), and then replaced with EX-Cell 302 (SAFC Biosciences) medium diluted to 25% in D / F medium. did. After culturing for 5 days, the culture supernatant was collected, filtered through a 0.22 μm filter (0.22 μm GP Express Membrane 500 ml; Japan Millipore, Japan), and then cooled at 4 ° C. (cold room). . When stored frozen, it was re-filtered through a 0.22 μm filter after thawing.
1-3. Purification and preparation of hsALK1Fc For purification of hsALK1Fc, an Antibody Affinity column (Hi Trap ProteinG HP 5 ml; GE Healthcare Biosciences, Japan) was used. The pretreated culture supernatant was applied to a column equilibrated with PBS, washed with a PBS solution containing PBS and 1.85 M NaCl, and then eluted with 20 mM sodium citrate, 50 mM NaCl, pH 2. The target protein was eluted using 7). For the separation and purification operation, AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used. Next, the eluate was subjected to sample concentration while substituting the solvent with PBS using an ultrafiltration membrane VIVASPIN20 10,000 MWCO PES (Sartorius Stedim Japan, Japan). After concentration, sterilization was performed using a 0.22 μm filter (Millex GV; Japan Millipore, Japan). The protein concentration was measured from A 280 nm and calculated from the molecular extinction coefficient (E 1%, 1 cm = 10).
インビトロでの精製ｈｓＡＬＫ１Ｆｃタンパク質の活性測定（インビトロ試験）
　ｈｓＡＬＫ１Ｆｃの活性評価にはＢＭＰシグナルを検出可能なレポーター・プラスミドを安定導入した細胞株を用いた。具体的には、ＢＭＰのシグナルを検出可能なレポーター・プラスミドである（ｐ（ＧＣＣＧ）１２−Ｌｕｃ／ｎｅｏ）を、ヒト肝癌細胞株であるＨｅｐＧ２（ＡＴＣＣより入手可能）に遺伝子導入し、レポーター・プラスミドの安定導入株ＨｅｐＧ２株（ｐ（ＧＣＣＧ）１２−Ｌｕｃ／ＨｅｐＧ２（３８−５））を作製した。なお、ＢＭＰシグナル検出用レポータープラスミド（ｐ（ＧＣＣＧ）１２−Ｌｕｃ／ｎｅｏ）には、ルシフェラーゼ遺伝子の上流に、ＢＭＰシグナル伝達因子であるＳｍａｄ１／５／８の結合配列をタンデムに１２個連結させたプラスミド（ｐ（ＧＣＣＧ）１２−Ｌｕｃ／ｎｅｏ）をＭｏｌ．Ｂｉｏｌ．Ｃｅｌｌ．，２０００，１１（２）：５５５−６５．のＫｕｓａｎａｇｉらの記載方法に準じて作製したものを用いた。
　精製したｈｓＡＬＫ１ＦｃのＢＭＰ９に対する中和活性測定は以下のようにして行った。上記の方法により作出した細胞株（ｐ（ＧＣＣＧ）１２−Ｌｕｃ／ＨｅｐＧ２（３８−５））に対し、３ｎｇ／ｍｌのＢＭＰ９タンパク質（Ｒ＆Ｄ社より購入）、および種々の濃度のｈｓＡＬＫ１Ｆｃタンパク質を含む溶液を添加し５時間培養した後、化学発光試薬（Ｓｔｅａｄｙ　Ｇｌｏ（商標）Ｌｕｃｉｆｅｒａｓｅ　ａｓｓａｙ　ｓｙｓｔｅｍ，日本国プロメガ株式会社より入手可能）を添加し、ｌｕｃｉｆｅｒａｓｅ活性を測定した。またコントールサンプルとして、ＢＭＰ９タンパク質およびｈｓＡＬＫ１Ｆｃの非添加サンプルを用意し、同様の測定を行った。図１のｈｓＡＬＫ１Ｆｃ添加時での値（％）は、ＢＭＰ９のみ添加したサンプルのｌｕｃｉｆｅｒａｓｅ値を１００％、ＢＭＰ９タンパク質およびｈｓＡＬＫ１Ｆｃ非添加サンプルのｌｕｃｉｆｅｒａｓｅ値を０％とした時の、％　ｏｆ　ｃｏｎｔｒｏｌを示している。結果、精製したｈｓＡＬＫ１Ｆｃタンパク質にはＢＭＰ９に対する中和作用があることが確認された。 Activity measurement of purified hsALK1Fc protein in vitro (in vitro test)
For evaluation of hsALK1Fc activity, a cell line into which a reporter plasmid capable of detecting a BMP signal was stably introduced was used. Specifically, a reporter plasmid (p (GCCG) 12-Luc / neo) that can detect a BMP signal is introduced into a human liver cancer cell line HepG2 (available from ATCC), and the reporter plasmid is introduced. A stably transfected strain HepG2 (p (GCCG) 12-Luc / HepG2 (38-5)) was prepared. The BMP signal detection reporter plasmid (p (GCCG) 12-Luc / neo) was linked in tandem with 12 binding sequences of Smad1 / 5/8, which is a BMP signaling factor, upstream of the luciferase gene. Plasmid (p (GCCG) 12-Luc / neo) was obtained from Mol. Biol. Cell. 2000, 11 (2): 555-65. Were prepared according to the method described by Kusanagi et al.
The neutralization activity of purified hsALK1Fc against BMP9 was measured as follows. A solution containing 3 ng / ml BMP9 protein (purchased from R & D) and various concentrations of hsALK1Fc protein for the cell line (p (GCCG) 12-Luc / HepG2 (38-5)) produced by the above method After culturing for 5 hours, a chemiluminescence reagent (Steady Glo ™ Luciferase assay system, available from Promega Corporation, Japan) was added, and luciferase activity was measured. Further, as a control sample, a sample not added with BMP9 protein and hsALK1Fc was prepared, and the same measurement was performed. The value (%) when hsALK1Fc is added in FIG. 1 indicates% of control when the luciferase value of the sample to which only BMP9 is added is 100%, and the luciferase value of the sample to which BMP9 protein and hsALK1Fc are not added is 0%. Yes. As a result, it was confirmed that the purified hsALK1Fc protein has a neutralizing effect on BMP9.
ＢＭＰ９の正常ヒト血管内皮細胞への添加試験（インビトロ試験）
　５Ｘ１０５個の正常ヒト大動脈内皮細胞（ＨＡＥＣ；日本国タカラバイオ株式会社より購入）を、３μｇ／ｍｌのｈｓＡＬＫ１Ｆｃタンパク質（実施例１にて記載）を含む内皮細胞増殖培地（２％　ＦＢＳ含有ＥＧＭＴＭ−２　ＢｕｌｌｅｔＫｉｔ（登録商標））に懸濁し、６ｃｍディッシュプレートに播種し、３７℃にて培養した。
　翌日、細胞上清を除去し、内皮細胞基本培地−２（ＥＢＭ（登録商標）−２）を用いて細胞洗浄した後、１０ｎｇ／ｍｌのＢＭＰ９タンパク質（Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社より購入）を含むＥＢＭ（登録商標）−２培地、もしくはＥＢＭ（登録商標）−２のみの培地を細胞に加え、３７℃にて７時間培養した。各条件につき、ｎ＝２で実施した。ＢＭＰ９刺激７時間後、上清を除去し、ＰＢＳにて念入りに２回洗浄した後、ＲＮｅａｓｙ　ｍｉｎｉ　ｋｉｔ（日本国株式会社キアゲン）の添付文書に従い、ＲＮＡを取得した。取得したＲＮＡはｉｌｌｕｍｉｎａ　Ｂｅａｄ　Ａｒｒａｙを用いたＧｅｎｅＣｈｉｐ解析に供するために、株式会社モリテックスに送付した。解析はモリテックス社「ＤＮＡ発現解析受託フロー」に従い、実施した。図２の値は、ＢＭＰ９添加による遺伝子発現変化（倍）を、ＢＭＰ９未刺激サンプルの値を１として算出したものである。
　その結果、ＢＭＰ９の血管内皮細胞への添加により、血管内皮障害のマーカーとされるＥ−ｓｅｌｅｃｔｉｎおよびＶＣＡＭ−１が強力に発現誘導されることが明らかになった（図２）。また、同様に、血管障害や炎症に関わるとされるＩＬ−８、ＣＯＸ−２といった遺伝子の強力な発現誘導も認められた（図２）。また反対に抗酸化作用、抗炎症作用に関わることが知られているＨｅｍｏ　ｏｘｙｇｅｒａｓｅ−１は、ＢＭＰ９刺激によりその発現量は約１／３に低下した。これらの結果より、ＢＭＰ９は血管内皮細胞に対して障害性因子として作用することが明らかになった。
　また、興味深いことに、高血圧症、腎疾患、糖尿病、糖尿病合併症、脂質異常症、動脈硬化性疾患、血栓症と深く関わることが知られているＡｐｅｌｉｎ、ａｄｒｅｎｏｍｅｄｕｌｌｉｎ、ＡＣＥ（ａｎｇｉｏｔｅｎｓｉｎ　Ｉ　ｃｏｎｖｅｒｔｉｎｇ　ｅｎｚｙｍｅ）、ＬＩＰＧ（Ｌｉｐａｓｅ，ｅｎｄｏｔｈｅｌｉａｌ）、ｐｌａｓｍｉｎｏｇｅｎ　ａｃｔｉｖａｔｏｒ、ｔｈｒｏｍｂｏｍｏｄｕｌｉｎといった遺伝子の発現変動も認められた（図２）。このことより、抗ＢＭＰ９療法は、血管障害性疾患、各種動脈硬化性疾患、炎症性疾患、高血圧症、各種腎疾患、糖尿病、糖尿病合併症、脂質異常症、血栓症に対する有効な治療法になりうる可能性が示された。 Addition test of BMP9 to normal human vascular endothelial cells (in vitro test)
5 × 10 5 normal human aortic endothelial cells (HAEC; purchased from Takara Bio Inc., Japan), endothelial cell growth medium (EGM ™ containing 2% FBS) containing 3 μg / ml of hsALK1Fc protein (described in Example 1) -2 BulletKit (registered trademark)), seeded on a 6 cm dish plate, and cultured at 37 ° C.
The next day, the cell supernatant was removed, the cells were washed with endothelial basic medium-2 (EBM (registered trademark) -2), and then EBM containing 10 ng / ml BMP9 protein (purchased from R & D Systems) (registered) (Trademark) -2 medium or a medium containing only EBM (registered trademark) -2 was added to the cells and cultured at 37 ° C. for 7 hours. For each condition, n = 2. After 7 hours of stimulation with BMP9, the supernatant was removed, washed carefully twice with PBS, and RNA was obtained according to the package insert of RNeasy mini kit (Qiagen, Japan). The obtained RNA was sent to Moritex Co., Ltd. for use in GeneChip analysis using an illumina bead array. The analysis was carried out according to the Moritex "DNA expression analysis commissioned flow". The values in FIG. 2 are obtained by calculating the gene expression change (fold) due to the addition of BMP9 with the value of the BMP9 unstimulated sample as 1.
As a result, it was revealed that the addition of BMP9 to vascular endothelial cells strongly induced expression of E-selectin and VCAM-1 which are markers for vascular endothelial injury (FIG. 2). Similarly, strong expression induction of genes such as IL-8 and COX-2, which are considered to be involved in vascular disorders and inflammation, was also observed (FIG. 2). On the other hand, the expression level of Hemo oxygerase-1, which is known to be involved in antioxidant and anti-inflammatory effects, was reduced to about ３ by BMP9 stimulation. From these results, it became clear that BMP9 acts as a damaging factor on vascular endothelial cells.
Also, interestingly, Apelin, adrenomedullin, ACE (angiotensin I converting enzyme), which is known to be deeply related to hypertension, kidney disease, diabetes, diabetic complications, dyslipidemia, arteriosclerotic disease, thrombosis, Changes in expression of genes such as LIPG (lipase, endothelial), plasminogen activator, and thrombomodulin were also observed (FIG. 2). Therefore, anti-BMP9 therapy is an effective treatment for vascular disorder diseases, various arteriosclerotic diseases, inflammatory diseases, hypertension, various renal diseases, diabetes, diabetic complications, dyslipidemia, and thrombosis. The possibility was shown.
ＵＳｍＢＭＰ９　ＫＩキメラマウスの作製及びコントロールキメラマウスの作製
　国際公開第ＷＯ　２００６／７８０７２号パンフレット記載の実施例に従い、マウスＢＭＰ９−ｃＤＮＡ（開始コドンから終止コドンを含む１２８７ｂｐ　配列番号７）よりｐＵＳｍＢＭＰ９　ＫＩベクターを作製した。
　以下に、配列番号７及び８におけるマウスＢＭＰ９シグナル配列、ｐｒｏ−ｒｅｇｉｏｎ、ｍａｔｕｒｅ体部分をそれぞれＧｅｎＢａｎｋアクセッション番号ＮＰ＿０６２３７９，Ｑ９ＷＶ５６の情
Figure JPOXMLDOC01-appb-I000015
配列番号７：
Figure JPOXMLDOC01-appb-I000016
Figure JPOXMLDOC01-appb-I000017
　以下に、配列番号７がコードするアミノ酸配列（４２８アミノ酸、配列番号８）を示す。
配列番号８：
Figure JPOXMLDOC01-appb-I000018
　ｐＵＳｍＢＭＰ９　ＫＩベクターｍＢＭＰ９発現ユニットの開始コドンから終止コドンまでのポリヌクレオチド配列（マウスＢＭＰ９シグナル配列を、イントロン領域を含んだマウスＩｇκシグナル配列〔下線部分〕に置換し、その下流にマウスＢＭＰ９　ｐｒｏ体配列を含む１５２２ｂｐ、配列番号９）、および該ｃＤＮＡがコードするアミノ酸配列（４２６アミノ酸、囲み線の部分はマウスＩｇκシグナル配列を示す、配列番号１０）を以下に示す。イントロン領域を含んだマウスＩｇκシグナル配列情報はＧｅｎＢａｎｋより取得したＭＵＳＩＧＫＶＲ１（アクセッション番号Ｋ０２１５９）をもとに、その上流のゲノム配列をＵＣＳＣマウスゲノムデータベースより取得した。
配列番号９
Figure JPOXMLDOC01-appb-I000019
Figure JPOXMLDOC01-appb-I000020
配列番号１０
Figure JPOXMLDOC01-appb-I000021
　ｐＵＳｍＢＭＰ９　ＫＩベクターを用い、国際公開第ＷＯ　２００６／７８０７２号パンフレットの実施例に従いＢ細胞特異的にマウスＢＭＰ９を発現するＵＳｍＢＭＰ９　ＫＩキメラマウスを作製した。更に下記実施例５及び６で用いられたマウスコントロール個体（コントロールキメラマウス）を、国際公開第ＷＯ　２００６／７８０７２号パンフレットの実施例１１記載の方法に従い作製した。 Preparation of USmBMP9 KI chimeric mouse and preparation of control chimeric mouse According to the examples described in WO 2006/78072 pamphlet, a pUSmBMP9 KI vector was prepared from mouse BMP9-cDNA (1287 bp SEQ ID NO: 7 including stop codon from start codon). did.
Below, the mouse BMP9 signal sequence, pro-region, and body part of SEQ ID NOs: 7 and 8 are represented by GenBank accession numbers NP_062379 and Q9WV56, respectively.
Figure JPOXMLDOC01-appb-I000015
SEQ ID NO: 7
Figure JPOXMLDOC01-appb-I000016
Figure JPOXMLDOC01-appb-I000017
The amino acid sequence encoded by SEQ ID NO: 7 (428 amino acids, SEQ ID NO: 8) is shown below.
SEQ ID NO: 8
Figure JPOXMLDOC01-appb-I000018
pUSmBMP9 KI vector The polynucleotide sequence from the start codon to the stop codon of the mBMP9 expression unit (the mouse BMP9 signal sequence was replaced with the mouse Igκ signal sequence [underlined portion] containing the intron region, and the mouse BMP9 pro body sequence was downstream of it. Including 1522 bp, SEQ ID NO: 9), and the amino acid sequence encoded by the cDNA (426 amino acids, the boxed portion indicates the mouse Igκ signal sequence, SEQ ID NO: 10) are shown below. Mouse Igκ signal sequence information including an intron region was obtained from the UCSC mouse genome database based on MUSIGKVR1 (accession number K02159) obtained from GenBank.
SEQ ID NO: 9
Figure JPOXMLDOC01-appb-I000019
Figure JPOXMLDOC01-appb-I000020
SEQ ID NO: 10
Figure JPOXMLDOC01-appb-I000021
Using the pUSmBMP9 KI vector, a USmBMP9 KI chimeric mouse that expresses mouse BMP9 in a B cell specific manner was prepared according to the examples of the pamphlet of International Publication No. WO 2006/78072. Further, mouse control individuals (control chimeric mice) used in Examples 5 and 6 below were prepared according to the method described in Example 11 of International Publication No. WO 2006/78072.
ＵＳｍＢＭＰ９　ＫＩキメラマウスの表現型解析
５−１．剖検所見
　上記実施例４で作製されたキメラマウスを用い、３週齢・４週齢・５週齢においてそれぞれ剖検を実施した。ＵＳｍＢＭＰ９　ＫＩキメラマウスにおいてのみ腹水、胸水、リンパ組織、小腸および膵臓に出血が原因と考えられる赤色化が認められた。それぞれの変化を観察した時期およびその個体数を以下に記す。
５−１−１．赤色化腹水貯留
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、腹水貯留は３週齢全ての個体で観察されなかったが、４週齢・５週齢では全ての個体で赤色化した腹水の貯留が認められた。
５−１−２．赤色化胸水貯留
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では１個体、４週齢・５週齢では全ての個体で赤色化した胸水の貯留が認められた。
５−１−３．赤色化腸間膜リンパ節
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢以降５週齢まで全ての個体において腸間膜リンパ節の赤色化が認められた。
５−１−４．赤色化顎下リンパ節
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢以降５週齢まで全ての個体において顎下リンパ節の赤色化が認められた。
５−１−５．赤色化肘リンパ節
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢以降５週齢まで全ての個体において肘リンパ節の赤色化が認められた。
５−１−６．赤色化鼠径リンパ節
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢以降５週齢まで全ての個体において鼠径リンパ節の赤色化が認められた。
５−１−７．赤色化膝窩リンパ節
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢の全個体、４週齢では８個体、５週齢の全個体で膝窩リンパ節の赤色化が認められた。
５−１−８．小腸の赤色化
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では５個体、４週齢・５週齢では全個体で小腸表面の一部に赤色点ないし赤色斑が認められた。
５−１−９．膵臓の赤色化
　３週齢では７個体、４週齢では９個体、５週齢では６個体ＵＳｍＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では２個体、４週齢では５個体、５週齢では４個体で膵臓表面の一部に赤色化が認められた。
　以上の結果より、ＢＭＰ９の過剰作用により、出血性変化が原因と考えられる腹水、胸水、リンパ組織、小腸および膵臓の赤色化が惹き起こされることが明らかになり、ＢＭＰ９の血管障害活性は、ｉｎ　ｖｉｔｒｏだけでなく、ｉｎ　ｖｉｖｏにおいても確認された。
５−２．病理所見
　３週齢のコントロールキメラマウスおよびＵＳｍＢＭＰ９　ＫＩキメラマウスから由来する腸間膜リンパ節および鼠径リンパ節のＨ＆Ｅ染色病理切片を用いた観察から、ＵＳｍＢＭＰ９　ＫＩキメラマウスにおいて洞内赤血球が特徴的に観察された。洞内赤血球は、リンパ管を介した血液吸収を示唆する像であり、周囲で出血性変化が起こった可能性が示された。ただし、肉眼・組織所見では明らかな出血巣は認められていないことから、血管外への赤血球漏出のような微小出血が起こっている可能性も示された。 Phenotypic analysis of USmBMP9 KI chimeric mice 5-1. Autopsy Findings Using the chimeric mice prepared in Example 4 above, necropsy was performed at 3 weeks, 4 weeks, and 5 weeks of age, respectively. Only in USmBMP9 KI chimera mice, ascites, pleural effusion, lymphoid tissue, small intestine and pancreas were reddened due to bleeding. The time when each change was observed and the number of individuals are described below.
5-1-1. Reddish ascites retention 7 mice at 3 weeks of age, 9 at 4 weeks of age, 6 at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, ascites retention was not observed in all the three-week-old individuals, but red-colored ascites retention was observed in all the individuals at 4 weeks and 5 weeks of age.
5-1-2. Red-colored pleural effusion retention 7 mice at 3 weeks of age, 9 at 4 weeks of age, 6 at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, one individual was observed at 3 weeks of age, and red pleural effusion was observed in all individuals at 4 weeks and 5 weeks of age.
5-1-3. Reddish mesenteric lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, red coloration of mesenteric lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age.
5-1-4. Reddish submandibular lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, reddening of the submandibular lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age.
5-1-5. Reddish elbow lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, reddening of the elbow lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age.
5-1-6. Reddened inguinal lymph node 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, reddening of the inguinal lymph nodes was observed in all individuals from 3 weeks of age to 5 weeks of age.
5-1-7. Red Popliteal Population Nodes 7 mice at 3 weeks of age, 9 at 4 weeks of age, 6 at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, reddening of the popliteal lymph nodes was observed in all 3 weeks old individuals, 4 weeks old, 8 individuals, and 5 weeks old all individuals.
5-1-8. Redness of small intestine Seven individuals at 3 weeks of age, 9 individuals at 4 weeks of age, and 6 individuals at 5 weeks of age were subjected to autopsy of USmBMP9 KI chimeric mice. As a result, 5 individuals at 3 weeks of age, red dots or red spots were observed on a part of the surface of the small intestine in all individuals at 4 weeks of age and 5 weeks of age.
5-1-9. Pancreas reddening 7 individuals at 3 weeks of age, 9 individuals at 4 weeks of age, 6 individuals at 5 weeks of age, USmBMP9 KI chimeric mice were necropsied. As a result, 2 individuals at 3 weeks of age, 5 individuals at 4 weeks of age, and 4 individuals at 5 weeks of age, redness was observed on a part of the pancreas surface.
From the above results, it is clarified that the excessive action of BMP9 causes reddening of ascites, pleural effusion, lymphoid tissue, small intestine and pancreas, which is thought to be caused by hemorrhagic changes. It was confirmed not only in vitro but also in vivo.
5-2. Pathological findings From observations of mesenteric and inguinal lymph nodes derived from 3-week-old control chimeric mice and USmBMP9 KI chimeric mice using H & E-stained pathological sections, sinusoidal erythrocytes were characteristically observed in USmBMP9 KI chimeric mice. It was done. Sinus erythrocytes are images suggesting blood absorption through lymphatic vessels, indicating the possibility of hemorrhagic changes around them. However, no obvious bleeding lesions were observed in the macroscopic and histological findings, suggesting the possibility of microhemorrhages such as erythrocyte leakage outside the blood vessels.
ＵＳｍＢＭＰ９　ＫＩキメラマウスの血管透過性試験
　実施例４で記載された方法に従って作製され、５週齢に達したコントロールキメラマウスおよびＵＳｍＢＭＰ９　ＫＩキメラマウスにエバンスブルー（日本国　株式会社エル・エス・エル）を３０ｍｇ／ｋｇの用量にてｉ．ｖ．投与した。６０分後に投与個体を剖検し、腹水・胸水・臓器の性状・変化を指標に血管透過性を評価した。その結果、エバンスブルーを投与したＵＳｍＢＭＰ９　ＫＩキメラマウスにおいてのみ、青く着色された腹水・胸水が認められた。また、コントロールキメラ個体に比べＵＳｍＢＭＰ９　ＫＩキメラマウスの肺、肝臓、空腸、回腸組織において青黒い濃染領域が高頻度で認められた。これらの結果から、ＵＳｍＢＭＰ９　ＫＩキメラマウスにおいて血管透過性が亢進し、それによって血中アルブミンが、短時間で腹腔・胸腔に漏出していることが示唆された。血管透過性の亢進は、血管内皮細胞障害と密接に関わることが知られており、ＢＭＰ９の過剰作用により、血管内皮細胞が障害され、その結果として、血管透過性が亢進としたものと推察される。 Vascular permeability test of USmBMP9 KI chimeric mouse Evans Blue (L.S., Inc., Japan) was prepared according to the method described in Example 4 and the control chimeric mouse and USmBMP9 KI chimeric mouse that reached 5 weeks of age. I. At a dose of 30 mg / kg. v. Administered. Sixty minutes later, the administered individuals were necropsied and vascular permeability was evaluated using ascites, pleural effusion and organ properties / changes as indicators. As a result, blue-colored ascites and pleural effusion were observed only in the USmBMP9 KI chimeric mice administered with Evans Blue. In addition, dark blue stained areas were frequently observed in the lung, liver, jejunum, and ileum tissues of USmBMP9 KI chimeric mice as compared to the control chimeric individuals. From these results, it was suggested that vascular permeability was enhanced in USmBMP9 KI chimeric mice, and that blood albumin leaked into the abdominal cavity and chest cavity in a short time. Increased vascular permeability is known to be closely related to vascular endothelial cell damage, and it is assumed that vascular endothelial cells are damaged by the excessive action of BMP9, resulting in increased vascular permeability. The
Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体の発現・調製
７−１．Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターの構築
７−１−１．ｐＬＮ１Ｖ５ベクターの構築
　５’末端にＢａｍＨＩ・ＮｈｅＩ・ＳａｌＩサイトを３’末端にＸｈｏＩサイトを持つ（Ｖ５タグ＋Ｓｔｏｐ　ｃｏｄｏｎ）センスオリゴＤＮＡおよびそれに対するアンチセンスオリゴＤＮＡ：Ｖ５ＳおよびＶ５ＡＳ（日本国　北海道システムサイエンス株式会社）を合成した。
Ｖ５Ｓ：ＧＡＴＣＣＧＣＴＡＧＣＧＴＣＧＡＣＧＧＴＡＡＧＣＣＴＡＴＣＣＣＴＡＡＣＣＣＴＣＴＣＣＴＣＧＧＴＣＴＣＧＡＴＴＣＴＡＣＧＴＧＡＣ（配列番号１１）
Ｖ５ＡＳ：ＴＣＧＡＧＴＣＡＣＧＴＡＧＡＡＴＣＧＡＧＡＣＣＧＡＧＧＡＧＡＧＧＧＴＴＡＧＧＧＡＴＡＧＧＣＴＴＡＣＣＧＴＣＧＡＣＧＣＴＡＧＣＧ（配列番号１２）
　上記合成オリゴＤＮＡを、掛田らの報告（Ｇｅｎｅ　Ｔｈｅｒ．１２：８５２−８５６，２００５）に記載されたｐＬＮ１ベクター上のＢａｍＨＩ−ＸｈｏＩサイトに導入し、ｐＬＮ１Ｖ５ベクターを構築した。
７−１−２．Ｎ末端にＨｉｓタグ配列を持つｍＢＭＰ９　ＤＮＡ断片の合成
マウスＢＭＰ９全長配列のＮ末端にＨｉｓタグを付加するためのＰＣＲプライマー
Figure JPOXMLDOC01-appb-I000022
　（配列番号１３）
Figure JPOXMLDOC01-appb-I000023
　（配列番号１４）
Figure JPOXMLDOC01-appb-I000024
　（配列番号１５）
Figure JPOXMLDOC01-appb-I000025
　（配列番号１６）
　Ｐｒｉｍｅ　ＳＴＡＲ　ＨＳ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（日本国タカラバイオ株式会社）を用い添付文書にしたがって反応液を調製し、５０μｌ反応液中に配列番号１３および１４、２種のプライマー各１０ｐｍｏｌ、鋳型としてマウスＢＭＰ９−ｃＤＮＡ（配列番号７）を添加し、９４℃５分保温した後、９８℃１０秒、５７℃５秒、および７２℃１分２０秒を１サイクルとして２５サイクル増幅し、得られた１０１ｂｐの増幅断片を０．８％ゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって増幅断片（ＮｈｅＩ　Ｈｉｓ　ｍＢＭＰ９）を回収した。
　Ｐｒｉｍｅ　ＳＴＡＲ　ＨＳ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（日本国タカラバイオ株式会社）を用い添付文書にしたがって反応液を調製し、５０μｌ反応液中に配列番号１５および１６、２種のプライマー各１０ｐｍｏｌ、鋳型としてマウスＢＭＰ９−ｃＤＮＡ（配列番号７）を添加し、９４℃５分保温した後、９８℃１０秒、５７℃５秒、および７２℃１分２０秒を１サイクルとして２５サイクル増幅し、得られた１２４９ｂｐの増幅断片を０．８％ゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって増幅断片（Ｈｉｓ　ｍＢＭＰ９　ＳａｌＩ）を回収した。
　上記２回のＰＣＲで得られたＤＮＡ増幅断片（ＮｈｅＩ　Ｈｉｓ　ｍＢＭＰ９）および（Ｈｉｓ　ｍＢＭＰ９　ＳａｌＩ）をＰｒｉｍｅＳＴＡＲ　ｂｕｆｆｅｒに加え全量を１００μｌとし、１００℃に１０分間加熱した後、室温に戻し、Ｈｉｓタグ領域をアニーリングさせた。その後、配列番号１３および１６、２種のプライマー各１０ｐｍｏｌ、Ｐｒｉｍｅ　ＳＴＡＲ　ＨＳ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（日本国タカラバイオ株式会社）を加え、伸長反応を７２℃３分間行い、次に、９８℃１０秒、５７℃５秒、および７２℃１分２０秒を１サイクルとして２０サイクル増幅し、最後に７２℃３分間インキュベート後、得られた１３３２ｂｐの増幅断片を０．８％ゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって増幅断片を回収した。
７−１−３．Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターの構築
　実施例７−１−２で回収されたＰＣＲ増幅断片をＮｈｅＩおよびＳａｌＩ（日本国ロシュ・ダイアグノスティックス株式会社）で酵素消化し、０．８％アガロースゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって酵素処理断片を回収した。得られた酵素処理断片を実施例７−１−１で作製されたｐＬＮ１Ｖ５ベクターのＮｈｅＩ・ＳａｌＩサイトに導入し、Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクター（図３）を構築した。
　以下にＮ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体ｃＤＮＡの開始コドンから終止コドンまでのポリヌクレオチド配列（１３０５ｂｐ、配列番号１７）、および該ｃＤＮＡがコードするｍＢＭＰ９のシグナル配列を含んだアミノ酸配列（４３４アミノ酸、配列番号１８）を示す。配列番号１７および１８において、下線部はマウスＢＭＰ９のシグナル配列部分を、囲み線はヒスチジンタグ部分を、イタリックはマウスＢＭＰ９　ｐｒｏ体部分を示す。
配列番号１７
Figure JPOXMLDOC01-appb-I000026
Figure JPOXMLDOC01-appb-I000027
配列番号１８　（ｍＢＭＰ９シグナル配列（下線部分）を含む）
Figure JPOXMLDOC01-appb-I000028
７−２．Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターを用いたＮ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘの一過的発現
７−２−１．遺伝子導入用発現ベクター調製
　実施例７−１−３．で取得されたＮ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターを大腸菌ＤＨ５αに導入し、得られた形質転換体よりＤＮＡをプラスミド精製キット（Ｑｉａｇｅｎ　ｐｌａｓｍｉｄ　Ｍａｘｉ　ｋｉｔ；日本国株式会社キアゲン）を用い調製した。
７−２−２．培養細胞へのベクター導入と分泌発現
　Ｆｒｅｅ　ｓｔｙｌｅ　２９３Ｆ細胞（日本国インビトロジェン株式会社）をＦｒｅｅ　ｓｔｙｌｅ　２９３　Ｅｘｐｒｅｓｓｉｏｎ　Ｍｅｄｉｕｍ（日本国インビトロジェン株式会社）を用い、３７℃、５％ＣＯ２、１２５ｒｐｍ条件下、細胞密度が１ｘ１０５から３ｘ１０６ｃｅｌｌｓ／ｍｌの範囲内で培養した。培地１Ｌを用いて培養した場合、発現ベクター１ｍｇに３５ｍｌのＯｐｔｉ−ＭＥＭ　Ｉ　Ｒｅｄｕｃｅｄ　Ｓｅｒｕｍ　Ｍｅｄｉｕｍ（日本国インビトロジェン株式会社）を加え、１．３ｍｌの２９３　ｆｅｃｔｉｎ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｒｅａｇｅｎｔ（日本国インビトロジェン株式会社）に３３．７ｍｌのＯｐｔｉ−ＭＥＭ　Ｉ　Ｒｅｄｕｃｅｄ　Ｓｅｒｕｍ　Ｍｅｄｉｕｍを加え、それぞれ５分間室温でインキュベートした。インキュベート後この２液を混合し、更に２０−３０分間室温でインキュベートした。その後、１ｘ１０９ｃｅｌｌｓ／ＬのＦｒｅｅ　ｓｔｙｌｅ　２９３Ｆ細胞を含む培地に前記方法で処理された発現ベクターを添加し、３日間培養した。
７−３．Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘの精製・調製
７−３−１．培養上清前処理
　培養後、上清を回収し０．２２μｍフィルター（０．２２μｍ　ＧＰ　Ｅｘｐｒｅｓｓ　Ｍｅｍｂｒａｎｅ　５００ｍｌ；日本国日本ミリポア株式会社）で濾過処理を行った後４℃（低温室）で冷却した。凍結保存した場合には融解後、再度０．２２μｍフィルターで濾過した。
７−３−２．Ｍｅｔａｌ　Ｃｈｅｌａｔｅ　Ａｆｆｉｎｉｔｙクロマトグラフィー
　前処理した１Ｌの培養上清をＰＢＳで平衡化したＮｉ　Ｓｅｐｈａｒｏｓｅカラム（Ｈｉｓ　Ｔｒａｐ　ＨＰ　５ｍｌ；日本国ＧＥヘルスケア　バイオサイエンス株式会社）にアプライした。その後、２５ｍｌのＰＢＳ、更に２Ｍ　ＮａＣｌを含むＰＢＳ　２５ｍｌでカラムを洗浄した。次に、２５ｍｌのＰＢＳ，３０ｍＭ　Ｉｍｉｄａｚｏｌｅを含むＰＢＳ　１０ｍｌ、４０ｍＭ　Ｉｍｉｄａｚｏｌｅを含むＰＢＳ　５０ｍｌの順番でカラムを洗浄した。洗浄操作終了後、カラムに６０ｍＭ　Ｉｍｉｄａｚｏｌｅを含むＰＢＳ　５０ｍｌを添加し目的タンパク質を回収した。上記分離精製操作にはＡＫＴＡｅｘｐｌｏｒｅｒ１０ｓ（日本国ＧＥヘルスケア　バイオサイエンス株式会社）を用いた。使用前にエンドトキシン除去処理を行った。
７−３−３．イオン交換クロマトグラフィー
　ＰＢＳで平衡化された強陰イオン交換カラム（Ｈｉ　Ｔｒａｐ　Ｑ　ＨＰ　１ｍＬ；日本国ＧＥヘルスケア　バイオサイエンス株式会社）に流速：１ｍＬ／ｍｉｎの条件下、実施例７−３−２で得られた目的タンパク質を添加した。ＰＢＳ　１５ｍｌ、８０ｍＭ　ＮａＣｌを含むＰＢＳ　１０ｍｌで順次カラムを洗浄した。洗浄操作終了後、１００ｍＭ　ＮａＣｌを含むＰＢＳ　２０ｍｌ、１４０ｍＭＮａＣｌを含むＰＢＳ　２５ｍｌを順次カラムにアプライし、目的タンパク質を回収した。上記分離精製操作にはＡＫＴＡｅｘｐｌｏｒｅｒ１０ｓ（日本国ＧＥヘルスケア　バイオサイエンス株式会社）を用いた。使用前にエンドトキシン除去処理を行った。
７−３−４．精製標品調製
　実施例７−３−３で得られた精製標品中の溶媒を限外濾過膜ＶＩＶＡＳＰＩＮ２０　１０，０００ＭＷＣＯ　ＰＥＳ（日本国ザルトリウス・ステディム・ジャパン株式会社）を用いてＰＢＳに置換後、サンプルを濃縮した。濃縮操作後、０．２２μｍフィルター（Ｍｉｌｌｅｘ　ＧＶ；日本国日本ミリポア株式会社）により濾過処理を行った。操作は可能な限りクリーンベンチ内で行った。実施例７−３で行われた全ての工程はクリーンベンチでの作業以外、低温室（＋４℃）ないしは氷上で実施した。最終精製品のＳＤＳ−ＰＡＧＥ（ＣＢＢ染色）より、非還元条件下ではｍａｔｕｒｅ２量体、少量のｐｒｏ２量体、ｐｒｏ−ｒｅｇｉｏｎが検出され、還元条件下ではｍａｔｕｒｅ単量体、ｐｒｏ−ｒｅｇｉｏｎ、少量のｐｒｏ単量体が検出された（図４）。この結果から、上記操作によって調製された精製標品にはｐｒｏ−ｒｅｇｉｏｎ２分子とｍａｔｕｒｅ２量体１分子が結合した複合体（ｃｏｍｐｌｅｘ体）が主に含まれていると考えられた。タンパク質濃度はＡ２８０ｎｍを測定し、分子吸光係数（Ｅ１％，１ｃｍ＝９．６）より算出した。
　抗Ｈｉｓ抗体Ｐｅｎｔａ　Ｈｉｓ　ＨＲＰ　Ｃｏｎｊｕｇａｔｅ（日本国株式会社キアゲン）を用いたウェスタンブロッティングによりｐｒｏ−ｒｅｇｉｏｎとｐｒｏ体を、抗ｈＢＭＰ９抗体（米国Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ，Ｉｎｃ．）を用いたウェスタンブロッティングによりｍａｔｕｒｅ体を確認した。 Expression and preparation of N-terminal His-type mBMP9 complex recombinant 7-1. Construction of N-terminal His-type mBMP9 complex recombinant expression vector 7-1-1. Construction of pLN1V5 vector Sense oligo DNA having a BamHI / NheI / SalI site at the 5 ′ end and an XhoI site at the 3 ′ end (V5 tag + Stop codon) and its antisense oligo DNA: V5S and V5AS (Hokkaido System Science Co., Ltd., Japan) Company).
V5S: GATCCGCTAGCGTCGACGGGTAAGCCCTCCTCAACCCTCTCCTGCGTCTCGATCTCGTGGAC (SEQ ID NO: 11)
V5AS: TCGAGTCACGTAGAGATCGAGACCGAGGAGAGGGTTTAGGATAGGGCTTACCGTCGACGCTAGCG (SEQ ID NO: 12)
The synthetic oligo DNA was introduced into the BamHI-XhoI site on the pLN1 vector described in a report by Kakeda et al. (Gene Ther. 12: 852-856, 2005) to construct a pLN1V5 vector.
7-1-2. Synthesis of mBMP9 DNA fragment having His tag sequence at N terminus PCR primer for adding His tag to N terminus of mouse full length sequence of BMP9
Figure JPOXMLDOC01-appb-I000022
(SEQ ID NO: 13)
Figure JPOXMLDOC01-appb-I000023
(SEQ ID NO: 14)
Figure JPOXMLDOC01-appb-I000024
(SEQ ID NO: 15)
Figure JPOXMLDOC01-appb-I000025
(SEQ ID NO: 16)
A reaction solution was prepared according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan), SEQ ID NOS: 13 and 14, 10 pmol of each of two primers in a 50 μl reaction solution, mouse BMP9-cDNA (as a template) SEQ ID NO: 7) was added and incubated at 94 ° C. for 5 minutes, followed by 25 cycles of amplification at 98 ° C. for 10 seconds, 57 ° C. for 5 seconds, and 72 ° C. for 1 minute 20 seconds. The resulting 101 bp amplified fragment was amplified Separated and recovered with 0.8% gel. An amplified fragment (NheI His mBMP9) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
A reaction solution was prepared according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan), SEQ ID NOS: 15 and 16, 10 pmol of each of the two primers in a 50 μl reaction solution, mouse BMP9-cDNA (as a template) SEQ ID NO: 7) was added and incubated at 94 ° C. for 5 minutes, followed by 25 cycles of amplification at 98 ° C. for 10 seconds, 57 ° C. for 5 seconds, and 72 ° C. for 1 minute 20 seconds. The resulting 1249 bp amplified fragment was amplified Separated and recovered with 0.8% gel. The amplified fragment (His mBMP9 SalI) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
The DNA amplified fragments (NheI His mBMP9) and (His mBMP9 SalI) obtained by the above two PCRs were added to PrimeSTAR buffer to a total volume of 100 μl, heated to 100 ° C. for 10 minutes, returned to room temperature, and the His tag region was removed. Annealed. Thereafter, SEQ ID NOs: 13 and 16, 2 ps of each primer, 10 pmol each, Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan) were added, an extension reaction was performed at 72 ° C. for 3 minutes, and then 98 ° C. for 10 seconds, 57 ° C. Amplification was carried out for 20 cycles with 5 seconds and 72 ° C. for 1 minute and 20 seconds, and finally, after incubation at 72 ° C. for 3 minutes, the obtained 1332 bp amplified fragment was separated and recovered on a 0.8% gel. The amplified fragment was recovered from the recovered gel according to the package insert using QIAquick Gel Extraction Kit (Qiagen, Japan).
7-1-3. Construction of N-terminal His-type mBMP9 complex recombinant expression vector The PCR-amplified fragment recovered in Example 7-1-2 was enzymatically digested with NheI and SalI (Roche Diagnostics, Japan). Separated and recovered on an 8% agarose gel. The enzyme-treated fragment was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert. The obtained enzyme-treated fragment was introduced into the NheI / SalI site of the pLN1V5 vector prepared in Example 7-1-1 to construct an N-terminal His-type mBMP9 complex recombinant expression vector (FIG. 3).
The following is a polynucleotide sequence (1305 bp, SEQ ID NO: 17) from the start codon to the stop codon of the N-terminal His-type mBMP9 complex recombinant cDNA, and an amino acid sequence (434 amino acids, including the signal sequence of mBMP9 encoded by the cDNA) SEQ ID NO: 18) is shown. In SEQ ID NOs: 17 and 18, the underlined portion represents the signal sequence portion of mouse BMP9, the boxed portion represents the histidine tag portion, and italic represents the mouse BMP9 pro body portion.
SEQ ID NO: 17
Figure JPOXMLDOC01-appb-I000026
Figure JPOXMLDOC01-appb-I000027
SEQ ID NO: 18 (including mBMP9 signal sequence (underlined portion))
Figure JPOXMLDOC01-appb-I000028
7-2. Transient expression of N-terminal His type mBMP9 complex using N-terminal His type mBMP9 complex recombinant expression vector 7-2-1. Preparation of expression vector for gene transfer Example 7-1-3. The N-terminal His-type mBMP9 complex recombinant expression vector obtained in 1) was introduced into E. coli DH5α, and DNA was prepared from the resulting transformant using a plasmid purification kit (Qiagen plasmid Maxi kit; Qiagen, Japan). .
7-2-2. Vector introduction and secretory expression into cultured cells Free style 293F cells (Invitrogen, Japan) were used with Free style 293 Expression Medium (Invitrogen, Japan) at 37 ° C, 5% CO 2 , 125 rpm, cell density Were cultured in the range of 1 × 10 5 to 3 × 10 6 cells / ml. When cultured using 1 L of medium, 35 ml of Opti-MEM I Reduced Serum Medium (Invitrogen, Japan) was added to 1 mg of the expression vector, and 33 ml of 1.3 ml of 293fectin Transfection Reagent (Invitrogen, Japan) was added. 7 ml of Opti-MEM I Reduced Serum Medium was added and incubated at room temperature for 5 minutes each. After incubation, the two solutions were mixed and incubated for another 20-30 minutes at room temperature. Thereafter, the expression vector treated by the above method was added to a medium containing 1 × 10 9 cells / L Free style 293F cells and cultured for 3 days.
7-3. Purification and preparation of N-terminal His-type mBMP9 complex 7-3-1. Pretreatment of culture supernatant After culture, the supernatant was collected, filtered through a 0.22 μm filter (0.22 μm GP Express Membrane 500 ml; Japan Millipore, Japan), and then cooled at 4 ° C. (cold room). When stored frozen, it was again thawed and filtered through a 0.22 μm filter.
7-3-2. Metal Chelate Affinity Chromatography 1 L of the pretreated culture supernatant was applied to a Ni Sepharose column (His Trap HP 5 ml; GE Healthcare Biosciences, Japan) equilibrated with PBS. Thereafter, the column was washed with 25 ml of PBS and further with 25 ml of PBS containing 2M NaCl. Next, the column was washed in the order of 25 ml of PBS, 10 ml of PBS containing 30 mM imidazole, and 50 ml of PBS containing 40 mM imidazole. After completion of the washing operation, 50 ml of PBS containing 60 mM imidazole was added to the column to recover the target protein. For the separation and purification operation, AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used. Endotoxin removal treatment was performed before use.
7-3-3. Ion Exchange Chromatography Example 7-3-2 under the conditions of a flow rate of 1 mL / min on a strong anion exchange column (Hi Trap Q HP 1 mL; GE Healthcare Biosciences, Japan) equilibrated with PBS. The obtained target protein was added. The column was washed sequentially with 15 ml of PBS and 10 ml of PBS containing 80 mM NaCl. After completion of the washing operation, 20 ml of PBS containing 100 mM NaCl and 25 ml of PBS containing 140 mM NaCl were sequentially applied to the column to recover the target protein. For the separation and purification operation, AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used. Endotoxin removal treatment was performed before use.
7-3-4. Purified sample preparation The solvent in the purified sample obtained in Example 7-3-3 was replaced with PBS using an ultrafiltration membrane VIVASPIN20 10,000 MWCO PES (Sartorius Stedim Japan, Japan). The sample was concentrated. After the concentration operation, filtration was performed with a 0.22 μm filter (Millex GV; Japan Millipore Corporation, Japan). The operation was performed in a clean bench as much as possible. All the processes performed in Example 7-3 were performed in a low temperature room (+ 4 ° C.) or on ice, except for work on a clean bench. SDS-PAGE (CBB staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and a small amount under reducing conditions. Pro monomer was detected (FIG. 4). From this result, it was considered that the purified preparation prepared by the above operation mainly contains a complex (complex body) in which two molecules of pro-region and one molecule of molecule dimer are bound. The protein concentration was measured from A 280 nm and calculated from the molecular extinction coefficient (E 1%, 1 cm = 9.6).
Pro-region and pro-body were confirmed by Western blotting using an anti-His antibody Penta His HRP Conjugate (Qiagen, Japan), and a mature body was confirmed by western blotting using an anti-hBMP9 antibody (US R & D Systems, Inc.). .
Ｎ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘのラット血圧に対する作用調査（インビボ試験）
　日本チャールズ・リバー社より購入の、４週齢の雄性のＳｐｒａｇｕｅ−Ｄａｗｌｅｙ系（Ｃｒｌ：ＣＤ（ＳＤ））２匹に対して１週間の予備飼育を行った後、５週齢にて血圧測定用テレメーター送信器（ＴＡ１１ＰＡ−Ｃ４０；Ｄａｔａ　Ｓｃｉｅｎｃｅｓ）の腹大動脈への埋め込み手術を施した。埋め込み手術は以下のように実施した。腹部正中線を約５ｃｍ開腹し、腹部血管を露出した後、腹大動脈と後大静脈を剥離し、腹大動脈を止血クリップではさんだ。止血クリップより１ｃｍ位下にディスポーザブル注射針（２１Ｇ、テルモ株式会社；針先を約２ｍｍ折り曲げたもの）を用いて腹大動脈にカテーテル挿入口を開け、テレメトリー送信器のカテーテル先端をディスポーザブル注射針の下を這わせるように挿入口に差込んだ。カテーテル挿入口付近の血液を脱脂綿等でふき取り、カテーテルと挿入口にボンド（３Ｍ、ＶｅｔｂｏｎｄＴＭ）を滴下した。ボンドが乾いた後、止血クリップをゆっくりと外し、出血がないことを確認した。腹部器官を腹腔内に戻し、筋層と送信器の固定部位を一緒に縫合した。
　送信器を埋め込み、１１週齢に達した２匹のラットに対して、コントロール溶液としてエンドトキシン含有ＰＢＳを静脈内投与し、テレメトリーシステムＤａｔａｑｕｅｓｔ　Ａ．Ｒ．Ｔ．を用いて、最高血圧、最低血圧、平均血圧及び心拍数を測定し、エンドトキシン含有ＰＢＳ投与により血圧変動がないことを確認した。次に、３日間の休薬期間を設けた後、実施例７の方法にて調整したＮ末Ｈｉｓ型ｍＢＭＰ９　ｃｏｍｐｌｅｘ体を、投与量０．５ｍｇ／ｋｇになるように静脈内投与し、最高血圧、最低血圧、平均血圧及び心拍数を測定した。更に、その３日後、同タンパク質を２．５ｍｇ／ｋｇの用量にて投与し、同様の測定を行った。
　結果、いずれの用量のｍＢＭＰ９　ｃｏｍｐｌｅｘ体投与においても、投与直後から、顕著な血圧（拡張期、収縮期及び平均血圧）上昇が認められ、その上昇は約２４時間継続した（図５）。もう一個体も同様の結果であった。また同時にｍＢＭＰ９　ｃｏｍｐｌｅｘ投与による心拍数の低下も認められた。これら結果より、ＢＭＰ９は昇圧作用を有することが判明し、抗ＢＭＰ９療法は降圧作用を有する可能性が示された。 Investigation of N-terminal His-type mBMP9 complex on rat blood pressure (in vivo study)
Two weeks old male Sprague-Dawley strain (Crl: CD (SD)) purchased from Charles River, Japan was preliminarily raised for 1 week and then used for blood pressure measurement at 5 weeks of age. A telemeter transmitter (TA11PA-C40; Data Sciences) was implanted into the abdominal aorta. Implantation was performed as follows. The abdominal midline was opened about 5 cm, the abdominal blood vessels were exposed, the abdominal aorta and the posterior vena cava were peeled off, and the abdominal aorta was sandwiched with a hemostatic clip. Open a catheter insertion port in the abdominal aorta using a disposable injection needle (21G, Terumo Corporation; needle tip bent about 2 mm) below the hemostatic clip, and place the tip of the telemetry transmitter catheter under the disposable injection needle It was inserted into the insertion slot so that The blood near the catheter insertion port was wiped off with absorbent cotton or the like, and a bond (3M, Vetbond ™ ) was dropped onto the catheter and the insertion port. After the bond had dried, the hemostatic clip was slowly removed to confirm no bleeding. The abdominal organ was returned into the abdominal cavity and the muscle layer and transmitter fixation site were sutured together.
Endotoxin-containing PBS was intravenously administered as a control solution to two rats implanted with a transmitter and reached 11 weeks of age, and the telemetry system Dataquest A. R. T. T. Was used to measure systolic blood pressure, diastolic blood pressure, mean blood pressure and heart rate, and it was confirmed that there was no blood pressure fluctuation by administration of endotoxin-containing PBS. Next, after a drug holiday period of 3 days, the N-terminal His-type mBMP9 complex body prepared by the method of Example 7 was intravenously administered so as to have a dosage of 0.5 mg / kg. Minimum blood pressure, mean blood pressure and heart rate were measured. Further, three days later, the same protein was administered at a dose of 2.5 mg / kg, and the same measurement was performed.
As a result, in any dose of mBMP9 complex body administration, a significant increase in blood pressure (diastolic, systolic and average blood pressure) was observed immediately after administration, and the increase continued for about 24 hours (FIG. 5). Another individual had similar results. At the same time, a decrease in heart rate due to administration of mBMP9 complex was also observed. From these results, it was found that BMP9 has a pressor action and the possibility that anti-BMP9 therapy has a hypotensive action was shown.
ＵＳｈＢＭＰ９　ＫＩキメラマウスの作製及びコントロールキメラマウスの作製
　国際公開第ＷＯ　２００６／７８０７２号パンフレット記載の実施例に従い、ヒトＢＭＰ９−ｃＤＮＡ（開始コドンから終止コドンを含む１２９０ｂｐ　配列番号１９）よりｐＵＳｈＢＭＰ９　ＫＩベクターを作製した。
　以下に、配列番号１９および２０におけるヒトＢＭＰ９シグナル配列、ｐｒｏ−ｒｅｇｉｏｎ、ｍａｔｕｒｅ体部分をそれぞれＧｅｎＢａｎｋアクセッション番号Ｑ９ＵＫ０５，ＡＡＤ５６９６０の情報を元に、下線、囲み線およびイタリック体で示す。
配列番号１９
Figure JPOXMLDOC01-appb-I000029
Figure JPOXMLDOC01-appb-I000030
　以下に、配列番号１９がコードするアミノ酸配列（４２９アミノ酸、配列番号２０）を示す。
配列番号２０
Figure JPOXMLDOC01-appb-I000031
　ｐＵＳｈＢＭＰ９　ＫＩベクターｈＢＭＰ９発現ユニットの開始コドンから終止コドンまでのポリヌクレオチド配列（ヒトＢＭＰ９シグナル配列を、イントロン領域を含んだマウスＩｇκシグナル配列〔下線部分〕に置換し、その下流にヒトＢＭＰ９　ｐｒｏ体配列を含む１５２５ｂｐ、配列番号２１）、および該ｃＤＮＡがコードするアミノ酸配列（４２７アミノ酸、囲み線の部分はマウスＩｇκシグナル配列を示す、配列番号２２）を以下に示す。イントロン領域を含んだマウスＩｇκシグナル配列情報はＧｅｎＢａｎｋより取得したＭＵＳＩＧＫＶＲ１（アクセッション番号Ｋ０２１５９）をもとに、その上流のゲノム配列をＵＣＳＣマウスゲノムデータベースより取得した。
配列番号２１
Figure JPOXMLDOC01-appb-I000032
配列番号２２
Figure JPOXMLDOC01-appb-I000033
　ｐＵＳｈＢＭＰ９　ＫＩベクターを用い、国際公開第ＷＯ　２００６／７８０７２号パンフレットの実施例に従いＢ細胞特異的にヒトＢＭＰ９を発現するＵＳｈＢＭＰ９　ＫＩキメラマウスを作製した。更に下記実施例１０及び１１で用いられたマウスコントロール個体（コントロールキメラマウス）を、国際公開第ＷＯ　２００６／７８０７２号パンフレットの実施例１１記載の方法に従い作製した。 Preparation of UShBMP9 KI chimeric mouse and preparation of control chimeric mouse In accordance with the examples described in WO 2006/78072 pamphlet, a pUShBMP9 KI vector was prepared from human BMP9-cDNA (1290 bp SEQ ID NO: 19 including stop codon from start codon). did.
In the following, the human BMP9 signal sequence, pro-region and nature body parts in SEQ ID NOS: 19 and 20 are shown in underlined, boxed and italicized based on the information of GenBank accession numbers Q9UK05 and AAD56960, respectively.
SEQ ID NO: 19
Figure JPOXMLDOC01-appb-I000029
Figure JPOXMLDOC01-appb-I000030
The amino acid sequence encoded by SEQ ID NO: 19 (429 amino acids, SEQ ID NO: 20) is shown below.
SEQ ID NO: 20
Figure JPOXMLDOC01-appb-I000031
pUShBMP9 KI vector The polynucleotide sequence from the start codon to the stop codon of the hBMP9 expression unit (the human BMP9 signal sequence was replaced with the mouse Igκ signal sequence [underlined portion] containing the intron region, and the human BMP9 pro body sequence was downstream of it. Including 1525 bp, SEQ ID NO: 21), and the amino acid sequence encoded by the cDNA (427 amino acids, the boxed portion indicates the mouse Igκ signal sequence, SEQ ID NO: 22) are shown below. Mouse Igκ signal sequence information including an intron region was obtained from the UCSC mouse genome database based on MUSIGKVR1 (accession number K02159) obtained from GenBank.
SEQ ID NO: 21
Figure JPOXMLDOC01-appb-I000032
SEQ ID NO: 22
Figure JPOXMLDOC01-appb-I000033
Using the pUShBMP9 KI vector, a UShBMP9 KI chimeric mouse that expresses human BMP9 specifically for B cells was prepared according to the example of the pamphlet of International Publication No. WO 2006/78072. Furthermore, mouse control individuals (control chimeric mice) used in Examples 10 and 11 below were prepared according to the method described in Example 11 of International Publication No. WO 2006/78072.
ＵＳｈＢＭＰ９　ＫＩキメラマウスの表現型解析
剖検所見
　上記実施例９で作製されたキメラマウスを用い、３週齢・４週齢・５週齢においてそれぞれ剖検を実施した。ＵＳｈＢＭＰ９　ＫＩキメラマウスにおいてのみ腹水・胸水、リンパ組織、小腸および膵臓に出血が原因と考えられる赤色化が認められた。それぞれの変化を観察した時期およびその個体数を以下に記す。
１０−１．赤色化腹水貯留
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では全個体で腹水貯留は観察されなかったが、４週齢では全ての個体で、５週齢では８個体で赤色化した腹水貯留が認められた。
１０−２．赤色化胸水貯留
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では１個体、４週齢では４個体、５週齢では７個体で赤色化した胸水貯留が認められた。
１０−３．腸間膜リンパ節赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢以降５週齢まで全ての個体において腸間膜リンパ節の赤色化が認められた。
１０−４．顎下リンパ節赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では７個体、以降５週齢まで全ての個体において顎下リンパ節の赤色化が認められた。
１０−５．肘リンパ節赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では７個体、以降５週齢まで全ての個体において肘リンパ節の赤色化が認められた。
１０−６．鼠径リンパ節赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では８個体、以降５週齢まで全ての個体において鼠径リンパ節の赤色化が認められた。
１０−７．膝窩リンパ節赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では８個体、以降５週齢まで全ての個体において膝窩リンパ節の赤色化が認められた。
１０−８．　胸腔内リンパ節赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では２個体、４週齢では３個体、５週齢では３個体で胸腔内リンパ節の赤色化が認められた。
１０−９．小腸赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では７個体、４週齢以降全ての個体で小腸表面の一部に赤色点ないし赤色斑が認められた。
１０−１０．膵臓赤色化
　３週齢では９個体、４週齢では８個体、５週齢では９個体ＵＳｈＢＭＰ９　ＫＩキメラマウスの剖検を実施した。その結果、３週齢では２個体、４週齢では３個体、５週齢では３個体で膵臓表面の一部に赤色化が認められた。
　以上より、ＵＳｈＢＭＰ９ＫＩキメラマウスにおいて観察された出血性変化は上記の実施例５で示されたＵＳｍＢＭＰ９ＫＩキメラマウスにおいて観察された表現型と極めて良く類似していることが明らかになった。 Phenotypic analysis of UShBMP9 KI chimeric mice Necropsy findings Using the chimeric mice prepared in Example 9 above, autopsy was performed at 3 weeks, 4 weeks, and 5 weeks of age, respectively. Only in UShBMP9 KI chimeric mice, ascites / pleural effusion, lymphoid tissue, small intestine and pancreas were reddened due to bleeding. The time when each change was observed and the number of individuals are described below.
10-1. Reddish ascites retention Nine individuals at 3 weeks of age, 8 individuals at 4 weeks of age, 9 individuals at 5 weeks of age, and UShBMP9 KI chimeric mice were necropsied. As a result, ascites retention was not observed in all individuals at 3 weeks of age, but ascites retention was red in all individuals at 4 weeks of age and 8 individuals at 5 weeks of age.
10-2. Red pleural effusion retention Nine individuals at 3 weeks of age, 8 individuals at 4 weeks of age, and 9 individuals at 5 weeks of age were subjected to autopsy of UShBMP9 KI chimeric mice. As a result, 1 individual at 3 weeks of age, 4 individuals at 4 weeks of age, 7 individuals at 5 weeks of age, and red pleural effusion were observed.
10-3. Mesenteric lymph node reddening 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, 9 individuals at 5 weeks of age, UShBMP9 KI chimeric mice were necropsied. As a result, red coloration of mesenteric lymph nodes was observed in all individuals from 3 weeks to 5 weeks of age.
10-4. Submandibular lymph node reddening 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, 9 individuals at 5 weeks of age, UShBMP9 KI chimeric mice were necropsied. As a result, reddening of the submandibular lymph nodes was observed in 7 individuals at 3 weeks of age and in all individuals until 5 weeks of age thereafter.
10-5. Elbow lymph node reddening 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, and 9 individuals at 5 weeks of age were subjected to necropsy of UShBMP9 KI chimeric mice. As a result, redness of elbow lymph nodes was observed in 7 individuals at 3 weeks of age and in all individuals until 5 weeks of age thereafter.
10-6. Reddening of the inguinal lymph nodes 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, and 9 individuals at 5 weeks of age were subjected to autopsy of UShBMP9 KI chimeric mice. As a result, redness of the inguinal lymph nodes was observed in 8 individuals at 3 weeks of age and in all individuals until 5 weeks of age thereafter.
10-7. Population lymph node reddening 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, 9 individuals at 5 weeks of age, UShBMP9 KI chimeric mice were necropsied. As a result, reddening of popliteal lymph nodes was observed in 8 individuals at 3 weeks of age and in all individuals until 5 weeks of age thereafter.
10-8. Intrathoracic lymph node reddening 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, 9 individuals at 5 weeks of age, UShBMP9 KI chimeric mice were necropsied. As a result, 2 individuals at 3 weeks of age, 3 individuals at 4 weeks of age, and 3 individuals at 5 weeks of age, reddening of intrathoracic lymph nodes was observed.
10-9. Redness of small intestine 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, and 9 individuals at 5 weeks of age were subjected to necropsy of UShBMP9 KI chimeric mice. As a result, red spots or red spots were observed on a part of the surface of the small intestine in 7 individuals at 3 weeks of age and in all individuals after 4 weeks of age.
10-10. Pancreatic reddening 9 individuals at 3 weeks of age, 8 individuals at 4 weeks of age, and 9 individuals at 5 weeks of age were subjected to autopsy of UShBMP9 KI chimeric mice. As a result, 2 individuals at 3 weeks of age, 3 individuals at 4 weeks of age, and 3 individuals at 5 weeks of age, redness was observed on a part of the surface of the pancreas.
From the above, it was revealed that the hemorrhagic changes observed in UShBMP9KI chimeric mice were very similar to the phenotypes observed in USmBMP9KI chimeric mice shown in Example 5 above.
ＵＳｈＢＭＰ９　ＫＩキメラマウスにおける血清生化学検査
　上記実施例９で記載された方法に従って作製され、５週齢に達したコントロールキメラマウスおよびＵＳｈＢＭＰ９　ＫＩキメラマウスの後大静脈より全採血した血清を用いて、自動分析装置７１８０（ＨＩＴＡＣＨＩ社製）による血液生化学解析を行った。その結果、ＵＳｈＢＭＰ９　ＫＩキメラマウスの血清ＬＤＨ、ＢＵＮ、ＬＤＬコレステロール、ＧＯＴ、ナトリウムの値は、コントロールキメラマウスに比して有意に高いことが分かった（図６）。また血清アルブミン、血清総タンパクの有意な低下も認められた（図６）。ＢＵＮは腎疾患の診断マーカーであり、慢性腎炎、浮腫、閉塞性尿路疾患と関連があること、ＬＤＬコレステロールは、脂質異常症および動脈硬化性疾患（狭心症、急性冠症候群、心筋梗塞、脳血栓、脳梗塞、ＡＳＯなど）と深く関わること、血清ＬＤＨおよびＧＯＴは、急性肝炎、慢性肝炎、肝臓がん、肝硬変、転移性肝臓がん、急性心筋梗塞、心不全と関連があること、血清ナトリウム上昇は高血圧症と関わること、また、血清アルブミンおよび血清総タンパクの低下は、肝機能障害、ネフローゼ症候群と関連があることが知られており、ＢＭＰ９過剰作用により、上記疾患が惹起されることが示唆された。すなわち、抗ＢＭＰ９療法は、慢性腎炎、浮腫、閉塞性尿路疾患、脂質異常症、動脈硬化性疾患、急性肝炎、慢性肝炎、肝臓がん、肝硬変、転移性肝臓がん、急性心筋梗塞、心不全、ネフローゼ症候群、高血圧症といった疾患に対する有効な治療法となりうる可能性が示された。 Serum biochemical test in UShBMP9 KI chimeric mice Using the serum collected from the posterior vena cava of control chimera mice and UShBMP9 KI chimeric mice, which were prepared according to the method described in Example 9 above, Blood biochemistry analysis was performed with an analyzer 7180 (manufactured by HITACHI). As a result, it was found that the serum LDH, BUN, LDL cholesterol, GOT, and sodium values of the UShBMP9 KI chimeric mice were significantly higher than those of the control chimeric mice (FIG. 6). In addition, a significant decrease in serum albumin and serum total protein was also observed (FIG. 6). BUN is a diagnostic marker for kidney disease and is associated with chronic nephritis, edema, obstructive urinary tract disease, LDL cholesterol is dyslipidemia and arteriosclerotic disease (angina, acute coronary syndrome, myocardial infarction, Serum LDH and GOT are associated with acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, acute myocardial infarction, heart failure, serum sodium It is known that an increase is associated with hypertension, and a decrease in serum albumin and serum total protein is known to be associated with liver dysfunction and nephrotic syndrome. It was suggested. That is, anti-BMP9 therapy is chronic nephritis, edema, obstructive urinary tract disease, dyslipidemia, arteriosclerotic disease, acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, acute myocardial infarction, heart failure It has been shown that it may be an effective treatment for diseases such as nephrotic syndrome and hypertension.
Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体の発現・調製
１２−１．Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターの構築
１２−１−１．Ｎ末端にＨｉｓタグ配列を持つｈＢＭＰ９　ＤＮＡ断片の合成
ヒトＢＭＰ９全長配列のＮ末端にＨｉｓタグを付加するためのＰＣＲプライマー
Figure JPOXMLDOC01-appb-I000034
　（配列番号２３）
Figure JPOXMLDOC01-appb-I000035
　（配列番号２４）
Figure JPOXMLDOC01-appb-I000036
　（配列番号２５）
Figure JPOXMLDOC01-appb-I000037
　（配列番号２６）
　Ｐｒｉｍｅ　ＳＴＡＲ　ＨＳ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（日本国タカラバイオ株式会社）を用い添付文書にしたがって反応液を調製し、５０μｌ反応液中に配列番号２３及び２４、２種のプライマー各１０ｐｍｏｌ、鋳型としてヒトＢＭＰ９−ｃＤＮＡ（配列番号１９）を添加し、９４℃５分保温した後、９８℃１０秒、５７℃５秒、及び７２℃２分を１サイクルとして２５サイクル増幅し、得られた１０１ｂｐの増幅断片を０．８％ゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって増幅断片（ＮｈｅＩ　Ｈｉｓ　ｈＢＭＰ９）を回収した。
　Ｐｒｉｍｅ　ＳＴＡＲ　ＨＳ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（日本国タカラバイオ株式会社）を用い添付文書にしたがって反応液を調製し、５０μｌ反応液中に配列番号２５及び２６、２種のプライマー各１０ｐｍｏｌ、鋳型としてヒトＢＭＰ９−ｃＤＮＡ（配列番号１９）を添加し、９４℃５分保温した後、９８℃１０秒、５７℃５秒、及び７２℃２分を１サイクルとして２５サイクル増幅し、得られた１２５２ｂｐの増幅断片を０．８％ゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって増幅断片（Ｈｉｓ　ｈＢＭＰ９　ＸｈｏＩ）を回収した。
　上記２回のＰＣＲで得られたＤＮＡ増幅断片（ＮｈｅＩ　Ｈｉｓ　ｈＢＭＰ９）及び（Ｈｉｓ　ｈＢＭＰ９　ＸｈｏＩ）をＰｒｉｍｅＳＴＡＲ　ｂｕｆｆｅｒに加え全量を１００μｌとし、１００℃に１０分間加熱した後、室温に戻し、Ｈｉｓタグ領域をアニーリングさせた。その後、配列番号２３及び２６、２種のプライマー各１０ｐｍｏｌ、Ｐｒｉｍｅ　ＳＴＡＲ　ＨＳ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（日本国タカラバイオ株式会社）を加え、伸長反応を７２℃３分間行い、次に、９８℃１０秒、５７℃５秒、及び７２℃３分を１サイクルとして２０サイクル増幅し、最後に７２℃３分間インキュベート後、得られた１３３５ｂｐの増幅断片を０．８％ゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって増幅断片を回収した。
１２−１−２．Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターの構築
　実施例１２−１−１で回収されたＰＣＲ増幅断片をＮｈｅＩ及びＸｈｏＩ（日本国ロシュ・ダイアグノスティックス株式会社）で酵素消化し、０．８％アガロースゲルで分離回収した。回収されたゲルからＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用い添付文書にしたがって酵素処理断片を回収した。得られた酵素処理断片をｐＬＮ１Ｖ５ベクターのＮｈｅＩ・ＸｈｏＩサイトに導入し、Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクター（図７）を構築した。
　以下にＮ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体ｃＤＮＡの開始コドンから終止コドンまでのポリヌクレオチド配列（１３０８ｂｐ、配列番号２７）、及び該ｃＤＮＡがコードするｈＢＭＰ９のシグナル配列を含んだアミノ酸配列（４３５アミノ酸、配列番号２８）を示す。配列番号２７及び２８において、下線部はヒトＢＭＰ９のシグナル配列部分を、囲み線はヒスチジンタグ部分を、イタリック体はヒトＢＭＰ９　ｐｒｏ体部分を示す。
配列番号２７：
Figure JPOXMLDOC01-appb-I000038
Figure JPOXMLDOC01-appb-I000039
配列番号２８（ｈＢＭＰ９シグナル配列〔下線部分〕を含む）：
Figure JPOXMLDOC01-appb-I000040
１２−２．Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターを用いたＮ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘの一過的発現
１２−２−１．遺伝子導入用発現ベクター調製
　実施例１２−１−２．で取得されたＮ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ組換え体発現ベクターを大腸菌ＤＨ５αに導入し、得られた形質転換体よりＤＮＡをプラスミド精製キット（Ｑｉａｇｅｎ　ｐｌａｓｍｉｄ　Ｍａｘｉ　ｋｉｔ；日本国株式会社キアゲン）を用い調製した。
１２−２−２．培養細胞へのベクター導入と分泌発現
　Ｆｒｅｅ　ｓｔｙｌｅ　２９３Ｆ細胞（日本国インビトロジェン株式会社）をＦｒｅｅ　ｓｔｙｌｅ　２９３　Ｅｘｐｒｅｓｓｉｏｎ　Ｍｅｄｉｕｍ（日本国インビトロジェン株式会社）を用い、３７℃、５％ＣＯ２、１２５ｒｐｍ条件下、細胞密度が１ｘ１０５から３ｘ１０６ｃｅｌｌｓ／ｍｌの範囲内で培養する。培地１Ｌを用いて培養した場合、発現ベクター１ｍｇに３５ｍｌのＯｐｔｉ−ＭＥＭ　Ｉ　Ｒｅｄｕｃｅｄ　Ｓｅｒｕｍ　Ｍｅｄｉｕｍ（日本国インビトロジェン株式会社）を加え、１．３ｍｌの２９３ｆｅｃｔｉｎ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｒｅａｇｅｎｔ（日本国インビトロジェン株式会社）に３３．７ｍｌのＯｐｔｉ−ＭＥＭ　Ｉ　Ｒｅｄｕｃｅｄ　Ｓｅｒｕｍ　Ｍｅｄｉｕｍを加え、それぞれ５分間室温でインキュベートした。インキュベート後この２液を混合し、更に２０−３０分間室温でインキュベートした。その後、１ｘ１０９　ｃｅｌｌｓ／ＬのＦｒｅｅ　ｓｔｙｌｅ　２９３Ｆ細胞を含む培地に前記方法で処理された発現ベクターを添加し、３日間培養した。
１２−３．Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘの精製・調製
１２−３−１．培養上清前処理
　培養後、上清を回収し０．２２μｍフィルター（０．２２μｍ　ＧＰ　Ｅｘｐｒｅｓｓ　Ｍｅｍｂｒａｎｅ　５００ｍｌ；日本国日本ミリポア株式会社）で濾過処理を行った後４℃（低温室）で冷却した。凍結保存した場合には融解後、再度０．２２μｍフィルターで濾過した。
１２−３−２．Ｍｅｔａｌ　Ｃｈｅｌａｔｅ　Ａｆｆｉｎｉｔｙクロマトグラフィー
　Ａ　ｂｕｆｆｅｒとしてＰＢＳ（Ｄｕｌｅｃｃｏ’ｓ　Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ；ＳＩＧＭＡ）、Ｂ　ｂｕｆｆｅｒとして０．５Ｍ　Ｉｍｉｄａｚｏｌｅを含むＰＢＳを用いた。
　前処理した１Ｌの培養上清をＰＢＳで平衡化したＮｉ　Ｓｅｐｈａｒｏｓｅカラム（Ｈｉｓ　Ｔｒａｐ　ＨＰ　５ｍｌ；日本国ＧＥヘルスケア　バイオサイエンス株式会社）にアプライした。その後、０％Ｂ　ｂｕｆｆｅｒ　２５ｍｌ、ＰＢＳにＮａＣｌを添加しＮａＣｌ濃度を１．８５Ｍに調製した緩衝液２５ｍｌ、０％Ｂ　ｂｕｆｆｅｒ　２５ｍｌ，９％Ｂ　ｂｕｆｆｅｒ　３０ｍｌ、１１％Ｂ　ｂｕｆｆｅｒ　４０ｍｌの順番でカラムを洗浄した。洗浄操作終了後、カラムに１６％Ｂ　ｂｕｆｆｅｒ　５０ｍｌを添加し目的蛋白質を回収した。上記分離精製操作にはＡＫＴＡｅｘｐｌｏｒｅｒ１０ｓ（日本国ＧＥヘルスケア　バイオサイエンス株式会社）を用いた。使用前にエンドトキシン除去処理を行った。
１２−３−３．イオン交換クロマトグラフィー
　Ａ　ｂｕｆｆｅｒとしてＰＢＳ、Ｂ　ｂｕｆｆｅｒとしてＰＢＳにＮａＣｌを添加しＮａＣｌ濃度を１．８５Ｍに調製した緩衝液を用いた。
　ＰＢＳで平衡化された強陰イオン交換カラム（Ｈｉ　Ｔｒａｐ　Ｑ　ＨＰ　１ｍＬ；日本国ＧＥヘルスケア　バイオサイエンス株式会社）に流速：１ｍＬ／ｍｉｎの条件下、実施例１２−３−２で得られた目的蛋白質を添加した。０％Ｂ　ｂｕｆｆｅｒ　２０ｍｌ、２％Ｂ　ｂｕｆｆｅｒ　１０ｍｌで順次カラムを洗浄した。洗浄操作終了後、７％Ｂ　ｂｕｆｆｅｒ　２０ｍｌをカラムにアプライし、目的蛋白質を回収した。上記分離精製操作にはＡＫＴＡｅｘｐｌｏｒｅｒ１０ｓ（日本国ＧＥヘルスケア　バイオサイエンス株式会社）を用いた。使用前にエンドトキシン除去処理を行った。
１２−３−４．精製標品調製
　実施例１２−３−３で得られた精製標品中の溶媒を限外濾過膜ＶＩＶＡＳＰＩＮ２０　１０，０００ＭＷＣＯ　ＰＥＳ（日本国ザルトリウス・ステディム・ジャパン株式会社）を用いてＰＢＳに置換後、サンプルを濃縮した。濃縮操作後、０．２２μｍフィルター（Ｍｉｌｌｅｘ　ＧＶ；日本国日本ミリポア株式会社）により濾過処理を行った。操作は可能な限りクリーンベンチ内で行った。実施例１２−３で行われた全ての工程はクリーンベンチでの作業以外、４℃（低温室ないしは氷上）で実施した。最終精製品のＳＤＳ−ＰＡＧＥ（銀染色）より、非還元条件下ではｍａｔｕｒｅ２量体、少量のｐｒｏ２量体、ｐｒｏ−ｒｅｇｉｏｎが検出され、還元条件下ではｍａｔｕｒｅ単量体、ｐｒｏ−ｒｅｇｉｏｎ、少量のｐｒｏ単量体が検出された（図８）。この結果から、上記操作によって調製された精製標品にはｐｒｏ−ｒｅｇｉｏｎ２分子とｍａｔｕｒｅ２量体１分子が結合した複合体（ｃｏｍｐｌｅｘ体）が主に含まれていると考えられた。蛋白質濃度はＡ２８０ｎｍを測定し、分子吸光係数（Ｅ１％，１ｃｍ＝９．６）より算出した。
　抗Ｈｉｓ抗体Ｐｅｎｔａ　Ｈｉｓ　ＨＲＰ　Ｃｏｎｊｕｇａｔｅ（日本国株式会社キアゲン）を用いたウェスタンブロッティングによりｐｒｏ−ｒｅｇｉｏｎとｐｒｏ体を、抗ｈＢＭＰ９抗体（米国Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ，Ｉｎｃ．）を用いたウェスタンブロッティングによりｍａｔｕｒｅ体を確認した。 Expression and preparation of N-terminal His-type hBMP9 complex recombinant 12-1. Construction of N-terminal His-type hBMP9 complex recombinant expression vector 12-1-1. Synthesis of hBMP9 DNA fragment having His tag sequence at N-terminus PCR primer for adding His tag to N-terminus of full-length human BMP9 sequence
Figure JPOXMLDOC01-appb-I000034
(SEQ ID NO: 23)
Figure JPOXMLDOC01-appb-I000035
(SEQ ID NO: 24)
Figure JPOXMLDOC01-appb-I000036
(SEQ ID NO: 25)
Figure JPOXMLDOC01-appb-I000037
(SEQ ID NO: 26)
Prepare a reaction solution according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan). In 50 μl reaction solution, SEQ ID NOS: 23 and 24, 10 pmol of each of the two primers, human BMP9-cDNA (as template) SEQ ID NO: 19) was added, and the mixture was incubated at 94 ° C. for 5 minutes, and then amplified for 25 cycles with 98 ° C. for 10 seconds, 57 ° C. for 5 seconds, and 72 ° C. for 2 minutes as one cycle. Separated and recovered with 8% gel. An amplified fragment (NheI His hBMP9) was recovered from the recovered gel using QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
Prepare a reaction solution according to the package insert using Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan). In 50 μl reaction solution, SEQ ID NOs: 25 and 26, 10 pmol of each of the two primers, human BMP9-cDNA (as template) SEQ ID NO: 19) was added and incubated at 94 ° C. for 5 minutes, followed by 25 cycles of amplification at 98 ° C. for 10 seconds, 57 ° C. for 5 seconds, and 72 ° C. for 2 minutes, and the resulting 1252 bp amplified fragment of 0. Separated and recovered with 8% gel. An amplified fragment (His hBMP9 XhoI) was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert.
The DNA amplified fragments (NheI His hBMP9) and (HisBMP9 XhoI) obtained by the above two PCRs were added to PrimeSTAR buffer to a total volume of 100 μl, heated to 100 ° C. for 10 minutes, returned to room temperature, and the His tag region was removed. Annealed. Thereafter, SEQ ID NOs: 23 and 26, 10 pmol each of two kinds of primers, Prime STAR HS DNA Polymerase (Takara Bio Inc., Japan) were added, an extension reaction was performed at 72 ° C. for 3 minutes, and then 98 ° C. for 10 seconds, 57 ° C. Amplification was carried out for 20 cycles with 5 seconds and 72 ° C. for 3 minutes as one cycle, and finally after incubation at 72 ° C. for 3 minutes, the obtained 1335 bp amplified fragment was separated and recovered on a 0.8% gel. The amplified fragment was recovered from the recovered gel according to the package insert using QIAquick Gel Extraction Kit (Qiagen, Japan).
12-1-2. Construction of N-terminal His-type hBMP9 complex recombinant expression vector The PCR-amplified fragment recovered in Example 12-1-1 was enzymatically digested with NheI and XhoI (Roche Diagnostics, Japan). Separated and recovered on an 8% agarose gel. The enzyme-treated fragment was recovered from the recovered gel using a QIAquick Gel Extraction Kit (Qiagen, Japan) according to the package insert. The obtained enzyme-treated fragment was introduced into the NheI / XhoI site of the pLN1V5 vector to construct an N-terminal His-type hBMP9 complex recombinant expression vector (FIG. 7).
The following is a polynucleotide sequence (1308 bp, SEQ ID NO: 27) from the start codon to the stop codon of the N-terminal His-type hBMP9 complex recombinant cDNA, and an amino acid sequence (435 amino acids, including the signal sequence of hBMP9 encoded by the cDNA) SEQ ID NO: 28). In SEQ ID NOs: 27 and 28, the underlined portion indicates the signal sequence portion of human BMP9, the surrounded line indicates the histidine tag portion, and the italic type indicates the human BMP9 pro body portion.
SEQ ID NO: 27
Figure JPOXMLDOC01-appb-I000038
Figure JPOXMLDOC01-appb-I000039
SEQ ID NO: 28 (including hBMP9 signal sequence [ underlined portion ]):
Figure JPOXMLDOC01-appb-I000040
12-2. Transient expression of N-terminal His-type hBMP9 complex using N-terminal His-type hBMP9 complex recombinant expression vector 12-2-1. Preparation of expression vector for gene transfer Example 12-1-2. The N-terminal His-type hBMP9 complex recombinant expression vector obtained in (1) was introduced into Escherichia coli DH5α, and DNA was prepared from the obtained transformant using a plasmid purification kit (Qiagen plasmid Maxi kit; Qiagen, Japan). .
12-2-2. Vector introduction and secretory expression into cultured cells Free style 293F cells (Invitrogen, Japan) were used with Free style 293 Expression Medium (Invitrogen, Japan) at 37 ° C, 5% CO 2 , 125 rpm, cell density Is cultured in the range of 1 × 10 5 to 3 × 10 6 cells / ml. When cultured in 1 L of medium, 35 ml of Opti-MEM I Reduced Serum Medium (Invitrogen, Japan) was added to 1 mg of the expression vector, and 33.7 ml in 1.3 ml of 293fectin Transfection Reagent (Invitrogen, Japan). Of Opti-MEM I Reduced Serum Medium was added and incubated at room temperature for 5 minutes each. After incubation, the two solutions were mixed and incubated for another 20-30 minutes at room temperature. Thereafter, the expression vector treated by the above method was added to a medium containing 1 × 10 9 cells / L Free style 293F cells and cultured for 3 days.
12-3. Purification and preparation of N-terminal His-type hBMP9 complex 12-3-1. Pretreatment of culture supernatant After culture, the supernatant was collected, filtered through a 0.22 μm filter (0.22 μm GP Express Membrane 500 ml; Japan Millipore, Japan), and then cooled at 4 ° C. (cold room). When stored frozen, it was again thawed and filtered through a 0.22 μm filter.
12-3-2. Metal Chelate Affinity Chromatography PBS (Dulecco's Phosphate Buffered Saline; SIGMA) was used as the A buffer, and PBS containing 0.5 M Imidazole was used as the B buffer.
The pretreated 1 L culture supernatant was applied to a Ni Sepharose column (His Trap HP 5 ml; GE Healthcare Biosciences, Japan) equilibrated with PBS. Thereafter, the column was washed in the order of 25 ml of 0% B buffer, 25 ml of buffer solution in which NaCl was added to PBS to adjust the NaCl concentration to 1.85 M, 25 ml of 0% B buffer, 30 ml of 9% B buffer, and 40 ml of 11% B buffer. did. After completion of the washing operation, 50 ml of 16% B buffer was added to the column to recover the target protein. For the separation and purification operation, AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used. Endotoxin removal treatment was performed before use.
12-3-3. Ion Exchange Chromatography A buffer was prepared by adding NaCl to PBS as A buffer and adjusting NaCl concentration to 1.85 M by adding NaCl to PBS as B buffer.
The purpose obtained in Example 12-3-2 on a strong anion exchange column equilibrated with PBS (Hi Trap Q HP 1 mL; GE Healthcare Biosciences, Japan) under a flow rate of 1 mL / min Protein was added. The column was washed sequentially with 20 ml of 0% B buffer and 10 ml of 2% B buffer. After the washing operation, 20 ml of 7% B buffer was applied to the column, and the target protein was recovered. For the separation and purification operation, AKTA explorer 10s (GE Healthcare Bioscience, Japan) was used. Endotoxin removal treatment was performed before use.
12-3-4. Purified sample preparation After replacing the solvent in the purified sample obtained in Example 12-3-3 with PBS using an ultrafiltration membrane VIVASPIN20 10,000 MWCO PES (Sartorius Stedim Japan, Japan), The sample was concentrated. After the concentration operation, filtration was performed with a 0.22 μm filter (Millex GV; Japan Millipore Corporation, Japan). The operation was performed in a clean bench as much as possible. All steps performed in Example 12-3 were performed at 4 ° C. (cold room or on ice) except for work on a clean bench. SDS-PAGE (silver staining) of the final purified product detects a nature dimer, a small amount of pro-dimer, and a pro-region under non-reducing conditions, and a monomer monomer, pro-region, and a small amount under reducing conditions. Pro monomer was detected (FIG. 8). From this result, it was considered that the purified preparation prepared by the above operation mainly contains a complex (complex body) in which two molecules of pro-region and one molecule of molecule dimer are bound. The protein concentration was measured from A 280 nm and calculated from the molecular extinction coefficient (E 1%, 1 cm = 9.6).
Pro-region and pro-body were confirmed by Western blotting using an anti-His antibody Penta His HRP Conjugate (Qiagen, Japan), and a mature body was confirmed by western blotting using an anti-hBMP9 antibody (US R & D Systems, Inc.). .
インビトロでのＮ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘの活性測定（インビトロ試験）
　ＢＭＰ９の活性評価は、実施例２に記載の方法に従って実施した。結果、精製したｈＢＭＰ９　ｃｏｍｐｌｅｘ体は、用量依存的にＬｕｃｉｆｅｒａｓｅ活性を上昇させ、ＢＭＰリガンド活性を有していることが確認された。 In vitro N-terminal His-type hBMP9 complex activity measurement (in vitro test)
BMP9 activity was evaluated according to the method described in Example 2. As a result, the purified hBMP9 complex body increased Luciferase activity in a dose-dependent manner, and was confirmed to have BMP ligand activity.
Ｎ末Ｈｉｓ型ｈＢＭＰ９　ｃｏｍｐｌｅｘ体のマウスへの投与試験（インビボ試験）
　４週齢の雌性のＢＡＬＢ／ｃＢｙＪＪｃ１マウスを３０匹日本クレア株式会社より購入し、ＣＥ−２飼料（日本クレア）にて２週間馴化した後、体重を指標に選抜したマウス１８匹を計６群（ｎ＝２~４）に群分けした。なお、群分け日をＤａｙ１とした。それぞれの群構成は以下の通り。
▲１▼群：１匹あたり３６０μｇのｈＢＭＰ９　ｃｏｍｐｌｅｘ体を投与した３６０μｇ／ｈｅａｄ投与群（ｎ＝３）
▲２▼群；▲１▼群のコントロール群（３６０μｇのｈＢＭＰ９　ｃｏｍｐｌｅｘ体投与液内に含まれるエンドトキシンを同量含むＰＢＳ溶液を投与した群）（ｎ＝２）
▲３▼群：１匹あたり１００μｇのｈＢＭＰ９　ｃｏｍｐｌｅｘ体を投与した１００μｇ／ｈｅａｄ投与群（ｎ＝３）、
▲４▼群：１匹あたり３０μｇのｈＢＭＰ９　ｃｏｍｐｌｅｘ体を投与した３０μｇ／ｈｅａｄ投与群（ｎ＝３）、
▲５▼群：▲３▼群のコントロール群（１００μｇのｈＢＭＰ９　ｃｏｍｐｌｅｘ体投与液内に含まれるエンドトキシンを同量含むＰＢＳ溶液を投与した群）（ｎ＝３）
▲６▼群：無処置群（ｎ＝４）
▲１▼、▲２▼群の２群については、各投与液をＤａｙ１，Ｄａｙ３にて尾静脈内投与し、Ｄａｙ４にて、後大静脈より全採血し、血清として調整した。
▲３▼−▲６▼群については、各投与液をＤａｙ１，Ｄａｙ３，Ｄａｙ５，Ｄａｙ８にて尾静脈内投与し、Ｄａｙ９にて、後大静脈より全採血し、血清として調整した。
　解析は、得られた血清を用いて自動分析装置７１８０（ＨＩＴＡＣＨＩ社製）にて血液生化学解析を行うとともに、日本チャールス・リバー株式会社に委託し、バイオマーカー調査を実施した。なお、バイオマーカー調査では、ＲＯＤＥＮＴＳ　ＭＵＬＴＩ−ＡＮＡＬＹＴＥ　ＰＲＯＦＩＬＥＳ（Ｒｏｄｅｎｔ　ＭＡＰ　Ｖ２．０　Ｐｌａｓｍａ　Ａｎｔｉｇｅｎ）を採用した。
　その結果、血液生化学解析では、１００μｇ／ｈｅａｄ投与群▲３▼群において、ＢＭＰ９投与によるＬＤＬコレステロール上昇、膵リパーゼ（ＬＩＰ）低下、血清アルブミン低下が認められ、いずれの変動もコントールの▲５▼群に比して有意なものであった（図９）。変動が見られたＬＤＬコレステロール上昇については脂質異常症および動脈硬化症との関連が、血清アルブミン低下については、ネフローゼ症候群、重症肝疾患、栄養失調、タンパク漏出性胃腸症、炎症と関連が、膵リパーゼ（ＬＩＰ）の低下では、慢性膵炎との関連が報告されており、ＢＭＰ９過剰作用は、脂質異常症、動脈硬化性疾患、ネフローゼ症候群、重症肝疾患、栄養失調、タンパク漏出性胃腸症、炎症、慢性膵炎といった疾患を惹起させうる可能性が示唆された。
　すなわち、抗ＢＭＰ９療法は、脂質異常症、動脈硬化性疾患、ネフローゼ症候群、重症肝疾患、栄養失調、タンパク漏出性胃腸症、炎症、慢性膵炎といった疾患の治療法になりうる可能性が示された。
　また、バイオマーカー調査からは、ＢＭＰ９投与により、ＣＤ４０リガンド、ＣＲＰ，Ｃｙｓｔａｔｉｎ−Ｃ、Ｅｎｄｏｔｈｅｌｉｎ−１、Ｆａｃｔｏｒ　ＶＩＩ、Ｈａｐｔｏｇｌｏｂｉｎ、ＩＬ−１０、ＩＬ−１８、ＩＬ−１ａｌｐｈａ、ＩＬ−５、ＭＣＰ−１、ＭＣＰ−３、ＭＣＰ−５、ＭＩＰ−１ｂｅｔａ、ＭＩＰ−１ｇａｍｍａ、ＭＩＰ−２、ＭＭＰ−９、ＭＰＯ、Ｏｓｔｅｏｐｏｎｔｉｎ、ＳＡＰ、ＴＩＭＰ−１、ＶＥＧＦ、ｖＷＦ（ｖｏｎ　Ｗｉｌｌｅｂｒａｎｄ　Ｆａｃｔｏｒ）が上昇、もしくは上昇傾向を示すことが明らかになった（図１０）。
　前記のバイオマーカーは以下のような疾患との関連が知られている。
Figure JPOXMLDOC01-appb-I000041
Figure JPOXMLDOC01-appb-I000042
　よって、抗ＢＭＰ９療法は、血栓症、動脈硬化性疾患（急性冠症候群、心筋梗塞、脳梗塞、閉塞性動脈硬化症（ＡＳＯ）など）、各種腎疾患（慢性腎疾患、急性腎障害、糸球体腎炎など）、高血圧症、血管障害性疾患（全身性エリテマトーデス（ＳＬＥ）などの自己免疫疾患、川崎病、血管炎症候群、敗血症など）、アレルギー性疾患（アレルギー性鼻炎、喘息、アトピー性皮膚炎など）、各種炎症性疾患（遅延型アレルギー、関節リウマチ、関節炎、肺疾患、肝炎、急性好中球性炎症、など）、心疾患（高血圧性心肥大、心筋症、ウイルス性心筋症などの慢性心不全）、がん、アルツハイマー、多発性硬化症、糖尿病性網膜症にみられる異常血管新生、レイノー症候群、クローン病に対する治療法となりうる可能性が示された。 N-terminal His-type hBMP9 complex administration test to mice (in vivo test)
Thirty four-week-old female BALB / cByJJc1 mice were purchased from Nippon Claire Co., Ltd., and acclimatized with CE-2 feed (CLEA Japan) for 2 weeks. They were divided into groups (n = 2 to 4). The grouping date was Day1. Each group composition is as follows.
(1) Group: 360 μg / head administration group (n = 3) administered with 360 μg of hBMP9 complex body per animal
(2) group; (1) group control group (group administered with a PBS solution containing the same amount of endotoxin contained in 360 μg of hBMP9 complex body administration solution) (n = 2)
(3) Group: 100 μg / head administration group (n = 3) in which 100 μg of hBMP9 complex was administered per animal,
(4) Group: 30 μg / head administration group (n = 3) in which 30 μg of hBMP9 complex was administered per animal,
(5) Group: (3) Control group (group in which PBS solution containing the same amount of endotoxin contained in 100 μg of hBMP9 complex body administration solution was administered) (n = 3)
(6) Group: no treatment group (n = 4)
For the two groups (1) and (2), each administration solution was administered into the tail vein via Day 1 and Day 3, and whole blood was collected from the posterior vena cava at Day 4 and prepared as serum.
For the groups (3) to (6), each administration solution was administered into the tail vein at Day 1, Day 3, Day 5, and Day 8, and whole blood was collected from the posterior vena cava at Day 9 and prepared as serum.
In the analysis, blood biochemistry analysis was performed using the obtained serum with an automatic analyzer 7180 (manufactured by HITACHI), and a biomarker survey was conducted by entrusting it to Charles River Japan Co., Ltd. In the biomarker survey, RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen) was employed.
As a result, blood biochemical analysis revealed that LMP cholesterol increased, pancreatic lipase (LIP) decreased, and serum albumin decreased by BMP9 administration in the 100 μg / head administered group (3), and all of these fluctuations were controlled by (5) It was significant compared to the group (FIG. 9). Increased LDL cholesterol was associated with dyslipidemia and arteriosclerosis, and decreased serum albumin was associated with nephrotic syndrome, severe liver disease, malnutrition, protein-losing gastroenteropathy, and inflammation. Reduced lipase (LIP) has been reported to be associated with chronic pancreatitis, and BMP9 overactivity is caused by dyslipidemia, arteriosclerotic disease, nephrotic syndrome, severe liver disease, malnutrition, protein-losing gastroenteropathy, inflammation This suggests the possibility of causing diseases such as chronic pancreatitis.
That is, it was shown that anti-BMP9 therapy may be a therapeutic method for diseases such as dyslipidemia, arteriosclerotic disease, nephrotic syndrome, severe liver disease, malnutrition, protein-losing gastroenteropathy, inflammation, and chronic pancreatitis. .
Further, from the biomarker survey, CD40 ligand, CRP, Cystatin-C, Endothelin-1, Factor VII, Haptoglobin, IL-10, IL-18, IL-1alpha, IL-5, MCP-1, MCP-3, MCP-5, MIP-1beta, MIP-1gamma, MIP-2, MMP-9, MPO, Osteopontin, SAP, TIMP-1, VEGF, vWF (von Willebrand Factor) show an upward trend or an upward trend It became clear (FIG. 10).
The biomarkers are known to be associated with the following diseases.
Figure JPOXMLDOC01-appb-I000041
Figure JPOXMLDOC01-appb-I000042
Therefore, anti-BMP9 therapy includes thrombosis, arteriosclerotic disease (acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.), various renal diseases (chronic kidney disease, acute kidney injury, glomerulus). Nephritis), hypertension, vascular disorder diseases (autoimmune diseases such as systemic lupus erythematosus (SLE), Kawasaki disease, vasculitis syndrome, sepsis, etc.), allergic diseases (allergic rhinitis, asthma, atopic dermatitis, etc.) ), Various inflammatory diseases (delayed allergy, rheumatoid arthritis, arthritis, lung disease, hepatitis, acute neutrophilic inflammation, etc.), heart diseases (hypertensive heart hypertrophy, cardiomyopathy, viral cardiomyopathy, etc.) ), Potential for treatment of cancer, Alzheimer's, multiple sclerosis, abnormal angiogenesis in Retinopathy of diabetics, Raynaud's syndrome, and Crohn's disease.
抗ＢＭＰ９中和抗体の正常マウスへの投与試験（インビボ試験）
　５週齢の雌性のＢＡＬＢ／ｃＢｙＪＪｃ１マウスを日本クレア株式会社より購入し、ＣＥ−２飼料（日本クレア株式会社）にて１週間馴化した後、体重を指標に汎用群分けソフト（株式会社ヴィジョンズ）を用いて、２群（Ａ、Ｂ；各５匹ずつ）に群分けした。群分けした日をＤａｙ１として、Ａ群にはＰＢＳを、Ｂ群にはＰＢＳにて希釈した抗ヒトＢＭＰ９モノクローナル抗体（Ｒ＆Ｄ　Ｓｙｓｔｅｍ，Ｉｎｃ、Ｃｌｏｎｅ　ＮＯ．：３６０１０７）を、２．５ｍｇ／ｋｇの投与量になるよう、腹腔内に投与した。なお、投与はＤａｙ１を含め、Ｄａｙ３，Ｄａｙ５，Ｄａｙ８，Ｄａｙ１０，Ｄａｙ１３の計６回行った。Ｄａｙ１５にて眼窩採血血液に対してＢａｙｅｒ　ＡＤＶＩＡ（登録商標）１２０　Ｈｅｍａｔｏｌｏｇｙ　Ｓｙｓｔｅｍ　Ａｎａｌｙｚｅｒを用いて血液学的検査を行うとともに、後大静脈より採血した血清を用いてバイオマーカー調査を実施した。なお、バイオマーカー調査（ＲＯＤＥＮＴＳ　ＭＵＬＴＩ−ＡＮＡＬＹＴＥ　ＰＲＯＦＩＬＥＳ（Ｒｏｄｅｎｔ　ＭＡＰ　Ｖ２．０　Ｐｌａｓｍａ　Ａｎｔｉｇｅｎ））は、日本チャールズ・リバー株式会社に委託し、実施した。
　その結果、血液学的検査ではＢＭＰ９中和抗体の投与により、血栓形成、炎症と関連のある血小板の数が約２５％有意に低下することが明らかになった（図１１）。
　また、バイオマーカー調査では、ＢＭＰ９中和抗体投与により、ＣＤ４０リガンド、ＣＲＰ、ＥＧＦ，Ｅｎｄｏｔｈｅｌｉｎ−１，Ｆａｃｔｏｒ　ＶＩＩ，Ｈａｐｔｏｇｌｏｂｉｎ，ＩＬ−１ａｌｐｈａ，ＩＬ−５，ＭＣＰ−１，ＭＣＰ−３，ＭＣＰ−５，ＭＩＰ−１ｂｅｔａ，ＭＩＰ−１ｇａｍｍａ，ＭＭＰ−９，Ｔｉｓｓｕｅ　Ｆａｃｔｏｒ，ＴＮＦ−ａ，ＴＩＭＰ−１，ＶＥＧＦ，ＲＡＮＴＥＳ，Ｌｙｍｐｈｏｔａｃｔｉｎ，Ｅｏｔａｘｉｎ，ＧＣＰ−２（Ｇｒａｎｕｌｏｃｙｔｅ　Ｃｈｅｍｏｔａｃｔｉｃ　Ｐｒｏｔｅｉｎ−２）といったバイオマーカーが低下傾向、もしくは有意に低下することが分かった（図１２）。また実施例１４で示された、ＢＭＰ９組換体投与により上昇することが確認されたバイオマーカーの大半は、ＢＭＰ９中和抗体を用いた抗ＢＭＰ９療法により、反対に低下することも確認された。
　前記のバイオマーカーは以下のような疾患との関連が知られている。
Figure JPOXMLDOC01-appb-I000043
Figure JPOXMLDOC01-appb-I000044
　よって、抗ＢＭＰ９療法は、各種血栓症、動脈硬化性疾患（急性冠症候群、心筋梗塞、脳梗塞、閉塞性動脈硬化症（ＡＳＯ）など）、各種腎疾患（慢性腎疾患、急性腎障害、糸球体腎炎など）、高血圧症、血管障害性疾患（全身性エリテマトーデス（ＳＬＥ）などの自己免疫疾患、川崎病、血管炎症候群、敗血症）、アレルギー性疾患（アレルギー性鼻炎、気道過敏症、喘息、アトピー性皮膚炎など）、各種炎症性疾患（遅延型アレルギー、関節リウマチ、関節症、肺疾患、肝炎、急性好中球性炎症、潰瘍性大腸炎など）、心疾患（高血圧性心肥大、心筋症、ウイルス性心筋症などの慢性心不全）、がん、糖尿病性網膜症、レイノー症候群、クローン病に対する治療法として有用である可能性が示された。 Test for administration of anti-BMP9 neutralizing antibody to normal mice (in vivo test)
After purchasing 5-week-old female BALB / cByJJc1 mice from Clea Japan Co., Ltd. and acclimatizing with CE-2 feed (Clea Japan Co., Ltd.) for 1 week, general-purpose grouping software (Visions Co., Ltd.) using body weight as an index Were grouped into 2 groups (A, B; 5 each). The day of grouping was set to Day 1, and the group A was administered PBS and the group B was administered with an anti-human BMP9 monoclonal antibody (R & D System, Inc, Clone NO .: 360107) diluted with PBS at a dose of 2.5 mg / kg. The dose was administered intraperitoneally to achieve an amount. In addition, administration was carried out 6 times in total including Day1, Day3, Day5, Day8, Day10, and Day13. At day 15, the blood collected from the orbit was subjected to hematological examination using Bayer ADVIA (registered trademark) 120 Hematology System Analyzer and biomarker investigation was performed using serum collected from the posterior vena cava. In addition, the biomarker survey (RODENTS MULTI-ANALYTE PROFILES (Rodent MAP V2.0 Plasma Antigen)) was commissioned to Charles River Japan Co., Ltd. and conducted.
As a result, hematological examination revealed that the administration of BMP9 neutralizing antibody significantly decreased the number of platelets related to thrombus formation and inflammation by about 25% (FIG. 11).
In the biomarker survey, CD40 ligand, CRP, EGF, Endothelin-1, Factor VII, Haptoglobin, IL-1alpha, IL-5, MCP-1, MCP-3, MCP-5, and BMP9 neutralizing antibody were administered. Biomarkers such as MIP-1 beta, MIP-1 gamma, MMP-9, Tissue Factor, TNF-a, TIMP-1, VEGF, RANTES, Lymphoactin, Eotaxin, GCP-2 (Granulocyte Chemical Protein-2) (Fig. 12). It was also confirmed that most of the biomarkers shown in Example 14 that were confirmed to be increased by administration of recombinant BMP9 were decreased by anti-BMP9 therapy using a BMP9 neutralizing antibody.
The biomarkers are known to be associated with the following diseases.
Figure JPOXMLDOC01-appb-I000043
Figure JPOXMLDOC01-appb-I000044
Therefore, anti-BMP9 therapy includes various thrombosis, arteriosclerotic diseases (acute coronary syndrome, myocardial infarction, cerebral infarction, obstructive arteriosclerosis (ASO), etc.), various renal diseases (chronic kidney disease, acute kidney injury, thread) Sphere nephritis), hypertension, vascular disorder diseases (autoimmune diseases such as systemic lupus erythematosus (SLE), Kawasaki disease, vasculitis syndrome, sepsis), allergic diseases (allergic rhinitis, airway hypersensitivity, asthma, atopy) Dermatitis, etc.), various inflammatory diseases (delayed allergy, rheumatoid arthritis, arthropathy, lung disease, hepatitis, acute neutrophilic inflammation, ulcerative colitis, etc.), heart disease (hypertensive cardiac hypertrophy, cardiomyopathy) , Chronic heart failure such as viral cardiomyopathy), cancer, diabetic retinopathy, Raynaud's syndrome, and the possibility of being useful as a treatment for Crohn's disease.
ｈｓＡＬＫ１ｍＦｃ組み換えタンパク質の取得・調製
１６−１．ｈｓＡＬＫ１ｍＦｃ発現ベクターの作製
　ｈｓＡＬＫ１ｍＦｃ発現ベクターはヒトＡＬＫ１の細胞外領域ｃＤＮＡ断片、およびｍＩｇＧ１のＦｃ領域ｃＤＮＡ断片を、ＥｃｏＲＩ、ＮｏｔＩ（ともに日本国ロシュ・ダイアグノスティックス株式会社）で酵素消化したＰＥＡＫ８発現ベクター（Ｅｄｇｅ　Ｂｉｏｓｙｓｔｅｍｓ社）に組み込むことにより作製した。
組み込まれたｈｓＡＬＫ１ｍＦｃのｃＤＮＡ配列は下記の通り（配列番号３５）。
Figure JPOXMLDOC01-appb-I000045
　以下に、配列番号３５がコードするアミノ酸配列（３４６アミノ酸、配列番号３６）を示す。
配列番号３６：
Figure JPOXMLDOC01-appb-I000046
　それぞれの断片は以下のようにして取得した。ヒトＡＬＫ１の細胞外領域ｃＤＮＡは、
ｈｓＡＬＫ１　ＦＷ：ａｇａａｔｔｃｃｃａｃｃａｔｇａｃｃｔｔｇｔｃｃｃｃｃａｇ（配列番号３）、
ｈｓＡＬＫ１　ＲＶ：ａａｃｔａｇｔｃｔｇｇｃｃａｔｃｔｇｔｔｃｃｃｇｇｃｔｇ（配列番号４）
を用いて、ヒト肺ｃＤＮＡライブラリーよりＰＣＲ法を用いてクローニングした。得られた断片をＥｃｏＲＩ、ＳｐｅＩで酵素消化した後、アガロースゲルにて電気泳動し、当該バンドを切り出した。アガロースからのＤＮＡの抽出にはＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用いた。
　一方、ｍＩｇＧ１のＦｃ領域ｃＤＮＡは、
ｍＩｇＧ１　ＦＰ：ａｃｔａｇｔｃｃｔａａｇｇｔｃａｃｇｔｇｔｇｔｔｇ（配列番号３７）
ｍＩｇＧ１　ＲＰ：ｇｃｇｇｃｃｇｃｔｔａｔｃａｔｔｔａｃｃａｇｇａｇａｇｔｇｇｇ（配列番号３８）
を用いてＰＣＲ法にてクローニングした。得られた断片をＳｐｅＩ、ＮｏｔＩ（ともに日本国ロシュ・ダイアグノスティックス株式会社）で酵素消化した後、アガロースゲルにて電気泳動し、当該バンドを切り出した。アガロースからのＤＮＡの抽出にはＱＩＡｑｕｉｃｋ　Ｇｅｌ　Ｅｘｔｒａｃｔｉｏｎ　Ｋｉｔ（日本国株式会社キアゲン）を用いた。
１６−２．ｈｓＡＬＫ１ｍＦｃ高発現株の作製・培養上清取得
　ｈｓＡＬＫ１ｍＦｃ高発現株の作製は、上記で作成した発現ベクターをＳｆｉ−Ｉ制限酵素を用いて線状化した後、その線状化したＤＮＡをＬｉｐｏｆｅｃｔａｍｉｎｅＴＭ　ＬＴＸ試薬（日本国インビトロジェン株式会社）を用いて添付文書に従い、ＣＨＯ　Ｒａｓ　ｃｌｏｎｅＩ細胞に遺伝子導入した。遺伝子導入２４時間後、６μｇ／ｍｌのｐｕｒｏｍｙｃｉｎｅおよび１０％血清（ＦＣＳ）を含むａｌｐｈａ　ＭＥＭを用いて遺伝子導入細胞を薬剤選抜した後、限外希釈法にて、シングルクローンの単離を行った。高発現株の選別には、ｍｏｕｓｅ　ＩｇＧ１　ＥＬＩＳＡ　ｋｉｔ（日本国コスモバイオ株式会社より購入）を用いた。
　得られた高発現株はスケールアップした後、ローラーボトルに播種し、３７℃にてコンフルエントになるまで培養した。コンフルエントになったことを確認後、ＰＢＳ（Ｄｕｌｅｃｃｏ’ｓ　Ｐｈｏｓｐｈａｔｅ　Ｂｕｆｆｅｒｅｄ　Ｓａｌｉｎｅ；ＳＩＧＭＡ）を用いて洗浄後、ＥＸ−Ｃｅｌｌ３０２（ＳＡＦＣ　Ｂｉｏｓｃｉｅｎｃｅｓ社）培地をＤＭＥＭ／Ｆ−１２混合培地にて２５％に希釈した培地に置換した。５~７日間培養した後、培養上清を回収し、０．２２μｍフィルター（０．２２μｍ　ＧＰ　Ｅｘｐｒｅｓｓ　Ｍｅｍｂｒａｎｅ　５００ｍｌ；日本国日本ミリポア株式会社）で濾過処理を行った後、４℃（低温室）で冷却した。凍結保存した場合には融解後０．２２μｍフィルターで再濾過した。
１６−３．ｈｓＡＬＫ１ｍＦｃ産生株培養上清の濃縮、調製
　大量取得したｈｓＡＬＫ１ｍＦｃの培養上清を、Ｐｅｌｌｉｃｏｎ　３カセット　ウルトラセルＰＬＣＧＣメンブレン装着、０．１１ｍ２（ミリポア製）を用いて濃縮し、適当な用量になったところで濃縮液を回収した。その後、流路をＰＢＳで共洗いした後、その共洗い液と先の濃縮液とを混ぜ合わせた。混ぜ合わせた濃縮液には、４Ｍ　ＮａＣｌを含む１Ｍ　Ｇｌｙｃｉｎｅ（ｐＨ８．５）溶液を等量加え、０．２２μｍフィルターでろ過した。
　ＡＫＴＡ　Ｐｒｉｍｅ（ＧＥヘルスケア・ジャパン社）に、約５０ｍｌのＰｒｏＳｅｐ　Ｕｌｔｒａ　Ｐｌｕｓ（ミリポア製）を充填したヴァンテージ−Ｌラボ用カラム（ミリポア社）を接続させた後、２Ｍ　ＮａＣｌを含む１Ｍ　Ｇｌｙｃｉｎｅ−ＮａＯＨ（ｐＨ８．５）溶液にてカラムを平衡化した。平衡化後、フィルターろ過した培養上清濃縮液をＰｒｏＳｅｐカラムにアプライした。アプライ後、２Ｍ　ＮａＣｌを含む１Ｍ　Ｇｌｙｃｉｎｅ−ＮａＯＨ（ｐＨ８．５）溶液にて洗浄し、ろ液の吸光度（ＯＤ２８０ｎｍ）の値がベースラインに戻ったことを確認後、更に１カラムボリューム分（５０ｍＬ）の同液にてカラムを洗浄した。洗浄後、５カラム容量の０．１Ｍクエン酸ナトリウム（ｐＨ５．０）溶液を用いてｈｓＡＬＫ１ｍＦｃタンパク質を溶出させた。溶出液の回収は、１ｍＬの０．２Ｍ　ＭＥＳ（ｐＨ６．７）溶液を予め加えておいたチューブに１０ｍＬずつで行った。
　次に、ＡＫＴＡ　ＥｘｐｌｏｒｅｒにＲＥＳＯＵＲＣＥ　Ｓ　６ｍｌ（ＧＥヘルスケア製）のカラムを接続し、Ａ１溶液に２０ｍＭ　ＭＥＳ（ｐＨ５．８）を、Ｂ１溶液に１Ｍ　ＮａＣｌを含む２０ｍＭ　ＭＥＳ（ｐＨ５．８）を用いて、カラムの平衡化を行った後、Ａ１溶液にて４−１０倍量に希釈したＰｒｏＳｅｐ溶出液を０．２２μｍフィルターでろ過後、カラムにアプライした。
　アプライ後、Ａ１を送液し、ＯＤ２８０ｎｍがベースラインに戻ったことを確認後、溶出プログラムを開始させた。溶出は、Ｂ１濃度０％で２カラム分洗浄した後、３５カラム分でＢ１濃度を０％→３０％に徐々に増加させ、溶出させた。溶出液の回収は６ｍＬ／ｔｕｂｅで行った。各フラクションを非還元条件下にてＳＤＳ−ＰＡＧＥした後、銀染色を行い、高分子ダイマーの含量が多く、かつ低分子ダイマーの含量が低いフラクションを選別し、選別液を混ぜ合わせた。
　次に１Ｍの（ＮＨ４）２ＳＯ４を含む０．１Ｍリン酸ナトリウム（ｐＨ７．２）にて平衡化したＲＥＳＯＵＲＣＥ　ＰＨＥ（ＧＥヘルスケア製）カラムに、上記のＲＥＳＯＵＲＣＥ　Ｓ溶出液混合液を２Ｍ（ＮＨ４）２ＳＯ４を含む０．１Ｍリン酸ナトリウムｐＨ７．２溶液にて２倍希釈しフィルターろ過したものを、アプライした。Ａ２溶液には、２Ｍ（ＮＨ４）２ＳＯ４を含む０．１Ｍリン酸ナトリウム（ｐＨ７．２）、Ｂ２溶液には、０．１Ｍリン酸ナトリウム（ｐＨ７．２）を用いた。アプライ後、Ｂ２濃度５０％の溶液にて洗浄後、２０カラム分にてＢ２濃度を５０→９０％に増加させ、溶出させた。各溶出フラクションを非還元条件下にてＳＤＳ−ＰＡＧＥした後、銀染色を行い、ダイマーのみを含むフラクションを選別し、混ぜ合わせた。
　次に希釈フラクションの濃縮を目的として、同カラム（ＲＥＳＯＵＲＣＥ　ＰＨＥ）に３ｒｄクロマトのフラクションの混和物中の硫安濃度を電気伝導度から推定し、硫安の終濃度が１Ｍを超えるように、混和物にＡ２を加えてから、カラムにアプライした。試料アプライ後、硫安濃度を１００~２００ｍＭまで一気に下げてステップワイズ溶出し、０．５ｍＬ／ｔｕｂｅで回収した。ＯＤ２８０ｎＭの吸光度より精製タンパクが含まれるフラクションを選別し混ぜ合わせた後、ＮＡＰ−２５（ＧＥヘルスケア製）を用いてＰＢＳにバッファー置換し、ろ過滅菌した。
　またサンプルの一部は、純度確認のため、還元下、非還元下にて８−１６％グラジエントゲルに泳動した後、コロイドＣＢＢ染色を行った。結果、精製したｈｓＡＬＫ１ｍＦｃタンパク質は還元下で５０ｋＤａ強、非還元下で９０~９５ｋＤａの移動度を示し、また高純度に精製されたタンパクであることを確認した（図１３）。 Acquisition and preparation of hsALK1mFc recombinant protein 16-1. Preparation of hsALK1mFc expression vector The hsALK1mFc expression vector is a PEAK8 expression vector obtained by enzymatic digestion of the extracellular region cDNA fragment of human ALK1 and the Fc region cDNA fragment of mIgG1 with EcoRI and NotI (both from Roche Diagnostics, Japan). It was prepared by incorporating into (Edge Biosystems).
The cDNA sequence of the incorporated hsALK1mFc is as follows (SEQ ID NO: 35).
Figure JPOXMLDOC01-appb-I000045
The amino acid sequence encoded by SEQ ID NO: 35 (346 amino acids, SEQ ID NO: 36) is shown below.
SEQ ID NO: 36:
Figure JPOXMLDOC01-appb-I000046
Each fragment was obtained as follows. The extracellular region cDNA of human ALK1 is
hsALK1 FW: agaattccccaccatgaccttgtccccccag (SEQ ID NO: 3),
hsALK1 RV: aacttagctgggcccatctgtttccccggctg (SEQ ID NO: 4)
Was cloned from a human lung cDNA library using the PCR method. The obtained fragment was enzymatically digested with EcoRI and SpeI, and then electrophoresed on an agarose gel to cut out the band. For extraction of DNA from agarose, QIAquick Gel Extraction Kit (Qiagen, Japan) was used.
On the other hand, the Fc region cDNA of mIgG1 is
mIgG1 FP: acttagcctaaggtcacgtgtgtgt (SEQ ID NO: 37)
mIgG1 RP: gcggccgcttattattattaccagaggaggggg (SEQ ID NO: 38)
Was cloned by PCR method. The obtained fragment was enzymatically digested with SpeI and NotI (both Roche Diagnostics, Japan), and then electrophoresed on an agarose gel to cut out the band. For extraction of DNA from agarose, QIAquick Gel Extraction Kit (Qiagen, Japan) was used.
16-2. Preparation of hsALK1mFc high expression strain and acquisition of culture supernatant The hsALK1mFc high expression strain was prepared by linearizing the expression vector prepared above using Sfi-I restriction enzyme and then converting the linearized DNA to Lipofectamine ™ LTX. Using a reagent (Invitrogen, Japan), the gene was introduced into CHO Ras clone I cells according to the package insert. Twenty-four hours after gene introduction, drug-selected cells were selected using alpha MEM containing 6 μg / ml puromycin and 10% serum (FCS), and then single clones were isolated by the limiting dilution method. A mouse IgG1 ELISA kit (purchased from Cosmo Bio Co., Ltd., Japan) was used for selection of high expression strains.
The obtained high expression strain was scaled up, seeded in a roller bottle, and cultured at 37 ° C. until it became confluent. After confirming that the cells were confluent, the cells were washed with PBS (Dulecco's Phosphate Buffered Saline; SIGMA), and then the EX-Cell 302 (SAFC Biosciences) medium was diluted to 25% with the DMEM / F-12 mixed medium. The medium was replaced. After culturing for 5 to 7 days, the culture supernatant was collected, filtered through a 0.22 μm filter (0.22 μm GP Express Membrane 500 ml; Japan Millipore, Japan), and then at 4 ° C. (cold room). Cooled down. When stored frozen, it was re-filtered through a 0.22 μm filter after thawing.
16-3. Concentration and preparation of culture supernatant of hsALK1mFc producing strain A large amount of the culture supernatant of hsALK1mFc obtained was concentrated using a Pellicon 3 cassette Ultracell PLCGC membrane, 0.11 m 2 (Millipore), and when the dosage reached an appropriate dose. The concentrate was collected. Then, after the channel was washed with PBS, the washed solution and the concentrated solution were mixed. To the combined concentrated solution, an equal amount of 1M Glycine (pH 8.5) solution containing 4M NaCl was added, followed by filtration with a 0.22 μm filter.
After connecting AKTA Prime (GE Healthcare Japan) to a Vantage-L laboratory column (Millipore) packed with approximately 50 ml of ProSep Ultra Plus (Millipore), 1M Glycine-NaOH containing 2M NaCl (Millipore) The column was equilibrated with a solution of pH 8.5). After equilibration, the filtered culture supernatant concentrate was applied to a ProSep column. After the application, it was washed with a 1M Glycine-NaOH (pH 8.5) solution containing 2M NaCl, and after confirming that the absorbance (OD 280 nm) of the filtrate returned to the baseline, another one column volume (50 mL) The column was washed with the same solution. After washing, hsALK1mFc protein was eluted using 5 column volumes of 0.1 M sodium citrate (pH 5.0) solution. The eluate was collected in 10 mL portions in a tube to which 1 mL of 0.2 M MES (pH 6.7) solution had been added in advance.
Next, a column of RESOURCE S 6 ml (manufactured by GE Healthcare) is connected to AKTA Explorer, 20 mM MES (pH 5.8) is used as the A1 solution, and 20 mM MES (pH 5.8) containing 1 M NaCl is used as the B1 solution. After equilibrating the column, the ProSep eluate diluted 4 to 10 times with the A1 solution was filtered through a 0.22 μm filter and applied to the column.
After the application, A1 was fed, and after confirming that OD280nm returned to the baseline, the elution program was started. For elution, after washing for 2 columns at a B1 concentration of 0%, the B1 concentration was gradually increased from 0% to 30% and eluted at 35 columns. The eluate was collected at 6 mL / tube. After each fraction was subjected to SDS-PAGE under non-reducing conditions, silver staining was performed to select a fraction having a high content of high molecular dimer and a low content of low molecular dimer, and the selection liquid was mixed.
Next, the above RESOURCE S eluate mixture was added to a RESOURCE PHE (GE Healthcare) column equilibrated with 0.1 M sodium phosphate (pH 7.2) containing 1 M (NH 4 ) 2 SO 4. A solution which was diluted 2-fold with 0.1 M sodium phosphate pH 7.2 solution containing (NH 4 ) 2 SO 4 and filtered was applied. 0.1M sodium phosphate (pH 7.2) containing 2M (NH 4 ) 2 SO 4 was used for the A2 solution, and 0.1M sodium phosphate (pH 7.2) was used for the B2 solution. After application, after washing with a solution having a B2 concentration of 50%, the B2 concentration was increased from 50 to 90% and eluted in 20 columns. Each eluted fraction was subjected to SDS-PAGE under non-reducing conditions and then subjected to silver staining, and the fraction containing only dimer was selected and mixed.
Next, for the purpose of concentrating the diluted fraction, the ammonium sulfate concentration in the mixture of the 3rd chromatographic fraction was estimated on the same column (RESOURCE PHE) from the electrical conductivity, and the final concentration of ammonium sulfate exceeded 1M. A2 was added and then applied to the column. After the sample was applied, the ammonium sulfate concentration was lowered to 100 to 200 mM at once, stepwise elution was performed, and the sample was collected at 0.5 mL / tube. The fraction containing the purified protein was selected and mixed based on the absorbance at OD 280 nM, and then the buffer was replaced with PBS using NAP-25 (manufactured by GE Healthcare), followed by filter sterilization.
In addition, a part of the sample was subjected to 8-16% gradient gel under reduced and non-reduced conditions for purity confirmation, and then colloidal CBB staining was performed. As a result, the purified hsALK1mFc protein showed a mobility of slightly over 50 kDa under reduction and 90-95 kDa under non-reduction, and was confirmed to be a highly purified protein (FIG. 13).
ＡＬＫ１阻害剤の肥満・高血圧自然発症ラットＳＨＲ−ＮＤを用いた心肥大、脂質異常に対する作用評価（インビボ試験）
　日本エスエルシー株式会社より７週齢の雄性のＳＨＲ／ＮＤｍｃ−ｃｐ（ｃｐ／ｃｐ）を３０匹、およびそのコントロールとして同週齢のＳＨＲ／ＮＤｍｃ−ｃｐ（＋／＋）を７匹を購入し、入荷後、固型飼料（ＦＲ−２、株式会社船橋農場製）にて１週間予備飼育した。本ＳＨＲ／ＮＤｍｃ−ｃｐ（ｃｐ／ｃｐ）は肥満を伴う高血圧自然発症ラットとして知られ、高血圧に伴い心肥大を起こす動物としても知られている（Ｍｅｔａｂｏｌｉｓｍ　４７，１１９９−１２０４（１９９８）。１週間後、飼育飼料を、ＦＲ−２からｃａｓｅｉｎ２０％，シュークロス２０％，ラード８％を含むラボＨスタンダードをベースとした特殊飼料（日本農産工業株式会社）に変更し、継続飼育した。
　飼料変更４週間後、代謝ケージにて約２４時間採尿した後、尾静脈からの採血を行った。得られた尿、血清を用いて、尿タンパク量、脂質（総コレステロール、トリグリセロール、血糖）の測定を行い、各群の総コレステロール、トリグリセロール、血糖値が均等になるように、ＳＨＲ／ＮＤｍｃ−ｃｐ（ｃｐ／ｃｐ）を３群（１群あたり７−８匹）に群分けした。群構成は、ＰＢＳ投与群（媒体投与群）、ＢＭＰ９中和抗体０．０４ｍｇ／ｋｇ投与群、ＢＭＰ９中和抗体０．２ｍｇ／ｋｇ投与群の３群の他、陰性対照として、ＳＨＲ／ＮＤｍｃ−ｃｐ（＋／＋）を１群設定した。
　群分け後、ヒトＢＭＰ９中和抗体（Ｒ＆Ｄ　Ｓｙｓｔｅｍ，Ｉｎｃ、Ｃｌｏｎｅ　ＮＯ．：３６０１０７）をＰＢＳにて０．１０ｍｇ／ｍＬ，０．０２０ｍｇ／ｍＬに希釈調製したものを、投与液量が２ｍＬ／ｋｇとなるように、投与前の体重を基に算出し、腹腔内に投与した。またコントロールであるＳＨＲ／ＮＤｍｃ−ｃｐ（＋／＋）にはＰＢＳを腹腔内に投与した。以降、週１回の頻度にて被験物質を投与した。体重は週１回、もしくは週２回測定し、採血は、被験物質投与１週間後より１週間おきに尾静脈より採血した。被験物質投与開始３２日目に、イソフルラン麻酔下に腹大動脈より全採血を行った後、腎臓および心臓を摘出し、各臓器重量を測定した。血液生化学解析は、試薬に協和メデックス株式販売のものを用いて、自動分析装置（日立社製７１７０）にて測定した。
　結果、ＢＭＰ９中和抗体の投与は体重増加に対して影響を与えなかった。全血血清を用いた血液生化学解析からは、総コレステロールおよびトリグリセロールの上昇が、ＢＭＰ９中和抗体の投与により抑制される傾向を示し（図１４）、ＢＭＰ９阻害は脂質異常症に対して有効である可能性が示された。また臓器重量測定では、ＢＭＰ９中和抗体は腎臓重量に対しては影響を及ぼさなかったものの、心臓重量に対しては用量依存的、かつ有意（Ｋｒｕｓｋａｌ−Ｗａｌｌｉｓ　ｔｅｓｔ＋Ｓｔｅｅｌ　ｔｅｓｔによる検定）に心肥大を抑制し（図１５）、ＢＭＰ９中和抗体は、心疾患で問題とされる心肥大に対して有効であること、すなわち、心疾患治療薬となりうる可能性が見出された。 Evaluation of effects of ALK1 inhibitor on cardiac hypertrophy and lipid abnormalities using spontaneously hypertensive rat SHR-ND (in vivo study)
Thirty male SHR / NDmc-cp (cp / cp) 7 weeks old and 7 SHR / NDmc-cp (+ / +) of the same week were purchased from Nippon SLC Co., Ltd. Then, after arrival, it was preliminarily raised for one week with solid feed (FR-2, manufactured by Funabashi Farm Co., Ltd.). This SHR / NDmc-cp (cp / cp) is known as a spontaneously hypertensive rat with obesity, and is also known as an animal that causes cardiac hypertrophy with hypertension (Metabolism 47, 1199-1204 (1998)). Thereafter, the breeding feed was changed from FR-2 to a special feed (Nippon Nosan Kogyo Co., Ltd.) based on Lab H Standard containing 20% casein, 20% shoecloth, and 8% lard.
Four weeks after the change of feed, urine was collected in a metabolic cage for about 24 hours, and then blood was collected from the tail vein. Using the obtained urine and serum, the amount of urine protein and lipid (total cholesterol, triglycerol, blood glucose) are measured, and SHR / NDmc so that the total cholesterol, triglycerol and blood glucose level of each group are equalized. -Cp (cp / cp) was divided into 3 groups (7-8 animals per group). The group composition includes PBS administration group (vehicle administration group), BMP9 neutralizing antibody 0.04 mg / kg administration group, BMP9 neutralizing antibody 0.2 mg / kg administration group, and SHR / NDmc− as a negative control. One group of cp (+ / +) was set.
After grouping, human BMP9 neutralizing antibody (R & D System, Inc, Clone NO .: 360107) diluted with PBS to 0.10 mg / mL and 0.020 mg / mL was prepared at a dose of 2 mL / kg. Thus, the weight was calculated based on the body weight before administration and administered intraperitoneally. Moreover, PBS was intraperitoneally administered to SHR / NDmc-cp (+ / +) which is control. Thereafter, the test substance was administered once a week. The body weight was measured once a week or twice a week, and blood was collected from the tail vein every other week from one week after administration of the test substance. On day 32 after the start of test substance administration, whole blood was collected from the abdominal aorta under isoflurane anesthesia, and then the kidney and heart were removed and the weight of each organ was measured. Blood biochemical analysis was performed with an automatic analyzer (7170 manufactured by Hitachi, Ltd.) using a reagent sold by Kyowa Medex Co., Ltd. as a reagent.
As a result, administration of the BMP9 neutralizing antibody had no effect on weight gain. From the blood biochemical analysis using whole blood serum, the increase in total cholesterol and triglycerol showed a tendency to be suppressed by administration of BMP9 neutralizing antibody (FIG. 14), and BMP9 inhibition is effective against dyslipidemia. The possibility of being was shown. In organ weight measurement, BMP9 neutralizing antibody had no effect on kidney weight, but suppressed heart hypertrophy in a dose-dependent and significant manner (test by Kruskal-Wallis test + Steel test) for heart weight. However, it was found that the neutralizing antibody for BMP9 is effective for cardiac hypertrophy, which is a problem in heart disease, that is, it may be a therapeutic agent for heart disease.
ＡＬＫ１阻害剤の動脈硬化進展に対する作用評価（インビボ試験）
　日本チャールズリバー株式会社より５週齢の雄性のＡＰＯＥ欠損マウス（Ｂ６．１２９Ｐ２−Ａｐｏｅ＜ｔｍ１Ｕｎｃ＞／Ｊ）３０匹、およびそのコントロールである同週齢のＣ５７ＢＬ／６Ｎ　ＣｒｌＣｒｌｊ　８匹を購入し、入荷後、固型飼料（ＦＲ−２、株式会社船橋農場製）で１週間予備飼育した。馴化後、ＡＰＯＥ欠損マウスを、体重を指標に、１群あたり７−８匹からなる３群に群分けした。群構成は、生理食塩水投与群（媒体投与群）、ＢＭＰ９中和抗体投与群、ｈｓＡＬＫ１ｍＦｃ投与群の３群の他、陰性対照としてＣ５７ＢＬ／６Ｎを１群設定した。
　群分け後、被験物質（生理食塩水、ＢＭＰ９中和抗体、ｈｓＡＬＫ１ｍＦｃ）を当該マウスに腹腔内投与した。またコントロールであるＣ５７ＢＬ／６マウスには生理食塩水を腹腔内投与した。ＢＭＰ９中和抗体、ｈｓＡＬＫ１ｍＦｃの投与量は、それぞれ１ｍｇ／ｋｇ、０．１ｍｇ／ｋｇ。飼育飼料は、群分け後より、動脈硬化が誘発し易いことが知られているｗｅｓｔｅｒｎ食（Ｆ２ＷＴＤ）（オリエンタル酵母社作製）に変更した。ＢＭＰ９中和抗体には、Ｒ＆Ｄ社から購入したもの（Ｃｌｏｎｅ：３６０１０７）を用い、ＢＭＰ９中和抗体、ｈｓＡＬＫ１ｍＦｃとも生理食塩水にて用事調製したものを投与した。
　以降、被験物質（生理食塩水、ＢＭＰ９中和抗体、ｈｓＡＬＫ１ｍＦｃ）は、用事調整したものを、週一回の頻度で腹腔内投与した。採血は、被験物質投与開始後、２週間もしくは４週間おきに経時的に採血した。血液生化学解析は、協和メデックス株式会社より販売の試薬を用いて自動分析装置（日立社製７１７０）にて行った。
　被験物質投与開始１２週目に、イソフルラン麻酔下に腹部大静脈より血液を全量採取した後、心臓直下より下行大動脈下部までの領域の大動脈を摘出した。その後、大動脈に付着する脂肪、結合組織を光学顕微鏡下でピンセット、ハサミを用いて除去した後、大動脈を前側および後ろ側から縦方向にハサミを用いて切断し切り開いた後、１０％ホルマリン液に浸し一晩固定した。固定した大動脈は、ＰＢＳで２回，６０％イソプロパノールで１回洗浄した後、１．８ｍｇ／ｍＬのオイルレッドＯ染色液に一晩浸した。
　翌日、６０％イソプロパノールで１回、ＰＢＳにて２回洗浄した後、大動脈外側に付着した余分な脂肪を光学顕微鏡下でピンセット、ハサミを用いて除去した後、大動脈サンプルをスライドグラス、カバーグラスを用いて挟み、画像解析に供した。画像解析では、摘出した大動脈の総面積、Ｏｉｌ　Ｒｅｄ　Ｏ染色された面積をそれぞれ算出し、それぞれのサンプルにおけるプラーク面積率を、Ｏｉｌ　Ｒｅｄ　Ｏ染色された面積を大動脈総面積で割ることにより算出した。なお、ｈｓＡＬＫｍＦｃおよび中和抗体投与群のプラーク形成抑制率（％）は、以下の式にて算出した。
（（ＡＰＯＥ欠損マウス生理食塩水投与群におけるプラーク面積率）—（ＡＰＯＥ欠損マウス被験物質投与群におけるプラーク面積率））÷（（ＡＰＯＥ欠損マウス生理食塩水投与群におけるプラーク面積率））—（Ｃ５７ＢＬ／６生理食塩水投与群におけるプラーク面積率））Ｘ１００
　結果、ＡＰＯＥ欠損マウスにおけるプラーク形成は、ｈｓＡＬＫｍＦｃ投与より約３２％、ＢＭＰ９中和抗体投与により約１５％抑制され（図１６）、ＢＭＰ９中和抗体を含むＡＬＫ１拮抗薬は、動脈硬化進展に対して有効である可能性が見出された。 Evaluation of the effects of ALK1 inhibitors on the progression of atherosclerosis (in vivo study)
Purchased 30 male 5-week-old male APOE-deficient mice (B6.129P2-Apoe <tm1Unc> / J) and 8 C57BL / 6N CrlCrlj mice of the same week as the control from Charles River Japan Thereafter, it was preliminarily raised for one week with a solid feed (FR-2, manufactured by Funabashi Farm Co., Ltd.). After acclimatization, APOE-deficient mice were divided into 3 groups consisting of 7-8 animals per group using body weight as an index. In addition to three groups, a physiological saline administration group (vehicle administration group), a BMP9 neutralizing antibody administration group, and an hsALK1mFc administration group, one group of C57BL / 6N was set as a negative control.
After grouping, test substances (saline, BMP9 neutralizing antibody, hsALK1mFc) were intraperitoneally administered to the mice. Further, physiological saline was intraperitoneally administered to C57BL / 6 mice as controls. The doses of the BMP9 neutralizing antibody and hsALK1mFc were 1 mg / kg and 0.1 mg / kg, respectively. The breeding feed was changed to a Western diet (F2WTD) (produced by Oriental Yeast Co., Ltd.), which is known to easily induce arteriosclerosis after grouping. As the BMP9 neutralizing antibody, a product purchased from R & D (Clone: 360107) was used, and both the BMP9 neutralizing antibody and hsALK1mFc were prepared in a normal saline solution.
Thereafter, test substances (physiological saline, BMP9 neutralizing antibody, hsALK1mFc) were administered intraperitoneally at a frequency of once a week. Blood was collected over time every 2 weeks or 4 weeks after the start of test substance administration. Blood biochemical analysis was performed with an automatic analyzer (Hitachi 7170) using reagents sold by Kyowa Medex Co., Ltd.
At 12 weeks from the start of administration of the test substance, the whole blood was collected from the abdominal vena cava under isoflurane anesthesia, and then the aorta in the region from directly under the heart to the lower descending aorta was removed. Then, after removing fat and connective tissue adhering to the aorta using tweezers and scissors under an optical microscope, the aorta is cut longitudinally from the anterior and posterior sides with scissors and then cut into 10% formalin solution. Soaked and fixed overnight. The fixed aorta was washed twice with PBS and once with 60% isopropanol, and then immersed in 1.8 mg / mL oil red O staining solution overnight.
The next day, after washing once with 60% isopropanol and twice with PBS, excess fat adhering to the outside of the aorta was removed using tweezers and scissors under an optical microscope. Used for image analysis. In the image analysis, the total area of the extracted aorta and the area stained with Oil Red O were calculated, and the plaque area ratio in each sample was calculated by dividing the area stained with Oil Red O by the total area of the aorta. The plaque formation inhibition rate (%) in the hsALKmFc and neutralizing antibody administration group was calculated by the following formula.
((Plaque area rate in the APOE-deficient mouse physiological saline administration group) − (Plaque area rate in the APOE-deficient mouse test substance administration group)) ÷ ((Plaque area rate in the APOE-deficient mouse physiological saline administration group)) — (C57BL / 6 plaque area ratio in saline administration group)) X100
As a result, plaque formation in APOE-deficient mice was suppressed by about 32% by hsALKmFc administration and by about 15% by BMP9 neutralizing antibody administration (FIG. 16). ALK1 antagonists containing BMP9 neutralizing antibody The possibility of being effective was found.
ＡＬＫ１阻害剤の血圧に対する作用評価（インビボ試験）
　日本エスエルシー社より購入の１６週齢の雄性のＳＨＲ／Ｉｚｍに対して予備飼育を行った後、１８週齢にて血圧測定用テレメーター送信器（ＴＡ１１ＰＡ−Ｃ４０；Ｄａｔａ　Ｓｃｉｅｎｃｅｓ）の腹大動脈への埋め込み手術を施した。埋め込み手術は以下のように実施した。腹部正中線を約５ｃｍ開腹し、腹部血管を露出した後、腹大動脈と後大静脈を剥離し、腹大動脈を止血クリップではさんだ。止血クリップより１ｃｍ位下にディスポーザブル注射針（２１Ｇ、テルモ株式会社；針先を約２ｍｍ折り曲げたもの）を用いて腹大動脈にカテーテル挿入口を開け、テレメトリー送信器のカテーテル先端をディスポーザブル注射針の下を這わせるように挿入口に差込んだ。カテーテル挿入口付近の血液を脱脂綿等でふき取り、カテーテルと挿入口に組織接着ボンドを滴下した。ボンドが乾いた後、止血クリップをゆっくりと外し、出血がないことを確認した。腹部器官を腹腔内に戻し、筋層と送信器の固定部位を一緒に縫合した。
　送信器を埋め込み、３３週齢に達したラットの拡張期血圧、収縮期血圧、及び心拍数をテレメトリーシステムＤａｔａｑｕｅｓｔ　Ａ．Ｒ．Ｔ．を用いて測定した後、それらの値が各群均等になるよう４群に群分けした。４群の構成は、ＰＢＳ投与群（陰性対照）、０．０４ｍｇ／ｋｇ　ＢＭＰ９中和抗体投与群、０．２ｍｇ／ｋｇ　ＢＭＰ９中和抗体投与群、０．２５ｍｇ／ｋｇ　ｈｓＡＬＫ１ｍＦｃ投与群からなる４群（ｎ＝５匹）。ＢＭＰ９中和抗体には、Ｒ＆Ｄ社から購入したもの（Ｃｌｏｎｅ：３６０１０７）を用いた。
　群分け後、抗体希釈用媒体（ＰＢＳ）を投与し、血圧等に変動がないことを確認した後、その翌日に、ＢＭＰ９中和抗体、ｈｓＡＬＫ１ｍＦｃなどの被験物質を腹腔内に投与し、拡張期血圧、収縮期血圧、及び心拍数の測定を行った。
　結果、ＢＭＰ９中和抗体には血圧に対する作用は認められなったものの、ｈｓＡＬＫ１ｍＦｃ投与群では、投与後約１４時間目より、拡張期血圧、収縮期血圧の低下が認められ、その低下は４日目まで継続した（図１７）。以上の結果より、ｈｓＡＬＫ１ｍＦｃは降圧作用を有することが明らかとなった。 Evaluation of effects of ALK1 inhibitor on blood pressure (in vivo test)
After preliminary breeding for 16-week-old male SHR / Izm purchased from Japan SLC, Inc. to the abdominal aorta of a blood pressure measurement telemeter transmitter (TA11PA-C40; Data Sciences) at 18 weeks of age An implant operation was performed. Implantation was performed as follows. The abdominal midline was opened about 5 cm, the abdominal blood vessels were exposed, the abdominal aorta and the posterior vena cava were peeled off, and the abdominal aorta was sandwiched with a hemostatic clip. Open a catheter insertion port in the abdominal aorta using a disposable injection needle (21G, Terumo Corporation; needle tip bent about 2 mm) below the hemostatic clip, and place the tip of the telemetry transmitter catheter under the disposable injection needle It was inserted into the insertion slot so that The blood in the vicinity of the catheter insertion port was wiped off with absorbent cotton or the like, and a tissue adhesive bond was dropped onto the catheter and the insertion port. After the bond had dried, the hemostatic clip was slowly removed to confirm no bleeding. The abdominal organ was returned into the abdominal cavity and the muscle layer and transmitter fixation site were sutured together.
The diastolic blood pressure, systolic blood pressure, and heart rate of a rat implanted with a transmitter and reaching the age of 33 weeks are measured by the telemetry system Dataquest A. R. T. T. After measuring using, the groups were divided into 4 groups so that their values were equal to each group. The four groups consist of 4 groups consisting of PBS administration group (negative control), 0.04 mg / kg BMP9 neutralizing antibody administration group, 0.2 mg / kg BMP9 neutralizing antibody administration group, and 0.25 mg / kg hsALK1mFc administration group. (N = 5). A BMP9 neutralizing antibody purchased from R & D (Clone: 360107) was used.
After grouping, the antibody dilution medium (PBS) was administered, and after confirming that there was no change in blood pressure etc., the test substance such as BMP9 neutralizing antibody and hsALK1mFc was administered intraperitoneally the next day. Blood pressure, systolic blood pressure, and heart rate were measured.
As a result, although BMP9 neutralizing antibody was not observed to have an effect on blood pressure, in the hsALK1mFc administration group, a decrease in diastolic blood pressure and systolic blood pressure was observed from about 14 hours after administration, and the decrease was observed on the 4th day. (FIG. 17). From the above results, it was revealed that hsALK1mFc has a hypotensive action.
ＷＫＹ抗ＧＢＭ腎炎モデルを用いたＢＭＰ９中和抗体の腎症進展に対する作用評価（インビボ試験）
　日本チャールズ・リバー社より購入の７週齢の雄性のＷＫＹ／ＮＣｒｌＣｒｌｊに対して１週間予備飼育を行った。その後、腎炎惹起を、ラット糸球体基底膜に対して作製した抗ＧＢＭウサギ血清（抗ＧＢＭ血清）をＰＢＳにて５倍希釈したものを３ｍＬ／ｋｇの用量にて静脈内投与することにより行った。またコントロールラットに対しては、正常ｒａｂｂｉｔ血清を５倍希釈したものを同様にして投与した。抗ＧＢＭ血清投与後Ｄａｙ７からＤａｙ８にかけて、代謝ケージにて２４時間採尿を行い、得られた尿の尿中タンパク量をマイクロＴＰ−テストワコー（和光純薬株式会社）を用いて添付文書に従い測定した。その値を基に、各群の尿中タンパク量の平均値ができるだけ均等になるように３群に群分けした（一群あたり７匹）。群構成は、生理食塩水投与群（媒体投与群）、０．０４ｍｇ／ｋｇ　ＢＭＰ９中和抗体投与群、０．２ｍｇ／ｋｇ　ＢＭＰ９中和抗体投与群の３群（１群あたり７匹）の他、陰性対照として、正常ｒａｂｂｉｔ血清投与群を１群設定した（ｎ＝５）。
　群分け後、被験物質（ＰＢＳ、ＢＭＰ９中和抗体）を当該ラットに腹腔内投与した。また陰性対照ラットにはＰＢＳを投与した。以降、週１回の頻度で被験物質を投与した。ＢＭＰ９中和抗体には、Ｒ＆Ｄ社から購入したもの（Ｃｌｏｎｅ：３６０１０７）を用いた。体重は週１回もしくは２回測定し、採血は、抗ＧＢＭ抗体投与後、Ｄａｙ１５、Ｄａｙ２２，Ｄａｙ２９，Ｄａｙ３５にて尾静脈より採血した。抗ＧＢＭ抗体投与後Ｄａｙ３７には、イソフルラン麻酔下にて腹大動脈より全採血を行った後、腎臓を摘出し、腎臓重量を測定した。血液生化学解析は、試薬に協和メデックス株式販売のものを用いて自動分析装置（日立社製７１７０）にて測定した。飼育飼料には試験を通じて固型飼料（ＦＲ−２、株式会社船橋農場製）を用いた。
　結果、経時的に採血した血清を用いた血液生化学解析において、腎疾患のマーカーであるＢＵＮの上昇は、ＢＭＰ９中和抗体の投与により抑制される傾向を示し（図１８）、抗ＢＭＰ９中和抗体は、腎疾患治療薬となりうる可能性が見出された。 Evaluation of the effect of BMP9 neutralizing antibody on the progression of nephropathy using WKY anti-GBM nephritis model (in vivo study)
A 7-week-old male WKY / NCrlCrlj purchased from Charles River Japan was preliminarily raised for one week. Thereafter, nephritis was induced by intravenously administering a 5-fold diluted anti-GBM rabbit serum (anti-GBM serum) prepared against rat glomerular basement membrane in PBS at a dose of 3 mL / kg. . For control rats, normal rabbit serum diluted 5-fold was administered in the same manner. From Day 7 to Day 8 after administration of anti-GBM serum, urine was collected in a metabolic cage for 24 hours, and the urinary protein content of the obtained urine was measured using MicroTP-Test Wako (Wako Pure Chemical Industries, Ltd.) according to the package insert. . Based on the value, the group was divided into 3 groups (7 animals per group) so that the average value of the urinary protein amount of each group was as uniform as possible. The group composition was 3 groups (7 mice per group): saline administration group (vehicle administration group), 0.04 mg / kg BMP9 neutralizing antibody administration group, 0.2 mg / kg BMP9 neutralizing antibody administration group As a negative control, one normal rabbit serum administration group was set (n = 5).
After grouping, the test substance (PBS, BMP9 neutralizing antibody) was intraperitoneally administered to the rats. PBS was administered to negative control rats. Thereafter, the test substance was administered once a week. A BMP9 neutralizing antibody purchased from R & D (Clone: 360107) was used. The body weight was measured once or twice a week, and blood was collected from the tail vein at Day 15, Day 22, Day 29, Day 35 after administration of the anti-GBM antibody. On Day 37 after administration of the anti-GBM antibody, whole blood was collected from the abdominal aorta under isoflurane anesthesia, and then the kidney was removed and the kidney weight was measured. Blood biochemical analysis was performed with an automatic analyzer (7170 manufactured by Hitachi, Ltd.) using a reagent sold by Kyowa Medex Co., Ltd. as a reagent. A solid feed (FR-2, manufactured by Funabashi Farm Co., Ltd.) was used as a breeding feed throughout the test.
As a result, in blood biochemical analysis using serum collected over time, the increase in BUN, which is a marker for renal disease, tends to be suppressed by administration of a BMP9 neutralizing antibody (FIG. 18), and anti-BMP9 neutralization It has been found that antibodies can be therapeutic drugs for kidney diseases.
　本発明の医薬組成物により、血管障害を抑制することができる。従って、血管障害および関連する様々な疾患又は障害を、治療することが可能となる。
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
With the pharmaceutical composition of the present invention, vascular injury can be suppressed. Thus, it is possible to treat vascular disorders and various related diseases or disorders.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (16)

  1. 　ＡＬＫ１阻害剤を有効成分とする血管障害を抑制するための医薬組成物。 A pharmaceutical composition for suppressing vascular injury comprising an ALK1 inhibitor as an active ingredient.
  2. 　ＡＬＫ１阻害剤が以下のいずれかから選択される、請求項１に記載の医薬組成物。
    （１）ＢＭＰ９に結合する抗体または該抗体断片
    （２）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質
    （３）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸
    （４）ＡＬＫ１細胞外領域ポリペプチドまたはその変異体、あるいはＡＬＫ１細胞外領域ポリペプチドまたはその変異体を含む蛋白質をコードする核酸を含む発現ベクター
    （５）ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片
    （６）ＢＭＰ９に対するアプタマーまたはＡＬＫ１細胞外領域ポリペプチドに対するアプタマー
    （７）ＡＬＫ１遺伝子を標的とするＲＮＡｉ分子、またはＢＭＰ９遺伝子を標的とするＲＮＡｉ分子
    （８）ＡＬＫ１のキナーゼ活性に対して阻害活性を有する低分子化合物
    （９）ＢＭＰ９とＡＬＫ１との結合、もしくはＢＭＰ９とタイプＩＩレセプターとの結合を阻害する活性を有する低分子化合物
    （１０）ＢＭＰ９阻害活性を有するＢＭＰ９アンタゴニスト蛋白質     The pharmaceutical composition according to claim 1, wherein the ALK1 inhibitor is selected from any of the following.
    (1) an antibody that binds to BMP9 or the antibody fragment (2) ALK1 extracellular region polypeptide or variant thereof, or ALK1 extracellular region polypeptide or protein containing the variant (3) ALK1 extracellular region polypeptide or Nucleic acid encoding a variant thereof, or an ALK1 extracellular region polypeptide or a protein comprising the variant (4) ALK1 extracellular region polypeptide or a variant thereof, or an ALK1 extracellular region polypeptide or a protein comprising the variant (5) An antibody that binds to an ALK1 extracellular region polypeptide or an antibody fragment thereof (6) An aptamer to BMP9 or an aptamer to an ALK1 extracellular region polypeptide (7) RNAi targeting the ALK1 gene Molecule, or BMP9 RNAi molecule targeting a gene (8) Low molecular weight compound having inhibitory activity against the kinase activity of ALK1 (9) It has the activity of inhibiting the binding of BMP9 and ALK1, or the binding of BMP9 and type II receptor Low molecular weight compound (10) BMP9 antagonist protein having BMP9 inhibitory activity
  3. 　（１）ＢＭＰ９に結合する抗体または該抗体断片、（２）ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片が、ヒト抗体、ヒト化抗体、またはその断片である、請求項２に記載の医薬組成物。 The antibody or the antibody fragment that binds to (1) BMP9, (2) the antibody or the antibody fragment that binds to an ALK1 extracellular region polypeptide is a human antibody, a humanized antibody, or a fragment thereof. Pharmaceutical composition.
  4. 　（１）ＢＭＰ９に結合する抗体または該抗体断片、（２）ＡＬＫ１細胞外領域ポリペプチドに結合する抗体または該抗体断片が、ポリクローナル抗体、ペプチド抗体、モノクローナル抗体またはその断片である、請求項３に記載の医薬組成物。 The antibody or the antibody fragment that binds to BMP9, (2) the antibody or the antibody fragment that binds to an ALK1 extracellular region polypeptide is a polyclonal antibody, a peptide antibody, a monoclonal antibody or a fragment thereof. The pharmaceutical composition as described.
  5. 　該抗体断片が、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、一本鎖抗体（ｓｃＦｖ）、二量体化Ｖ領域（Ｄｉａｂｏｄｙ）、ジスルフィド安定化Ｖ領域（ｄｓＦｖ）およびＣＤＲを含むペプチドから選ばれる抗体断片である、請求項３または４に記載の医薬組成物。 The antibody fragment is selected from peptides comprising Fab, Fab ′, F (ab ′) 2, single chain antibody (scFv), dimerized V region (Diabody), disulfide stabilized V region (dsFv) and CDR. The pharmaceutical composition according to claim 3 or 4, which is an antibody fragment.
  6. 　ＡＬＫ１細胞外領域ポリペプチドまたはその変異体が、哺乳動物由来免疫グロブリンＦｃ蛋白質又はその変異体との融合蛋白質である、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein the ALK1 extracellular region polypeptide or a variant thereof is a fusion protein with a mammal-derived immunoglobulin Fc protein or a variant thereof.
  7. 　血管障害を伴う疾患に対して用いられる、請求項１~６のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 6, which is used for diseases associated with vascular disorders.
  8. 　血管障害を伴う疾患が、腎疾患、動脈硬化性疾患、高血圧症、肺性高血圧症、心疾患、糖尿病、糖尿病合併症、血栓症、脂質異常症などの生活習慣病、血管炎を伴う疾患またはＢＭＰ９の関与の可能性が示された疾患から選択される、請求項７に記載の医薬組成物。 Diseases with vascular disorders are renal diseases, arteriosclerotic diseases, hypertension, pulmonary hypertension, heart diseases, diabetes, diabetic complications, thrombosis, dyslipidemia and other lifestyle diseases, diseases with vasculitis or The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is selected from diseases in which the possibility of involvement of BMP9 has been shown.
  9. 　腎疾患が、ＩｇＡ腎症を含む慢性糸球体腎炎、糖尿病性腎症、ループス腎炎、腎硬化症または急速進行性糸球体腎炎である、請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8, wherein the renal disease is chronic glomerulonephritis including IgA nephropathy, diabetic nephropathy, lupus nephritis, nephrosclerosis or rapidly progressive glomerulonephritis.
  10. 　動脈硬化性疾患が、脳血管障害（脳卒中、ラクナ梗塞も含む脳梗塞、脳血栓、脳出血、クモ膜下出血など）、虚血性心疾患（心筋梗塞や狭心症など）、大動脈瘤、大動脈解離、腎硬化症または閉塞性動脈硬化症である、請求項８に記載の医薬組成物。 Atherosclerotic diseases include cerebrovascular disorders (cerebral infarction including stroke, lacunar infarction, cerebral thrombus, cerebral hemorrhage, subarachnoid hemorrhage, etc.), ischemic heart disease (such as myocardial infarction and angina), aortic aneurysm, aortic dissection, The pharmaceutical composition according to claim 8, which is nephrosclerosis or obstructive arteriosclerosis.
  11. 　心疾患が、心臓弁膜症、心筋梗塞、狭心症、心筋症である、請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8, wherein the heart disease is valvular heart disease, myocardial infarction, angina pectoris, or cardiomyopathy.
  12. 　糖尿病合併症が、動脈硬化症、糖尿病性網膜症、糖尿病性腎症、糖尿病性神経障害、糖尿病性壊疽、慢性感染症または白内障である、請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8, wherein the diabetic complication is arteriosclerosis, diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, diabetic gangrene, chronic infection or cataract.
  13. 　血栓症が、肺血栓塞栓症、脳梗塞、心筋梗塞、下肢急性動脈血栓症、腸壊死（上腸間膜動脈血栓症）である、請求項８に記載の医薬組成物。 The pharmaceutical composition according to claim 8, wherein the thrombosis is pulmonary thromboembolism, cerebral infarction, myocardial infarction, lower limb acute arterial thrombosis, intestinal necrosis (upper mesenteric artery thrombosis).
  14. 　血管炎を伴う疾患が、高安動脈炎、巨細胞動脈炎（側頭動脈炎）、結節性多発動脈炎、ウェゲナー肉芽腫症、チャーグ・ストラウス症候群、川崎病、ヘノッホ・シェーンライン紫斑病、過敏性血管炎、全身性エリテマトーデスまたは関節リウマチである、請求項８に記載の医薬組成物。 Diseases with vasculitis are Takayasu's arteritis, giant cell arteritis (temporal arteritis), polyarteritis nodosa, Wegener's granulomatosis, Churg-Strauss syndrome, Kawasaki disease, Henoch-Schönlein purpura, hypersensitivity The pharmaceutical composition according to claim 8, which is vasculitis, systemic lupus erythematosus or rheumatoid arthritis.
  15. 　ＢＭＰ９の関与の可能性が示された疾患が、肝疾患（急性肝炎、慢性肝炎、肝臓がん、肝硬変、転移性肝臓がんなど）、癌性腹水・胸水を伴うがん、慢性膵炎、アレルギー性疾患（アレルギー性鼻炎、喘息、気道過敏症、アトピー性皮膚炎など）、炎症性疾患（遅延型アレルギー、関節リウマチ、関節炎、肺疾患、肝炎、潰瘍性大腸炎など）、アルツハイマー、多発性硬化症、糖尿病性網膜症、レイノー症候群、クローン病またはがんである、請求項８に記載の医薬組成物。 Diseases that may be involved in BMP9 include liver disease (acute hepatitis, chronic hepatitis, liver cancer, cirrhosis, metastatic liver cancer, etc.), cancer with cancerous ascites / pleural effusion, chronic pancreatitis, allergy Inflammatory diseases (allergic rhinitis, asthma, airway hypersensitivity, atopic dermatitis, etc.), inflammatory diseases (delayed allergy, rheumatoid arthritis, arthritis, lung disease, hepatitis, ulcerative colitis, etc.), Alzheimer, multiple sclerosis 9. The pharmaceutical composition according to claim 8, wherein the composition is diabetic, diabetic retinopathy, Raynaud's syndrome, Crohn's disease or cancer.
  16. 　請求項１~１５のいずれか１項に記載の医薬組成物を用いた血管障害を伴う疾患の治療法。 A method for treating a disease associated with a vascular disorder using the pharmaceutical composition according to any one of claims 1 to 15.
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