WO2014051109A1 - Anticorps anti-bmp9 humain et agent thérapeutique pour une maladie d'ossification ectopique contenant ledit anticorps comme principe actif - Google Patents

Anticorps anti-bmp9 humain et agent thérapeutique pour une maladie d'ossification ectopique contenant ledit anticorps comme principe actif Download PDF

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WO2014051109A1
WO2014051109A1 PCT/JP2013/076411 JP2013076411W WO2014051109A1 WO 2014051109 A1 WO2014051109 A1 WO 2014051109A1 JP 2013076411 W JP2013076411 W JP 2013076411W WO 2014051109 A1 WO2014051109 A1 WO 2014051109A1
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antibody
human
bmp9
seq
amino acid
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PCT/JP2013/076411
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清 清水
山崎　雄司
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協和発酵キリン株式会社
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

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  • the present invention binds to human BMP9 (Bone morphogenetic protein-9), has binding inhibitory activity between human BMP9 and ALK1, and selectively transmits a signal derived from FOP-ALK2 among human BMP type I receptors.
  • the present invention also relates to a pharmaceutical composition containing the antibody or the antibody fragment as an active ingredient for use in the treatment of ectopic ossification disease and a method for treating ectopic ossification disease using the same.
  • BMP9 is an abbreviation for Bone Morphogenetic Protein 9 and is also called GDF2.
  • BMP9 belongs to about 20 types of BMP (bone morphogenetic protein) family molecules, and human BMP9 is a secreted protein consisting of 429 amino acids (Non-patent Document 1).
  • BMP9 is mainly expressed in the spinal cord or interstitial membrane in the fetal period and in the liver in the adult period (Non-Patent Documents 2, 3, and 4), and human BMP9 is present in the blood at a concentration of 2-12 ng / mL. It is known to be a circulating factor in blood (Non-patent Document 5).
  • Non-patent Documents 6, 7, 8 expression of promoting action on the formation of hypertrophic chondrocytes or differentiation from mesenchymal cells to cartilage
  • Non-Patent Documents 9 expression of promoting action on blood precursor cell production or colony formation
  • Ectopic ossification is a general term for bone formation that occurs in soft tissues (muscles, tendons, ligaments, organs) where bone formation does not occur, and is also called ectopic bone formation.
  • FOP progressive ossification fibrodysplasia
  • fibers such as skeletal muscle, fascia, tendon or ligament throughout the body from childhood Sexual tissue is progressively ossified, which results in decreased range of motion or stiffness of the limb joints, or decreased mobility or deformation of the trunk.
  • This disease is caused by a genetic mutation (617G> A; R206H) of activin-like kinase receptor 2 (sometimes also referred to as ALK2, ACVR1), which is a type I receptor for BMP (Bone morphogenic protein) ( Non-patent document 10).
  • This gene mutation is considered to be a constitutively activated mutation in which ossification occurs when a signal is transmitted into the cell even if BMP5, BMP6, BMP7, BMP9, and activin, which are ligands of ALK2, do not bind. (Non-Patent Document 10).
  • FOP-ALK2 ALK2 having this gene mutation
  • BMP6 BMP6
  • R & D antibody As an anti-BMP9 monoclonal antibody, a monoclonal antibody having a neutralizing activity against BMP9 (R & D Systems, clone No. 360107, hereinafter referred to as R & D antibody) is commercially available from R & D Systems, but other antibodies are known. Not.
  • Non-Patent Document 12 Low molecular weight compounds having kinase inhibitory activity against ALK2 have been shown to be effective for FOP treatment (Non-Patent Document 12), there are concerns about side effects due to non-selective kinase inhibitory activity. Against this background, development of new therapeutic agents is desired for FOP.
  • the object of the present invention is to provide a therapeutic agent comprising an anti-human BMP9 antibody as an active ingredient, which can be used for the treatment of ectopic ossification diseases including FOP.
  • the present inventors tried to obtain an anti-BMP9 antibody using a BMP9-deficient mouse, and succeeded in obtaining an anti-BMP9 antibody with significantly improved binding to BMP9 as compared to the R & D antibody.
  • the obtained anti-BMP9 antibody recognized an epitope different from that of the R & D antibody, and was an antibody having an inhibitory effect on binding to the BMP type I receptor, which is not found in the R & D antibody.
  • this anti-BMP9 antibody was an antibody capable of selectively inhibiting bone differentiation based on the FOP-ALK2-derived signal with respect to the ALK1-derived signal by BMP9.
  • an anti-BMP9 antibody as an active ingredient can be provided with a therapeutic agent for ectopic ossification disease, and completed the present invention.
  • the gist of the present invention is as follows. (1) Competing with one antibody selected from the following (a) to (d) and binding to human BMP9, inhibiting the binding of human BMP type I receptor and human BMP9, and human BMP type I receptor A monoclonal antibody or an antibody fragment thereof that selectively inhibits a FOP-ALK2-derived signal among body-derived signals.
  • CDR Complementary determining region 1 to 3 each include an antibody heavy chain (hereinafter referred to as H chain) comprising the amino acid sequence represented by SEQ ID NOs: 54 to 56, respectively.
  • An antibody comprising a variable region (hereinafter referred to as VL) (d) A VH of an antibody comprising the amino acid sequence represented by SEQ ID NO: 51 and / or an VL of an antibody comprising the amino acid sequence represented by SEQ ID NO: 53
  • the monoclonal antibody or the antibody fragment according to (1) which is a monoclonal antibody that binds to the same epitope as the one antibody selected from (a) to (d).
  • the monoclonal antibody or the antibody fragment thereof according to (1) or (2) which is a recombinant antibody.
  • the monoclonal antibody or the antibody fragment according to (3) which is a recombinant antibody selected from a human chimeric antibody, a humanized antibody and a human antibody.
  • CDRs 1 to 3 each contain the heavy chain of an antibody comprising the amino acid sequence represented by SEQ ID NOs: 54 to 56, and / or CDRs 1 to 3 each comprise an amino acid sequence represented by SEQ ID NOs: 57 to 59
  • the antibody fragment (b) CDR1-3 comprises the H chain of an antibody comprising the amino acid sequence represented by SEQ ID NOs: 60-62, respectively, and / or CDR1-3 comprise SEQ ID NO: 63, respectively.
  • a monoclonal antibody comprising an L chain of an antibody comprising the amino acid sequence represented by ⁇ 65 and the antibody fragment (6) contained in the 1st to 74th amino acid sequences of the human BMP9 nature region represented by SEQ ID NO: 67
  • the monoclonal antibody or the antibody fragment thereof according to any one of (1) to (5), which binds to an amino acid.
  • An antibody selected from a peptide comprising Fab, Fab ′, F (ab ′) 2 , a single chain antibody (scFv), a dimerized V region (diabody), a disulfide stabilized V region (dsFv) and a CDR
  • the antibody fragment according to any one of (1) to (6), which is a fragment.
  • the transformed strain according to (10) is cultured in a medium, and the monoclonal antibody or the antibody fragment according to any one of (1) to (7) is produced and accumulated in the culture, The method for producing a monoclonal antibody or the antibody fragment according to any one of (1) to (7), wherein the antibody or the antibody fragment is collected.
  • a pharmaceutical composition comprising the monoclonal antibody or the antibody fragment according to any one of (1) to (7) and a pharmacologically acceptable carrier.
  • a monoclonal antibody that binds to human BMP9, inhibits binding between human BMP type I receptor and human BMP9, and selectively inhibits FOP-ALK2-derived signals among human BMP type I receptors, or A method for treating an ectopic ossification disease involving human BMP9, comprising administering an antibody fragment.
  • the monoclonal antibody and the antibody fragment of the present invention recognize an epitope different from that of the R & D antibody and have an inhibitory effect on binding to the BMP type I receptor, which is not found in the R & D antibody.
  • the monoclonal antibody and the antibody fragment of the present invention have the advantage that the bone differentiation based on the FOP-ALK2-derived signal can be selectively inhibited with respect to the ALK1-derived signal by BMP9 and there is less concern about side effects. Therefore, according to the monoclonal antibody and the antibody fragment of the present invention, it is possible to provide a therapeutic agent for ectopic ossification disease including FOP, which comprises the antibody and the antibody fragment as active ingredients.
  • FIG. 1 is a diagram showing the structure of a mouse BMP9 gene knockout vector.
  • mouse 5 'genome is, BMP9 gene knockout vector 5' regions of homology
  • Neo r is the neomycin resistance gene
  • the mouse 3 'genome is BMP9 gene knockout vector 3' homologous region
  • DT-A is a diphtheria toxin A chain gene
  • T3 represents a T3 promoter
  • T7 represents a T7 promoter
  • pBluescript represents a cloning vector.
  • FIG. 2 is a view showing the binding specificity of the obtained anti-human BMP9 monoclonal antibody to human BMP9 by enzyme-linked immunosorbent assay (ELISA).
  • the vertical axis represents absorbance (450-570 nm).
  • FIG. 3 is a diagram showing the binding of the obtained anti-human BMP9 monoclonal antibody to human BMP9 by ELISA.
  • the horizontal axis represents antibody concentration (ng / mL), and the vertical axis represents absorbance (450-570 nm) at each antibody concentration.
  • no antibody is indicated by ⁇
  • 3B7-3-3 antibody is indicated by ⁇
  • 3C7-3-1 antibody is indicated by ⁇
  • R & D antibody is indicated by ⁇ .
  • FIG. 4 shows the inhibitory action of the obtained anti-human BMP9 monoclonal antibody on the binding of human BMP9 and labeled R & D antibody.
  • the horizontal axis represents the concentration of unlabeled antibody (ng / mL), and the vertical axis represents the inhibitory activity (%) of each antibody against the binding of BMP9 / R & D antibody.
  • the 3B7-3-3 antibody is indicated by ⁇
  • the 3C7-3-1 antibody is indicated by ⁇
  • the R & D antibody is indicated by ⁇ .
  • FIG. 5 is a diagram in which the inhibitory action on binding between each labeled antibody and human BMP9 by the unlabeled antibody of the existing and acquired anti-BMP9 monoclonal antibody was detected by enzyme-linked immunosorbent assay (ELISA).
  • the horizontal axis represents the biotin-labeled antibody used for detection, and the vertical axis represents the absorbance (450-570 nm).
  • the 3B7-3-3 antibody is shown in black, the 3C7-3-1 antibody is shown in diagonal lines, the R & D antibody is shown in a grid, and no antibody is shown in white.
  • FIG. 6 shows the inhibitory action of the obtained anti-human BMP9 monoclonal antibody on the binding of human BMP9 and human ALK1.
  • the horizontal axis represents antibody concentration (ng / mL), and the vertical axis represents the inhibitory activity (%) of each antibody against BMP9 / ALK1 binding.
  • the 3B7-3-3 antibody is indicated by ⁇
  • the 3C7-3-1 antibody is indicated by ⁇
  • the R & D antibody is indicated by ⁇ .
  • FIG. 7 is a diagram showing the inhibitory action of the obtained anti-human BMP9 monoclonal antibody on the BMP9-dependent ALK1-derived signal.
  • the horizontal axis represents antibody concentration (ng / mL), and the vertical axis represents the inhibitory activity (%) of each antibody against the BMP9-dependent ALK1-derived signal.
  • FIG. 8 is a diagram comparing the effect of serum (FCS) concentration on bone differentiation of FOP-ALK2 gene-introduced C2C12 cells at 5% and 20%.
  • the vertical axis represents the alkaline phosphatase activity value in each cell; absorbance (414 nm).
  • the FOP-ALK2 gene is shown in black, the wild-type hALK2 gene is hatched, and the control vector is shown in white.
  • FIG. 9 shows the effect of R & D antibody on bone differentiation of FOP-ALK2 gene-introduced C2C12 cells by addition of high serum (FCS) concentration.
  • the vertical axis represents the alkaline phosphatase activity value in each cell; absorbance (414 nm).
  • FCS high serum
  • the horizontal axis represents the concentration of human BMP9 recombinant protein added (ng / mL), and the vertical axis represents the alkaline phosphatase activity value in each cell; absorbance (414 nm).
  • the wild-type ALK2 gene-introduced 2C12 cell line is indicated by ⁇
  • the FOP-ALK2 gene-introduced C2C12 cell line is indicated by ⁇
  • FIG. 11 is a graph showing the effect of an anti-human BMP9 monoclonal antibody on bone differentiation (increased alkaline phosphatase activity) of a FOP-ALK2 gene-introduced C2C12 cell line by adding a human BMP9 recombinant protein.
  • the horizontal axis represents the antibody concentration (ng / mL), and the vertical axis represents the inhibitory activity (%) of each antibody against bone differentiation caused by the addition of the BMP9 recombinant protein.
  • the 3C7-3-1 antibody is indicated by ⁇
  • the R & D antibody is indicated by ⁇ .
  • the present invention relates to a monoclonal antibody that binds to human BMP9.
  • an antibody that competes with an antibody and binds to human BMP9 has an epitope (also referred to as an antigenic determinant) identical or partially identical to that of the monoclonal antibody of the present invention, and binds to the epitope.
  • Antibody The antibody that binds to the same epitope to which the monoclonal antibody of the present invention binds refers to an antibody that recognizes and binds to the same sequence as the amino acid sequence of human BMP9 recognized by the monoclonal antibody of the present invention.
  • Human BMP9 is synthesized as a single-chain precursor protein protein (Pre-Pro body) having the amino acid sequence represented by SEQ ID NO: 66.
  • This single-chain Pre-Pro body is composed of the amino acid sequence represented by SEQ ID NO: 66, and after the 1st to 22nd signal peptide regions have been cleaved in the Golgi apparatus, A dimer (Pro dimer) is formed through a disulfide bond between groups.
  • the furin-like protease cleaves between the 319th and 320th amino acid residues of the amino acid sequence represented by SEQ ID NO: 66, and the propeptide region (SEQ ID NO: 66) having no disulfide bond. And a C-terminal fragment (mature region) consisting of the amino acid sequence represented by SEQ ID NO: 67 and a peptide comprising the 23rd amino acid to the 319th amino acid).
  • the shape region forms a dimer through a disulfide bond between the cysteine residues remaining at position 73 of the amino acid sequence represented by SEQ ID NO: 67 even after cleavage of the propeptide region (hereinafter referred to as “mature dimer”). write).
  • BMP9 refers to the involvement of BMP9 in intracellular signal transduction.
  • intracellular signal transduction when BMP9 binds to two receptors of BMP type I and BMP type II belonging to the TGF ⁇ superfamily, the receptor is activated, and Smad1 / 5/8 is phosphorylated, Furthermore, Smad1 / 5/8 activated by phosphorylation forms a complex with Smad4, then moves into the nucleus and functions as a transcription factor.
  • BMP type I receptor examples include ALK1 and ALK2.
  • ALK2 also includes FOP-ALK2 having a gene mutation (617G> A; R206H) that is responsible for FOP.
  • BMP type II receptor examples include BMP type II receptor (BMPRII), activin type IIa receptor (ActRIIa), and activin type IIb receptor (ActRIIb).
  • the number of amino acids to be deleted, substituted or added is not particularly limited, but is preferably 1 to several tens, for example 1 to 20, more preferably 1 to several, for example 1 to 5 amino acids. It is.
  • the gene encoding human BMP9 examples include the nucleotide sequence represented by SEQ ID NO: 68 or GenBank accession number NM_016204.
  • the base sequence represented by SEQ ID NO: 69 corresponding to the feature region comprises a base sequence in which one or more bases are deleted, substituted or added, and includes a DNA encoding a polypeptide having the function of human BMP9 From a gene, a base sequence having at least 60% homology with the base sequence shown in SEQ ID NO: 69, preferably a base sequence having 80% or more homology, more preferably a base sequence having 95% or more homology
  • a DNA having the base sequence represented by SEQ ID NO: 69 as a probe As the DNA that hybridizes under stringent conditions, a DNA having the base sequence represented by SEQ ID NO: 69 as a probe, a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or It means a hybridizable DNA obtained by a DNA microarray method or the like.
  • 0.7 to 1.0 mol / L of sodium chloride is present using a DNA or DNA derived from a hybridized colony or plaque, or a filter or slide glass on which a PCR product or oligo DNA having the sequence is immobilized.
  • the hybridizable DNA is DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 69, preferably DNA having 80% homology or more, more preferably 95% homology or more. Can be mentioned.
  • the gene used in the present invention includes a gene in which a small-scale mutation has occurred in the nucleotide sequence due to such a polymorphism, and is included in the gene encoding BMP9 of the present invention.
  • the numerical value of homology in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art unless otherwise specified.
  • BLAST J. Mol. Biol. 215, 403 (1990)
  • the like are calculated using default parameters.
  • BLAST2 Nucleic Acids Res. 25, 3389 (1997), Genome Res. 7, 649 (1997), http: // www. ncbi. nlm. nih. gov / Education / BLASTinfo / information3. numerical values calculated using default parameters in [html].
  • the default parameters are 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, and 1 if it is an amino acid sequence.
  • -Q (Penalty for nucleotide mismatch) is -3
  • -r (reward for nucleotide match) is 1
  • -e (expect value) is 10
  • 11 residues when -W (wordsize) is a base sequence
  • -y [Dropoff (X) for blast extensions in bits] is 20 when blastn, 7 for programs other than blastn
  • -X X dropoff value for 50 if the gap alignment in bits
  • Z final X dropoff value for gapment alignment in bits
  • a polypeptide consisting of a partial sequence of the amino acid sequence represented by SEQ ID NO: 66 or GenBank accession number NP_057288 can be prepared by methods known to those skilled in the art, for example, a DNA encoding the amino acid sequence represented by SEQ ID NO: 66 Can be prepared by culturing a transformant into which an expression vector containing this is introduced.
  • polypeptide or DNA produced by the above method one or more amino acids are deleted in the partial sequence of the amino acid sequence represented by SEQ ID NO: 66 or GenBank accession number NP_057288 by the same method as described above.
  • a polypeptide having a substituted or added amino acid sequence can be obtained.
  • polypeptide consisting of a partial sequence of the amino acid sequence represented by SEQ ID NO: 66 or GenBank accession number NP_057288, or one or more amino acids deleted in the partial sequence of the amino acid sequence represented by SEQ ID NO: 66 or GenBank accession number NP_057288
  • a polypeptide having a substituted or added amino acid sequence can also be produced by a chemical synthesis method such as a fluorenylmethyloxycarbonyl (Fmoc) method or a t-butyloxycarbonyl (tBoc) method.
  • the monoclonal antibody of the present invention (hereinafter also referred to as the antibody of the present invention) is an antibody or an antibody fragment thereof that recognizes and binds to the amino acid sequence of human BMP9, or a three-dimensional structure thereof, and has human BMP type I receptor and It has the property of inhibiting binding of human BMP9 and selectively inhibiting FOP-ALK2-derived signals among human BMP type I receptor-derived signals.
  • Examples of the amino acid sequence of human BMP9 in the present invention include those containing two amino acid sequences of the human BMP9 nature region represented by SEQ ID NO: 67, and the 73rd cysteine residue forming a disulfide bond. It is done.
  • human BMP9 containing the amino acid sequence represented by SEQ ID NO: 66, GenBank accession number NP_057288 or SEQ ID NO: 67 has a structure equivalent to that which can be taken in the natural state. Any structure may be used.
  • the three-dimensional structure that human BMP9 can take in the natural state refers to the natural three-dimensional structure of human BMP9.
  • human BMP type I receptor in the present invention examples include human ALK1 and human ALK2.
  • human ALK1 include a polypeptide comprising the amino acid sequence from the 22nd to the 118th amino acid sequence corresponding to the extracellular region in the amino acid sequence represented by SEQ ID NO: 18 or GenBank accession number AAH42637.
  • SEQ ID NO: 71 a polypeptide containing the amino acid sequence from the 22nd to the 118th corresponding to the extracellular region can be mentioned.
  • the antibody include the following monoclonal antibodies (i) and (ii) and antibody fragments thereof.
  • Monoclonal antibody comprising a chain and antibody fragment thereof (ii) CDR1-3 comprise the heavy chain of an antibody comprising the amino acid sequence represented by SEQ ID NO: 60-62, respectively, and CDR1-3 are represented by SEQ ID NO: 63-65, respectively.
  • Monoclonal antibodies containing an L chain of an antibody comprising the amino acid sequence and antibody fragments thereof include the following monoclonal antibodies and antibody fragments thereof (a) and (b): It is done.
  • a monoclonal antibody comprising the antibody VH comprising the amino acid sequence represented by SEQ ID NO: 50 and comprising the VL of the antibody comprising the amino acid sequence represented by SEQ ID NO: 52, and an antibody fragment thereof (b) A monoclonal antibody comprising the antibody VH comprising the amino acid sequence represented and the antibody VL comprising the amino acid sequence represented by SEQ ID NO: 53, and an antibody fragment thereof
  • examples of the monoclonal antibody of the present invention include a monoclonal antibody that binds to the same epitope as that present in human BMP9 to which the monoclonal antibody binds, and an antibody fragment thereof.
  • the binding of the antibody of the present invention or the antibody fragment thereof to the amino acid sequence of human BMP9 or its three-dimensional structure expressed human BMP9 or human BMP9, such as enzyme-linked immunosorbent assay (ELISA) using solid phase antigen can be confirmed by a known immunological detection method for a tissue, a method capable of examining the binding properties of a specific antigen and an antibody to the specific antigen, and the like.
  • ELISA enzyme-linked immunosorbent assay
  • Examples include surface plasmon resonance using a Biacore system (GE Healthcare) and isothermal titration calorimetry using ITC (manufactured by DKSH).
  • the binding dissociation constant (Kd value) of the antibody to the antigen is determined by performing analysis according to the Scatchard plot or the package insert of each apparatus from any of ELISA, surface plasmon resonance, and isothermal titration calorimetry. be able to.
  • the tissue expressing human BMP9 may be any tissue as long as it expresses BMP9, and examples thereof include blood and liver.
  • That the antibody of the present invention or an antibody fragment thereof inhibits the binding between human BMP type I receptor and human BMP9 is the result of inhibiting human BMP9 or human BMP9, such as enzyme-linked immunosorbent assay (ELISA) using solid phase antigen. It can be confirmed by a known immunological detection method for the expressed tissue. For example, it can be confirmed that human ALK1 or human ALK2 does not bind to solid-phased human BMP9 in the presence of the antibody of the present invention or an antibody fragment thereof.
  • ELISA enzyme-linked immunosorbent assay
  • the selective inhibition of FOP-ALK2-derived signals among human BMP type I receptor signals by the antibody of the present invention or an antibody fragment thereof produces human ALK1-expressing cells and human FOP-ALK2-expressing cells, This can be confirmed by using a known detection method such as an assay. For example, the cell response observed by adding human BMP9 to the seeded human FOP-ALK2-expressing cells is compared to the cell response observed by adding human BMP9 to the seeded human ALK1-expressing cells. This can be confirmed by the stronger inhibition of the antibody of the present invention or the antibody fragment thereof.
  • the human ALK1-expressing cell may be any cell as long as it is a cell transformed with a vector containing the gene encoding the ALK1 and a protein necessary for detection. The same applies to human FOP-ALK2-expressing cells.
  • Examples of the monoclonal antibody of the present invention include an antibody produced by a hybridoma, or a recombinant antibody produced by a transformant transformed with an expression vector containing an antibody gene.
  • a monoclonal antibody is an antibody that is secreted by an antibody-producing cell of a single clone, recognizes only one epitope (also referred to as an antigenic determinant), and has a uniform amino acid sequence (primary structure) constituting the monoclonal antibody. It is a feature.
  • epitopes include a single amino acid sequence that is recognized and bound by a monoclonal antibody, a three-dimensional structure composed of amino acid sequences, a three-dimensional structure composed of amino acid sequences bound to sugar chains, and a three-dimensional structure composed of amino acid sequences bound to sugar chains.
  • the monoclonal antibody of the present invention preferably binds to the amino acid sequence of human BMP9.
  • the epitope to which the monoclonal antibody of the present invention binds is preferably contained in the amino acid sequence of human BMP9.
  • the hybridoma is prepared, for example, by preparing human BMP9 as an antigen, inducing antibody-producing cells having antigen specificity from an animal immunized with the antigen, and further fusing the antibody-producing cells and myeloma cells.
  • An anti-BMP9 monoclonal antibody can be obtained by culturing the hybridoma or administering the hybridoma cell to an animal to cause ascites cancer, and separating and purifying the culture medium or ascites.
  • Any animal can be used as the animal to immunize with the antigen as long as it can produce a hybridoma.
  • a mouse, a rat, a hamster, a chicken or a rabbit is used.
  • An antibody produced by a hybridoma prepared by obtaining a cell capable of producing an antibody from such an animal, immunizing the cell in vitro, and then fusing it with a myeloma cell is also an antibody of the present invention. Is included.
  • the gene recombinant antibody in the present invention includes an antibody produced by gene recombination such as a human chimeric antibody, a human CDR grafted antibody, a human antibody or an antibody fragment.
  • a recombinant antibody having characteristics of a monoclonal antibody, low antigenicity, and extended blood half-life is preferable as a therapeutic agent.
  • Examples of the recombinant antibody include those obtained by modifying the monoclonal antibody of the present invention using a gene recombination technique.
  • the human chimeric antibody refers to an antibody comprising VH and VL of a non-human animal antibody and a heavy chain constant region (hereinafter referred to as CH) and light chain constant region (hereinafter referred to as CL) of a human antibody.
  • CH heavy chain constant region
  • CL light chain constant region
  • the human chimeric antibody of the present invention is obtained by obtaining cDNAs encoding VH and VL from the above hybridoma and inserting them into expression vectors for animal cells having genes encoding CH and CL of the human antibody, respectively.
  • An antibody expression vector can be constructed and expressed by being introduced into animal cells.
  • the CH of the human chimeric antibody may be any CH as long as it belongs to human immunoglobulin (hereinafter referred to as hIg), but preferably the hIgG class is used, and hIgG1, hIgG2, and hIgG3 belonging to the hIgG class. Alternatively, any subclass such as hIgG4 can be used.
  • the CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of ⁇ class or ⁇ class can be used.
  • a chimeric antibody comprising VH of an antibody comprising the amino acid sequence represented by SEQ ID NO: 50 and comprising VL of an antibody comprising the amino acid sequence represented by SEQ ID NO: 52 Can be mentioned.
  • a chimeric antibody containing the VH of an antibody comprising the amino acid sequence represented by SEQ ID NO: 51 and the VL of an antibody comprising the amino acid sequence represented by SEQ ID NO: 53 can be mentioned.
  • the human CDR-grafted antibody is sometimes referred to as a humanized antibody, and refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to appropriate positions of the human antibodies VH and VL.
  • the human CDR-grafted antibody of the present invention is a non-human antibody produced from a hybridoma that specifically recognizes human BMP9 and produces a monoclonal antibody of a non-human animal that binds to the amino acid sequence of human BMP9 or its three-dimensional structure.
  • a cDNA encoding the V region obtained by grafting the VH and VL CDR amino acid sequences of the animal antibodies of the above to the VH and VL framework regions (hereinafter referred to as FR) of any human antibody was constructed.
  • a human CDR-grafted antibody expression vector can be constructed by inserting it into an animal cell expression vector having a gene encoding CH and CL, and expressed and produced by introducing it into an animal cell.
  • the CH of the human CDR-grafted antibody may be any as long as it belongs to hIg, but preferably the hIgG class is used, and any subclass such as hIgG1, hIgG2, hIgG3, or hIgG4 belonging to the hIgG class should be used. Can do.
  • the CL of the human CDR-grafted antibody may be any CL as long as it belongs to hIg, and those of ⁇ class or ⁇ class can be used.
  • the human CDR-grafted antibody of the present invention includes VH of an antibody comprising the amino acid sequences represented by SEQ ID NOs: 54 to 56, respectively, and CDRs 1 to 3 are SEQ ID NOs: 57 to 59, respectively. And humanized antibodies that contain the VL of the antibody comprising the amino acid sequence represented.
  • CDR1 to CDR3 contain the VH of an antibody comprising the amino acid sequence represented by SEQ ID NOs: 60 to 62, respectively, and CDR1 to 3 comprise the VL of an antibody comprising the amino acid sequence represented by SEQ ID NOs: 63 to 65, respectively.
  • a humanized antibody is mentioned.
  • a human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library and a human antibody-producing transgene produced by recent advances in genetic engineering, cell engineering, and developmental engineering. Also included are antibodies obtained from transgenic animals.
  • the antibody naturally present in the human body can be cultured by, for example, isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the lymphocytes that produce the antibody.
  • the antibody can be further purified.
  • the human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting antibody genes prepared from human B cells into the phage genes. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.
  • a human antibody-producing transgenic animal means an animal in which a human antibody gene is incorporated into cells.
  • a human antibody-producing transgenic mouse can be produced by introducing a human antibody gene into a mouse ES cell, transplanting the ES cell into an early mouse embryo, and generating it.
  • human antibody-producing hybridomas are obtained and cultured using normal hybridoma production methods performed in non-human animals to produce human antibodies in the culture supernatant. It can be produced by accumulating.
  • the number of amino acids to be deleted, substituted, inserted and / or added is 1 or more, and the number is not particularly limited, but site-specific mutagenesis [Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols, Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)], and the like, and the number can be deleted, substituted or added.
  • the number is preferably 1 to several tens, more preferably 1 to 20, further preferably 1 to 10, and particularly preferably 1 to 5.
  • deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence of the above antibody indicates the following. That is, it means that there is a deletion, substitution, insertion or addition of one or a plurality of amino acid residues in any one and a plurality of amino acid sequences in the same sequence.
  • deletion, substitution, insertion or addition may occur simultaneously, and the amino acid residue to be substituted, inserted or added may be either a natural type or a non-natural type.
  • natural amino acid residues include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, or L-cysteine.
  • amino acid residues contained in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
  • Group C asparagine, glutamine
  • D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
  • Group E proline, 3 -Hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoser
  • antibody fragments include Fab, F (ab ′) 2 , Fab ′, single chain antibody (scFv), dimerization V region (diabody), disulfide stabilized V region (dsFv) and CDR. Examples thereof include peptides.
  • 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 the monoclonal antibody of the present invention 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 expressing the vector by introducing the vector into a prokaryotic or eukaryotic organism. it can.
  • F (ab ′) 2 is obtained by decomposing the lower part of the disulfide bond in the hinge region of IgG with pepsin, a proteolytic enzyme. It is a fragment having a large antigen-binding activity.
  • F (ab ′) 2 of the present invention can be obtained by treating the monoclonal antibody of the present invention with pepsin.
  • Fab ′ described below can be produced by thioether bond or disulfide bond.
  • Fab ′ is an antibody fragment having an antigen binding activity of about 50,000 molecular weight obtained by cleaving a disulfide bond in the hinge region of F (ab ′) 2 .
  • the Fab ′ of the present invention can be obtained by treating the F (ab ′) 2 of the present invention 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 ′. You can also
  • scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P), and has antigen-binding activity. It is an antibody fragment having
  • the scFv of the present invention obtains cDNA encoding the VH and VL of the monoclonal antibody of the present invention, 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 it into a prokaryotic or eukaryotic organism.
  • Diabody is an antibody fragment obtained by dimerizing scFv 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 the VH and VL of the monoclonal antibody of the present invention, 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 and 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 cDNA encoding the VH and VL of the monoclonal antibody of the present invention, constructs a DNA encoding dsFv, and inserts the DNA into a prokaryotic expression vector or a eukaryotic expression vector.
  • the expression vector can be expressed and produced by introducing it into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR is configured to contain 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 the DNA encoding the VH and VL CDRs of the monoclonal antibody of the present invention, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector Can be expressed and produced by introducing it into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR can also be manufactured by chemical synthesis methods, such as Fmoc method or tBoc method.
  • the monoclonal antibody of the present invention includes a derivative of an antibody in which a radioisotope, a low molecular drug, a high molecular drug, a protein or the like is chemically or genetically bound to the monoclonal antibody of the present invention or an antibody fragment thereof. To do.
  • an antibody derivative is used as a detection method, a quantification method, a detection reagent, or a quantification reagent, as a drug that binds to the monoclonal antibody of the present invention or an antibody fragment thereof, a conventional immunological detection or measurement method can be used.
  • the label used is mentioned.
  • the derivative of the antibody in the present invention includes the N-terminal side or C-terminal side of the H chain or L chain of the monoclonal antibody of the present invention or an antibody fragment thereof, an appropriate substituent or side chain in the antibody or the antibody fragment thereof, and By binding a radioisotope, a low molecular weight drug, a high molecular weight drug, a protein, etc. to a sugar chain or the like in a monoclonal antibody or an antibody fragment thereof by a chemical method [Introduction to antibody engineering, Jishinshokan (1994)]. Can be manufactured.
  • the derivative of the antibody in the present invention is obtained by ligating the DNA encoding the monoclonal antibody or antibody fragment of the present invention and the DNA encoding the protein to be bound, and inserting the expression vector into an appropriate host. It can be produced by genetic engineering techniques that are introduced into cells and expressed.
  • radioisotope examples include 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu, and 211 At.
  • the radioisotope can be directly bound to the antibody by the chloramine T method or the like. Further, a substance that chelates a radioisotope may be bound to the antibody.
  • the chelating agent include 1-isothiocyanate benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA).
  • low molecular weight agents examples include luminescent materials such as acridinium esters or lophine, and fluorescent materials such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).
  • luminescent materials such as acridinium esters or lophine
  • fluorescent materials such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (RITC).
  • Examples of a method for binding a low molecular weight drug to an antibody include, for example, a method of binding between the drug and the amino group of the antibody via glutaraldehyde, or a drug amino group and the antibody carboxyl group via a water-soluble carbodiimide. And the like.
  • polymer drug examples include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polyvinyl pyrrolidone, pyran copolymer, and hydroxypropyl methacrylamide.
  • PEG polyethylene glycol
  • albumin dextran
  • polyoxyethylene polyoxyethylene
  • styrene maleic acid copolymer polyoxyethylene
  • polyvinyl pyrrolidone polyvinyl pyrrolidone
  • pyran copolymer examples include hydroxypropyl methacrylamide.
  • Examples of a method for binding PEG to an antibody include a method of reacting with a PEGylation modifying reagent [Bioconjugate pharmaceutical, Yodogawa Shoten (1993)].
  • Examples of the PEGylation modifying reagent include a modifier for ⁇ -amino group of lysine (Japanese Unexamined Patent Publication No. 61-178926), a modifier for the carboxyl group of aspartic acid and glutamic acid (Japanese Unexamined Patent Publication No. 56-23587). And a modifier for guanidino group of arginine (Japanese Patent Laid-Open No. 2-117920).
  • Examples of the protein include enzymes such as alkaline phosphatase, peroxidase, and luciferase.
  • the present invention also relates to a therapeutic agent for ectopic ossification disease involving BMP9 containing the monoclonal antibody or antibody fragment thereof as an active ingredient.
  • Examples of ectopic ossification diseases involving BMP9 include FOP.
  • the therapeutic agent of the present invention contains the above-described monoclonal antibody of the present invention or the antibody fragment as an active ingredient.
  • the therapeutic agent containing the antibody or the antibody fragment or derivative thereof of the present invention may contain only the antibody or the antibody fragment or derivative thereof as an active ingredient, but is usually pharmacological. It is desirable to provide a pharmaceutical formulation prepared by any method known in the pharmaceutical arts, mixed with one or more pharmaceutically acceptable carriers.
  • oral administration or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular or intravenous is preferable, and intravenous administration or subcutaneous administration is preferable.
  • administration forms include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age and weight, but is usually 10 ⁇ g / kg to 10 mg / kg per day for an adult.
  • the present invention relates to an immunological detection or measurement method for BMP9, which contains a monoclonal antibody or an antibody fragment thereof that specifically recognizes and binds to the amino acid sequence of BMP9 or its three-dimensional structure as an active ingredient.
  • any known method may be used as a method for detecting or measuring the amount of BMP9.
  • immunological detection or measurement methods can be mentioned.
  • the immunological detection or measurement method is a method for detecting or measuring an antibody amount or an antigen amount using a labeled antigen or antibody.
  • Examples of the immunological detection or measurement method include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay method (EIA or ELISA), fluorescent immunoassay method (FIA), luminescent immunoassay method, western immunoassay, western Examples thereof include a blotting method and a physicochemical method.
  • BMP9 or BMP9 as an antigen is expressed in an expression vector containing cDNA encoding BMP9 full length or a partial length thereof, such as Escherichia coli, yeast, insect cells, or animal cells. It can be obtained by introducing into In addition, BMP9 can be purified and obtained from human tissue expressing BMP9 in a large amount. In addition, the tissue or the like can be used as an antigen as it is. Furthermore, a synthetic peptide having a partial sequence of BMP9 can be prepared by a chemical synthesis method such as the Fmoc method or the tBoc method and used as an antigen.
  • BMP9 used in the present invention is used in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) or Current Protocols in BioJulge 19 and the like. By the following method, DNA encoding the BMP9 can be expressed in a host cell and produced.
  • a recombinant vector is prepared by inserting a full-length cDNA containing a portion encoding BMP9 downstream of a promoter of an appropriate expression vector.
  • a DNA fragment of an appropriate length containing a polypeptide-encoding portion prepared based on the full-length cDNA may be used.
  • a transformant producing the polypeptide can be obtained by introducing the obtained recombinant vector into a host cell suitable for the expression vector.
  • Any expression vector can be used so long as it can be autonomously replicated in the host cell to be used or integrated into the chromosome, and contains an appropriate promoter at a position where the DNA encoding the polypeptide can be transcribed. Can do.
  • any microorganism that belongs to the genus Escherichia such as Escherichia coli, yeast, insect cells, or animal cells can be used so long as it can express the target gene.
  • the recombinant vector When a prokaryote such as E. coli is used as a host cell, the recombinant vector is capable of autonomous replication in a prokaryote, and at the same time, contains a promoter, a ribosome binding sequence, DNA containing a BMP9-encoding portion, and a transcription termination sequence. It is preferable that it is a vector containing.
  • the recombinant vector does not necessarily require a transcription termination sequence, but it is preferable to place the transcription termination sequence immediately below the structural gene.
  • the recombinant vector may contain a gene that controls the promoter.
  • the recombinant vector it is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence (also referred to as SD sequence), which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • SD sequence also referred to as SD sequence
  • start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • the base sequence of the DNA encoding the BMP9 can be substituted with a base so as to be an optimal codon for expression in the host, thereby improving the production rate of the target BMP9.
  • Any expression vector can be used as long as it can function in the host cell to be used.
  • pBTrp2, pBTac1, pBTac2 above, Roche Diagnostics
  • pKK233-2 Pharmacia
  • pSE280 manufactured by Invitrogen
  • pGEMEX-1 manufactured by Promega
  • pQE-8 manufactured by Qiagen
  • pKYP10 Japanese Unexamined Patent Publication No. 58-110600
  • pKYP200 Agricultural Biological Chemistry, 48, 669 (1984)]
  • pLSA1 Agric Biol. Chem. 53, 277 (1989)
  • pGEL1 Proc. Natl.
  • any promoter can be used as long as it can function in the host cell to be used.
  • promoters derived from Escherichia coli or phage such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, or T7 promoter can be mentioned.
  • An artificially designed and modified promoter such as a tandem promoter, tac promoter, lacT7 promoter, or let I promoter in which two Ptrps are connected in series can also be used.
  • Examples of host cells include E. coli XL-1 Blue, E. coli XL2-Blue, E. coli DH1, E. coli MC1000, E. coli KY3276, E. coli W1485, E. coli JM109, E. coli HB101, E. coli No. 49, E. coli W3110, E. coli NY49, or E. coli DH5 ⁇ .
  • Any method can be used for introducing a recombinant vector into a host cell as long as it is a method for introducing DNA into the host cell to be used.
  • a method using calcium ions Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].
  • any expression vector can be used as long as it can function in animal cells.
  • Any promoter can be used as long as it can function in animal cells. Examples include cytomegalovirus (CMV) immediate early (IE) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SR ⁇ promoter, or Moloney murine leukemia virus promoter or enhancer. . In addition, an enhancer of human CMV IE gene may be used together with a promoter.
  • CMV cytomegalovirus
  • IE immediate early
  • SV40 early promoter SV40 early promoter
  • retrovirus promoter metallothionein promoter
  • heat shock promoter metallothionein promoter
  • SR ⁇ promoter heat shock promoter
  • Moloney murine leukemia virus promoter or enhancer Moloney murine leukemia virus promoter or enhancer.
  • an enhancer of human CMV IE gene may be used together with a promoter.
  • host cells examples include human leukemia cells Namalwa cells, monkey cells COS cells, Chinese hamster ovary cells CHO cells (Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Sci.USA, 77, 4216 (1980); Proc.Natl.Acad.Sci.USA, 60, 1275 (19 8); Cell, 6, 121 (1975); Molecular Cell genetics, Appendix I, II (pp.
  • CHO / DG44 CHO-K1 (ATCC number: CCL-61), DUkXB11 (ATCC number: CCL) -9096), Pro-5 (ATCC number: CCL-1781), CHO-S (Life Technologies, Cat # 11619), Pro-3, rat myeloma cells YB2 / 3HL. P2. G11.16 Ag. 20 (also referred to as YB2 / 0), mouse myeloma cell NSO, mouse myeloma cell SP2 / 0-Ag14, Syrian hamster cell BHK or HBT5637 (Japanese Unexamined Patent Publication No. 63-000299).
  • Any method can be used for introducing a recombinant vector into a host cell as long as it is a method for introducing DNA into animal cells.
  • electroporation method [Cytotechnology 3, 133 (1990)]
  • calcium phosphate method Japanese Patent Laid-Open No. 227075/1990
  • lipofection method Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • a microorganism having a recombinant vector incorporating the DNA encoding BMP9 obtained as described above, or a transformant derived from an animal cell or the like is cultured in a medium, and the BMP9 is produced and accumulated in the culture. By collecting from the culture, BMP9 can be produced.
  • the method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
  • BMP9 When expressed in cells derived from eukaryotes, BMP9 with an added sugar or sugar chain can be obtained.
  • an inducer may be added to the medium as necessary.
  • cultivating a microorganism transformed with a recombinant vector using the lac promoter cultivate a microorganism transformed with isopropyl- ⁇ -D-thiogalactopyranoside or the like using a recombinant vector using the trp promoter.
  • indole acrylic acid or the like may be added to the medium.
  • Examples of a medium for culturing a transformant obtained using an animal cell as a host include, for example, a commonly used RPMI1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)], 199 medium [Proc. Soc. Exp. Biol. Med. 73, 1 (1950)], Iscove's Modified Dulbecco's Medium (IMDM) medium, or a medium obtained by adding fetal bovine serum (FBS) or the like to these mediums.
  • RPMI1640 medium The Journal of the American Medical Association, 199, 519 (1967)]
  • Eagle's MEM medium Science 122, 501 (1952)]
  • Dulbecco's modified MEM medium [Virology, 8, 396 (1959)]
  • 199 medium Proc. Soc. Exp. Biol. Med.
  • the culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and 5% CO 2 .
  • a method for producing BMP9 there are a method of producing it in a host cell, a method of secreting it outside the host cell, or a method of producing it on the outer membrane of the host cell, and changing the structure of the host cell to be used or the BMP9 to be produced. Thus, an appropriate method can be selected.
  • the production amount of BMP9 can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like (Japanese Patent Laid-Open No. 2-227075).
  • the obtained BMP9 can be isolated and purified as follows, for example.
  • BMP9 When BMP9 is expressed in a dissolved state in the cells, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then used with an ultrasonic crusher, French press, Manton Gaurin homogenizer, or dynomill. The cells are disrupted to obtain a cell-free extract.
  • a normal protein isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical), cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resins such as butyl sepharose, phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, or electrophoresis such as isoelectric focusing Can be used alone or in combination to obtain purified preparations. Can.
  • a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufact
  • BMP9 When BMP9 is expressed in the form of an insoluble substance in the cell, the cell is collected and crushed in the same manner as described above, and centrifuged to collect the insoluble substance of BMP9 as a precipitate fraction.
  • the recovered insoluble matter of BMP9 is solubilized with a protein denaturant.
  • the BMP9 is returned to a normal three-dimensional structure, and then a purified polypeptide preparation can be obtained by the same isolation and purification method as described above.
  • the derivative such as BMP9 or a sugar-modified product thereof can be recovered from the culture supernatant.
  • a soluble fraction can be obtained by treating the culture by a method such as centrifugation as described above, and a purified preparation can be obtained from the soluble fraction by using the same isolation and purification method as described above. it can.
  • BMP9 used in the present invention can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • chemical chemistry is performed using peptide synthesizers such as Advanced Chemtech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecel-Vega, Perceptive, or Shimadzu. It can also be synthesized.
  • Immunization is performed by administering the antigen subcutaneously, intravenously or intraperitoneally to the animal together with an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine.
  • an appropriate adjuvant such as Freund's complete adjuvant or aluminum hydroxide gel and pertussis vaccine.
  • the antigen is a partial peptide
  • a conjugate with a carrier protein such as BSA (bovine serum albumin) or KLH (Keyhole Limothemoyanin) is prepared and used as an immunogen.
  • BSA bovine serum albumin
  • KLH Keyhole Limothemoyanin
  • the antigen is administered 2 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood is collected from the fundus venous plexus, and the antibody titer of the serum is measured using an enzyme immunoassay [Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. An animal whose serum shows a sufficient antibody titer against the antigen used for immunization is used as a source of antibody producing cells for fusion.
  • tissues containing antibody-producing cells such as the spleen are removed from the immunized animal, and antibody-producing cells are collected.
  • the spleen is shredded and loosened, and then centrifuged, and the erythrocytes are removed to obtain antibody producing cells for fusion.
  • a myeloma cell a cell line obtained from a mouse is used.
  • U1 Current Topics in Microbiology and Immunology, 18, 1 (1978)]
  • P3-NS1 / 1 Ag41 NS-1
  • SP2 SP2 / 0-Ag14
  • SP-2 SP2 / 0-Ag14
  • SP-2 SP2 / 0-Ag14
  • SP-2 SP2 / 0-Ag14
  • P3-X63-Ag8653 6
  • P3-X63-Ag8 (X63) Nature, 256, 495 (1975)] or the like is used.
  • the myeloma cells are subcultured in normal medium [RPMI 1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, FBS, and 8-azaguanine], and subcultured into normal medium 3 to 4 days before cell fusion. On the day of fusion, secure a cell count of 2 ⁇ 10 7 or more.
  • MEM Minimum Essential Medium
  • PBS disodium phosphate 1. 83 g, monopotassium phosphate 0.21 g, sodium chloride 7.65 g, distilled water 1 liter, pH 7.2
  • a mixture of polyethylene glycol-1000 (PEG-1000), MEM medium and dimethyl sulfoxide is added at 37 ° C. with stirring.
  • the cells are gently suspended in the HAT medium [normal medium supplemented with hypoxanthine, thymidine, and aminopterin] on the antibody-producing cells for fusion. This suspension is cultured for 7-14 days at 37 ° C. in a 5% CO 2 incubator.
  • a part of the culture supernatant is extracted, and a cell group that reacts with an antigen containing BMP9 and does not react with an antigen not containing BMP9 is selected by a hybridoma selection method such as a binding assay described later.
  • cloning was repeated twice by the limiting dilution method (first time was HT medium (medium obtained by removing aminopterin from HAT medium), second time normal medium was used), and a stable and strong antibody titer was observed.
  • One is selected as a monoclonal antibody-producing hybridoma.
  • the monoclonal antibody-producing hybridoma obtained in (4) is cultured in an RPMI1640 medium supplemented with 10% FBS, the supernatant is removed by centrifugation, and the suspension is suspended in a Hybridoma-SFM medium and cultured for 3 to 7 days. To do.
  • the obtained cell suspension is centrifuged, and purified using a protein A-column or protein G-column from the obtained supernatant, and the IgG fraction is collected to obtain a purified monoclonal antibody.
  • 5% Daigo GF21 can be added to the Hybridoma-SFM medium.
  • the antibody subclass is determined by enzyme immunoassay using a sub-clustering kit.
  • the amount of protein is calculated from the Raleigh method or absorbance at 280 nm.
  • the monoclonal antibody is selected by a binding assay by the enzyme immunoassay shown below and a kinetic analysis by Biacore.
  • (6-a) Binding assay As an antigen, a gene-introduced cell or a recombinant obtained by introducing an expression vector containing cDNA encoding BMP9 obtained in (1) into E. coli, yeast, insect cells, animal cells, etc. A protein or a purified polypeptide or partial peptide obtained from human tissue is used. When the antigen is a partial peptide, a conjugate with a carrier protein such as BSA or KLH may be prepared and used.
  • a carrier protein such as BSA or KLH
  • a test substance such as serum, hybridoma culture supernatant or purified monoclonal antibody is dispensed as the first antibody and allowed to react.
  • an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent substance, a radiation compound or the like is dispensed and reacted as a second antibody.
  • a reaction is performed according to the labeling substance of the second antibody, and a monoclonal antibody that specifically reacts with the immunogen is selected.
  • the monoclonal antibody of the present invention can be obtained by adding a test antibody to the above-described binding assay system and allowing it to react. That is, by screening for an antibody that inhibits the binding of the monoclonal antibody when the test antibody is added, a monoclonal antibody that competes with the acquired monoclonal antibody for binding to the amino acid sequence of BMP9 or its three-dimensional structure is obtained. be able to.
  • an antibody that binds to the same epitope as the epitope recognized by the monoclonal antibody of the present invention is an epitope of the antibody obtained by the above-mentioned binding assay system, and a partially synthetic peptide of the identified epitope, or It can be obtained by preparing a synthetic peptide or the like mimicking the three-dimensional structure of the epitope and immunizing it.
  • ⁇ Kinetics analysis is performed on the obtained data using the software attached to the device using a 1: 1 binding model, and various parameters are acquired.
  • human BMP9 is immobilized on a sensor chip by, for example, an amine coupling method, and then purified monoclonal antibodies having a plurality of known concentrations are flowed to measure binding and dissociation.
  • the obtained data is subjected to kinetic analysis using a software that is included with the device, using a binding binding model, and various parameters are acquired.
  • a recombinant antibody expression vector is an animal cell expression vector into which DNAs encoding human antibodies CH and CL are incorporated, and the animal cell expression vector is human. It can be constructed by cloning DNAs encoding antibody CH and CL, respectively.
  • Any human antibody CH and CL can be used for the C region of a human antibody.
  • ⁇ 1 subclass CH and ⁇ class CL of human antibodies are used.
  • cDNA is used for the DNA encoding CH and CL of the human antibody, chromosomal DNA consisting of exons and introns can also be used.
  • Any expression vector for animal cells can be used as long as it can incorporate and express a gene encoding the C region of a human antibody.
  • pAGE107 [Cytotechnol. 3, 133 (1990)]
  • pAGE103 [J. Biochem. , 101, 1307 (1987)]
  • pHSG274 [Gene, 27, 223 (1984)]
  • pKCR Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)]
  • pSG1bd2-4 [Cytotechnol. 4, 173 (1990)]
  • pSE1UK1Sed1-3 [Cytotechnol. 13, 79 (1993)].
  • the promoter and enhancer include the SV40 early promoter [J. Biochem. 101, 1307 (1987)], Moloney murine leukemia virus LTR [Biochem. Biophys. Res. Commun. 149, 960 (1987)], or an immunoglobulin heavy chain promoter [Cell, 41, 479 (1985)] and an enhancer [Cell, 33, 717 (1983)].
  • Recombinant antibody expression vectors balance the ease of construction of recombinant antibody expression vectors, the ease of introduction into animal cells, and the balance of expression levels of antibody H and L chains in animal cells.
  • a vector for expressing a recombinant antibody of a type (tandem type) in which the antibody H chain and L chain are present on the same vector [J. Immunol. Methods, 167, 271 (1994)]
  • pKANTEX93 International Publication No. 97/10354
  • pEE18 Hybridoma, 17, 559 (1998)
  • MRNA is extracted from hybridoma cells producing non-human antibodies, and cDNA is synthesized.
  • the synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library.
  • Recombinant phages or recombinant plasmids having cDNA encoding VH or VL are isolated from the library using DNA encoding the C region portion or V region portion of the mouse antibody as a probe.
  • the entire base sequence of VH or VL of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, respectively, and the total amino acid sequence of VH or VL is estimated from the base sequence, respectively.
  • non-human animal for producing a hybridoma cell producing a non-human antibody a mouse, rat, hamster, rabbit or the like is used, but any animal can be used as long as it can produce a hybridoma cell. .
  • RNA easy kit manufactured by Qiagen
  • oligo (dT) -immobilized cellulose column method For preparation of mRNA from total RNA, oligo (dT) -immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989)], or Oligo-dT> SurSpurP Use a kit such as (Takara Bio).
  • mRNA can be prepared from hybridoma cells using a kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or QuickPrep mRNA Purification Kit (manufactured by Pharmacia).
  • any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma cell as a template is incorporated.
  • Any Escherichia coli into which a cDNA library constructed by a phage or plasmid vector is introduced can be used as long as it can introduce, express and maintain the cDNA library.
  • PCR method Polymerase Chain Reaction method
  • the selected cDNA is cleaved with an appropriate restriction enzyme and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and the nucleotide sequence of the cDNA is determined by a commonly used nucleotide sequence analysis method.
  • a base sequence analysis method include the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], etc., followed by ABI PRISM 3700 (manufactured by PE Biosystems) or A.P. L. F. An automatic base sequence analyzer such as a DNA sequencer (Pharmacia) is used.
  • VH and VL of an antibody including a secretory signal sequence all amino acid sequences of known antibody VH and VL [A. L. F. DNA, US Dept. By comparing with Health and Human Services (1991)], the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and further, the subgroup to which they belong can be known.
  • the amino acid sequences of CDRs of VH and VL are also the amino acid sequences of VH and VL of known antibodies [A. L. F. DNA, US Dept. It can be found by comparing with Health and Human Services (1991)].
  • VH and VL complete amino acid sequences for example, BLAST method [J. Mol. Biol. 215, 403 (1990)] and the like, and whether the complete amino acid sequences of VH and VL are novel can be confirmed.
  • the base sequence of the linking portion encodes an appropriate amino acid, and VH and VL cDNAs designed to have appropriate restriction enzyme recognition sequences are prepared.
  • the prepared VH and VL cDNAs are expressed in an appropriate form upstream of each gene encoding the human antibody CH or CL of the human CDR-grafted antibody expression vector obtained in (1). Each is cloned to construct a human chimeric antibody expression vector.
  • a recombinant antibody expression vector obtained in (1) by amplifying cDNA encoding non-human antibody VH or VL by a PCR method using a synthetic DNA having an appropriate restriction enzyme recognition sequence at both ends, respectively. can also be cloned.
  • a cDNA encoding VH or VL of human CDR-grafted antibody can be constructed as follows.
  • the amino acid sequence of the VH or VL FR of the human antibody to be transplanted with the VH or VL CDR amino acid sequence of the non-human antibody is selected. Any amino acid sequence can be used as long as it is derived from a human antibody.
  • FR amino acid sequences of human antibodies registered in databases such as Protein Data Bank, or common amino acid sequences of each subgroup of FRs of human antibodies [A. L. F. DNA, US Dept. Health and Human Services (1991)] are used.
  • an FR amino acid sequence having as high a homology as possible (at least 60% or more) with the VH or VL FR amino acid sequence of the original antibody is selected.
  • the amino acid sequence of the CDR of the original antibody is transplanted to the amino acid sequence of the FR of VH or VL of the selected human antibody, and the amino acid sequence of VH or VL of the human CDR-grafted antibody is designed.
  • Frequency of codon usage of the designed amino acid sequence in the base sequence of the antibody gene [A. L. F. DNA, US Dept. Considering Health and Human Services (1991)]
  • the DNA sequence is converted into a DNA sequence, and a DNA sequence encoding the amino acid sequence of VH or VL of the human CDR-grafted antibody is designed.
  • the human CDR-grafted antibody VH can be easily added to the human CDR-grafted antibody expression vector obtained in (1).
  • cDNA encoding VL can be cloned.
  • each H chain and L chain full-length synthetic DNA synthesized as one DNA based on the designed DNA sequence.
  • the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the base sequence is determined by the same method as described in (2), and the desired human CDR is obtained.
  • a plasmid having a DNA sequence encoding the amino acid sequence of VH or VL of the transplanted antibody is obtained.
  • a human CDR-grafted antibody can be obtained by transplanting only non-human antibody VH and VL CDRs into human antibody VH and VL FRs. The binding activity is reduced compared to the original non-human antibody [BIO / TECHNOLOGY, 9, 266 (1991)].
  • amino acid residues directly involved in binding to the antigen amino acid residues that interact with the CDR amino acid residues, and Decreased by maintaining the three-dimensional structure of the antibody, identifying amino acid residues indirectly involved in antigen binding, and substituting those amino acid residues with the amino acid residues of the original non-human antibody Antigen binding activity can be increased.
  • the amino acid residues of FR of human antibody VH and VL can be modified by performing the PCR reaction described in (4) using the synthetic DNA for modification.
  • the base sequence is determined by the method described in (2) and it is confirmed that the target modification has been performed.
  • a recognition sequence for an appropriate restriction enzyme at the 5 ′ end of the synthetic DNA located at both ends are introduced into the human CDR-grafted antibody expression vector obtained in (1), and cloned so that they are expressed in an appropriate form upstream of each gene encoding CH or CL of the human antibody.
  • Any host cell capable of expressing the recombinant antibody can be used as the host cell into which the expression vector is introduced.
  • COS-7 cells ATCC number: CRL1651 are used [Methods in Nucleic Acids. Res. , CRC Press, 283 (1991)].
  • the expression amount and antigen binding activity of the recombinant antibody in the culture supernatant are measured by enzyme immuno-antibody method [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-ALaboratory Cold Spring Harbor Laboratory (1988), monoclonal antibody experiment manual, Kodansha Scientific (1987)] and the like.
  • Any host cell capable of expressing a recombinant antibody can be used as a host cell into which the recombinant antibody expression vector is introduced.
  • CHO-K1 ATCC number: CCL-61
  • DUkXB11 ATCC number: CCL-9096
  • Pro-5 ATCC number: CCL-1781
  • CHO-S Life Technologies, Cat # 11619
  • mice myeloma cell NSO mouse myeloma cell SP2 / 0-Ag14
  • mouse P3-X63-Ag8653 cell ATCC number: CRL1580
  • dihydrofolate reductase gene Deficient CHO cells [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], Lec13 [Somatic Cell and Molecular Genetics, 12, 55 (1986)] that has acquired lectin resistance, CHO cells deficient in the ⁇ 1,6-fucose transferase gene (International Publication No. 2005 / 035586, WO 02/31140), rat YB2 / 3HL. P2. G11.16 Ag. 20 cells (ATCC number: CRL1662) are used.
  • a transformant that stably expresses the recombinant antibody is selected by culturing in an animal cell culture medium containing a drug such as G418 sulfate (Japanese Patent Laid-Open No. 2-257891).
  • the medium for animal cell culture include RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nippon Pharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM medium ( Invitrogen) or a medium obtained by adding various additives such as FBS to these mediums.
  • the obtained transformant is cultured in a medium to express and accumulate the recombinant antibody in the culture supernatant.
  • the expression level and antigen-binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or the like.
  • the transformed strain can increase the expression level of the recombinant antibody using a DHFR amplification system (Japanese Patent Laid-Open No. 2-257891).
  • the recombinant antibody is purified from the culture supernatant of the transformed strain using a protein A-column [Monoclonal Antibodies-Principles and Practice, Third edition, Academic Press (1996), Antibodies-A LaboratoryLaboratoryLaboratoryLaboratoryLaboratoryLaboratoryLaboratory. (1988)].
  • methods used in protein purification such as gel filtration, ion exchange chromatography, and ultrafiltration can be combined.
  • the molecular weight of the purified recombinant antibody H chain, L chain, or whole antibody molecule is determined by polyacrylamide gel electrophoresis [Nature, 227, 680 (1970)] or Western blotting [Monoclonal Antibodies-Principles and practicies, Third]. edition, Academic Press (1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  • Activity evaluation of purified monoclonal antibody or antibody fragment thereof The activity evaluation of the purified monoclonal antibody or antibody fragment thereof of the present invention can be performed as follows.
  • the binding activity to BMP9 and BMP9-expressing tissue is as described in 1. Measurement is performed using a surface plasmon resonance method using a binding assay described in (6-a) and a Biacore system described in (6-b). In addition, the fluorescent antibody method [Cancer Immunol. Immunother. 36, 373 (1993)].
  • the monoclonal antibody or antibody fragment thereof of the present invention can be used for the treatment of diseases involving ectopic ossification involving BMP9.
  • the therapeutic agent containing the monoclonal antibody of the present invention or an antibody fragment thereof, or a derivative thereof may contain only the antibody or the antibody fragment, or a derivative thereof as an active ingredient. It is provided as a pharmaceutical preparation produced by a method known in the technical field of pharmaceutics, mixed with one or more physically acceptable carriers.
  • Examples of the administration route include oral administration and parenteral administration such as intraoral, intratracheal, rectal, subcutaneous, intramuscular or intravenous.
  • Examples of the dosage form include sprays, capsules, tablets, powders, granules, syrups, emulsions, suppositories, injections, ointments, or tapes.
  • preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, and granules.
  • Liquid preparations such as emulsions or syrups include sugars such as water, sucrose, sorbitol or fructose, glycols such as polyethylene glycol or propylene glycol, oils such as sesame oil, olive oil or soybean oil, p-hydroxybenzoic acid Manufactured using preservatives such as esters or flavors such as strawberry flavor or peppermint as additives.
  • Capsules, tablets, powders or granules include excipients such as lactose, glucose, sucrose or mannitol, disintegrants such as starch or sodium alginate, lubricants such as magnesium stearate or talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose or gelatin, a surfactant such as fatty acid ester, or a plasticizer such as glycerin is used as an additive.
  • preparations suitable for parenteral administration include injections, suppositories, and sprays.
  • Injection is manufactured using a carrier composed of a salt solution, a glucose solution, or a mixture of both.
  • Suppositories are manufactured using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the propellant is manufactured using a carrier that does not irritate the recipient's oral cavity and airway mucosa, disperses the monoclonal antibody of the present invention or an antibody fragment thereof as fine particles, and facilitates absorption.
  • a carrier for example, lactose or glycerin is used. It can also be produced as an aerosol or dry powder.
  • Example 1 Construction of targeting vector for mouse BMP9 gene knockout 1-1) Construction of cassette vector pBlueLAB-LoxP-Neo-DT-A
  • the cassette vector pBlueLAB-LoxP-Neo-DT-A which is a basic vector for creating a knockout (KO) targeting vector, has a restriction enzyme site added to pBluescript, followed by a LoxP sequence at both ends of the neomycin resistance marker gene expression unit.
  • a vector in which LoxP-Neo having a diphtheria and a diphtheria toxin A chain gene (DT-A) are inserted, is the same as the vector described in Example 7 of WO 2006/078072. The outline of the vector production is described below.
  • LinkA1 SEQ ID NO: 1
  • LinkA2 SEQ ID NO: 2
  • LinkB1 SEQ ID NO: 3
  • LinkB2 SEQ ID NO: 4
  • PBluescript II SK (-) was treated with restriction enzymes SalI and XhoI, and the plasmid fragment was extracted with phenol / chloroform from the reaction solution and ethanol precipitated.
  • restriction enzymes SalI and XhoI restriction enzymes SalI and XhoI
  • a linker consisting of LinkA1 and LinkA2 was inserted and introduced into E. coli DH5 ⁇ . Plasmid pBlueLA was obtained from the resulting transformant.
  • PBlueLA was treated with restriction enzymes NotI and EcoRI, and the plasmid fragment was extracted from the reaction solution with phenol / chloroform and ethanol precipitated.
  • restriction enzymes NotI and EcoRI restriction enzymes NotI and EcoRI
  • SalI restriction enzymes
  • a linker consisting of LinkB1 and LinkB2 was inserted and introduced into E. coli DH5 ⁇ . Plasmid pBlueLAB was obtained from the obtained transformant.
  • the plasmid pLoxP-STneo described in International Publication No. 00/10383 was enzymatically digested with XhoI to obtain a Neo resistance gene (LoxP-Neo) having a LoxP sequence at both ends. Both ends of LoxP-Neo were blunted using T4 DNA polymerase to obtain LoxP-Neo-B.
  • PBlueLAB was enzymatically digested with EcoRV, and the plasmid fragment was extracted with phenol / chloroform from the reaction solution, followed by ethanol precipitation. LoxP-Neo-B was inserted into the obtained plasmid fragment and introduced into E. coli DH5 ⁇ . Plasmid pBlueLAB-LoxP-Neo was obtained from the obtained transformant.
  • PMC1DT-A (manufactured by Lifetech Oriental) was enzymatically digested with XhoI and SalI, and then a fragment containing the DT-A gene was collected using QIAquick Gel Extraction Kit (manufactured by Qiagen).
  • PBlueLAB-LoxP-Neo was enzymatically digested with XhoI, and the plasmid fragment was extracted with phenol / chloroform from the reaction solution and ethanol precipitated. A fragment containing the DT-A gene was inserted into the obtained plasmid fragment and introduced into E. coli DH5 ⁇ . A cassette vector pBlueLAB-LoxP-Neo-DT-A was obtained from the obtained transformant.
  • mouse BMP9 gene 3 'genomic region fragment Primer (SEQ ID NOs: 5 and 6) is designed from the genomic DNA sequence (accession number: ENSMUSG0000007625) containing the mouse BMP9 (GDF2) gene acquired from Ensemble Genome Browser did.
  • primers of SEQ ID NOS: 5 and 6 were each 10 pmol, KOD-plus- (Toyobo Co., Ltd.) Prepared) and kept at 94 ° C. for 3 minutes, followed by 35 cycles of PCR at 98 ° C. for 10 seconds and 68 ° C. for 5 minutes.
  • the resulting PCR amplified fragment was subjected to agarose gel electrophoresis, and then a 2.1 kbp fragment was recovered using QIAquick Gel Extraction Kit (manufactured by Qiagen).
  • the recovered PCR amplified fragment was enzymatically digested with ClaI and AscI, subjected to agarose gel electrophoresis, and then an enzyme-treated fragment (ClaI-AscI fragment) was recovered using QIAquick Gel Extraction Kit.
  • PBlueLAB was enzymatically digested with ClaI and AscI and subjected to Shrim Alkaline Phosphatase (SAP), and then this ClaI-AscI fragment was extracted with phenol / chloroform and ethanol precipitated.
  • SAP Shrim Alkaline Phosphatase
  • the ClaI-AscI fragment recovered above was inserted and introduced into E. coli DH5 ⁇ .
  • a clone having an inserted gene free from mutation caused by PCR was selected from the obtained transformants.
  • This plasmid DNA was enzymatically digested with ClaI and AscI, subjected to agarose gel electrophoresis, and then an enzyme-treated fragment containing the 3'-side genomic sequence of the 2.1 kbp mouse BMP9 gene was collected using QIAquick Gel Extraction Kit.
  • the obtained PCR amplified fragment was subjected to agarose gel electrophoresis, and then a 5.1 kbp fragment was recovered using QIAquick Gel Extraction Kit (manufactured by Qiagen).
  • the recovered PCR amplified fragment was enzymatically digested with PacI and FseI, subjected to agarose gel electrophoresis, and then an enzyme-treated fragment (PacI-FseI fragment) was recovered using QIAquick Gel Extraction Kit.
  • PBlueLAB was enzymatically digested with PacI and FseI and treated with SAP, and this PacI-FseI fragment was extracted with phenol / chloroform and ethanol precipitated.
  • the PacI-FseI fragment recovered above was inserted and introduced into E. coli DH5 ⁇ .
  • a clone having an inserted gene free from mutation caused by PCR was selected from the obtained transformants.
  • the plasmid DNA was enzymatically digested with PacI and FseI, subjected to agarose gel electrophoresis, and then an enzyme-treated fragment containing the 5.1 kbp mouse BMP9 gene 5'-side genomic sequence was recovered using QIAquick Gel Extraction Kit.
  • mice BMP9 gene 3 'genomic region fragment into cassette vector pBlueLAB-Lox-Neo-DT-A obtained in Example 1-1 was enzymatically digested with ClaI and AscI, and agarose gel electrophoresis Then, the DNA fragment was recovered using a QIAquick Gel Extraction Kit.
  • the enzyme-treated fragment obtained in Example 1-2 was inserted into the collected DNA fragment of about 7.6 kbp, and then introduced into E. coli DH5 ⁇ . A clone having a DNA fragment inserted therein was selected from the obtained transformant. It was confirmed that there was no problem in the base sequence of the ligation part.
  • Example 1-4 Insertion of mouse BMP9 gene 5 'genomic region fragment into a cassette vector having mouse BMP9 gene 3' genomic region fragment.
  • the plasmid obtained in Example 1-4 was enzymatically digested with PacI and FseI, and agarose After being subjected to gel electrophoresis, the enzyme-treated fragment was recovered using QIAquick Gel Extraction Kit.
  • the enzyme-treated fragment prepared in Example 1-3 was inserted into the recovered 9.7 kbp DNA fragment and then introduced into E. coli DH5 ⁇ .
  • a clone having a DNA fragment inserted was selected from the obtained transformant. After confirming that there was no problem in the base sequence of the ligation part, a targeting vector for mouse BMP9 gene knockout, pBluemBmp9-Lox-Neo-DT-A-3′KO-5′KO (FIG. 1) was obtained.
  • Example 2 Preparation of mouse BMP9 gene targeting vector 60 ⁇ g of pBluemBmp9-Lox-Neo-DT-A-3′KO-5′KO obtained in Example 1-5 was added to spermidine (Sigma) to a final concentration of 1 mmol / L. The enzyme was digested with NotI in H buffer for restriction enzymes (Roche Diagnostics) adjusted to pH 7.0.
  • the vector fragment was extracted with phenol / chloroform and ethanol precipitated.
  • PH 7.0 HBS solution [5 liters of HEPES, 8 g of NaCl, 0.37 g of KCl, 0.125 g of Na 2 HPO 4 ⁇ 2H 2 O, Dextrose (D--) so that the DNA concentration becomes 0.5 ⁇ g / ⁇ L.
  • Glucose) solution containing 1 g] and storing for 1 hour at room temperature to prepare electroporation mouse BMP9 gene targeting vector pBluemBmp9-Lox-Neo-DT-A-3′KO-5′KO-NotI did.
  • Example 3 Preparation of probe for genomic Southern blot analysis 3-1) Preparation of probe for confirming 5 ′ genome of mouse BMP9 gene Based on the base sequence information of BAC clone RP23-181N8, about 500 bp of 5 ′ genome region of mouse BMP9 gene Primers (SEQ ID NOs: 9, 10) were designed to obtain the containing probe.
  • the obtained PCR amplified fragment was subjected to agarose gel electrophoresis, and then a probe for 5'-side genomic Southern blot (5'KO-probe) of about 500 bp was recovered using QIAquick Gel Extraction Kit (manufactured by Qiagen).
  • the obtained PCR amplified fragment was subjected to agarose gel electrophoresis, and then about 500 bp of 3′-side genomic Southern blot probe (3′KO-probe) was recovered using QIAquick Gel Extraction Kit (manufactured by Qiagen).
  • Example 4 Obtaining mouse BMP9 KO ES cell line
  • mouse BMP9 gene KO ES cells using homologous recombination according to established methods (Shinichi Aizawa, Biomanual Series 8, Gene Targeting, Yodosha, 1995) PBluemBmp9-Lox-Neo-DT-A-3′KO-5′KO-NotI prepared in Example 2 was introduced into mouse ES cell TT2 (Yagi et al., Analytical Biochem., 214: 70, 1993). A detailed method is described below.
  • G418-resistant primary cultured cells manufactured by Invitrogen
  • mitomycin C manufactured by Sigma
  • vegetative cells were used to grow TT2 cells under conditions of 37 ° C. and 5% CO 2 .
  • TT2 cells were trypsinized and suspended in the HBS solution described in Example 2 to 3 ⁇ 10 7 cells / mL.
  • the electroporated cells were treated with ES medium [180 mL of fetal bovine serum (FBS), 3.5 g of D-glucose, 10 g of Dulbecco's modified Eagle medium powder (manufactured by Invitrogen), a non-essential amino acid solution (100-fold concentrated solution).
  • FBS fetal bovine serum
  • D-glucose Dulbecco's modified Eagle medium powder
  • Dulbecco's modified Eagle medium powder manufactured by Invitrogen
  • a non-essential amino acid solution 100-fold concentrated solution.
  • a solution containing 10 mL and 1.9 g of sodium bicarbonate suspended in 10 mL, and seeded on one 100 mm plastic petri dish for tissue culture (manufactured by Falcon) previously seeded with the above-mentioned vegetative cells.
  • ES medium containing 200 ⁇ g / mL neomycin (manufactured by Sigma). Colonies generated after 7 days were picked up and seeded in 24-well plates. After growing to confluence, one-third of the cells were seeded in a 12-well gelatin-coated plate and cultured for 2 days, so that genomic DNA was purified from 10 6 to 10 7 cells using Puregene DNA Isolation Kits ( (Manufactured by Gentra System). These neomycin-resistant TT2 cell genomic DNAs were enzymatically digested with EcoRI and subjected to agarose gel electrophoresis.
  • Example 3-2 Southern blotting using 3'KO-probe obtained in Example 3-2 was performed.
  • One band (about 15.5 kbp) was detected in wild-type TT2 cells, and two bands (about 11.5 kbp and about 15.5 kbp) were detected in homologous recombinants.
  • the genomic DNA of clones confirmed to have homologous recombination was enzymatically digested with NcoI and subjected to agarose gel electrophoresis.
  • Example 3-1 Southern blotting using 5'KO-probe obtained in Example 3-1 was performed.
  • One band (about 13.4 kbp) was detected in wild-type TT2 cells, and two bands (about 8.5 kbp and about 13.4 kbp) were detected in homologous recombinants.
  • Example 5 Production of BMP9 KO heterozygous mice ES cells obtained in Example 4 are TT2 cells derived from dark brown CBA ⁇ C57BL / 6 F1 mice (Yagi et al., Analytical Biochemistry, 214: 70-76, 1993). . Therefore, in order to easily distinguish between a chimeric mouse having an ES cell-derived gene and a host mouse not having an ES cell-derived gene, an ICR mouse having a white coat color was selected as the host mouse.
  • mice of mouse BMP9 gene KO ES cells that were confirmed to have normal karyotype in Example 4 were transferred to 8 cell stage embryos obtained by mating male and female mice of the ICR strain. 8-10 were injected. After that, the embryos were developed overnight using ES medium, so that the injection embryos were developed up to the blastocyst, and then the uterus on one side was placed on the uterus of the temporary parent ICR mouse (CLEA Japan) 2.5 days after the pseudopregnancy treatment. Injection embryos that had been developed to about 10 blastocysts per each were transplanted.
  • mice As a result of transplanting a total of 260 injection embryos that had developed to blastocysts, 102 offspring chimeric mice were born. Of the 102 mice born, 89 mice had dark brown hair color, that is, chimeric mice in which ES cells contributed. Of these 89 mice, 40 were mice whose hair color was all dark brown and did not contain a white portion, that is, chimeric mice with a high chimera rate derived from ES cells. Next, as a result of mating these male chimeric mice with a high chimera rate with C57BL / 6 females, a brown mouse was born, confirming the transmission of the ES cell genome to the germline It was done.
  • mBMP9_FW5915 which is a primer specific for the neomycin resistance gene region in the mouse BMP9 gene knockout vector pBluemBmp9-Lox-Neo-DT-A-3′KO-5′KO prepared in Example 1, is used. 13) and mBMP9_RV17165 (SEQ ID NO: 14) were prepared.
  • Example 6 Production of BMP9KO homozygous mice Male and female individuals of BMP9KO (+/-) produced in Example 5 were mated to obtain offspring mice. In the same manner as in Example 5, genomic DNA was extracted from the tail of the mouse using Puregene DNA Isolation Kit. PCR was performed in the same manner as in Example 5 using this genomic DNA as a template. Furthermore, BMP9KO heterozygous [BMP9KO (+/ ⁇ )] and homozygous [BMP9KO ( ⁇ / ⁇ )] were selected by performing agarose electrophoresis.
  • a reaction solution containing 10 pmol each of the primers of SEQ ID NOS: 15 and 16 and EX Taq (manufactured by Takara Shuzo Co., Ltd.) was prepared using the genomic DNA of the selected individual as a template, 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, And 35-cycle PCR was performed with one minute at 72 ° C. as one cycle.
  • Example 7 Preparation of anti-human BMP9 monoclonal antibody 7-1) Preparation of immunogen The human BMP9 recombinant protein used as the immunogen was prepared according to the method described in Example 12 of WO2010 / 126169.
  • Example 7-2 Immunization to animals and preparation of antibody-producing cells 9 pairs of human BMPs prepared in Example 7-1 using RIBI adjuvant (Corixa) or Titer Max Gold (Titer Max) as an adjuvant
  • RIBI adjuvant Corixa
  • Titer Max Gold Titer Max
  • the suspension was added to KO mice so that 25 ⁇ g of human BMP9 recombinant protein was administered per mouse. It was administered intraperitoneally.
  • the suspension is KO so that 25 ⁇ g of human BMP9 recombinant protein is administered per animal. Mice were administered subcutaneously. Immunization was performed three times in total including the final boost. The spleen was removed 3 days after the last dose.
  • the excised spleen was chopped in PBS (phosphate-buffered saline), and the spleen cells were collected by centrifugation (1500 rpm, 3 minutes). Since the obtained spleen cell fraction contains red blood cells, RED Blood Cell Lysing Buffer (manufactured by Sigma) was added, and the red blood cells were removed by processing on ice. The obtained splenocytes were washed twice with DMEM (Dulbecco's modified Eagle's Medium; manufactured by Invitrogen) and then subjected to cell fusion.
  • PBS phosphate-buffered saline
  • mice myeloma cells A mouse myeloma cell line (Sp2 / 0, ATCC: CRL1581) was cultured in DMEM supplemented with 10% fetal bovine serum and used as a parent strain for cell fusion.
  • Example 7-4) Preparation of hybridoma
  • the mouse spleen cells obtained in Example 7-2 and the myeloma cells obtained in Example 7-3 were mixed at a ratio of 5: 1 and centrifuged (1500 rpm, 3 minutes). did.
  • DMEM medium containing FCS 10 v / v%, ⁇ -mercaptoethanol 50 ⁇ mol / L, insulin 50 ⁇ g / mL, and IL-6 10 ng / mL. After suspending so that the number became 1 ⁇ 10 6 cells / mL, 100 ⁇ L each was seeded in a 96-well plate.
  • HAT media supplement Sigma, Cat # H0262-10VL
  • the culture was performed under the following conditions. The medium was exchanged three times a week using a complete medium containing a final concentration of HATmedia supplement recommended by the manufacturer until the number of cells in the well was suitable for screening.
  • ALK1 / BMP9 sandwich ELISA is an anti-human BMP9 mouse monoclonal antibody (R & D antibody) and human ALK1 extracellular region human IgG1 Fc known as type I receptor for human BMP9.
  • the region fusion protein (hsALK1-Fc) was used to construct as follows.
  • hsALK1-Fc prepared according to the method described in Example 1 of WO2010 / 126169 was prepared on a 96-well plate for ELISA (F96 MAXISORP NUNC-IMMUNO PLATE, manufactured by Thermo Fisher Scientific, Cat # 439454).
  • 50 mmol / L NaHCO 3 buffer (Wako, Cat # 191-01305) adjusted to 0.05 ⁇ g / mL was dispensed at 100 ⁇ L / well and allowed to stand overnight at 4 ° C. for adsorption. .
  • human nature dimer BMP9 recombinant protein R & D Systems Cat # 3209-BP
  • SuperBlock and PBS-T at a ratio of 1: 9 at a concentration of 1 ng / mL in 10% SuperBlock in PBS-T.
  • the prepared solution was dispensed at 100 ⁇ L / well, allowed to stand at room temperature for 1 hour, and washed 4 times with PBS-T.
  • the plate was washed 4 times with PBS-T, and then Streptavidin-polyHRP80 (Stereospecific Detection Technologies, Cat # SP80D50) diluted 500 times with 10% SuperBlock in PBS-T was dispensed at 100 ⁇ L / well at room temperature. It was allowed to stand for 30 minutes to 1 hour.
  • TMB coloring solution TMB + Substrate-Chromogen, Dako, Cat # S1599.
  • TMB coloring solution TMB + Substrate-Chromogen, Dako, Cat # S1599.
  • 1N sulfuric acid was obtained.
  • a solution (Wako, Cat # 192-04755) was added at 100 ⁇ L / well, and absorbance at 450 nm was measured using ARVO (Perkin Elmer).
  • the well in which the color development was suppressed was judged to be positive, and hsALK1-Fc and human nature dimer BMP9 recombinant protein or human nature dimer BMP9 recombinant protein and R & D antibody Hybridoma strains producing antibodies that inhibit the interaction were selected.
  • the selected hybridomas were subjected to limiting dilution in a complete medium containing HT (manufactured by Sigma, Cat # H0137-10VL), seeded in a 96-well plate, and cloned. Cloning was performed a total of three times for well-derived hybridomas that were judged positive in the first round. Through the above operation, hybridoma strains producing 3B7-3-3 antibody and 3C7-3-1 antibody having human BMP9 neutralizing activity were isolated.
  • the hybridomas producing the 3B7-3-3 antibody and the 3C7-3-1 antibody obtained in Example 7-6 were scaled up using a complete medium for antibody acquisition, and then the medium was mixed with CD hybridoma medium (Invitrogen). And cultured at 37 ° C. for 6 days.
  • the cells were removed from the culture supernatant by filtration.
  • the anti-human BMP9 antibody was purified from the supernatant from which the cells had been removed, using an open column filled with Protein G Sepharose 4 Fast Flow (GE Healthcare).
  • the obtained antibody was sterilized using a 0.22 ⁇ m filter and then subjected to an in vitro test.
  • a human well dimer BMP9 recombinant protein (R & D Systems, Cat # 3209-BP) or a 96-well ELISA plate (F96 MAXISORP NUNC-IMMUNO PLATE, Thermo Fisher Scientific, Cat # 442404)
  • a human nature dimer BMP10 recombinant protein (R & D Systems Cat # 2926-BP) prepared with 50 mmol / L NaHCO 3 buffer (Wako, Cat # 191-01305) at 100 ng / mL was prepared at 50 ⁇ L / mL.
  • the wells were dispensed and allowed to stand overnight at 4 ° C. for adsorption.
  • Super Block (Thermo SCIENTIFIC, Cat # 37535) was added at 200 ⁇ L / well, allowed to stand at room temperature for 1 hour for blocking, and TBST (Tris Buffered Saline with Tween 20; SANTA CRUZ) Manufactured by SC-24953).
  • TBST Tris Buffered Saline with Tween 20; SANTA CRUZ
  • 3B7-3-3 antibody, 3C7-3-1 antibody, R & D antibody, or anti-BMP10 antibody (R & D Systems, Inc.) labeled with biotin using Biotin Labeling Kit-NH2 (Cat # LK03, manufactured by Cosmo Bio) Cat # MAB2926) was adjusted to 100 ng / mL with 10% Super Block in TBST in which SuperBlock and TBST were mixed at a ratio of 1: 9, and then dispensed at 50 ⁇ L / well as the primary antibody for 1 hour at room temperature. Left to stand.
  • Streptavidin-polyHRP80 (Stereospecific Detection Technologies, Cat # SP80D50) diluted 500-fold with 10% Super Block in TBST was dispensed at 100 ⁇ L / well for 1 hour at room temperature. I put it.
  • TMB substrate solution TMB + Substrate-Chromogen, Dako, Cat # S1599
  • 1N sulfuric acid solution 50 ⁇ L / well of Wako, Cat # 192-04755
  • the absorbance at a sample wavelength of 450 nm and a reference wavelength of 570 nm was measured using a plate reader (Spectra Max, manufactured by Molecular Devices).
  • a 96-well plate for ELISA F96 MAXISORP NUNC-IMMUNO PLATE, Thermo Fisher Scientific, Cat # 439454
  • human mature dimer BMP9 recombinant protein R & D Systems, Cat # 3209-BP
  • a solution adjusted to 100 ng / mL with / L NaHCO 3 buffer was dispensed at 50 ⁇ L / well and allowed to stand overnight at 4 ° C. for adsorption.
  • Super Block (Thermo SCIENTIFIC, Cat # 37535) was added at 200 ⁇ L / well, allowed to stand at room temperature for 1 hour, blocked, and washed 3 times with TBST.
  • biotin-labeled 3B7-3-3 antibody, 3C7-3-1 antibody or R & D antibody adjusted to 10% Super Block in TBST to be 1, 5, 20, 100, and 300 ng / mL, respectively.
  • Streptavidin-polyHRP80 diluted 500-fold with 10% Super Block in TBST was dispensed at 100 ⁇ L / well and allowed to stand at room temperature for 1 hour.
  • TMB substrate solution TMB + Substrate-Chromogen, Dako, Cat # S1599
  • 1N sulfuric acid solution (Wako) Cat # 192-0455) was added at 50 ⁇ L / well, and the absorbance (450 nm-570 nm) at a sample wavelength of 450 nm and a reference wavelength of 570 nm was measured using a plate reader (Spectra Max, manufactured by Molecular Devices).
  • Example 10 Action of Acquired Antibody on Binding of Human BMP9 and R & D Antibody Based on the characteristics of the system used in Example 7, the obtained antibody is an interaction between hsALK1-Fc and human nature dimeric BMP9 recombinant protein, or human nature dimeric BMP9 recombination. It is considered that the antibody inhibits any interaction between the protein and the R & D antibody. Therefore, first, a binding assay system between human BMP9 and R & D antibody was constructed, and the action of the obtained antibody on the binding between human BMP9 and R & D antibody was examined.
  • a 96-well ELISA plate (F96 MAXISORP NUNC-IMMUNO PLATE, Thermo Fisher Scientific, Cat # 439454) was prepared by adding 100 ng / L of the human BMP9 recombinant protein prepared in Example 7-1 with 50 mmol / L NaHCO 3 buffer. What was adjusted to mL was dispensed at 100 ⁇ L / well and allowed to stand at room temperature for 1 hour for adsorption.
  • This plate was washed 4 times with PBS-T, and then Streptavidin-polyHRP80 (Stereospecific Detection Technologies, Cat # SP80D50) diluted 500 times with 10% Super Block in PBS-T was dispensed at 100 ⁇ L / well. For 30 minutes to 1 hour.
  • TMB coloring solution TMB + Substrate-Chromogen, manufactured by Dako, Cat # S1599.
  • TMB coloring solution TMB + Substrate-Chromogen, manufactured by Dako, Cat # S1599.
  • a solution Wako, Cat # 192-04755 was added at 100 ⁇ L / well, and absorbance at 450 nm was measured using ARVO (Perkin Elmer). The results are shown in FIG.
  • Example 11 Epitope analysis of acquired antibody by enzyme-linked immunosorbent assay (ELISA) using solid phase antigen In order to further clarify the difference in the properties of the acquired antibody, it was acquired using enzyme-linked immunosorbent assay (ELISA) The epitope analysis of the antibody was performed. Specifically, when the biotin-labeled antibody was reacted with the solid phase BMP9, an excess amount of each non-biotin-labeled antibody was added simultaneously.
  • the reaction when the reaction is attenuated by the unlabeled antibody, it is suggested that the epitopes of the labeled antibody and the unlabeled antibody are the same or in the vicinity. Furthermore, it is suggested that the properties of labeled and unlabeled antibodies are the same or similar.
  • the assay was performed in the same manner as in Example 9. However, based on the results of Example 9, biotin-labeled 3B7-3-3 antibody, 3C7-3-1 antibody, and R & D antibody were used so that the final absorbance was about 1.0 to 1.5. Reactions were made at final concentrations of 30.0, 4.0 and 5.0 ng / mL, respectively. Furthermore, simultaneously with the addition of these labeled antibodies, unlabeled antibodies were added at a final concentration of 10 ⁇ g / mL, respectively.
  • Example 12 The effect of the obtained antibody on the binding of human BMP9 and human ALK1 From the results of Example 10, the 3B7-3-3 antibody and the 3C7-3-1 antibody are not an interaction between the human nature dimer BMP9 recombinant protein and the R & D antibody. This suggests the possibility of an antibody that suppresses the interaction between human nature dimer BMP9 recombinant protein and hsALK1-Fc. Therefore, a binding assay system for human BMP9 and human ALK1 was constructed, and the action of the obtained antibody on human BMP9 and human ALK1 binding was examined.
  • a 96-well ELISA plate (F96 MAXISORP NUNC-IMMUNO PLATE, Thermo Fisher Scientific, Cat # 439454) was prepared by adding 100 ng / L of the human BMP9 recombinant protein prepared in Example 7-1 with 50 mmol / L NaHCO 3 buffer. What was diluted to mL was dispensed at 100 ⁇ L / well and allowed to stand at room temperature for 1 hour for adsorption. After removing the solid phase, Super Block was added at 250 ⁇ L / well, allowed to stand at room temperature for 1 hour to block, and washed 3 times with PBS-T.
  • This plate was washed 4 times with PBS-T, and then Streptavidin-polyHRP80 (Stereospecific Detection Technologies, Cat # SP80D50) diluted 500 times with 10% Super Block in PBS-T was dispensed at 100 ⁇ L / well. For 30 minutes to 1 hour.
  • TMB coloring solution TMB + Substrate-Chromogen, manufactured by Dako, Cat # S1599.
  • TMB coloring solution TMB + Substrate-Chromogen, manufactured by Dako, Cat # S1599.
  • 1N sulfuric acid was obtained.
  • a solution (Wako, Cat # 192-04755) was added at 100 ⁇ L / well, and absorbance at 450 nm was measured using ARVO (Perkin Elmer).
  • the inhibitory activity (%) of each antibody was calculated by setting the absorbance of the well to which only biotin-labeled human ALK1-Fc was added to 0% and the absorbance of the well to which only the diluent was added was 100%. The results are shown in FIG.
  • the binding between human BMP9 and human ALK1-Fc was inhibited by the addition of 3B7-3-3 antibody and 3C7-3-1 antibody.
  • addition of R & D antibody was not inhibited.
  • the 3B7-3-3 antibody and the 3C7-3-1 antibody are anti-BMP9 antibodies that inhibit the binding between human BMP9 and human ALK1, and the R & D antibody inhibits the binding between human BMP9 and human ALK1.
  • the anti-BMP9 antibody does not.
  • Binding activity of the obtained antibody to human BMP9 recombinant protein (Biacore analysis) The binding activity (affinity) of the 3C7-3-1 antibody and R & D antibody to human BMP9 recombinant protein was examined using BIAcore 2000 (GE Healthcare). An anti-mouse IgG antibody was immobilized on the sensor chip CM5 using a Mouse Antibody Capture Kit (manufactured by GE Healthcare), and then the anti-human BMP9 antibody was bound to an RU value of 700 to 1100.
  • the human BMP9 recombinant protein prepared in Example 7-1 was 0.152 nmol / L, 0.457 nmol / L, 1.37 nmol / L, 4.1 nmol using an HBS-EP solution (GE manufactured by Healthcare).
  • / L, 12.3 nmol / L, 36.89 nmol / L, 110.67 nmol / L, and 322 nmol / L were added as analytes, and the bond strength was measured.
  • the bond dissociation constant (Kd value) was calculated by a single cycle kinetics calculation method (BIAevaluation Software ver.3, manufactured by GE Healthcare) based on the obtained value. The results are listed in Table 1.
  • the Kd value of the R & D antibody was 6.16 ⁇ 10 ⁇ 10 mol / L, whereas the Kd value of the 3C7-3-1 antibody was 2.64 ⁇ 10 ⁇ 11 mol / L. It was revealed that the binding activity of the 3-1 antibody to human BMP9 was 23.3 times as high as that of the R & D antibody.
  • Example 14 Inhibitory action of the obtained antibody on BMP9-dependent ALK1-derived signal A human ALK1-expressing reporter cell line was prepared, and the inhibitory action of the obtained antibody on the human BMP9-dependent human ALK1-derived signal was evaluated.
  • a human ALK1 expression reporter cell is a BMP signal detection strain described in Example 2 of WO2010 / 126169 [p (GCCG) 12-Luc / HepG2 (38. 5)] was forcibly expressed by the full-length human ALK1 gene [ALK1 / p (GCCG) 12-Luc / HepG2 (38.5)].
  • the human ALK1 expression vector was prepared by integrating a full-length cDNA of human ALK1 having EcoRI and NotI restriction enzyme sites at both ends with EcoRI and NotI into a pEAK8 expression vector (manufactured by Edge Biosystems).
  • Human ALK1 full-length cDNA (cDNA Genbank accession number: BC042637.1, SEQ ID NO: 17, amino acid sequence GenBank accession number: AAH42637, SEQ ID NO: 18) is a human lung cDNA library and primers (SEQ ID NOs: 19 and 20). ).
  • Bif9-dependent ALK1-derived signal of each antibody with Luciferase activity of wells containing only human nature dimer BMP9 recombinant protein as 0%, Luciferase activity of wells containing only 0.1% BSA-containing DMEM medium as 100% Inhibitory activity (%) was calculated by the following formula.
  • BMP9-dependent ALK1-derived signal inhibitory activity [(Luciferase activity in wells containing only human nature dimer BMP9 recombinant protein) ⁇ (well in human well dimer BMP9 recombinant protein and anti-human BMP9 antibody added) Luciferase activity)] / [(Luciferase activity in wells containing only human nature dimer BMP9 recombinant protein)-(Luciferase activity in wells containing only 0.1% BSA-containing DMEM medium)] ⁇ 100
  • the 3C7-3-1 antibody like the R & D antibody, suppresses the BMP9-dependent ALK1-derived signal and increases its neutralizing activity against human BMP9 as it is added at a higher concentration.
  • the inhibitory activity was as weak as about 1/16 compared to the R & D antibody.
  • the 3C7-3-1 antibody has a higher affinity for human BMP9 than the R & D antibody (Example 13), recognizes an epitope different from that of the R & D antibody (Examples 10 and 11), and R & D. It was revealed that the inhibitory activity against the signal derived from human BMP9-dependent ALK1 was weaker than that of the antibody.
  • PCR was performed using the human ALK2 gene as a template and hALK2 RP (SEQ ID NO: 23) and hALK2 G617A FP (SEQ ID NO: 24) as primers.
  • hALK2 RP SEQ ID NO: 23
  • hALK2 G617A FP SEQ ID NO: 24
  • the obtained PCR fragment was ligated with a pGEMT-easy vector (manufactured by Promega) and then introduced into E. coli DH5 ⁇ strain.
  • the plasmid vector was recovered from this transformed clone, and the insertion of the PCR fragment was confirmed. Furthermore, substitution of the mutation site was confirmed by sequence analysis.
  • FOP-ALK2 cDNA, wild-type hALK2 cDNA, and pTracerCMV vector manufactured by Invitrogen whose substitution was confirmed were subjected to restriction enzyme treatment using EcoRI and NotI, and then subjected to agarose gel electrophoresis, and each fragment was subjected to QIAquick. It refine
  • the obtained FOP-ALK2 cDNA fragment and wild type hALK2 cDNA were each ligated with the pTracerCMV vector and then introduced into DH5 ⁇ E. coli.
  • Each clone (FOP-ALK2 / pTracerCMV or wild type hALK2 / pTracerCMV) that was confirmed to contain the target cDNA was prepared in large quantities and subjected to the following experiment.
  • Example 16 Effect of serum concentration on bone differentiation of FOP-ALK2 gene-introduced C2C12 cells (in vitro test) C2C12 cells were seeded in 96-well plates at 4 ⁇ 10 3 cells / well. The next day, first, 0.2 ⁇ g of FOP-ALK2 / pTracerCMV, wild type hALK2 / pTracerCMV, or control vector (pTracerCMV) was diluted in 20 ⁇ L of Opti-MEM I reduced serum medium (manufactured by Invitrogen), and after 0 minutes .05 ⁇ L of PLUS reagent (Invitrogen) was added and left at room temperature for 5 minutes.
  • Opti-MEM I reduced serum medium manufactured by Invitrogen
  • PLUS reagent Invitrogen
  • Lipofectamine LTX manufactured by Invitrogen
  • the medium was replaced with a DMEM medium containing 5% or 20% FCS, and cultured for about 90 hours. Then, after removing the supernatant and washing with 200 ⁇ L of TBS [20 mM Tris (pH 7.4), 150 mM NaCl], 100 ⁇ L of 0.1 M Tris solution (pH 7.4) containing 0.1% Triton X-100 was added, The cells were lysed by stirring for 10 minutes with a plate mixer.
  • the cell lysate was collected in a 96-well plate, centrifuged at 3000 rpm for 10 minutes, and then the supernatant was collected so as not to suck the precipitate fraction, and subjected to alkaline phosphatase (ALP) activity measurement.
  • ALP alkaline phosphatase
  • ALP activity ALP kit (manufactured by WAKO) was used for ALP activity measurement. Specifically, 100 ⁇ L of the reaction solution attached to the ALP kit was added to 20 ⁇ L of the collected supernatant solution and reacted for 30 minutes, and then the absorbance at 414 nm was measured with a microplate reader. The results are shown in FIG.
  • Example 17 Effect of anti-human BMP9 neutralizing antibody (R & D antibody) on bone differentiation of FOP-ALK2 transgenic C2C12 cells (in vitro test) Involvement of BMP9 in bone differentiation of FOP-ALK2 gene-introduced C2C12 cells by adding 3 ⁇ g / mL R & D antibody in the presence of 20% FCS in which increased ALP activity (induction of bone differentiation) occurs in FOP-ALK2 gene-introduced C2C12 cells I investigated. The results are shown in FIG.
  • Example 18 Effect of anti-human BMP9 antibody on bone differentiation of C2C12 cells stably expressing FOP-ALK2 (in vitro test) 18-1) Preparation of FOP-ALK2 stably expressing C2C12 cell line and examination of reactivity to BMP9 ALP increase in the presence of 20% FCS was observed only in C2C12 cells into which FOP-ALK2 was introduced. This suggests that FOP-ALK2 may be more responsive to BMP9 than wild-type hALK2. Therefore, C2C12 cells that stably express FOP-ALK2 were first prepared.
  • the FOP-ALK2 / pTracerCMV, wild type hALK2 / pTracerCMV and pTracerCMV prepared in Example 15 were enzymatically digested with ScaI, and each 8 ⁇ g of the single-stranded plasmid was 400 ⁇ L of Opti-MEM I reduced serum medium (Invitrogen). Dilute to After 5 minutes, 2.0 ⁇ L of PLUS reagent (manufactured by Invitrogen) was added, and the mixture was further left at room temperature for 5 minutes.
  • Lipofectamine LTX manufactured by Invitrogen
  • 4.8 ⁇ L of Lipofectamine LTX was added, allowed to stand at room temperature for 30 minutes, and then added about 440 ⁇ L each to a 6-well plate seeded at 1.5 ⁇ 10 5 C2C12 cells. And incubated at 37 ° C. for 24 hours.
  • the cells were collected from the plate and suspended in DMEM medium containing 150 ⁇ g / mL Zeocin (manufactured by Invitrogen), 1 ⁇ g / mL hsALK1-Fc and 10% FCS. Each suspension cell was seeded on a new plate, and drug selection was performed.
  • each cell was suspended in DMEM medium containing 5% FCS and seeded in a 96-well plate at 4 ⁇ 10 3 cells / well.
  • ALP activity ALP kit (manufactured by WAKO) was used for ALP activity measurement. Specifically, 100 ⁇ L of the reaction solution attached to the ALP kit was added to 20 ⁇ L of the above-mentioned supernatant and reacted for 30 minutes, and then the absorbance at 414 nm was measured with a microplate reader. The results are shown in FIG.
  • FOP-ALK2 stably expressing C2C12 cells were found to have enhanced responsiveness to human BMP9 compared to cells introduced with control vectors and cells transfected with wild-type hALK2.
  • FOP-ALK2 stably expressing C2C12 cells were suspended in DMEM medium containing 1 ⁇ g / mL hsALK1-Fc and 5% FCS, and seeded at 4 ⁇ 10 3 cells / well in a 96-well plate.
  • ALP activity ALP kit (manufactured by WAKO) was used for ALP activity measurement. Specifically, 100 ⁇ L of the reaction solution attached to the ALP kit was added to 20 ⁇ L of the above-mentioned supernatant and reacted for 30 minutes, and then the absorbance at 414 nm was measured with a microplate reader. The results are shown in FIG.
  • the 3C7-3-1 antibody selectively suppresses bone differentiation based on BMP9-dependent FOP-ALK2-derived signals among BMP9-dependent BMP type I receptor-derived signals. It became clear.
  • ALK1 is an important receptor for maintaining the function of the vascular endothelium
  • the 3C7-3-1 antibody is less susceptible to side effects than the R & D antibody, which has no selectivity for the ALK1-derived signal and the FOP-ALK2-derived signal. This suggests the possibility of being useful as a therapeutic agent.
  • Example 19 Isolation of gene sequences encoding VH and VL of anti-human BMP9 monoclonal antibody 19-1) Preparation of total RNA from hybridoma cells producing anti-human BMP9 monoclonal antibody 3B7-3-3 antibody and 3C7- described in Example 7 Total RNA was prepared from 1 ⁇ 10 6 hybridomas producing 3-1 antibody using RNAeasy Mini kit (QIAGEN, Cat # 74104) and QIA shredder (QIAGEN, Cat # 79654).
  • primers specific to mouse IgG1 SEQ ID NOs: 25 and 26
  • primers specific to mouse IgG2a SEQ ID NOs: 27 and 28
  • primers specific to mouse IgG2b SEQ ID NOs: 29 and 30
  • VH cDNA of each antibody was amplified.
  • PCR was also carried out by combining mouse Ig ( ⁇ ) -specific primers (SEQ ID NOs: 34 and 35) or mouse Ig ( ⁇ ) -specific primers (SEQ ID NOs: 36 and 37) with universal primer A, respectively.
  • the VL cDNA fragment of each antibody was amplified.
  • PCR consists of 5 reaction cycles consisting of 94 ° C for 30 seconds and 72 ° C for 3 minutes, 94 ° C for 30 seconds, 70 ° C for 30 seconds, 72 ° C for 3 minutes, 5 cycles at 94 ° C.
  • a reaction cycle consisting of 30 seconds, 68 ° C. for 30 seconds and 72 ° C. for 3 minutes was performed 25 times.
  • the 3B7-3-3 antibody-producing hybridoma-derived cDNA yielded a PCR amplification product when a specific primer encoding the IgG2c heavy chain constant region was used.
  • a PCR amplification product was obtained when a specific primer encoding the IgG1 heavy chain constant region was used.
  • cDNAs derived from both hybridomas also yielded PCR amplification products when using mouse Ig ( ⁇ ) specific primers.
  • Each PCR amplification product was purified using Gel Extraction Kit (QIAEX II, manufactured by QIAGEN, Cat # 20021).
  • the obtained gene fragment was inserted into a pCR4 vector (manufactured by Invitrogen) using Zero Blunt TOPO PCR Cloning Kit for Sequencing (manufactured by Invitrogen, Cat # K287540SP).
  • the resulting plasmid was introduced into E. coli DH5 ⁇ strain.
  • a plasmid was extracted from the obtained transformant using an automatic plasmid extractor (manufactured by Kurabo Industries), and the nucleotide sequence was analyzed. As a result, it was confirmed that full-length VH cDNA and VL cDNA having an ATG sequence presumed to be the initiation codon at the 5 'end of the cDNA were obtained.
  • the base sequence obtained by removing the signal sequence from SEQ ID NOs: 38 and 39 is shown in SEQ ID NOs: 46 and 47
  • the base sequence obtained by removing the signal sequence from SEQ ID NOs: 42 and 43 is shown in SEQ ID NOs: 48 and 49
  • the amino acid sequences excluding the signal sequence are shown in SEQ ID NOs: 50 and 51
  • the amino acid sequences obtained by removing the signal sequence from SEQ ID NOs: 44 and 45 are shown in SEQ ID NOs: 52 and 53, respectively.
  • the CDRs of VH and VL of each monoclonal antibody were identified by comparing with the amino acid sequences of known antibodies.
  • the amino acid sequences of CDR1, CDR2, and CDR3 of VH of the 3B7-3-3 antibody are shown in SEQ ID NOs: 54, 55, and 56, and the amino acid sequences of CDR1, CDR2, and CDR3 of VL are shown in SEQ ID NOs: 57, 58, and 59, respectively.
  • amino acid sequences of VH CDR1, CDR2 and CDR3 of the 3C7-3-1 antibody are shown in SEQ ID NOs: 60, 61 and 62, and the amino acid sequences of CDR, CDR2 and CDR3 of VL are shown in SEQ ID NOs: 63, 64 and 65, respectively.
  • Example 20 Epitope analysis of anti-human BMP9 antibody 20-1) Preparation of recombinant 3C7-3-1 antibody
  • the nucleotide sequence of the light chain and heavy chain variable region (including signal sequence) of 3C7-3-1 antibody was determined using mouse light chain ( ⁇ Chain) and heavy chain (IgG1) constant regions, respectively, and subcloned into an antibody expression vector.
  • primers comprising the nucleotide sequences represented by SEQ ID NOs: 72 to 75 using the vector prepared in Example 19 as a template PCR was performed using
  • Amplification of the base sequence of the mouse light chain ( ⁇ chain) and heavy chain (IgG1) constant region was performed using the base sequences shown by SEQ ID NOs: 80 and 81 prepared by artificial gene synthesis (Takara Bio Inc.) as templates, respectively. PCR was performed using primers consisting of the nucleotide sequences shown in SEQ ID NOs: 76 to 79.
  • PCRs were performed using PrimeSTAR HS (Premix) (Takara Bio, R040A), kept at 96 ° C. for 2 minutes, then 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 to 2 minutes. The reaction was carried out for 30 cycles (adjusted according to the size of the amplified product) as one cycle.
  • the obtained PCR amplification step was subjected to agarose gel electrophoresis and then purified using a QIAquick Gel Extraction Kit to obtain an insert.
  • N5KG1 vector Biogen IDEC
  • BglII and EcoRI was enzymatically treated with BglII and EcoRI and purified by the same procedure as above to obtain an insert insertion vector.
  • the inserts of the light chain variable region and mouse light chain constant region of the 3C7-3-1 antibody were incorporated into an N5KG1 vector that had been enzymatically treated using In-fusion Advantage PCR Cloning Kit (manufactured by Takara Bio Inc.) and introduced into Escherichia coli.
  • plasmid DNA into which the correct sequence was inserted was selected.
  • This plasmid DNA was enzymatically digested with SalI and BamHI and purified by the same procedure as described above.
  • the inserts of the heavy chain variable region and mouse heavy chain constant region of the 3C7-3-1 antibody were incorporated into the above-described vector in which the light chain had already been inserted, and introduced into E. coli. From the obtained transformant, an antibody expression vector in which both a light chain and a heavy chain were inserted was selected. By sequencing, it was confirmed that the 3C7-3-1 antibody (mouse IgG1, ⁇ ) sequence was inserted into this vector.
  • Escherichia coli was transformed with the prepared 3C7-3-1 antibody expression vector, and a vector was prepared by NucleoBond Xtra Maxi (manufactured by Takara Bio Inc., U0414B). FreeStyle 293 Expression System (manufactured by Life Technologies Co., Ltd. was used for transient expression to express the recombinant antibody.
  • the culture supernatant was obtained from the culture solution by centrifugation and filtration using a 0.22 ⁇ m filter.
  • the antibody was purified by affinity chromatography using Protein G Sepharose 4 Fast Flow (manufactured by GE Healthcare).
  • the buffer was replaced with a citrate buffer (10 mM citrate-NaOH (pH 6.0), 150 mM NaCl) using a NAP-25 column (manufactured by GE Healthcare).
  • the concentration was determined by measuring the absorbance at 280 nm. As the molecular extinction coefficient, 1.4 mL / (mg ⁇ cm) was used.
  • the human BMP9 feature region (SEQ ID NO: 67) was divided into three regions and defined as human BMP9 feature regions A, B, and C, respectively.
  • human BMP9 feature region A is the amino acid sequence from the first to the 37th amino acid sequence of human BMP9 feature region represented by SEQ ID NO: 67
  • human BMP9 feature region B is the human sequence represented by SEQ ID NO: 67.
  • the amino acid sequence is from the 38th to the 74th amino acid sequence of the BMP9 nature region
  • the human BMP9 feature region C is the 75th to 110th amino acid sequence of the human BMP9 feature region represented by SEQ ID NO: 67.
  • three corresponding regions (BMP10 feature regions A, B, and C) were defined based on the homology of the primary structure with BMP9.
  • human BMP10 feature region A is the first to 36th amino acid sequence of human BMP10 feature region represented by SEQ ID NO: 96
  • human BMP10 feature region B is human represented by SEQ ID NO: 96.
  • the amino acid sequence from the 37th to the 73rd position of the BMP10 feature region, and the human BMP10 feature region C is the amino acid sequence from the 74th to the 108th position of the human BMP10 feature region represented by SEQ ID NO: 96.
  • the amino acids contained in each region of human BMP9 and BMP10 are represented by SEQ ID NOs: 82 to 87.
  • the chimeric proteins A, B and C were designed by substituting the amino acid residues of regions A, B and C of the BMP9 nature region with the amino acid residues of BMP10 corresponding to BMP9.
  • the amino acid sequences of BMP9 / 10 chimeric proteins A, B and C designed in this way are shown in SEQ ID NOs: 88 to 90.
  • a human BMP10 expression vector was prepared by the following procedure.
  • An N-terminal His-type hBMP9 complex recombinant expression vector (hereinafter referred to as pLN1V5_hBMP9) described in Example 12 of International Publication No. 2010/126169 and FIG. 7 is treated with NheI and XhoI, and subjected to agarose gel electrophoresis. Then, it was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
  • PCR was performed using a combination of Fwd-1 / Rv-1 and Fwd-2 / Rv-2 primers shown in SEQ ID NOs: 91 to 94, using Human Heart PCR-Ready cDNA (Ambion, 3326) as a template. .
  • PCR was performed using PrimeSTAR HS (Premix) and incubated at 96 ° C. for 2 minutes, followed by 32 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 2 minutes.
  • the obtained two PCR-amplified fragments were subjected to agarose electrophoresis and then purified using QIAquick Gel Extraction Kit (manufactured by Qiagen) to obtain an insert.
  • QIAquick Gel Extraction Kit manufactured by Qiagen
  • the insert was inserted into the previously purified pLN1V5 vector and subcloned to obtain a pLN1V5_hBMP10 vector.
  • SEQ ID NOs: 95 to 98 show the amino acid sequence and base sequence of human BMP10 and its nature region.
  • SEQ ID NOs: 99 and 100 show the nucleotide sequence inserted between the NheI and XhoI recognition sites of pLN1V5_hBMP10 and the amino acid sequence of the expressed protein.
  • BMP9 / 10 chimeric protein A, B and C expression vectors were prepared by the following procedure.
  • PrimeSTAR HS was used and incubated at 96 ° C. for 2 minutes, followed by 32 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 2 minutes.
  • the obtained PCR fragment was purified by agarose gel electrophoresis.
  • inserts A, B and C were prepared. It is as follows when it describes similarly to the above-mentioned DNA fragment. ⁇ Insert A, SEQ ID NO: 101/106, mixture of A-1, A-2 and A-3 purified fragments ⁇ ⁇ Insert B, SEQ ID NO: 101/106, mixture of B-1, B-2 and B-3 purified fragments ⁇ ⁇ InsertC, SEQ ID NO: 101/113, mixture of C-1 and C-2 purified fragments ⁇
  • the obtained PCR amplified fragments were cleaved with restriction enzymes NheI and XhoI, respectively, and purified by agarose gel electrophoresis to obtain inserts A, B, and C. These were transferred into a pLN1V5 digested vector digested with NheI and XhoI, and DNA Ligation Kit Ver. 2.1 (Takara Bio, 6022) was used to obtain pLN1V5_hBMP9 / 10A, pLN1V5_hBMP9 / 10B, and pLN1V5_hBMP9 / 10C, respectively.
  • pLN1V5_hBMP9 / 10A, pLN1V5_hBMP9 / 10B and pLN1V5_hBMP9 / 10C are respectively a signal sequence of human BMP9, a His6 tag sequence, a human BMP9 propeptide region, and a part of the mature region of human BMP9 (region A, B or C, respectively) Is a human BMP9 / 10 chimeric protein expression vector in which a human BMP9 feature region sequence in which is substituted with the corresponding sequence of BMP10 is inserted into pLN1V5.
  • the pLN1V5_hBMP9 vector, the pLN1V5_hBMP10 vector, the pLN1V5_hBMP9 / 10A vector, the pLN1V5_hBMP9 / 10B vector, and the pLN1V5_hBMP9 / 10C vector that were prepared in the previous section were used to transform Escherichia coli DH5 ⁇ . Prepared.
  • Transient expression was performed using FreeStyle 293 Expression System (manufactured by Life Technologies), and human BMP9, human BMP10, and human BMP9 / 10 chimeric proteins A, B, and C were expressed in the culture solution.
  • the cell supernatant was obtained from the culture by centrifugation and filtration using a 0.22 ⁇ m filter.
  • each protein was purified using Ni-NTA Agarose (manufactured by QIAGEN). 20 mM HEPES-NaOH (pH 7.4) and 500 mM NaCl 40 mM Imidazole were used as the binding buffer, and 20 mM HEPES-NaOH (pH 7.4) and 500 mM NaCl 200 mM Imidazole were used as the elution buffer.
  • the buffer was replaced with PBS using a NAP-25 column (manufactured by GE Healthcare).
  • the absorbance at 280 nm was measured to determine the concentration of each protein solution.
  • molecular extinction coefficients human BMP9, human BMP10, human BMP9 / 10 chimera A, B and C use 1.05, 0.96, 1.12, 1.08 and 1.06 mL / (mg ⁇ cm), respectively. did.
  • 3C7-3-1 antibody (prepared in Example 20-1) adjusted to 1000 ng / mL with 1% BSA-PBS, R & D antibody (mouse IgG2b), BMP10 antibody (R & D, Cat # MAB2926) Mouse IgG2a), BMP9 goat polyclonal antibody (R & D, Cat # AF3209), negative control mouse IgG1, IgG2b monoclonal antibody (R & D, Cat # MAB002, Cat # MAB004) were added at 50 ⁇ L / well, and 1 at room temperature. Let stand for hours.
  • TMB color developing solution TMB + Substrate-Chromogen, Dako, Cat # S1599
  • 1N sulfuric acid solution Wako
  • absorbance at 450 nm and 570 nm was measured using Multiscan Spectrum (manufactured by Thermo Labsystems).
  • the 3C7-3-1 antibody specifically bound to human BMP9 and human BMP9 / 10 chimeric protein C, but did not bind to human BMP9 / 10 chimeric protein A and B. Furthermore, the R & D antibody specifically bound to human BMP9 and human BMP9 / 10 chimeric proteins A and B, but did not bind to human BMP9 / 10 chimeric protein C.
  • the 3C7-3-1 antibody recognizes an epitope that exists in regions other than human BMP9, that is, regions A and B, and that the R & D antibody recognizes an epitope in region C of human BMP9. Indicates that it is recognized. From the above, it was revealed that the 3C7-3-1 antibody is an antibody that recognizes an epitope of BMP9 different from the existing R & D antibody.
  • SEQ ID NO: 1 Base sequence of LinkA SEQ ID NO: 2: Base sequence of LinkA2 SEQ ID NO: 3: Base sequence of LinkB1 SEQ ID NO: 4: Base sequence of LinkB2 SEQ ID NO: 5: Base sequence of Bmp9KOClaI-3′Fw SEQ ID NO: 6: Bmp9KOAscI ⁇ 3′Rv nucleotide sequence SEQ ID NO: 7: Bmp9KOPacI-5′Fw nucleotide sequence SEQ ID NO: 8: Bmp9KOFseI-5′Rv nucleotide sequence SEQ ID NO: 9: Bmp9 KO5 ′ probe FW2 nucleotide sequence SEQ ID NO: 10: Bmp9 KO5 ′ probe RV2 nucleotide sequence SEQ ID NO: 11: Bmp9 KO3 ′ probe FW2 nucleotide sequence SEQ ID NO: 12: Bmp9 KO3 ′ probe RV2 nucleotide sequence SEQ ID NO: 13: m

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Abstract

La présente invention concerne : un anticorps monoclonal anti-BMP9 qui se lie à BMP9 humain (protéine morphogénétique osseuse 9) ou un fragment d'anticorps de celui-ci ; un hybridome qui produit l'anticorps ou le fragment d'anticorps ; de l'ADN qui code pour l'anticorps ou le fragment d'anticorps ; un vecteur contenant l'ADN ; un transformant obtenu par l'introduction du vecteur ; un procédé de production de l'anticorps ou du fragment d'anticorps à l'aide de l'hybridome ou du transformant ; et un agent thérapeutique contenant, en tant que principe actif, l'anticorps ou le fragment d'anticorps. La présente invention concerne une composition médicinale pour l'utilisation dans le traitement d'une maladie d'ossification ectopique, contenant comme principe actif l'anticorps ou le fragment d'anticorps, et fournit une méthode de traitement d'une maladie d'ossification ectopique à l'aide de cette composition.
PCT/JP2013/076411 2012-09-28 2013-09-27 Anticorps anti-bmp9 humain et agent thérapeutique pour une maladie d'ossification ectopique contenant ledit anticorps comme principe actif WO2014051109A1 (fr)

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CN107207604A (zh) * 2015-01-30 2017-09-26 学校法人埼玉医科大学 抗alk2抗体
JP2018518461A (ja) * 2015-04-29 2018-07-12 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 進行性骨化性線維異形成症の治療
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CN113817743A (zh) * 2021-09-30 2021-12-21 魏伟 IgDR基因、IgDR单克隆抗体及其制备方法与应用
US11292848B2 (en) 2019-10-24 2022-04-05 Prometheus Biosciences, Inc. Humanized antibodies to TNF-like ligand 1A (TL1A) and uses thereof
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11407822B2 (en) 2014-09-12 2022-08-09 Regeneron Pharmaceuticals, Inc. Treatment of fibrodysplasia ossificans progressiva
CN107207604B (zh) * 2015-01-30 2021-10-26 学校法人埼玉医科大学 抗alk2抗体
EP3252074A4 (fr) * 2015-01-30 2018-07-11 Saitama Medical University Anticorps anti-alk2
US11447554B2 (en) 2015-01-30 2022-09-20 Saitama Medical University Anti-ALK2 antibody
CN107207604A (zh) * 2015-01-30 2017-09-26 学校法人埼玉医科大学 抗alk2抗体
US10428148B2 (en) 2015-01-30 2019-10-01 Saitama Medical University Anti-ALK2 antibody
US11312776B2 (en) 2015-01-30 2022-04-26 Saitama Medical University Anti-ALK2 antibody
US20190153085A1 (en) * 2015-04-29 2019-05-23 Regeneron Pharmaceuticals, Inc. Treatment of fibrodysplasia ossificans progressiva
JP2018518461A (ja) * 2015-04-29 2018-07-12 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 進行性骨化性線維異形成症の治療
US10047155B2 (en) 2015-06-05 2018-08-14 Novartis Ag Antibodies targeting bone morphogenetic protein 9 (BMP9) and methods therefor
CN111432839A (zh) * 2017-10-30 2020-07-17 约翰霍普金斯大学 异位性骨化及治疗方法
US10689439B2 (en) 2018-04-25 2020-06-23 Prometheus Biosciences, Inc. Optimized anti-TL1A antibodies
WO2019209995A3 (fr) * 2018-04-25 2019-12-12 Prometheus Biosciences, Inc. Anticorps anti-tl1a optimisés
US11440954B2 (en) 2018-04-25 2022-09-13 Prometheus Biosciences, Inc. Optimized anti-TL1A antibodies
US11292848B2 (en) 2019-10-24 2022-04-05 Prometheus Biosciences, Inc. Humanized antibodies to TNF-like ligand 1A (TL1A) and uses thereof
CN113817743A (zh) * 2021-09-30 2021-12-21 魏伟 IgDR基因、IgDR单克隆抗体及其制备方法与应用
CN113817743B (zh) * 2021-09-30 2023-10-03 魏伟 IgDR基因、IgDR单克隆抗体及其制备方法与应用
US11999789B2 (en) 2022-02-28 2024-06-04 Prometheus Biosciences, Inc. Humanized antibodies to TNF-like ligand 1A (TL1A) and uses thereof

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