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WO2019009407A1 - Anti-protein c antibody - Google Patents

Anti-protein c antibody Download PDF

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Publication number
WO2019009407A1
WO2019009407A1 PCT/JP2018/025679 JP2018025679W WO2019009407A1 WO 2019009407 A1 WO2019009407 A1 WO 2019009407A1 JP 2018025679 W JP2018025679 W JP 2018025679W WO 2019009407 A1 WO2019009407 A1 WO 2019009407A1
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amino acid
seq
acid sequence
antibody
set forth
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PCT/JP2018/025679
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French (fr)
Japanese (ja)
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壯俊 古郡
健介 中村
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第一三共株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to an anti-protein C antibody that specifically recognizes protein C and activated protein C, a method for producing the anti-protein C antibody, a pharmaceutical composition containing the antibody, and the like.
  • Hemophilia is a hemorrhagic disease caused by congenital defects or dysfunction of blood coagulation factor VIII (FVIII) or blood coagulation factor IX (FIX).
  • the former is referred to as hemophilia type A (hereinafter also referred to as “hemophilia A”), and the latter is referred to as hemophilia type B (hereinafter referred to as "hemophilia B").
  • hemophilia type A hereinafter also referred to as “hemophilia A
  • hemophilia B hemophilia type B
  • Most of the onset patients are males because all genes exist on the X chromosome and are sexually recessive. The prevalence rate is about 9 per 100,000 males, and the ratio of hemophilia A to hemophilia B is about 5: 1.
  • the main bleeding symptoms include subcutaneous hemorrhage, abnormal traumatic hemorrhage, intracranial hemorrhage, joint hemorrhage, intraoral hemorrhage, intranasal hemorrhage, intranasal hemorrhage, iliopsoas hemorrhage, gastrointestinal tract hemorrhage and the like.
  • Repeated hemorrhage to a specific joint causes chronic synovitis, which causes irreversible changes in articular cartilage, which leads to arthritic joint disorder, hemophilic arthropathy with difficulty walking, and finally
  • joint replacement surgery may be required, which is a major factor that significantly reduces the quality of life of hemophiliacs.
  • the severity of hemophilia is based on FVIII activity or FIX activity in blood, with less than 1% active patients as severe, 1% or more less than 5% patients moderate, 5% or more less than 40% patients as mild It specifies. More than 60% of haemophilia patients are considered severe. The moderate and mild cases of haemophilia differ only by a few percent of the coagulation factor activity from the severe, but the bleeding frequency is clearly less, so maintaining the FVIII activity or FIX activity at 1% or more is a bleeding event It is considered effective in reducing the frequency.
  • Blood coagulation factor preparations (FVIII preparations, FIX preparations) purified from plasma or produced by genetic engineering techniques are mainly used for the prevention and / or treatment of hemorrhage in hemophiliacs. Since blood coagulation factor has a short half-life of several hours to several tens of hours, its persistence of efficacy is short, and blood coagulation factor preparations are intravenously administered about 3 times a week to prevent bleeding. It is necessary to maintain FVIII activity or FIX activity) at 1% or more. This blood coagulation factor replacement therapy reduces the frequency of bleeding and consequently contributes significantly to the improvement of QOL of hemophilia patients.
  • blood glucose factor preparations are periodically added to maintain a certain level of coagulation factor activity for a certain period of time to prevent complete hemostasis and rebleeding.
  • blood coagulation factor formulations are limited to intravenous administration. Intravenous administration is technically difficult, especially in pediatric and lean patients, where the veins are narrow and more difficult, requiring careful administration.
  • antibodies have high stability in blood, can be administered subcutaneously, and have high target selectivity, so they have recently been applied and marketed as pharmaceuticals.
  • the antibody has a long half-life, which can reduce the frequency of administration, and because it can be administered subcutaneously, is easy to administer and is not affected by the presence or absence of an inhibitor, so it is suitable for drug discovery for hemophilia it is conceivable that.
  • PC Protein C
  • EPCR endotherial protein receptor
  • aPC activated protein C
  • Non-patent Document 1 an inhibitor of aPC partially restores the reduction of thrombin generation in FVIII-deficient plasma in vitro.
  • Non-patent Document 2 an antibody that inhibits the activity of aPC inhibits the anticoagulant activity of aPC, exhibits a blood coagulation effect, and is effective for the treatment of hemophilia.
  • Non-patent Document 3 mutant FVa having degradation resistance to aPC reduced the amount of bleeding in a FVIII-deficient mouse bleeding model. Furthermore, it has been reported that an antibody that inhibits PC activation and / or aPC activity also reduces the bleeding scar area in a puncture bleeding model of FVIII-deficient nude mice (Patent Document 2).
  • Non-patent Document 4 There is a report that the aPC antibody suppressed hemorrhage in a monkey hemorrhage model (Non-patent Document 4).
  • An object of the present invention is to provide a novel medicament for treating hemorrhagic disease having an excellent blood coagulation effect, a method for treating hemorrhagic disease and the like using the medicament.
  • the present inventors diligently studied to achieve the above problems, and obtain an antibody that binds to both protein C and activated protein C, and inhibits activation of protein C and activity of activated protein C.
  • the inventors have found that the antibody has a high hemostatic effect and is effective in the treatment of hemorrhagic diseases, particularly hemophilia A and / or hemophilia B, and completed the present invention.
  • the present invention provides an antibody that inhibits activation of protein C and activity of activated protein C, and a novel medicament for treating hemorrhagic disease having an excellent blood coagulation effect, which comprises the antibody as an active ingredient, the medicine Provided a method for treating hemorrhagic disease using
  • the present invention (1) An antibody having a competitive inhibitory activity with the antibody according to any one of the following groups (a) to (e), for binding to protein C or activated protein C: Functional fragments of antibodies: (A) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 33 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 237 of SEQ ID NO. (B) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO.
  • a polypeptide comprising a peptide consisting of the amino acid sequence shown by amino acid numbers 43 to 153 of SEQ ID NO: 3 in the sequence listing and a peptide consisting of the amino acid sequence shown by amino acid numbers 154 to 412 linked by SS bond
  • the peptide consisting of the amino acid sequence shown by amino acid numbers 43 to 153 of SEQ ID NO: 3 in the sequence listing and the peptide consisting of the amino acid sequence shown by amino acid numbers 166 to 412 are linked by SS bond
  • the antibody according to any one of (1) to (3) or a functional fragment of the antibody, which is a polypeptide.
  • the antibody according to any one of (1) to (4) or a functional fragment of the antibody characterized in that the protein C is activated and / or the activity of the activated protein C is inhibited.
  • the antibody according to any one of (1) to (5) or the functionality of the antibody characterized by having at least one property selected from the following (a) to (d): fragment: (A) specifically bind to protein C and activated protein C; (B) specifically bind to protein C and inhibit protein C activation; (C) It specifically binds to activated protein C and causes decomposition and / or inactivation of activated blood coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa) by activated protein C Inhibit; (D) restore thrombin generation, (7) The antibody according to any one of (1) to (6) or a functional fragment of the antibody, wherein CDRH2 consists of the amino acid sequence shown in SEQ ID NO: 8, (8) The antibody according to any one of (1) to (6) or
  • the antibody according to any one of (7) and (9) or a functional fragment of the antibody (11) The antibody according to any one of (1) to (10), wherein the constant region is a human-derived constant region, or a functional fragment of the antibody, (12) The antibody according to any one of (1) to (11) or a functional fragment of the antibody, which is humanized.
  • the antibody according to (12) or the functional fragment of the antibody which has a light chain variable region consisting of the amino acid sequence according to any one of: (A) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16, (B) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18; (C) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, (D) an amino acid sequence having at least 95% or more homology to a sequence of a framework region other than each CDR sequence in the sequences of (a) to (c), (E) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequences of (a) to (d), (F) the amino acid sequence set forth in amino acid
  • a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20 and a light chain variable flow region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO. 13) or the antibody according to (14) or a functional fragment of the antibody,
  • a method for producing the antibody or functional fragment of the antibody (30) An antibody characterized by being obtained by the production method according to (29) or a functional fragment of the antibody, (31) The functional fragment of the antibody according to (30), wherein the functional fragment is selected from the group consisting of Fab, F (ab) 2, Fab ′ and Fv.
  • (32) Glycosylation to N-linkage, Glycosylation to O-linkage, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, methionine residue at N-terminal (1) to (22), (30) comprising one or more modifications selected from the group consisting of addition of groups, amidation of proline residues and deletion of one or two amino acids at the heavy chain carboxyl terminus Or a functional fragment of the antibody according to any one of (31) and (31) (33) The antibody according to (32), wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.
  • composition (39) The pharmaceutical composition according to (38), wherein the hemorrhagic disease is hemophilia A and / or hemophilia B, (40) An antibody selected from (1) to (22), and (30) to (35) or a functional fragment of the antibody, a salt thereof, or a hydrate thereof is administered to an individual For treating hemorrhagic diseases, characterized in that (41) The treatment according to (40), wherein the hemorrhagic disease is at least one selected from hemophilia A, hemophilia B, acquired hemophilia and von Willebrand's disease.
  • FIG. 1A Concentration dependency of thrombin generation (TG) recovery action by administration of rat anti-human PC antibody purified in plasma from hemophilia A patient (FVIII-deficient plasma).
  • FIG. 1B Concentration dependency of thrombin generation (TG) recovery action by rat anti-human PC antibody administration in hemophiliac B patient-derived plasma (FIX deficient plasma). The method of calculating the recovery rate of thrombin production (%) was calculated according to the following equation.
  • Underlining in the amino acid sequence indicates a CDR sequence.
  • Underlining in the amino acid sequence indicates a CDR sequence.
  • “•” indicates the same amino acid residue as cR74_L, and the place where the amino acid residue is described indicates a substituted amino acid residue.
  • "-" Indicates the place where the corresponding amino acid residue is missing.
  • FIG. 14A Thrombin production (TG) recovery effect of human chimerized anti-human PC antibody cR74_IgG1-LALA, R74, positive control polyclonal antibody administration in hemophilia A patient-derived plasma (FVIII-deficient plasma).
  • FIG. 14B Thrombin production (TG) recovery action by human chimeric anti-human PC antibody cR74_IgG1-LALA, R74, positive control polyclonal antibody administration in hemophilia B patient-derived plasma (FVIX-deficient plasma).
  • FIG. 16A shows the thrombin generation (TG) recovery action of hR74_H1 / L1 and BAY1896502 in hemophilia A patient-derived plasma (FVIII-deficient plasma).
  • FIG. 16B Thrombin production (TG) recovery action of hR74_H1 / L1 and BAY1896502 in hemophiliac B patient-derived plasma (FIX deficient plasma).
  • the calculation method of the subcutaneous bleeding area (%) was calculated according to the following formula.
  • the present invention has the activity of binding to both PC and aPC, and inhibits the activation of PC and the activity of aPC, thereby activating a coagulation factor VIII (FVIIIa) together with protein S of which aPC is its cofactor.
  • anti-PC antibody which can be used for treatment of hemorrhagic disease which inhibits degradation and inactivation of activated blood coagulation factor V (FVa), and a pharmaceutical composition etc. containing the antibody as an active ingredient. .
  • the term "gene” includes not only DNA but also its mRNA, cDNA and its cRNA.
  • polynucleotide is used interchangeably with nucleic acid and includes DNA, RNA, probes, oligonucleotides, and primers.
  • polypeptide and “protein” are used without distinction.
  • cell also includes cells in an animal individual and cultured cells.
  • PC is used in the same meaning as protein C.
  • aPC is used in the same meaning as activated protein C.
  • aPC is an active PC produced by removing the activation peptide consisting of 12 amino acids present in PC by the thrombin cleavage site.
  • the “hemorrhagic disease” is not particularly limited as long as it is a hemorrhagic disease caused by deficiency, deficiency or functional failure of a blood coagulation factor, for example, blood coagulation factor VIII (FVIII) Hemophilia A, which is a hemorrhagic disease caused by birth defects or dysfunction, Hemophilia B, a hemorrhagic disease caused by birth defects or dysfunction of blood coagulation factor IX (FIX), and the like Acquired hemophilia, von Willebrand's disease can be mentioned.
  • a blood coagulation factor VIII FVIII
  • Hemophilia A which is a hemorrhagic disease caused by birth defects or dysfunction
  • Hemophilia B a hemorrhagic disease caused by birth defects or dysfunction of blood coagulation factor IX (FIX)
  • FIX blood coagulation factor IX
  • epitope means a partial peptide or partial conformation of PC and / or aPC to which a specific anti-PC antibody binds.
  • the epitope which is a partial peptide of the above PC and / or aPC can be determined by methods well known to those skilled in the art, such as immunoassays.
  • various partial structures of the antigen are prepared. In the preparation of partial structures, known oligonucleotide synthesis techniques can be used.
  • bind to the same epitope means antibodies that bind to a common epitope. If the second antibody binds to the partial peptide or partial conformation to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope. Also, by confirming that the second antibody competes with the binding of the first antibody to the antigen (ie, the second antibody interferes with the binding of the antigen to the first antibody), the sequence of the specific epitope Alternatively, even if the structure is not determined, it can be determined that the first antibody and the second antibody bind to the same epitope.
  • the second antibody has similar activity. It can be expected to have.
  • the first antibody and the second antibody are PC and PC by confirming that the second anti-PC antibody competes with the partial peptide to which the first antibody's anti-PC binds. It can be determined as an antibody that binds to the same epitope of aPC.
  • CDR complementarity determining region
  • the CDRs also called hypervariable regions, are in the variable regions of the heavy and light chains of the antibody and are the sites of particularly high variability of the primary structure, and the heavy and light chain polypeptide chains In the primary structure, they are separated into three locations.
  • the CDRs of the antibody the CDRs of the heavy chain are denoted as CDRH1, CDRH2 and CDRH3 from the amino terminal side of the heavy chain amino acid sequence
  • the CDRs of the light chain are CDRL1 from the amino terminal side of the light chain amino acid sequence. , CDRL2 and CDRL3. These sites are close to each other on the conformation to determine their specificity for the binding antigen.
  • hybridize under stringent conditions refers to hybridization at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (Clontech) or using a filter on which DNA is immobilized. After hybridization at 68 ° C. in the presence of 7-1.0 M NaCl, using a 0.1-2 fold concentration SSC solution (with 1-fold concentration SSC consisting of 150 mM NaCl, 15 mM sodium citrate), It refers to hybridization under conditions that can be identified by washing at 68 ° C. or conditions equivalent thereto.
  • PC Protein C
  • aPC activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa).
  • the PC and / or aPC used in the present invention may be purified directly from human and non-human mammalian (rat, mouse etc.) PC and / or aPC expressing cells, or the cell membrane fraction of the cells may be prepared. It can be used as such and can be obtained by synthesizing PC and / or aPC in vitro or by causing it to be produced in host cells by genetic engineering.
  • partial peptides containing partial sequences of PC and / or aPC protein can also be used as long as anti-PC antibody can be obtained.
  • an aPC partial peptide obtained by treating the polypeptide with thrombin can also be used as a suitable antigen.
  • PC and / or aPC expressing cells by genetic engineering or cell lines expressing PC and / or aPC as PC and / or aPC protein.
  • an expression vector incorporating PC or PC partial peptide cDNA may be directly administered to the animal to be immunized to express PC, aPC or a partial peptide of the peptide in the body of the animal to be immunized.
  • accession numbers such as (Accession No. NP — 000303 of NCBI Protein Database).
  • amino acid sequence of PC one or several amino acids consist of an amino acid sequence substituted, deleted and / or added, and a protein having biological activity equivalent to the protein is also included in PC.
  • the amino acid sequence of human PC is described in SEQ ID NO: 1 of the sequence listing.
  • the amino acid sequence shown in amino acid numbers 1 to 18 in SEQ ID NO: 1 is a signal sequence, 19 to 42 are prepro sequences, and the amino acid sequence shown in amino acid numbers 200 to 211 (DTEDQEDQVDPR) shows the sequence of the activation peptide. .
  • aPC used in the present invention is a polypeptide in which a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 in the sequence listing and a peptide consisting of amino acid numbers 166 to 412 are linked by SS bond.
  • an antibody that recognizes the higher-order structure of the polypeptide shown in SEQ ID NO: 1 in the sequence listing can be mentioned.
  • Another example of the anti-PC antibody of the present invention is an antibody that recognizes the higher-order structure of a polypeptide consisting of the amino acid sequence shown in amino acid numbers 43 to 460 of SEQ ID NO: 3 in the sequence listing.
  • Another example of the anti-PC antibody of the present invention is a polypeptide in which a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and a peptide consisting of amino acid numbers 154 to 412 are linked by SS bond.
  • a polypeptide comprising a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and a peptide consisting of amino acid numbers 166 to 412 linked by SS bond And antibodies that recognize the higher order structure of
  • a polypeptide comprising a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and a peptide consisting of amino acid numbers 154 to 412 linked by SS bond Of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and the conformation of the polypeptide in which the peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 and the peptide consisting of amino acid numbers 166 to 412 are bound by SS bond
  • the anti-PC antibody of the present invention may be derived from any species, preferably human, rat, mouse and rabbit. When derived from species other than human, it is preferable to chimerize or humanize using well-known techniques.
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.
  • the anti-PC antibody of the present invention is an antibody capable of targeting PC and aPC, that is, properties capable of recognizing PC and aPC, activity to inhibit PC activation, protein S with which aPC is a cofactor, and activated blood. It has an activity to inhibit the action of degrading and / or inactivating coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa).
  • the anti-PC antibody of the present invention has an activity to restore the production of thrombin, has a blood coagulation effect, exhibits a hemostatic effect in a subject administered with the antibody, and can be a therapeutic agent for hemorrhagic disease.
  • the anti-PC antibody of the present invention is an antibody that recognizes aPC in which a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 in the sequence listing and a peptide consisting of amino acid numbers 166 to 412 are linked by SS bond. is there.
  • the binding of the antibody to PC and / or aPC can be confirmed using, for example, flow cytometry.
  • the anti-PC antibody can be obtained by immunizing an animal with a polypeptide to be an antigen, and collecting and purifying the antibody produced in vivo, using methods commonly practiced in the art.
  • the origin of the antigen is not limited to human, and the animal can be immunized with an antigen derived from a nonhuman animal such as a mouse or a rat.
  • antibodies that can be applied to human diseases can be selected by testing the cross-reactivity between the human antigen and the antibody that binds to the obtained heterologous antigen.
  • a hybridoma can be established by fusing an antibody-producing cell producing an antibody against an antigen with a myeloma cell to obtain a monoclonal antibody.
  • An antigen can be obtained by genetically producing a gene encoding an antigen protein in a host cell. Specifically, a vector capable of expressing an antigen gene may be prepared, introduced into a host cell to express the gene, and the expressed antigen may be purified.
  • an antibody for example, thrombin
  • an antibody is further treated as an aPC to obtain an antibody by immunizing an animal with an antigen-expressing cell by the above-mentioned genetic manipulation or a cell line expressing an antigen. it can.
  • the cDNA of the antigen protein may be incorporated into an expression vector and administered to the animal to be immunized without using the antigen protein, and the antigen protein may be expressed in the body of the animal to be immunized to produce antibodies against the antigen protein. It can be acquired.
  • the anti-PC antibody used in the present invention is not particularly limited, but preferably used, for example, the antibody specified by the amino acid sequence shown in the sequence listing of the present application. it can.
  • As the anti-PC antibody used in the present invention those having the following characteristics are desirable.
  • A-3 It specifically binds to aPC and inhibits the decomposition and / or inactivation of activated blood coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa) by aPC.
  • (A-4) restore thrombin generation.
  • (B) The antibody or the antibody according to the above (a), wherein PC and aPC are human PC and human aPC.
  • the method for obtaining an antibody to PC of the present invention is not particularly limited as long as an anti-PC antibody can be obtained.
  • the following may be mentioned as an example of specific monoclonal antibody acquisition.
  • the cDNA of PC is incorporated into an expression vector (for example, pcDNA3.3-TOPO / LaxZ (ThermoFisher SCIENTOFIC) and transiently expressed by transfecting FreeStyle 293F cells (ThermoFisher SCIENTOFIC), from the culture supernatant
  • the fraction containing PC is collected,
  • the collected PC is thrombin-treated to form aPC, which is administered to an animal (eg, female of WKY / Izm rat) which induces an immune response.
  • a tissue (eg, lymph node) containing antibody-producing cells is collected from the above-described animal in which an immune response has been induced.
  • C Preparation of myeloma cells (hereinafter referred to as "myelomas") (eg, mouse myeloma SP2 / 0-ag14 cells)
  • D cell fusion between antibody-producing cells and myeloma
  • E Selection of hybridomas producing the target antibody
  • F Division into single cell clones (cloning)
  • H Physiological activity of the thus produced monoclonal antibody and examination of its binding specificity, or assay of properties as a labeling reagent
  • flow cytometry or Cell-ELISA methods can be mentioned but are not limited to these methods.
  • anti-PC antibody-producing hybridoma R74 can be mentioned.
  • an antibody produced by anti-PC antibody-producing hybridoma R74 is referred to as "R74 antibody” or simply "R74".
  • the R74 antibody has an activity of binding to both PC and aPC.
  • the heavy chain variable region of the R74 antibody has the amino acid sequence shown in SEQ ID NO: 6 in the Sequence Listing.
  • the variable region is CDRH1 consisting of an amino acid sequence consisting of the 26th to 35th amino acid residues in SEQ ID NO: 6 in the sequence listing, CDRH2 consisting of an amino acid sequence consisting of the 50th to 58th amino acid residues It has CDRH3 consisting of an amino acid sequence consisting of amino acid residues.
  • the CDRH1 of the R74 antibody has the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing
  • the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing
  • the amino acid sequence of CDRH3 shows the SEQ ID NO in the sequence listing It has the amino acid sequence shown in 9.
  • the amino acid sequences of the heavy chain variable region, CDRH1, CDRH2 and CDRH3 of R74 antibody are described in FIG.
  • variable region of the light chain of R74 antibody has the amino acid sequence shown in SEQ ID NO: 11 in the Sequence Listing.
  • the variable region is CDRL 1 consisting of an amino acid sequence consisting of amino acid residues 23 to 37 in SEQ ID NO: 11 in the sequence listing, CDR L 2 consisting of an amino acid sequence consisting of amino acid residues 53 to 59 It has CDRL3 consisting of an amino acid sequence consisting of amino acid residues.
  • CDR L1 of the R74 antibody has the amino acid sequence shown in SEQ ID NO: 12 in the Sequence Listing
  • the amino acid sequence of CDR L2 has the amino acid sequence shown in SEQ ID NO: 13 in the Sequence Listing
  • the amino acid sequence of CDR L3 has the SEQ ID NO in SEQ ID NO: It has the amino acid sequence shown in 14.
  • the amino acid sequences of the light chain variable region, CDRL1, CDRL2 and CDRL3 of the R74 antibody are described in FIG.
  • amino acid sequence of the heavy chain variable region of the R74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing. The sequence of SEQ ID NO: 5 is described in FIG.
  • the nucleotide sequence of the light chain variable region of the R74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 10 of the sequence listing. Also, the sequence of SEQ ID NO: 10 is described in FIG.
  • the monoclonal antibody competes with the binding of the R74 antibody to PC or aPC (ie, the monoclonal antibody prevents the binding of the R74 antibody to PC or aPC)
  • the specific epitope Even if the sequence or structure has not been determined, it can be determined that the monoclonal antibody binds to the same epitope as the anti-PC antibody.
  • the epitopes are confirmed to be identical, it is strongly expected that the monoclonal antibody has the same antigen binding ability and biological activity as the R74 antibody.
  • the antibodies of the present invention include genetically engineered antibodies, for example, chimera (Chimeric), which are artificially modified for the purpose of reducing heterologous antigenicity to humans, etc., in addition to the monoclonal antibodies against PC. Also included are antibodies, humanized antibodies, human antibodies and the like. These antibodies can be produced using known methods.
  • chimeric antibodies include antibodies in which the variable region of the antibody and the constant region are heterologous to each other, such as a chimeric antibody in which the variable region of a mouse- or rat-derived antibody is conjugated to a constant region derived from human (Proc. Natl. Acad) Sci.U.S.A., 81, 6851-6855, (1984)).
  • a chimeric antibody derived from a rat anti-human PC antibody R74 antibody is an antibody consisting of a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain comprising a light chain variable region shown in SEQ ID NO: 11. And may have any human-derived constant region.
  • chimeric antibody cR74 derived from rat anti-human PC antibody R74 antibody can be mentioned.
  • the amino acid sequence of the cR74 antibody is comprised of a heavy chain having an amino acid sequence consisting of the 20th to 467th amino acid residues of SEQ ID NO: 33 in the Sequence Listing and a light chain having an amino acid sequence consisting of 21 to 237 of SEQ ID NO: 30 in the Sequence Listing Become.
  • the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
  • the amino acid sequence consisting of amino acid residues 20 to 137 is a variable region
  • the amino acid sequence consisting of residues 138 to 467 is a constant region.
  • the sequence of SEQ ID NO: 33 is set forth in FIG.
  • the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence
  • the amino acid sequence consisting of amino acid residues 21 to 132 is a variable region
  • the amino acid sequence consisting of amino acid residues 133 to 237 is a constant region.
  • the sequence of SEQ ID NO: 30 is set forth in FIG.
  • the heavy chain amino acid sequence of the cR74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 32 in the Sequence Listing.
  • the nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 32 in the sequence listing encodes the signal sequence of the cR74 antibody
  • the nucleotide sequence consisting of nucleotides 58 to 411 is the heavy chain of the cR74 antibody
  • a nucleotide sequence encoding a variable region sequence and consisting of nucleotides 412 to 1401 encodes a heavy chain constant region of cR74 antibody.
  • the sequence of SEQ ID NO: 32 is set forth in FIG.
  • the light chain amino acid sequence of the cR74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 29 in the Sequence Listing.
  • the nucleotide sequence consisting of the 1st to 60th nucleotides of the nucleotide sequence shown in SEQ ID NO: 29 in the sequence listing encodes the signal sequence of the cR74 antibody
  • the nucleotide sequence consisting of the 61st to 396th nucleotides consists of the light chain of the cR74 antibody
  • a nucleotide sequence encoding a variable region sequence and consisting of nucleotides 397 to 711 encodes a light chain constant region of cR74 antibody.
  • the sequence of SEQ ID NO: 29 is set forth in FIG.
  • a humanized antibody an antibody in which only a complementarity determining region (CDR) is incorporated into a human-derived antibody (see Nature (1986) 321, p. 522-525), CDR sequences by CDR grafting method
  • CDR sequences by CDR grafting method
  • some framework amino acid residues have also been grafted onto human antibodies (WO 90/07861), and antibodies in which the amino acid sequences of some CDRs have been modified while maintaining their ability to bind to the antigen. Can be mentioned.
  • humanized antibody derived from R74 antibody it is not limited to a specific humanized antibody as long as it retains the CDR sequences of all six types of R74 antibody and has an activity to recover thrombin generation, It is not limited to a specific humanized antibody as long as it has an ability to bind to PC and aPC while altering the amino acid sequence of the CDRs of SEQ ID NO: 1 and has an activity to restore thrombin generation.
  • the heavy chain variable region of the above-mentioned humanized antibody comprises CDRH1 (GFSLTGYGVS) consisting of the amino acid sequence shown in SEQ ID NO: 7, CDRH2 (AVWRGGSKD) consisting of the amino acid sequence shown in SEQ ID NO: 8 and amino acid sequence shown in SEQ ID NO: 9 Possess CDRH3 (SGPEGTPFDY) consisting of
  • the light chain variable region of the above human antibody comprises the CDRL1 (KTNQNVDFDFYGNSYIH) consisting of the amino acid sequence shown in SEQ ID NO: 12, the CDRL2 (SASNLAS) consisting of the amino acid sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14 Possess CDRL3 (QQSRNLPNT).
  • a humanized antibody of rat antibody R74 (1) an amino acid sequence consisting of the 20th to 137th amino acid residues of SEQ ID NO: 16 or 18 in the sequence listing, (2) an amino acid sequence having at least 95% or more homology to the amino acid sequence of (1) above; And (3) a heavy chain comprising a heavy chain variable region consisting of any one of the amino acid sequences wherein one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above, and (4) the sequence An amino acid sequence consisting of the 21st to 133rd amino acid residues of No.
  • humanized antibody of rat antibody R74 is a heavy chain consisting of amino acid residues 20 to 467 of SEQ ID NO: 16 in the sequence listing and amino acids 21 to 238 of SEQ ID NO: 23 of the sequence listing.
  • the antibody of the present invention also includes an antibody in which the viscosity of the antibody has been reduced by further introducing mutations into the CDRs of the above-mentioned humanized antibody.
  • CDR modifications can include CDRH2 (AVYTGGSKD: SEQ ID NO: 21) in which the third and fourth amino acid residues of CDRH2 derived from R74 antibody (AVWRGGSKD: SEQ ID NO: 8) are modified. That is, in the above-mentioned humanized antibody, a humanized antibody in which only the sequence of CDRH2 is substituted with CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 21 is also included in the humanized antibody of the present invention.
  • an antibody include an amino acid sequence consisting of the 20th to 137th amino acid residues of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, (2) at least the amino acid sequence of (1) above.
  • a heavy chain variable comprising an amino acid sequence having 95% or more homology and (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above
  • a heavy chain including the region and (4) an amino acid sequence consisting of amino acid residues 21 to 133 of SEQ ID NO: 23, 25 or 27, (5) a homology of at least 95% or more to the amino acid sequence of (4) above
  • An antibody (hR74_H12 / L2) consisting of a light chain consisting of amino acid residues, and a heavy chain consisting of the amino acid residues 20 to 467 of SEQ ID NO: 20 in the Sequence Listing and a heavy chain consisting of amino acid residues 21 to An antibody (hR74_H12 / L4) consisting of a light chain consisting of the 238th amino acid residue can be mentioned.
  • an antibody in which one of the heavy chain or the light chain is humanized and the other is a light chain or a heavy chain of a rat antibody or a chimeric antibody can also be used.
  • “several” means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 To 3 or 1 or 2 means.
  • amino acid substitution is preferable as the amino acid substitution in the present specification.
  • Conservative amino acid substitutions are those that occur within a group of amino acids that are related to amino acid side chains.
  • Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402).
  • Blast algorithm is available on the Internet at www. ncbi. nlm. nih. It can also be used by accessing gov / blast.
  • the antibodies of the present invention can further include human antibodies that bind to PC and aPC.
  • the anti-PC human antibody means a human antibody having only the gene sequence of the antibody derived from human chromosome.
  • the anti-PC human antibody is a method using a human antibody-producing mouse having a human chromosomal fragment containing genes of heavy and light chains of human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133). Kuroda, Y. et. Al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. Al., Animal Cell Technology: Basic and Applied Aspects vol. p.
  • human antibody-producing mouse specifically, loci of endogenous immunoglobulin heavy chains and light chains are destroyed, and instead, human immunoglobulins are obtained via a yeast artificial chromosome (YAC) vector or the like.
  • YAC yeast artificial chromosome
  • knockout animals and transgenic animals can be produced by crossing them.
  • eukaryotic cells are transformed with cDNA encoding each of heavy and light chains of such human antibody, preferably the vector containing the cDNA by genetic recombination technology to produce recombinant human monoclonal antibody.
  • This antibody can also be obtained from the culture supernatant by culturing transformed cells.
  • eukaryotic cells preferably CHO cells
  • mammalian cells such as lymphocytes and myelomas
  • a method for obtaining a phage display-derived human antibody selected from a human antibody library (Wormstone, IM et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Mé, S. et. Al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. Al., Ophthalmology (2002) 109 (3), p. 427-431, etc.) are also known.
  • a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which the variable region of human antibody is expressed on the phage surface as a single chain antibody (scFv) and phages that bind to the antigen are selected. -1116) can be used.
  • the DNA sequence encoding the variable region of human antibody binding to the antigen can be determined.
  • a human antibody can be obtained by preparing an expression vector having the sequence, and introducing it into an appropriate host for expression (WO 92/01047).
  • a newly generated human antibody binds to a partial peptide or partial conformation to which the R74 antibody described herein binds, it can be determined that the human antibody binds to the same epitope as the R74 antibody. Also, by confirming that the human antibody competes for the binding of the R74 antibody to PC or aPC (ie, the human antibody prevents the binding of the R74 antibody to PC or aPC), a specific epitope can be obtained. It can be determined that the human antibody binds to the same epitope as the R74 antibody, even though the sequence or structure of is not determined. When the epitopes are confirmed to be identical, it is strongly expected that the human antibody has biological activity equivalent to that of the R74 antibody.
  • the chimeric antibody, the humanized antibody or the human antibody obtained by the above method can be evaluated for the binding to the antigen by a known method and the like, and a suitable antibody can be selected.
  • DSC Differential scanning calorimetry
  • Tm thermal denaturation midpoint
  • the differences in thermal stability can be compared by measuring Tm values using DSC and comparing the values.
  • the storage stability of the antibody is known to show some correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p. 265-273).
  • Suitable antibodies can be selected on the basis of stability.
  • Other indicators for selecting antibodies can include high yields in appropriate host cells and low aggregation in aqueous solution. For example, since the antibody with the highest yield does not necessarily exhibit the highest thermostability, it is necessary to select the antibody most suitable for human administration, judging comprehensively on the basis of the index described above. .
  • the antibodies of the present invention also include modified antibodies.
  • modified form means that the antibody of the present invention is chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like.
  • post-translational modification eg, glycosylation to N- or O-link, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation
  • those obtained by adding a methionine residue at the N-terminus by expression using a prokaryotic host cell e.g, glycosylation to N- or O-link, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation
  • modified products of the antibody of the present invention are useful for improvement of antibody stability and retention in blood, reduction of antigenicity, detection or isolation of antibody or antigen, and the like.
  • WO 1999/54342, WO 2000/61739, WO 2002/31140, WO 2007/133855, etc. are known as modulation techniques for antibody sugar chain modification, but are limited thereto. It is not a thing.
  • the antibodies of the present invention also include antibodies in which the sugar chain modification has been adjusted.
  • a combination of a suitable host and an expression vector can be used.
  • antibody genes mention may be made of a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence.
  • the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there.
  • animal cells When eukaryotic cells are used as a host, animal cells, plant cells, and eukaryotic microorganisms can be used.
  • animal cells mammalian cells, for example, cells of monkeys such as COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblast NIH 3 T 3 (ATCC No. 4). CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61), dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad. Sci. U. S. A. (1980) 77, p. 4126-4220), FreeStyle 293F cells (Invitrogen) can be mentioned.
  • COS cells Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650
  • mouse fibroblast NIH 3 T 3 ATCC No
  • prokaryotic cells for example, E. coli and Bacillus subtilis can be mentioned.
  • the antibody gene of interest is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain an antibody.
  • the yield may differ depending on the sequence of the antibody, and it is possible to select among the antibodies having the same binding activity, those which can be easily produced as medicaments using the yield as an index. Therefore, the antibody of the present invention is characterized by comprising the steps of culturing the above-mentioned transformed host cell, and collecting the target antibody or a functional fragment of the antibody from the culture obtained in the step. Also included are antibodies obtained by the method for producing the antibody.
  • the antibody according to the present invention also includes the antibody that has been modified and functional fragments of the antibody, a deleted form in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and amidated
  • the deletion body for example, a heavy chain in which a proline residue at the carboxyl terminal site is amidated
  • deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above-mentioned type, as long as the antigen binding ability and effector function are maintained.
  • the two heavy chains constituting the antibody according to the present invention may be any one kind of heavy chain selected from the group consisting of full length and the above-mentioned deletion product, or any two kinds thereof are combined. It may be one.
  • both main chains of the antibody according to the present invention may be carboxyl in both of two heavy chains. There may be mentioned the case where one terminal amino acid residue is deleted.
  • IgG immunoglobulin G
  • IgG1, IgG2, IgG3, IgG4 immunoglobulin G
  • IgG1 or IgG2 can be mentioned.
  • the biological activity of the antibody generally includes antigen binding activity, activity to inhibit the activity of the antigen by binding to the antigen, activity to neutralize the activity of the antigen, activity to enhance the activity of the antigen, antibody dependency
  • cytotoxicity ADCC
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa) are bound together with protein S which binds to aPC and aPC is its cofactor. And the activity to inhibit deactivating and inactivating.
  • Other preferable activities of the anti-PC antibody of the present invention include an activity to inhibit the anticoagulant action by aPC, blood coagulation activity or thrombin generation recovery activity.
  • the resulting antibodies can be purified to homogeneity.
  • separation and purification methods used for ordinary proteins may be used.
  • antibodies can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric focusing electrophoresis etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory ( 988)) it is not intended to be limited thereto.
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography and the like.
  • chromatographies can be performed using liquid chromatographies, such as HPLC and FPLC.
  • a protein A column and a protein G column can be mentioned.
  • Hyper D Hyper D
  • POROS Sepharose F. F. (Pharmacia) and the like.
  • Production of anti-PC antibody” and the examples above a polynucleotide comprising a nucleotide sequence encoding these amino acid sequences, expression
  • a drug comprising at least one of a vector and a host cell as an active ingredient inhibits PC activation and suppresses aPC production and / or binds to aPC and inhibits aPC activity, resulting in thrombin Production (TG) is recovered, blood clotting effect is shown, and it is used as a therapeutic agent for hemorrhagic diseases such as hemophilia, hemophilia A, hemophilia B, etc. alone or in combination with other drugs.
  • TG thrombin Production
  • the anti-PC antibody of the present invention can be used for detection of PC in plasma.
  • the anti-PC antibody of the present invention may become a hydrate due to absorption of water or attachment of adsorbed water, etc., by leaving it in the air, or performing recrystallization or purification operation.
  • Such water-containing compounds or pharmacologically acceptable salts are also included in the present invention.
  • acid addition salts for example, hydrohalic acid salts such as hydrogen fluoride, hydrochloride, hydrobromide, hydroiodide, etc .; nitrates, perchlorates, sulfates, phosphates Inorganic acid salts such as; lower sulfonates such as methane sulfonate, trifluoromethane sulfonate, ethane sulfonate; aryl sulfonates such as benzene sulfonate, p-toluene sulfonate; formate , Acetates, trifluoroacetates, malates, fumarates, succinates, citrates, tartrates, oxalates, organic salts such as maleates; or ornitrates, glutamates, asparagine
  • Such base addition salts include, for example, alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; inorganic salts such as ammonium salts; dibenzylamine salts, morpholine Salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, diethylamine salts, triethylamine salts, cyclohexylamine salts, dicyclohexylamine salts, N, N'-dibenzylethylenediamine salts, diethanolamine salts, N-benzyl-N And organic amine salts such as-(2-phenylethoxy) amine salt, piperazine salt, tetramethylammonium salt, tris (hydroxy
  • the invention can also encompass anti-PC antibodies in which one or more of the atoms that make up the antibody are replaced with an isotope of that atom.
  • isotopes There are two types of isotopes, radioactive isotopes and stable isotopes, and examples of isotopes include, for example, isotopes of hydrogen ( 2 H and 3 H) and isotopes of carbon ( 11 C, 13 C and 14 C), nitrogen isotopes ( 13 N and 15 N), oxygen isotopes ( 15 O, 17 O and 18 O), fluorine isotopes ( 18 F) and the like can be mentioned.
  • composition containing the isotope-labeled antibody is useful as, for example, a therapeutic agent, a prophylactic agent, a research reagent, an assay reagent, a diagnostic agent, an in vivo diagnostic imaging agent, and the like.
  • Isotopically labeled antibodies and mixtures of any proportions of isotopically labeled antibodies are also encompassed by the present invention.
  • the isotope-labeled antibody can be produced by a method known in the art, for example, by using an isotope-labeled raw material instead of the raw material in the production method of the present invention described later.
  • TG recovery activity can be obtained, for example, by adding anti-PC antibody at various concentrations to plasma derived from hemophilia A or hemophilia B patients, and combining with recombinant tissue factor (rTF). Incubate for an appropriate time, then add appropriate amount of FluCa-kit (2.5 mM Z-Gly Gly-Arg-aminomethylcoumarin (Z-GGR-AMC), 100 mM CaCl2) to perform reaction, and examine TG recovery action It can confirm by that.
  • rTF tissue factor
  • the therapeutic effect on hemophilia using experimental animals in vivo can be obtained, for example, by administering an anti-PC antibody to a cynomolgus monkey that has developed a transient hemophilia A by administering an anti-human FVIII neutralizing antibody, Wounds under anesthesia can be confirmed by observing the area of subcutaneous hemorrhage of the wound over time and comparing with the case where anti-PC antibody was not administered.
  • the type of hemorrhagic disease to which the anti-PC antibody of the present invention is applied is not particularly limited as long as it is a disease to which PC and / or aPC are involved in the treatment subject and hemorrhagic disease. Hemophilia B, as well as acquired haemophilia and von Willebrand's disease.
  • the anti-PC of the present invention can be suitably administered to mammals, but is more preferably human.
  • the substance used in the pharmaceutical composition containing the anti-PC antibody of the present invention can be appropriately selected and applied from the formulation additives and the like usually used in the field in dosage amount and concentration.
  • the anti-PC antibodies of the invention can be administered as pharmaceutical compositions comprising one or more pharmaceutically compatible components.
  • the pharmaceutical composition is typically one or more pharmaceutical carriers (eg, sterile liquids (eg, water and oils (eg, petroleum, animal, vegetable, or oils of synthetic origin (eg, peanut oil) , Soybean oil, mineral oil, sesame oil, etc.)) water is a more representative carrier when the above-mentioned pharmaceutical composition is administered intravenously, saline solution, and aqueous dextrose solution and aqueous glycerol solution.
  • Liquid carriers may also be used as liquid carriers, in particular for injectable solutions Suitable pharmaceutical excipients are known in the art. Or an emulsifying agent, or a pH buffering agent Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Science" by E. W. Martin. Described ". The formulations correspond to the mode of administration.
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous routes. Administration may be, for example, by injection or bolus injection. In certain preferred embodiments, administration of the antibody is by injection. Parenteral administration is a preferred route of administration.
  • the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the medicament may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of injection.
  • the above components may be combined separately, or in unit dosage form, as a dry lyophilized powder or as an anhydrous concentrate, for example in a sealed sealed container such as in an ampoule or sachette indicating the amount of active agent
  • a sealed sealed container such as in an ampoule or sachette indicating the amount of active agent
  • the drug can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided, for example, such that the components can be mixed prior to administration.
  • the pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the anti-PC antibody of the present invention, or a pharmaceutical composition containing the anti-PC antibody and at least one other therapeutic agent for hemorrhagic disease, It is also good.
  • the anti-PC antibody of the present invention can also be administered together with other therapeutic agents for hemorrhagic diseases, which can enhance the therapeutic effect on hemorrhagic diseases.
  • the other therapeutic agents for hemorrhagic disease used for such purpose may be administered to the individual simultaneously, separately or sequentially with the antibody, or may be administered at different intervals of administration. .
  • hemorrhagic diseases examples include hemophiliacs (coagulation factor preparations, antibodies, nucleic acid pharmaceuticals, gene therapy preparations, etc.), tranexamic acid, adrenaline, etc. If it is, it will not be limited.
  • Such a pharmaceutical composition may be formulated as a lyophilized formulation or a liquid formulation as a formulation having a selected composition and the required purity.
  • a lyophilised formulation it may be a formulation comprising suitable formulation additives used in the art.
  • a liquid preparation it can be formulated as a liquid preparation containing various preparation additives used in this field.
  • the anti-PC antibody contained in the pharmaceutical composition of the present invention has an affinity for the antigen of the antibody, that is, a dissociation constant (Kd value) for the antigen.
  • Kd value dissociation constant
  • the dose can also be set based on the state of affinity between the antibody and the antigen.
  • the antibody of the present invention is administered to human, for example, about 0.001 to 100 mg / kg may be administered once or plural times at intervals of 1 to 180 days.
  • 0.1 to 50 mg / kg, more preferably 1 to 15 mg / kg may be administered several times at intervals of once every 2 to 3 weeks.
  • the fraction containing activated human PC (aPC) is concentrated with AmiconUltra (MERCK MILLIPORE), then gel filtration is performed with HiLoad 16/600 Superdex 75 pg equilibrated with PBS, 1 mM CaCl 2 to obtain activated human PC
  • the fractions containing it were collected, concentrated to about 2 mg / mL with Amicon Ultra (MERCK MILLIPORE), and used as a purified sample.
  • Hybridoma culture supernatant is screened using a system that uses human PC-deficient plasma as a marker for recovery from the TG suppression effect of human aPC did. 90 ⁇ L of hybridoma culture supernatant, 70 ⁇ L of human PC-depleted plasma with or without 2 nM human aPC, 20 ⁇ L of recombinant tissue factor (rTF) (final concentration 3 pM) and adding at 10 ° C at 37 ° C Incubated for a minute.
  • rTF tissue factor
  • Hybridoma SFM Thermo Fisher SFM (Thermo Fisher), in which a rat anti-PC monoclonal antibody R74-producing hybridoma is grown to a sufficient amount with ClonaCell-HY Selection Medium E (manufactured by StemCell Technologies), and 20% of Ultra Low IgG FBS (manufactured by Thermo Fisher Scienftific) is added. After changing the medium to Fisher Scienftific), 8 to 9 ⁇ 10 7 hybridoma cells were seeded in a 1272 cm 2 flask (Corning) and cultured for 7 days. The main culture supernatant was collected by centrifugation and sterilized through a 0.45 ⁇ m filter (manufactured by Corning).
  • Antibodies were purified from the culture supernatant of hybridomas by Protein G affinity chromatography.
  • the antibody was adsorbed on a Protein G column (GE Healthcare Bioscience), and the column was washed with PBS and eluted with 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7).
  • pH 7.0 to 7.5 After adjusting the pH to 7.0 to 7.5 by adding 1 M Tris-HCl (pH 9.0) to the eluate, buffer with PBS (-) with Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cut off UF 30 K, Sartorius) While performing substitution, concentration of the antibody was performed, and the antibody concentration was adjusted to 1.8 mg / mL.
  • the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
  • Example 2 -3 TG recovery action using plasma from hemophiliac patient-2
  • concentration dependency of the TG recovery action of the antibody purified in Example 2) -1 was examined using plasma from hemophilia A patient (FVIII deficient plasma) and plasma from hemophilia B patient (FIX deficient plasma) .
  • Hemophilia A patient-derived plasma: 5 pM rTF, 10 nM recombinant thrombomodulin (rTM), each antibody concentration: 0.3 ⁇ g / mL to 30 ⁇ g / mL, type B hemophilia patient-derived plasma Evaluation was performed under the conditions of 2.5 pM rTF, 10 nM TM, and each antibody concentration of 0.1 ⁇ g / mL to 10 ⁇ g / mL. R74 recovered TG concentration-dependently in hemophilia A patient-derived plasma, and showed a TG recovery effect equal to or higher than the positive control (anti-human aPC rabbit polyclonal antibody) (FIG. 1A). Similar results were obtained with hemophilia B patient-derived plasma (FIG. 1B).
  • Example 3 Determination of nucleotide sequence of cDNA encoding variable region of rat anti-human PC antibody 3) -1 Preparation of total RNA of R74-producing hybridoma In order to amplify cDNA encoding variable region of R74, R74-producing hybridoma Total RNA was prepared using TRIzol Reagent (Ambion).
  • CDNA encoding the variable region of heavy chain amplified by 5'-RACE PCR was cloned into a plasmid, and then sequence analysis of the nucleotide sequence of cDNA encoding variable region of heavy chain was performed.
  • the nucleotide sequence of the cDNA encoding the variable region of the heavy chain of R74 determined is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.
  • the amino acid sequence of CDRH1 according to the definition of Abm is shown in SEQ ID NO: 7
  • the amino acid sequence of CDRH2 is shown in SEQ ID NO: 8
  • the amino acid sequence of CDRH3 is shown in SEQ ID NO: 9.
  • the nucleotide and amino acid sequences of the variable region of R74 heavy chain, and the amino acid sequences of CDRH1, CDRH2 and CDRH3 are also shown in FIG.
  • the nucleotide sequence of the cDNA encoding the variable region of R74 light chain is shown in SEQ ID NO: 10, and the amino acid sequence of the variable region of R74 light chain is shown in SEQ ID NO: 11.
  • the amino acid sequence of CDRL1 according to the definition of Abm is shown in SEQ ID NO: 12
  • the amino acid sequence in CDRL2 is shown in SEQ ID NO: 13
  • the amino acid sequence of CDRL3 is shown in SEQ ID NO: 14.
  • the nucleotide and amino acid sequences of the variable region of R74 light chain, and the amino acid sequences of CDRL1, CDRL2 and CDRL3 are also shown in FIG.
  • R74 R74 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-1 0033 (1989)).
  • the heavy chain full-length amino acid sequence of hR74_H1 is set forth in SEQ ID NO: 16.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is set forth in SEQ ID NO: 15.
  • the heavy chain full-length amino acid sequence of hR74_H2 is set forth in SEQ ID NO: 18.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 is set forth in SEQ ID NO: 17.
  • the heavy chain full-length amino acid sequence of hR74_H12 is set forth in SEQ ID NO: 20.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 20 is set forth in SEQ ID NO: 19.
  • hR74_H1 and hR74_H2 the amino acid sequence of each CDR as defined by Abm was identical to R74.
  • the amino acid sequences of the CDRs of hR74_H12 according to the Abm definition were identical to R74 in CDRH1, CDRH3, CDRL1, CDRL2 and CDRL3, but the amino acid sequences of CDRH2 were different.
  • the amino acid sequence of CDRH2 of hR74_H12 according to the Abm definition is shown in SEQ ID NO: 21.
  • the nucleotide and amino acid sequences of the humanized antibody hR74_H1 are shown in FIG.
  • the nucleotide and amino acid sequences of hR74_H2 are shown in FIG.
  • the nucleotide and amino acid sequences of hR74_H12 and the amino acid sequence of CDRH2 are shown in FIG.
  • the light chain full-length amino acid sequence of hR74_L1 is set forth in SEQ ID NO: 22.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23 is set forth in SEQ ID NO: 22.
  • the light chain full-length amino acid sequence of hR74_L2 is set forth in SEQ ID NO: 25.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25 is set forth in SEQ ID NO: 24.
  • the light chain full-length amino acid sequence of hR74_L4 is set forth in SEQ ID NO: 27.
  • the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 27 is set forth in SEQ ID NO: 26.
  • hR74_L1 the amino acid sequence of each CDR as defined by Abm was identical to R74.
  • the nucleotide and amino acid sequences of humanized antibody hR74_L1 are shown in FIG.
  • the nucleotide and amino acid sequences of hR74_L2 are shown in FIG.
  • the nucleotide and amino acid sequences of hR74_L4 are shown in FIG.
  • PCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.
  • Example 3 Using the cDNA encoding the variable region of R74 light chain obtained in -3 as a template, PCR is carried out using a primer designed for in-fusion cloning, thereby including the cDNA encoding the light chain variable region The DNA fragment was amplified.
  • the cR74_L expression vector was constructed by inserting the amplified DNA fragment at the site where pCMA-LK was cleaved with restriction enzyme BsiWI using In-Fusion HD PCR cloning kit (Clontech).
  • the nucleotide sequence of cR74_L and the amino acid sequence of the light chain are shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
  • PCR is carried out using a primer designed for In-fusion cloning, thereby including the cDNA encoding the heavy chain variable region
  • the DNA fragment was amplified.
  • the cR74_H expression vector was constructed by inserting the amplified DNA fragment into a site where pCMA-G1-LALA was cleaved with restriction enzyme BlpI using In-Fusion HD PCR cloning kit (Clontech).
  • the nucleotide sequence of cR74_H and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 32 and SEQ ID NO: 33, respectively.
  • Example 5-3 Purification of Human Chimerized Anti-Human PC Antibody
  • the culture supernatant obtained in Example 5-3 was purified by a one-step process of rProtein A affinity chromatography.
  • the culture supernatant was applied to a column (manufactured by GE Healthcare Bioscience) packed with PBS equilibrated with MabSelectSuRe, and then the column was washed with 2 column volumes or more of PBS. It was then eluted with 2 M arginine hydrochloride solution (pH 4.0), and the fractions containing the antibody were collected.
  • the fraction was subjected to buffer substitution to PBS (-) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette).
  • the antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractional molecular weight UF10K, Sartorius) to adjust the IgG concentration to 1 mg / mL or more. Finally, the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
  • VIVASPIN 20 fractional molecular weight UF10K, Sartorius
  • Example 6 In vitro activity of human chimerized anti-human PC antibody 6) -1 TG recovery action by human chimerized anti-human PC antibody using hemophiliac patient-derived plasma Friend of human chimerized anti-human PC antibody cR74_IgG1-LALA The TG recovery action using the plasma derived from the disease A patient and the plasma derived from the hemophilia B patient was compared with the original human anti-PC rat antibody and the positive control polyclonal antibody. The method was carried out according to the method described in 2) -2. cR74_IgG1-LALA showed a TG recovery effect at least equivalent to that of the original human anti-PC rat antibody (R74) in plasma from hemophilia A patients (FIG. 14A).
  • the type B hemophilia patient-derived plasma also showed almost the same results as the hemophilia A patient-derived plasma (FIG. 14B).
  • Example 7 Preparation of humanized anti-human PC antibody 7)-1 Construction of humanized anti-human PC antibody light chain hR74_L expression vector 7)-1-1 Construction of hR74_L1 expression vector Nucleotide sequence of hR74_L1 shown in SEQ ID NO: 11 The DNA fragments shown in nucleotide numbers 37 to 414 of SEQ ID NO: 1 were synthesized (GENEART). The hR74_L1 expression vector was constructed by inserting the synthesized DNA fragment into the site where the chimeric and humanized antibody light chain expression vector pCMA-LK was digested with restriction enzyme BsiWI using the In-Fusion HD PCR cloning kit (Clontech). did.
  • hR74_L2 Expression Vector A DNA fragment containing the DNA sequence shown in nucleotide numbers 37 to 414 of the nucleotide sequence of hR74_L2 shown in SEQ ID NO: 23 was synthesized (GENEART).
  • Example 7 hR74_L2 expression vector was constructed in the same manner as in -1-1.
  • hR74_L4 expression vector was constructed in the same manner as in -1-1.
  • hR74_H1 Expression Vector Construction of hR74_H1 Expression Vector A DNA fragment shown in nucleotide numbers 36 to 428 of the nucleotide sequence of hR74_H1 shown in SEQ ID NO: 15 is synthesized Yes (GENEART). Using the In-Fusion HD PCR cloning kit (Clontech), the hR74_H1 expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G1-LALA was digested with restriction enzyme BlpI.
  • hR74_H12 expression vector was constructed by introducing a mutation using KOD-Plus-Mutagenesis Kit (TOYOBO) using hR74_H1 as a template.
  • the nucleotide sequence of hR74_H12 is shown in SEQ ID NO: 19.
  • Example 7 -3-2 Purification of Humanized Antibody
  • the antibody was purified from the culture supernatant obtained in Example 7) by a two-step process of rProtein A affinity chromatography and ceramic hydroxyapatite.
  • the culture supernatant was applied to a column (manufactured by GE Healthcare Bioscience) packed with PBS equilibrated MabSelectSuRe, and then the column was washed with 2 column volumes or more of PBS.
  • the antibody was then eluted with 2 M arginine hydrochloride solution (pH 4.0).
  • the fractions containing the antibody were subjected to buffer substitution into PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) and diluted 5-fold with a buffer of 5 mM sodium phosphate / 50 mM MES / pH 7.0 Later, it was applied to a ceramic hydroxyapatite column (Bio-Scale CHT Type-1 Hydroxyapatite Column, Japan Bio-Rad) equilibrated with a buffer of 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0. Linear gradient elution with sodium chloride was performed to collect fractions containing the antibody.
  • the fraction was subjected to buffer substitution to HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette).
  • the antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractional molecular weight UF10K, Sartorius) to adjust the IgG concentration to 40 mg / mL or more.
  • the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
  • BAY 1896502 was prepared with reference to WO 2014/085527 A1 and WO 2015/179435 A1.
  • BAY 1896502 light chain expression vector (Murakami, throne) The DNA fragment shown in nucleotide numbers 37 to 414 of the nucleotide sequence of BAY 1896502 light chain shown in SEQ ID NO: 34 was synthesized (GENEART).
  • SEQ ID NO: 34 The DNA fragment shown in nucleotide numbers 37 to 414 of the nucleotide sequence of BAY 1896502 light chain shown in SEQ ID NO: 34 was synthesized (GENEART).
  • Example 7 An expression vector of BAY 1896502 light chain was constructed in the same manner as in-1-1. The amino acid sequence of BAY 1896502 light chain is shown in SEQ ID NO: 35.
  • the DNA fragment shown in nucleotide numbers 36 to 428 of the nucleotide sequence of BAY 1896502 heavy chain shown in SEQ ID NO: 37 was synthesized (GENEART).
  • a BAY 1896502 heavy chain expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G2 was digested with restriction enzyme BlpI.
  • the amino acid sequence of BAY 1896502 heavy chain is shown in SEQ ID NO: 38.
  • Example 9 In Vitro Activity of Humanized Anti-Human PC Antibody 9) -1 Antigen Binding Activity of Humanized Anti-Human PC Antibody by SPR The binding of the antibody to the antigen was determined using Biacore 4000 (GE Healthcare Biosciences Co., Ltd.) ) was used to capture (capture) the antibody as a ligand to the immobilized anti-human IgG Fc antibody, and the antigen was measured by a capture method that measures as an analyte. Human PC (Enzyme Research Laboratories) was used as an antigen.
  • Anti-human IgG Fc antibody Human Antibody Capture Kit, GE Healthcare Biosciences, Inc.
  • CM5 GE Healthcare Biosciences, Inc.
  • HBS-EP + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as a running buffer.
  • Viscosity measurement of humanized anti-PC antibody The sample is concentrated using Vivapore 5 (Sartorius), adjusted to 150 mg / mL with 25 mM histidine, 5% sorbitol, pH 6.0 buffer, The measurement was performed using m-VROC (RheoSense) at 25 ° C. and a flow rate of 75 ⁇ L / min. As a result, the viscosity was about 40 cP for hR74_H1 / L1, and about 50% for hR74_H12 / L1 and hR74_H12 / L4 (Table 2).
  • the method was carried out according to the method described in 9) -3-1.
  • the TG recovery effect using hemophilia A patient-derived plasma (FIG. 16A) and hemophilia B patient-derived plasma (FIG. 16B) was compared between hR74_H1 / L1 and BAY 1896502. Although both antibodies recovered TG in a concentration-dependent manner, the TG recovery effect of hR74_H1 / L1 clearly exceeded that of BAY 1896502 in both types of hemophiliac patient-derived plasma.
  • Example 10 In vivo activity of humanized anti-human PC antibody The subcutaneous hemorrhage suppressing effect of hR74_H1 / L1 in a transient hemophilia A cynomolgus monkey hemorrhage model was examined.
  • Anti-human FVIII neutralizing antibody (HAEMATOLOGIC TECHNOLOGIES, sheep polyclonal antibody) alone (18,000 Bethesda Unit (BU) / kg, iv) alone or hR74_H1 / L1 (3 or 1.5 mg / kg) from the superficial vein under awakening , Iv) and after 2 hours isoflurane (2-3% when introduced, 1.5-2.5% maintenance) under anesthesia BD Microtainer (Catalog No. 366594, Blue, 1.5 mm wide blade, Using a depth of 2.0 mm, a large vein was avoided, and 24 locations (4 locations on each upper arm, 2 locations on each forearm, 4 locations on the inside of each thigh, 2 locations on each groin) .
  • the day of wounding was designated as Day 0. After administration of the analgesic, he was awakened from anesthesia. Dosage regimen: lepetan (as buprenorphine) 0.01 mg / kg, SC and metacam (as meloxicam) 0.2 mg / kg, SC, 24, 48 and 72 hours later as metacam (as meloxicam) on the day of the experiment It was 0.1 mg / kg, SC.
  • the subcutaneous bleeding site was photographed with a digital camera on Day 1, 2 and 3 (24, 48 and 72 hours after administration of FVIII neutralizing antibody), and the area was measured the day after by analysis software.
  • the humanized anti-PC antibody of the present invention has a stronger thrombin generation activity than known antibodies, and can be a preventive and / or therapeutic agent for hemophilia.
  • Sequence number 2 Peptide tag sequence number 3: Sequence X Sequence number 4: Sequence Y SEQ ID NO: 5: Nucleotide sequence of variable region of heavy chain of R74 SEQ ID NO: 6: Amino acid sequence of variable region of heavy chain of R74 SEQ ID NO: 7: CDRH1 SEQ ID NO: 8: CDRH2 Sequence number 9: CDRH3 SEQ ID NO: 10: Nucleotide sequence of variable region of light chain of R74 SEQ ID NO: 11: Amino acid sequence of variable region of light chain of R74 SEQ ID NO: 12: CDR L1 SEQ ID NO: 13: CDRL2 SEQ ID NO: 14: CDRL3 SEQ ID NO: 15: Nucleotide sequence encoding hR74_H1 amino acid sequence of hR74_H1 SEQ ID NO: 17: nucleotide sequence encoding hR74_H2 amino acid sequence of hR74_H2 SEQ ID NO: 19 nucleotide sequence

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Abstract

The problem addressed by the present invention is to provide a novel drug for treating haemorrhagic disorders which has a superior blood-coagulating effect, and a method for treating haemorrhagic disorders which uses said drug. Provided are: an antibody which bonds to both Protein C and activated-Protein C, and inhibits Protein C activation and activated-Protein C activity; an antigen-binding fragment of said antibody; a chimeric antibody of said antibody; a pharmaceutical compound comprising said antibody; and the like.

Description

抗プロテインＣ抗体Anti-protein C antibody
　本発明は、プロテインＣ及び活性化プロテインＣを特異的に認識する、抗プロテインＣ抗体、該抗プロテインＣ抗体の製造方法、並びに該抗体を含む医薬組成物等に関する。 The present invention relates to an anti-protein C antibody that specifically recognizes protein C and activated protein C, a method for producing the anti-protein C antibody, a pharmaceutical composition containing the antibody, and the like.
　血友病は血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩ）又は血液凝固第ＩＸ因子（ＦＩＸ）の先天的欠損又は機能不全に起因する出血性疾患である。前者は血友病タイプＡ（以下、「血友病Ａ」とも記載する。）、後者は血友病タイプＢ（以下、「血友病Ｂ」とも記載する。）と呼ばれている。いずれの遺伝子もＸ染色体上に存在し、伴性劣性遺伝であることから発症患者のほとんどが男性である。有病率は男性１０万人あたり約９人であり、血友病Ａと血友病Ｂの比率は約５：１である。 Hemophilia is a hemorrhagic disease caused by congenital defects or dysfunction of blood coagulation factor VIII (FVIII) or blood coagulation factor IX (FIX). The former is referred to as hemophilia type A (hereinafter also referred to as "hemophilia A"), and the latter is referred to as hemophilia type B (hereinafter referred to as "hemophilia B"). Most of the onset patients are males because all genes exist on the X chromosome and are sexually recessive. The prevalence rate is about 9 per 100,000 males, and the ratio of hemophilia A to hemophilia B is about 5: 1.
　主な出血症状としては、皮下出血、異常外傷性出血、頭蓋内出血、関節内出血、口腔内出血、鼻腔内出血、腸腰筋出血、消化管出血などがある。特定の関節に出血を繰り返すことにより慢性滑膜炎が起こり、これが持続することにより関節軟骨に不可逆的な変化が生じ、関節障害、歩行困難を伴う血友病性関節症に進展し、最終的には関節置換術が必要になる場合もあり、血友病患者のＱＯＬを著しく低下させる大きな要因となっている。 The main bleeding symptoms include subcutaneous hemorrhage, abnormal traumatic hemorrhage, intracranial hemorrhage, joint hemorrhage, intraoral hemorrhage, intranasal hemorrhage, intranasal hemorrhage, iliopsoas hemorrhage, gastrointestinal tract hemorrhage and the like. Repeated hemorrhage to a specific joint causes chronic synovitis, which causes irreversible changes in articular cartilage, which leads to arthritic joint disorder, hemophilic arthropathy with difficulty walking, and finally In some cases, joint replacement surgery may be required, which is a major factor that significantly reduces the quality of life of hemophiliacs.
　血友病の重症度は、血液中のＦＶＩＩＩ活性あるいはＦＩＸ活性により、１％未満の活性患者を重症、１％以上５％未満の患者を中等症、５％以上４０％未満の患者を軽症と規定している。血友病患者の６０％以上が重症とされている。血友病の中等症・軽症は、重症とは数％程度の凝固因子活性の相違しかないが、出血頻度が明らかに少ないことから、ＦＶＩＩＩ活性あるいはＦＩＸ活性を１％以上に維持することが出血頻度の減少に有効と考えられている。 The severity of hemophilia is based on FVIII activity or FIX activity in blood, with less than 1% active patients as severe, 1% or more less than 5% patients moderate, 5% or more less than 40% patients as mild It specifies. More than 60% of haemophilia patients are considered severe. The moderate and mild cases of haemophilia differ only by a few percent of the coagulation factor activity from the severe, but the bleeding frequency is clearly less, so maintaining the FVIII activity or FIX activity at 1% or more is a bleeding event It is considered effective in reducing the frequency.
　血友病患者における出血の予防及び／又は治療には、血漿から精製された、又は遺伝子組換え技術によって作製された血液凝固因子製剤（ＦＶＩＩＩ製剤、ＦＩＸ製剤）が主に使用されている。血液凝固因子は血中半減期が数時間から数十時間と短いために薬効の持続性が短く、出血の予防には血液凝固因子製剤を週に３回程度静注投与し、凝固因子活性（ＦＶＩＩＩ活性あるいはＦＩＸ活性）を１％以上に維持する必要がある。この血液凝固因子製剤の定期補充療法により、出血の頻度を減少させ、その結果血友病患者のＱＯＬ改善に大きく寄与している。 Blood coagulation factor preparations (FVIII preparations, FIX preparations) purified from plasma or produced by genetic engineering techniques are mainly used for the prevention and / or treatment of hemorrhage in hemophiliacs. Since blood coagulation factor has a short half-life of several hours to several tens of hours, its persistence of efficacy is short, and blood coagulation factor preparations are intravenously administered about 3 times a week to prevent bleeding. It is necessary to maintain FVIII activity or FIX activity) at 1% or more. This blood coagulation factor replacement therapy reduces the frequency of bleeding and consequently contributes significantly to the improvement of QOL of hemophilia patients.
　また出血時の治療（補充療法）においても、完全な止血、再出血の防止のために一定期間、一定以上の凝固因子活性を維持するため血液凝固因子製剤の定期的な追加投与を行なう。 In addition, in hemorrhage treatment (replacement therapy), blood glucose factor preparations are periodically added to maintain a certain level of coagulation factor activity for a certain period of time to prevent complete hemostasis and rebleeding.
　さらに血液凝固因子製剤は投与経路が静脈内投与に限られる。静脈内投与は技術的に難しく、特に小児患者や痩身な患者においては静脈が細く、より困難であり、投与時の慎重さが求められる。 Furthermore, blood coagulation factor formulations are limited to intravenous administration. Intravenous administration is technically difficult, especially in pediatric and lean patients, where the veins are narrow and more difficult, requiring careful administration.
　前述の予防及び／又は治療の投与では、多くの場合、家庭療法や自己注射が行なわれるが、頻回投与が必要であり、さらに投与に技術的困難さが伴う。このことは、投与に際して患者に苦痛を与えるのみならず、家庭療法や自己注射の普及を妨げる要因となり、アドヒアランスの低下をもたらしている。 The administration of the aforementioned prophylaxis and / or treatment often involves home remedies and self-injection, but requires frequent administration and further technical difficulties associated with administration. This not only causes pain to patients upon administration, but also causes the spread of home remedies and self-injection, resulting in decreased adherence.
　従って、既存の凝固因子製剤に比較して半減期が長く、投与頻度を軽減できる薬剤、あるいは投与が簡単で患者のアドヒアランスの向上に繋がる薬剤が強く求められていた。 Therefore, there has been a strong demand for a drug having a long half life and reducing the frequency of administration as compared to the existing coagulation factor formulations, or a drug that is easy to administer and leads to improvement in patient adherence.
　さらに、血友病患者ではインヒビターと呼ばれる凝固因子製剤に対する中和抗体が少なくない確率で発生する。特に重症患者ではその確率は３０％とも言われている。インヒビターが発生すると、凝固因子製剤の効果がインヒビターにより中和され、その効果が減少、最悪の場合消失する。その結果、活性型プロトロンビン複合体製剤や遺伝子組換え活性型第ＶＩＩ因子製剤を用いるバイパス療法が実施される。しかし、いずれの場合も定期補充療法は確立しておらず、また患者に対する止血管理が非常に困難である。よって、インヒビターの有無に影響を受けない薬剤が強く求められていた。 Furthermore, in hemophiliacs, neutralizing antibodies against coagulation factor preparations called inhibitors occur with a considerable probability. The probability is said to be as high as 30%, especially in critically ill patients. When an inhibitor is generated, the effect of the coagulation factor preparation is neutralized by the inhibitor, and the effect is reduced, and in the worst case disappears. As a result, bypass therapy using an active prothrombin complex preparation or a recombinant active factor VII preparation is performed. However, in neither case has fixed prophylaxis has been established, and hemostasis management for the patient is very difficult. Therefore, there has been a strong demand for drugs that are not affected by the presence or absence of inhibitors.
　さて、抗体は血中での安定性が高く、皮下投与が可能であり、標的の選択性も高いことから、近年医薬品として応用され、上市されている。抗体は、半減期が長いことから投与頻度が軽減でき、皮下投与が可能であることから投与が簡便であり、インヒビターの有無に影響を受けないことから、血友病の創薬に適していると考えられる。 Now, antibodies have high stability in blood, can be administered subcutaneously, and have high target selectivity, so they have recently been applied and marketed as pharmaceuticals. The antibody has a long half-life, which can reduce the frequency of administration, and because it can be administered subcutaneously, is easy to administer and is not affected by the presence or absence of an inhibitor, so it is suitable for drug discovery for hemophilia it is conceivable that.
　プロテインＣ（以下、「ＰＣ」とも記載する。）は血液凝固におけるネガティブフィードバック機構において主要な役割を担っている。ＰＣは血管内皮に発現するｅｎｄｏｔｈｅｒｉａｌ　Ｐｒｏｔｅｉｎ　Ｃ　ｒｅｃｅｐｔｏｒ（ＥＰＣＲ）に結合し、トロンビン／トロンボモデュリン複合体により活性化され、活性化プロテインＣ（以下、「ａＰＣ」とも記載する。）となる。ａＰＣはその補因子であるプロテインＳとともに、活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び活性化血液凝固第Ｖ因子（ＦＶａ）を分解、不活化する。従って、ＰＣを阻害し、ネガティブフィードバック機構を阻害することで凝固の促進が期待できる。ホモ接合体先天性ＰＣ欠乏症患者では血栓性の電撃性紫斑病を発症する事が知られていることも上記の考えを支持している。 Protein C (hereinafter also referred to as "PC") plays a major role in the negative feedback mechanism in blood coagulation. PC binds to endotherial protein receptor (EPCR) expressed on vascular endothelium and is activated by thrombin / thrombomodulin complex to become activated protein C (hereinafter also described as "aPC"). aPC, together with its cofactor, protein S, degrades and inactivates activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa). Therefore, the inhibition of PC and the inhibition of the negative feedback mechanism can be expected to promote coagulation. The fact that homozygous congenital PC deficiency patients are known to develop thrombotic shock purpura also supports the above idea.
　実際、ｉｎ　ｖｉｔｒｏでａＰＣの阻害物質がＦＶＩＩＩ欠乏血漿におけるトロンビン産生の低下を部分的に回復させることが報告されている（非特許文献１、非特許文献２）。また、ａＰＣの活性を阻害する抗体がａＰＣの抗凝血活性を阻害し、血液凝固効果を示し、血友病の治療に有効である可能性が報告されている（特許文献１）。 In fact, it has been reported that an inhibitor of aPC partially restores the reduction of thrombin generation in FVIII-deficient plasma in vitro (Non-patent Document 1, Non-patent Document 2). In addition, it has been reported that an antibody that inhibits the activity of aPC inhibits the anticoagulant activity of aPC, exhibits a blood coagulation effect, and is effective for the treatment of hemophilia (Patent Document 1).
　またａＰＣに対する分解抵抗性を有する変異ＦＶａが、ＦＶＩＩＩ欠乏マウス出血モデルにおいて出血量を軽減させたことが報告されている（非特許文献３）。さらにＰＣの活性化を阻害する及び／又はａＰＣの活性を阻害する抗体もＦＶＩＩＩ欠損ヌードマウスの穿刺出血モデルにおいて出血痕面積を減少させたことが報告されている（特許文献２）。 In addition, it has been reported that mutant FVa having degradation resistance to aPC reduced the amount of bleeding in a FVIII-deficient mouse bleeding model (Non-patent Document 3). Furthermore, it has been reported that an antibody that inhibits PC activation and / or aPC activity also reduces the bleeding scar area in a puncture bleeding model of FVIII-deficient nude mice (Patent Document 2).
　ａＰＣ抗体がサル出血モデルで出血を抑制したという報告がある（非特許文献４）。 There is a report that the aPC antibody suppressed hemorrhage in a monkey hemorrhage model (Non-patent Document 4).
　ａＰＣに対しモデュレーターとしての機能を有すると考えられる抗体が報告されている（特許文献３、４）。しかしながら、非げっ歯類の血友病モデルにおいてＰＣ及びａＰＣの双方に結合し得る抗体の止血効果は確認されていない。 An antibody which is considered to have a function as a modulator for aPC has been reported (Patent Documents 3 and 4). However, the hemostatic effect of an antibody capable of binding to both PC and aPC has not been confirmed in a non-rodent hemophiliac model.
特開２０１４－２３７６５１号特許公報JP, 2014-237651, A patent gazette ＷＯ２０１５／０４１３１０号国際公開公報WO2015 / 041310 International Publication ＷＯ２０１４／０８５５２７号国際公開公報WO2014 / 085525 International Publication ＷＯ２０１５／１７９４３５号国際公開公報WO2015 / 179435 International Publication
　本発明の課題は、優れた血液凝固効果を有する新規な出血性疾患治療用医薬、該医薬を使用した出血性疾患の治療方法等を提供することである。 An object of the present invention is to provide a novel medicament for treating hemorrhagic disease having an excellent blood coagulation effect, a method for treating hemorrhagic disease and the like using the medicament.
　本発明者らは、上記課題を達成するために鋭意検討し、プロテインＣ及び活性化プロテインＣの双方に結合し、プロテインＣの活性化及び活性化プロテインＣの活性を阻害する抗体を取得し該抗体が高い止血効果を有し、出血性疾患、特に血友病Ａ及び／又は血友病Ｂの治療に効果を有することを見出し、本発明を完成させた。 The present inventors diligently studied to achieve the above problems, and obtain an antibody that binds to both protein C and activated protein C, and inhibits activation of protein C and activity of activated protein C. The inventors have found that the antibody has a high hemostatic effect and is effective in the treatment of hemorrhagic diseases, particularly hemophilia A and / or hemophilia B, and completed the present invention.
　本発明により、プロテインＣの活性化及び活性化プロテインＣの活性を阻害する抗体が提供され、該抗体を有効成分として含有する優れた血液凝固効果を有する新規な出血性疾患治療用医薬、該医薬を使用した出血性疾患の治療方法等が提供された。 The present invention provides an antibody that inhibits activation of protein C and activity of activated protein C, and a novel medicament for treating hemorrhagic disease having an excellent blood coagulation effect, which comprises the antibody as an active ingredient, the medicine Provided a method for treating hemorrhagic disease using
　すなわち本願発明は、
（１）プロテインＣ又は活性化プロテインＣへの結合に対して、以下の（ａ）乃至（ｅ）からなる群から選択されるいずれか１項に記載の抗体と競合阻害活性を有する抗体又は当該抗体の機能性断片：
（ａ）配列番号３３のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号３０のアミノ酸番号２１乃至２３７に示されるアミノ酸配列からなる軽鎖を有する抗体、
（ｂ）配列番号１６のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２３のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体、
（ｃ）配列番号２０のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２５のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体、
（ｄ）配列番号２０のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２３のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体、及び
（ｅ）配列番号２０のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２７のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体、
（２）配列番号７に示されるアミノ酸配列からなるＣＤＲＨ１、配列番号８又は配列番号２１に示されるアミノ酸配列からなるＣＤＲＨ２及び配列番号９に示されるアミノ酸配列からなるＣＤＲＨ３を含む重鎖、並びに、配列番号１２に示されるアミノ酸配列からなるＣＤＲＬ１、配列番号１３に示されるアミノ酸配列からなるＣＤＲＬ２及び配列番号１４に示されるアミノ酸配列からなるＣＤＲＬ３を含む軽鎖からなり、プロテインＣ及び活性化プロテインＣに特異的に結合することを特徴とする抗体又は当該抗体の機能性断片、
（３）プロテインＣ及び活性化プロテインＣがヒト由来であることを特徴とする、（１）又は（２）に記載の抗体又は当該抗体の機能性断片、
（４）プロテインＣが配列表の配列番号３のアミノ酸番号４３乃至１５３に示されるアミノ酸配列からなるペプチドとアミノ酸番号１５４乃至４１２に示されるアミノ酸配列からなるペプチドがＳＳ結合で結合しているポリペプチドであり、活性化プロテインＣが配列表の配列番号３のアミノ酸番号４３乃至１５３に示されるアミノ酸配列からなるペプチドとアミノ酸番号１６６乃至４１２に示されるアミノ酸配列からなるペプチドがＳＳ結合で結合しているポリペプチドであることを特徴とする、（１）乃至（３）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（５）プロテインＣの活性化及び／又は活性化プロテインＣの活性を阻害することを特徴とする、（１）乃至（４）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（６）以下の（ａ）乃至（ｄ）から選択される少なくとも１つの特性を有することを特徴とする、（１）乃至（５）のいずれか１項に記載の抗体又は当該抗体の機能性断片：
（ａ）プロテインＣ及び活性化プロテインＣに特異的に結合する；
（ｂ）プロテインＣに特異的に結合しプロテインＣの活性化を阻害する；
（ｃ）活性化プロテインＣに特異的に結合し、活性化プロテインＣによる活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び／又は活性化血液凝固第Ｖ因子（ＦＶａ）の分解及び／又は不活化を阻害する；
　（ｄ）トロンビン産生を回復する、
（７）ＣＤＲＨ２が配列番号８に示されるアミノ酸配列からなることを特徴とする、（１）乃至（６）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（８）ＣＤＲＨ２が配列番号２１に示されるアミノ酸配列からなることを特徴とする、（１）乃至（６）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（９）配列番号６に記載の重鎖可変領域、及び配列番号１１に記載の軽鎖可変領域を含むことからなる、（１）乃至（６）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（１０）配列番号３３のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号３０のアミノ酸番号２１乃至２３７に示されるアミノ酸配列からなる軽鎖を含むことからなる、（１）乃至（７）、及び（９）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（１１）定常領域がヒト由来定常領域である（１）乃至（１０）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（１２）ヒト化されている（１）乃至（１１）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（１３）以下の（ａ）乃至（ｅ）からなる群から選択されるいずれか１項に記載のアミノ酸配列からなる重鎖可変領域、及び（ｆ）乃至（ｊ）からなる群から選択されるいずれか１項に記載のアミノ酸配列からなる軽鎖可変領域を有する（１２）に記載の抗体又は当該抗体の機能性断片：
（ａ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列、
（ｂ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列、
（ｃ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列、
（ｄ）（ａ）乃至（ｃ）の配列において各ＣＤＲ　配列以外のフレームワーク領域の配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、
（ｅ）（ａ）乃至（ｄ）の配列における各ＣＤＲ　配列以外のフレームワーク領域の配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、
（ｆ）配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列、
（ｇ）配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列、
（ｈ）配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列、
（ｉ）（ｆ）乃至（ｈ）の配列において各ＣＤＲ　配列以外のフレームワーク領域の配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、及び
（ｊ）　（ｆ）乃至（ｉ）の配列における各ＣＤＲ　配列以外のフレームワーク領域の配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、
（１４）以下の（ａ）乃至（ｉ）からなる群から選択されるいずれか１項に記載の重鎖可変領域及び軽鎖可変領域を含む（１３）に記載の抗体又は当該抗体の機能性断片：
（ａ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域、
（ｂ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる、重鎖可変領域および配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（ｃ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる、重鎖可変領域および配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（ｄ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（ｅ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（ｆ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（ｇ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（ｈ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（ｉ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
（１５）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域を含む（１３）又は（１４）に記載の抗体又は当該抗体の機能性断片、
（１６）配列番号配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域を含む（１３）又は（１４）に記載の抗体又は当該抗体の機能性断片、
（１７）配列番号配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域を含む（１３）又は（１４）に記載の抗体又は当該抗体の機能性断片、 That is, the present invention
(1) An antibody having a competitive inhibitory activity with the antibody according to any one of the following groups (a) to (e), for binding to protein C or activated protein C: Functional fragments of antibodies:
(A) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 33 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 237 of SEQ ID NO.
(B) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO.
(C) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 20 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO.
(D) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 20 and a light chain consisting of the amino acid sequences shown by amino acid numbers 21 to 238 of SEQ ID NO. An antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of 20 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO.
(2) A heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 21 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 9, Consisting of a light chain comprising CDRL1 consisting of the amino acid sequence shown in No. 12, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO. 13 and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO. Or a functional fragment of the antibody, characterized in that
(3) The antibody according to (1) or (2) or a functional fragment of the antibody, wherein the protein C and the activated protein C are derived from human.
(4) A polypeptide comprising a peptide consisting of the amino acid sequence shown by amino acid numbers 43 to 153 of SEQ ID NO: 3 in the sequence listing and a peptide consisting of the amino acid sequence shown by amino acid numbers 154 to 412 linked by SS bond And the peptide consisting of the amino acid sequence shown by amino acid numbers 43 to 153 of SEQ ID NO: 3 in the sequence listing and the peptide consisting of the amino acid sequence shown by amino acid numbers 166 to 412 are linked by SS bond The antibody according to any one of (1) to (3) or a functional fragment of the antibody, which is a polypeptide.
(5) The antibody according to any one of (1) to (4) or a functional fragment of the antibody, characterized in that the protein C is activated and / or the activity of the activated protein C is inhibited.
(6) The antibody according to any one of (1) to (5) or the functionality of the antibody, characterized by having at least one property selected from the following (a) to (d): fragment:
(A) specifically bind to protein C and activated protein C;
(B) specifically bind to protein C and inhibit protein C activation;
(C) It specifically binds to activated protein C and causes decomposition and / or inactivation of activated blood coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa) by activated protein C Inhibit;
(D) restore thrombin generation,
(7) The antibody according to any one of (1) to (6) or a functional fragment of the antibody, wherein CDRH2 consists of the amino acid sequence shown in SEQ ID NO: 8,
(8) The antibody according to any one of (1) to (6) or a functional fragment of the antibody, wherein CDRH2 consists of the amino acid sequence shown in SEQ ID NO: 21;
(9) The antibody according to any one of (1) to (6), which comprises the heavy chain variable region of SEQ ID NO: 6 and the light chain variable region of SEQ ID NO: 11 or the antibody Functional fragments of,
(10) A heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 33 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 237 of SEQ ID NO. The antibody according to any one of (7) and (9) or a functional fragment of the antibody,
(11) The antibody according to any one of (1) to (10), wherein the constant region is a human-derived constant region, or a functional fragment of the antibody,
(12) The antibody according to any one of (1) to (11) or a functional fragment of the antibody, which is humanized.
(13) A heavy chain variable region consisting of the amino acid sequence according to any one of the following (a) to (e) and a group selected from the group consisting of (f) to (j) The antibody according to (12) or the functional fragment of the antibody, which has a light chain variable region consisting of the amino acid sequence according to any one of:
(A) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16,
(B) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18;
(C) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20,
(D) an amino acid sequence having at least 95% or more homology to a sequence of a framework region other than each CDR sequence in the sequences of (a) to (c),
(E) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequences of (a) to (d),
(F) the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23,
(G) the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25;
(H) the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27;
(I) an amino acid sequence having at least 95% or more homology with a sequence of a framework region other than each CDR sequence in the sequence of (f) to (h), and (j) (f) to (i) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of framework regions other than each CDR sequence in the sequence,
(14) The antibody according to (13), which comprises the heavy chain variable region and the light chain variable region according to any one of the following (a) to (i): fragment:
(A) a heavy chain variable region consisting of the amino acid sequence as set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16, and a light chain variable flow region consisting of the amino acid sequence as set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23
(B) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25
(C) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27;
(D) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23;
(E) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25;
(F) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27
(G) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23
(H) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25;
(I) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27
(15) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable flow region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 (13) Or the antibody according to (14) or a functional fragment of the antibody,
(16) SEQ ID NO: 20 comprises a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20 and a light chain variable flow region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO. 13) or the antibody according to (14) or a functional fragment of the antibody,
(17) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20 and a light chain variable flow region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO. 13) or the antibody according to (14) or a functional fragment of the antibody,
（１８）以下の（ａ）乃至（ｉ）からなる群から選択されるいずれか１項に記載の重鎖及び軽鎖を含む（１３）乃至（１７）のいずれか１項に記載の抗体又は抗体の機能性断片：
（ａ）配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｂ）配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２５においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｃ）配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｄ）配列番号１８においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｅ）配列番号１８においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２５においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｆ）配列番号１８においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｇ）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｈ）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２５においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（ｉ）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
（１９）配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖を含む（１３）乃至（１８）のいずれか１項に記載の抗体又は当該抗体の機能性断片。
（２０）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖を含む（１３）乃至（１８）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（２１）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖を含む（１３）乃至（１８）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（２２）機能性断片がＦａｂ、Ｆ（ａｂ）２、Ｆａｂ’及びＦｖからなる群から選択される（１）乃至（２１）のいずれか１項に記載の抗体の機能性断片、
（２３）（１）乃至（２２）のいずれか１項に記載の抗体又は当該抗体の機能性断片をコードするポリヌクレオチド、
（２４）配列番号７に記載のアミノ酸配列からなるＣＤＲＨ１、配列番号８又は２１に記載のアミノ酸配列からなるＣＤＲＨ２及び配列番号９に記載のアミノ酸配列からなるＣＤＲＨ３をコードするポリヌクレオチド、並びに配列番号１２に記載のアミノ酸配列からなるＣＤＲＬ１、配列番号１３に記載のアミノ酸配列からなるＣＤＲＬ２及び配列番号１４に記載のアミノ酸配列からなるＣＤＲＬ３をコードするポリヌクレオチドを含む（２３）に記載のポリヌクレオチド、
（２５）以下の（ａ）乃至（ｊ）からなる群から選択されるいずれか１項に記載のポリヌクレオチドを含む（２３）又は（２４）に記載のポリヌクレオチド：
（ａ）配列番号６に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号１１に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド；
（ｂ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域をコードするポリヌクレオチド；
（ｃ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、および配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド；
（ｄ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド，および配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド；
（ｅ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド；
（ｆ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド；
（ｇ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド；
（ｈ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド；
（ｉ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド；
（ｊ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
（２６）（２３）乃至（２５）のいずれか１項に記載のポリヌクレオチドを含有する発現ベクター、
（２７）（２６）に記載の発現ベクターにより形質転換された宿主細胞、
（２８）宿主細胞が真核細胞である（２６）に記載の宿主細胞、
（２９）（２７）又は（２８）に記載の宿主細胞を培養する工程、及び当該工程で得られた培養物から目的の抗体又は当該抗体の機能性断片を採取する工程を含むことを特徴とする当該抗体又は当該抗体の機能性断片の製造方法、
（３０）（２９）に記載の製造方法により得られることを特徴とする抗体又は当該抗体の機能性断片、
（３１）機能性断片がＦａｂ、Ｆ（ａｂ）２、Ｆａｂ’及びＦｖからなる群から選択される（３０）に記載の抗体の機能性断片、
（３２）Ｎ－結合への糖鎖付加、Ｏ－結合への糖鎖付加、Ｎ末のプロセッシング、Ｃ末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化、Ｎ末にメチオニン残基の付加、プロリン残基のアミド化及び重鎖カルボキシル末端における１つ又は２つのアミノ酸の欠失からなる群より選択される１又は２以上の修飾を含む（１）乃至（２２）、（３０）及び（３１）のいずれか１項に記載の抗体又は当該抗体の機能性断片、
（３３）重鎖のカルボキシル末端において１つ又は２つのアミノ酸が欠失している（３２）に記載の抗体、
（３４）２本の重鎖の双方でカルボキシル末端において１つのアミノ酸が欠失している（３３）に記載の抗体、
（３５）重鎖のカルボキシル末端のプロリン残基が更にアミド化されている（３２）乃至（３４）のいずれか１項に記載の抗体、 (18) The antibody according to any one of (13) to (17), which comprises the heavy chain and the light chain according to any one of the following (a) to (i): Functional fragments of antibodies:
(A) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23
(B) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 25;
(C) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27
(D) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 18 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23;
(E) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 18 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 25;
(F) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 18 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27;
(G) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23;
(H) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 25;
(I) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27;
(19) A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23 (13) to (18) The antibody according to any one of or a functional fragment of the antibody.
(20) A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23 (13) to (18) An antibody according to any one of the claims or a functional fragment of the antibody,
(21) A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27 (13) to (18) An antibody according to any one of the claims or a functional fragment of the antibody,
(22) The functional fragment of the antibody according to any one of (1) to (21), wherein the functional fragment is selected from the group consisting of Fab, F (ab) 2, Fab ′ and Fv.
(23) An antibody according to any one of (1) to (22) or a polynucleotide encoding a functional fragment of the antibody,
(24) A polynucleotide encoding CDRH1 consisting of the amino acid sequence of SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence of SEQ ID NO: 8 or 21 and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 9, The polynucleotide according to (23), which comprises a polynucleotide encoding the CDRL1 consisting of the amino acid sequence described in; the CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 13 and the CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 14
(25) The polynucleotide according to (23) or (24), which comprises a polynucleotide according to any one of the following groups (a) to (j):
(A) a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 6, and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11;
(B) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence as set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable watershed consisting of the amino acid sequence as set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 A polynucleotide encoding
(C) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25 A polynucleotide encoding
(D) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27 A polynucleotide encoding
(E) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 A polynucleotide encoding
(F) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25 A polynucleotide encoding
(G) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27 A polynucleotide encoding
(H) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 A polynucleotide encoding
(I) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25 A polynucleotide encoding
(J) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27 A polynucleotide encoding
(26) An expression vector comprising the polynucleotide according to any one of (23) to (25),
(27) A host cell transformed with the expression vector according to (26),
(28) The host cell according to (26), wherein the host cell is a eukaryotic cell,
(29) A step of culturing the host cell according to (27) or (28), and a step of collecting a target antibody or a functional fragment of the antibody from the culture obtained in the step. A method for producing the antibody or functional fragment of the antibody,
(30) An antibody characterized by being obtained by the production method according to (29) or a functional fragment of the antibody,
(31) The functional fragment of the antibody according to (30), wherein the functional fragment is selected from the group consisting of Fab, F (ab) 2, Fab ′ and Fv.
(32) Glycosylation to N-linkage, Glycosylation to O-linkage, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, methionine residue at N-terminal (1) to (22), (30) comprising one or more modifications selected from the group consisting of addition of groups, amidation of proline residues and deletion of one or two amino acids at the heavy chain carboxyl terminus Or a functional fragment of the antibody according to any one of (31) and (31)
(33) The antibody according to (32), wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.
(34) The antibody according to (33), wherein one amino acid is deleted at the carboxyl terminus of both of the two heavy chains.
(35) The antibody according to any one of (32) to (34), wherein a proline residue at the carboxyl terminus of the heavy chain is further amidated.
（３６）（１）乃至（２２）、（３０）乃至（３５）のいずれかひとつに記載の抗体又は当該抗体の機能性断片、その塩、それらの水和物、（２３）乃至（２５）のいずれかひとつに記載のポリヌクレオチド、（２６）に記載の発現ベクター、（２７）又は（２８）に記載の宿主細胞からなる群から選択されるいずれかを有効成分として含むことを特徴とする医薬組成物、
（３７）出血性疾患治療薬であることを特徴とする、（３６）に記載の医薬組成物、
（３８）出血性疾患が血友病Ａ、血友病Ｂ、後天性血友病及びｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病から選択される少なくともいずれか一つであることを特徴とする、（３７）に記載の医薬組成物、
（３９）出血性疾患が血友病Ａ及び／又は血友病Ｂであることを特徴とする、（３８）に記載の医薬組成物、
（４０）（１）乃至（２２）、及び（３０）乃至（３５）に記載の抗体又は当該抗体の機能性断片、その塩、又はそれらの水和物から選択されるいずれかを個体に投与することを特徴とする出血性疾患の治療方法、
（４１）出血性疾患が血友病Ａ、血友病Ｂ、後天性血友病及びｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病から選択される少なくともいずれか一つであることを特徴とする、（４０）に記載の治療方法、
（４２）出血性疾患が血友病Ａ及び／又は血友病Ｂであることを特徴とする、（４１）に記載の治療方法、（４３）（１）乃至（２２）、及び（３０）乃至（３５）に記載の抗体又は当該抗体の機能性断片、その塩、又はそれらの水和物から選択される少なくとも一つ、を含むことからなる医薬組成物及び少なくとも一つの出血性疾患治療薬を、同時に、別々に又は連続して個体に投与することを特徴とする出血性疾患の治療方法、
（４４）出血性疾患が血友病Ａ、血友病Ｂ、後天性血友病及びｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病からなる群から選択される少なくともいずれか一つであることを特徴とする、（４３）に記載の治療方法。
（４５）出血性疾患が血友病Ａ及び／又は血友病Ｂであることを特徴とする、（４４）に記載の治療方法、
からなる (36) The antibody according to any one of (1) to (22) and (30) to (35) or a functional fragment of the antibody, a salt thereof, a hydrate thereof, (23) to (25) Characterized in that it contains as an active ingredient any one selected from the group consisting of the polynucleotide according to any one of the above, the expression vector according to (26), and the host cell according to (27) or (28) A pharmaceutical composition,
(37) The pharmaceutical composition according to (36), which is a therapeutic agent for hemorrhagic disease,
(38) The medicament according to (37), wherein the hemorrhagic disease is at least one selected from hemophilia A, hemophilia B, acquired hemophilia and von Willebrand's disease. Composition,
(39) The pharmaceutical composition according to (38), wherein the hemorrhagic disease is hemophilia A and / or hemophilia B,
(40) An antibody selected from (1) to (22), and (30) to (35) or a functional fragment of the antibody, a salt thereof, or a hydrate thereof is administered to an individual For treating hemorrhagic diseases, characterized in that
(41) The treatment according to (40), wherein the hemorrhagic disease is at least one selected from hemophilia A, hemophilia B, acquired hemophilia and von Willebrand's disease. Method,
(42) The treatment method according to (41), wherein the hemorrhagic disease is hemophilia A and / or hemophilia B, (43) (1) to (22), and (30) (35) a pharmaceutical composition comprising the antibody according to (35) or at least one selected from functional fragments of the antibody, a salt thereof, or a hydrate thereof, and at least one therapeutic agent for hemorrhagic disease At the same time, separately or sequentially to the individual, a method of treating hemorrhagic disease,
(44) The hemorrhagic disease is at least one selected from the group consisting of hemophilia A, hemophilia B, acquired hemophilia and von Willebrand's disease, (43) Therapeutic method described.
(45) The treatment method according to (44), wherein the hemorrhagic disease is hemophilia A and / or hemophilia B,
Consists of
図１Ａ：血友病Ａ患者由来血漿（ＦＶＩＩＩ欠乏血漿）において精製したラット抗ヒトＰＣ抗体投与によるトロンビン産生（ＴＧ）回復作用の濃度依存性を示す図。図１Ｂ：血友病Ｂ患者由来血漿（ＦＩＸ欠乏血漿）においてラット抗ヒトＰＣ抗体投与によるトロンビン産生（ＴＧ）回復作用の濃度依存性を示す図。トロンビン産生回復率（％）の算出方法は以下の式に従って算出した。FIG. 1A: Concentration dependency of thrombin generation (TG) recovery action by administration of rat anti-human PC antibody purified in plasma from hemophilia A patient (FVIII-deficient plasma). FIG. 1B: Concentration dependency of thrombin generation (TG) recovery action by rat anti-human PC antibody administration in hemophiliac B patient-derived plasma (FIX deficient plasma). The method of calculating the recovery rate of thrombin production (%) was calculated according to the following equation.
　１００×（（Ｒ７４の血友病患者由来血漿でのＴＧ－Ｒ７４非添加の血友病患者由来血漿でのＴＧ）／（Ｒ７４非添加の正常血漿でのＴＧ－Ｒ７４非添加の血友病患者由来血漿でのＴＧ））
Ｒ７４の重鎖可変領域のヌクレオチド配列、アミノ酸配列、ＣＤＲＨ１、ＣＤＲＨ２、ＣＤＲＨ３のアミノ酸配列を示す図。ヌクレオチド配列及びアミノ酸配列における下線はＣＤＲ配列を示す。 Ｒ７４の軽鎖の可変領域をコードするｃＤＮＡのヌクレオチド配列、アミノ酸配列、ＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３のアミノ酸配列を示す図。ヌクレオチド配列及びアミノ酸配列における下線はＣＤＲ配列を示す。 ヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４の重鎖であるｃＲ７４＿Ｈ、ヒト化抗体ｈＲ７４＿Ｈ１、ｈＲ７４＿Ｈ２及びｈＲ７４＿Ｈ１２のアミノ酸配列の比較を示す図。「・」はｃＲ７４＿Ｈと同一のアミノ酸残基を示し、アミノ酸残基が記載されている箇所は置換されたアミノ酸残基を示す。 ｈＲ７４＿Ｈ１のヌクレオチド配列及びアミノ酸配列を示す図。アミノ酸配列における下線はＣＤＲ配列を示す。 ｈＲ７４＿Ｈ２のヌクレオチド配列及びアミノ酸配列を示す図。アミノ酸配列における下線はＣＤＲ配列を示す。 ｈＲ７４＿Ｈ１２のヌクレオチド配列、アミノ酸配列及びＣＤＲＨ２のアミノ酸配列を示す図。アミノ酸配列における下線はＣＤＲ配列を示す。 ヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４の軽鎖であるｃＲ７４＿Ｌ、ヒト化抗体ｈＲ７４＿Ｌ１、ｈＲ７４＿Ｌ２及びｈＲ７４＿Ｌ４のアミノ酸配列の比較を示す図。「・」はｃＲ７４＿Ｌと同一のアミノ酸残基を示し、アミノ酸残基が記載されている箇所は置換されたアミノ酸残基を示す。「－」は対応するアミノ酸残基が欠落している箇所を示す。 ｈＲ７４＿Ｌ１のヌクレオチド配列及びアミノ酸配列を示す図。アミノ酸配列における下線はＣＤＲ配列を示す。 ｈＲ７４＿Ｌ２のヌクレオチド配列及びアミノ酸配列を示す図。アミノ酸配列における下線はＣＤＲ配列を示す。 ｈＲ７４＿Ｌ４のヌクレオチド配列及びアミノ酸配列を示す図。アミノ酸配列における下線はＣＤＲ配列を示す。 ヒトキメラ化Ｒ７４重鎖ｃＲ７４＿Ｈをコードするヌクレオチド配列及びヒトキメラ化Ｒ７４重鎖ｃＲ７４＿Ｈのアミノ酸配列を示す図。 ヒトキメラ化Ｒ７４軽鎖ｃＲ７４＿Ｌをコードするヌクレオチド配列及びヒトキメラ化Ｒ７４軽鎖ｃＲ７４＿Ｌのアミノ酸配列を示す図。 図１４Ａ：血友病Ａ患者由来血漿（ＦＶＩＩＩ欠乏血漿）においてヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４＿ＩｇＧ１－ＬＡＬＡ、Ｒ７４、陽性対照ポリクローナル抗体投与によるトロンビン産生（ＴＧ）回復作用を示す図。図１４Ｂ：血友病Ｂ患者由来血漿（ＦＶＩＸ欠乏血漿）においてヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４＿ＩｇＧ１－ＬＡＬＡ、Ｒ７４、陽性対照ポリクローナル抗体投与によるトロンビン産生（ＴＧ）回復作用を示す図。 血友病Ａ患者由来血漿（ＦＶＩＩＩ欠乏血漿）においてｈＲ７４＿Ｈ１２／Ｌ１、ｈＲ７４＿Ｈ１２／Ｌ４、及びｈＲ７４＿Ｈ１／Ｌ１のトロンビン産生（ＴＧ）回復作用を示す図。 図１６Ａ：血友病Ａ患者由来血漿（ＦＶＩＩＩ欠乏血漿）においてｈＲ７４＿Ｈ１／Ｌ１及びＢＡＹ１８９６５０２のトロンビン産生（ＴＧ）回復作用を示す図。図１６Ｂ：血友病Ｂ患者由来血漿（ＦＩＸ欠乏血漿）においてｈＲ７４＿Ｈ１／Ｌ１及びＢＡＹ１８９６５０２のトロンビン産生（ＴＧ）回復作用を示す図。 一過性血友病Ａカニクイザル出血モデルにおけるｈＲ７４＿Ｈ１／Ｌ１投与による皮下出血抑制効果を示す図。皮下出血面積（％）の算出方法は以下の式に従って算出した。 100 × ((TG-R74 with hemophilia patient-derived plasma and TG with R-74 non-supplemented hemophilia patient-derived plasma) / (R74 non-loaded normal plasma without TG-R74-added hemophilia patient TG with derived plasma))
The figure which shows the nucleotide sequence of the heavy chain variable region of R74, the amino acid sequence, the amino acid sequence of CDRH1, CDRH2, and CDRH3. Underlines in the nucleotide and amino acid sequences indicate CDR sequences. The figure which shows the nucleotide sequence of the cDNA which codes the variable region of R74 light chain, an amino acid sequence, the amino acid sequence of CDRL1, CDRL2, and CDRL3. Underlines in the nucleotide and amino acid sequences indicate CDR sequences. The figure which shows the comparison of the amino acid sequence of cR74_H which is a heavy chain of human chimerized anti-human PC antibody cR74, humanized antibody hR74_H1, hR74_H2, and hR74_H12. “•” indicates the same amino acid residue as cR74_H, and the place where the amino acid residue is described indicates a substituted amino acid residue. The figure which shows the nucleotide sequence and amino acid sequence of hR74_H1. Underlining in the amino acid sequence indicates a CDR sequence. The figure which shows the nucleotide sequence and amino acid sequence of hR74_H2. Underlining in the amino acid sequence indicates a CDR sequence. The figure which shows the nucleotide sequence of hR74_H12, the amino acid sequence, and the amino acid sequence of CDRH2. Underlining in the amino acid sequence indicates a CDR sequence. The figure which shows the comparison of the amino acid sequence of cR74_L which is a light chain of human chimerized anti-human PC antibody cR74, humanized antibody hR74_L1, hR74_L2, and hR74_L4. “•” indicates the same amino acid residue as cR74_L, and the place where the amino acid residue is described indicates a substituted amino acid residue. "-" Indicates the place where the corresponding amino acid residue is missing. The figure which shows the nucleotide sequence and amino acid sequence of hR74_L1. Underlining in the amino acid sequence indicates a CDR sequence. The figure which shows the nucleotide sequence and amino acid sequence of hR74_L2. Underlining in the amino acid sequence indicates a CDR sequence. The figure which shows the nucleotide sequence and amino acid sequence of hR74_L4. Underlining in the amino acid sequence indicates a CDR sequence. The figure which shows the nucleotide sequence which codes human chimerized R74 heavy chain cR74_H, and the amino acid sequence of human chimerized R74 heavy chain cR74_H. The figure which shows the nucleotide sequence which codes human chimerization R74 light chain cR74_L, and the amino acid sequence of human chimerization R74 light chain cR74_L. FIG. 14A: Thrombin production (TG) recovery effect of human chimerized anti-human PC antibody cR74_IgG1-LALA, R74, positive control polyclonal antibody administration in hemophilia A patient-derived plasma (FVIII-deficient plasma). FIG. 14B: Thrombin production (TG) recovery action by human chimeric anti-human PC antibody cR74_IgG1-LALA, R74, positive control polyclonal antibody administration in hemophilia B patient-derived plasma (FVIX-deficient plasma). The figure which shows the thrombin generation (TG) recovery | restoration effect | action of hR74_H12 / L1, hR74_H12 / L4, and hR74_H1 / L1 in the blood plasma (FVIII deficient plasma) from hemophilia A patient. FIG. 16A shows the thrombin generation (TG) recovery action of hR74_H1 / L1 and BAY1896502 in hemophilia A patient-derived plasma (FVIII-deficient plasma). FIG. 16B: Thrombin production (TG) recovery action of hR74_H1 / L1 and BAY1896502 in hemophiliac B patient-derived plasma (FIX deficient plasma). The figure which shows the subcutaneous hemorrhage inhibitory effect by hR74_H1 / L1 administration in a transient hemophilia A cynomolgus monkey hemorrhage model. The calculation method of the subcutaneous bleeding area (%) was calculated according to the following formula.
　１００×皮下出血面積／Ｃｏｎｔｒｏｌ群のＤａｙ３の皮下出血面積の平均値（％） Average value (%) of subcutaneous bleeding area of 100 × subcutaneous bleeding area / control group on Day 3
　本発明はＰＣ及びａＰＣの双方に結合する活性を有し、ＰＣの活性化及びａＰＣの活性を阻害し、もってａＰＣがその補因子であるプロテインＳとともに、活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び活性化血液凝固第Ｖ因子（ＦＶａ）を分解、不活化するのを阻害する出血性疾患の治療に用いることが出来る抗ＰＣ抗体及び該抗体を有効成分として含有する医薬組成物等を提供する。 The present invention has the activity of binding to both PC and aPC, and inhibits the activation of PC and the activity of aPC, thereby activating a coagulation factor VIII (FVIIIa) together with protein S of which aPC is its cofactor. And anti-PC antibody which can be used for treatment of hemorrhagic disease which inhibits degradation and inactivation of activated blood coagulation factor V (FVa), and a pharmaceutical composition etc. containing the antibody as an active ingredient. .
　以下、本発明を実施するための好適な形態について図面を参照しながら説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. The embodiment described below shows an example of a representative embodiment of the present invention, and the scope of the present invention is not narrowly interpreted.
　本明細書中において、「遺伝子」という語には、ＤＮＡのみならずそのｍＲＮＡ、ｃＤＮＡ及びそのｃＲＮＡも含まれる。 As used herein, the term "gene" includes not only DNA but also its mRNA, cDNA and its cRNA.
　本明細書中において、「ポリヌクレオチド」という語は核酸と同じ意味で用いており、ＤＮＡ、ＲＮＡ、プローブ、オリゴヌクレオチド、及びプライマーも含まれる。 As used herein, the term "polynucleotide" is used interchangeably with nucleic acid and includes DNA, RNA, probes, oligonucleotides, and primers.
　本明細中においては、「ポリペプチド」と「タンパク質」は区別せずに用いている。 In the present specification, "polypeptide" and "protein" are used without distinction.
　本明細書中において、「細胞」には、動物個体内の細胞、培養細胞も含んでいる。 As used herein, "cell" also includes cells in an animal individual and cultured cells.
　本明細書中において、「ＰＣ」は、プロテインＣと同じ意味で用いている。 In the present specification, "PC" is used in the same meaning as protein C.
　本明細書において「ａＰＣ」は、活性化プロテインＣと同じ意味で用いている。ａＰＣはトロンビン切断部位によりＰＣに存在する１２アミノ酸からなる活性化ペプチドが除去されることによって生成される活性型のＰＣである。 In the present specification, "aPC" is used in the same meaning as activated protein C. aPC is an active PC produced by removing the activation peptide consisting of 12 amino acids present in PC by the thrombin cleavage site.
　本明細書において「出血性疾患」とは、血液凝固因子の欠損、不足又は機能的不全に起因する出血性の疾患であれば特に制限はないが、例えば、血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩ）の先天的欠損又は機能不全に起因する出血性疾患である血友病Ａ及び、血液凝固第ＩＸ因子（ＦＩＸ）の先天的欠損又は機能不全に起因する出血性疾患である血友病Ｂ、さらには後天性血友病、ｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病を挙げることができる。 In the present specification, the “hemorrhagic disease” is not particularly limited as long as it is a hemorrhagic disease caused by deficiency, deficiency or functional failure of a blood coagulation factor, for example, blood coagulation factor VIII (FVIII) Hemophilia A, which is a hemorrhagic disease caused by birth defects or dysfunction, Hemophilia B, a hemorrhagic disease caused by birth defects or dysfunction of blood coagulation factor IX (FIX), and the like Acquired hemophilia, von Willebrand's disease can be mentioned.
　本明細書において、「エピトープ」とは、特定の抗ＰＣ抗体の結合するＰＣ及び／又はａＰＣの部分ペプチド又は部分立体構造を意味する。前記のＰＣ及び／又はａＰＣの部分ペプチドであるエピトープは免疫アッセイ法等当業者にはよく知られている方法によって、決定することができる。まず、抗原の様々な部分構造を作製する。部分構造の作製にあたっては、公知のオリゴヌクレオチド合成技術を用いることができる。例えば、ＰＣ及び／又はａＰＣのＣ末端又はＮ末端から適当な長さで順次短くした一連のポリペプチドを当業者に周知の遺伝子組み換え技術を用いて作製した後、それらに対する抗体の反応性を検討し、大まかな認識部位を決定した後に、更に短いペプチドを合成してそれらのペプチドとの反応性を検討することによって、エピトープを決定することができる。 As used herein, “epitope” means a partial peptide or partial conformation of PC and / or aPC to which a specific anti-PC antibody binds. The epitope which is a partial peptide of the above PC and / or aPC can be determined by methods well known to those skilled in the art, such as immunoassays. First, various partial structures of the antigen are prepared. In the preparation of partial structures, known oligonucleotide synthesis techniques can be used. For example, after producing a series of polypeptides which are sequentially shortened by an appropriate length from the C terminus or N terminus of PC and / or aPC using genetic recombination technology well known to those skilled in the art, the reactivity of antibodies against them is examined Then, after determining rough recognition sites, epitopes can be determined by synthesizing shorter peptides and examining their reactivity with the peptides.
　本明細書において、「同一のエピトープに結合する」とは、共通のエピトープに結合する抗体を意味している。第一の抗体の結合する部分ペプチド又は部分立体構造に第二の抗体が結合すれば、第一の抗体と第二の抗体が同一のエピトープに結合すると判定することができる。また、第一の抗体の抗原に対する結合に第二の抗体が競合する（即ち、第二の抗体が第一の抗体と抗原の結合を妨げる）ことを確認することによって、具体的なエピトープの配列又は構造が決定されていなくても、第一の抗体と第二の抗体が同一のエピトープに結合すると判定することができる。さらに、第一の抗体と第二の抗体が同一のエピトープに結合し、かつ第一の抗体が抗トロンビン産生回復や血液凝固等の特殊な効果を有する場合、第二の抗体も同様の活性を有することが期待できる。従って、抗ＰＣ抗体については、第一の抗体の抗ＰＣ抗体の結合する部分ペプチドに第二の抗ＰＣ抗体が競合することを確認することによって、第一の抗体と第二の抗体がＰＣ及び／又はａＰＣの同一のエピトープに結合する抗体と判定することができる。 As used herein, "bind to the same epitope" means antibodies that bind to a common epitope. If the second antibody binds to the partial peptide or partial conformation to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope. Also, by confirming that the second antibody competes with the binding of the first antibody to the antigen (ie, the second antibody interferes with the binding of the antigen to the first antibody), the sequence of the specific epitope Alternatively, even if the structure is not determined, it can be determined that the first antibody and the second antibody bind to the same epitope. Furthermore, if the first antibody and the second antibody bind to the same epitope, and the first antibody has special effects such as antithrombin recovery and blood coagulation, the second antibody has similar activity. It can be expected to have. Thus, for the anti-PC antibody, the first antibody and the second antibody are PC and PC by confirming that the second anti-PC antibody competes with the partial peptide to which the first antibody's anti-PC binds. It can be determined as an antibody that binds to the same epitope of aPC.
　本明細書における「ＣＤＲ」とは、相補性決定領域（ＣＤＲ：Ｃｏｍｐｌｅｍｅｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）を意味する。抗体の重鎖及び軽鎖にはそれぞれ３箇所のＣＤＲがあることが知られている。ＣＤＲは、超可変領域（ｈｙｐｅｒｖａｒｉａｂｌｅ　ｒｅｇｉｏｎ）とも呼ばれ、抗体の重鎖及び軽鎖の可変領域内にあって、一次構造の変異性が特に高い部位であり、重鎖及び軽鎖のポリペプチド鎖の一次構造上において、それぞれ３ヶ所に分離している。本明細書中においては、抗体のＣＤＲについて、重鎖のＣＤＲを重鎖アミノ酸配列のアミノ末端側からＣＤＲＨ１、ＣＤＲＨ２、ＣＤＲＨ３と表記し、軽鎖のＣＤＲを軽鎖アミノ酸配列のアミノ末端側からＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３と表記する。これらの部位は立体構造の上で相互に近接し、結合する抗原に対する特異性を決定している。 The term "CDR" as used herein means a complementarity determining region (CDR). It is known that there are three CDRs in each of the heavy chain and light chain of the antibody. The CDRs, also called hypervariable regions, are in the variable regions of the heavy and light chains of the antibody and are the sites of particularly high variability of the primary structure, and the heavy and light chain polypeptide chains In the primary structure, they are separated into three locations. In the present specification, with regard to the CDRs of the antibody, the CDRs of the heavy chain are denoted as CDRH1, CDRH2 and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the CDRs of the light chain are CDRL1 from the amino terminal side of the light chain amino acid sequence. , CDRL2 and CDRL3. These sites are close to each other on the conformation to determine their specificity for the binding antigen.
　本発明において、「ストリンジェントな条件下でハイブリダイズする」とは、市販のハイブリダイゼーション溶液ＥｘｐｒｅｓｓＨｙｂ　Ｈｙｂｒｉｄｉｚａｔｉｏｎ　Ｓｏｌｕｔｉｏｎ（クロンテック社）中、６８℃でハイブリダイズすること、又はＤＮＡを固定したフィルターを用いて０．７－１．０ＭのＮａＣｌ存在下６８℃でハイブリダイゼーションを行った後、０．１－２倍濃度のＳＳＣ溶液（１倍濃度ＳＳＣとは１５０ｍＭ　ＮａＣｌ、１５ｍＭクエン酸ナトリウムからなる）を用い、６８℃で洗浄することによって同定することができる条件又はそれと同等の条件でハイブリダイズすることをいう。 In the present invention, “hybridize under stringent conditions” refers to hybridization at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (Clontech) or using a filter on which DNA is immobilized. After hybridization at 68 ° C. in the presence of 7-1.0 M NaCl, using a 0.1-2 fold concentration SSC solution (with 1-fold concentration SSC consisting of 150 mM NaCl, 15 mM sodium citrate), It refers to hybridization under conditions that can be identified by washing at 68 ° C. or conditions equivalent thereto.
　１．プロテインＣ（ＰＣ）
　ＰＣは血液凝固におけるネガティブフィードバック機構において主要な役割を担っている４６１残基からなるタンパク質である。ＰＣは分泌発現することによりＳｉｇｎａｌが外れて、更に、内在性のＦｕｒｉｎで切断されてＬｉｇｈｔ　ＣｈａｉｎとＨｅａｖｙ　Ｃｈａｉｎに分かれたヘテロダイマーになる。これをＰＣの非活性体（ｉｎａｃｔｉｖｅ　ｆｏｒｍ）という。ＰＣの非活性体がトロンビンで活性化すると活性化ペプチド（Ａｃｔｉｖａｔｉｏｎ　ｐｅｐｔｉｄｅ）が外れ活性型ＰＣ（ａＰＣ）になる。ａＰＣはその補因子であるプロテインＳとともに、活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び活性化血液凝固第Ｖ因子（ＦＶａ）を分解、不活化する。 1. Protein C (PC)
PC is a 461 residue protein that plays a major role in the negative feedback mechanism in blood coagulation. Secretory expression of PC results in the removal of Signal, and cleavage by endogenous Furin into a heterodimer divided into Light Chain and Heavy Chain. This is called PC's inactive form. When an inactive form of PC is activated with thrombin, an activation peptide (release peptide) is released to become active PC (aPC). aPC, together with its cofactor, protein S, degrades and inactivates activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa).
　本発明で用いるＰＣ及び／又はａＰＣは、ヒト、非ヒト哺乳動物（ラット、マウス等）のＰＣ及び／又はａＰＣ発現細胞から直接精製して使用するか、あるいは当該細胞の細胞膜画分を調製して使用することができ、また、ＰＣ及び／又はａＰＣをｉｎ　ｖｉｔｒｏにて合成する、あるいは遺伝子操作によって宿主細胞に産生させることによって得ることができる。またＰＣタンパク質の全長のタンパク質の他、ＰＣ及び／又はａＰＣタンパク質の一部の配列を含む部分ペプチドも抗ＰＣ抗体を得ることができる限りにおいて用いることが出来る。そのような部分ペプチドの例としては、配列表の配列番号３に記載のポリペプチドを挙げることができる。さらに、当該ポリペプチドをトロンビンによって処理したａＰＣ部分ペプチドを好適な抗原として用いることも出来る。 The PC and / or aPC used in the present invention may be purified directly from human and non-human mammalian (rat, mouse etc.) PC and / or aPC expressing cells, or the cell membrane fraction of the cells may be prepared. It can be used as such and can be obtained by synthesizing PC and / or aPC in vitro or by causing it to be produced in host cells by genetic engineering. In addition to full-length PC protein, partial peptides containing partial sequences of PC and / or aPC protein can also be used as long as anti-PC antibody can be obtained. As an example of such partial peptide, mention may be made of the polypeptide set forth in SEQ ID NO: 3 of the sequence listing. Furthermore, an aPC partial peptide obtained by treating the polypeptide with thrombin can also be used as a suitable antigen.
　また、前記の遺伝子操作によるＰＣ及び／又はａＰＣ発現細胞、あるいはＰＣ及び／又はａＰＣを発現している細胞株をＰＣ及び／又はａＰＣタンパク質として使用することも可能である。また、ＰＣ又はＰＣの部分ペプチドのｃＤＮＡを組み込んだ発現ベクターを直接被免疫動物に投与し被免疫動物の体内でＰＣ、ａＰＣ又は該ペプチドの部分ペプチドを発現させることもできる。 Moreover, it is also possible to use PC and / or aPC expressing cells by genetic engineering or cell lines expressing PC and / or aPC as PC and / or aPC protein. Alternatively, an expression vector incorporating PC or PC partial peptide cDNA may be directly administered to the animal to be immunized to express PC, aPC or a partial peptide of the peptide in the body of the animal to be immunized.
　ヒトＰＣのＤＮＡ配列及びアミノ酸配列は公的データベース上に公開されており、例えば（ＮＣＢＩの蛋白データベースのＡＣＣＥＳＳＩＯＮ番号ＮＰ＿０００３０３等のアクセッション番号により参照可能である。 The DNA and amino acid sequences of human PC are publicly available on public databases, and can be referred to by, for example, accession numbers such as (Accession No. NP — 000303 of NCBI Protein Database).
　また、上記ＰＣのアミノ酸配列において、１又は数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、当該タンパク質と同等の生物活性を有するタンパク質もＰＣに含まれる。ヒトＰＣのアミノ酸配列は、配列表の配列番号１に記載されている。配列番号１においてアミノ酸番号1乃至１８に示されるアミノ酸配列はシグナル配列であり、１９乃至４２はプレプロ配列であり、アミノ酸番号２００乃至２１１に示されるアミノ酸配列（ＤＴＥＤＱＥＤＱＶＤＰＲ）は活性化ペプチドの配列を示す。また本発明で用いたａＰＣは配列表の配列番号３に示すポリペプチドのアミノ酸番号４３乃至１５３からなるペプチドとアミノ酸番号１６６乃至４１２からなるペプチドがＳＳ結合で結合しているポリペプチドである。 Moreover, in the amino acid sequence of PC, one or several amino acids consist of an amino acid sequence substituted, deleted and / or added, and a protein having biological activity equivalent to the protein is also included in PC. The amino acid sequence of human PC is described in SEQ ID NO: 1 of the sequence listing. The amino acid sequence shown in amino acid numbers 1 to 18 in SEQ ID NO: 1 is a signal sequence, 19 to 42 are prepro sequences, and the amino acid sequence shown in amino acid numbers 200 to 211 (DTEDQEDQVDPR) shows the sequence of the activation peptide. . Further, aPC used in the present invention is a polypeptide in which a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 in the sequence listing and a peptide consisting of amino acid numbers 166 to 412 are linked by SS bond.
　２．抗ＰＣ抗体の製造
　本発明の抗ＰＣ抗体の一例として、配列表の配列番号１に示すポリペプチドの高次構造を認識する抗体を挙げることができる。本発明の抗ＰＣ抗体の他の一例としては、配列表の配列番号３のアミノ酸番号４３乃至４６０に示されるアミノ酸配列からなるポリペプチドの高次構造を認識する抗体を挙げることができる。本発明の抗ＰＣ抗体の他の一例としては、配列表の配列番号３に示すポリペプチドのアミノ酸番号４３乃至１５３からなるペプチドとアミノ酸番号１５４乃至４１２からなるペプチドがＳＳ結合で結合しているポリペプチドの高次構造を認識する抗体を挙げることができる。本発明の抗ＰＣ抗体の他の一例として、配列表の配列番号３に示すポリペプチドのアミノ酸番号４３乃至１５３からなるペプチドとアミノ酸番号１６６乃至４１２からなるペプチドがＳＳ結合で結合しているポリペプチドの高次構造を認識する抗体を挙げることができる。本発明の抗ＰＣ抗体の他の一例として、配列表の配列番号３に示すポリペプチドのアミノ酸番号４３乃至１５３からなるペプチドとアミノ酸番号１５４乃至４１２からなるペプチドがＳＳ結合で結合しているポリペプチドの高次構造及び、配列表の配列番号３に示すポリペプチドのアミノ酸番号４３乃至１５３からなるペプチドとアミノ酸番号１６６乃至４１２からなるペプチドがＳＳ結合で結合しているポリペプチドの高次構造を認識する抗体を挙げることができる。 2. Production of Anti-PC Antibody As an example of the anti-PC antibody of the present invention, an antibody that recognizes the higher-order structure of the polypeptide shown in SEQ ID NO: 1 in the sequence listing can be mentioned. Another example of the anti-PC antibody of the present invention is an antibody that recognizes the higher-order structure of a polypeptide consisting of the amino acid sequence shown in amino acid numbers 43 to 460 of SEQ ID NO: 3 in the sequence listing. Another example of the anti-PC antibody of the present invention is a polypeptide in which a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and a peptide consisting of amino acid numbers 154 to 412 are linked by SS bond. Mention may be made of antibodies which recognize the higher order structure of the peptide. As another example of the anti-PC antibody of the present invention, a polypeptide comprising a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and a peptide consisting of amino acid numbers 166 to 412 linked by SS bond And antibodies that recognize the higher order structure of As another example of the anti-PC antibody of the present invention, a polypeptide comprising a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and a peptide consisting of amino acid numbers 154 to 412 linked by SS bond Of the polypeptide shown in SEQ ID NO: 3 of the sequence listing and the conformation of the polypeptide in which the peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 and the peptide consisting of amino acid numbers 166 to 412 are bound by SS bond Antibodies can be mentioned.
　本発明の抗ＰＣ抗体は、いずれの種に由来してもよいが、好ましくは、ヒト、ラット、マウス及びウサギを例示できる。ヒト以外の種に由来する場合は、周知の技術を用いて、キメラ化又はヒト化することが好ましい。本発明の抗体は、ポリクローナル抗体であっても、モノクローナル抗体であってもよいが、モノクローナル抗体が好ましい。 The anti-PC antibody of the present invention may be derived from any species, preferably human, rat, mouse and rabbit. When derived from species other than human, it is preferable to chimerize or humanize using well-known techniques. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.
　本発明の抗ＰＣ抗体はＰＣ及びａＰＣを標的にできる抗体であり、すなわちＰＣ及びａＰＣを認識できる特性、ＰＣの活性化を阻害する活性、ａＰＣがその補因子であるプロテインＳとともに、活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び／又は活性化血液凝固第Ｖ因子（ＦＶａ）の分解及び／又は不活化する働きを阻害する活性を有する。かくして、本発明の抗ＰＣ抗体はトロンビンの産生を回復する活性を有し、血液凝固効果を有し、該抗体を投与された被験者において止血効果を示し、出血性疾患の治療剤となり得る。更に本発明の抗ＰＣ抗体は配列表の配列番号３に示すポリペプチドのアミノ酸番号４３乃至１５３からなるペプチドとアミノ酸番号１６６乃至４１２からなるペプチドがＳＳ結合で結合しているａＰＣを認識する抗体である。 The anti-PC antibody of the present invention is an antibody capable of targeting PC and aPC, that is, properties capable of recognizing PC and aPC, activity to inhibit PC activation, protein S with which aPC is a cofactor, and activated blood. It has an activity to inhibit the action of degrading and / or inactivating coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa). Thus, the anti-PC antibody of the present invention has an activity to restore the production of thrombin, has a blood coagulation effect, exhibits a hemostatic effect in a subject administered with the antibody, and can be a therapeutic agent for hemorrhagic disease. Furthermore, the anti-PC antibody of the present invention is an antibody that recognizes aPC in which a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 in the sequence listing and a peptide consisting of amino acid numbers 166 to 412 are linked by SS bond. is there.
　抗体のＰＣ及び／又はａＰＣへの結合性は、例えばフローサイトメトリーを用いて確認できる。 The binding of the antibody to PC and / or aPC can be confirmed using, for example, flow cytometry.
　抗ＰＣ抗体は、この分野で通常実施される方法を用いて、抗原となるポリペプチドを動物に免疫し、生体内に産生される抗体を採取、精製することによって得ることができる。 The anti-PC antibody can be obtained by immunizing an animal with a polypeptide to be an antigen, and collecting and purifying the antibody produced in vivo, using methods commonly practiced in the art.
　抗原の由来はヒトに限定されず、マウス、ラット等のヒト以外の動物に由来する抗原を動物に免疫することもできる。この場合には、取得された異種抗原に結合する抗体とヒト抗原との交差性を試験することによって、ヒトの疾患に適用可能な抗体を選別できる。 The origin of the antigen is not limited to human, and the animal can be immunized with an antigen derived from a nonhuman animal such as a mouse or a rat. In this case, antibodies that can be applied to human diseases can be selected by testing the cross-reactivity between the human antigen and the antibody that binds to the obtained heterologous antigen.
　また、公知の方法（例えば、Ｋｏｈｌｅｒ　ａｎｄ　Ｍｉｌｓｔｅｉｎ，　Ｎａｔｕｒｅ（１９７５）２５６，　ｐ．４９５－４９７、Ｋｅｎｎｅｔ，　Ｒ．ｅｄ．，　Ｍｏｎｏｃｌｏｎａｌ　Ａｎｔｉｂｏｄｉｅｓ，　ｐ．３６５－３６７，　Ｐｌｅｎｕｍ　Ｐｒｅｓｓ，　Ｎ．Ｙ．（１９８０））に従って、抗原に対する抗体を産生する抗体産生細胞とミエローマ細胞とを融合させることによってハイブリドーマを樹立し、モノクローナル抗体を得ることもできる。 Also, known methods (for example, Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N. Y. (1980). According to the above, a hybridoma can be established by fusing an antibody-producing cell producing an antibody against an antigen with a myeloma cell to obtain a monoclonal antibody.
　以下、具体的にＰＣに対する抗体の取得方法を説明する。
（１）抗原の調製
　抗原は抗原タンパク質をコードする遺伝子を遺伝子操作によって宿主細胞に産生させることによって得ることができる。具体的には、抗原遺伝子を発現可能なベクターを作製し、これを宿主細胞に導入して該遺伝子を発現させ、発現した抗原を精製すればよい。ａＰＣを抗原として用いる場合には更に酵素（例えばトロンビン）処理によってａＰＣとして上記の遺伝子操作による抗原発現細胞、あるいは抗原を発現している細胞株を動物に免疫する方法を用いることによっても抗体を取得できる。 Hereinafter, a method for obtaining an antibody against PC will be specifically described.
(1) Preparation of Antigen An antigen can be obtained by genetically producing a gene encoding an antigen protein in a host cell. Specifically, a vector capable of expressing an antigen gene may be prepared, introduced into a host cell to express the gene, and the expressed antigen may be purified. When aPC is used as an antigen, an antibody (for example, thrombin) is further treated as an aPC to obtain an antibody by immunizing an animal with an antigen-expressing cell by the above-mentioned genetic manipulation or a cell line expressing an antigen. it can.
　また、抗原タンパク質を用いずに、抗原タンパク質のｃＤＮＡを発現ベクターに組み込んで被免疫動物に投与し、被免疫動物の体内で抗原タンパク質を発現させ、抗原タンパク質に対する抗体を産生させることによっても抗体を取得することができる。 Alternatively, the cDNA of the antigen protein may be incorporated into an expression vector and administered to the animal to be immunized without using the antigen protein, and the antigen protein may be expressed in the body of the animal to be immunized to produce antibodies against the antigen protein. It can be acquired.
　（２）抗ＰＣモノクローナル抗体の製造
　本発明で使用される抗ＰＣ抗体は、特に制限はないが、例えば、本願の配列表で示されたアミノ酸配列で特定される抗体を好適に使用することができる。本発明において使用される抗ＰＣ抗体としては、以下の特性を有するものが望ましい。
（ａ）以下の（ａ－１）乃至（ａ－４）から選択される少なくとも１つの特性を有することを特徴とする抗体；
　（ａ－１）ＰＣ及びａＰＣに特異的に結合する
　（ａ－２）ＰＣに特異的に結合しＰＣの活性化を阻害する。 (2) Production of Anti-PC Monoclonal Antibody The anti-PC antibody used in the present invention is not particularly limited, but preferably used, for example, the antibody specified by the amino acid sequence shown in the sequence listing of the present application. it can. As the anti-PC antibody used in the present invention, those having the following characteristics are desirable.
(A) An antibody characterized by having at least one property selected from the following (a-1) to (a-4):
(A-1) specifically bind to PC and aPC (a-2) bind specifically to PC and inhibit activation of PC.
　（ａ－３）ａＰＣに特異的に結合し、ａＰＣによる活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び／又は活性化血液凝固第Ｖ因子（ＦＶａ）の分解及び／又は不活化を阻害する。 (A-3) It specifically binds to aPC and inhibits the decomposition and / or inactivation of activated blood coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa) by aPC.
　（ａ－４）トロンビン産生を回復する。
（ｂ）ＰＣ及びａＰＣがヒトＰＣ及びヒトａＰＣである上記（ａ）に記載の抗体又は当該抗体。
（ｃ）ヒトＰＣの配列番号３に示すポリペプチドのアミノ酸番号４３乃至１５３からなるペプチドとアミノ酸番号１６６乃至４１２からなるペプチドがＳＳ結合で結合している高次構造を認識する（ａ）又は（ｂ）に記載の抗体。 (A-4) restore thrombin generation.
(B) The antibody or the antibody according to the above (a), wherein PC and aPC are human PC and human aPC.
(C) Recognize a higher-order structure in which a peptide consisting of amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of human PC and a peptide consisting of amino acid numbers 166 to 412 are linked by SS bond (a) or The antibody as described in b).
　本発明のＰＣに対する抗体の取得方法は、抗ＰＣ抗体を取得できる限りにおいて、特に制限されない
　具体的なモノクローナル抗体取得の例としては、以下を挙げることもできる。
（ａ）ＰＣのｃＤＮＡを発現ベクター（例えば、ｐｃＤＮＡ３．３－ＴＯＰＯ／ＬａｘＺ（ＴｈｅｒｍｏＦｉｓｈｅｒ　ＳＣＩＥＮＴＯＦＩＣ）に組み込み、ＦｒｅｅＳｔｙｌｅ　２９３Ｆ　ｃｅｌｌｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ　ＳＣＩＥＮＴＯＦＩＣ）にトランスフェクションすることで一過性に発現させ、培養上清よりＰＣの含まれる画分を回収する。回収されたＰＣをトロンビン処理しａＰＣとし、免疫反応を誘起させる動物(たとえばＷＫＹ／Ｉｚｍラットの雌)に投与する。
（ｂ）免疫反応を誘起された上述の動物より、抗体産生細胞を含む組織（例えばリンパ節）を採取する。
（ｃ）骨髄腫細胞（以下「ミエローマ」という）（例えば、マウスミエローマＳＰ２／０－ａｇ１４細胞）の調製、
（ｄ）抗体産生細胞とミエローマとの細胞融合、
（ｅ）目的とする抗体を産生するハイブリドーマ群の選別、
（ｆ）単一細胞クローンへの分割（クローニング）、
（ｇ）場合によっては、モノクローナル抗体を大量に製造するためのハイブリドーマの培養、又はハイブリドーマを移植した動物の飼育、
（ｈ）このようにして製造されたモノクローナル抗体の生理活性、及びその結合特異性の検討、あるいは標識試薬としての特性の検定
　ここで用いられる抗体価の測定法としては、例えば、フローサイトメトリー又はＣｅｌｌ－ＥＬＩＳＡ法を挙げることができるがこれらの方法に制限されない。 The method for obtaining an antibody to PC of the present invention is not particularly limited as long as an anti-PC antibody can be obtained. The following may be mentioned as an example of specific monoclonal antibody acquisition.
(A) The cDNA of PC is incorporated into an expression vector (for example, pcDNA3.3-TOPO / LaxZ (ThermoFisher SCIENTOFIC) and transiently expressed by transfecting FreeStyle 293F cells (ThermoFisher SCIENTOFIC), from the culture supernatant The fraction containing PC is collected, The collected PC is thrombin-treated to form aPC, which is administered to an animal (eg, female of WKY / Izm rat) which induces an immune response.
(B) A tissue (eg, lymph node) containing antibody-producing cells is collected from the above-described animal in which an immune response has been induced.
(C) Preparation of myeloma cells (hereinafter referred to as "myelomas") (eg, mouse myeloma SP2 / 0-ag14 cells)
(D) cell fusion between antibody-producing cells and myeloma,
(E) Selection of hybridomas producing the target antibody
(F) Division into single cell clones (cloning),
(G) optionally culturing a hybridoma to produce a large amount of monoclonal antibody, or rearing a hybridoma-transplanted animal,
(H) Physiological activity of the thus produced monoclonal antibody and examination of its binding specificity, or assay of properties as a labeling reagent As a method of measuring the antibody titer used here, for example, flow cytometry or Cell-ELISA methods can be mentioned but are not limited to these methods.
　このようにして樹立されたハイブリドーマ株の例としては、抗ＰＣ抗体産生ハイブリドーマＲ７４を挙げることができる。なお、本明細書中においては、抗ＰＣ抗体産生ハイブリドーマＲ７４が産生する抗体を、「Ｒ７４抗体」又は単に「Ｒ７４」と記載する。Ｒ７４抗体はＰＣ及びａＰＣの双方に結合する活性を有する。 As an example of the hybridoma strain thus established, anti-PC antibody-producing hybridoma R74 can be mentioned. In the present specification, an antibody produced by anti-PC antibody-producing hybridoma R74 is referred to as "R74 antibody" or simply "R74". The R74 antibody has an activity of binding to both PC and aPC.
　Ｒ７４抗体の重鎖可変領域は、配列表の配列番号６に示されるアミノ酸配列を有する。当該可変領域は、配列表の配列番号６において、２６乃至３５番目のアミノ酸残基からなるアミノ酸配列からなるＣＤＲＨ１、５０乃至５８番目のアミノ酸残基からなるアミノ酸配列からなるＣＤＲＨ２、９８乃至１０７番目のアミノ酸残基からなるアミノ酸配列からなるＣＤＲＨ３を有する。Ｒ７４抗体のＣＤＲＨ１は配列表の配列番号７に示されるアミノ酸配列を有し、ＣＤＲＨ２のアミノ酸配列は配列表の配列番号８に示されるアミノ酸配列を有し、ＣＤＲＨ３のアミノ酸配列は配列表の配列番号９に示されるアミノ酸配列を有する。また、Ｒ７４抗体の重鎖可変領域、ＣＤＲＨ１、ＣＤＲＨ２及びＣＤＲＨ３のアミノ酸配列は図２に記載されている。 The heavy chain variable region of the R74 antibody has the amino acid sequence shown in SEQ ID NO: 6 in the Sequence Listing. The variable region is CDRH1 consisting of an amino acid sequence consisting of the 26th to 35th amino acid residues in SEQ ID NO: 6 in the sequence listing, CDRH2 consisting of an amino acid sequence consisting of the 50th to 58th amino acid residues It has CDRH3 consisting of an amino acid sequence consisting of amino acid residues. The CDRH1 of the R74 antibody has the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing, the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing, and the amino acid sequence of CDRH3 shows the SEQ ID NO in the sequence listing It has the amino acid sequence shown in 9. Also, the amino acid sequences of the heavy chain variable region, CDRH1, CDRH2 and CDRH3 of R74 antibody are described in FIG.
　Ｒ７４抗体の軽鎖の可変領域は、配列表の配列番号１１に示されるアミノ酸配列を有する。当該可変領域は、配列表の配列番号１１において、２３乃至３７番目のアミノ酸残基からなるアミノ酸配列からなるＣＤＲＬ１、５３乃至５９番目のアミノ酸残基からなるアミノ酸配列からなるＣＤＲＬ２、９２乃至１００番目のアミノ酸残基からなるアミノ酸配列からなるＣＤＲＬ３を有する。Ｒ７４抗体のＣＤＲＬ１は配列表の配列番号１２に示されるアミノ酸配列を有し、ＣＤＲＬ２のアミノ酸配列は配列表の配列番号１３に示されるアミノ酸配列を有し、ＣＤＲＬ３のアミノ酸配列は配列表の配列番号１４に示されるアミノ酸配列を有する。また、Ｒ７４抗体の軽鎖可変領域、ＣＤＲＬ１、ＣＤＲＬ２及びＣＤＲＬ３のアミノ酸配列は図３に記載されている。 The variable region of the light chain of R74 antibody has the amino acid sequence shown in SEQ ID NO: 11 in the Sequence Listing. The variable region is CDRL 1 consisting of an amino acid sequence consisting of amino acid residues 23 to 37 in SEQ ID NO: 11 in the sequence listing, CDR L 2 consisting of an amino acid sequence consisting of amino acid residues 53 to 59 It has CDRL3 consisting of an amino acid sequence consisting of amino acid residues. CDR L1 of the R74 antibody has the amino acid sequence shown in SEQ ID NO: 12 in the Sequence Listing, the amino acid sequence of CDR L2 has the amino acid sequence shown in SEQ ID NO: 13 in the Sequence Listing, and the amino acid sequence of CDR L3 has the SEQ ID NO in SEQ ID NO: It has the amino acid sequence shown in 14. Also, the amino acid sequences of the light chain variable region, CDRL1, CDRL2 and CDRL3 of the R74 antibody are described in FIG.
　Ｒ７４抗体の重鎖可変領域のアミノ酸配列は、配列表の配列番号５に示されるヌクレオチド配列によってコードされている。配列番号５の配列は図２に記載されている。 The amino acid sequence of the heavy chain variable region of the R74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing. The sequence of SEQ ID NO: 5 is described in FIG.
　Ｒ７４抗体の軽鎖可変領域のヌクレオチド配列は、配列表の配列番号１０に示されるヌクレオチド配列によってコードされている。また、配列番号１０の配列は図３に記載されている。 The nucleotide sequence of the light chain variable region of the R74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 10 of the sequence listing. Also, the sequence of SEQ ID NO: 10 is described in FIG.
　さらに、「３．抗ＰＣ抗体の製造」（ａ）乃至（ｈ）の工程を再度実施して別途に独立してモノクローナル抗体を取得した場合や他の方法によって別途にモノクローナル抗体を取得した場合においても、Ｒ７４抗体と同等の活性を有する抗体を取得することが可能である。このような抗体の一例として、Ｒ７４抗体と同一のエピトープに結合する抗体を挙げることができる。新たに作製されたモノクローナル抗体が、Ｒ７４抗体の結合する部分ペプチド又は部分立体構造に結合すれば、該モノクローナル抗体がＲ７４抗体と同一のエピトープに結合すると判定することができる。また、Ｒ７４抗体のＰＣ又はａＰＣに対する結合に対して該モノクローナル抗体が競合する（即ち、該モノクローナル抗体が、Ｒ７４抗体とＰＣ又はａＰＣの結合を妨げる）ことを確認することによって、具体的なエピトープの配列又は構造が決定されていなくても、該モノクローナル抗体が抗ＰＣ抗体と同一のエピトープに結合すると判定することができる。エピトープが同一であることが確認された場合、該モノクローナル抗体がＲ７４抗体と同等の抗原結合能、生物活性を有していることが強く期待される。 Furthermore, in the case where the steps of "3. production of anti-PC antibody" (a) to (h) are performed again and the monoclonal antibody is separately obtained independently or when the monoclonal antibody is separately obtained by other methods. It is also possible to obtain an antibody having an activity equivalent to that of the R74 antibody. An example of such an antibody is an antibody that binds to the same epitope as the R74 antibody. If a newly generated monoclonal antibody binds to a partial peptide or partial conformation to which the R74 antibody binds, it can be determined that the monoclonal antibody binds to the same epitope as the R74 antibody. Also, by confirming that the monoclonal antibody competes with the binding of the R74 antibody to PC or aPC (ie, the monoclonal antibody prevents the binding of the R74 antibody to PC or aPC), the specific epitope Even if the sequence or structure has not been determined, it can be determined that the monoclonal antibody binds to the same epitope as the anti-PC antibody. When the epitopes are confirmed to be identical, it is strongly expected that the monoclonal antibody has the same antigen binding ability and biological activity as the R74 antibody.
（３）その他の抗体
　本発明の抗体には、上記ＰＣに対するモノクローナル抗体に加え、ヒトに対する異種抗原性を低下させること等を目的として人為的に改変した遺伝子組換え型抗体、例えば、キメラ（Ｃｈｉｍｅｒｉｃ）抗体、ヒト化（Ｈｕｍａｎｉｚｅｄ）抗体、ヒト抗体等も含まれる。これらの抗体は、既知の方法を用いて製造することができる。 (3) Other Antibodies The antibodies of the present invention include genetically engineered antibodies, for example, chimera (Chimeric), which are artificially modified for the purpose of reducing heterologous antigenicity to humans, etc., in addition to the monoclonal antibodies against PC. Also included are antibodies, humanized antibodies, human antibodies and the like. These antibodies can be produced using known methods.
　キメラ抗体としては、抗体の可変領域と定常領域が互いに異種である抗体、例えばマウス又はラット由来抗体の可変領域をヒト由来の定常領域に接合したキメラ抗体を挙げることができる（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．，８１，６８５１－６８５５，（１９８４）参照）。 Examples of chimeric antibodies include antibodies in which the variable region of the antibody and the constant region are heterologous to each other, such as a chimeric antibody in which the variable region of a mouse- or rat-derived antibody is conjugated to a constant region derived from human (Proc. Natl. Acad) Sci.U.S.A., 81, 6851-6855, (1984)).
　ラット抗ヒトＰＣ抗体Ｒ７４抗体由来のキメラ抗体は、配列番号６に示されるアミノ酸配列からなる重鎖可変領域を含む重鎖及び配列番号１１に示される軽鎖可変領域を含む軽鎖からなる抗体であり、任意のヒト由来の定常領域を有していて良い。 A chimeric antibody derived from a rat anti-human PC antibody R74 antibody is an antibody consisting of a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 6 and a light chain comprising a light chain variable region shown in SEQ ID NO: 11. And may have any human-derived constant region.
　また、ラット抗ヒトＰＣ抗体Ｒ７４抗体由来のキメラ抗体の具体例として、ラット抗ヒトＰＣ抗体Ｒ７４抗体由来のキメラ抗体ｃＲ７４を挙げることができる。ｃＲ７４抗体のアミノ酸配列は、配列表の配列番号３３の２０乃至４６７番目のアミノ酸残基からなるアミノ酸配列を有する重鎖及び配列表の配列番号３０の２１乃至２３７からなるアミノ酸配列を有する軽鎖からなる。なお、配列表の配列番号３３に示される重鎖配列中で、１乃至１９番目のアミノ酸残基からなるアミノ酸配列はシグナル配列であり、２０乃至１３７番目のアミノ酸残基からなるアミノ酸配列は可変領域であり、１３８乃至４６７番目の残基からなるアミノ酸配列は定常領域である。配列番号３３の配列は図１２に記載されている。また、配列表の配列番号３０に示される軽鎖配列中で、１乃至２０番目のアミノ酸残基からなるアミノ酸配列はシグナル配列であり、２１乃至１３２番目のアミノ酸残基からなるアミノ酸配列は可変領域であり、１３３乃至２３７番目のアミノ酸残基からなるアミノ酸配列は定常領域である。配列番号３０の配列は図１３に記載されている。 Further, as a specific example of the chimeric antibody derived from rat anti-human PC antibody R74 antibody, chimeric antibody cR74 derived from rat anti-human PC antibody R74 antibody can be mentioned. The amino acid sequence of the cR74 antibody is comprised of a heavy chain having an amino acid sequence consisting of the 20th to 467th amino acid residues of SEQ ID NO: 33 in the Sequence Listing and a light chain having an amino acid sequence consisting of 21 to 237 of SEQ ID NO: 30 in the Sequence Listing Become. In the heavy chain sequence shown in SEQ ID NO: 33 in the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence, and the amino acid sequence consisting of amino acid residues 20 to 137 is a variable region The amino acid sequence consisting of residues 138 to 467 is a constant region. The sequence of SEQ ID NO: 33 is set forth in FIG. Also, in the light chain sequence shown in SEQ ID NO: 30 in the sequence listing, the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence, and the amino acid sequence consisting of amino acid residues 21 to 132 is a variable region The amino acid sequence consisting of amino acid residues 133 to 237 is a constant region. The sequence of SEQ ID NO: 30 is set forth in FIG.
　ｃＲ７４抗体の重鎖アミノ酸配列は、配列表の配列番号３２に示されるヌクレオチド配列によってコードされている。配列表の配列番号３２に示されるヌクレオチド配列の１乃至５７番目のヌクレオチドからなるヌクレオチド配列はｃＲ７４抗体のシグナル配列をコードしており、５８乃至４１１番目のヌクレオチドからなるヌクレオチド配列はｃＲ７４抗体の重鎖可変領域配列をコードしており、４１２乃至１４０１番目のヌクレオチドからなるヌクレオチド配列はｃＲ７４抗体の重鎖定常領域をコードしている。配列番号３２の配列は図１２に記載されている。 The heavy chain amino acid sequence of the cR74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 32 in the Sequence Listing. The nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 32 in the sequence listing encodes the signal sequence of the cR74 antibody, and the nucleotide sequence consisting of nucleotides 58 to 411 is the heavy chain of the cR74 antibody A nucleotide sequence encoding a variable region sequence and consisting of nucleotides 412 to 1401 encodes a heavy chain constant region of cR74 antibody. The sequence of SEQ ID NO: 32 is set forth in FIG.
　ｃＲ７４抗体の軽鎖アミノ酸配列は、配列表の配列番号２９に示されるヌクレオチド配列によってコードされている。配列表の配列番号２９に示されるヌクレオチド配列の１乃至６０番目のヌクレオチドからなるヌクレオチド配列はｃＲ７４抗体のシグナル配列をコードしており、６１乃至３９６番目のヌクレオチドからなるヌクレオチド配列はｃＲ７４抗体の軽鎖可変領域配列をコードしており、３９７乃至７１１番目のヌクレオチドからなるヌクレオチド配列はｃＲ７４抗体の軽鎖定常領域をコードしている。配列番号２９の配列は図１３に記載されている。 The light chain amino acid sequence of the cR74 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 29 in the Sequence Listing. The nucleotide sequence consisting of the 1st to 60th nucleotides of the nucleotide sequence shown in SEQ ID NO: 29 in the sequence listing encodes the signal sequence of the cR74 antibody, and the nucleotide sequence consisting of the 61st to 396th nucleotides consists of the light chain of the cR74 antibody A nucleotide sequence encoding a variable region sequence and consisting of nucleotides 397 to 711 encodes a light chain constant region of cR74 antibody. The sequence of SEQ ID NO: 29 is set forth in FIG.
　ヒト化抗体としては、相補性決定領域（ＣＤＲ；ｃｏｍｐｌｅｍｅｎｔａｒｉｔｙ　ｄｅｔｅｒｍｉｎｉｎｇ　ｒｅｇｉｏｎ）のみをヒト由来の抗体に組み込んだ抗体（Ｎａｔｕｒｅ（１９８６）３２１，ｐ．５２２－５２５参照）、ＣＤＲ移植法によって、ＣＤＲの配列に加え一部のフレームワークのアミノ酸残基もヒト抗体に移植した抗体（国際公開第９０／０７８６１号）、更に、抗原に対する結合能を維持しつつ、一部のＣＤＲのアミノ酸配列を改変した抗体を挙げることができる。 As a humanized antibody, an antibody in which only a complementarity determining region (CDR) is incorporated into a human-derived antibody (see Nature (1986) 321, p. 522-525), CDR sequences by CDR grafting method In addition to the above, some framework amino acid residues have also been grafted onto human antibodies (WO 90/07861), and antibodies in which the amino acid sequences of some CDRs have been modified while maintaining their ability to bind to the antigen. Can be mentioned.
　但し、Ｒ７４抗体由来のヒト化抗体としては、Ｒ７４抗体の６種全てのＣＤＲ配列を保持し、トロンビン産生を回復する活性を有している限り特定のヒト化抗体に限定されず、更に一部のＣＤＲのアミノ酸配列を改変しつつＰＣ及びａＰＣに対する結合能を有し、トロンビン産生を回復する活性を有する限り、特定のヒト化抗体に限定されない。前記のヒト化抗体の重鎖可変領域は、配列番号７に示されるアミノ酸配列からなるＣＤＲＨ１（ＧＦＳＬＴＧＹＧＶＳ）、配列番号８に示されるアミノ酸配列からなるＣＤＲＨ２（ＡＶＷＲＧＧＳＫＤ）及び配列番号９に示されるアミノ酸配列からなるＣＤＲＨ３（ＳＧＰＥＧＴＰＦＤＹ）を保有している。前記のヒト抗体の軽鎖可変領域は、配列番号１２に示されるアミノ酸配列からなるＣＤＲＬ１（ＫＴＮＱＮＶＤＦＹＧＮＳＹＩＨ）、配列番号１３に示されるアミノ酸配列からなるＣＤＲＬ２（ＳＡＳＮＬＡＳ）及び配列番号１４に示されるアミノ酸配列からなるＣＤＲＬ３（ＱＱＳＲＮＬＰＮＴ）を保有している。 However, as a humanized antibody derived from R74 antibody, it is not limited to a specific humanized antibody as long as it retains the CDR sequences of all six types of R74 antibody and has an activity to recover thrombin generation, It is not limited to a specific humanized antibody as long as it has an ability to bind to PC and aPC while altering the amino acid sequence of the CDRs of SEQ ID NO: 1 and has an activity to restore thrombin generation. The heavy chain variable region of the above-mentioned humanized antibody comprises CDRH1 (GFSLTGYGVS) consisting of the amino acid sequence shown in SEQ ID NO: 7, CDRH2 (AVWRGGSKD) consisting of the amino acid sequence shown in SEQ ID NO: 8 and amino acid sequence shown in SEQ ID NO: 9 Possess CDRH3 (SGPEGTPFDY) consisting of The light chain variable region of the above human antibody comprises the CDRL1 (KTNQNVDFDFYGNSYIH) consisting of the amino acid sequence shown in SEQ ID NO: 12, the CDRL2 (SASNLAS) consisting of the amino acid sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO: 14 Possess CDRL3 (QQSRNLPNT).
　ラット抗体Ｒ７４のヒト化抗体の実例としては、
（１）配列表の配列番号１６又は１８の２０乃至１３７番目のアミノ酸残基からなるアミノ酸配列、（２）上記（１）のアミノ酸配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、及び（３）上記（１）のアミノ酸配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる重鎖可変領域を含む重鎖、並びに（４）配列番号２３、２５又は２７の２１乃至１３３番目のアミノ酸残基からなるアミノ酸配列、（５）上記（４）のアミノ酸配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、及び（６）上記（４）のアミノ酸配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる軽鎖可変領域を含む軽鎖の任意の組合せを挙げることができる。 As an example of a humanized antibody of rat antibody R74,
(1) an amino acid sequence consisting of the 20th to 137th amino acid residues of SEQ ID NO: 16 or 18 in the sequence listing, (2) an amino acid sequence having at least 95% or more homology to the amino acid sequence of (1) above; And (3) a heavy chain comprising a heavy chain variable region consisting of any one of the amino acid sequences wherein one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above, and (4) the sequence An amino acid sequence consisting of the 21st to 133rd amino acid residues of No. 23, 25 or 27, (5) an amino acid sequence having at least 95% or more homology to the amino acid sequence of (4) above, and (6) above Any combination of light chains comprising a light chain variable region consisting of any one of amino acid sequences wherein one or several amino acids are deleted, substituted or added in the amino acid sequence of (4); It can gel.
　更に好適なラット抗体Ｒ７４のヒト化抗体の実例としては、配列表の配列番号１６のアミノ酸番号２０乃至４６７番目のアミノ酸残基からなる重鎖及び配列表の配列番号２３のアミノ酸番号２１乃至２３８番目のアミノ酸残基からなる軽鎖からなる抗体（ｈＲ７４＿Ｈ１／Ｌ１）を挙げることができる。 Further illustrative of the humanized antibody of rat antibody R74 is a heavy chain consisting of amino acid residues 20 to 467 of SEQ ID NO: 16 in the sequence listing and amino acids 21 to 238 of SEQ ID NO: 23 of the sequence listing. An antibody (hR74_H1 / L1) consisting of a light chain consisting of amino acid residues of
　本発明の抗体は、上記のヒト化抗体のＣＤＲに更に変異を導入し、抗体の粘度を低減させた抗体も含まれる。そのようなＣＤＲの改変の具体例としてはＲ７４抗体由来のＣＤＲＨ２（ＡＶＷＲＧＧＳＫＤ：配列番号８）の３番目と４番目のアミノ酸残基を改変したＣＤＲＨ２（ＡＶＹＴＧＧＳＫＤ：配列番号２１）を挙げることができる。即ち上記ヒト化抗体において、ＣＤＲＨ２の配列のみを配列番号２１に示されるアミノ酸配列からなるＣＤＲＨ２に置換したヒト化抗体も本発明のヒト化抗体に含まれる。 The antibody of the present invention also includes an antibody in which the viscosity of the antibody has been reduced by further introducing mutations into the CDRs of the above-mentioned humanized antibody. Specific examples of such CDR modifications can include CDRH2 (AVYTGGSKD: SEQ ID NO: 21) in which the third and fourth amino acid residues of CDRH2 derived from R74 antibody (AVWRGGSKD: SEQ ID NO: 8) are modified. That is, in the above-mentioned humanized antibody, a humanized antibody in which only the sequence of CDRH2 is substituted with CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 21 is also included in the humanized antibody of the present invention.
　そのような抗体の更に具体例としては、配列表の配列番号２０に示されるアミノ酸配列の２０乃至１３７番目のアミノ酸残基からなるアミノ酸配列、（２）上記（１）のアミノ酸配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、及び（３）上記（１）のアミノ酸配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる重鎖可変領域を含む重鎖、並びに（４）配列番号２３、２５又は２７の２１乃至１３３番目のアミノ酸残基からなるアミノ酸配列、（５）上記（４）のアミノ酸配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、及び（６）上記（４）のアミノ酸配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる軽鎖可変領域を含む軽鎖の任意の組合せを挙げることができる。 Further specific examples of such an antibody include an amino acid sequence consisting of the 20th to 137th amino acid residues of the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing, (2) at least the amino acid sequence of (1) above. A heavy chain variable comprising an amino acid sequence having 95% or more homology and (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above A heavy chain including the region, and (4) an amino acid sequence consisting of amino acid residues 21 to 133 of SEQ ID NO: 23, 25 or 27, (5) a homology of at least 95% or more to the amino acid sequence of (4) above Amino acid sequence having one or more amino acids, and (6) any one of the amino acid sequences of the above (4) in which one or several amino acids are deleted, substituted or added It may include any combination of the light chain comprising a Ranaru light chain variable region.
　更に具体的なヒト化抗体の例としては、配列表の配列番号２０のアミノ酸番号２０乃至４６７番目のアミノ酸残基からなる重鎖及び配列表の配列番号２３のアミノ酸番号２１乃至２３８番目のアミノ酸残基からなる軽鎖からなる抗体（ｈＲ７４＿Ｈ１２／Ｌ１）、配列表の配列番号２０のアミノ酸番号２０乃至４６７番目のアミノ酸残基からなる重鎖及び配列表の配列番号２５のアミノ酸番号２１乃至２３８番目のアミノ酸残基からなる軽鎖からなる抗体（ｈＲ７４＿Ｈ１２／Ｌ２）、並びに配列表の配列番号２０のアミノ酸番号２０乃至４６７番目のアミノ酸残基からなる重鎖及び配列表の配列番号２７のアミノ酸番号２１乃至２３８番目のアミノ酸残基からなる軽鎖からなる抗体（ｈＲ７４＿Ｈ１２／Ｌ４）を挙げることができる。 As a further specific example of the humanized antibody, a heavy chain consisting of the amino acid residues 20 to 467 of SEQ ID NO: 20 in the sequence listing and the amino acid residue 21 to 238 of amino acid residues of SEQ ID NO: 23 of the sequence listing Group consisting of a light chain (hR74_H12 / L1), a heavy chain consisting of the 20th to 467th amino acid residues of SEQ ID NO: 20 in the Sequence Listing, and a 21st to 238th amino acid residues of SEQ ID NO: 25 in the Sequence Listing An antibody (hR74_H12 / L2) consisting of a light chain consisting of amino acid residues, and a heavy chain consisting of the amino acid residues 20 to 467 of SEQ ID NO: 20 in the Sequence Listing and a heavy chain consisting of amino acid residues 21 to An antibody (hR74_H12 / L4) consisting of a light chain consisting of the 238th amino acid residue can be mentioned.
　また、重鎖又は軽鎖の一方をヒト化し、他方をラット抗体やキメラ抗体の軽鎖又は重鎖とした抗体も用いることができる。 In addition, an antibody in which one of the heavy chain or the light chain is humanized and the other is a light chain or a heavy chain of a rat antibody or a chimeric antibody can also be used.
　なお、本明細書中における「数個」とは、１乃至１０個、１乃至９個、１乃至８個、１乃至７個、１乃至６個、１乃至５個、１乃至４個、１乃至３個、又は１若しくは２個を意味する。 In the present specification, “several” means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 To 3 or 1 or 2 means.
　また、本明細書中におけるアミノ酸の置換としては保存的アミノ酸置換が好ましい。保存的アミノ酸置換とは、アミノ酸側鎖に関連のあるアミノ酸グループ内で生じる置換である。好適なアミノ酸グループは、以下のとおりである：酸性グループ＝アスパギン酸、グルタミン酸；塩基性グループ＝リシン、アルギニン、ヒスチジン；非極性グループ＝アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン；及び非帯電極性ファミリー＝グリシン、アスパラギン、グルタミン、システイン、セリン、スレオニン、チロシン。他の好適なアミノ酸グループは次のとおりである：脂肪族ヒドロキシグループ＝セリン及びスレオニン；アミド含有グループ＝アスパラギン及びグルタミン；脂肪族グループ＝アラニン、バリン、ロイシン及びイソロイシン；並びに芳香族グループ＝フェニルアラニン、トリプトファン及びチロシン。かかるアミノ酸置換は元のアミノ酸配列を有する物質の特性を低下させない範囲で行うのが好ましい。 In addition, conservative amino acid substitution is preferable as the amino acid substitution in the present specification. Conservative amino acid substitutions are those that occur within a group of amino acids that are related to amino acid side chains. Preferred amino acid groups are as follows: acidic group = aspagic acid, glutamic acid; basic group = lysine, arginine, histidine; nonpolar group = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; And uncharged polar families = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other preferred amino acid groups are: aliphatic hydroxy groups = serine and threonine; amide containing groups = asparagine and glutamine; aliphatic groups = alanine, valine, leucine and isoleucine; and aromatic groups = phenylalanine, tryptophan And tyrosine. It is preferable to carry out such amino acid substitution within the range that does not reduce the properties of the substance having the original amino acid sequence.
　上記の重鎖アミノ酸配列及び軽鎖アミノ酸配列と高い相同性を示す配列を組み合わせることによって、上記の各抗体と同等の生物活性を有する抗体を選択することが可能である。このような相同性は、一般的には８０％以上の相同性であり、好ましくは９０％以上の相同性であり、より好ましくは９５％以上の相同性であり、最も好ましくは９９％以上の相同性である。また、重鎖又は軽鎖のアミノ酸配列に１乃至数個のアミノ酸残基が置換、欠失又は付加されたアミノ酸配列を組み合わせることによっても、上記の各抗体と同等の生物活性を有する抗体を選択することが可能である。 By combining the above-mentioned heavy chain amino acid sequence and light chain amino acid sequence with a sequence showing high homology, it is possible to select an antibody having biological activity equivalent to each of the above-mentioned antibodies. Such homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, most preferably 99% or more. It is homologous. Also, by combining an amino acid sequence in which one to several amino acid residues are substituted, deleted or added to the amino acid sequence of heavy chain or light chain, an antibody having biological activity equivalent to each of the above-mentioned antibodies is selected. It is possible.
　二種類のアミノ酸配列間の相同性は、Ｂｌａｓｔ　ａｌｇｏｒｉｔｈｍ　ｖｅｒｓｉｏｎ　２．２．２（Ａｌｔｓｃｈｕｌ，　Ｓｔｅｐｈｅｎ　Ｆ．，Ｔｈｏｍａｓ　Ｌ．Ｍａｄｄｅｎ，Ａｌｅｊａｎｄｒｏ　Ａ．Ｓｃｈａｆｆｅｒ，　Ｊｉｎｇｈｕｉ　Ｚｈａｎｇ，　Ｚｈｅｎｇ　Ｚｈａｎｇ，　Ｗｅｂｂ　Ｍｉｌｌｅｒ，　ａｎｄ　Ｄａｖｉｄ　Ｊ．Ｌｉｐｍａｎ（１９９７），「Ｇａｐｐｅｄ　ＢＬＡＳＴ　ａｎｄ　ＰＳＩ－ＢＬＡＳＴ：ａ　ｎｅｗ　ｇｅｎｅｒａｔｉｏｎ　ｏｆ　ｐｒｏｔｅｉｎ　ｄａｔａｂａｓｅ　ｓｅａｒｃｈ　ｐｒｏｇｒａｍｓ」，Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．２５：３３８９－３４０２）のデフォルトパラメーターを使用することによって決定することができる。Ｂｌａｓｔ　ａｌｇｏｒｉｔｈｍは、インターネットでｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ｂｌａｓｔにアクセスすることによっても使用することができる。 The homology between the two amino acid sequences is shown in Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402). Blast algorithm is available on the Internet at www. ncbi. nlm. nih. It can also be used by accessing gov / blast.
　本発明の抗体としては、さらに、ＰＣ及びａＰＣに結合する、ヒト抗体を挙げることができる。抗ＰＣヒト抗体とは、ヒト染色体由来の抗体の遺伝子配列のみを有するヒト抗体を意味する。抗ＰＣヒト抗体は、ヒト抗体の重鎖と軽鎖の遺伝子を含むヒト染色体断片を有するヒト抗体産生マウスを用いた方法（Ｔｏｍｉｚｕｋａ，Ｋ．ｅｔ　ａｌ．，Ｎａｔｕｒｅ　Ｇｅｎｅｔｉｃｓ（１９９７）１６，ｐ．１３３－１４３，；Ｋｕｒｏｉｗａ，Ｙ．ｅｔ．ａｌ．，Ｎｕｃｌ．Ａｃｉｄｓ　Ｒｅｓ．（１９９８）２６，ｐ．３４４７－３４４８；Ｙｏｓｈｉｄａ，Ｈ．ｅｔ．ａｌ．，Ａｎｉｍａｌ　Ｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｙ：Ｂａｓｉｃ　ａｎｄ　Ａｐｐｌｉｅｄ　Ａｓｐｅｃｔｓ　ｖｏｌ．１０，ｐ．６９－７３（Ｋｉｔａｇａｗａ，Ｙ．，Ｍａｔｓｕｄａ，Ｔ．ａｎｄ　Ｉｉｊｉｍａ，Ｓ．ｅｄｓ．），Ｋｌｕｗｅｒ　Ａｃａｄｅｍｉｃ　Ｐｕｂｌｉｓｈｅｒｓ，１９９９．；Ｔｏｍｉｚｕｋａ，Ｋ．ｅｔ．ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ（２０００）９７，ｐ．７２２－７２７等を参照。）によって取得することができる。 The antibodies of the present invention can further include human antibodies that bind to PC and aPC. The anti-PC human antibody means a human antibody having only the gene sequence of the antibody derived from human chromosome. The anti-PC human antibody is a method using a human antibody-producing mouse having a human chromosomal fragment containing genes of heavy and light chains of human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133). Kuroda, Y. et. Al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yoshida, H. et. Al., Animal Cell Technology: Basic and Applied Aspects vol. p. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999 .; Tomizuka, K. et. al., P. oc.Natl.Acad.Sci.USA (2000) 97, can be obtained by reference.) The p.722-727 like.
　このようなヒト抗体産生マウスは、具体的には、内在性免疫グロブリン重鎖及び軽鎖の遺伝子座が破壊され、代わりに酵母人工染色体（Ｙｅａｓｔ　ａｒｔｉｆｉｃｉａｌ　ｃｈｒｏｍｏｓｏｍｅ，ＹＡＣ）ベクター等を介してヒト免疫グロブリン重鎖及び軽鎖の遺伝子座が導入された遺伝子組み換え動物として、ノックアウト動物及びトランスジェニック動物の作製及びこれらの動物同士を掛け合わせることによって作り出すことができる。 In such a human antibody-producing mouse, specifically, loci of endogenous immunoglobulin heavy chains and light chains are destroyed, and instead, human immunoglobulins are obtained via a yeast artificial chromosome (YAC) vector or the like. As genetically modified animals into which heavy chain and light chain loci have been introduced, knockout animals and transgenic animals can be produced by crossing them.
　また、遺伝子組換え技術によって、そのようなヒト抗体の重鎖及び軽鎖の各々をコードするｃＤＮＡ、好ましくは該ｃＤＮＡを含むベクターによって真核細胞を形質転換し、遺伝子組換えヒトモノクローナル抗体を産生する形質転換細胞を培養することによって、この抗体を培養上清中から得ることもできる。 In addition, eukaryotic cells are transformed with cDNA encoding each of heavy and light chains of such human antibody, preferably the vector containing the cDNA by genetic recombination technology to produce recombinant human monoclonal antibody. This antibody can also be obtained from the culture supernatant by culturing transformed cells.
　ここで、宿主としては例えば真核細胞、好ましくはＣＨＯ細胞、リンパ球やミエローマ等の哺乳動物細胞を用いることができる。 Here, as a host, for example, eukaryotic cells, preferably CHO cells, mammalian cells such as lymphocytes and myelomas can be used.
　また、ヒト抗体ライブラリーより選別したファージディスプレイ由来のヒト抗体を取得する方法（Ｗｏｒｍｓｔｏｎｅ，Ｉ．Ｍ．ｅｔ．ａｌ，Ｉｎｖｅｓｔｉｇａｔｉｖｅ　Ｏｐｈｔｈａｌｍｏｌｏｇｙ　＆　Ｖｉｓｕａｌ　Ｓｃｉｅｎｃｅ．（２００２）４３（７），ｐ．２３０１－２３０８；Ｃａｒｍｅｎ，Ｓ．ｅｔ．ａｌ．，Ｂｒｉｅｆｉｎｇｓ　ｉｎ　Ｆｕｎｃｔｉｏｎａｌ　Ｇｅｎｏｍｉｃｓ　ａｎｄ　Ｐｒｏｔｅｏｍｉｃｓ（２００２），１（２），ｐ．１８９－２０３；Ｓｉｒｉｗａｒｄｅｎａ，Ｄ．ｅｔ．ａｌ．，Ｏｐｈｔｈａｌｍｏｌｏｇｙ（２００２）１０９（３），ｐ．４２７－４３１等参照。）も知られている。 Also, a method for obtaining a phage display-derived human antibody selected from a human antibody library (Wormstone, IM et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Carmen, S. et. Al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. Al., Ophthalmology (2002) 109 (3), p. 427-431, etc.) are also known.
　例えば、ヒト抗体の可変領域を一本鎖抗体（ｓｃＦｖ）としてファージ表面に発現させて、抗原に結合するファージを選択するファージディスプレイ法（Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５），２３，（９），ｐ．１１０５－１１１６）を用いることができる。 For example, a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which the variable region of human antibody is expressed on the phage surface as a single chain antibody (scFv) and phages that bind to the antigen are selected. -1116) can be used.
　抗原に結合することで選択されたファージの遺伝子を解析することによって、抗原に結合するヒト抗体の可変領域をコードするＤＮＡ配列を決定することができる。 By analyzing the gene of the selected phage by binding to the antigen, the DNA sequence encoding the variable region of human antibody binding to the antigen can be determined.
　抗原に結合するｓｃＦｖのＤＮＡ配列が明らかになれば、当該配列を有する発現ベクターを作製し、適当な宿主に導入して発現させることによってヒト抗体を取得することができる（国際公開第９２／０１０４７号、同９２／２０７９１号、同９３／０６２１３号、同９３／１１２３６号、同９３／１９１７２号、同９５／０１４３８号、同９５／１５３８８号、Ａｎｎｕ．Ｒｅｖ．Ｉｍｍｕｎｏｌ（１９９４）１２，ｐ．４３３－４５５、Ｎａｔｕｒｅ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（２００５）２３（９），ｐ．１１０５－１１１６）。 Once the DNA sequence of the scFv that binds to the antigen is clarified, a human antibody can be obtained by preparing an expression vector having the sequence, and introducing it into an appropriate host for expression (WO 92/01047). No. 92/20791 No. 93/062213 No. 93/11236 No. 93/19172 No. 95/01438 No. 95/15388 No. Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), pages 1105-1116).
　新たに作製されたヒト抗体が、本明細書に記載のＲ７４抗体の結合する部分ペプチド又は部分立体構造に結合すれば、該ヒト抗体がＲ７４抗体と同一のエピトープに結合すると判定することができる。また、Ｒ７４抗体のＰＣ又はａＰＣに対する結合に対して該ヒト抗体が競合する（すなわち、該ヒト抗体が、Ｒ７４抗体のＰＣ又はａＰＣへの結合を妨げる）ことを確認することによって、具体的なエピトープの配列又は構造が決定されていなくても、該ヒト抗体がＲ７４抗体と同一のエピトープに結合すると判定することができる。エピトープが同一であることが確認された場合、該ヒト抗体がＲ７４抗体と同等の生物活性を有していることが強く期待される。 If a newly generated human antibody binds to a partial peptide or partial conformation to which the R74 antibody described herein binds, it can be determined that the human antibody binds to the same epitope as the R74 antibody. Also, by confirming that the human antibody competes for the binding of the R74 antibody to PC or aPC (ie, the human antibody prevents the binding of the R74 antibody to PC or aPC), a specific epitope can be obtained. It can be determined that the human antibody binds to the same epitope as the R74 antibody, even though the sequence or structure of is not determined. When the epitopes are confirmed to be identical, it is strongly expected that the human antibody has biological activity equivalent to that of the R74 antibody.
　以上の方法によって得られたキメラ抗体、ヒト化抗体、又はヒト抗体は、公知の方法等によって抗原に対する結合性を評価し、好適な抗体を選抜することができる。 The chimeric antibody, the humanized antibody or the human antibody obtained by the above method can be evaluated for the binding to the antigen by a known method and the like, and a suitable antibody can be selected.
　抗体の性質を比較する際の別の指標の一例としては、抗体の安定性を挙げることができる。示差走査カロリメトリー（ＤＳＣ）は、蛋白の相対的構造安定性のよい指標となる熱変性中点（Ｔｍ）を素早く、また正確に測定することができる装置である。ＤＳＣを用いてＴｍ値を測定し、その値を比較することによって、熱安定性の違いを比較することができる。抗体の保存安定性は、抗体の熱安定性とある程度の相関を示すことが知られており（Ｌｏｒｉ　Ｂｕｒｔｏｎ，ｅｔ．ａｌ．，Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｄｅｖｅｌｏｐｍｅｎｔ　ａｎｄ　Ｔｅｃｈｎｏｌｏｇｙ（２００７）１２，ｐ．２６５－２７３）、熱安定性を指標に、好適な抗体を選抜することができる。抗体を選抜するための他の指標としては、適切な宿主細胞における収量が高いこと、及び水溶液中での凝集性が低いことを挙げることができる。例えば収量の最も高い抗体が最も高い熱安定性を示すとは限らないので、以上に述べた指標に基づいて総合的に判断して、ヒトへの投与に最も適した抗体を選抜する必要がある。 As an example of another index in comparing the properties of antibodies, the stability of antibodies can be mentioned. Differential scanning calorimetry (DSC) is a device that can quickly and accurately measure the thermal denaturation midpoint (Tm), which is a good indicator of the relative structural stability of proteins. The differences in thermal stability can be compared by measuring Tm values using DSC and comparing the values. The storage stability of the antibody is known to show some correlation with the thermal stability of the antibody (Lori Burton, et. Al., Pharmaceutical Development and Technology (2007) 12, p. 265-273). Suitable antibodies can be selected on the basis of stability. Other indicators for selecting antibodies can include high yields in appropriate host cells and low aggregation in aqueous solution. For example, since the antibody with the highest yield does not necessarily exhibit the highest thermostability, it is necessary to select the antibody most suitable for human administration, judging comprehensively on the basis of the index described above. .
　本発明の抗体には抗体の修飾体も含まれる。当該修飾体とは、本発明の抗体に化学的又は生物学的な修飾が施されてなるものを意味する。化学的な修飾体には、アミノ酸骨格への化学部分の結合、Ｎ－結合又はＯ－結合炭水化物鎖の化学修飾体等が含まれる。生物学的な修飾体には、翻訳後修飾（例えば、Ｎ－結合又はＯ－結合への糖鎖付加、Ｎ末又はＣ末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化）されたもの、原核生物宿主細胞を用いて発現させることによってＮ末にメチオニン残基が付加したもの等が含まれる。また、本発明の抗体又は抗原の検出又は単離を可能にするために標識されたもの、例えば、酵素標識体、蛍光標識体、アフィニティ標識体もかかる修飾物の意味に含まれる。このような本発明の抗体の修飾物は、抗体の安定性及び血中滞留性の改善、抗原性の低減、抗体又は抗原の検出又は単離等に有用である。 The antibodies of the present invention also include modified antibodies. The term "modified form" means that the antibody of the present invention is chemically or biologically modified. Chemical modifications include attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like. For biological modification, post-translational modification (eg, glycosylation to N- or O-link, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation) And those obtained by adding a methionine residue at the N-terminus by expression using a prokaryotic host cell. In addition, those labeled so as to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme labels, fluorescent labels, affinity labels are also included in the meaning of such modified products. Such modified products of the antibody of the present invention are useful for improvement of antibody stability and retention in blood, reduction of antigenicity, detection or isolation of antibody or antigen, and the like.
　また、本発明の抗体に結合している糖鎖修飾を調節すること（グリコシル化、脱フコース化等）によって、抗体依存性細胞傷害活性を増強することが可能である。抗体の糖鎖修飾の調節技術としては、国際公開第１９９９／５４３４２号、同２０００／６１７３９号、同２００２／３１１４０号、国際公開第２００７／１３３８５５号等が知られているが、これらに限定されるものではない。本発明の抗体には当該糖鎖修飾を調節された抗体も含まれる。 In addition, it is possible to enhance antibody-dependent cytotoxic activity by modulating the glycosylation (glycosylation, defucosification, etc.) bound to the antibody of the present invention. WO 1999/54342, WO 2000/61739, WO 2002/31140, WO 2007/133855, etc. are known as modulation techniques for antibody sugar chain modification, but are limited thereto. It is not a thing. The antibodies of the present invention also include antibodies in which the sugar chain modification has been adjusted.
　抗体遺伝子を一旦単離した後、適当な宿主に導入して抗体を作製する場合には、適当な宿主と発現ベクターの組み合わせを使用することができる。抗体遺伝子の具体例としては、本明細書に記載された抗体の重鎖配列をコードする遺伝子、及び軽鎖配列をコードする遺伝子を組み合わせたものを挙げることができる。宿主細胞を形質転換する際には、重鎖配列遺伝子と軽鎖配列遺伝子は、同一の発現ベクターに挿入されていることが可能であり、又別々の発現ベクターに挿入されていることも可能である。 Once the antibody gene has been isolated and then introduced into a suitable host to produce an antibody, a combination of a suitable host and an expression vector can be used. As specific examples of antibody genes, mention may be made of a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence. When transforming host cells, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there.
　真核細胞を宿主として使用する場合、動物細胞、植物細胞、真核微生物を用いることができる。特に動物細胞としては、哺乳類細胞、例えば、サルの細胞であるＣＯＳ細胞（Ｇｌｕｚｍａｎ，Ｙ．Ｃｅｌｌ（１９８１）２３，ｐ．１７５－１８２、ＡＴＣＣ　ＣＲＬ－１６５０）、マウス線維芽細胞ＮＩＨ３Ｔ３（ＡＴＣＣ　Ｎｏ．ＣＲＬ－１６５８）やチャイニーズ・ハムスター卵巣細胞（ＣＨＯ細胞、ＡＴＣＣ　ＣＣＬ－６１）のジヒドロ葉酸還元酵素欠損株（Ｕｒｌａｕｂ，Ｇ．ａｎｄ　Ｃｈａｓｉｎ，Ｌ．Ａ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．（１９８０）７７，ｐ．４１２６－４２２０）、ＦｒｅｅＳｔｙｌｅ　２９３Ｆ細胞（Ｉｎｖｉｔｒｏｇｅｎ社）を挙げることができる。 When eukaryotic cells are used as a host, animal cells, plant cells, and eukaryotic microorganisms can be used. Particularly, as animal cells, mammalian cells, for example, cells of monkeys such as COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblast NIH 3 T 3 (ATCC No. 4). CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61), dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad. Sci. U. S. A. (1980) 77, p. 4126-4220), FreeStyle 293F cells (Invitrogen) can be mentioned.
　原核細胞を使用する場合は、例えば、大腸菌、枯草菌を挙げることができる。 When prokaryotic cells are used, for example, E. coli and Bacillus subtilis can be mentioned.
　これらの細胞に目的とする抗体遺伝子を形質転換によって導入し、形質転換された細胞をｉｎ　ｖｉｔｒｏで培養することによって抗体が得られる。当該培養においては抗体の配列によって収量が異なる場合があり、同等な結合活性を持つ抗体の中から収量を指標に医薬としての生産が容易なものを選別することが可能である。よって、本発明の抗体には、上記形質転換された宿主細胞を培養する工程、及び当該工程で得られた培養物から目的の抗体又は当該抗体の機能性断片を採取する工程を含むことを特徴とする当該抗体の製造方法によって得られる抗体も含まれる。 The antibody gene of interest is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain an antibody. In the culture, the yield may differ depending on the sequence of the antibody, and it is possible to select among the antibodies having the same binding activity, those which can be easily produced as medicaments using the yield as an index. Therefore, the antibody of the present invention is characterized by comprising the steps of culturing the above-mentioned transformed host cell, and collecting the target antibody or a functional fragment of the antibody from the culture obtained in the step. Also included are antibodies obtained by the method for producing the antibody.
　なお、哺乳類培養細胞で生産される抗体の重鎖のカルボキシル末端のリシン残基が欠失することが知られており（Ｊｏｕｒｎａｌ　ｏｆ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ　Ａ，７０５：１２９－１３４（１９９５））、また、同じく重鎖カルボキシル末端のグリシン、リシンの２アミノ酸残基が欠失し、新たにカルボキシル末端に位置するプロリン残基がアミド化されることが知られている（Ａｎａｌｙｔｉｃａｌ　Ｂｉｏｃｈｅｍｉｓｔｒｙ，３６０：７５－８３（２００７））。しかし、これらの重鎖配列の欠失及び修飾は、抗体の抗原結合能及びエフェクター機能（補体の活性化や抗体依存性細胞傷害作用等）には影響を及ぼさない。従って、本発明に係る抗体には、当該修飾を受けた抗体及び当該抗体の機能性断片も含まれ、重鎖カルボキシル末端において１又は２つのアミノ酸が欠失した欠失体、及びアミド化された当該欠失体（例えば、カルボキシル末端部位のプロリン残基がアミド化された重鎖）等も包含される。但し、抗原結合能及びエフェクター機能が保たれている限り、本発明に係る抗体の重鎖のカルボキシル末端の欠失体は上記の種類に限定されない。本発明に係る抗体を構成する２本の重鎖は、完全長及び上記の欠失体からなる群から選択される重鎖のいずれか一種であってもよいし、いずれか二種を組み合わせたものであってもよい。各欠失体の量比は本発明に係る抗体を産生する哺乳類培養細胞の種類及び培養条件に影響を受け得るが、本発明に係る抗体の主成分としては２本の重鎖の双方でカルボキシル末端の１つのアミノ酸残基が欠失している場合を挙げることができる。 In addition, it is known that the lysine residue at the carboxyl terminus of the heavy chain of the antibody produced in mammalian cultured cells is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and It is known that two amino acid residues of glycine and lysine at the carboxyl terminus of the chain are deleted, and a proline residue newly located at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007) ). However, deletion and modification of these heavy chain sequences do not affect the antigen binding ability and effector function (such as complement activation and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, the antibody according to the present invention also includes the antibody that has been modified and functional fragments of the antibody, a deleted form in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and amidated The deletion body (for example, a heavy chain in which a proline residue at the carboxyl terminal site is amidated) and the like are also included. However, deletion of the carboxyl terminus of the heavy chain of the antibody according to the present invention is not limited to the above-mentioned type, as long as the antigen binding ability and effector function are maintained. The two heavy chains constituting the antibody according to the present invention may be any one kind of heavy chain selected from the group consisting of full length and the above-mentioned deletion product, or any two kinds thereof are combined. It may be one. Although the quantitative ratio of each deletion body may be influenced by the type and culture conditions of mammalian cultured cells producing the antibody according to the present invention, both main chains of the antibody according to the present invention may be carboxyl in both of two heavy chains. There may be mentioned the case where one terminal amino acid residue is deleted.
　本発明の抗体のアイソタイプとしては、例えばＩｇＧ（ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４）等を挙げることができるが、好ましくはＩｇＧ１又はＩｇＧ２を挙げることができる。 As the isotype of the antibody of the present invention, for example, IgG (IgG1, IgG2, IgG3, IgG4) and the like can be mentioned, and preferably, IgG1 or IgG2 can be mentioned.
　抗体の生物活性としては、一般的には抗原結合活性、抗原と結合することによって該抗原の活性を阻害する活性、抗原の活性を中和する活性、抗原の活性を増強する活性、抗体依存性細胞傷害（ＡＤＣＣ）活性、補体依存性細胞傷害（ＣＤＣ）活性及び抗体依存性細胞媒介食作用（ＡＤＣＰ）を挙げることができるが、本発明に係る抗ＰＣ抗体が有する機能は、ＰＣ及びａＰＣに対する結合活性であり、好ましくはＰＣと結合することによってＰＣの活性化を阻害する活性である。さらに本発明の抗ＰＣ抗体の好ましい活性としては、ａＰＣに結合し、ａＰＣがその補因子であるプロテインＳとともに、活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び活性化血液凝固第Ｖ因子（ＦＶａ）を分解、不活化することを阻害する活性を挙げることができる。本発明の抗ＰＣ抗体の他の好ましい活性としては、ａＰＣによる抗凝血作用を阻害する活性、血液凝固活性又はトロンビン産生回復活性を挙げることができる。 The biological activity of the antibody generally includes antigen binding activity, activity to inhibit the activity of the antigen by binding to the antigen, activity to neutralize the activity of the antigen, activity to enhance the activity of the antigen, antibody dependency Although there may be mentioned cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity and antibody-dependent cell-mediated phagocytosis (ADCP), the function of the anti-PC antibody according to the present invention is PC and aPC. , And preferably an activity to inhibit the activation of PC by binding to PC. Furthermore, as a preferable activity of the anti-PC antibody of the present invention, activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa) are bound together with protein S which binds to aPC and aPC is its cofactor. And the activity to inhibit deactivating and inactivating. Other preferable activities of the anti-PC antibody of the present invention include an activity to inhibit the anticoagulant action by aPC, blood coagulation activity or thrombin generation recovery activity.
　得られた抗体は、均一にまで精製することができる。抗体の分離、精製は通常のタンパク質で使用されている分離、精製方法を使用すればよい。例えばカラムクロマトグラフィー、フィルター濾過、限外濾過、塩析、透析、調製用ポリアクリルアミドゲル電気泳動、等電点電気泳動等を適宜選択、組み合わせれば、抗体を分離、精製することができる（Ｓｔｒａｔｅｇｉｅｓ　ｆｏｒ　Ｐｒｏｔｅｉｎ　Ｐｕｒｉｆｉｃａｔｉｏｎ　ａｎｄ　Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｃｏｕｒｓｅ　Ｍａｎｕａｌ，Ｄａｎｉｅｌ　Ｒ．Ｍａｒｓｈａｋ　ｅｔ　ａｌ．ｅｄｓ．，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ（１９９６）；Ａｎｔｉｂｏｄｉｅｓ：Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ．Ｅｄ　Ｈａｒｌｏｗ　ａｎｄ　Ｄａｖｉｄ　Ｌａｎｅ，Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ（１９８８））が、これらに限定されるものではない。 The resulting antibodies can be purified to homogeneity. For separation and purification of antibodies, separation and purification methods used for ordinary proteins may be used. For example, antibodies can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric focusing electrophoresis etc. (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory ( 988)) it is not intended to be limited thereto.
　クロマトグラフィーとしては、アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水性クロマトグラフィー、ゲル濾過クロマトグラフィー、逆相クロマトグラフィー、吸着クロマトグラフィー等を挙げることができる。 Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, adsorption chromatography and the like.
　これらのクロマトグラフィーは、ＨＰＬＣやＦＰＬＣ等の液体クロマトグラフィーを用いて行うことができる。 These chromatographies can be performed using liquid chromatographies, such as HPLC and FPLC.
　アフィニティークロマトグラフィーに用いるカラムとしては、プロテインＡカラム、プロテインＧカラムを挙げることができる。例えばプロテインＡカラムを用いたカラムとして、Ｈｙｐｅｒ　Ｄ，ＰＯＲＯＳ，Ｓｅｐｈａｒｏｓｅ　Ｆ．Ｆ．（ファルマシア）等を挙げることができる。 As columns used for affinity chromatography, a protein A column and a protein G column can be mentioned. For example, as columns using protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like.
　また抗原を固定化した担体を用いて、抗原への結合性を利用して抗体を精製することも可能である。 In addition, it is also possible to purify an antibody using its antigen-binding ability using a carrier on which the antigen is immobilized.
　４．医薬
　上記、「２．抗ＰＣ抗体の製造」の項及び実施例に記載された本発明の抗ＰＣ抗体及び当該抗体の機能性断片、これらのアミノ酸配列をコードするヌクレオチド配列を含むポリヌクレオチド、発現ベクター、宿主細胞のいずれか一つ以上を有効成分として含む医薬は、ＰＣの活性化を阻害しａＰＣの産生を抑制し、及び／又はａＰＣに結合してａＰＣの活性を阻害し、結果としてトロンビン産生（ＴＧ）を回復し、血液凝固効果を示し、単独であるいは他の薬剤と組み合わせて、例えば、血友病、血友病Ａ、血友病Ｂ等の出血性疾患の治療剤として用いることができる。 4. The anti-PC antibody of the present invention and the functional fragment of the antibody described in the section “2. Production of anti-PC antibody” and the examples above, a polynucleotide comprising a nucleotide sequence encoding these amino acid sequences, expression A drug comprising at least one of a vector and a host cell as an active ingredient inhibits PC activation and suppresses aPC production and / or binds to aPC and inhibits aPC activity, resulting in thrombin Production (TG) is recovered, blood clotting effect is shown, and it is used as a therapeutic agent for hemorrhagic diseases such as hemophilia, hemophilia A, hemophilia B, etc. alone or in combination with other drugs. Can.
　また、本発明の抗ＰＣ抗体は血漿中のＰＣの検出に用いることができる。 In addition, the anti-PC antibody of the present invention can be used for detection of PC in plasma.
　本発明の抗ＰＣ抗体は、大気中に放置したり、又は再結晶や精製操作をしたりすることにより、水分を吸収し、あるいは吸着水が付着するなどして、水和物になる場合があり、そのような水を含む化合物又は薬理学的に許容され得る塩も本発明に包含される。 The anti-PC antibody of the present invention may become a hydrate due to absorption of water or attachment of adsorbed water, etc., by leaving it in the air, or performing recrystallization or purification operation. Such water-containing compounds or pharmacologically acceptable salts are also included in the present invention.
　本発明の抗ＰＣ抗体が、アミノ基などの塩基性基を有する場合、所望により薬理学的に許容され得る酸付加塩を形成することができる。そのような酸付加塩としては、例えばフッ化水素酸塩、塩酸塩、臭化水素酸塩、ヨウ化水素酸塩などのハロゲン化水素酸塩；硝酸塩、過塩素酸塩、硫酸塩、燐酸塩などの無機酸塩；メタンスルホン酸塩、トリフルオロメタンスルホン酸塩、エタンスルホン酸塩などの低級アルカンスルホン酸塩；ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩などのアリ－ルスルホン酸塩；蟻酸塩、酢酸塩、トリフルオロ酢酸塩、りんご酸塩、フマル酸塩、コハク酸塩、クエン酸塩、酒石酸塩、シュウ酸塩、マレイン酸塩などの有機酸塩；又はオルニチン酸塩、グルタミン酸塩、アスパラギン酸塩などのアミノ酸塩などを挙げることができる。 When the anti-PC antibody of the present invention has a basic group such as an amino group, it is possible to optionally form a pharmaceutically acceptable acid addition salt. As such acid addition salts, for example, hydrohalic acid salts such as hydrogen fluoride, hydrochloride, hydrobromide, hydroiodide, etc .; nitrates, perchlorates, sulfates, phosphates Inorganic acid salts such as; lower sulfonates such as methane sulfonate, trifluoromethane sulfonate, ethane sulfonate; aryl sulfonates such as benzene sulfonate, p-toluene sulfonate; formate , Acetates, trifluoroacetates, malates, fumarates, succinates, citrates, tartrates, oxalates, organic salts such as maleates; or ornitrates, glutamates, asparagine Examples include amino acid salts such as acid salts.
　本発明の抗ＰＣ抗体が、カルボキシ基などの酸性基を有する場合、所望により薬理学的に許容され得る塩基付加塩を形成することができる。そのような塩基付加塩としては、例えばナトリウム塩、カリウム塩、リチウム塩などのアルカリ金属塩；カルシウム塩、マグネシウム塩などのアルカリ土類金属塩；アンモニウム塩などの無機塩；ジベンジルアミン塩、モルホリン塩、フェニルグリシンアルキルエステル塩、エチレンジアミン塩、Ｎ－メチルグルカミン塩、ジエチルアミン塩、トリエチルアミン塩、シクロヘキシルアミン塩、ジシクロヘキシルアミン塩、Ｎ，Ｎ’－ジベンジルエチレンジアミン塩、ジエタノールアミン塩、Ｎ－ベンジル－Ｎ－（２－フェニルエトキシ）アミン塩、ピペラジン塩、テトラメチルアンモニウム塩、トリス（ヒドロキシメチル）アミノメタン塩などの有機アミン塩などを挙げることができる。　 When the anti-PC antibody of the present invention has an acidic group such as a carboxy group, it is possible to optionally form a pharmaceutically acceptable base addition salt. Such base addition salts include, for example, alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; inorganic salts such as ammonium salts; dibenzylamine salts, morpholine Salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts, diethylamine salts, triethylamine salts, cyclohexylamine salts, dicyclohexylamine salts, N, N'-dibenzylethylenediamine salts, diethanolamine salts, N-benzyl-N And organic amine salts such as-(2-phenylethoxy) amine salt, piperazine salt, tetramethylammonium salt, tris (hydroxymethyl) aminomethane salt and the like.
　本発明はまた、抗体を構成する原子の１以上が、その原子の同位体で置換された抗ＰＣ抗体を包含し得る。同位体には放射性同位体及び安定同位体の２種類が存在し、同位体の例としては、例えば、水素の同位体（２Ｈ及び３Ｈ）、炭素の同位体（１１Ｃ、１３Ｃ及び１４Ｃ）、窒素の同位体（１３Ｎ及び１５Ｎ）、酸素の同位体（１５Ｏ、１７Ｏ及び１８Ｏ）、フッ素の同位体（１８Ｆ）などを挙げることができる。同位体で標識された抗体を含む組成物は、例えば、治療剤、予防剤、研究試薬、アッセイ試薬、診断剤、インビボ画像診断剤などとして有用である。同位体で標識された抗体、及び、同位体で標識された抗体の任意の割合の混合物もすべて本発明に包含される。同位体で標識された抗体は、当該分野で公知の方法により、例えば、後述する本発明の製造方法における原料の代わりに同位体で標識された原料を用いることにより、製造することができる。 The invention can also encompass anti-PC antibodies in which one or more of the atoms that make up the antibody are replaced with an isotope of that atom. There are two types of isotopes, radioactive isotopes and stable isotopes, and examples of isotopes include, for example, isotopes of hydrogen ( 2 H and 3 H) and isotopes of carbon ( 11 C, 13 C and 14 C), nitrogen isotopes ( 13 N and 15 N), oxygen isotopes ( 15 O, 17 O and 18 O), fluorine isotopes ( 18 F) and the like can be mentioned. The composition containing the isotope-labeled antibody is useful as, for example, a therapeutic agent, a prophylactic agent, a research reagent, an assay reagent, a diagnostic agent, an in vivo diagnostic imaging agent, and the like. Isotopically labeled antibodies and mixtures of any proportions of isotopically labeled antibodies are also encompassed by the present invention. The isotope-labeled antibody can be produced by a method known in the art, for example, by using an isotope-labeled raw material instead of the raw material in the production method of the present invention described later.
　ｉｎ　ｖｉｔｒｏでのＴＧ回復活性は、例えば、血友病Ａ又は血友病Ｂ患者由来の血漿に、種々の濃度で抗ＰＣ抗体を添加して、組み換え型組織因子（ｒｅｃｏｍｂｉｎａｎｔ　ｔｉｓｓｕｅ　ｆａｃｔｏｒ：ｒＴＦ）と適当な時間インキュベーションさせ、次いで適当量のＦｌｕＣａ－ｋｉｔ（２．５　ｍＭ　Ｚ－Ｇｌｙ　Ｇｌｙ－Ａｒｇ－ａｍｉｎｏｍｅｔｈｙｌｃｏｕｍａｒｉｎ（Ｚ－ＧＧＲ－ＡＭＣ），１００　ｍＭ　ＣａＣｌ２）を加えて反応を行い、ＴＧ回復作用を調べることで確認することができる。
　ｉｎ　ｖｉｖｏでの実験動物を用いた血友病に対する治療効果は、例えば、抗ヒトＦＶＩＩＩ中和抗体を投与して一過性の血友病Ａを発症させたカニクイザルに抗ＰＣ抗体を投与し、麻酔下で創傷させ、創傷部の皮下出血の面積を経時的に観察し、抗ＰＣ抗体を投与しなかった場合と比較することによって確認することができる。 In vitro TG recovery activity can be obtained, for example, by adding anti-PC antibody at various concentrations to plasma derived from hemophilia A or hemophilia B patients, and combining with recombinant tissue factor (rTF). Incubate for an appropriate time, then add appropriate amount of FluCa-kit (2.5 mM Z-Gly Gly-Arg-aminomethylcoumarin (Z-GGR-AMC), 100 mM CaCl2) to perform reaction, and examine TG recovery action It can confirm by that.
The therapeutic effect on hemophilia using experimental animals in vivo can be obtained, for example, by administering an anti-PC antibody to a cynomolgus monkey that has developed a transient hemophilia A by administering an anti-human FVIII neutralizing antibody, Wounds under anesthesia can be confirmed by observing the area of subcutaneous hemorrhage of the wound over time and comparing with the case where anti-PC antibody was not administered.
　本発明の抗ＰＣ抗体が適用される出血性疾患の種類としては、治療対象と出血性疾患においてＰＣ及び／又はａＰＣが関与している疾患であれば特に制限されないが、例えば、血友病Ａ、血友病Ｂ、さらには後天性血友病、ｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病を挙げることができる。本発明の抗ＰＣは、哺乳動物に対して好適に投与することができるが、より好ましくはヒトである。 The type of hemorrhagic disease to which the anti-PC antibody of the present invention is applied is not particularly limited as long as it is a disease to which PC and / or aPC are involved in the treatment subject and hemorrhagic disease. Hemophilia B, as well as acquired haemophilia and von Willebrand's disease. The anti-PC of the present invention can be suitably administered to mammals, but is more preferably human.
　本発明の抗ＰＣ抗体が含まれる医薬組成物において使用される物質としては、投与量や投与濃度において、この分野において通常使用される製剤添加物その他から適宜選択して適用することができる。 The substance used in the pharmaceutical composition containing the anti-PC antibody of the present invention can be appropriately selected and applied from the formulation additives and the like usually used in the field in dosage amount and concentration.
　本発明の抗ＰＣ抗体は、１種以上の薬学的に適合性の成分を含む薬学的組成物として投与され得る。例えば、上記薬学的組成物は、代表的には、１種以上の薬学的キャリア（例えば、滅菌した液体（例えば、水及び油（石油、動物、植物、又は合成起源の油（例えば、ラッカセイ油、大豆油、鉱油、ごま油など）を含む））を含む。水は、上記薬学的組成物が静脈内投与される場合に、より代表的なキャリアである。食塩水溶液、並びにデキストロース水溶液及びグリセロール水溶液もまた、液体キャリアとして、特に、注射用溶液のために使用され得る。適切な薬学的賦形剤は、当該分野で公知である。上記組成物はまた、所望であれば、微量の湿潤剤もしくは乳化剤、又はｐＨ緩衝化剤を含み得る。適切な薬学的キャリアの例は、Ｅ．Ｗ．Ｍａｒｔｉｎによる「Ｒｅｍｉｎｇｔｏｎ’ｓ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｓｃｉｅｎｃｅｓ」に記載される。その処方は、投与の態様に対応する。 The anti-PC antibodies of the invention can be administered as pharmaceutical compositions comprising one or more pharmaceutically compatible components. For example, the pharmaceutical composition is typically one or more pharmaceutical carriers (eg, sterile liquids (eg, water and oils (eg, petroleum, animal, vegetable, or oils of synthetic origin (eg, peanut oil) , Soybean oil, mineral oil, sesame oil, etc.)) water is a more representative carrier when the above-mentioned pharmaceutical composition is administered intravenously, saline solution, and aqueous dextrose solution and aqueous glycerol solution. Liquid carriers may also be used as liquid carriers, in particular for injectable solutions Suitable pharmaceutical excipients are known in the art. Or an emulsifying agent, or a pH buffering agent Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Science" by E. W. Martin. Described ". The formulations correspond to the mode of administration.
　種々の送達システムが公知であり、本発明の抗ＰＣ抗体を投与するために使用され得る。導入方法としては、皮内、筋肉内、腹腔内、静脈内、及び皮下の経路が挙げられるが、これらに限定されない。投与は、例えば、注入又はボーラス注射によるものであり得る。特定の好ましい実施形態において、上記抗体の投与は、注入によるものである。非経口的投与は、好ましい投与経路である。 Various delivery systems are known and can be used to administer the anti-PC antibodies of the invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous routes. Administration may be, for example, by injection or bolus injection. In certain preferred embodiments, administration of the antibody is by injection. Parenteral administration is a preferred route of administration.
　代表的実施形態において、上記薬学的組成物は、ヒトへの静脈内投与に適合した薬学的組成物として、常習的手順に従って処方される。代表的には、静脈内投与のための組成物は、滅菌の等張性の水性緩衝液中の溶液である。必要である場合、上記医薬はまた、可溶化剤及び注射部位での疼痛を和らげるための局所麻酔剤（例えば、リグノカイン）を含み得る。一般に、上記成分は、（例えば、活性剤の量を示すアンプル又はサシェなどに密封してシールされた容器中の乾燥凍結乾燥粉末又は無水の濃縮物として、別個に、又は単位剤形中で一緒に混合して、のいずれかで供給される。上記医薬が注入によって投与される予定である場合、それは、例えば、滅菌の製薬グレードの水又は食塩水を含む注入ボトルで投薬され得る。上記医薬が注射によって投与される場合、注射用滅菌水又は食塩水のアンプルは、例えば、上記成分が投与前に混合され得るように、提供され得る。 In an exemplary embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the medicament may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of injection. In general, the above components may be combined separately, or in unit dosage form, as a dry lyophilized powder or as an anhydrous concentrate, for example in a sealed sealed container such as in an ampoule or sachette indicating the amount of active agent If the drug is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. When is administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, such that the components can be mixed prior to administration.
　本発明の医薬組成物には本願の抗ＰＣ抗体のみを含む医薬組成物であってもよいし、抗ＰＣ抗体及び少なくとも一つのこれ以外の出血性疾患用治療剤を含む医薬組成物であってもよい。本発明の抗ＰＣ抗体は、他の出血性疾患用治療剤と共に投与することもでき、これによって出血性疾患に対する治療効果を増強させることができる。このような目的で使用される他の出血性疾患用治療剤は、抗体と同時に、別々に、あるいは連続して個体に投与されてもよいし、それぞれの投与間隔を変えて投与してもよい。このような出血性疾患用治療剤として、血友病治療薬（凝固因子製剤、抗体、核酸医薬品、遺伝子治療製剤など）、トラネキサム酸、アドレナリン等を挙げることができるが、出血防止作用を有する薬剤であれば限定されることはない。 The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the anti-PC antibody of the present invention, or a pharmaceutical composition containing the anti-PC antibody and at least one other therapeutic agent for hemorrhagic disease, It is also good. The anti-PC antibody of the present invention can also be administered together with other therapeutic agents for hemorrhagic diseases, which can enhance the therapeutic effect on hemorrhagic diseases. The other therapeutic agents for hemorrhagic disease used for such purpose may be administered to the individual simultaneously, separately or sequentially with the antibody, or may be administered at different intervals of administration. . Examples of therapeutic agents for hemorrhagic diseases include hemophiliacs (coagulation factor preparations, antibodies, nucleic acid pharmaceuticals, gene therapy preparations, etc.), tranexamic acid, adrenaline, etc. If it is, it will not be limited.
　このような医薬組成物は、選択された組成と必要な純度を持つ製剤として、凍結乾燥製剤あるいは液状製剤として製剤化すればよい。凍結乾燥製剤として製剤化する際には、この分野において使用される適当な製剤添加物が含まれる製剤であってもよい。また液剤においても同様にして、この分野において使用される各種の製剤添加物を含む液状製剤として製剤化することができる。 Such a pharmaceutical composition may be formulated as a lyophilized formulation or a liquid formulation as a formulation having a selected composition and the required purity. When formulated as a lyophilised formulation, it may be a formulation comprising suitable formulation additives used in the art. Similarly, in the case of a liquid preparation, it can be formulated as a liquid preparation containing various preparation additives used in this field.
　医薬組成物の組成及び濃度は投与方法によっても変化するが、本発明の医薬組成物に含まれる抗ＰＣ抗体は、抗体の抗原に対する親和性、すなわち、抗原に対する解離定数（Ｋｄ値）の点において、親和性が高い（Ｋｄ値が低い）ほど、少量の投与量であっても薬効を発揮させことができる。したがって、抗体の投与量の決定に当たっては、抗体と抗原との親和性の状況に基づいて投与量を設定することもできる。本発明の抗体をヒトに対して投与する際には、例えば、約０．００１～１００ｍｇ／ｋｇを１回あるいは１～１８０日間に１回の間隔で複数回投与すればよい。好適には、０．１～５０ｍｇ／ｋｇを、さらに好適には、１乃至１５　ｍｇ／ｋｇを２～３週間に１回の間隔で複数回投与すればよい。 Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the anti-PC antibody contained in the pharmaceutical composition of the present invention has an affinity for the antigen of the antibody, that is, a dissociation constant (Kd value) for the antigen. The higher the affinity (the lower the Kd value), the smaller the dose the drug can exert. Therefore, when determining the dose of the antibody, the dose can also be set based on the state of affinity between the antibody and the antigen. When the antibody of the present invention is administered to human, for example, about 0.001 to 100 mg / kg may be administered once or plural times at intervals of 1 to 180 days. Preferably, 0.1 to 50 mg / kg, more preferably 1 to 15 mg / kg may be administered several times at intervals of once every 2 to 3 weeks.
　以下に示す実施例によって本発明を具体的に説明するが、本発明はこれらに限定されるものではない。また、これらはいかなる意味においても限定的に解釈されるものではない。なお、下記実施例において遺伝子操作に関する各操作は特に明示がない限り、「モレキュラークローニング（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ）」（Ｓａｍｂｒｏｏｋ，Ｊ．，Ｆｒｉｔｓｃｈ，Ｅ．Ｆ．及びＭａｎｉａｔｉｓ，Ｔ．著，Ｃｏｌｄ　ＳｐｒｉｎｇＨａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓより１９８９年発刊）に記載の方法及びその他の当業者が使用する実験書に記載の方法により行うか、又は、市販の試薬やキットを用いる場合には市販品の指示書に従って行った。また、本明細書において、特に記載のない試薬、溶媒及び出発材料は、市販の供給源から容易に入手可能である。 The present invention will be specifically described by way of the following examples, but the present invention is not limited thereto. Moreover, these are not to be interpreted as limiting in any sense. In the following examples, unless otherwise specified, each operation relating to genetic manipulation is “Molecular cloning” (Sambrook, J., Fritsch, EF and Maniatis, T., Cold Spring Harbor Laboratory Press) (1989) and the procedures described in the experimental documents used by those skilled in the art, or in the case of using commercially available reagents and kits, according to the instructions of the commercially available products. Also, reagents, solvents and starting materials not specifically described herein are readily available from commercial sources.
　［実施例１］ラット抗ヒトプロテインＣ（ＰＣ）抗体の作製
　１）－１　免疫
　１）－１－１　ヒトＰＣ発現ベクターの構築
　ヒトＰＣ（ＮＣＢＩの蛋白データベースのＡＣＣＥＳＳＩＯＮ番号ＮＰ＿０００３０３のアミノ酸配列（配列表の配列番号１）の１乃至４２番目と８９乃至４５８番目のアミノ酸残基を連結したポリペプチド）のＣ末端側に配列番号２に示すペプチドタグ（ＬＶＰＲＧＳＭＤＹＫＤＤＤＤＫＮＳＡＶＤＭＨＨＨＨＨＨＨ）（配列番号２）を連結したポリペプチド（配列Ｘ：配列番号３）をコードするＤＮＡ（配列Ｙ：配列番号４））と制限酵素ＸｂａＩとＰｍｅＩで消化したベクターｐｃＤＮＡ３．３－ＴＯＰＯ／ＬａｘＺ（ＴｈｅｒｍｏＦｉｓｈｅｒ　ＳＣＩＥＮＴＩＦＩＣ）をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　Ｃｌｏｎｉｎｇ　Ｋｉｔ（ＣＬＯＮＴＥＣＨ）を用いて結合することにより、ヒトＰＣ発現ベクターを作製した。 [Example 1] Preparation of rat anti-human protein C (PC) antibody 1) -1 Immunization 1) -1-1 Construction of human PC expression vector Human PC (amino acid sequence of ACCESSION number NP_000303 of protein database of NCBI (sequence list A polypeptide in which the peptide tag (LVPRGSMDYKDDDDKNSAVDMHHHHHH) (SEQ ID NO: 2) shown in SEQ ID NO: 2 is linked to the C-terminal side of the polypeptide in which the 1st to 42nd and 89th to 458th amino acid residues of SEQ ID NO: 1) are linked (Sequence X: SEQ ID NO: 3) (Sequence Y: SEQ ID NO: 4)) and vector pcDNA3.3-TOPO / LaxZ (ThermoFisher SCIENTIFIC) digested with restriction enzymes XbaI and PmeI, and used in-fusion HD cloning By binding to using it (CLONTECH), to prepare the human PC expression vector.
　１）－１－２　ヒトＰＣの発現、精製
　ヒトＰＣ発現ベクターをＦｒｅｅＳｔｙｌｅ　２９３Ｆ　ｃｅｌｌｓ（ＴｈｅｒｍｏＦｉｓｈｅｒ　ＳＣＩＥＮＴＩＦＩＣ）にトランスフェクションすることで一過性に発現させた。培養上清をＡＮＴＩ－ＦＬＡＧ　Ｍ２　Ａｆｆｉｎｉｔｙ　Ｇｅｌ（ＳＩＧＭＡ－ＡＬＤＲＩＣＨ）に吸着させて、カラム容量３０倍の２０　ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ８．０，１５０　ｍＭ　ＮａＣｌ，５％　Ｇｌｙｃｅｒｏｌでカラム洗浄したのち、２０　ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ　８．０，１５０　ｍＭ　ＮａＣｌ，５％　Ｇｌｙｃｅｒｏｌ，１００　ｕｇ／ｍＬ　３ｘＦｌａｇ　ｐｅｐｔｉｄｅ（ＳＩＧＭＡ－ＡＬＤＲＩＣＨ）で溶出してヒトＰＣの含まれる画分を回収した。 1) -1-2 Expression and Purification of Human PC Human PC expression vector was transiently expressed by transfecting FreeStyle 293F cells (ThermoFisher SCIENTIFIC). The culture supernatant is adsorbed to ANTI-FLAG M2 Affinity Gel (SIGMA-ALDRICH), and after column washing with a column volume of 30 mM 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% Glycerol, 20 mM Tris -The fraction containing human PC was recovered by eluting with HCl pH 8.0, 150 mM NaCl, 5% Glycerol, 100 ug / mL 3x Flag peptide (SIGMA-ALDRICH).
　１）－１－３　ヒトＰＣの活性化
　ヒトＰＣにＴｈｒｏｍｂｉｎ（トロンビン液モチダソフトボトル１万、持田製薬）を添加し、室温で１時間反応させたのち終濃度５　ｍＭ　ＥＤＴＡを添加することで反応を停止させた。反応液は透析により２０　ｍＭ　Ｔｒｉｓ（ｐＨ７．５）へバッファー交換し、２０　ｍＭ　Ｔｒｉｓ（ｐＨ７．５）で平衡化させたＨｉｓＴｒａｐ　Ｑ　ＨＰ（ＧＥヘルスケアバイオサイエンス）にアプライした。カラム容量１０倍の２０　ｍＭ　Ｔｒｉｓ（ｐＨ７．５）でカラムを洗浄した。塩化ナトリウムによる直線的濃度勾配溶出を実施し、活性化ヒトＰＣを含む画分を回収した。活性化ヒトＰＣ（ａＰＣ）を含む画分をＡｍｉｃｏｎＵｌｔｒａ（ＭＥＲＣＫ　ＭＩＬＬＩＰＯＲＥ）で濃縮後、ＰＢＳ，１　ｍＭ　ＣａＣｌ２で平衡化したＨｉＬｏａｄ　１６／６００　Ｓｕｐｅｒｄｅｘ　７５　ｐｇでゲルろ過を実施し、活性化ヒトＰＣを含む画分を回収し、ＡｍｉｃｏｎＵｌｔｒａ（ＭＥＲＣＫ　ＭＩＬＬＩＰＯＲＥ）で２　ｍｇ／ｍＬ程度まで濃縮し精製サンプルとした。 1) -1-3 Activation of human PC Add Thrombin (Thrombin liquid Motida soft bottle 10,000, Mochida Pharmaceutical) to human PC, react at room temperature for 1 hour, and then add final concentration of 5 mM EDTA. Stopped. The reaction solution was buffer-exchanged to 20 mM Tris (pH 7.5) by dialysis, and applied to HisTrap Q HP (GE Healthcare Biosciences) equilibrated with 20 mM Tris (pH 7.5). The column was washed with 10 column volumes of 20 mM Tris (pH 7.5). A linear gradient elution with sodium chloride was performed to collect fractions containing activated human PC. The fraction containing activated human PC (aPC) is concentrated with AmiconUltra (MERCK MILLIPORE), then gel filtration is performed with HiLoad 16/600 Superdex 75 pg equilibrated with PBS, 1 mM CaCl 2 to obtain activated human PC The fractions containing it were collected, concentrated to about 2 mg / mL with Amicon Ultra (MERCK MILLIPORE), and used as a purified sample.
　１）－１－４　免疫
　免疫にはＷＫＹ／Ｉｚｍラットの雌（日本エスエルシー社）を使用した。１）－１－３で作製した抗原蛋白とＦｒｅｕｎｄ‘ｓ　Ｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（和光純薬社製）を混合したものを尾根部に投与したラットのリンパ節及び脾臓を採取しハイブリドーマ作製に用いた。 1) -1-4 Immunization Females of WKY / Izm rats (Japan SLC) were used for immunization. 1) Lymph nodes and spleens of rats administered to the ridge portion were collected by using a mixture of the antigen protein prepared in -1-3 and Freund's Complete Adjuvant (manufactured by Wako Pure Chemical Industries, Ltd.) and used for preparation of hybridomas.
　１）－２　ハイブリドーマ作製
　リンパ節細胞あるいは脾臓細胞とマウスミエローマＳＰ２／０－ａｇ１４細胞（ＡＴＣＣ：ＣＲＬ－１５８１）とをＬＦ３０１－Ｃｅｌｌ　Ｆｕｓｉｏｎ　Ｕｎｉｔ（ＢＥＸ社製）を用いて電気細胞融合し、ＣｌｏｎａＣｅｌｌ－ＨＹ　Ｓｅｌｅｃｔｉｏｎ　Ｍｅｄｉｕｍ　Ｄ（ＳｔｅｍＣｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）に希釈して培養した。出現したハイブリドーマコロニーを回収することでモノクローンハイブリドーマを作製した。回収された各ハイブリドーマコロニーを培養し、得られたハイブリドーマ培養上清用いて抗ＰＣ抗体産生ハイブリドーマのスクリーニングを実施した。 1) -2 Preparation of hybridomas Lymph node cells or spleen cells and mouse myeloma SP2 / 0-ag14 cells (ATCC: CRL-1581) are subjected to electric cell fusion using LF301-Cell Fusion Unit (manufactured by BEX), and ClonaCell- The cells were cultured after dilution in HY Selection Medium D (manufactured by StemCell Technologies). A monoclonal hybridoma was produced by collecting the appearing hybridoma colonies. The recovered hybridoma colonies were cultured, and the obtained hybridoma culture supernatant was used to screen for anti-PC antibody-producing hybridomas.
　１）－３　ハイブリドーマ培養上清を用いたトロンビン産生（ＴＧ）による一次スクリーニング
　ハイブリドーマ培養上清をヒトＰＣ欠乏血漿を用いて、ヒトａＰＣによるＴＧ抑制効果に対する回復作用を指標とする系によりスクリーニングを実施した。
　ハイブリド－マ培養上清９０　μＬに２　ｎＭヒトａＰＣ含有又は非含有ヒトＰＣ欠乏血漿７０　μＬ、組み換え型組織因子（ｒｅｃｏｍｂｉｎａｎｔ　ｔｉｓｓｕｅ　ｆａｃｔｏｒ：ｒＴＦ）２０　μＬ（最終濃度３　ｐＭ）を加えて３７℃で１０分間インキュベーションした。次いでＦｌｕＣａ－ｋｉｔ（２．５　ｍＭ　Ｚ－Ｇｌｙ　Ｇｌｙ－Ａｒｇ－ａｍｉｎｏｍｅｔｈｙｌｃｏｕｍａｒｉｎ（Ｚ－ＧＧＲ－ＡＭＣ），１００　ｍＭ　ＣａＣｌ２）を２０　μＬ（最終濃度Ｚ－ＧＧＲ－ＡＭＣ：２５０　μＭ，ＣａＣｌ２　１０　ｍＭ）加えて反応を開始した。蛍光度（ｅｘ．３９０　ｎｍ，ｅｍ．４６０　ｎｍ）を２０秒ごとに５０分間測定した。ａＰＣによるＴＧの抑制をより強く回復させたハイブリドーマを抗ヒトＰＣ抗体産生陽性として選択した。 1) -3 Primary Screening by Thrombin Production (TG) Using Hybridoma Culture Supernatant Hybridoma culture supernatant is screened using a system that uses human PC-deficient plasma as a marker for recovery from the TG suppression effect of human aPC did.
90 μL of hybridoma culture supernatant, 70 μL of human PC-depleted plasma with or without 2 nM human aPC, 20 μL of recombinant tissue factor (rTF) (final concentration 3 pM) and adding at 10 ° C at 37 ° C Incubated for a minute. Then 20 μL of FluCa-kit (2.5 mM Z-Gly Gly-Arg-aminomethylcoumarin (Z-GGR-AMC), 100 mM CaCl 2 ) (final concentration Z-GGR-AMC: 250 μM, CaCl 2 10 mM) In addition, the reaction was started. The fluorescence (ex. 390 nm, em. 460 nm) was measured every 20 seconds for 50 minutes. The hybridoma which recovered the suppression of TG by aPC more strongly was selected as anti-human PC antibody production positive.
　１）－４　血友病患者由来血漿を用いたＴＧ回復作用による二次スクリーニング
　実施例１）－３で陽性と判定されたハイブリドーマが産生する抗体の二次スクリーニングとして、血友病Ａ（ＦＶＩＩＩ欠乏血漿）血友病患者由来血漿（Ｇｅｏｒｇｅ　Ｋｉｎｇ　Ｂｉｏ－Ｍｅｄｉｃａｌ社）及び血友病Ｂ（ＦＩＸ欠乏血漿）血友病患者由来血漿（Ｇｅｏｒｇｅ　Ｋｉｎｇ　Ｂｉｏ－Ｍｅｄｉｃａｌ社）を用いて、実施例１）－３と同様の方法でＴＧ回復作用を検討した。結果として血友病Ａ及び血友病Ｂの両方の血友病患者由来の血漿で効果を示したハイブリドーマが３６個取得された。 1) -4 Secondary screening by TG recovery using hemophiliac patient-derived plasma Example 1) Hemophilia A (FVIII deficiency) as secondary screening of antibodies produced by hybridomas determined as positive in -3 Plasma) Hemophilia patient-derived plasma (George King Bio-Medical) and Hemophilia B (FIX-deficient plasma) Hemophilia patient-derived plasma (George King Bio-Medical) Example 1) -3 The TG recovery action was examined in the same manner as in. As a result, 36 hybridomas that show an effect on plasma from hemophiliacs of both hemophilia A and hemophilia B were obtained.
　［実施例２］ラット抗ヒトＰＣ抗体Ｒ７４のｉｎ　ｖｉｔｒｏ評価
２）－１　ラットハイブリドーマ培養上清からの抗ヒトＰＣ抗体の精製
　ラット抗ＰＣモノクローナル抗体Ｒ７４は、ハイブリドーマ培養上清から精製した。 Example 2 In Vitro Evaluation of Rat Anti-Human PC Antibody R74 2) Purification of Anti-Human PC Antibody from Rat Hybridoma Culture Supernatant Rat anti-PC monoclonal antibody R74 was purified from hybridoma culture supernatant.
　まず、ラット抗ＰＣモノクローナル抗体Ｒ７４産生ハイブリドーマをＣｌｏｎａＣｅｌｌ－ＨＹ　Ｓｅｌｅｃｔｉｏｎ　Ｍｅｄｉｕｍ　Ｅ（ＳｔｅｍＣｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）で十分量まで増殖させ、Ｕｌｔｒａ　Ｌｏｗ　ＩｇＧ　ＦＢＳ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｆｔｉｆｉｃ社製）を２０％添加したＨｙｂｒｉｄｏｍａ　ＳＦＭ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｆｔｉｆｉｃ社製）に培地交換した後、８～９×１０７個のハイブリドーマ細胞を１２７２　ｃｍ２フラスコ（Ｃｏｒｎｉｎｇ社製）に播種して７日間培養した。本培養上清を遠心により回収し０．４５μｍのフィルター（Ｃｏｒｎｉｎｇ社製）を通して滅菌した。 First, Hybridoma SFM (Thermo Fisher SFM (Thermo Fisher), in which a rat anti-PC monoclonal antibody R74-producing hybridoma is grown to a sufficient amount with ClonaCell-HY Selection Medium E (manufactured by StemCell Technologies), and 20% of Ultra Low IgG FBS (manufactured by Thermo Fisher Scienftific) is added. After changing the medium to Fisher Scienftific), 8 to 9 × 10 7 hybridoma cells were seeded in a 1272 cm 2 flask (Corning) and cultured for 7 days. The main culture supernatant was collected by centrifugation and sterilized through a 0.45 μm filter (manufactured by Corning).
　ハイブリドーマの培養上清から抗体をＰｒｏｔｅｉｎ　Ｇアフィニティークロマトグラフィーで精製した。Ｐｒｏｔｅｉｎ　Ｇカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社）に抗体を吸着させ、ＰＢＳでカラムを洗浄後に０．１Ｍ　グリシン／塩酸水溶液（ｐＨ２．７）で溶出した。溶出液に１Ｍ　Ｔｒｉｓ－ＨＣｌ（ｐＨ９．０）を加えてｐＨ７．０～７．５に調整した後に、Ｃｅｎｔｒｉｆｕｇａｌ　ＵＦ　Ｆｉｌｔｅｒ　Ｄｅｖｉｃｅ　ＶＩＶＡＳＰＩＮ２０（分画分子量ＵＦ３０Ｋ、Ｓａｒｔｏｒｉｕｓ社）にてＰＢＳ（－）へのバッファー置換を行うとともに抗体の濃縮を行い、抗体濃度を１．８　ｍｇ／ｍＬに調製した。最後にＭｉｎｉｓａｒｔ－Ｐｌｕｓ　ｆｉｌｔｅｒ（Ｓａｒｔｏｒｉｕｓ社）でろ過し、精製サンプルとした。 Antibodies were purified from the culture supernatant of hybridomas by Protein G affinity chromatography. The antibody was adsorbed on a Protein G column (GE Healthcare Bioscience), and the column was washed with PBS and eluted with 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7). After adjusting the pH to 7.0 to 7.5 by adding 1 M Tris-HCl (pH 9.0) to the eluate, buffer with PBS (-) with Centrifugal UF Filter Device VIVASPIN 20 (molecular weight cut off UF 30 K, Sartorius) While performing substitution, concentration of the antibody was performed, and the antibody concentration was adjusted to 1.8 mg / mL. Finally, the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
　２）－２　血友病患者由来血漿を用いたＴＧ回復作用－１
　ラット抗ヒトＰＣ抗体の濃度を１０　μｇ／ｍＬに固定し、血友病Ａ患者由来血漿（ＦＶＩＩＩ欠乏血漿）及び血友病Ｂ患者由来血漿（ＦＩＸ欠乏血漿）を用いて、実施例１）－３と同様の方法でＴＧ回復作用を検討した。Ｒ７４は、血友病Ａ患者由来血漿において、陽性対照（ヒトａＰＣポリクローナル抗体）と同等以上の活性を示した。血友病Ｂ患者由来血漿でも同様の結果だった。 2) -2 TG recovery action using plasma from hemophiliac patient
The concentration of rat anti-human PC antibody is fixed at 10 μg / mL, and using hemophilia A patient-derived plasma (FVIII deficient plasma) and hemophilia B patient-derived plasma (FIX deficient plasma), Example 1)- The TG recovery action was examined in the same manner as in 3. R74 showed an activity equal to or higher than that of the positive control (human aPC polyclonal antibody) in plasma from hemophilia A patients. Similar results were obtained with hemophiliac B patient-derived plasma.
　２）－３　血友病患者由来血漿を用いたＴＧ回復作用－２
　血友病Ａ患者由来血漿（ＦＶＩＩＩ欠乏血漿）及び血友病Ｂ患者由来血漿（ＦＩＸ欠乏血漿）を用いて、実施例２）－１で精製した抗体のＴＧ回復作用の濃度依存性を検討した。血友病Ａ患者由来血漿では５　ｐＭ　ｒＴＦ，１０　ｎＭ　ｒｅｃｏｍｂｉｎａｎｔ　トロンボモデュリン（ｒＴＭ），各抗体濃度０．３　μｇ／ｍＬ～３０　μｇ／ｍＬの条件で、タイプＢ血友病患者由来血漿では２．５　ｐＭ　ｒＴＦ，１０　ｎＭ　ＴＭ，各抗体濃度０．１　μｇ／ｍＬ～１０　μｇ／ｍＬの条件で評価した。Ｒ７４は、血友病Ａ患者由来血漿において濃度依存的にＴＧを回復し、陽性対照（抗ヒトａＰＣウサギポリクローナル抗体）と同等以上のＴＧ回復効果を示した（図１Ａ）。血友病Ｂ患者由来血漿でも同様の結果が得られた（図１Ｂ）。 2) -3 TG recovery action using plasma from hemophiliac patient-2
The concentration dependency of the TG recovery action of the antibody purified in Example 2) -1 was examined using plasma from hemophilia A patient (FVIII deficient plasma) and plasma from hemophilia B patient (FIX deficient plasma) . Hemophilia A patient-derived plasma: 5 pM rTF, 10 nM recombinant thrombomodulin (rTM), each antibody concentration: 0.3 μg / mL to 30 μg / mL, type B hemophilia patient-derived plasma Evaluation was performed under the conditions of 2.5 pM rTF, 10 nM TM, and each antibody concentration of 0.1 μg / mL to 10 μg / mL. R74 recovered TG concentration-dependently in hemophilia A patient-derived plasma, and showed a TG recovery effect equal to or higher than the positive control (anti-human aPC rabbit polyclonal antibody) (FIG. 1A). Similar results were obtained with hemophilia B patient-derived plasma (FIG. 1B).
　［実施例３］ラット抗ヒトＰＣ抗体の可変領域をコードするｃＤＮＡのヌクレオチド配列の決定
　３）－１　Ｒ７４産生ハイブリドーマのｔｏｔａｌ　ＲＮＡの調製
　Ｒ７４の可変領域をコードするｃＤＮＡを増幅するため、Ｒ７４産生ハイブリドーマよりＴＲＩｚｏｌ　Ｒｅａｇｅｎｔ（Ａｍｂｉｏｎ社）を用いてｔｏｔａｌ　ＲＮＡを調製した。 [Example 3] Determination of nucleotide sequence of cDNA encoding variable region of rat anti-human PC antibody 3) -1 Preparation of total RNA of R74-producing hybridoma In order to amplify cDNA encoding variable region of R74, R74-producing hybridoma Total RNA was prepared using TRIzol Reagent (Ambion).
　３）－２　５’－ＲＡＣＥ　ＰＣＲによるＲ７４の重鎖可変領域をコードするｃＤＮＡの増幅と配列の決定
　重鎖可変領域を含むｃＤＮＡの増幅は、実施例３）－１で調製したｔｏｔａｌ　ＲＮＡの約１　μｇとＳＭＡＲＴｅｒ　ＲＡＣＥ　５’／３’　Ｋｉｔ（Ｃｌｏｎｔｅｃｈ社）を用いて実施した。Ｒ７４の重鎖遺伝子の可変領域をコードするｃＤＮＡをＰＣＲで増幅するためのプライマーとして、ＵＰＭ　（Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ａ　Ｍｉｘ：ＳＭＡＲＴｅｒ　ＲＡＣＥ　５’／３’　Ｋｉｔに付属）、及び公知のラット重鎖の定常領域の配列から設計したプライマーを用いた。 3) -2 Amplification and sequencing of cDNA encoding R74 heavy chain variable region by 5'-RACE PCR Amplification of cDNA containing heavy chain variable region is the same as that of the total RNA prepared in Example 3) -1. It implemented using 1 microgram and SMARTer RACE 5 '/ 3' Kit (Clontech). UPM (Universal Primer A Mix: supplied with SMARTer RACE 5 '/ 3' Kit) as a primer for PCR amplification of the cDNA encoding the variable region of R74 heavy chain gene, and the known constant region of rat heavy chain Primers designed from the sequence of
　５’－ＲＡＣＥ　ＰＣＲで増幅した重鎖の可変領域をコードするｃＤＮＡをプラスミドにクローニングし、次に重鎖の可変領域をコードするｃＤＮＡのヌクレオチド配列のシークエンス解析を実施した。 CDNA encoding the variable region of heavy chain amplified by 5'-RACE PCR was cloned into a plasmid, and then sequence analysis of the nucleotide sequence of cDNA encoding variable region of heavy chain was performed.
　決定されたＲ７４の重鎖の可変領域をコードするｃＤＮＡのヌクレオチド配列を配列番号５に示し、アミノ酸配列を配列番号６に示す。また、Ａｂｍの定義によるＣＤＲＨ１のアミノ酸配列を配列番号７に示し、ＣＤＲＨ２のアミノ酸配列を配列番号８に示し、ＣＤＲＨ３のアミノ酸配列を配列番号９に示す。Ｒ７４重鎖の可変領域のヌクレオチド配列及びアミノ酸配列、ＣＤＲＨ１、ＣＤＲＨ２、ＣＤＲＨ３のアミノ酸配列は図２にも示されている。 The nucleotide sequence of the cDNA encoding the variable region of the heavy chain of R74 determined is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6. The amino acid sequence of CDRH1 according to the definition of Abm is shown in SEQ ID NO: 7, the amino acid sequence of CDRH2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDRH3 is shown in SEQ ID NO: 9. The nucleotide and amino acid sequences of the variable region of R74 heavy chain, and the amino acid sequences of CDRH1, CDRH2 and CDRH3 are also shown in FIG.
　３）－３　５’－ＲＡＣＥ　ＰＣＲによるＲ７４の軽鎖可変領域をコードするｃＤＮＡの増幅と配列の決定
　実施例３）－２と同様の方法で実施した。ただし、Ｒ７４の軽鎖遺伝子の可変領域をコードするｃＤＮＡをＰＣＲで増幅するためのプライマーとして、ＵＰＭ　（Ｕｎｉｖｅｒｓａｌ　Ｐｒｉｍｅｒ　Ａ　Ｍｉｘ：ＳＭＡＲＴｅｒ　ＲＡＣＥ　５’／３’　Ｋｉｔに付属）、及び公知のラット軽鎖の定常領域の配列から設計したプライマーを用いた。 3) -3 Amplification and Sequence Determination of cDNA Encoding the Light Chain Variable Region of R74 by 5'-RACE PCR The procedure was performed in the same manner as in Example 3) -2. However, UPM (Universal Primer A Mix: supplied with SMARTer RACE 5 '/ 3' Kit) as a primer for PCR amplification of the cDNA encoding the variable region of the R74 light chain gene, and a known rat light chain Primers designed from the constant region sequences were used.
　決定されたＲ７４の軽鎖の可変領域をコードするｃＤＮＡのヌクレオチド配列を配列番号１０に示し、Ｒ７４の軽鎖の可変領域のアミノ酸配列を配列番号１１に示す。また、Ａｂｍの定義によるＣＤＲＬ１のアミノ酸配列を配列番号１２に示し、ＣＤＲＬ２にアミノ酸配列を配列番号１３に示し、ＣＤＲＬ３のアミノ酸配列を配列番号１４に示す。Ｒ７４軽鎖の可変領域のヌクレオチド配列及びアミノ酸配列、ＣＤＲＬ１、ＣＤＲＬ２、ＣＤＲＬ３のアミノ酸配列は図３にも示されている。 The nucleotide sequence of the cDNA encoding the variable region of R74 light chain is shown in SEQ ID NO: 10, and the amino acid sequence of the variable region of R74 light chain is shown in SEQ ID NO: 11. The amino acid sequence of CDRL1 according to the definition of Abm is shown in SEQ ID NO: 12, the amino acid sequence in CDRL2 is shown in SEQ ID NO: 13, and the amino acid sequence of CDRL3 is shown in SEQ ID NO: 14. The nucleotide and amino acid sequences of the variable region of R74 light chain, and the amino acid sequences of CDRL1, CDRL2 and CDRL3 are also shown in FIG.
　［実施例４］ヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４のヒト化バージョンｈＲ７４の設計
４）－１　Ｒ７４のヒト化体デザイン
４）－１－１　Ｒ７４の可変領域の分子モデリング
　Ｒ７４の可変領域の分子モデリングは、ホモロジーモデリングとして公知の方法（Ｍｅｔｈｏｄｓ　ｉｎ　Ｅｎｚｙｍｏｌｏｇｙ，２０３，１２１－１５３，（１９９１））を利用した。Ｒ７４の重鎖と軽鎖の可変領域に対して高い配列同一性を有するＰｒｏｔｅｉｎ　Ｄａｔａ　Ｂａｎｋ（Ｎｕｃ．Ａｃｉｄ　Ｒｅｓ．３５，Ｄ３０１－Ｄ３０３（２００７））に登録されている構造（ＰＤＢ　ＩＤ：３ＩＹ７）を鋳型に、市販のタンパク質立体構造解析プログラムＢｉｏＬｕｍｉｎａｔｅ（Ｓｃｈｒｏｄｉｎｇｅｒ社製）を用いて行った。 [Example 4] Design of humanized version hR74 of human chimerized anti-human PC antibody cR74 4) -1 Human body design of R74 4) -1-1 Molecular modeling of R74 variable region Molecular modeling of R74 variable region A method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)) was used. Structure (PDB ID: 3IY7) registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) having high sequence identity to the variable regions of heavy chain and light chain of R74 A commercially available protein conformation analysis program BioLuminate (manufactured by Schrodinger) was used as a template.
　４）－１－２　Ｒ７４のヒト化設計方法
　Ｒ７４は、ＣＤＲグラフティング（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８６，１００２９－１００３３（１９８９））によりヒト化した。ＫＡＢＡＴ　ｅｔ　ａｌ．（Ｓｅｑｕｅｎｃｅｓ　ｏｆ　Ｐｒｏｔｅｉｎｓ　ｏｆ　Ｉｍｍｕｎｏｌｏｇｉｃａｌ　Ｉｎｔｅｒｅｓｔ，５ｔｈ　Ｅｄ．Ｐｕｂｌｉｃ　Ｈｅａｌｔｈ　Ｓｅｒｖｉｃｅ　Ｎａｔｉｏｎａｌ　Ｉｎｓｔｉｔｕｔｅｓ　ｏｆ　Ｈｅａｌｔｈ，Ｂｅｔｈｅｓｄａ，ＭＤ．（１９９１））において既定されるヒトのｋａｐｐａ鎖サブグループ３及び４のコンセンサス配列とＩＭＧＴ（ＴＨＥ　ＩＮＴＥＲＮＡＴＩＯＮＡＬ　ＩＭＭＵＮＯＧＥＮＥＴＩＣＳ　ＩＮＦＯＲＭＡＴＩＯＮ　ＳＹＳＴＥＭ（登録商標））において規定されるヒトｇａｍｍａ鎖のＩＧＨＶ４－３０－４＊０２とＩＧＨＪ１＊０１が、Ｒ７４のフレームワーク領域に対して高い同一性を有することから、それぞれ、軽鎖と重鎖のアクセプターとして選択された。アクセプター上に移入すべきドナー残基は、Ｑｕｅｅｎ　ｅｔ　ａｌ．（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　８６，１００２９－１００３３（１９８９））によって与えられる基準などを参考に三次元モデルを分析することで選択された。 4) -1-2 Humanization Design Method of R74 R74 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-1 0033 (1989)). KABAT et al. Consensus sequences of human kappa chain subgroups 3 and 4 as defined in (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) and IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION Since IGHV4-30-4 * 02 and IGHJ1 * 01 of human gamma chain defined in SYSTEM (registered trademark) have high identity to the framework region of R74, light chain and heavy chain respectively. Was selected as the acceptor of Donor residues to be transferred onto the acceptor are described by Queen et al. It was selected by analyzing the three-dimensional model with reference to the criteria given by (Proc. Natl. Acad. Sci. USA 86, 10029-1 0033 (1989)).
　４）－２　Ｒ７４重鎖のヒト化
　設計された３種のヒト化された重鎖をｈＲ７４＿Ｈ１、ｈＲ７４＿Ｈ２及びｈＲ７４＿Ｈ１２と命名した。実施例５で示されるヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４の重鎖であるｃＲ７４＿Ｈ、ヒト化重鎖ｈＲ７４＿Ｈ１、ｈＲ７４＿Ｈ２及びｈＲ７４＿Ｈ１２のアミノ酸配列の比較を図４に示す。ｈＲ７４＿Ｈ１、ｈＲ７４＿Ｈ２及びｈＲ７４＿Ｈ１２の配列において「・」はｃＲ７４＿Ｈと同一のアミノ酸残基を示し、アミノ酸残基が記載されている箇所は置換されたアミノ酸残基を示す。ｈＲ７４＿Ｈ１の重鎖全長アミノ酸配列を配列番号１６に記載する。配列番号１６のアミノ酸配列をコードするヌクレオチド配列を配列番号１５に記載する。ｈＲ７４＿Ｈ２の重鎖全長アミノ酸配列を配列番号１８に記載する。配列番号１８のアミノ酸配列をコードするヌクレオチド配列を配列番号１７に記載する。ｈＲ７４＿Ｈ１２の重鎖全長アミノ酸配列を配列番号２０に記載する。配列番号２０のアミノ酸配列をコードするヌクレオチド配列を配列番号１９に記載する。ｈＲ７４＿Ｈ１及びｈＲ７４＿Ｈ２においてはＡｂｍの定義による各ＣＤＲのアミノ酸配列はＲ７４と同一であった。一方、Ａｂｍの定義によるｈＲ７４＿Ｈ１２のＣＤＲのアミノ酸配列はＣＤＲＨ１、ＣＤＲＨ３、ＣＤＲＬ１、ＣＤＲＬ２及びＣＤＲＬ３はＲ７４と同一であったが、ＣＤＲＨ２のアミノ酸配列は異なっていた。Ａｂｍの定義によるｈＲ７４＿Ｈ１２のＣＤＲＨ２のアミノ酸配列を配列番号２１に示す。ヒト化抗体ｈＲ７４＿Ｈ１のヌクレオチド配列及びアミノ酸配列を図５に示す。ｈＲ７４＿Ｈ２のヌクレオチド配列及びアミノ酸配列を図６に示す。ｈＲ７４＿Ｈ１２のヌクレオチド配列及びアミノ酸配列並びにＣＤＲＨ２のアミノ酸配列を図７に示す。 4) -2 Humanization of R74 Heavy Chain The three humanized heavy chains designed were named hR74_H1, hR74_H2 and hR74_H12. A comparison of the amino acid sequences of the heavy chain cR74_H, humanized heavy chains hR74_H1, hR74_H2 and hR74_H12 of the human chimerized anti-human PC antibody cR74 shown in Example 5 is shown in FIG. In the sequences of hR74_H1, hR74_H2 and hR74_H12, “•” indicates the same amino acid residue as cR74_H, and the place where the amino acid residue is described indicates the substituted amino acid residue. The heavy chain full-length amino acid sequence of hR74_H1 is set forth in SEQ ID NO: 16. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is set forth in SEQ ID NO: 15. The heavy chain full-length amino acid sequence of hR74_H2 is set forth in SEQ ID NO: 18. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18 is set forth in SEQ ID NO: 17. The heavy chain full-length amino acid sequence of hR74_H12 is set forth in SEQ ID NO: 20. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 20 is set forth in SEQ ID NO: 19. In hR74_H1 and hR74_H2, the amino acid sequence of each CDR as defined by Abm was identical to R74. On the other hand, the amino acid sequences of the CDRs of hR74_H12 according to the Abm definition were identical to R74 in CDRH1, CDRH3, CDRL1, CDRL2 and CDRL3, but the amino acid sequences of CDRH2 were different. The amino acid sequence of CDRH2 of hR74_H12 according to the Abm definition is shown in SEQ ID NO: 21. The nucleotide and amino acid sequences of the humanized antibody hR74_H1 are shown in FIG. The nucleotide and amino acid sequences of hR74_H2 are shown in FIG. The nucleotide and amino acid sequences of hR74_H12 and the amino acid sequence of CDRH2 are shown in FIG.
　４）－３　Ｒ７４軽鎖のヒト化
　設計された３種の軽鎖をｈＲ７４＿Ｌ１、ｈＲ７４＿Ｌ２及びｈＲ７４＿Ｌ４と命名した。実施例５で示されるヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４の軽鎖であるヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４＿Ｌ、ヒト化抗体ｈＲ７４＿Ｌ１、ｈＲ７４＿Ｌ２及びｈＲ７４＿Ｌ４のアミノ酸配列の比較を図８に示す。ｈＲ７４＿Ｌ１、ｈＲ７４＿Ｌ２及びｈＲ７４＿Ｌ４の配列において、「・」はｃＲ７４＿Ｌと同一のアミノ酸残基を示し、アミノ酸残基が記載されている箇所は置換されたアミノ酸残基を示し、「－」は対応するアミノ酸残基が欠落している箇所を示す。 4) -3 Humanization of R74 Light Chain The three designed light chains were named hR74_L1, hR74_L2 and hR74_L4. A comparison of the amino acid sequences of human chimeric anti-human PC antibody cR74_L, which is the light chain of human chimeric anti-human PC antibody cR74 shown in Example 5, and humanized antibodies hR74_L1, hR74_L2 and hR74_L4 is shown in FIG. In the sequences of hR74_L1, hR74_L2 and hR74_L4, "." indicates the same amino acid residue as cR74_L, the position where the amino acid residue is described indicates the substituted amino acid residue, and "-" indicates the corresponding amino acid residue Indicates where the group is missing.
　ｈＲ７４＿Ｌ１の軽鎖全長アミノ酸配列を配列番号２２に記載する。配列番号２３のアミノ酸配列をコードするヌクレオチド配列を配列番号２２に記載する。ｈＲ７４＿Ｌ２の軽鎖全長アミノ酸配列を配列番号２５に記載する。配列番号２５のアミノ酸配列をコードするヌクレオチド配列は配列番号２４に記載する。ｈＲ７４＿Ｌ４の軽鎖全長アミノ酸配列を配列番号２７に記載する。配列番号２７のアミノ酸配列をコードするヌクレオチド配列は配列番号２６に記載する。ｈＲ７４＿Ｌ１、ｈＲ７４＿Ｌ２及びＬ４においてはＡｂｍの定義による各ＣＤＲのアミノ酸配列はＲ７４と同一であった。ヒト化抗体ｈＲ７４＿Ｌ１のヌクレオチド配列及びアミノ酸配列を図９に示す。ｈＲ７４＿Ｌ２のヌクレオチド配列及びアミノ酸配列を図１０に示す。ｈＲ７４＿Ｌ４のヌクレオチド配列及びアミノ酸配列を図１１に示す。 The light chain full-length amino acid sequence of hR74_L1 is set forth in SEQ ID NO: 22. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 23 is set forth in SEQ ID NO: 22. The light chain full-length amino acid sequence of hR74_L2 is set forth in SEQ ID NO: 25. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 25 is set forth in SEQ ID NO: 24. The light chain full-length amino acid sequence of hR74_L4 is set forth in SEQ ID NO: 27. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 27 is set forth in SEQ ID NO: 26. In hR74_L1, hR74_L2 and L4, the amino acid sequence of each CDR as defined by Abm was identical to R74. The nucleotide and amino acid sequences of humanized antibody hR74_L1 are shown in FIG. The nucleotide and amino acid sequences of hR74_L2 are shown in FIG. The nucleotide and amino acid sequences of hR74_L4 are shown in FIG.
　［実施例５］ヒトキメラ化抗ヒトＰＣ抗体の作製
　５）－１　ヒトキメラ化Ｒ７４軽鎖ｃＲ７４＿Ｌ発現ベクターの構築
　プラスミドｐｃＤＮＡ３．３－ＴＯＰＯ／ＬａｃＺ（Ｉｎｖｉｔｒｏｇｅｎ社）を制限酵素ＸｂａＩ及びＰｍｅＩで消化して得られる約５．４ｋｂのフラグメントと、配列番号２８に示すヒト軽鎖シグナル配列及びヒトκ鎖定常領域をコードするＤＮＡ配列を含むＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて結合して、ｐｃＤＮＡ３．３／ＬＫを作製した。 [Example 5] Preparation of human chimeric anti-human PC antibody 5) -1 Construction of human chimeric R74 light chain cR74_L expression vector Obtained by digesting plasmid pcDNA3.3-TOPO / LacZ (Invitrogen) with restriction enzymes XbaI and PmeI The fragment of about 5.4 kb and a DNA fragment containing a DNA sequence encoding a human light chain signal sequence shown in SEQ ID NO: 28 and a human κ chain constant region are linked using In-Fusion HD PCR cloning kit (Clontech) Then, pcDNA3.3 / LK was prepared.
　ｐｃＤＮＡ３．３／ＬＫからネオマイシン発現ユニットを除去することによりｐＣＭＡ－ＬＫを構築した。 PCMA-LK was constructed by removing the neomycin expression unit from pcDNA3.3 / LK.
　実施例３）－３で得られたＲ７４軽鎖の可変領域をコードするｃＤＮＡをテンプレートとして、Ｉｎ－ｆｕｓｉｏｎクローニング用に設計したプライマーでＰＣＲを行うことにより軽鎖の可変領域をコードするｃＤＮＡを含むＤＮＡ断片を増幅した。ｐＣＭＡ－ＬＫを制限酵素ＢｓｉＷＩで切断した箇所に、Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて、増幅したＤＮＡ断片を挿入することによりｃＲ７４＿Ｌ発現ベクターを構築した。ｃＲ７４＿Ｌのヌクレオチド配列及び該軽鎖のアミノ酸配列を、配列番号２９及び配列番号３０にそれぞれ示す。 Example 3) Using the cDNA encoding the variable region of R74 light chain obtained in -3 as a template, PCR is carried out using a primer designed for in-fusion cloning, thereby including the cDNA encoding the light chain variable region The DNA fragment was amplified. The cR74_L expression vector was constructed by inserting the amplified DNA fragment at the site where pCMA-LK was cleaved with restriction enzyme BsiWI using In-Fusion HD PCR cloning kit (Clontech). The nucleotide sequence of cR74_L and the amino acid sequence of the light chain are shown in SEQ ID NO: 29 and SEQ ID NO: 30, respectively.
　５）－２　ヒトキメラ化Ｒ７４重鎖ｃＲ７４＿Ｈ発現ベクターの構築
　ｐＣＭＡ－ＬＫをＸｂａＩ及びＰｍｅＩで消化してヒト軽鎖シグナル配列及びヒトκ鎖定常領域を取り除いたＤＮＡ断片と、配列番号３１で示されるヒト重鎖シグナル配列及びヒトＩｇＧ１－ＬＡＬＡタイプ定常領域をコードするＤＮＡ配列を含むＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて結合して、ｐＣＭＡ－Ｇ１－ＬＡＬＡを構築した。 5) -2 Construction of Human Chimeric R74 Heavy Chain cR74_H Expression Vector A DNA fragment from which pCMA-LK was digested with XbaI and PmeI to remove the human light chain signal sequence and human κ chain constant region, and the human shown by SEQ ID NO: 31 A DNA fragment containing a heavy chain signal sequence and a DNA sequence encoding human IgG1-LALA type constant region was ligated using In-Fusion HD PCR cloning kit (Clontech) to construct pCMA-G1-LALA.
　実施例３）－２で得られたＲ７４重鎖の可変領域をコードするｃＤＮＡをテンプレートとして、Ｉｎ－ｆｕｓｉｏｎクローニング用に設計したプライマーでＰＣＲを行うことにより重鎖の可変領域をコードするｃＤＮＡを含むＤＮＡ断片を増幅した。ｐＣＭＡ－Ｇ１－ＬＡＬＡを制限酵素ＢｌｐＩで切断した箇所に、Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて、増幅したＤＮＡ断片を挿入することによりｃＲ７４＿Ｈ発現ベクターを構築した。ｃＲ７４＿Ｈのヌクレオチド配列及び該重鎖のアミノ酸配列を、配列番号３２及び配列番号３３にそれぞれ示す。 Using the cDNA encoding the variable region of R74 heavy chain obtained in Example 3) -2 as a template, PCR is carried out using a primer designed for In-fusion cloning, thereby including the cDNA encoding the heavy chain variable region The DNA fragment was amplified. The cR74_H expression vector was constructed by inserting the amplified DNA fragment into a site where pCMA-G1-LALA was cleaved with restriction enzyme BlpI using In-Fusion HD PCR cloning kit (Clontech). The nucleotide sequence of cR74_H and the amino acid sequence of the heavy chain are shown in SEQ ID NO: 32 and SEQ ID NO: 33, respectively.
　５）－３　ヒトキメラ化抗ヒトＰＣ抗体の調製
　５）－３－１　ヒトキメラ化抗ヒトＰＣ抗体の生産
　ＦｒｅｅＳｔｙｌｅ　２９３Ｆ細胞（Ｉｎｖｉｔｒｏｇｅｎ社）はマニュアルに従い、継代、培養をおこなった。対数増殖期の１．２×１０９個のＦｒｅｅＳｔｙｌｅ　２９３Ｆ細胞（Ｉｎｖｉｔｒｏｇｅｎ社）を３Ｌ　Ｆｅｒｎｂａｃｈ　Ｅｒｌｅｎｍｅｙｅｒ　Ｆｌａｓｋ（ＣＯＲＮＩＮＧ社）に播種し、ＦｒｅｅＳｔｙｌｅ２９３　ｅｘｐｒｅｓｓｉｏｎ　ｍｅｄｉｕｍ　（Ｉｎｖｉｔｒｏｇｅｎ社）で希釈して２．０×１０６細胞／ｍＬに調製した。４０　ｍＬのＯｐｔｉ－Ｐｒｏ　ＳＦＭ培地（Ｉｎｖｉｔｒｏｇｅｎ社）に０．２４　ｍｇの重鎖発現ベクターと０．３６　ｍｇの軽鎖発現ベクターと１．８　ｍｇのＰｏｌｙｅｔｈｙｌｅｎｅｉｍｉｎｅ（Ｐｏｌｙｓｃｉｅｎｃｅ　＃２４７６５）を加えて穏やかに攪拌し、さらに５分間放置した後にＦｒｅｅＳｔｙｌｅ　２９３Ｆ細胞に添加した。３７℃、８％ＣＯ２インキュベーターで４時間、９０　ｒｐｍで振とう培養後に６００　ｍＬのＥＸ－ＣＥＬＬ　ＶＰＲＯ培地（ＳＡＦＣ　Ｂｉｏｓｃｉｅｎｃｅｓ社）、１８　ｍＬのＧｌｕｔａＭＡＸ　Ｉ（ＧＩＢＣＯ社）、及び３０　ｍＬのＹｅａｓｔｏｌａｔｅ　Ｕｌｔｒａｆｉｌｔｒａｔｅ（ＧＩＢＣＯ社）を添加し、３７℃、８％ＣＯ２インキュベーターで７日間、９０　ｒｐｍで振とう培養して得られた培養上清をＤｉｓｐｏｓａｂｌｅ　Ｃａｐｓｕｌｅ　Ｆｉｌｔｅｒ　（Ａｄｖａｎｔｅｃ　＃ＣＣＳ－０４５－Ｅ１Ｈ）でろ過した。 5) -3 Preparation of Human Chimerized Anti-Human PC Antibody 5) -3-1 Production of Human Chimerized Anti-Human PC Antibody FreeStyle 293F cells (Invitrogen) were passaged and cultured according to the manual. 1.2 × 10 9 FreeStyle 293F cells (Invitrogen) in logarithmic growth phase are seeded in 3 L Fernbach Erlenmeyer Flask (CORNING) and diluted with FreeStyle 293 expression medium (Invitrogen) to 2.0 × 10 6 cells It was adjusted to / mL. In 40 mL of Opti-Pro SFM medium (Invitrogen), 0.24 mg of heavy chain expression vector, 0.36 mg of light chain expression vector and 1.8 mg of Polyethyleneimine (Polyscience # 24765) were added and gently stirred And left for another 5 minutes before adding to FreeStyle 293F cells. After shaking culture at 37 ° C., 8% CO 2 incubator for 4 hours at 90 rpm, 600 mL of EX-CELL VPRO medium (SAFC Biosciences), 18 mL of GlutaMAX I (GIBCO), and 30 mL of Yeastolate Ultrafiltrate ( GIBCO) was added, and the culture supernatant obtained by shaking culture at 90 rpm for 7 days at 37 ° C. in an 8% CO 2 incubator was filtered through a Disposable Capsule Filter (Advantec # CCS-045-E1H).
　取得されたヒトキメラ化抗ヒトＰＣ抗体を「ｃＲ７４」と命名した。 The obtained human chimerized anti-human PC antibody was named "cR74".
　５）－３－２　ヒトキメラ化抗ヒトＰＣ抗体の精製
　実施例５）－３－１で得られた培養上清から抗体をｒＰｒｏｔｅｉｎ　Ａアフィニティークロマトグラフィーの１段階工程で精製した。培養上清をＰＢＳで平衡化したＭａｂＳｅｌｅｃｔＳｕＲｅが充填されたカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製）にアプライしたのちに、カラム容量の２倍以上のＰＢＳでカラムを洗浄した。次に２　Ｍ　アルギニン塩酸塩溶液（ｐＨ４．０）で溶出し、抗体の含まれる画分を集めた。その画分を透析（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ社、Ｓｌｉｄｅ－Ａ－Ｌｙｚｅｒ　Ｄｉａｌｙｓｉｓ　Ｃａｓｓｅｔｔｅ）によりＰＢＳ（－）へのバッファー置換を行った。Ｃｅｎｔｒｉｆｕｇａｌ　ＵＦ　Ｆｉｌｔｅｒ　Ｄｅｖｉｃｅ　ＶＩＶＡＳＰＩＮ２０（分画分子量ＵＦ１０Ｋ，Ｓａｒｔｏｒｉｕｓ社）で抗体を濃縮し、ＩｇＧ濃度を１　ｍｇ／ｍＬ以上に調製した。最後にＭｉｎｉｓａｒｔ－Ｐｌｕｓ　ｆｉｌｔｅｒ（Ｓａｒｔｏｒｉｕｓ社）でろ過し、精製サンプルとした。 5) -3-2 Purification of Human Chimerized Anti-Human PC Antibody The culture supernatant obtained in Example 5-3 was purified by a one-step process of rProtein A affinity chromatography. The culture supernatant was applied to a column (manufactured by GE Healthcare Bioscience) packed with PBS equilibrated with MabSelectSuRe, and then the column was washed with 2 column volumes or more of PBS. It was then eluted with 2 M arginine hydrochloride solution (pH 4.0), and the fractions containing the antibody were collected. The fraction was subjected to buffer substitution to PBS (-) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractional molecular weight UF10K, Sartorius) to adjust the IgG concentration to 1 mg / mL or more. Finally, the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
　［実施例６］ヒトキメラ化抗ヒトＰＣ抗体のｉｎ　ｖｉｔｒｏ活性
　６）－１　血友病患者由来血漿を用いたヒトキメラ化抗ヒトＰＣ抗体によるＴＧ回復作用
　ヒトキメラ化抗ヒトＰＣ抗体ｃＲ７４＿ＩｇＧ１－ＬＡＬＡの血友病Ａ患者由来血漿及び血友病Ｂ患者由来血漿を用いたＴＧ回復作用を元のヒト抗ＰＣラット抗体並びに陽性対照ポリクローナル抗体と比較検討した。方法は２）－２記載の方法に準じて実施した。
ｃＲ７４＿ＩｇＧ１－ＬＡＬＡは、血友病Ａ患者由来血漿で元のヒト抗ＰＣラット抗体（Ｒ７４）と同等以上のＴＧ回復効果を示した（図１４Ａ）。 [Example 6] In vitro activity of human chimerized anti-human PC antibody 6) -1 TG recovery action by human chimerized anti-human PC antibody using hemophiliac patient-derived plasma Friend of human chimerized anti-human PC antibody cR74_IgG1-LALA The TG recovery action using the plasma derived from the disease A patient and the plasma derived from the hemophilia B patient was compared with the original human anti-PC rat antibody and the positive control polyclonal antibody. The method was carried out according to the method described in 2) -2.
cR74_IgG1-LALA showed a TG recovery effect at least equivalent to that of the original human anti-PC rat antibody (R74) in plasma from hemophilia A patients (FIG. 14A).
　タイプＢ血友病患者由来血漿においても、血友病Ａ患者由来血漿とほぼ同様の結果を示した（図１４Ｂ）。 The type B hemophilia patient-derived plasma also showed almost the same results as the hemophilia A patient-derived plasma (FIG. 14B).
　［実施例７］ヒト化抗ヒトＰＣ抗体の作製
　７）－１ヒト化抗ヒトＰＣ抗体軽鎖ｈＲ７４＿Ｌ発現ベクターの構築
　７）－１－１　ｈＲ７４＿Ｌ１発現ベクターの構築
　配列番号１１に示すｈＲ７４＿Ｌ１のヌクレオチド配列のヌクレオチド番号３７乃至４１４に示されるＤＮＡ断片を合成した（ＧＥＮＥＡＲＴ社）。Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて、キメラ及びヒト化抗体軽鎖発現ベクターｐＣＭＡ－ＬＫを制限酵素ＢｓｉＷＩで切断した箇所に合成したＤＮＡ断片を挿入することによりｈＲ７４＿Ｌ１発現ベクターを構築した。 [Example 7] Preparation of humanized anti-human PC antibody 7)-1 Construction of humanized anti-human PC antibody light chain hR74_L expression vector 7)-1-1 Construction of hR74_L1 expression vector Nucleotide sequence of hR74_L1 shown in SEQ ID NO: 11 The DNA fragments shown in nucleotide numbers 37 to 414 of SEQ ID NO: 1 were synthesized (GENEART). The hR74_L1 expression vector was constructed by inserting the synthesized DNA fragment into the site where the chimeric and humanized antibody light chain expression vector pCMA-LK was digested with restriction enzyme BsiWI using the In-Fusion HD PCR cloning kit (Clontech). did.
　７）－１－２　ｈＲ７４＿Ｌ２発現ベクターの構築
　配列番号２３に示すｈＲ７４＿Ｌ２のヌクレオチド配列のヌクレオチド番号３７乃至４１４に示されるＤＮＡ配列を含むＤＮＡ断片を合成した（ＧＥＮＥＡＲＴ社）。実施例７）－１－１と同様の方法でｈＲ７４＿Ｌ２発現ベクターを構築した。 7) -1-2 Construction of hR74_L2 Expression Vector A DNA fragment containing the DNA sequence shown in nucleotide numbers 37 to 414 of the nucleotide sequence of hR74_L2 shown in SEQ ID NO: 23 was synthesized (GENEART). Example 7 hR74_L2 expression vector was constructed in the same manner as in -1-1.
　７）－１－３　ｈＲ７４＿Ｌ４発現ベクターの構築
　配列番号２５に示すｈＲ７４＿Ｌ４のヌクレオチド配列のヌクレオチド番号３７乃至４１４に示されるＤＮＡ断片を合成した（ＧＥＮＥＡＲＴ社）。実施例７）－１－１と同様の方法でｈＲ７４＿Ｌ４発現ベクターを構築した。 7) -1-3 Construction of hR74_L4 Expression Vector The DNA fragment shown in nucleotide numbers 37 to 414 of the nucleotide sequence of hR74_L4 shown in SEQ ID NO: 25 was synthesized (GENEART). Example 7 hR74_L4 expression vector was constructed in the same manner as in -1-1.
　７）－２　ヒト化抗ヒトＰＣ抗体重鎖ｈＲ７４＿Ｈ発現ベクターの構築
　７）－２－１　ｈＲ７４＿Ｈ１発現ベクターの構築
　配列番号１５に示すｈＲ７４＿Ｈ１のヌクレオチド配列のヌクレオチド番号３６乃至４２８に示されるＤＮＡ断片を合成した（ＧＥＮＥＡＲＴ社）。Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて、ｐＣＭＡ－Ｇ１－ＬＡＬＡを制限酵素ＢｌｐＩで切断した箇所に合成したＤＮＡ断片を挿入することによりｈＲ７４＿Ｈ１発現ベクターを構築した。 7) -2 Construction of Humanized Anti-Human PC Antibody Heavy Chain hR74_H Expression Vector 7) Construction of hR74_H1 Expression Vector A DNA fragment shown in nucleotide numbers 36 to 428 of the nucleotide sequence of hR74_H1 shown in SEQ ID NO: 15 is synthesized Yes (GENEART). Using the In-Fusion HD PCR cloning kit (Clontech), the hR74_H1 expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G1-LALA was digested with restriction enzyme BlpI.
　７）－２－２　ｈＲ７４＿Ｈ２発現ベクターの構築
　配列番号１７に示すｈＲ７４＿Ｈ２のヌクレオチド配列のヌクレオチド番号３６乃至４２８に示されるＤＮＡ断片を合成した（ＧＥＮＥＡＲＴ社）。実施例７）－２－１と同様の方法でｈＲ７４＿Ｈ２発現ベクターを構築した。 7) Construction of -2-2 hR74_H2 Expression Vector The DNA fragment shown in nucleotide numbers 36 to 428 of the nucleotide sequence of hR74_H2 shown in SEQ ID NO: 17 was synthesized (GENEART). Example 7 hR74_H2 expression vector was constructed in the same manner as -2-1.
　７）－２－３　ｈＲ７４＿Ｈ１２発現ベクターの構築
　ｈＲ７４＿Ｈ１をテンプレートとして、ＫＯＤ　－Ｐｌｕｓ－　Ｍｕｔａｇｅｎｅｓｉｓ　Ｋｉｔ（ＴＯＹＯＢＯ社）を用いて変異を導入することによりｈＲ７４＿Ｈ１２発現ベクターを構築した。ｈＲ７４＿Ｈ１２のヌクレオチド配列を配列番号１９に示す。 7) -2-3 Construction of hR74_H12 Expression Vector A hR74_H12 expression vector was constructed by introducing a mutation using KOD-Plus-Mutagenesis Kit (TOYOBO) using hR74_H1 as a template. The nucleotide sequence of hR74_H12 is shown in SEQ ID NO: 19.
　７）－３　ヒト化抗ヒトＰＣ抗体ｈＲ７４の調製
　７）－３－１　ヒト化抗体の生産
　実施例５）－３－１と同様の方法で生産した。ｈＲ７４＿Ｈ１発現ベクターとｈＲ７４＿Ｌ１発現ベクターの組み合わせで取得したヒト化抗体を「ｈＲ７４＿Ｈ１／Ｌ１」と命名した。ｈＲ７４＿Ｈ１２発現ベクターとｈＲ７４＿Ｌ１発現ベクターの組み合わせで取得したヒト化抗体を「ｈＲ７４＿Ｈ１２／Ｌ１」と命名した。ｈＲ７４＿Ｈ１２発現ベクターとｈＲ７４＿Ｌ４発現ベクターの組み合わせで取得したヒト化抗体を「ｈＲ７４＿Ｈ１２／Ｌ４」と命名した。 7) -3 Preparation of Humanized Anti-Human PC Antibody hR74 7) -3-1 Production of Humanized Antibody The antibody was produced in the same manner as in Example 5) -3-1. The humanized antibody obtained by the combination of hR74_H1 expression vector and hR74_L1 expression vector was designated as “hR74_H1 / L1”. The humanized antibody obtained by the combination of hR74_H12 expression vector and hR74_L1 expression vector was designated as "hR74_H12 / L1". The humanized antibody obtained by the combination of hR74_H12 expression vector and hR74_L4 expression vector was named "hR74_H12 / L4".
　７）－３－２　ヒト化抗体の精製
　実施例７）－３－１で得られた培養上清から抗体をｒＰｒｏｔｅｉｎ　Ａアフィニティークロマトグラフィーとセラミックハイドロキシアパタイトの２段階工程で精製した。培養上清をＰＢＳで平衡化したＭａｂＳｅｌｅｃｔＳｕＲｅが充填されたカラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏｓｃｉｅｎｃｅ社製）にアプライした後に、カラム容量の２倍以上のＰＢＳでカラムを洗浄した。次に２　Ｍアルギニン塩酸塩溶液（ｐＨ４．０）で抗体を溶出した。抗体の含まれる画分を透析（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ社、Ｓｌｉｄｅ－Ａ－Ｌｙｚｅｒ　Ｄｉａｌｙｓｉｓ　Ｃａｓｓｅｔｔｅ）によりＰＢＳへのバッファー置換を行い、５　ｍＭリン酸ナトリウム／５０　ｍＭ　ＭＥＳ／ｐＨ７．０のバッファーで５倍希釈した後に、５　ｍＭ　ＮａＰｉ／５０　ｍＭ　ＭＥＳ／３０　ｍＭ　ＮａＣｌ／ｐＨ７．０のバッファーで平衡化したセラミックハイドロキシアパタイトカラム（日本バイオラッド、Ｂｉｏ－Ｓｃａｌｅ　ＣＨＴ　Ｔｙｐｅ―１　Ｈｙｄｒｏｘｙａｐａｔｉｔｅ　Ｃｏｌｕｍｎ）にアプライした。塩化ナトリウムによる直線的濃度勾配溶出を実施し、抗体の含まれる画分を集めた。その画分を透析（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ社、Ｓｌｉｄｅ－Ａ－Ｌｙｚｅｒ　Ｄｉａｌｙｓｉｓ　Ｃａｓｓｅｔｔｅ）によりＨＢＳｏｒ（２５　ｍＭ　ヒスチジン／５％　ソルビトール、ｐＨ６．０）へのバッファー置換を行った。Ｃｅｎｔｒｉｆｕｇａｌ　ＵＦ　Ｆｉｌｔｅｒ　Ｄｅｖｉｃｅ　ＶＩＶＡＳＰＩＮ２０（分画分子量ＵＦ１０Ｋ，Ｓａｒｔｏｒｉｕｓ社）にて抗体を濃縮し、ＩｇＧ濃度を４０　ｍｇ／ｍＬ以上に調製した。最後にＭｉｎｉｓａｒｔ－Ｐｌｕｓ　ｆｉｌｔｅｒ（Ｓａｒｔｏｒｉｕｓ社）でろ過し、精製サンプルとした。 7) -3-2 Purification of Humanized Antibody The antibody was purified from the culture supernatant obtained in Example 7) by a two-step process of rProtein A affinity chromatography and ceramic hydroxyapatite. The culture supernatant was applied to a column (manufactured by GE Healthcare Bioscience) packed with PBS equilibrated MabSelectSuRe, and then the column was washed with 2 column volumes or more of PBS. The antibody was then eluted with 2 M arginine hydrochloride solution (pH 4.0). The fractions containing the antibody were subjected to buffer substitution into PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) and diluted 5-fold with a buffer of 5 mM sodium phosphate / 50 mM MES / pH 7.0 Later, it was applied to a ceramic hydroxyapatite column (Bio-Scale CHT Type-1 Hydroxyapatite Column, Japan Bio-Rad) equilibrated with a buffer of 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0. Linear gradient elution with sodium chloride was performed to collect fractions containing the antibody. The fraction was subjected to buffer substitution to HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated with Centrifugal UF Filter Device VIVASPIN 20 (fractional molecular weight UF10K, Sartorius) to adjust the IgG concentration to 40 mg / mL or more. Finally, the resultant was filtered with Minisart-Plus filter (Sartorius) to obtain a purified sample.
　［実施例８］　ＢＡＹ１８９６５０２の作製
　ＢＡＹ１８９６５０２はＷＯ２０１４／０８５５２７　Ａ１並びにＷＯ２０１５／１７９４３５　Ａ１を参照して作製した。 Example 8 Preparation of BAY 1896502 BAY 1896502 was prepared with reference to WO 2014/085527 A1 and WO 2015/179435 A1.
　８）－１　ヒト化抗ヒトａＰＣ抗体　ＢＡＹ１８９６５０２軽鎖発現ベクターの構築（村上、御座）　
　配列番号３４に示すＢＡＹ１８９６５０２軽鎖のヌクレオチド配列のヌクレオチド番号３７乃至４１４に示されるＤＮＡ断片を合成した（ＧＥＮＥＡＲＴ社）。実施例７）－１－１と同様の方法でＢＡＹ１８９６５０２軽鎖の発現ベクターを構築した。ＢＡＹ１８９６５０２軽鎖のアミノ酸配列を配列番号３５に示す。 8) -1 Construction of a humanized anti-human aPC antibody BAY 1896502 light chain expression vector (Murakami, throne)
The DNA fragment shown in nucleotide numbers 37 to 414 of the nucleotide sequence of BAY 1896502 light chain shown in SEQ ID NO: 34 was synthesized (GENEART). Example 7) An expression vector of BAY 1896502 light chain was constructed in the same manner as in-1-1. The amino acid sequence of BAY 1896502 light chain is shown in SEQ ID NO: 35.
　８）－２　ヒト化抗ヒトａＰＣ抗体　ＢＡＹ１８９６５０２重鎖発現ベクターの構築
　ｐＣＭＡ－ＬＫをＸｂａＩ及びＰｍｅＩで消化して軽鎖シグナル配列及びヒトκ鎖定常領域を取り除いたＤＮＡ断片と、配列番号３６で示されるヒト重鎖シグナル配列及びヒトＩｇＧ２定常領域のアミノ酸をコードするＤＮＡ配列を含むＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて結合して、ｐＣＭＡ－Ｇ２を構築した。 8) -2 Construction of humanized anti-human aPC antibody BAY1896502 heavy chain expression vector A DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the light chain signal sequence and human κ chain constant region, as shown by SEQ ID NO: 36 A DNA fragment containing the human heavy chain signal sequence and the DNA sequence encoding the amino acid of human IgG2 constant region was ligated using In-Fusion HD PCR cloning kit (Clontech) to construct pCMA-G2.
　配列番号３７に示すＢＡＹ１８９６５０２重鎖のヌクレオチド配列のヌクレオチド番号３６乃至４２８に示されるＤＮＡ断片を合成した（ＧＥＮＥＡＲＴ社）。Ｉｎ－Ｆｕｓｉｏｎ　ＨＤ　ＰＣＲクローニングキット（Ｃｌｏｎｔｅｃｈ社）を用いて、ｐＣＭＡ－Ｇ２を制限酵素ＢｌｐＩで切断した箇所に合成したＤＮＡ断片を挿入することによりＢＡＹ１８９６５０２重鎖発現ベクターを構築した。ＢＡＹ１８９６５０２重鎖のアミノ酸配列を配列番号３８に示す。 The DNA fragment shown in nucleotide numbers 36 to 428 of the nucleotide sequence of BAY 1896502 heavy chain shown in SEQ ID NO: 37 was synthesized (GENEART). Using the In-Fusion HD PCR cloning kit (Clontech), a BAY 1896502 heavy chain expression vector was constructed by inserting the synthesized DNA fragment into the site where pCMA-G2 was digested with restriction enzyme BlpI. The amino acid sequence of BAY 1896502 heavy chain is shown in SEQ ID NO: 38.
　８）－３　ヒト化抗ヒトａＰＣ抗体　ＢＡＹ１８９６５０２の調製
８）－３－１　ヒト化抗ヒトａＰＣ抗体　ＢＡＹ１８９６５０２の生産
　実施例５）－３－１と同様の方法で生産した。 8) -3 Preparation of humanized anti-human aPC antibody BAY1896502 8) -3-1 Production of humanized anti-human aPC antibody BAY1896502 It was produced in the same manner as in Example 5) -3-1.
　８）－３－２　ヒト化抗ヒトａＰＣ抗体　ＢＡＹ１８９６５０２の精製
　実施例５）－３－２と同様の方法で精製した。 8) -3-2 Purification of humanized anti-human aPC antibody BAY 1896502 The purification was carried out in the same manner as in Example 5) -3-2.
　［実施例９］ヒト化抗ヒトＰＣ抗体のｉｎ　ｖｉｔｒｏ活性
　９）－１　ＳＰＲによるヒト化抗ヒトＰＣ抗体の抗原結合活性
　抗体と抗原との結合は、Ｂｉａｃｏｒｅ　４０００（ＧＥヘルスケアバイオサイエンス　（株））を使用し、固定化した抗ヒトＩｇＧ　Ｆｃ抗体に抗体をリガンドとして捕捉（キャプチャー）し、抗原をアナライトとして測定するキャプチャー法にて測定した。抗原には、ヒトＰＣ（Ｅｎｚｙｍｅ　Ｒｅｓｅａｒｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ）を使用した。抗ヒトＩｇＧ　Ｆｃ抗体（Ｈｕｍａｎ　Ａｎｔｉｂｏｄｙ　Ｃａｐｔｕｒｅ　Ｋｉｔ、ＧＥヘルスケアバイオサイエンス（株））は、センサーチップＣＭ５（ＧＥヘルスケアバイオサイエンス（株））へアミンカップリング法にて約２０００ＲＵ共有結合させた。リファレンスセルにも同様に固定化した。ランニングバッファーとしてＨＢＳ－ＥＰ＋（１０　ｍＭ　ＨＥＰＥＳ　ｐＨ７．４、０．１５　Ｍ　ＮａＣｌ、３　ｍＭ　ＥＤＴＡ、０．０５％Ｓｕｒｆａｃｔａｎｔ　Ｐ２０）を用いた。抗ヒトＩｇＧ　Ｆｃ抗体を固定化したチップ上に、抗体を約１分間添加しリガンドとして捕捉した後、０．１　ｎＭ～１００　ｎＭの抗原を流速３０　μＬ／分で３００秒間添加し、続いてランニングバッファーを６００秒間添加し、抗原の結合解離をモニターした。再生溶液として、３　Ｍ　ＭｇＣｌ２を流速　１０　μＬ／分で３０秒間を２回添加した。解析には、１：１結合の理論式を用いた。その結果、ｈＲ７４＿Ｈ１／Ｌ１では一桁ｎＭのＫＤ値を示し、ほかの２サンプルでは約１０倍程度の高いＫＤ値を示した（表１）。 Example 9 In Vitro Activity of Humanized Anti-Human PC Antibody 9) -1 Antigen Binding Activity of Humanized Anti-Human PC Antibody by SPR The binding of the antibody to the antigen was determined using Biacore 4000 (GE Healthcare Biosciences Co., Ltd.) ) Was used to capture (capture) the antibody as a ligand to the immobilized anti-human IgG Fc antibody, and the antigen was measured by a capture method that measures as an analyte. Human PC (Enzyme Research Laboratories) was used as an antigen. Anti-human IgG Fc antibody (Human Antibody Capture Kit, GE Healthcare Biosciences, Inc.) was covalently coupled to the sensor chip CM5 (GE Healthcare Biosciences, Inc.) by amine coupling method for about 2000 RU. It was fixed to the reference cell as well. HBS-EP + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used as a running buffer. After adding the antibody for about 1 minute and capturing it as a ligand on a chip on which anti-human IgG Fc antibody is immobilized, 0.1 nM to 100 nM of antigen is added for 300 seconds at a flow rate of 30 μL / minute, and then running Buffer was added for 600 seconds to monitor binding dissociation of the antigen. As a regeneration solution, 3 M MgCl 2 was added twice for 30 seconds at a flow rate of 10 μL / min. The theoretical formula of 1: 1 binding was used for the analysis. As a result, hR74_H1 / L1 showed a KD value of one digit nM, and the other two samples showed a KD value as high as about 10 times (Table 1).
Figure JPOXMLDOC01-appb-T000001
  
Figure JPOXMLDOC01-appb-T000001
  
　９）－２　ヒト化抗ＰＣ抗体の粘性測定
　サンプルはＶｉｖａｐｏｒｅ　５（Ｓａｒｔｏｒｉｕｓ社）を使用して濃縮し、２５　ｍＭヒスチジン、５％ソルビトール、ｐＨ６．０のバッファーにて１５０　ｍｇ／ｍＬに調製し、ｍ－ＶＲＯＣ（ＲｈｅｏＳｅｎｓｅ）を用いて２５℃、流速７５　μＬ／ｍｉｎの条件で測定した。その結果、ｈＲ７４＿Ｈ１／Ｌ１では約４０　ｃＰ、またｈＲ７４＿Ｈ１２／Ｌ１及びｈＲ７４＿Ｈ１２／Ｌ４ではその約５０％の粘性を示した（表２）。 9) -2 Viscosity measurement of humanized anti-PC antibody The sample is concentrated using Vivapore 5 (Sartorius), adjusted to 150 mg / mL with 25 mM histidine, 5% sorbitol, pH 6.0 buffer, The measurement was performed using m-VROC (RheoSense) at 25 ° C. and a flow rate of 75 μL / min. As a result, the viscosity was about 40 cP for hR74_H1 / L1, and about 50% for hR74_H12 / L1 and hR74_H12 / L4 (Table 2).
Figure JPOXMLDOC01-appb-T000002
  
　
Figure JPOXMLDOC01-appb-T000002
  
　
９）－３
　９）－３－１　血友病患者由来血漿を用いたヒト化抗ヒトＰＣ抗体によるＴＧ回復作用
　ヒト化抗ヒトＰＣ抗体の血友病Ａ患者由来血漿を用いたＴＧ回復作用を評価した。方法は２）－２記載の方法に準じて実施した。ヒトＦＶＩＩＩ欠乏血漿７５　μＬ（１０．７　ｎＭ　ｒＴＭ（最終濃度１０　ｎＭ　ｉｎ　ｐｌａｓｍａ）に種々濃度のヒト化抗ヒトＰＣ抗体５　μＬ、３０　ｐＭ　ｒＴＦ　２０　μＬ（最終濃度５　ｐＭ）を加えて３７℃で１０分間インキュベーションした。次いでＦｌｕＣａ－ｋｉｔ　（２．５　ｍＭ　Ｚ－Ｇｌｙ－Ｇｌｙ－Ａｒｇ－ａｍｉｎｏｍｅｔｈｙｌｃｏｕｍａｒｉｎ，１００　ｍＭ　ＣａＣｌ２）を２０　μＬ（最終濃度Ｚ－Ｇｌｙ－Ｇｌｙ－Ａｒｇ－ａｍｉｎｏｍｅｔｈｙｌｃｏｕｍａｒｉｎ：４１７　μＭ，ＣａＣｌ２　１６．７　ｍＭ）加えて反応を開始した。蛍光度（ｅｘ．３９０　ｎｍ，　ｅｍ．４６０　ｎｍ）を２０秒ごとに４０分間測定した。ＴＧはｔｈｒｏｍｂｉｎｏｓｃｏｐｅ　ｓｏｆｔｗａｒｅ（Ｔｈｒｏｍｂｉｎｏｓｃｏｐｅ　ＢＶ）にて算出した。ｈＲ７４＿Ｈ１２／Ｌ１、ｈＲ７４＿Ｈ１２／Ｌ４、及びｈＲ７４＿Ｈ１２／Ｌ１のいずれの抗体も濃度依存的にＴＧを回復し、その程度もほぼ同等であった（図１５）。 9) -3
9) -3-1 TG recovery action by humanized anti-human PC antibody using hemophiliac patient-derived plasma The TG recovery action using hemophiliac A patient-derived plasma was evaluated using humanized anti-human PC antibody. The method was carried out according to the method described in 2) -2. 5 μl of human FVIII-deficient plasma (10.7 nM rTM (final concentration 10 nM in plasma) and 5 μl of various concentrations of humanized anti-human PC antibody and 20 μl of 30 pM rTF (final concentration 5 pM) at 37 ° C After incubation for 10 minutes, 20 μL of FluCa-kit (2.5 mM Z-Gly-Gly-Arg-aminomethylcoumarin, 100 mM CaCl 2 ) (final concentration Z-Gly-Gly-Arg-aminomethylcoumarin: 417 μM, CaCl 2) The reaction was started by adding 16.7 mM) The fluorescence (ex. 390 nm, em. 460 nm) was measured every 20 seconds for 40 minutes TG was added to thromnoscope software (Thrombinoscope BV) Calculated .hR74_H12 / L1, hR74_H12 / L4, and also the concentration-dependent manner to recover the TG any antibody of hR74_H12 / L1, the degree was also approximately equivalent (FIG. 15).
　９）－３－２　血友病患者由来血漿を用いたＢＡＹ１８９６５０２とのＴＧ回復作用との比較
　バイエル社特許記載のヒト化抗ヒトａＰＣ抗体ＢＡＹ１８９６５０２とヒト化抗ヒトＰＣ抗体（ｈＲ７４＿Ｈ１／Ｌ１）によるＴＧ回復作用の比較を行なった。ＢＡＹ１８９６５０２はａＰＣのみを阻害するのに対し、ｈＲ７４＿Ｈ１／Ｌ１はＰＣ並びにａＰＣの両方を阻害するという特徴の違いがある。 9) Comparison with TG recovery action with BAY 1896502 using plasma derived from hemophilia patients TG by TG with humanized anti-human aPC antibody BAY 1896502 described in Bayer AG and humanized anti-human PC antibody (hR74_H1 / L1) A comparison of recovery was made. While BAY 1896502 only inhibits aPC, hR74_H1 / L1 has a characteristic difference in that it inhibits both PC and aPC.
　方法は９）－３－１記載の方法に準じて実施した。 The method was carried out according to the method described in 9) -3-1.
　血友病Ａ患者由来血漿（図１６Ａ）並びに血友病Ｂ患者由来血漿（図１６Ｂ）を用いたＴＧ回復効果をｈＲ７４＿Ｈ１／Ｌ１とＢＡＹ１８９６５０２で比較した。両抗体ともに濃度依存的にＴＧを回復しが、いずれのタイプの血友病患者由来血漿においてもｈＲ７４＿Ｈ１／Ｌ１のＴＧ回復効果はＢＡＹ１８９６５０２のそれを明らかに上回った。 The TG recovery effect using hemophilia A patient-derived plasma (FIG. 16A) and hemophilia B patient-derived plasma (FIG. 16B) was compared between hR74_H1 / L1 and BAY 1896502. Although both antibodies recovered TG in a concentration-dependent manner, the TG recovery effect of hR74_H1 / L1 clearly exceeded that of BAY 1896502 in both types of hemophiliac patient-derived plasma.
　［実施例１０］ヒト化抗ヒトＰＣ抗体のｉｎ　ｖｉｖｏ活性
　一過性血友病Ａカニクイザル出血モデルにおけるｈＲ７４＿Ｈ１／Ｌ１の皮下出血抑制効果を検討した。 [Example 10] In vivo activity of humanized anti-human PC antibody The subcutaneous hemorrhage suppressing effect of hR74_H1 / L1 in a transient hemophilia A cynomolgus monkey hemorrhage model was examined.
　覚醒下にて表在静脈から抗ヒトＦＶＩＩＩ中和抗体（ＨＡＥＭＡＴＯＬＯＧＩＣ　ＴＥＣＨＮＯＬＯＧＩＥＳ，ヒツジポリクローナル抗体）単独（１８，０００　Ｂｅｔｈｅｓｄａ　Ｕｎｉｔ　（ＢＵ）／ｋｇ，ｉｖ）又はｈＲ７４＿Ｈ１／Ｌ１（３　ｏｒ　１．５　ｍｇ／ｋｇ，ｉｖ）と併用投与し、２時間後にイソフルラン（導入時２－３％、維持１．５－２．５％）麻酔下で、ＢＤ　Ｍｉｃｒｏｔａｉｎｅｒ　（カタログ番号３６６５９４，ブルー，１．５　ｍｍ幅刃，深さ２．０　ｍｍ）を用いて、大きな静脈を避け、２４ヶ所（各上腕に４ヶ所、各前腕に２ヶ所、各大腿の内側に４ヶ所、各腓腹部に２ヶ所）を創傷させた。創傷した日をＤａｙ０とした。鎮痛剤を投与後、麻酔から覚醒させた。投与用法・用量は、実験当日はレペタン（ブプレノルフィンとして）０．０１　ｍｇ／ｋｇ，ＳＣ及びメタカム（メロキシカムとして）０．２　ｍｇ／ｋｇ，ＳＣ、２４，４８，７２時間後はメタカム（メロキシカムとして）０．１　ｍｇ／ｋｇ，ＳＣとした。
Ｄａｙ１、２、３（ＦＶＩＩＩ中和抗体投与２４，４８，７２時間後）に皮下出血部位をデジカメで撮影し、後日解析ソフトにて面積を測定した。 Anti-human FVIII neutralizing antibody (HAEMATOLOGIC TECHNOLOGIES, sheep polyclonal antibody) alone (18,000 Bethesda Unit (BU) / kg, iv) alone or hR74_H1 / L1 (3 or 1.5 mg / kg) from the superficial vein under awakening , Iv) and after 2 hours isoflurane (2-3% when introduced, 1.5-2.5% maintenance) under anesthesia BD Microtainer (Catalog No. 366594, Blue, 1.5 mm wide blade, Using a depth of 2.0 mm, a large vein was avoided, and 24 locations (4 locations on each upper arm, 2 locations on each forearm, 4 locations on the inside of each thigh, 2 locations on each groin) . The day of wounding was designated as Day 0. After administration of the analgesic, he was awakened from anesthesia. Dosage regimen: lepetan (as buprenorphine) 0.01 mg / kg, SC and metacam (as meloxicam) 0.2 mg / kg, SC, 24, 48 and 72 hours later as metacam (as meloxicam) on the day of the experiment It was 0.1 mg / kg, SC.
The subcutaneous bleeding site was photographed with a digital camera on Day 1, 2 and 3 (24, 48 and 72 hours after administration of FVIII neutralizing antibody), and the area was measured the day after by analysis software.
　ＦＶＩＩＩ中和抗体単独静注群（Ｃｏｎｔｒｏｌ）では時間経過とともに皮下出血部位の面積が増加した（図１７）。一方、ｈＲ７４＿Ｈ１／Ｌ１投与群はいずれの用量においてもほとんど皮下出血が認められなかった。以上のことから、抗ＰＣ抗体（ｈＲ７４＿Ｈ１／Ｌ１）は血友病Ａの出血症状に対して止血効果を有することが示された。 In the FVIII neutralizing antibody alone intravenous administration group (Control), the area of the subcutaneous bleeding site increased with time (FIG. 17). On the other hand, in the hR74_H1 / L1 administration group, almost no subcutaneous bleeding was observed at any dose. From the above, it was shown that the anti-PC antibody (hR74_H1 / L1) has a hemostatic effect on hemorrhagic symptoms of hemophilia A.
　本発明のヒト化抗ＰＣ抗体は、公知の抗体より強力なトロンビン産生作用を有しており、血友病の予防及び／又は治療剤となり得る。 The humanized anti-PC antibody of the present invention has a stronger thrombin generation activity than known antibodies, and can be a preventive and / or therapeutic agent for hemophilia.
配列番号２：ペプチドタグ
配列番号３：配列Ｘ
配列番号４：配列Ｙ
配列番号５：Ｒ７４の重鎖の可変領域のヌクレオチド配列
配列番号６：Ｒ７４の重鎖の可変領域のアミノ酸配列
配列番号７：ＣＤＲＨ１
配列番号８：ＣＤＲＨ２
配列番号９：ＣＤＲＨ３
配列番号１０：Ｒ７４の軽鎖の可変領域のヌクレオチド配列
配列番号１１：Ｒ７４の軽鎖の可変領域のアミノ酸配列
配列番号１２：ＣＤＲＬ１
配列番号１３：ＣＤＲＬ２
配列番号１４：ＣＤＲＬ３
配列番号１５：ｈＲ７４＿Ｈ１をコードするヌクレオチド配列
配列番号１６：ｈＲ７４＿Ｈ１のアミノ酸配列
配列番号１７：ｈＲ７４＿Ｈ２をコードするヌクレオチド配列
配列番号１８：ｈＲ７４＿Ｈ２のアミノ酸配列
配列番号１９：ｈＲ７４＿Ｈ１２をコードするヌクレオチド配列
配列番号２０：ｈＲ７４＿Ｈ１２のアミノ酸配列
配列番号２１：ｈＲ７４＿Ｈ１２のＣＤＲＨ２
配列番号２２：ｈＲ７４＿Ｌ１をコードするヌクレオチド配列
配列番号２３：ｈＲ７４＿Ｌ１のアミノ酸配列
配列番号２４：ｈＲ７４＿Ｌ２をコードするヌクレオチド配列
配列番号２５：ｈＲ７４＿Ｌ２のアミノ酸配列
配列番号２６：ｈＲ７４＿Ｌ４をコードするヌクレオチド配列
配列番号２７：ｈＲ７４＿Ｌ４のアミノ酸配列
配列番号２８：ヒト軽鎖シグナル配列及びヒトκ鎖定常領域のアミノ酸配列をコードする配列を含むＤＮＡ断片のヌクレオチド配列
配列番号２９：ヒトキメラ化Ｒ７４軽鎖ｃＲ７４＿Ｌをコードするヌクレオチド配列
配列番号３０：ヒトキメラ化Ｒ７４軽鎖ｃＲ７４＿Ｌのアミノ酸配列
配列番号３１：ヒト重鎖シグナル配列及びヒトＩｇＧ１－ＬＡＬＡタイプ定常領域のアミノ酸をコードする配列を含むＤＮＡ断片のヌクレオチド配列
配列番号３２：ヒトキメラ化Ｒ７４重鎖ｃＲ７４＿Ｈをコードするヌクレオチド配列
配列番号３３：ヒトキメラ化Ｒ７４重鎖ｃＲ７４＿Ｈのアミノ酸配列
配列番号３４：ＢＡＹ１８９６５０２軽鎖をコードするヌクレオチド配列
配列番号３５：ＢＡＹ１８９６５０２軽鎖のアミノ酸配列
配列番号３６：ヒト重鎖シグナル配列及びヒトＩｇＧ２定常領域のアミノ酸をコードする配列を含むＤＮＡ断片のヌクレオチド配列
配列番号３７：ＢＡＹ１８９６５０２重鎖をコードするヌクレオチド配列
配列番号３８：ＢＡＹ１８９６５０２重鎖のアミノ酸配列 Sequence number 2: Peptide tag sequence number 3: Sequence X
Sequence number 4: Sequence Y
SEQ ID NO: 5: Nucleotide sequence of variable region of heavy chain of R74 SEQ ID NO: 6: Amino acid sequence of variable region of heavy chain of R74 SEQ ID NO: 7: CDRH1
SEQ ID NO: 8: CDRH2
Sequence number 9: CDRH3
SEQ ID NO: 10: Nucleotide sequence of variable region of light chain of R74 SEQ ID NO: 11: Amino acid sequence of variable region of light chain of R74 SEQ ID NO: 12: CDR L1
SEQ ID NO: 13: CDRL2
SEQ ID NO: 14: CDRL3
SEQ ID NO: 15: Nucleotide sequence encoding hR74_H1 amino acid sequence of hR74_H1 SEQ ID NO: 17: nucleotide sequence encoding hR74_H2 amino acid sequence of hR74_H2 SEQ ID NO: 19 nucleotide sequence encoding hR74_H12 SEQ ID NO: 20 Amino acid sequence of hR74_H12 SEQ ID NO: 21: CDRH2 of hR74_H12
SEQ ID NO: 22: Nucleotide sequence coding for hR74_L1 Amino acid sequence for hR74_L1 Nucleotide sequence for hR74_L2 SEQ ID NO: 25 amino acid sequence for hR74_L2 SEQ ID NO: 26: Nucleotide sequence encoding hR74_L4 SEQ ID NO: 27 Amino acid sequence of hR74_L4 SEQ ID NO: 28: Nucleotide sequence of a DNA fragment containing the sequence encoding the amino acid sequence of human light chain signal sequence and human κ chain constant region SEQ ID NO: 29: Nucleotide sequence encoding human chimerized R74 30: Amino acid sequence of human chimerized R74 light chain cR74_L SEQ ID NO: 31: DNA fragment containing human heavy chain signal sequence and a sequence encoding an amino acid of human IgG1-LALA type constant region Nucleotide sequence SEQ ID NO: 32: Human chimerized R74 heavy chain cR74_H encoding nucleotide sequence SEQ ID NO: 33: human chimerized R74 heavy chain cR74_H amino acid sequence SEQ ID NO: 34: BAY 1896502 light chain encoding nucleotide sequence SEQ ID NO: 35: BAY 1896502 light chain Amino acid sequence SEQ ID NO: 36: Nucleotide sequence of a DNA fragment comprising a human heavy chain signal sequence and a sequence encoding a human IgG2 constant region amino acid sequence SEQ ID NO: 37: BAY 1896502 heavy chain encoding nucleotide sequence SEQ ID NO 38: BAY 1896502 heavy chain amino acid Array

Claims (45)

  1. 　プロテインＣ又は活性化プロテインＣへの結合に対して、以下の（ａ）乃至（ｅ）からなる群から選択されるいずれか１項に記載の抗体と競合阻害活性を有する抗体又は当該抗体の機能性断片：
    （ａ）配列番号３３のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号３０のアミノ酸番号２１乃至２３７に示されるアミノ酸配列からなる軽鎖を有する抗体、
    （ｂ）配列番号１６のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２３のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体、
    （ｃ）配列番号２０のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２５のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体、
    （ｄ）配列番号２０のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２３のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体、及び
    （ｅ）配列番号２０のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号２７のアミノ酸番号２１乃至２３８に示されるアミノ酸配列からなる軽鎖を有する抗体。     An antibody having competitive inhibitory activity with the antibody according to any one of the following groups (a) to (e) or a function of the antibody for binding to protein C or activated protein C: Sex fragment:
    (A) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 33 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 237 of SEQ ID NO.
    (B) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 16 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO.
    (C) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 20 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO.
    (D) an antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 20 and a light chain consisting of the amino acid sequences shown by amino acid numbers 21 to 238 of SEQ ID NO. 20. An antibody having a heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of 20 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO.
  2. 　配列番号７に示されるアミノ酸配列からなるＣＤＲＨ１、配列番号８又は配列番号２１に示されるアミノ酸配列からなるＣＤＲＨ２及び配列番号９に示されるアミノ酸配列からなるＣＤＲＨ３を含む重鎖、並びに、配列番号１２に示されるアミノ酸配列からなるＣＤＲＬ１、配列番号１３に示されるアミノ酸配列からなるＣＤＲＬ２及び配列番号１４に示されるアミノ酸配列からなるＣＤＲＬ３を含む軽鎖からなり、プロテインＣ及び活性化プロテインＣに特異的に結合することを特徴とする抗体又は当該抗体の機能性断片。 A heavy chain comprising CDRH1 consisting of the amino acid sequence shown in SEQ ID NO: 7, CDRH2 consisting of the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 21 and CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 9, It consists of a light chain comprising CDRL1 consisting of the amino acid sequence shown, CDRL2 consisting of the amino acid sequence shown in SEQ ID NO: 13 and CDR L3 consisting of the amino acid sequence shown in SEQ ID NO: 14 and specifically binding to protein C and activated protein C Or a functional fragment of the antibody.
  3. 　プロテインＣ及び活性化プロテインＣがヒト由来であることを特徴とする、請求項１又は２に記載の抗体又は当該抗体の機能性断片。 The antibody according to claim 1 or 2, or a functional fragment of the antibody, wherein protein C and activated protein C are derived from human.
  4. 　プロテインＣが配列表の配列番号３のアミノ酸番号４３乃至１５３に示されるアミノ酸配列からなるペプチドとアミノ酸番号１５４乃至４１２に示されるアミノ酸配列からなるペプチドがＳＳ結合で結合しているポリペプチドであり、活性化プロテインＣが配列表の配列番号３のアミノ酸番号４３乃至１５３に示されるアミノ酸配列からなるペプチドとアミノ酸番号１６６乃至４１２に示されるアミノ酸配列からなるペプチドがＳＳ結合で結合しているポリペプチドであることを特徴とする、請求項１乃至３のいずれか１項に記載の抗体又は当該抗体の機能性断片。 A peptide in which the protein C is a peptide consisting of the amino acid sequence shown in amino acid numbers 43 to 153 of SEQ ID NO: 3 and a peptide consisting of the amino acid sequence shown in amino acid numbers 154 to 412 are linked by SS bond; A polypeptide comprising a peptide consisting of the amino acid sequence set forth in amino acid numbers 43 to 153 of SEQ ID NO: 3 in the sequence listing and a peptide consisting of the amino acid sequence set forth on amino acid numbers 166 to 412 in the SS list The antibody according to any one of claims 1 to 3, or a functional fragment of the antibody, characterized in that
  5. 　プロテインＣの活性化及び／又は活性化プロテインＣの活性を阻害することを特徴とする、請求項１乃至４のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The antibody according to any one of claims 1 to 4 or a functional fragment of the antibody, which inhibits protein C activation and / or activity of activated protein C.
  6. 　以下の（ａ）乃至（ｄ）から選択される少なくとも１つの特性を有することを特徴とする、請求項１乃至５のいずれか１項に記載の抗体又は当該抗体の機能性断片：
    （ａ）プロテインＣ及び活性化プロテインＣに特異的に結合する；
    （ｂ）プロテインＣに特異的に結合しプロテインＣの活性化を阻害する；
    （ｃ）活性化プロテインＣに特異的に結合し、活性化プロテインＣによる活性化血液凝固第ＶＩＩＩ因子（ＦＶＩＩＩａ）及び／又は活性化血液凝固第Ｖ因子（ＦＶａ）の分解及び／又は不活化を阻害する；
    　（ｄ）トロンビン産生を回復する、     The antibody according to any one of claims 1 to 5, or a functional fragment of the antibody, characterized in having at least one property selected from the following (a) to (d):
    (A) specifically bind to protein C and activated protein C;
    (B) specifically bind to protein C and inhibit protein C activation;
    (C) It specifically binds to activated protein C and causes decomposition and / or inactivation of activated blood coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa) by activated protein C Inhibit;
    (D) restore thrombin generation,
  7. 　ＣＤＲＨ２が配列番号８に示されるアミノ酸配列からなることを特徴とする、請求項１乃至６のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The antibody according to any one of claims 1 to 6, or a functional fragment of the antibody, wherein CDRH2 consists of the amino acid sequence shown in SEQ ID NO: 8.
  8. 　ＣＤＲＨ２が配列番号２１に示されるアミノ酸配列からなることを特徴とする、請求項１乃至６のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The antibody according to any one of claims 1 to 6, or a functional fragment of the antibody, wherein CDRH2 consists of the amino acid sequence shown in SEQ ID NO: 21.
  9. 　配列番号６に記載の重鎖可変領域、及び配列番号１１に記載の軽鎖可変領域を含むことからなる、請求項１乃至６のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The antibody according to any one of claims 1 to 6, or a functional fragment thereof, comprising the heavy chain variable region shown in SEQ ID NO: 6 and the light chain variable region shown in SEQ ID NO: 11.
  10. 　配列番号３３のアミノ酸番号２０乃至４６７に示されるアミノ酸配列からなる重鎖及び配列番号３０のアミノ酸番号２１乃至２３７に示されるアミノ酸配列からなる軽鎖を含むことからなる、請求項１乃至７、及び９のいずれか１項に記載の抗体又は当該抗体の機能性断片。 A heavy chain consisting of the amino acid sequence shown by amino acid numbers 20 to 467 of SEQ ID NO: 33 and a light chain consisting of the amino acid sequence shown by amino acid numbers 21 to 237 of SEQ ID NO. 9. The antibody according to any one of 9 or a functional fragment of the antibody.
  11. 　定常領域がヒト由来定常領域である請求項１乃至１０のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The antibody according to any one of claims 1 to 10 or a functional fragment of the antibody, wherein the constant region is a human-derived constant region.
  12. 　ヒト化されている請求項１乃至１１のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The antibody according to any one of claims 1 to 11 or a functional fragment of the antibody, which is humanized.
  13. 　以下の（ａ）乃至（ｅ）からなる群から選択されるいずれか１項に記載のアミノ酸配列からなる重鎖可変領域、及び（ｆ）乃至（ｊ）からなる群から選択されるいずれか１項に記載のアミノ酸配列からなる軽鎖可変領域を有する請求項１２に記載の抗体又は当該抗体の機能性断片：
    （ａ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列、
    （ｂ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列、
    （ｃ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列、
    （ｄ）（ａ）乃至（ｃ）の配列において各ＣＤＲ　配列以外のフレームワーク領域の配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、
    （ｅ）（ａ）乃至（ｄ）の配列における各ＣＤＲ　配列以外のフレームワーク領域の配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、
    （ｆ）配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列、
    （ｇ）配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列、
    （ｈ）配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列、
    （ｉ）（ｆ）乃至（ｈ）の配列において各ＣＤＲ　配列以外のフレームワーク領域の配列に対して少なくとも９５％以上の相同性を有するアミノ酸配列、及び
    （ｊ）　（ｆ）乃至（ｉ）の配列における各ＣＤＲ　配列以外のフレームワーク領域の配列において１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。     A heavy chain variable region consisting of the amino acid sequence according to any one of the following (a) to (e), and any one selected from the group consisting of (f) to (j): 13. The antibody according to claim 12, or a functional fragment of the antibody, which has a light chain variable region consisting of the amino acid sequence described in paragraph:
    (A) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16,
    (B) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18;
    (C) the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20,
    (D) an amino acid sequence having at least 95% or more homology to a sequence of a framework region other than each CDR sequence in the sequences of (a) to (c),
    (E) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequences of (a) to (d),
    (F) the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23,
    (G) the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25;
    (H) the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27;
    (I) an amino acid sequence having at least 95% or more homology with a sequence of a framework region other than each CDR sequence in the sequence of (f) to (h), and (j) (f) to (i) An amino acid sequence in which one or several amino acids have been deleted, substituted or added in the sequence of framework regions other than each CDR sequence in the sequence.
  14. 　以下の（ａ）乃至（ｉ）からなる群から選択されるいずれか１項に記載の重鎖可変領域及び軽鎖可変領域を含む請求項１３に記載の抗体又は当該抗体の機能性断片：
    （ａ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域、
    （ｂ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる、重鎖可変領域および配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
    （ｃ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる、重鎖可変領域および配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
    （ｄ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
    （ｅ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
    （ｆ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
    （ｇ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
    （ｈ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域、
    （ｉ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域。     The antibody according to claim 13, which comprises the heavy chain variable region and the light chain variable region according to any one of the following (a) to (i):
    (A) a heavy chain variable region consisting of the amino acid sequence as set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16, and a light chain variable flow region consisting of the amino acid sequence as set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23
    (B) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25
    (C) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27;
    (D) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23;
    (E) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25;
    (F) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27
    (G) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23
    (H) a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25;
    (I) A heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27
  15. 　配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域を含む請求項１３又は１４に記載の抗体又は当該抗体の機能性断片。 The heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and the light chain variable flow region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO. The described antibody or a functional fragment of the antibody.
  16. 　配列番号配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域を含む請求項１３又は１４に記載の抗体又は当該抗体の機能性断片。 The heavy chain variable region consisting of the amino acid sequence as set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and the light chain variable region consisting of the amino acid sequence as set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 14. The antibody according to 14 or a functional fragment of the antibody.
  17. 　配列番号配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域、および配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域を含む請求項１３又は１４に記載の抗体又は当該抗体の機能性断片。 The heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and the light chain variable flow region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO. 14. The antibody according to 14 or a functional fragment of the antibody.
  18. 　以下の（ａ）乃至（ｉ）からなる群から選択されるいずれか１項に記載の重鎖及び軽鎖を含む請求項１３乃至１７のいずれか１項に記載の抗体又は抗体の機能性断片：
    （ａ）配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｂ）配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２５においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｃ）配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｄ）配列番号１８においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｅ）配列番号１８においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２５においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｆ）配列番号１８においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｇ）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｈ）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２５においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖、
    （ｉ）配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖。     The antibody or the functional fragment of the antibody according to any one of claims 13 to 17, comprising the heavy chain and the light chain according to any one of the following (a) to (i): :
    (A) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23
    (B) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 25;
    (C) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27
    (D) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 18 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23;
    (E) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 18 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 25;
    (F) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 18 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27;
    (G) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23;
    (H) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 25;
    (I) A heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27.
  19. 　配列番号１６においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖を含む請求項１３乃至１８のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The heavy chain consisting of the amino acid sequence as set forth in amino acid numbers 20 to 467 in SEQ ID NO: 16 and the light chain consisting of the amino acid sequence as set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23 Or the functional fragment of the antibody.
  20. 　配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２３においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖を含む請求項１３乃至１８のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The heavy chain comprising the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20, and the light chain composed of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 23 Or the functional fragment of the antibody.
  21. 　配列番号２０においてアミノ酸番号２０乃至４６７に記載のアミノ酸配列からなる重鎖、および配列番号２７においてアミノ酸番号２１乃至２３８に記載のアミノ酸配列からなる軽鎖を含む請求項１３乃至１８のいずれか１項に記載の抗体又は当該抗体の機能性断片。 The heavy chain comprising the amino acid sequence set forth in amino acid numbers 20 to 467 in SEQ ID NO: 20, and the light chain composed of the amino acid sequence set forth in amino acid numbers 21 to 238 in SEQ ID NO: 27 Or the functional fragment of the antibody.
  22. 　機能性断片がＦａｂ、Ｆ（ａｂ）２、Ｆａｂ’及びＦｖからなる群から選択される請求項１乃至２１のいずれか１項に記載の抗体の機能性断片。 22. The functional fragment of the antibody according to any one of claims 1 to 21, wherein the functional fragment is selected from the group consisting of Fab, F (ab) 2, Fab 'and Fv.
  23. 　請求項１乃至２２のいずれか１項に記載の抗体又は当該抗体の機能性断片をコードするポリヌクレオチド。 A polynucleotide encoding the antibody according to any one of claims 1 to 22 or a functional fragment of the antibody.
  24. 　配列番号７に記載のアミノ酸配列からなるＣＤＲＨ１、配列番号８又は２１に記載のアミノ酸配列からなるＣＤＲＨ２及び配列番号９に記載のアミノ酸配列からなるＣＤＲＨ３をコードするポリヌクレオチド、並びに配列番号１２に記載のアミノ酸配列からなるＣＤＲＬ１、配列番号１３に記載のアミノ酸配列からなるＣＤＲＬ２及び配列番号１４に記載のアミノ酸配列からなるＣＤＲＬ３をコードするポリヌクレオチドを含む請求項２３に記載のポリヌクレオチド。 A polynucleotide encoding a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 7, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 8 and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 9, The polynucleotide according to claim 23, comprising a polynucleotide encoding a CDRL1 consisting of an amino acid sequence, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 13 and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 14.
  25. 　以下の（ａ）乃至（ｊ）からなる群から選択されるいずれか１項に記載のポリヌクレオチドを含む請求項２３又は２４に記載のポリヌクレオチド：
    （ａ）配列番号６に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号１１に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
    （ｂ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、および配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変流域をコードするポリヌクレオチド、
    （ｃ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、および配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
    （ｄ）配列番号１６においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド，および配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
    （ｅ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
    （ｆ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
    （ｇ）配列番号１８においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
    （ｈ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２３においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
    （ｉ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２５においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド、
    （ｊ）配列番号２０においてアミノ酸番号２０乃至１３７に記載のアミノ酸配列からなる重鎖可変領域をコードするポリヌクレオチド、及び配列番号２７においてアミノ酸番号２１乃至１３３に記載のアミノ酸配列からなる軽鎖可変領域をコードするポリヌクレオチド。     The polynucleotide according to claim 23 or 24, comprising the polynucleotide according to any one of the following selected from the group consisting of (a) to (j):
    (A) a polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 6, and a polynucleotide encoding a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 11
    (B) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence as set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable watershed consisting of the amino acid sequence as set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 A polynucleotide encoding
    (C) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25 A polynucleotide encoding
    (D) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 16 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27 A polynucleotide encoding
    (E) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 A polynucleotide encoding
    (F) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25 A polynucleotide encoding
    (G) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27 A polynucleotide encoding
    (H) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 23 A polynucleotide encoding
    (I) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 25 A polynucleotide encoding
    (J) A polynucleotide encoding a heavy chain variable region consisting of the amino acid sequence set forth in amino acid numbers 20 to 137 in SEQ ID NO: 20, and a light chain variable region consisting of the amino acid sequence set forth in amino acid numbers 21 to 133 in SEQ ID NO: 27 Polynucleotide encoding
  26. 　請求項２３乃至２５のいずれか１項に記載のポリヌクレオチドを含有する発現ベクター。 An expression vector comprising the polynucleotide according to any one of claims 23 to 25.
  27. 　請求項２６に記載の発現ベクターにより形質転換された宿主細胞。 A host cell transformed by the expression vector according to claim 26.
  28. 　宿主細胞が真核細胞である請求項２６に記載の宿主細胞。 27. The host cell of claim 26, wherein the host cell is a eukaryotic cell.
  29. 　請求項２７又は２８に記載の宿主細胞を培養する工程、及び当該工程で得られた培養物から目的の抗体又は当該抗体の機能性断片を採取する工程を含むことを特徴とする当該抗体又は当該抗体の機能性断片の製造方法。 A method of culturing the host cell according to claim 27 or 28, and a step of collecting the target antibody or a functional fragment of the antibody from the culture obtained in the step, or the antibody Methods of producing functional fragments of antibodies.
  30. 　請求項２９に記載の製造方法により得られることを特徴とする抗体又は当該抗体の機能性断片。 An antibody obtained by the production method according to claim 29, or a functional fragment of the antibody.
  31. 　機能性断片がＦａｂ、Ｆ（ａｂ）２、Ｆａｂ’及びＦｖからなる群から選択される請求項３０に記載の抗体の機能性断片。 31. The functional fragment of an antibody according to claim 30, wherein the functional fragment is selected from the group consisting of Fab, F (ab) 2, Fab 'and Fv.
  32. 　Ｎ－結合への糖鎖付加、Ｏ－結合への糖鎖付加、Ｎ末のプロセッシング、Ｃ末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化、Ｎ末にメチオニン残基の付加、プロリン残基のアミド化及び重鎖カルボキシル末端における１つ又は２つのアミノ酸の欠失からなる群より選択される１又は２以上の修飾を含む請求項１乃至２２、３０及び３１のいずれか１項に記載の抗体又は当該抗体の機能性断片。 Glycosylation to N-linkage, glycosylation to O-linkage, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, N-terminal addition of methionine residue 32. Any one of claims 1 to 22, 30 and 31 comprising one or more modifications selected from the group consisting of amidification of proline residues and deletion of one or two amino acids at the heavy chain carboxyl terminus. The antibody according to item or a functional fragment of the antibody.
  33. 　重鎖のカルボキシル末端において１つ又は２つのアミノ酸が欠失している請求項３２に記載の抗体。 33. The antibody of claim 32, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.
  34. 　２本の重鎖の双方でカルボキシル末端において１つのアミノ酸が欠失している請求項３３に記載の抗体。 34. The antibody according to claim 33, wherein one amino acid is deleted at the carboxyl terminus of both of the two heavy chains.
  35. 重鎖のカルボキシル末端のプロリン残基が更にアミド化されている請求項３２乃至３４のいずれか１項に記載の抗体。
    35. The antibody according to any one of claims 32-34, wherein the proline residue at the carboxyl terminus of the heavy chain is further amidated.
  36. 　請求項１乃至２２、３０乃至３５のいずれかひとつに記載の抗体又は当該抗体の機能性断片、その塩、それらの水和物、請求項２３乃至２５のいずれかひとつに記載のポリヌクレオチド、請求項２６に記載の発現ベクター、請求項２７又は２８に記載の宿主細胞からなる群から選択されるいずれかを有効成分として含むことを特徴とする医薬組成物。 The antibody according to any one of claims 1 to 22, 30 to 35, or a functional fragment of the antibody, a salt thereof, a hydrate thereof, the polynucleotide according to any one of claims 23 to 25, A pharmaceutical composition comprising, as an active ingredient, any one selected from the group consisting of the expression vector of claim 26 and the host cell of claim 27 or 28.
  37. 　出血性疾患治療薬であることを特徴とする、請求項３６に記載の医薬組成物。 The pharmaceutical composition according to claim 36, which is a therapeutic agent for hemorrhagic disease.
  38. 　出血性疾患が血友病Ａ、血友病Ｂ、後天性血友病及びｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病から選択される少なくともいずれか一つであることを特徴とする、請求項３７に記載の医薬組成物。 The pharmaceutical composition according to claim 37, wherein the hemorrhagic disease is at least one selected from hemophilia A, hemophilia B, acquired hemophilia and von Willebrand's disease.
  39. 　出血性疾患が血友病Ａ及び／又は血友病Ｂであることを特徴とする、請求項３８に記載の医薬組成物。 The pharmaceutical composition according to claim 38, wherein the hemorrhagic disease is hemophilia A and / or hemophilia B.
  40. 請求項１乃至２２、及び３０乃至３５に記載の抗体又は当該抗体の機能性断片、その塩、又はそれらの水和物から選択されるいずれかを個体に投与することを特徴とする出血性疾患の治療方法。 A hemorrhagic disease characterized by administering any one selected from the antibody according to any one of claims 1 to 22 and 30 to 35 or a functional fragment of the antibody, a salt thereof, or a hydrate thereof Treatment method.
  41. 出血性疾患が血友病Ａ、血友病Ｂ、後天性血友病及びｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病から選択される少なくともいずれか一つであることを特徴とする、請求項４０に記載の治療方法。 The therapeutic method according to claim 40, wherein the hemorrhagic disease is at least one selected from hemophilia A, hemophilia B, acquired hemophilia and von Willebrand's disease.
  42. 　出血性疾患が血友病Ａ及び／又は血友病Ｂであることを特徴とする、請求項４１に記載の治療方法。 42. The method according to claim 41, wherein the bleeding disorder is hemophilia A and / or hemophilia B.
  43. 　請求項１乃至２２、及び３０乃至３５に記載の抗体又は当該抗体の機能性断片、その塩、又はそれらの水和物から選択される少なくとも一つ、を含むことからなる医薬組成物及び少なくとも一つの出血性疾患治療薬を、同時に、別々に又は連続して個体に投与することを特徴とする出血性疾患の治療方法。 A pharmaceutical composition comprising the antibody according to any one of claims 1 to 22 and 30 to 35, or a functional fragment of the antibody, a salt thereof, or a hydrate thereof, A method of treating hemorrhagic disease, which comprises administering two hemorrhagic disease therapeutic agents simultaneously, separately or sequentially to an individual.
  44. 　出血性疾患が血友病Ａ、血友病Ｂ、後天性血友病及びｖｏｎ Ｗｉｌｌｅｂｒａｎｄ病からなる群から選択される少なくともいずれか一つであることを特徴とする、請求項４３に記載の治療方法。 The treatment according to claim 43, wherein the hemorrhagic disease is at least one selected from the group consisting of hemophilia A, hemophilia B, acquired hemophilia and von Willebrand's disease. Method.
  45. 　出血性疾患が血友病Ａ及び／又は血友病Ｂであることを特徴とする、請求項４４に記載の治療方法。 The treatment method according to claim 44, wherein the hemorrhagic disease is hemophilia A and / or hemophilia B.
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JPH0638743A (en) * 1983-10-18 1994-02-15 Fujisawa Pharmaceut Co Ltd Protein c-resistant monoclonal antibody and hybridoma producing the same
WO2009055669A2 (en) * 2007-10-26 2009-04-30 Oklahoma Medical Research Foundation Monoclonal antibodies against activated and unactivated protein c
WO2015041310A1 (en) * 2013-09-20 2015-03-26 中外製薬株式会社 Treatment for hemorrhagic diseases by anti-protein-c antibody

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0638743A (en) * 1983-10-18 1994-02-15 Fujisawa Pharmaceut Co Ltd Protein c-resistant monoclonal antibody and hybridoma producing the same
WO2009055669A2 (en) * 2007-10-26 2009-04-30 Oklahoma Medical Research Foundation Monoclonal antibodies against activated and unactivated protein c
WO2015041310A1 (en) * 2013-09-20 2015-03-26 中外製薬株式会社 Treatment for hemorrhagic diseases by anti-protein-c antibody

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