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WO2019009407A1 - Anticorps anti-protéine c - Google Patents

Anticorps anti-protéine c 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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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

Le problème abordé par la présente invention est de fournir un nouveau médicament pour le traitement de troubles hémorragiques qui a un effet de coagulation sanguine supérieur, et une méthode de traitement de troubles hémorragiques qui utilise ledit médicament. L'invention concerne : un anticorps qui se lie à la fois à la Protéine C et à la Protéine C activée, et inhibe l'activation de la protéine C et l'activité de la Protéine C activée ; un fragment de liaison à l'antigène dudit anticorps ; un anticorps chimérique dudit anticorps ; un composé pharmaceutique comprenant ledit anticorps ; et similaire.
PCT/JP2018/025679 2017-07-07 2018-07-06 Anticorps anti-protéine c WO2019009407A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
JPH0638743A (ja) * 1983-10-18 1994-02-15 Fujisawa Pharmaceut Co Ltd 抗プロテインｃモノクローナル抗体およびそれを産生するハイブリドーマ
WO2009055669A2 (fr) * 2007-10-26 2009-04-30 Oklahoma Medical Research Foundation Anticorps monoclonaux contre la protéine c activée
WO2015041310A1 (fr) * 2013-09-20 2015-03-26 中外製薬株式会社 Traitement de maladies hémorragiques par anticorps anti-protéine-c

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0638743A (ja) * 1983-10-18 1994-02-15 Fujisawa Pharmaceut Co Ltd 抗プロテインｃモノクローナル抗体およびそれを産生するハイブリドーマ
WO2009055669A2 (fr) * 2007-10-26 2009-04-30 Oklahoma Medical Research Foundation Anticorps monoclonaux contre la protéine c activée
WO2015041310A1 (fr) * 2013-09-20 2015-03-26 中外製薬株式会社 Traitement de maladies hémorragiques par anticorps anti-protéine-c

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