TW201906868A - Anti-protein C antibody - Google Patents

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

   Anti-protein C antibody   

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, and a pharmaceutical composition containing the antibody.

Hemophilia is a bleeding disorder caused by congenital defect or insufficiency 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, also referred to as "hemophilia B"). Because any gene exists on the X chromosome and is sex-linked recessive inheritance, almost all the patients are male. The prevalence rate is about 9 per 100,000 men, and the ratio of hemophilia A to hemophilia B is about 5: 1.

As the main bleeding symptoms, there are subcutaneous bleeding, abnormal traumatic bleeding, intracranial hemorrhage, intraarticular bleeding, intraoral bleeding, intranasal bleeding, iliopsoas bleeding, and gastrointestinal bleeding. Chronic synovitis caused by repeated bleeding in specific joints, and irreversible changes in articular cartilage due to its continuous progress, progress to joint disease, hemophilic arthropathy with difficulty walking, and eventually become needed The situation of joint replacement surgery has become an important factor that significantly reduces the quality of life (QOL) of patients with hemophilia.

The severity of hemophilia is based on the FVIII activity or FIX activity in the blood, and less than 1% of the active patients are defined as severe, more than 1% and less than 5% of patients are classified as moderate, and more than 5% and less than 40% of patients are prescribed as mild. More than 60% of patients with hemophilia are considered severe. Hemophilia's moderate and minor coagulation factor activity differs from severe cases by only a few percent, but because the frequency of bleeding is significantly less, it can be considered that maintaining FVIII activity or FIX activity above 1% is effective in reducing the frequency of bleeding.

For the prevention and / or treatment of bleeding in hemophilia patients, blood coagulation factor preparations (FVIII preparations, FIX preparations) that are refined from plasma or produced by genetic recombination technology are mainly used. The blood coagulation factor has a short half-life in the blood of several hours to tens of hours, so the persistence of the drug effect is short. For the prevention of bleeding, the blood coagulation factor preparation needs to be administered intravenously about 3 times a week to coagulate the factor. Activity (FVIII activity or FIX activity) was maintained above 1%. The regular replacement therapy of blood coagulation factor preparations reduces the frequency of bleeding, and as a result, it greatly contributes to the improvement of QOL in patients with hemophilia.

In addition, in the treatment at the time of bleeding (complementary therapy), in order to completely stop bleeding and prevent rebleeding, regular additional administration of blood coagulation factor preparations is performed to maintain the activity of coagulation factors for a certain period or more.

The route of administration of the blood coagulation factor preparation is limited to intravenous administration. Intravenous administration is technically difficult, especially for pediatric patients or thin patients, and the veins are more difficult. Therefore, caution in administration is required.

In the aforementioned prevention and / or treatment administration, although home remedies or self-injection are performed in most cases, frequent administration is required. Furthermore, administration is accompanied by technical difficulties. This condition not only causes patient pain during administration, but also becomes a factor that hinders the popularization of home therapy or self-injection, leading to a decrease in adherence.

Therefore, there is a strong demand for an agent that has a longer half-life and can reduce the frequency of administration compared to an existing coagulation factor preparation, or an agent that is simple to be administered and related to the improvement of patient compliance.

Furthermore, in patients with hemophilia, neutralizing antibodies for coagulation factor preparations called inhibitors are produced with a high probability. Especially in critically ill patients, the probability is said to be 30%. When an inhibitor is produced, the effect of the coagulation factor preparation is neutralized by the inhibitor, the effect is reduced, and the worst case disappears. As a result, a bypass therapy using an active prothrombin complex preparation or a genetically recombinant active factor VII preparation is performed. However, in either case, regular complementary therapies have not been established and the management of hemostasis in patients is very difficult. Therefore, there is a strong demand for an agent that is not affected by the presence or absence of an inhibitor.

In addition, antibodies have high stability in blood, can be administered subcutaneously, and have high selectivity of the target. Therefore, they have been applied and marketed as pharmaceuticals in recent years. Antibodies can be administered less frequently due to their long half-life, they can be administered easily by subcutaneous administration, and because they are not affected by the presence or absence of inhibitors, they are considered to be suitable for drug development in hemophilia.

Protein C (hereinafter also referred to as "PC") plays a major role in the negative feedback mechanism of blood coagulation. PC binds to endothelial protein C receptor (EPCR) expressed by vascular endothelium, and is activated by the thrombin / thrombomodulin complex to become activated protein C (hereinafter, also described "APC"). The aPC system decomposes and deactivates protein S, which is a cofactor, and activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa). Therefore, it is expected to promote coagulation by suppressing PC and suppressing the negative feedback mechanism. It is known that patients with homozygous congenital PC deficiency develop thrombotic purpura fulminans, and this also supports the above idea.

In fact, it has been reported that aPC inhibitors partially restore the reduction of thrombin production in FVIII-deficient plasma in vitro (non-patent document 1, non-patent document 2). In addition, there have been reports that an anti-system that inhibits the activity of aPC inhibits the anticoagulant activity of aPC, shows a blood coagulation effect, and is effective for the treatment of hemophilia (Patent Document 1).

In addition, it has been reported that a mutant FVa having anti-decomposition resistance to aPC reduces the amount of bleeding in a bleeding pattern in FVIII-deficient mice (Non-Patent Document 3). Furthermore, it has been reported that antibodies that inhibit the activation of PC and / or the activity of aPC also reduce the area of bleeding marks in the puncture bleeding mode of FVIII-deficient nude mice (Patent Document 2).

APC antibodies have been reported to suppress bleeding in monkey bleeding mode (Non-Patent Document 4).

It has been reported that an antibody is considered to have a function as a modulator for aPC (Patent Documents 3 and 4). However, in a non-rodent hemophilia model, the hemostatic effect of antibodies capable of binding to both PC and aPC has not been confirmed.

Prior art literature Patent literature

Patent Document 1 Japanese Patent Application Publication No. 2014-237651

Patent Document 2 WO2015 / 041310

Patent Document 3 WO2014 / 085527

Patent Document 4 WO2015 / 179435

Non-patent literature

Non-Patent Literature 1 Brummel-Ziedins KE et al., J Thromb Haemost 2011; 9: 2262-2267.

Non-Patent Literature 2 Bach P et al., Bioorg Med Chem Lett 2014; 24 (3), 821-827

Non-Patent Document 3 von Drygalski A et al., J Thromb Haemost 2014; 12: 363-372

Non-Patent Document 4 Xian-Yan Zhao et al., ASH 58th Annual Meeting & Exposition, December 3-6, 2016, Session: 321, “80 Targeted Inhibition of Activated Protein C Anticoagulant Activity By Monoclonal Antibody HAPC1573 for Treatment of Hemophilia”

Summary of invention

An object of the present invention is to provide a novel medicine for the treatment of bleeding disorders having an excellent blood coagulation effect, a method for treating a bleeding disorder using the medicine, and the like.

In order to achieve the above-mentioned subject, the present inventors conducted intensive studies to obtain an antibody that binds to both protein C and activated protein C and inhibits the activation of protein C and the activity of activated protein C, and found that the antibody has a high hemostatic effect and is effective for bleeding Treatment of sexual diseases, especially hemophilia A and / or hemophilia B is effective, and the present invention has been completed.

According to the present invention, an antibody that inhibits the activation of protein C and the activity of activated protein C is provided, and a novel medicine for the treatment of bleeding disorders with excellent blood coagulation effect containing the antibody as an active ingredient, and a bleeding disorder using the medicine are provided. Treatment methods, etc.

That is, the present invention includes:

(1) An antibody or a functional fragment of the antibody having binding to protein C or activated protein C and having an antibody selected from the group consisting of any one of the following groups (a) to (e) Competitive inhibitory activity: (a) A heavy chain comprising an amino acid sequence represented by amino acid numbers 20 to 467 of sequence identification number 33 and an amino acid sequence represented by amino acid numbers 21 to 237 including sequence identification number 30 An antibody having a light chain of an amino acid sequence, (b) a heavy chain having an amino acid sequence represented by amino acid numbers 20 to 467 of sequence identification number 16, and an amino acid number 21 including sequence identification number 23 Antibodies having a light chain of an amino acid sequence shown to 238, (c) a heavy chain having an amino acid sequence shown in amino acid numbers 20 to 467 of sequence identification number 20, and Antibodies having a light chain of an amino acid sequence represented by amino acid numbers 21 to 238, (d) a heavy chain having an amino acid sequence represented by amino acid numbers 20 to 467 of sequence identification number 20, and The light chain antibody of the amino acid sequence shown by amino acid numbers 21 to 238 of SEQ ID NO: 23, and (e) has an inclusion sequence The amino acid sequence shown alias of amino acid numbers 20 to 467 of the heavy chain 20, and the number of amino acids comprising SEQ ID NO 27 of the antibody light chain amino acid sequence shown in 21-238;

(2) An antibody or a functional fragment of the antibody, comprising a heavy chain and a light chain, and specifically binding to protein C and activated protein C; the heavy chain includes: an amine group shown in sequence identification number 7 CDRH1 of the acid sequence, CDRH2 containing the amino acid sequence shown in sequence identification number 8 or sequence identification number 21, and CDRH3 containing the amino acid sequence shown in sequence identification number 9; the light chain contains: contains the sequence identification number 12 CDRL1 of the amino acid sequence shown in the figure, CDRL2 containing the amino acid sequence shown in sequence identification number 13 and CDRL3 containing the amino acid sequence shown in sequence identification number 14;

(3) the antibody or the functional fragment of the antibody according to (1) or (2), characterized in that protein C and activated protein C are derived from humans;

(4) The antibody or the functional fragment of the antibody according to any one of (1) to (3), characterized in that the protein C is an amino acid number 43 to 153 including the sequence identification number 3 of the sequence listing The peptide shown in the amino acid sequence and the peptide containing the amino acid sequence shown in amino acid numbers 154 to 412 are polypeptides bound by SS bonds. The activated protein C is the amino acid number containing sequence identification number 3 of the sequence listing. A polypeptide in which the peptide of the amino acid sequence shown in 43 to 153 and the peptide containing the amino acid sequence shown in amino acid numbers 166 to 412 are bound by an SS bond;

(5) the antibody or the functional fragment of the antibody according to any one of (1) to (4), which is characterized by inhibiting the activation of protein C and / or the activity of activated protein C;

(6) The antibody or the functional fragment of the antibody according to any one of (1) to (5), characterized by having at least one characteristic selected from the following (a) to (d): (a) and Protein C and activated protein C specifically bind, (b) specifically bind to protein C and inhibit activation of protein C, (c) specifically bind to activated protein C, inhibit activated blood coagulation caused by activated protein C. Section VIII Decomposition and / or deactivation of factor (FVIIIa) and / or activated blood coagulation factor V (FVa), (d) restoration of thrombin production;

(7) the antibody or the functional fragment of the antibody according to any one of (1) to (6), wherein CDRH2 includes an amino acid sequence represented by sequence identification number 8;

(8) the antibody or the functional fragment of the antibody according to any one of (1) to (6), characterized in that CDRH2 includes an amino acid sequence represented by sequence identification number 21;

(9) The antibody or the functional fragment of the antibody according to any one of (1) to (6), which includes the heavy chain variable region described in sequence identification number 6 and the sequence identification number 11 The light chain variable region;

(10) The antibody or the functional fragment of the antibody according to any one of (1) to (7) and (9), which is composed of the following heavy and light chains: A heavy chain of the amino acid sequence shown by amino acid numbers 20 to 467, and a light chain including the amino acid sequence of amino acid numbers 21 to 237 of sequence identification number 30;

(11) the antibody or the functional fragment of the antibody according to any one of (1) to (10), wherein the constant region is a human-derived constant region;

(12) the antibody or the functional fragment of the antibody according to any one of (1) to (11), which is humanized;

(13) The antibody or the functional fragment of the antibody according to (12), which has a heavy chain comprising an amino acid sequence selected from the group consisting of any one of the following groups (a) to (e) A variable region, and a light chain variable region comprising an amino acid sequence selected from the group consisting of (f) to (j): (a) amino acid number 20 in sequence identification number 16 The amino acid sequences described in 131 to 137, the amino acid sequences described in (b) amino acid numbers 20 to 137 in sequence identification number 18, and the amino acid sequences 20 to 137 in (c) sequence identification number 20 Amino acid sequence, (d) an amino acid sequence having at least 95% homology to a sequence of a framework region other than each CDR sequence in the sequences (a) to (c), (e ) Amino acid sequence in which one or more amino acids have been deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequences of (a) to (d), (f) in sequence identification number 23 The amino acid sequence described in amino acid numbers 21 to 133, (g) the amino acid sequence described in amino acid number 21 to 133, (h) the amino acid number described in sequence identification number 27 21 to 133 Amino acid sequence, (i) an amino acid sequence having at least 95% homology to a sequence in a framework region other than each CDR sequence in the sequences (f) to (h), and (j) in (f) An amino acid sequence in which one or more amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequence of (i);

(14) The antibody or the functional fragment of the antibody according to (13), which comprises a heavy chain variable region and a light chain selected from the group consisting of any one of the following groups (a) to (i): Variable region: (a) a heavy chain variable region comprising an amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16 and an amino acid number 21 to 133 included in sequence identification number 23 The light chain variable region of the amino acid sequence, (b) the heavy chain variable region including the amino acid sequence of amino acid numbers 20 to 137 in SEQ ID NO: 16, and the amine sequence of 25 The light chain variable region of the amino acid sequence described in amino acid numbers 21 to 133, (c) the heavy chain variable region including the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16, And a light chain variable region comprising the amino acid sequences described in amino acid numbers 21 to 133 in sequence identification number 27, (d) the amino acid sequences described in amino acid numbers 20 to 137 in sequence identification number 18 The heavy chain variable region of the sequence, and the light chain variable region including the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 23, (e) including the amine in sequence identification number 18 The heavy chain variable region of the amino acid sequence described in amino acid numbers 20 to 137, and the light chain variable region of the amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 25, (f ) A heavy chain variable region comprising an amino acid sequence described in amino acid numbers 20 to 137 in SEQ ID NO: 18 and an amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 27 The light chain variable region, (g) the heavy chain variable region including the amino acid sequences described in amino acid numbers 20 to 137 in sequence identification number 20, and the amino acid number 21 to 133 including amino acid numbers in sequence identification number 23 The light chain variable region of the described amino acid sequence, (h) the heavy chain variable region including the amino acid sequence described in amino acid numbers 20 to 137 of sequence identification number 20, and the sequence identification number 25 The light chain variable region of the amino acid sequence described in the amino acid numbers 21 to 133, (i) The heavy chain including the amino acid sequence described in the amino acid numbers 20 to 137 of the sequence identification number 20 is variable Region, and a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 27;

(15) The antibody or the functional fragment of the antibody according to (13) or (14), comprising: the heavy chain including the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16 may A variable region, and a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 23;

(16) The antibody or the functional fragment of the antibody according to (13) or (14), comprising: an antibody comprising the amino acid sequence described in amino acid numbers 20 to 137 in the sequence identification number sequence identification number 20; A heavy chain variable region and a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 23;

(17) The antibody or the functional fragment of the antibody according to (13) or (14), comprising: an antibody comprising the amino acid sequence described in amino acid numbers 20 to 137 in the sequence identification number sequence identification number 20; A heavy chain variable region and a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 27;

(18) The antibody or the functional fragment of an antibody according to any one of (13) to (17), which comprises a substance selected from the group consisting of the following (a) to (i): Chain and light chain: (a) A heavy chain containing the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 16 and an amine described in amino acid number 21 to 238 in sequence identification number 23 The light chain of the amino acid sequence, (b) the heavy chain containing the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 16, and the heavy chain containing the amino acid sequence 21 to 238 in sequence identification number 25 The light chain of the amino acid sequence, (c) the heavy chain containing the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 16, and the amino acid sequence 21 to 238 including the amino acid sequence in sequence identification number 27 The light chain of the amino acid sequence described, (d) the heavy chain containing the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 18, and the amino acid sequence 21 of the amino acid sequence number 23 The light chain of the amino acid sequence described in 238 to 238, (e) The heavy chain including the amino acid sequence described in amino acid number 20 to 467 in sequence identification number 18, and the containing sequence The light chain of the amino acid sequence described in amino acid numbers 21 to 238 in identification number 25, (f) the heavy chain including the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 18, and A light chain containing the amino acid sequences described in amino acid numbers 21 to 238 in SEQ ID NO: 27, and (g) a heavy chain containing the amino acid sequences described in amino acid numbers 20 to 467 in sequence identification 20 And a light chain containing the amino acid sequences described in amino acid numbers 21 to 238 in sequence identification number 23, (h) a light chain containing the amino acid sequences described in amino acid numbers 20 to 467 in sequence identification number 20 Heavy chain and light chain containing amino acid sequences described in amino acid numbers 21 to 238 in SEQ ID NO: 25, (i) containing amino acids described in amino acid numbers 20 to 467 in serial identification number 20 The heavy chain of the sequence and the light chain comprising the amino acid sequence described in amino acid numbers 21 to 238 in sequence identification number 27;

(19) The antibody or the functional fragment of the antibody according to any one of (13) to (18), comprising the amino acid sequence described in amino acid number 20 to 467 in sequence identification number 16 And a light chain including the amino acid sequences described in amino acid numbers 21 to 238 in SEQ ID NO: 23.

(20) The antibody or the functional fragment of the antibody according to any one of (13) to (18), comprising the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 20 Heavy chain, and a light chain comprising the amino acid sequence described in amino acid numbers 21 to 238 in sequence identification number 23;

(21) The antibody or the functional fragment of the antibody according to any one of (13) to (18), comprising the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 20 Heavy chain, and a light chain comprising the amino acid sequence described in amino acid numbers 21 to 238 in sequence identification number 27;

(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) A polynucleotide encoding the antibody or the functional fragment of the antibody according to any one of (1) to (22);

(24) The polynucleotide according to (23), which comprises a CDRH1 encoding an amino acid sequence described in SEQ ID NO: 7, a CDRH2 including an amino acid sequence described in SEQ ID NO: 8 or 21. A polynucleotide comprising CDRH3 comprising the amino acid sequence described in SEQ ID NO: 9; and CDRL2 comprising the amino acid sequence described in SEQ ID NO: 12 and CDRL2 including the amino acid sequence described in SEQ ID NO: 13 And a polynucleotide comprising CDRL3 comprising the amino acid sequence described in SEQ ID NO: 14;

(25) The polynucleotide according to (23) or (24), which comprises a polynucleotide selected from any one of the group consisting of the following (a) to (j): (a) an encoding comprising The polynucleotide of the variable region of the heavy chain of the amino acid sequence of SEQ ID NO: 6 and the polynucleotide of the variable region of the light chain comprising the amino acid sequence of SEQ ID NO: 11, ( b) A polynucleotide encoding a heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16, and encoding a sequence including amino acid numbers 21 to 133 in sequence identification number 23 Polynucleotide of the light chain variable region of the described amino acid sequence, (c) Polynucleoside encoding a heavy chain variable region comprising the amino acid sequence of amino acid numbers 20 to 137 in SEQ ID NO: 16 An acid, and a polynucleotide encoding a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 25, (d) encoding the amino acid number 20 including amino acid number in sequence identification 16 Polynucleotides of the heavy chain variable region of the amino acid sequence described in 137 to 137, and encoding the amino acid sequence described in SEQ ID NO: 27 including amino acid numbers 21 to 133 A polynucleotide of the light chain variable region of the amino acid sequence, (e) a polynucleotide encoding a heavy chain variable region comprising the amino acid sequence of amino acid numbers 20 to 137 in sequence identification number 18, And a polynucleotide encoding a light chain variable region comprising an amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 23, (f) encoding a sequence including amino acid numbers 20 to 137 in sequence identification number 18 Polynucleotide of the heavy chain variable region of the described amino acid sequence, and polynucleotide of the light chain variable region encoding the amino acid sequence of amino acid numbers 21 to 133 in SEQ ID NO: 25 (G) a polynucleotide encoding a heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 18, and encoding a sequence including amino acid numbers 21 to 19 in sequence identification number 27 Polynucleotide of the light chain variable region of the amino acid sequence of 133, (h) encoding of the heavy chain variable region of the amino acid sequence of amino acid numbers 20 to 137 in the sequence identification number 20 Polynucleotide and variable light chain encoding amino acid sequences comprising amino acid numbers 21 to 133 in SEQ ID NO: 23 Polynucleotide, (i) a polynucleotide encoding a heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in SEQ ID NO: 20, and encoding an amino group comprising the amino acid sequence in SEQ ID NO: 25 Polynucleotide of the light chain variable region of the amino acid sequence described in acid numbers 21 to 133, (j) A heavy chain encoding the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 20 A polynucleotide of a variable region, and a polynucleotide encoding a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 27;

(26) A performance vector containing the polynucleotide according to any one of (23) to (25);

(27) A host cell transformed by the expression vector according to (26);

(28) The host cell according to (26), wherein the host cell is a eukaryotic cell;

(29) A method for producing an antibody or a functional fragment of the antibody, comprising a step of culturing the host cell according to (27) or (28), and taking the target antibody from the culture obtained in the step Or a step of a functional fragment of the antibody;

(30) an antibody or a functional fragment of the antibody, characterized in that it is obtained by the production method according to (29);

(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) The antibody or the functional fragment of the antibody according to any one of (1) to (22), (30), and (31), comprising one or two or more selected from the group consisting of Modifications: N-bond glycosylation, O-bond glycosylation, N-terminal processing, C-terminal processing, deamination amination, aspartic acid isomerization, methionine oxidation, The addition of methionine residues at the N-terminus, the amination of proline residues, and the absence of one or two amino acids at the carboxy terminus of the heavy chain;

(33) the antibody according to (32), wherein one or two amino acids are deleted at the carboxy terminus of the heavy chain;

(34) the antibody according to (33), wherein one amino acid is deleted at the carboxyl terminus on both sides of the two heavy chains;

(35) the antibody according to any one of (32) to (34), wherein the proline residue at the carboxy terminus of the heavy chain is further amidated;

(36) A medicinal composition comprising as an active ingredient any one selected from the group consisting of: as described in any one of (1) to (22), (30) to (35) An antibody or a functional fragment of the antibody, a salt thereof, such a hydrate, a polynucleotide described in any one of (23) to (25), a performance vector described in (26), such as (27 ) Or the host cell of (28);

(37) The pharmaceutical composition according to (36), characterized in that it is a therapeutic agent for bleeding disorders;

(38) The pharmaceutical composition according to (37), wherein the hemorrhagic disease is at least one member selected from hemophilia A, hemophilia B, acquired hemophilia, and Von Willebrand's disease One

(39) The pharmaceutical composition according to (38), characterized in that the hemorrhagic disease is hemophilia A and / or hemophilia B;

(40) A method for treating a bleeding disorder, which comprises selecting an antibody or a functional fragment of the antibody, a salt thereof, or the antibody as described in (1) to (22) and (30) to (35). Any one of the hydrates is administered to the individual;

(41) The treatment method according to (40), characterized in that the hemorrhagic disease is at least any one selected from hemophilia A, hemophilia B, acquired hemophilia, and von Willich's disease;

(42) The treatment method according to (41), wherein the hemorrhagic disease is hemophilia A and / or hemophilia B;

(43) A method for treating a hemorrhagic disease, which comprises an antibody selected from the group consisting of the antibodies described in (1) to (22) and (30) to (35), a functional fragment of the antibody, a salt thereof, or A pharmaceutical composition made of at least one of these hydrates, and at least one therapeutic agent for bleeding disorders, simultaneously, individually or continuously administered to an individual;

(44) The treatment method according to (43), wherein the hemorrhagic disease is at least any one selected from the group consisting of hemophilia A, hemophilia B, acquired hemophilia, and von Willich's disease .

(45) The treatment method according to (44), wherein the hemorrhagic disease is hemophilia A and / or hemophilia B.

Figure 1A in Figure 1 shows the concentration-dependent recovery of thrombin generation (TG) in plasma derived from hemophilia A patients (FVIII-deficient plasma) by administration of purified rat anti-human PC antibody. Illustration. FIG. 1B is a graph showing the concentration-dependent recovery of thrombin generation (TG) caused by the administration of rat anti-human PC antibody in plasma (FIX-deficient plasma) derived from a patient with hemophilia B. FIG. The calculation method of the thrombin production recovery rate (%) is calculated according to the following formula.

100 × ((TG in plasm of hemophilia patients with R74-TG in plasm of hemophilia patients without R74) / (TG in normal plasma without R74- TG in plasma from hemophilia patients with R74 added))

FIG. 2 is a diagram showing the nucleotide sequence, amino acid sequence, amino acid sequence of CDRH1, CDRH2, and CDRH3 of the heavy chain variable region of R74. The underline in the nucleotide and amino acid sequences indicates the CDR sequence.

Figure 3 is a diagram showing the nucleotide sequence, amino acid sequence, amino acid sequence of CDRL1, CDRL2, CDRL3 of the cDNA encoding the light chain variable region of R74. The underline in the nucleotide and amino acid sequences indicates the CDR sequence.

FIG. 4 shows a comparison of amino acid sequences of cR74_H of the heavy chain of human chimeric anti-human PC antibody cR74, humanized antibodies hR74_H1, hR74_H2, and hR74_H12. "‧" represents the same amino acid residue as cR74_H, and the place where the amino acid residue is described represents a substituted amino acid residue.

Figure 5 is a diagram showing the nucleotide sequence and amino acid sequence of hR74_H1. The underline in the amino acid sequence indicates the CDR sequence.

Figure 6 is a diagram showing the nucleotide sequence and amino acid sequence of hR74_H2. The underline in the amino acid sequence indicates the CDR sequence.

FIG. 7 is a diagram showing the nucleotide sequence, amino acid sequence, and amino acid sequence of CDRH2 of hR74_H12. The underline in the amino acid sequence indicates the CDR sequence.

FIG. 8 is a diagram showing a comparison of amino acid sequences of cR74_L of the light chain of human chimeric anti-human PC antibody cR74, humanized antibodies hR74_L1, hR74_L2, and hR74_L4. "‧" represents the same amino acid residue as cR74_L, and the place where the amino acid residue is described represents a substituted amino acid residue. "-" Indicates the absence of the corresponding amino acid residue.

Figure 9 is a diagram showing the nucleotide sequence and amino acid sequence of hR74_L1. The underline in the amino acid sequence indicates the CDR sequence.

FIG. 10 is a diagram showing the nucleotide sequence and amino acid sequence of hR74_L2. The underline in the amino acid sequence indicates the CDR sequence.

FIG. 11 is a diagram showing the nucleotide sequence and amino acid sequence of hR74_L4. The underline in the amino acid sequence indicates the CDR sequence.

FIG. 12 is a diagram showing the nucleotide sequence encoding the human chimeric R74 heavy chain cR74_H and the amino acid sequence of the human chimeric R74 heavy chain cR74_H.

Figure 13 is a diagram showing the nucleotide sequence encoding the human chimeric R74 light chain cR74_L and the amino acid sequence of the human chimeric R74 light chain cR74_L.

Figure 14A of Figure 14 shows the thrombin generation caused by the administration of human chimeric anti-human PC antibodies cR74_IgG1-LALA, R74, and positive control antibodies in plasma derived from hemophilia A patients (FVIII-deficient plasma). (TG) Diagram of recovery action. Figure 14B shows the thrombin generation (TG) caused by the administration of human chimeric anti-human PC antibodies cR74_IgG1-LALA, R74, and positive control antibodies in plasma derived from hemophilia B patients (FVIX-deficient plasma). Diagram of recovery action.

Figure 15 is a graph showing the thrombin generation (TG) recovery effect of hR74_H12 / L1, hR74_H12 / L4, and hR74_H1 / L1 in plasma (FVIII-deficient plasma) derived from a patient with hemophilia A.

FIG. 16A in FIG. 16 is a graph showing the thrombin generation (TG) recovery effect of hR74_H1 / L1 and BAY1896502 in plasma (FVIII-deficient plasma) derived from a patient with hemophilia A. FIG. 16B is a graph showing the thrombin generation (TG) recovery effect of hR74_H1 / L1 and BAY1896502 in plasma (FIX-deficient plasma) derived from a patient with hemophilia B. FIG.

FIG. 17 is a graph showing the effect of suppressing subcutaneous hemorrhage caused by the administration of hR74_H1 / L1 in the hemorrhagic A cynomolgus monkey hemorrhagic model.

The calculation method of the subcutaneous bleeding area (%) is calculated according to the following formula.

100 × subcutaneous hemorrhage area / average of subcutaneous hemorrhage area (Control group) on day 3 (%)

A form for implementing the invention

The present invention provides an anti-PC antibody and a pharmaceutical composition containing the antibody as an active ingredient. The anti-PC antibody has an activity of binding to both PC and aPC, and inhibits the activation of PC and the activity of aPC. Therefore, the anti-PC antibody can be used for bleeding diseases. Treatment, the treatment of this bleeding disorder is to inhibit aPC from decomposing and deactivating protein S, which is its cofactor, and activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa).

Hereinafter, a preferred embodiment for implementing the present invention will be described with reference to the drawings. The embodiments described below are examples of representative embodiments of the present invention, and the scope of the present invention is not limited to the embodiments.

In the present specification, the term "gene" includes not only DNA but also mRNA, cDNA, and cRNA thereof.

In this specification, the term "polynucleotide" is used in the same meaning as a nucleic acid, and also includes DNA, RNA, a probe, an oligonucleotide, and a primer.

In this specification, "polypeptide" and "protein" are used without distinction.

In the present specification, "cell" also includes cells in animal animals and cultured cells.

In this specification, "PC" is used in the same meaning as protein C.

In this specification, "aPC" is used in the same meaning as activated protein C. aPC is an active PC produced by removing the activated peptide containing 12 amino acids present in the PC by a thrombin cleavage site.

In the present specification, the "hemorrhagic disease" is not particularly limited as long as it is a hemorrhagic disease caused by a defect, deficiency, or functional insufficiency of the blood coagulation factor, but examples include blood coagulation factor VIII (FVIII) Hemophilia A with congenital defect or insufficiency of hemorrhagic disease, Hemophilia B with congenital defect or insufficiency of hemorrhagic disease due to blood coagulation factor IX (FIX), and acquired blood Friendship disease, Feng Willi's disease.

In the present specification, "epitope" means a partial peptide or a partial three-dimensional structure of PC and / or aPC to which a specific anti-PC antibody binds. The epitopes of the aforementioned partial peptides of PC and / or aPC can be determined by methods known to those having ordinary knowledge in the technical field such as immunoassay. First, various partial structures of the antigen are prepared. In the preparation of partial structures, well-known oligonucleotide synthesis techniques can be used. For example, after using a genetic recombination technique known to those skilled in the art to produce a series of polypeptides that are shortened in sequence from the C- or N-terminus of PC and / or aPC by an appropriate length, the authors discuss After recognizing the recognition site of the antibody, a shorter peptide is synthesized, and the reactivity with these peptides is examined to determine the epitope.

In the present specification, "binding to the same epitope" means antibodies that bind to a common epitope. If the second antibody is bound to a partial peptide or a partial three-dimensional structure bound to the first antibody, it can be determined that the first antibody and the second antibody can bind to the same epitope. Also, by confirming that the second antibody system competes for the binding of the first antibody to the antigen (that is, the second antibody prevents the binding of the first antibody to the antigen), even if the sequence or structure of the specific epitope is not determined, It is determined that the first antibody and the second antibody bind to the same epitope. Furthermore, when the first antibody and the second antibody bind to the same epitope, and the first antibody has special effects such as restoration of antithrombin production or blood coagulation, the second antibody can be expected to have the same activity. Therefore, for the anti-PC antibody, by confirming that the second anti-PC antibody competes with the partial peptide bound by the anti-PC antibody of the first antibody, it can be determined that the first antibody and the second antibody system bind to the same table of PC and / or aPC Antibodies.

The “CDR” in the present specification means a complementarity determining region (CDR). There are three known CDRs in the heavy and light chains of the antibody. CDRs are also known as hypervariable regions. They are located in the variable regions of the heavy and light chains of the antibody. They are particularly highly volatile primary structures. They are at the first level of the heavy and light polypeptide chains. Structurally, they are separated in 3 places. In the present specification, with respect to the CDRs of an antibody, the CDRs of the heavy chain are labeled as CDRH1, CDRH2, and CDRH3 from the amine end of the heavy chain amino acid sequence, and the CDRs of the light chain are from the amino terminal of the light chain amino acid sequence. They are labelled CDRL1, CDRL2, CDRL3 from the side. These sites are close to each other in the three-dimensional structure and determine the specificity for the bound antigen.

In the present invention, "hybridization under severe conditions" refers to hybridization under the following conditions or equivalent conditions: hybridization is performed at 68 ° C in a commercially available hybridization solution ExpressHyb Hybridization Solution (Clontech) Or use a DNA-immobilized filter in the presence of 0.7-1.0M NaCl to perform hybridization at 68 ° C, and then use a 0.1-2 times concentration SSC solution (the so-called 1-time concentration SSC, containing 150mM NaCl, 15mM Sodium citrate), washed at 68 ° C. to identify the conditions.

    1. Protein C (PC)    

The PC line contains 461 residues of proteins that play a major role in the negative feedback mechanism in blood coagulation. The PC is detached from the signal by secretory expression, and is further cleaved by the intrinsic furin to become a heterodimer divided into a light chain and a heavy chain. This is called an inactive form of PC. When a non-activated type of PC is activated with thrombin, an activation peptide is detached to become an active PC (aPC). aPC decomposes and deactivates protein S, its cofactor, and activated blood coagulation factor VIII (FVIIIa) and activated blood coagulation factor V (FVa).

The PC and / or aPC used in the present invention can be directly purified and used from PC and / or aPC expressing cells of human and non-human mammals (rats, mice, etc.); or a cell membrane fraction of the cells can be prepared (fraction) ) And can be obtained by synthesizing PC and / or aPC in vitro or generating host cells by genetic manipulation. In addition to a full-length protein of a PC protein, a partial peptide including a partial sequence of a PC and / or aPC protein may be used as long as an anti-PC antibody is available. Examples of such partial peptides include the polypeptides described in SEQ ID NO: 3 in the Sequence Listing. Furthermore, an aPC partial peptide obtained by treating the polypeptide with thrombin can also be used as a suitable antigen.

In addition, the PC and / or aPC expressing cells or the cell lines expressing PC and / or aPC caused by the genetic manipulation can be used as the PC and / or aPC protein. Alternatively, the expression vector of the cDNA incorporated into the PC or a partial peptide of the PC may be directly administered to the animal to be immunized, and PC, aPC or a partial peptide of the peptide may be expressed in the body of the animal to be immunized.

The DNA sequence and amino acid sequence of a human PC are disclosed on a public database. For example, a reference number such as the accession number of the protein database of NCBI (NP_000303) can be referred to.

In addition, the PC also includes a protein including an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence of the PC, and has a biological activity equivalent to the protein. The amino acid sequence of human PC is described in SEQ ID NO: 1 in the Sequence Listing. The amino acid sequences shown in amino acid numbers 1 to 18 in sequence identification number 1 are message sequences, 19 to 42 are prepro-sequences, and the amino acid sequences shown in amino acid numbers 200 to 211 ( DTEDQEDQVDPR) shows the sequence of the activated peptide. In addition, the aPC used in the present invention is a polypeptide comprising amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the Sequence Listing and a peptide including amino acid numbers 166 to 412 with SS bonds.

    2. Manufacture of anti-PC antibodies    

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 sequence identification number 1 of the sequence listing can be mentioned. As another example of the anti-PC antibody of the present invention, an antibody that recognizes a higher-order structure of a polypeptide including an amino acid sequence represented by amino acid numbers 43 to 460 of sequence identification number 3 of the sequence listing can be mentioned. As another example of the anti-PC antibody of the present invention, peptides containing amino acid numbers 43 to 153 that recognize the polypeptide shown in sequence identification number 3 of the Sequence Listing and peptides containing amino acid numbers 154 to 412 can be listed as SS bonds Antibodies of higher order to the bound polypeptide. As another example of the anti-PC antibody of the present invention, peptides containing amino acid numbers 43 to 153 that recognize the polypeptide shown in sequence identification number 3 of the sequence listing and peptides containing amino acid numbers 166 to 412 can be listed as SS bonds. Antibodies of higher order to the bound polypeptide. As another example of the anti-PC antibody of the present invention, peptides containing amino acid numbers 43 to 153 that recognize the polypeptide shown in sequence identification number 3 of the Sequence Listing and peptides containing amino acid numbers 154 to 412 can be listed as SS bonds. The higher order structure of the bound polypeptide, and the peptides containing amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 of the Sequence Listing and the peptides containing amino acid numbers 166 to 412 with SS bonds Substructure antibodies.

The anti-PC antibody of the present invention may be derived from any species, but preferably includes humans, rats, mice, and rabbits. When it is derived from a species other than humans, chimerization or humanization is preferably performed using a known technique. The antibody of the present invention may be a multiple antibody or a single antibody, but is preferably a single antibody.

The anti-PC anti-system of the present invention can use PC and aPC as the target antibodies, that is, it can recognize the characteristics of PC and aPC, inhibit the activation of PC, inhibit the protein S that aPC will be its cofactor, and activate blood coagulation. Factor (FVIIIa) and / or activated blood coagulation factor V (FVa) activity to decompose and / or deactivate. As described above, the anti-PC antibody of the present invention has an activity of restoring thrombin production, has a blood coagulation effect, shows a hemostatic effect in a subject administered the antibody, and can be used as a therapeutic agent for bleeding disorders. Furthermore, the anti-PC antibody system of the present invention recognizes an aPC antibody, which comprises a peptide having amino acid numbers 43 to 153 of the polypeptide shown in sequence identification number 3 of the Sequence Listing and a peptide containing amino acid numbers 166 to 412. The peptide is bound to aPC by the SS bond.

The binding of the antibody to PC and / or aPC can be confirmed using, for example, flow cytometry.

An anti-PC antibody can be obtained by immunizing an animal with a polypeptide that becomes an antigen by using a method generally performed in this field, and taking and purifying the antibody produced in the body.

The source of the antigen is not limited to humans, and animals derived from animals other than humans such as mice and rats may be immunized. In this case, antibodies that can be applied to human diseases can be screened by testing the cross-reactivity between the antibody bound to the obtained heterogeneous antigen and the human antigen.

It is also possible to follow well-known methods (for example, Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennet, R.ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY ( 1980)), the antibody-producing cells that produce antibodies to the antigen are fused with myeloma cells, and a fusion tumor is established to obtain a single antibody.

Hereinafter, a method for obtaining antibodies to PC will be specifically described.

    (1) Preparation of antigen    

Antigens can be obtained by genetically manipulating genes encoding antigen proteins to produce antigens in host cells. Specifically, it is only necessary to prepare a vector capable of expressing an antigen gene, introduce it into a host cell, express the gene, and refine the expressed antigen. In the case of using aPC as the antigen, the antibody can also be obtained by using the following method: further treating with an enzyme (such as thrombin), and using the antigen-expressing cells or the cell line expressing the antigen as aPC by the above-mentioned genetic manipulation The method of immunizing animals.

In addition, the cDNA of the antigen protein may be incorporated into a expression vector without using the antigen protein, and administered to the animal to be immunized, the antigen protein may be expressed in the body of the animal to be immunized, and antibodies may be obtained for the antigen protein.

    (2) Manufacturing of anti-PC monoclonal antibodies    

The anti-PC antibody used in the present invention is not particularly limited, but an antibody specified by, for example, an amino acid sequence shown in the sequence listing of the present application can be preferably used. The anti-PC antibody used in the present invention is preferably one having the following properties.

(a) An antibody characterized by having at least one characteristic selected from the following (a-1) to (a-4): (a-1) specifically binds to PC and aPC; (a-2) is specific to PC (A-3) specifically binds to aPC and inhibits the decomposition of activated blood coagulation factor VIII (FVIIIa) and / or activated blood coagulation factor V (FVa) caused by aPC and / Or deactivation; (a-4) restore thrombin production.

(b) The antibody or the antibody according to (a) above, wherein PC and aPC are human PC and human aPC.

(c) The antibody as described in (a) or (b), which recognizes human PCs containing amino acid numbers 43 to 153 of the polypeptide shown in SEQ ID NO: 3 and peptides containing amino acid numbers 166 to 412 Higher order structure of peptides bound by SS bonds.

The method for obtaining an antibody against PC of the present invention is not particularly limited as long as an anti-PC antibody can be obtained.

Specific examples of obtaining monoclonal antibodies include the following.

(a) Assemble the cDNA of the PC into a expression vector (for example, pcDNA3.3-TOPO / LaxZ (ThermoFisher SCIENTOFIC)) and transfect it into FreeStyle 293F cells (ThermoFisher SCIENTOFIC), thereby temporarily expressing it from the culture supernatant. Fractions containing PC were recovered, and the recovered PC was treated with thrombin to form aPC, which was administered to an animal to induce an immune response (for example, WKY / Izm female rats).

(b) From the above-mentioned animal that has induced an immune response, a tissue (for example, a lymph node) containing antibody-producing cells is taken.

(c) Preparation of myeloma cells (hereinafter referred to as "myeloma") (for example, mouse myeloma SP2 / 0-ag14 cells).

(d) Fusion of antibody-producing cells with myeloma cells.

(e) Screening of a fusion tumor population that produces an antibody of interest.

(f) Segmentation (cloning) to a single cell clone.

(g) Depending on the circumstances, the culture of fusion tumors used for the production of a single antibody in large quantities, or the breeding of animals with transplanted fusion tumors.

(h) Investigation of the physiological activity and binding specificity of the single-body antibody thus produced, or examination of the characteristics as a labeled reagent.

Examples of the measurement method of the antibody valence used herein include, but are not limited to, such methods as flow cytometry and Cell-ELISA.

As an example of the fused tumor strain thus established, an anti-PC antibody produces a fusion tumor R74. In this specification, an antibody produced by an anti-PC antibody producing fusion tumor R74 is described as "R74 antibody" or only "R74". R74 antibody has the 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. This variable region has a CDRH1 comprising an amino acid sequence consisting of the 26th to 35th amino acid residues in the sequence identification number 6 of the Sequence Listing, and comprises 50 to 58 amino acid residues. CDRH2 of the amino acid sequence, CDRH3 including the amino acid sequence consisting of the 98th to 107th amino acid residues. The CDRH1 of the R74 antibody has the amino acid sequence shown in sequence identification number 7 of the sequence listing, the amino acid sequence of CDRH2 has the amino acid sequence shown in sequence identification number 8 of the sequence listing, and the amino acid sequence of CDRH3 has the sequence The amino acid sequence shown in Sequence ID 9 is listed. The amino acid sequences of the heavy chain variable region, CDRH1, CDRH2, and CDRH3 of the R74 antibody are shown in FIG. 2.

The variable region of the light chain of the R74 antibody has the amino acid sequence shown in SEQ ID NO: 11 in the Sequence Listing. This variable region has a CDRL1 including an amino acid sequence consisting of the 23rd to 37th amino acid residues in the sequence identification number 11 of the sequence listing, and includes a sequence consisting of the 53rd to 59th amino acid residues. CDRL2 of amino acid sequence, CDRL3 including amino acid sequence consisting of 92 to 100 amino acid residues. The CDRL1 of the R74 antibody has the amino acid sequence shown in Sequence ID No. 12 in the Sequence Listing, the amino acid sequence of CDRL2 has the amino acid sequence shown in Sequence ID No. 13 in the Sequence Listing, and the amino acid sequence of CDRL3 has the sequence The amino acid sequence shown in Sequence ID No. 14 is listed. The amino acid sequences of the light chain variable region, CDRL1, CDRL2, and CDRL3 of the R74 antibody are shown in FIG. 3.

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 the sequence identification number 5 is described in FIG. 2.

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 in the Sequence Listing. The sequence of the sequence identification number 10 is shown in FIG. 3.

Furthermore, in the case where the steps of (3) to (h) of "3. Production of anti-PC antibody" are performed again and a single antibody is obtained separately or when a single antibody is obtained by another method, An antibody having the same activity as the R74 antibody can be obtained. Examples of such antibodies include antibodies that bind to the same epitope as the R74 antibody. As long as the newly produced single antibody binds to a partial peptide or a partial three-dimensional structure bound to the R74 antibody, it can be determined that the single antibody and the R74 antibody bind to the same epitope. Also, by confirming that the monoclonal antibody competes with R74 antibody for binding to PC or aPC (that is, the monoclonal antibody prevents the binding of R74 antibody to PC or aPC), even if the sequence of a specific epitope or The structure can also be determined that the monoclonal antibody and the anti-PC antibody bind to the same epitope. When the epitopes are confirmed to be the same, the monoclonal antibody is strongly expected to have the same antigen-binding ability and biological activity as the R74 antibody.

    (3) Other antibodies    

The antibodies of the present invention include not only the above-mentioned monoclonal antibodies to PC, but also genetically recombinant antibodies that have been artificially altered for the purpose of reducing heterogeneous antigenicity to humans, such as chimeric antibodies, humans, and the like. Humanized antibodies, human antibodies, and the like. These antibodies can be produced using known methods.

Examples of the chimeric antibody include antibodies in which the variable region and the constant region of the antibody are mutually different. For example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is joined to a human-derived constant region (See Proc. Natl. Acad. Sci. USA, 81,6851-6855, (1984)).

A chimeric antibody derived from a rat anti-human PC antibody R74 antibody is an antibody comprising the following heavy and light chains, and may have any human-derived constant region: a heavy chain comprising a heavy chain variable region, the The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 6; and the light chain including the light chain variable region, which contains the amino acid sequence shown in SEQ ID NO: 11.

A specific example of the chimeric antibody derived from the rat anti-human PC antibody R74 antibody includes a chimeric antibody cR74 derived from the rat anti-human PC antibody R74 antibody. The amino acid sequence of the cR74 antibody includes: a heavy chain having an amino acid sequence including the 20th to 467 amino acid residues including sequence identification number 33 of the sequence listing, and 21 having a sequence identification number 30 of the sequence listing Light chain to amino acid sequence to 237. In addition, in the heavy chain sequence shown by sequence identification number 33 in the Sequence Listing, the amino acid sequence consisting of the 1 to 19 amino acid residues is a message sequence, and the 20 to 137 amino acid residues The amino acid sequence composed of the amino group is a variable region, and the amino acid sequence composed of the 138th to 467th residues is a constant region. The sequence of the sequence identification number 33 is shown in FIG. In the light chain sequence shown by sequence identification number 30 in the Sequence Listing, the amino acid sequence composed of the 1st to 20th amino acid residues is a message sequence, and the 21st to 132th amino acid residues The amino acid sequence composed of amino groups is a variable region, and the amino acid sequence composed of 133 to 237 amino acid residues is a constant region. The sequence of the sequence identification number 30 is described in FIG. 13.

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 the 1st to 57th nucleotides of the nucleotide sequence shown in SEQ ID NO: 32 in the Sequence Listing is a message sequence encoding the cR74 antibody and is composed of 58th to 411th nucleotides. The nucleotide sequence is the heavy chain variable region sequence of the cR74 antibody, and the nucleotide sequence consisting of 412 to 1401 nucleotides is the heavy chain constant region of the cR74 antibody. The sequence of the sequence identification number 32 is shown 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 is a message sequence encoding the cR74 antibody and is composed of the 61st to 396th nucleotides The nucleotide sequence is the light chain variable region sequence of the cR74 antibody, and the nucleotide sequence consisting of nucleotides 397 to 711 is the light chain constant region of the cR74 antibody. The sequence of the sequence identification number 29 is described in FIG. 13.

Examples of humanized antibodies include antibodies that combine only complementarity determining regions (CDRs) with human-derived antibodies (see Nature (1986) 321, p. 522-525); other than CDR sequences A portion of the amino acid residues of the framework was also transplanted to the antibody of the human antibody by the CDR transplantation method (International Publication No. 90/07861); and a portion of the amino acid residues were changed while maintaining the ability to bind to the antigen. Antibodies to CDR amino acid sequences.

However, as a humanized antibody derived from an R74 antibody, as long as it retains all six CDR sequences of the R74 antibody and has the activity of restoring thrombin production, it is not limited to a specific humanized antibody; moreover, only a part of it needs to be changed At the same time as the amino acid sequence of the CDR, it has the ability to bind to PC and aPC and has the activity of restoring thrombin production, it is not limited to a specific humanized antibody. The heavy chain variable region of the aforementioned humanized antibody retains CDRH1 (GFSLTGYGVS) composed of the amino acid sequence shown in SEQ ID NO: 7 and CDRH2 composed of the amino acid sequence shown in SEQ ID NO: 8 (AVWRGGSKD) and CDRH3 (SGPEGTPFDY) consisting of the amino acid sequence shown in SEQ ID NO: 9. The light chain variable region of the aforementioned human antibody retains CDRL1 (KTNQNVDFYGNSYIH) composed of the amino acid sequence shown in SEQ ID NO: 12 and CDRL2 (SASNLAS) composed of the amino acid sequence shown in SEQ ID NO: 13 ) And CDRL3 (QQSRNLPNT) consisting of the amino acid sequence shown in SEQ ID NO: 14.

Examples of humanized antibodies to rat antibody R74 include any combination of the following heavy and light chains: the heavy chain includes a heavy chain variable region, and the heavy chain variable region includes any of the following: (1) the amino acid sequence including the 20 to 137 amino acid residues of the sequence identification number 16 or 18 of the sequence listing, (2) the amino acid sequence of the above (1) has at least 95% identity A source amino acid sequence, and (3) the amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (1) above; the light chain includes a light chain variable region The light chain variable region comprises any of the following: (4) an amino acid sequence comprising the 21st to 133th amino acid residues of the sequence identification number 23, 25 or 27, (5) for the above ( 4) an amino acid sequence having at least 95% homology to the amino acid sequence, and (6) one or more amino acids in the amino acid sequence of (4) above being deleted, substituted or added Amino acid sequence.

Examples of a more suitable humanized antibody for the rat antibody R74 include antibodies (hR74_H1 / L1) containing the following heavy and light chains: amino acid numbers 20 to 467 including the sequence identification number 16 of the sequence listing A heavy chain of amino acid residues, a light chain containing amino acid numbers 21 to 238 of amino acid residues including sequence identification number 23 of the Sequence Listing.

The antibody of the present invention also includes an antibody that further introduces a mutation into the CDR of the humanized antibody to reduce the viscosity of the antibody. As a specific example of such a change in CDR, it is possible to cite changes in the third and fourth amino acid residues of CDRH2 (AVWRGGSKD: sequence identification number 8) derived from R74 antibody (AVYTGGSKD: sequence identification number 21). . That is, the humanized antibody of the present invention also includes a humanized antibody in which only the sequence of CDRH2 is replaced with a sequence of CDRH2 composed of the amino acid sequence shown in SEQ ID NO: 21 in the humanized antibody. .

As further specific examples of such antibodies, any combination of the following heavy and light chains may be mentioned; the heavy chain includes a heavy chain variable region, and the heavy chain variable region includes any one of the following: a sequence including a sequence listing The amino acid sequence of the 20th to 137th amino acid residues of the amino acid sequence shown in the identification number 20, (2) has at least 95% homology to the amino acid sequence of the above (1) The amino acid sequence, and (3) the amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of (1) above; the light chain includes a light chain variable region, and the light chain The chain variable region includes any of: (4) an amino acid sequence including the 21st to 133th amino acid residues of sequence identification numbers 23, 25, or 27, (5) An amino acid sequence having at least 95% homology to the amino acid sequence, and (6) the amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (4) above sequence.

Examples of a more specific humanized antibody include a heavy chain including an amino acid number 20 to 467 amino acid residues including the sequence identification number 20 of the sequence listing, and a sequence identification number 23 of the sequence listing. Antibody with a light chain of amino acid residues 21 to 238 (hR74_H12 / L1); contains an amino acid residue containing the amino acid sequence number 20 to 467 of the sequence listing Heavy chain and an amino acid numbering sequence of SEQ ID NO: 25 with an amino acid number of 21 to 238 amino acid residues of the light chain antibody (hR74_H12 / L2); and an amine containing the sequence number of SEQ ID NO: 20 Antibody to heavy chain of amino acid residues 20 to 467 amino acid residues and light chain of amino acid residues 21 to 238 amino acid residues containing sequence identification number 27 of the Sequence Listing (hR74_H12 / L4) .

Furthermore, an antibody that humanizes one of the heavy or light chains and uses the other as the light or heavy chain of a rat or chimeric antibody can also be used.

In addition, the term "several" in this specification 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.

As the substitution of the amino acid in the present specification, a substituted amino acid is preferred. The so-called retained amino acid substitution refers to a substitution generated in an amino acid group associated with an amino acid side chain. Suitable amino acid groups are as follows: acidic group = aspartic acid, glutamic acid; basic group = lysine, arginine, histamine; non-polar group = alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Methionine, Tryptophan; and Non-Polarized Family = Glycine, Aspartic Acid, Glutamine , Cysteine, serine, threonine, tyrosine. Other suitable amino acid groups are described below: Aliphatic hydroxyl group = serine and threonine; groups containing ammonium = aspartic acid and glutamic acid; aliphatic groups = Alanine, valine, leucine, and isoleucine; and aromatic groups = phenylalanine, tryptophan, and tyrosine. Such amino acid substitution is preferably performed within a range that does not reduce the characteristics of a substance having an ortho amino acid sequence.

By combining sequences that exhibit high homology with the heavy-chain amino acid sequence and the light-chain amino acid sequence, it is possible to select an antibody having a biological activity equivalent to that of each of the antibodies. Such homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more . In addition, by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, or added in the amino acid sequence of the heavy or light chain, it is also possible to select an antibody having the same amino acid sequence as that of each of the above antibodies. Biologically active antibodies.

The homology between the two amino acid sequences can be achieved by using 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) were determined by default parameters. The Blast algorithm can also be used by accessing www.ncbi.nlm.nih.gov/blast over the Internet.

Examples of the antibody of the present invention include human antibodies that bind to PC and aPC. The so-called anti-PC human antibody means a human antibody having only a gene sequence of an antibody derived from a human chromosome. Anti-PC human antibodies can be obtained by using a human antibody-producing mouse that has human chromosome fragments containing genes of the heavy and light chains of human antibodies (see Tomizika, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y. et.al., Nucl. Acids Res. (1998) 26, p. 3447-3448; Yosida, 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, etc.).

In such human antibody-producing mice, specifically, the loci that are intrinsic immunoglobulin heavy and light chains are destroyed, and human immunoglobulin is introduced via a yeast artificial chromosome (YAC) vector or the like instead. Genetically modified animals made up of heavy chain and light chain loci can be made by making knockout animals and transgenic animals, and mating these animals with each other.

In addition, by means of genetic recombination technology and cDNAs encoding the heavy and light chains of such human antibodies, preferably vectors containing the cDNA, the eukaryotic cells are transformed and cultured to produce recombinant humans. The transformed cells of a single antibody can also be used to obtain this antibody from the culture supernatant.

Here, as the host, for example, eukaryotic cells can be used, preferably mammalian cells such as CHO cells, lymphocytes, or myeloma.

In addition, a method for obtaining human antibodies derived from phage display screened from a human antibody library is also known (see Wormstone, Imet.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.).

For example, a phage display method can be used, which displays the variable region of a human antibody as a single chain antibody (scFv) on the surface of the phage and selects phages that bind to the antigen (Nature Biotechnology (2005), 23, (9), p .1105-1116).

The genes of the phage selected by binding to the antigen will be analyzed to determine the DNA sequence encoding the variable region of the human antibody that binds to the antigen.

If the DNA sequence of the scFv bound to the antigen becomes clear, a human antibody can be obtained by preparing a expression vector having the sequence and introducing it into an appropriate host for its expression (International Publication No. 92/01047, International Publication No. 92/20791, International Publication No. 93/06213, International Publication No. 93/11236, International Publication No. 93/19172, International Publication No. 95/01438, International Publication No. 95/15388, Annu. Rev Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116).

As long as the newly produced human antibody binds to a partial peptide or a partial three-dimensional structure bound to the R74 antibody described in this specification, it can be determined that the human anti-system and the R74 antibody bind to the same epitope. Also, by confirming that the binding of the human antibody to the R74 antibody to PC or aPC is competitive (that is, the human antibody prevents the binding of the R74 antibody to PC or aPC), even if the sequence or structure of the specific epitope is not determined It can also be determined that the human antibody and the R74 antibody bind to the same epitope. When the epitopes are confirmed to be the same, it is strongly expected that the human antibody has the same biological activity as that of the R74 antibody.

The chimeric antibody, humanized antibody, or human antibody obtained by the above method can be used to evaluate the binding property to the antigen by a well-known method and the like, and select a preferable antibody.

As an example of other indexes when comparing the properties of antibodies, the stability of antibodies can be cited. The differential scanning calorimetry (DSC) is a device that can quickly and accurately measure the thermally modified midpoint (Tm). This thermally modified midpoint (Tm) is a suitable indicator of the relative structural stability of the protein. The DSM is used to measure the Tm value and compare the values to compare the differences in thermal stability. It is known that the storage stability of antibodies shows a certain degree of correlation with the thermal stability of antibodies (Lori Burton, et.al., Pharmaceutical Development and Technology (2007) 12, p. 265-273), and thermal stability can be used as an indicator And pick the better antibody. Other indicators for selecting antibodies include high yield in an appropriate host cell and low agglutination in an aqueous solution. For example, the antibody with the highest yield does not necessarily show the highest thermal stability. Therefore, it is necessary to make a comprehensive judgment based on the above-mentioned indicators and select the antibody that is most suitable for administration to humans.

The antibodies of the present invention also include modified products of the antibodies. The modification refers to a chemically or biologically modified antibody of the present invention. The chemical modification includes a combination of a chemical moiety of an amino acid skeleton, a chemical modification of an N-bond or an O-bond carbohydrate chain, and the like. Biological modifications include post-translational modifications (e.g., N- or O-bond glycosylation, N- or C-terminal processing, deamination, aspartic acid isomerization, formazan Oxidizing thioic acid), using a prokaryotic host cell to express it, and adding a methionine residue at the N-terminus. In addition, those who are labeled so that the antibody or antigen of the present invention can be detected or separated, for example, an enzyme label, a fluorescent label, and an affinity label are also included in the meaning of this modifier. Such a modified product of the antibody of the present invention is useful for stability of the antibody, improvement in blood retention, reduction of antigenicity, detection or separation of the antibody or antigen, and the like.

In addition, by regulating the sugar chain modification (saccharification, de-saccharification, etc.) that binds to the antibody of the present invention, the antibody-dependent cytotoxic activity can be enhanced. As techniques for regulating sugar chain modification of antibodies, International Publication No. 1999/54342, International Publication No. 2000/61739, International Publication No. 2002/31140, International Publication No. 2007/133855, and the like are known, but are not limited to Wait. The antibodies of the present invention also include antibodies that modulate the sugar chain modification.

In the case where the antibody gene is temporarily isolated and then introduced into an appropriate host to prepare an antibody, a combination of an appropriate host and a expression vector can be used. Specific examples of the antibody gene include a combination of a gene encoding a heavy chain sequence of an antibody described in the present specification and a gene encoding a light chain sequence. When the host cell is transformed, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or they can be inserted into different expression vectors, respectively.

In the case of using eukaryotic cells as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used. In particular, mammalian cells are exemplified as animal cells, for example, COS cells (Gluzman, Y. Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblast cells NIH3T3 (ATCC No.CRL-1658), a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Urlaub, G. and Chasin, LAProc. Natl. Acad. Sci. USA (1980) 77 p. 4126-4220), FreeStyle 293F cells (Invitrogen).

When a prokaryotic cell is used, for example, E. coli and Bacillus subtilis are mentioned.

The target antibody gene is introduced into these cells by transformation, and the transformed cells are cultured in vitro to obtain antibodies. In this culture, the yield may vary depending on the sequence of the antibody, and it may be easier to select the yield as an indicator from antibodies with equivalent binding activity and use it as an index for the production of medicine. Therefore, the antibody of the present invention also includes an antibody obtainable by a method for producing an antibody, and the method for producing the antibody is characterized by including the following steps: a step of culturing the transformed host cell described above; and the obtained from this step The culture takes the steps of the antibody of interest or a functional fragment of the antibody.

In addition, it is known that amino acid residues at the carboxyl terminus of the heavy chain of antibodies produced by mammalian culture cells are deleted (Journal of Chromatography A, 705: 129-134 (1995)). Two amino acid residues of glycine and lysine were deleted, and the proline residue newly located at the carboxyl terminal was aminated with amidine (Analytical Biochemistry, 360: 75-83 (2007)). However, the deletion and modification of these heavy chain sequences does not affect the antibody's antigen-binding ability and effector function (activation of complement, antibody-dependent cellular cytotoxicity, etc.). Therefore, the antibody of the present invention also includes the antibody that accepts the modification and the functional fragment of the antibody, and also includes the deletion body that lacks one or two amino acids at the carboxy terminus of the heavy chain, and the deletion body that has been amidated. (For example, a sulfonated heavy chain of a proline residue at the carboxyl terminal site). However, as long as the antigen-binding ability and effector function are retained, the carboxy-terminal deletion of the heavy chain of the antibody of the present invention is not limited to the above-mentioned species. The two heavy chains constituting the antibody of the present invention may be any one selected from the group consisting of a full length and the above-mentioned deletion body, or any combination of the two. The amount ratio of each deletion body may be affected by the type and culture conditions of the mammalian culture cells that produce the antibody of the present invention, but the main component of the antibody of the present invention includes one amine group at the carboxyl terminus of both heavy chains. A case where an acid residue is missing.

Examples of the isotype of the antibody used in the present invention include IgG (IgG1, IgG2, IgG3, IgG4), and the like, and preferably, IgG1 or IgG2 is mentioned.

The biological activity of an antibody generally includes an antigen-binding activity, an activity that inhibits the antigen activity by binding to the antigen, an activity that neutralizes the antigen activity, an activity that enhances the antigen activity, and an antibody-dependent cytotoxicity (ADCC) activity. , Complement-dependent cytotoxicity (CDC) activity and antibody-dependent cellular phagocytosis (ADCP), etc., the function of the anti-PC antibody of the present invention is the binding activity to PC and aPC, preferably In order to inhibit PC activation by binding to PC. In addition, as a preferred activity of the anti-PC antibody of the present invention, there can be mentioned protein S that binds to aPC and inhibits aPC as its cofactor, and activates blood coagulation factor VIII (FVIIIa) and activates blood coagulation factor V (FVa ) Decomposition and deactivation activity. Other preferable activities of the anti-PC antibody of the present invention include an activity that inhibits an anticoagulant effect caused by aPC, a blood coagulation activity, or a thrombin production restoration activity.

The obtained antibodies can be purified to homogeneity. The isolation and purification of the antibodies may be performed by using the isolation and purification methods commonly used for proteins. For example, as long as column chromatography, filter filtration, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric point electrophoresis, etc. are appropriately selected and combined, 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)) , But not limited to these.

Examples of the chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.

These chromatography methods can be performed using liquid chromatography methods such as HPLC or FPLC.

Examples of the column used in the affinity chromatography include a protein A column and a protein G column. Examples of the column using a protein A column include Hyper D, POROS, Sepharose F.F. (Pharmacia), and the like.

Alternatively, an antibody can be purified by using an antigen-immobilized carrier and utilizing its binding property to the antigen.

    4. Medicine    

The anti-PC antibody of the present invention described in the item "2. Production of anti-PC antibody" and the functional fragments of the antibody as described in the examples, and a polynucleotide comprising a nucleotide sequence encoding these amino acid sequences Medicines containing any one or more of expression vectors and host cells as active ingredients, inhibits PC activation and inhibits aPC production, and / or binds to aPC and inhibits aPC activity. As a result, thrombin production (TG) is restored and blood is displayed. The coagulation effect can be used alone or in combination with other agents as a therapeutic agent for hemorrhagic diseases such as hemophilia, hemophilia A, and hemophilia B.

The anti-PC antibody of the present invention can be used for the detection of PC in plasma.

The anti-PC antibody of the present invention may be hydrated by absorbing moisture or adhering to water due to being left in the air or undergoing recrystallization or purification operations. The present invention also includes such compounds containing water or Pharmacologically acceptable salt.

When the anti-PC antibody of the present invention has a basic group such as an amine group, it is expected that a pharmacologically acceptable acid addition salt can be formed. Examples of such acid addition salts include hydrohalates such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodate; inorganic salts such as nitrate, perchlorate, sulfate, and phosphate Acid salts; lower alkane sulfonates such as mesylate, triflate, ethanesulfonate; aryl sulfonates such as benzene sulfonate, p-toluene sulfonate; formate, acetate, Organic acid salts such as trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate; or ornithine, bran Amino acid salts such as amine acid salts and aspartic acid salts.

When the anti-PC antibody of the present invention has an acidic group such as a carboxyl group, it is expected that a pharmacologically acceptable base addition salt can be formed. Examples of such alkali addition salts include alkali metal salts such as sodium, potassium, and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; inorganic salts such as ammonium salts; dibenzylamine salts, Porphyrin salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine Amine salt, N, N'-dibenzyl vinylamine salt, diethanolamine salt, N-benzyl-N- (2-phenylethoxy) amine salt, piperazine Organic amine salts such as salts, tetramethylammonium salts, and gins (hydroxymethyl) aminomethane salts.

The present invention can further include an anti-PC antibody in which one or more atoms constituting the antibody are substituted with isotopes of the atoms. There are two types of radioisotopes and stable isotopes. Examples of isotopes include hydrogen isotopes ( ² H and ³ H), carbon isotopes ( ¹¹ C, ¹³ C, and ¹⁴ C), and nitrogen isotopes ( ¹³ N and ¹⁵ N), oxygen isotopes ( ¹⁵ O, ¹⁷ O, and ¹⁸ O), fluorine isotopes ( ¹⁸ F), and the like. The composition containing an antibody labeled with an isotope is useful as, for example, a therapeutic agent, a preventive agent, a research reagent, an analytical reagent, a diagnostic agent, an in-vivo imaging diagnostic agent, and the like. All isotopically labeled antibodies and mixtures of the isotopically labeled antibodies in any ratio are also included in the present invention. An isotope-labeled antibody can be produced by a method known in the art, for example, by using an isotope-labeled raw material in place of the raw material in the production method of the present invention described later.

The TG recovery activity in vitro can be confirmed, for example, by adding anti-PC antibodies at various concentrations to the plasma derived from patients with hemophilia A or hemophilia B to recombinant Tissue factor: rTF) was cultured for an appropriate period of time, and then an appropriate amount of FluCa-kit (2.5 mM Z-Gly Gly-Arg-amine methylcoumarin (Z-GGR-AMC), 100 mM CaCl ₂ ) was added for reaction, and TG was investigated. Recovery effect.

The therapeutic effect of experimental animals in vivo on hemophilia can be confirmed by, for example, the following: for the onset of temporary hemophilia A caused by the administration of anti-human FVIII neutralizing antibodies Cynomolgus macaques were administered anti-PC antibodies, and they were wounded under anesthesia. The area of subcutaneous bleeding in the wound was observed over time, and compared with the case where anti-PC antibodies were not administered.

The type of bleeding disease to which the anti-PC antibody of the present invention is applied is not particularly limited as long as it is a disease related to PC and / or aPC in the subject to be treated and the bleeding disease, but examples thereof include hemophilia A, blood Friend B, as well as acquired hemophilia, Feng Willie's disease. Although the anti-PC of the present invention can be suitably administered to mammals, it 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 other formulation additives commonly used in this field in the amount and concentration to be administered.

The anti-PC antibody of the present invention can be administered as a pharmaceutical composition containing one or more pharmaceutically suitable ingredients. For example, the pharmaceutical composition described above typically includes more than one pharmaceutical carrier (e.g., a sterilized liquid (e.g., water and oil (including oil derived from petroleum, animal, plant, or synthetic) (e.g., Peanut oil, soybean oil, mineral oil, sesame oil, etc.))). In the case of intravenous administration of the above pharmaceutical composition, water is a more representative carrier. Aqueous saline solution, and dextrose aqueous solution and glycerol aqueous solution It can also be used as a liquid carrier, especially for injectable solutions. Appropriate pharmaceutical excipients are well known in the art. Moreover, the above-mentioned composition can contain trace amounts of wetting or emulsifying agents, if desired. Or pH buffer. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. The formulation corresponds to the state of administration.

Various delivery systems are well known and can be used for administering the anti-PC antibody of the present invention. Examples of the introduction method include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In a specific preferred embodiment, the antibody is administered by infusion. Non-oral administration is the preferred route.

In a typical embodiment, the pharmaceutical composition is formulated as a pharmaceutical composition suitable for intravenous administration to humans, and is formulated according to a customary procedure. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. Where necessary, the above medicine may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) for relieving pain at the injection site. In general, the above ingredients are, for example, ampoules or sachets sealed to show the amount of active agent, etc., as dry freeze-dried powders or anhydrous concentrates in sealed containers, individually or in unit dosage forms Either mix it together. In the case where the above medicine is intended to be administered by infusion, it can be administered, for example, in an infusion bottle containing sterilized pharmaceutical-grade water or saline. When the above medicine is administered by injection, an ampoule of sterilized water for injection or saline can be provided, for example, so that the above-mentioned ingredients can be mixed before administration.

The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the anti-PC antibody of the present case, or a pharmaceutical composition containing the anti-PC antibody and at least one other therapeutic agent for bleeding disorders. The anti-PC antibody of the present invention can also be administered together with other therapeutic agents for bleeding disorders, thereby enhancing the therapeutic effect on bleeding disorders. Other therapeutic agents for bleeding disorders used for this purpose may be administered to an individual simultaneously, separately, or continuously with the antibodies, or the administration intervals may be changed and administered. Examples of therapeutic agents for such bleeding disorders include hemophilia therapeutic agents (coagulation factor preparations, antibodies, nucleic acid medicines, gene therapy preparations, etc.), tranexamic acid, epinephrine, etc. The agent for preventing bleeding is not limited.

Such a pharmaceutical composition may be formulated as a lyophilized preparation or a liquid preparation as a preparation having a selected composition and necessary purity. When it is formulated as a freeze-dried preparation, it may be a preparation containing an appropriate preparation additive used in this field. In addition, it can be similarly performed in a liquid preparation, and can be formulated into a liquid preparation containing various preparation additives used in this field.

The composition and concentration of the pharmaceutical composition also vary depending on the method of administration, but the anti-PC antibody contained in the pharmaceutical composition of the present invention is based on the affinity of the antibody for the antigen, that is, the dissociation constant (Kd value) for the antigen On the other hand, the higher the affinity (the lower the Kd value), the more effective the drug will be even in a small amount. Therefore, when determining the administration amount of an antibody, the administration amount can also be set based on the state of the affinity of an antibody and an antigen. When the antibody of the present invention is administered to humans, for example, about 0.001 to 100 mg / kg may be administered once, or multiple administrations may be performed at intervals of 1 to 180 days. It is preferably 0.1 to 50 mg / kg, more preferably 1 to 15 mg / kg, and multiple administrations may be performed at intervals of 2 to 3 weeks.

Examples

The present invention is specifically explained by the following examples, but the present invention is not limited to these. Again, these are not to be interpreted restrictively in any sense. In addition, in the following examples, as long as each operation related to genetic manipulation is not specifically stated, it is based on "Molecular Cloning" (Sambrook, J., Fritsch, EF and Maniatis, T., by Cold Spring Harbor Laboratory Press published in 1989) and other methods described in experimental books used by those with ordinary knowledge in the technical field to which the present invention pertains, or when using commercially available reagents or kits, Follow the instructions for commercially available products. Note that reagents, solvents, and starting materials not specifically described in this specification can be easily obtained from commercially available sources.

    [Example 1] Preparation of rat anti-human protein C (PC) antibody         1) -1 immunity         1) -1-1 Construction of human PC expression vector    

Using the In-Fusion HD Cloning Kit (CLONTECH), the 1st to 42nd and 89th sequences of amino acid sequences (serial identification number 1 of the sequence listing) encoded in a human PC (linked to the NCBI protein database accession number NP_000303) A peptide of 458 amino acid residues) is linked to the C-terminal side of a peptide (sequence X: sequence identification number 3) of the peptide (sequence X: sequence identification number 3) of the peptide tag (LVPRGSMDYKDDDDKNSAVDMHHHHHH) (SEQ ID NO: 2) Y: sequence identification number 4)), and combined with the vector pcDNA3.3-TOPO / LaxZ (ThermoFisher SCIENTIFIC) digested with restriction enzymes XbaI and PmeI, thereby preparing a human PC expression vector.

    1) -1-2 Human PC performance and refinement    

The human PC expression vector was transfected into FreeStyle 293F cells (ThermoFisher SCIENTIFIC) to temporarily express it. The culture supernatant was adsorbed on ANTI-FLAG M2 Affinity Gel (SIGMA-ALDRICH), and the column was washed with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% Glycerol 30 times the column capacity, and then washed with 20 mM Tris- HCl pH 8.0, 150 mM NaCl, 5% Glycerol, 100 ug / mL 3xFlag peptide (SIGMA-ALDRICH) was eluted and the fraction containing human PC was recovered.

    1) -1-3 activation of human PC    

Thrombin (Thrombin liquid Mochida Soft Bottle 10000, Moda Pharmaceutical Co., Ltd.) was added to a human PC and reacted at room temperature for 1 hour, and then the reaction was stopped by adding a final concentration of 5 mM EDTA. The reaction solution was subjected to buffer exchange to 20 mM Tris (pH 7.5) by dialysis, and was supplied to HisTrapQ HP (GE Healthcare Bioscience) equilibrated with 20 mM Tris (pH 7.5). Wash the column with 20 mM Tris (pH 7.5), which is 10 times the column capacity. A linear concentration gradient elution with sodium chloride was performed to recover a portion containing activated human PC. After concentrating the part containing activated human PC (aPC) with AmiconUltra (MERCK MILLIPORE), gel filtration was performed with HiLoad 16/600 Superdex 75 pg equilibrated with PBS and 1mM CaCl _{2 to} recover the part containing activated human PC. (MERCK MILLIPORE) was concentrated to about 2 mg / mL to prepare a refined sample.

    1) -1-4 immunity    

For the immune system, WKY / Izm female rats (Japan SLC) were used. The lymph node and spleen of the rat that has been administered to the root of the tail are prepared by mixing the antigen protein prepared in 1) -1-3 with Freund's Complete Adjuvant (manufactured by Wako Pure Chemical Industries, Ltd.). For fusion tumor production.

    1) -2 fusion tumor production    

Using LF301-Cell Fusion Unit (manufactured by BEX Corporation), lymph node cells or spleen callus cells were fused with mouse myeloma SP2 / 0-ag14 cells (ATCC: CRL-1581) by electric cell fusion, and cloned in ClonaCell-HY Selection Medium D ( StemCell Technologies). The emerged colony was recovered, thereby producing a single plant fusion. The recovered fusion tumor communities were cultured, and the obtained fusion tumor culture supernatant was used to perform screening of anti-PC antibody-producing fusion tumors.

    1) -3 Primary screening of thrombin generation (TG) using fusion tumor culture supernatant    

Screening of fusion tumor culture supernatants was performed using a system in which human PC deficiency plasma was used as a parameter to restore the effect of TG inhibition by human aPC.

To 90 μL of the fusion tumor culture supernatant, add 2 nM of human PC with or without human aPC to lack plasma 70 μL, 20 μL of recombinant tissue factor (rTF) (final concentration 3 pM), and incubate at 37 ° C for 10 minutes . Next, 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) was added. And start to react. Fluorescence (ex.390nm, em.460nm) was measured every 20 seconds for 50 minutes. Fusion tumors that will more strongly restore the inhibition of TG by aPC are selected as positive for anti-human PC antibody production.

    1) -4 Secondary screening using TG recovery using plasma from patients with hemophilia    

As a secondary screening of antibodies produced by fusion tumors that were determined to be positive in Example 1) -3, plasma derived from hemophilia patients with hemophilia A (FVIII-deficient plasma) (George King Bio-Medical) And plasma derived from hemophilia patients (George King Bio-Medical) derived from hemophilia B (FIX-deficient plasma), and the TG recovery effect was examined in the same manner as in Example 1) -3. As a result, 36 fusion tumors showing effects in the plasma of hemophilia patients derived from both hemophilia A and hemophilia B were obtained.

    [Example 2] In vitro evaluation of rat anti-human PC antibody R74         2) -1 Purification of anti-human PC antibody from rat fusion tumor culture supernatant    

Rat anti-PC monoclonal antibody R74 was purified from a fusion tumor culture supernatant.

First, rat anti-PC monoclonal antibody R74 was fused to proliferate with ClonaCell-HY Selection Medium E (manufactured by StemCell Technologies) to a sufficient amount, and the medium was exchanged with 20% Ultra Low IgG FBS (Thermo Fisher Scienftific). (Hybridoma SFM (manufactured by Thermo Fisher Scienftific)), 8-9 × 10 ⁷ fusion tumor cells were seeded into a 1272 cm ² flask (manufactured by Corning) and cultured for 7 days. This culture supernatant was recovered by centrifugation, and passed through a 0.45 μm filter (manufactured by Corning) to be sterilized.

The antibody was purified from the culture supernatant of the fusion tumor by protein G affinity chromatography. The antibody was adsorbed on a protein G column (GE Healthcare Bioscience), the column was washed with PBS, and then eluted with a 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7). 1M Tris-HCl (pH 9.0) was added to the eluent, and the pH was adjusted to 7.0 to 7.5. Then, Centrifugal UF Filter Device VIVASPIN20 (molecular weight cutoff UF30K, Sartorius) was used to replace the buffer solution with PBS (-) was used to concentrate the antibodies at the same time to adjust the antibody concentration to 1.8 mg / mL. Finally, a Minisart-Plus filter (Sartorius) was used for filtering to prepare a refined sample.

    2) -2 TG recovery using plasma from hemophilia patients-1    

The concentration of rat anti-human PC antibody was fixed at 10 μg / mL, and plasma derived from hemophilia A patients (FVIII-deficient plasma) and plasma derived from hemophilia B patients (FIX-deficient plasma) were used in the same manner as in the examples. 1) -3 In the same way, explore the role of TG recovery. R74 showed activity equal to or higher than that of the positive control (human aPC polyclonal antibody) in the plasma of a patient with hemophilia A. The same results were obtained in plasma from patients with hemophilia B.

    2) -3 TG recovery using plasma from hemophilia patients-2    

Using plasma derived from hemophilia A patients (FVIII-deficient plasma) and plasma derived from hemophilia B patients (FIX-deficient plasma), the concentration dependence of the TG recovery of the antibody purified in Example 2) -1 was investigated. . Plasma lines derived from patients with hemophilia A were evaluated under conditions of 5 pM rTF, 10 nM recombinant thrombomodulin (rTM), and antibody concentrations of 0.3 μg / mL to 30 μg / mL. Plasma of philiatric patients was evaluated 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 restored TG in a concentration-dependent manner in the plasma derived from patients with hemophilia A, and showed a TG recovery effect equal to or higher than that of the positive control (anti-human aPC rabbit polyclonal antibody) (FIG. 1A). The same results were obtained in plasma from patients with hemophilia B (Figure 1B).

    [Example 3] Determination of the nucleotide sequence of cDNA encoding the variable region of rat anti-human PC antibody         3) -1 Preparation of total RNA (total RNA) produced by R74 fusion tumor    

In order to amplify the cDNA encoding the variable region of R74, TRIzol Reagent (Ambion) was used to generate a total RNA from a fusion tumor produced by R74.

    3) -2 Amplification and sequence determination of cDNA encoding the heavy chain variable region of R74 by 5’-RACE PCR    

Amplification of the cDNA containing the heavy chain variable region was performed using approximately 1 µg of the total RNA prepared in Example 3) -1 and SMARTer RACE 5 '/ 3'Kit (Clontech). As a primer for amplifying the cDNA encoding the variable region of the heavy chain gene of R74 by PCR, UPM (affiliated with Universal Primer A Mix: SMARTer RACE 5 '/ 3'Kit) and a well-known method for rat weight Primers designed for the sequence of the constant region of the strand.

The 5'-RACE PCR-amplified cDNA encoding the variable region of the heavy chain was cloned into plastids, followed by sequence analysis of the nucleotide sequence of the cDNA encoding the variable region of the heavy chain.

The nucleotide sequence of the determined cDNA encoding the variable region of the heavy chain of R74 is shown in sequence identification number 5, and the amino acid sequence is shown in sequence identification number 6. In addition, the amino acid sequence of CDRH1 according to the definition of Abm is shown in sequence identification number 7, the amino acid sequence of CDRH2 is shown in sequence identification number 8, and the amino acid sequence of CDRH3 is shown in sequence identification number 9. The nucleotide sequence and amino acid sequence of the heavy region of R74 heavy chain, the amino acid sequence of CDRH1, CDRH2, and CDRH3 are also shown in FIG. 2.

    3) -3 Amplification and sequence determination of cDNA encoding light chain variable region of R74 by 5’-RACE PCR    

It carried out by the same method as Example 3) -2. However, as a primer for amplifying the cDNA encoding the variable region of the light chain gene of R74 by PCR, UPM (Attached to the Universal Primer A Mix: SMARTer RACE 5 '/ 3'Kit), and a well-known gene, Primers designed for the sequence of the constant region of a mouse light chain.

The nucleotide sequence of the determined cDNA encoding the variable region of the light chain of R74 is shown in sequence identification number 10, and the amino acid sequence of the variable region of the light chain of R74 is shown in sequence identification number 11. The amino acid sequence of CDRL1 according to the definition of Abm is shown as sequence identification number 12, the amino acid sequence of CDRL2 is shown as sequence identification number 13, and the amino acid sequence of CDRL3 is shown as sequence identification number 14. The nucleotide sequence and amino acid sequence of the variable region of the R74 light chain, amino acid sequences of CDRL1, CDRL2, and CDRL3 are also shown in FIG. 3.

    [Example 4] Design of humanized version of human chimeric anti-human PC antibody cR74 hR74         4) -1 Humanized design of R74         4) -1-1 Molecular simulation of the variable region of R74    

The molecular simulation of the variable region of R74 uses the well-known method (Methods in Enzymology, 203, 121-153, (1991)) as the homology modeling method. Using the structure (PDB ID: 3IY7) registered in the Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) with high sequence identity for the variable regions of the heavy and light chains of R74 as a model, A commercially available protein stereostructure analysis program BioLuminate (manufactured by Schrodinger) was used.

    4) -1-2 R74 humanization design method    

R74 was humanized by CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Because of the common sequence of the human kappa chain subgroups 3 and 4 as defined in KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)) and IMGT The IGHV4-30-4 * 02 and IGHJ1 * 01 of the human gamma chain specified in (THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (registered trademark)) have high consistency for the framework region of R74, so they are selected as light chains respectively. Acceptor with heavy chain. Based on the reference given by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)), the three-dimensional model is analyzed to select the prosthetic residues to be transferred to the recipient ( donor residue).

    4) -2 Humanization of R74 heavy chain    

The three humanized heavy chains were named hR74_H1, hR74_H2 and hR74_H12. A comparison of the amino acid sequences of cR74_H, humanized heavy chains hR74_H1, hR74_H2, and hR74_H12 of the human chimeric anti-human PC antibody cR74 shown in Example 5 is shown in FIG. 4. In the sequences of hR74_H1, hR74_H2, and hR74_H12, "‧" represents the same amino acid residue as cR74_H, and where the amino acid residue is described represents a substituted amino acid residue. The full-length amino acid sequence of hR74_H1 is described in SEQ ID NO: 16. The nucleotide sequence encoding the amino acid sequence of sequence identification number 16 is described in sequence identification number 15. The full-length amino acid sequence of hR74_H2 is described in SEQ ID NO: 18. The nucleotide sequence encoding the amino acid sequence of sequence identification number 18 is described in sequence identification number 17. The full-length amino acid sequence of hR74_H12 is described in SEQ ID NO: 20. The nucleotide sequence of the amino acid sequence of the coding sequence identification number 20 is described in sequence identification number 19. In hR74_H1 and hR74_H2, the amino acid sequence of each CDR according to the definition of Abm is the same as R74. On the other hand, according to the definition of Abm, the amino acid sequence of CDRs of hR74_H12, CDRH1, CDRH3, CDRL1, CDRL2, and CDRL3 are the same as R74, but the amino acid sequence of CDRH2 is different. The amino acid sequence of CDRH2 of hR74_H12 according to the definition of Abm is shown in sequence identification number 21. The nucleotide sequence and amino acid sequence of the humanized antibody hR74_H1 are shown in FIG. 5. The nucleotide sequence and amino acid sequence of hR74_H2 are shown in FIG. 6. The nucleotide sequence and amino acid sequence of hR74_H12 and the amino acid sequence of CDRH2 are shown in FIG. 7.

    4) -3 Humanization of R74 light chain    

The three light chains designed are named hR74_L1, hR74_L2 and hR74_L4. A comparison of the amino acid sequences of the human chimeric anti-human PC antibody cR74_L, humanized antibodies hR74_L1, hR74_L2, and hR74_L4, which are the light chains of the human chimeric anti-human PC antibody cR74 shown in Example 5, is shown in the figure. 8. In the sequences of hR74_L1, hR74_L2 and hR74_L4, "‧" represents the same amino acid residue as cR74_L, where the amino acid residue is described represents a substituted amino acid residue, and "-" represents the corresponding amino group Where acid residues are missing.

The full-length amino acid sequence of hR74_L1 is described in SEQ ID NO: 22. The nucleotide sequence encoding the amino acid sequence of sequence identification number 23 is described in sequence identification number 22. The full-length amino acid sequence of hR74_L2 is described in SEQ ID NO: 25. The nucleotide sequence encoding the amino acid sequence of sequence identification number 25 is described in sequence identification number 24. The full-length amino acid sequence of hR74_L4 is described in SEQ ID NO: 27. The nucleotide sequence encoding the amino acid sequence of sequence identification number 27 is described in sequence identification number 26. In hR74_L1, hR74_L2, and L4, the amino acid sequence of each CDR according to the definition of Abm is the same as R74. The nucleotide sequence and amino acid sequence of the humanized antibody hR74_L1 are shown in FIG. 9. The nucleotide sequence and amino acid sequence of hR74_L2 are shown in FIG. 10. The nucleotide sequence and amino acid sequence of hR74_L4 are shown in FIG. 11.

    [Example 5] Preparation of human chimeric anti-human PC antibody         5) -1 Construction of human chimeric R74 light chain cR74_L expression vector    

Using the In-Fusion HD PCR colony kit (Clontech), a 5.4 kb fragment obtained by digesting pcDNA3.3-TOPO / LacZ (Invitrogen) with restriction enzymes XbaI and PmeI, and containing a sequence identification number The human light chain message sequence shown in 28 and the DNA fragment encoding the DNA sequence of the human κ chain constant region were combined to make pcDNA3.3 / LK.

The neomycin expression unit was removed from pcDNA3.3 / LK, thereby constructing pCMA-LK.

The cDNA encoding the variable region of the R74 light chain obtained in Example 3) -3 was used as a template, and PCR was performed with primers designed for in-fusion breeding. DNA fragments are amplified. Where pCMA-LK was cleaved with the restriction enzyme BsiWI, the amplified DNA fragment was inserted using the In-Fusion HD PCR selection kit (Clontech) to construct the cR74_L expression vector. The nucleotide sequence of cR74_L and the amino acid sequence of the light chain are shown in sequence identification number 29 and sequence identification number 30, respectively.

    5) Construction of human chimeric R74 heavy chain cR74_H expression vector    

Using the In-Fusion HD PCR colony kit (Clontech), pCMA-LK will be digested with XbaI and PmeI to remove the human light chain message sequence and the human κ chain constant region DNA fragment, as shown in SEQ ID NO: 31. The human heavy chain message sequence and a DNA fragment encoding a human IgG1-LALA type constant region DNA sequence are combined to construct pCMA-G1-LALA.

The cDNA encoding the variable region of the R74 heavy chain obtained in Example 3) -2 was used as a template, and PCR was performed with primers designed for in-fusion breeding. DNA fragments are amplified. Where pCMA-G1-LALA was cleaved with the restriction enzyme BlpI, the amplified DNA fragment was inserted using the In-Fusion HD PCR selection kit (Clontech) to construct the cR74_H expression vector. The nucleotide sequence of cR74_H and the amino acid sequence of the heavy chain are shown in sequence identification number 32 and sequence identification number 33, respectively.

    5) -3 Preparation of human chimeric anti-human PC antibody         5) -3-1 Production of human chimeric anti-human PC antibodies    

FreeStyle 293F cells (Invitrogen) were subcultured and cultured according to the manual. 1.2 × 10 ⁹ FreeStyle 293F cells (Invitrogen) in the logarithmic growth phase were seeded into 3L Fernbach Erlenmeyer Flask (CORNING), and diluted with FreeStyle293 expression medium (Invitrogen) to prepare 2.0 × 10 ⁶ cells / mL. In 40 mL of Opti-Pro SFM medium (Invitrogen), 0.24 mg of a heavy chain expression vector, 0.36 mg of a light chain expression vector, and 1.8 mg of polyethyleneimine (Polyscience # 24765) were added, and gently stirred, After 5 minutes of rest, add to FreeStyle 293F cells. After incubator at 37 ° C, 8% CO ₂ incubator, shaking at 90 rpm for 4 hours, 600 mL of EX-CELL VPRO medium (SAFC Biosciences), 18 mL of GlutaMAX I (GIBCO), and 30 mL of Yeastolate Ultrafiltrate were added. (GIBCO), cultured at 37 ° C, 8% CO ₂ incubator, and shaken at 90 rpm for 7 days, and the obtained culture supernatant was filtered with a Disposable Capsule Filter (Advantec # CCS-045-E1H).

The obtained human chimeric anti-human PC antibody was named "cR74".

    5) -3-2 Purification of human chimeric anti-human PC antibody    

From the culture supernatant obtained in Example 5) -3-1, antibodies were purified by a one-step procedure of rProtein A affinity chromatography. The culture supernatant was supplied to a MabSelectSuRe-equipped column (manufactured by GE Healthcare Bioscience) equilibrated with PBS, and then the column was washed with PBS twice the column capacity or more. Then, it was eluted with a 2M spermine hydrochloride solution (pH 4.0), and a fraction containing the antibody was collected. This portion was replaced with PBS (-) by buffering with dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated with Centrifugal UF Filter Device VIVASPIN20 (cutoff molecular weight UF10K, Sartorius), and the IgG concentration was adjusted to 1 mg / mL or more. Finally, a Minisart-Plus filter (Sartorius) was used for filtering to prepare a refined sample.

    [Example 6] In vitro activity of human chimeric anti-human PC antibody         6) -1 TG recovery by human chimeric anti-human PC antibody using plasma derived from hemophilia patients    

The use of human chimeric anti-human PC antibody cR74_IgG1-LALA in the recovery of TG from plasma of patients with hemophilia A and plasma of patients with hemophilia B has more TG recovery effects than the original human anti-PC rat antibodies and positive controls Strain antibodies were compared. The method is implemented in accordance with the method described in 2) -2.

cR74_IgG1-LALA showed a TG recovery effect equal to or higher than that of the original human anti-PC rat antibody (R74) in the plasma of patients with hemophilia A (FIG. 14A).

Plasma derived from patients with hemophilia B also showed almost the same results as those derived from patients with hemophilia A (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    

DNA fragments shown by nucleotide numbers 37 to 414 of the nucleotide sequence of hR74_L1 shown by sequence identification number 11 were synthesized (GENEART). Using the In-Fusion HD PCR colony kit (Clontech), hR74_L1 was constructed by inserting the synthesized DNA fragment at a place where the chimeric and humanized antibody light chain expression vector pCMA-LK was cut with the restriction enzyme BsiWI. Performance vector.

    7) -1-2 Construction of hR74_L2 expression vector    

A DNA fragment (GENEART) containing a DNA sequence shown in nucleotide numbers 37 to 414 of the nucleotide sequence of hR74_L2 shown in sequence identification number 23 was synthesized. The hR74_L2 expression vector was constructed in the same manner as in Example 7) -1-1.

    7) -1-3 Construction of hR74_L4 expression vector    

DNA fragments shown by nucleotide numbers 37 to 414 of the nucleotide sequence of hR74_L4 shown by sequence identification number 25 (GENEART) were synthesized. The hR74_L4 expression vector was constructed in the same manner as in Example 7) -1-1.

    7) Construction of humanized anti-human PC antibody heavy chain hR74_H expression vector         7) -2-1 Construction of hR74_H1 expression vector    

DNA fragments shown by nucleotide numbers 36 to 428 of the nucleotide sequence of hR74_H1 shown by sequence identification number 15 (GENEART) were synthesized. The hR74_H1 expression vector was constructed by inserting the synthesized DNA fragment using the In-Fusion HD PCR colony kit (Clontech) where the pCMA-G1-LALA was cleaved with the restriction enzyme BlpI.

    7) Construction of 2-2 hR74_H2 expression vector    

DNA fragments shown by nucleotide numbers 36 to 428 of the nucleotide sequence of hR74_H2 shown by sequence identification number 17 (GENEART) were synthesized. The hR74_H2 expression vector was constructed in the same manner as in Example 7) -2-1.

    7) -2-3 Construction of hR74_H12 expression vector    

Using hR74_H1 as a template, a mutation was introduced using the KOD-Plus-Mutagenesis Kit (TOYOBO) to construct the hR74_H12 expression vector. The nucleotide sequence of hR74_H12 is shown in SEQ ID NO: 19.

    7) -3 Preparation of humanized anti-human PC antibody hR74         7) -3-1 Production of humanized antibodies    

Production was carried out in the same manner as in Example 5) -3-1. The humanized antibody obtained by combining the hR74_H1 expression vector and the hR74_L1 expression vector was named "hR74_H1 / L1". The humanized antibody obtained by combining the hR74_H12 expression vector and the hR74_L1 expression vector was named "hR74_H12 / L1". The humanized antibody obtained by combining the hR74_H12 expression vector and the hR74_L4 expression vector was named "hR74_H12 / L4".

    7) Purification of 3-2 humanized antibody    

From the culture supernatant obtained in Example 7) -3-1, the antibody was purified by a two-step procedure of rProtein A affinity chromatography and ceramic hydroxyapatite. The culture supernatant was supplied to a MabSelectSuRe-equipped column (manufactured by GE Healthcare Bioscience) equilibrated with PBS, and then the column was washed with PBS twice the column capacity or more. Next, the antibody was eluted with a 2M spermine hydrochloride solution (pH 4.0). The antibody-containing fraction was replaced with PBS by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette) and the buffer was replaced with 5 mM sodium phosphate / 50 mM MES / pH 7.0 buffer. Then, it was supplied to a ceramic hydroxide apatite column (Japan Bio-Rad, Bio-Scale CHT Type-1 Hydroxyapatite Column) equilibrated with a buffer solution of 5 mM NaPi / 50 mM MES / 30 mM NaCl / pH 7.0. A linear concentration gradient elution with sodium chloride was performed to collect the fraction containing the antibody. This portion was replaced with HBSor (25 mM histidine / 5% sorbitol, pH 6.0) by buffering the portion by dialysis (Thermo Scientific, Slide-A-Lyzer Dialysis Cassette). The antibody was concentrated with Centrifugal UF Filter Device VIVASPIN20 (cutoff molecular weight UF10K, Sartorius), and the IgG concentration was adjusted to 40 mg / mL or more. Finally, a Minisart-Plus filter (Sartorius) was used for filtering to prepare a refined sample.

    [Example 8] Production of BAY1896502    

BAY1896502 is produced with reference to WO2014 / 085527 A1 and WO2015 / 179435 A1.

    8) -1 Construction of a humanized anti-human aPC antibody BAY1896502 light chain expression vector (Murakami, Throne)    

DNA fragments shown by nucleotide numbers 37 to 414 of the nucleotide sequence of the BAY1896502 light chain shown by sequence identification number 34 were synthesized (GENEART). The expression vector for the light chain BAY1896502 was constructed in the same manner as in Example 7) -1-1. The amino acid sequence of the BAY1896502 light chain is shown in SEQ ID NO: 35.

    8) -2 Construction of humanized anti-human aPC antibody BAY1896502 heavy chain expression vector    

Using the In-Fusion HD PCR selection kit (Clontech), pCMA-LK will be digested with XbaI and PmeI to remove the light chain message sequence and the DNA fragment of the constant region of the human κ chain, as well as the human including the sequence ID 36 The heavy chain message sequence and the DNA fragment encoding the amino acid DNA sequence of the human IgG2 constant region are combined to construct pCMA-G2.

A DNA fragment shown by nucleotide numbers 36 to 428 of the nucleotide sequence of the BAY1896502 heavy chain shown by sequence identification number 37 was synthesized (GENEART). A BAY1896502 heavy chain expression vector was constructed by inserting the synthesized DNA fragment by using the In-Fusion HD PCR selection kit (Clontech) to cleave pCMA-G2 with the restriction enzyme BlpI. The amino acid sequence of the BAY1896502 heavy chain is shown in SEQ ID NO: 38.

    8) -3 Preparation of humanized anti-human aPC antibody BAY1896502         8) -3-1 Production of humanized anti-human aPC antibody BAY1896502    

It was produced by the same method as in Example 5) -3-1.

    8) Purification of 3-2 humanized anti-human aPC antibody BAY1896502    

It was purified by the same method as in Example 5) -3-2.

    [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 antibody-antigen binding system was measured using the Biacore 4000 (GE Healthcare Bioscience (stock)) using a capture method based on an immobilized anti-human IgG Fc antibody, and the antibody was captured (captured) as a ligand. And measure the antigen as an analyte. For the antigenic system, human PC (Enzyme Research Laboratories) was used. The anti-human IgG Fc antibody (Human Antibody Capture Kit, GE Healthcare Bioscience (stock)) was covalently bound to the sensor wafer CM5 (GE Healthcare Bioscience (stock)) by using an amine coupling method to approximately 2000 RU. The reference cell is fixed in the same manner. As the electrophoresis buffer, HBS-EP + (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% Surfactant P20) was used. After the anti-human IgG Fc antibody has been immobilized on the wafer, the antibody is added for about 1 minute and captured as a ligand, and then the antigen is added at a flow rate of 30 μL / min for 0.1 seconds to 100 nM for 300 seconds, and then electrophoresis buffer 600 is added. In seconds, the binding dissociation of the antigen is monitored. As a playback solution, 3 M MgCl _{2 was added} twice at a flow rate of 10 μL / min for 30 seconds. The analysis system uses a 1: 1 combination of theoretical formulas. As a result, a single-digit KM value of nM was displayed in hR74_H1 / L1, and a high KD value of about 10 times was shown in the other two samples (Table 1).

    9) -2 Viscosity measurement of humanized anti-PC antibody    

Samples were concentrated using Vivapore 5 (Sartorius), prepared to a concentration of 150 mg / mL with a 25 mM histidine, 5% sorbitol, and pH 6.0 buffer, using m-VROC (RheoSense) at 25 ° C and a flow rate of 75 μL / min. Conditions. As a result, it showed about 40 cP in hR74_H1 / L1 and about 50% of its viscosity in hR74_H12 / L1 and hR74_H12 / L4 (Table 2).

    9) -3         9) -3-1 TG recovery by humanized anti-human PC antibody using plasma derived from hemophiliacs    

The use of humanized anti-human PC antibodies to evaluate the TG recovery of plasma derived from hemophilia A patients. The method is implemented in accordance with the method described in 2) -2. 75 μL of human FVIII-deficient plasma (10.7 nM rTM (final concentration of 10 nM) in plasma) was added with humanized anti-human PC antibodies at various concentrations of 5 μL, 30 pM rTF20 μL (final concentration of 5 pM), and cultured at 37 ° C. for 10 minutes. Next, 20 μL of FluCa-kit (2.5 mM Z-Gly-Gly-Arg-amine methylcoumarin, 100 mM CaCl ₂ ) (final concentration Z-Gly-Gly-Arg-amine methylcoumarin: 417 μM, CaCl ₂ 16.7 mM). Fluorescence (ex.390nm, em.460nm) was measured every 20 seconds for 40 minutes. TG is calculated using the thrombinoscope software (Thrombinoscope BV). Any of hR74_H12 / L1, hR74_H12 / L4, and hR74_H12 / L1 antibodies restored TG in a concentration-dependent manner to the same extent (Figure 15).

    9) Comparison of TG recovery effect of 3-2 and BAY1896502 using plasma derived from hemophiliacs    

A comparison of the TG recovery effect of the humanized anti-human aPC antibody BAY1896502 and the humanized anti-human PC antibody (hR74_H1 / L1) described in BAYER's patent was performed. Compared to BAY1896502, which only inhibits aPC, hR74_H1 / L1 has the characteristics of so-called inhibition of PC and aPC.

The method is implemented in accordance with the method described in 9) -3-1.

The TG recovery effect using plasma from patients with hemophilia A (Figure 16A) and plasma from patients with hemophilia B (Figure 16B) was compared with hR74_H1 / L1 and BAY1896502. Both antibodies restored TG in a concentration-dependent manner, but the TG recovery effect of hR74_H1 / L1 was significantly greater than that of BAY1896502 in the plasma of any type of hemophilia patients.

    [Example 10] In vivo activity of humanized anti-human PC antibody    

To investigate the suppression effect of hR74_H1 / L1 subcutaneous hemorrhage in hemorrhagic A cynomolgus monkey hemorrhagic model.

Under awakening, anti-human FVIII neutralizing antibodies (HAEMATOLOGIC TECHNOLOGIES, sheep polyclonal antibodies) alone (18,000 Bethesda Unit (BU) / kg, iv) or with hR74_H1 / L1 (3 or 1.5) from superficial veins mg / kg, iv) and administered in combination. After 2 hours, use BD Microtainer (catalog number 366594, blue, under anesthesia of isoflurane (2-3% at the time of introduction, maintaining 1.5-2.5%)). 1.5mm wide blade, 2.0mm in depth), avoiding large veins and receiving 24 (4 on each upper arm, 2 on each forearm, 4 on the inside of each thigh, 2 on each gastrocnemius) trauma. The trauma day is set to day 0 (Day 0). After administering an analgesic agent, they are awakened from anesthesia. Dosage and Administration: The dosage is Lepetan (as Buprenorphine) 0.01mg / kg, SC and Metacam (as Meloxicam) 0.2mg / kg, SC on the day of the experiment on 24, 48, 72 After 1 hour, Metacam (as meloxicam) was 0.1 mg / kg and SC.

On days 1, 2, and 3 (24, 48, and 72 hours after FVIII neutralizing antibody administration), the subcutaneous bleeding site was photographed with a digital camera, and then the area was measured with analysis software.

In the FVIII neutralizing antibody alone intravenous injection group (Control), the area of the subcutaneous bleeding site increased over time (Fig. 17). On the other hand, in the hR74_H1 / L1 administration group, almost no subcutaneous hemorrhage was recognized in any dosage. From the above, it was shown that the anti-PC antibody (hR74_H1 / L1) has a hemostatic effect on the bleeding symptoms of hemophilia A.

Industrial availability

The humanized anti-PC antibody of the present invention has a stronger thrombin generation effect than well-known antibodies, and can be a preventive and / or therapeutic agent for hemophilia.

    [Sequence list non-keyword text]    

SEQ ID NO: 2: Peptide Tag

Sequence ID 3: Sequence X

Sequence ID 4: Sequence Y

SEQ ID NO: 5: Nucleotide sequence of the variable region of the heavy chain of R74

SEQ ID NO: 6 Amino acid sequence of variable region of heavy chain of R74

Sequence identification number 7: CDRH1

Sequence identification number 8: CDRH2

Sequence identification number 9: CDRH3

SEQ ID NO: 10: Nucleotide sequence of the variable region of the light chain of R74

SEQ ID NO: 11 Amino acid sequence of variable region of light chain of R74

Sequence identification number 12: CDRL1

Sequence identification number 13: CDRL2

Sequence identification number 14: CDRL3

Sequence identification number 15: a nucleotide sequence encoding hR74_H1

SEQ ID NO: 16: Amino acid sequence of hR74_H1

SEQ ID NO: 17: Nucleotide sequence encoding hR74_H2

SEQ ID NO: 18: 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

Sequence identification number 21: CDR2 of hR74_H12

SEQ ID NO: 22: Nucleotide sequence encoding hR74_L1

SEQ ID NO: 23: Amino acid sequence of hR74_L1

SEQ ID NO: 24: Nucleotide sequence encoding hR74_L2

SEQ ID NO: 25: Amino acid sequence of 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 a human light chain message sequence and a sequence encoding an amino acid sequence of a human κ chain constant region

SEQ ID NO: 29: Nucleotide sequence encoding human chimeric R74 light chain cR74_L

SEQ ID NO: 30: Amino acid sequence of human chimeric R74 light chain cR74_L

SEQ ID NO: 31: A nucleotide sequence of a DNA fragment containing a human heavy chain message sequence and a sequence encoding an amino acid of a human IgG1-LALA constant region

SEQ ID NO: 32: Nucleotide sequence encoding human chimeric R74 heavy chain cR74_H

SEQ ID NO: 33: Amino acid sequence of human chimeric R74 heavy chain cR74_H

SEQ ID NO: 34: Nucleotide sequence encoding BAY1896502 light chain

SEQ ID NO: 35: Amino acid sequence of BAY1896502 light chain

SEQ ID NO: 36: Nucleotide sequence of a DNA fragment containing a human heavy chain message sequence and a sequence encoding an amino acid of a human IgG2 constant region

SEQ ID NO: 37: Nucleotide sequence encoding BAY1896502 heavy chain

SEQ ID NO: 38: Amino acid sequence of BAY1896502 heavy chain

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<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> Human chimeric R74 heavy chain cR74_H nucleotide sequence

<220>

<221> Message peptide coding sequence

<222> (1) .. (57)

<400> 32

<210> 33

<211> 467

<212> PRT

<213> Artificial sequence

<220>

<223> Human chimeric R74 heavy chain cR74_H amino acid sequence

<220>

<221> Scope of the message sequence

<222> (1) .. (19)

<400> 33

<210> 34

<211> 714

<212> DNA

<213> Artificial sequence

<220>

<223> BAY1896502 light chain nucleotide sequence

<220>

<221> Message peptide coding sequence

<222> (1) .. (60)

<400> 34

<210> 35

PD1184727 (9)

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> BAY1896502 light chain amino acid sequence

<220>

<221> Scope of the message sequence

<222> (1) .. (20)

<400> 35

<210> 36

<211> 1111

<212> DNA

<213> Artificial sequence

<220>

<223> Human heavy chain message sequence and IgG2 constant region nucleotide sequence

<400> 36

<210> 37

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> BAY1896502 heavy chain nucleotide sequence

<220>

<221> Message peptide coding sequence

<222> (1) .. (57)

<400> 37

<210> 38

<211> 463

<212> PRT

<213> Artificial sequence

<220>

<223> BAY1896502 heavy chain amino acid sequence

<220>

<221> Scope of the message sequence

<222> (1) .. (19)

<400> 38

Claims (45)

    An antibody or a functional fragment of the antibody that competitively inhibits binding to protein C or activated protein C with an antibody selected from the group consisting of any one of the following groups (a) to (e) Activity: (a) A heavy chain having an amino acid sequence represented by amino acid numbers 20 to 467 of sequence identification number 33 and an amino acid represented by amino acid numbers 21 to 237 of sequence identification number 30 An antibody against the light chain of the sequence; (b) a heavy chain comprising an amino acid sequence represented by amino acid numbers 20 to 467 of sequence identification number 16 and an amino acid number 21 to 238 including sequence identification number 23 (C) a heavy chain comprising an amino acid sequence represented by amino acid numbers 20 to 467 of the amino acid sequence number 20, and an amino acid comprising sequence amino acid number 25; Antibodies having a light chain of an amino acid sequence represented by numbers 21 to 238; (d) a heavy chain having an amino acid sequence shown by amino acid numbers 20 to 467 of sequence identification number 20, and a sequence identification number An antibody having a light chain of an amino acid sequence represented by amino acid numbers 21 to 238 of 23; and (e) having a sequence recognition An antibody having the heavy chain of the amino acid sequence shown by the amino acid number 20 to 467 of No. 20 and the light chain of the amino acid sequence shown by the amino acid number 21 of the sequence number 27.         An antibody or a functional fragment of the antibody, characterized in that it comprises a heavy chain and a light chain, and specifically binds to protein C and activated protein C; the heavy chain comprises: an amino acid sequence including the amino acid sequence shown in SEQ ID NO: 7 CDRH1, CDRH2 containing the amino acid sequence shown in sequence identification number 8 or sequence identification number 21, and CDRH3 containing the amino acid sequence shown in sequence identification number 9; the light chain contains: CDRL1 of the amino acid sequence, CDRL2 including the amino acid sequence shown in SEQ ID NO: 13, and CDRL3 including the amino acid sequence shown in SEQ ID NO: 14.         The antibody or functional fragment of the antibody according to claim 1 or 2, wherein protein C and activated protein C are derived from humans.         The antibody or the functional fragment thereof according to any one of claims 1 to 3, wherein protein C is a peptide comprising the amino acid sequence shown in amino acid numbers 43 to 153 of sequence identification number 3 of the sequence listing and A peptide comprising an amino acid sequence represented by amino acid numbers 154 to 412, a polypeptide bound by an SS bond, and activated protein C is an amino acid represented by amino acid numbers 43 to 153 of sequence listing number 3 The peptide of the sequence is a polypeptide in which an amino acid sequence shown in amino acid numbers 166 to 412 is bound by an SS bond.         The antibody or the functional fragment of the antibody according to any one of claims 1 to 4, which inhibits the activation of protein C and / or the activity of activated protein C.         The antibody or functional fragment of any one of claims 1 to 5, which has at least one characteristic selected from the following (a) to (d): (a) specifically binds to protein C and activated protein C ; (B) specifically bind to protein C and inhibit activation of protein C; (c) specifically bind to activated protein C to inhibit activated blood coagulation factor VIII (FVIIIa) and / or activated blood caused by activated protein C Decomposition and / or deactivation of coagulation factor V (FVa); (d) restoration of thrombin production.         The antibody or the functional fragment of the antibody according to any one of claims 1 to 6, wherein CDRH2 comprises an amino acid sequence represented by SEQ ID NO: 8.         The antibody or the functional fragment of the antibody according to any one of claims 1 to 6, wherein CDRH2 comprises an amino acid sequence represented by SEQ ID NO: 21.         The antibody or the functional fragment of the antibody according to any one of claims 1 to 6, which includes the heavy chain variable region described in sequence identification number 6 and the light chain variable region described in sequence identification number 11 and to make.         For example, the antibody or the functional fragment of the antibody according to any one of claims 1 to 7, and 9, comprising the following heavy and light chains: amino acid numbers 20 to 467 including the sequence identification number 33 The heavy chain of the amino acid sequence and the light chain including the amino acid sequence shown by amino acid numbers 21 to 237 of SEQ ID NO: 30.         The antibody or functional fragment of any one of claims 1 to 10, wherein the constant region is a human-derived constant region.         The antibody of any one of claims 1 to 11 or a functional fragment of the antibody, which is humanized.         The antibody or the functional fragment thereof according to claim 12, which has a heavy chain variable region comprising an amino acid sequence selected from the group consisting of any one of the following groups (a) to (e), and A light chain variable region comprising an amino acid sequence described in any one of the group consisting of (f) to (j): (a) described in amino acid numbers 20 to 137 in sequence identification number 16 Amino acid sequences; (b) the amino acid sequences described in amino acid numbers 20 to 137 in sequence identification number 18; (c) the amino acid sequences described in amino acid numbers 20 to 137 in sequence identification number 20 (D) an amino acid sequence having at least 95% homology to a sequence of a framework region other than each of the CDR sequences in the sequences of (a) to (c); (e) in (a) The amino acid sequence in which one or more amino acids are deleted, substituted or added in the sequence of the framework region other than each CDR sequence in the sequence of (d); (f) the amino acid number 21 in the sequence identification number 23 (G) the amino acid sequence described in amino acid number 21 to 133 in sequence identification number 25; (h) the amino acid sequence described in amino acid number 21 to 133 in sequence identification number 25 Amine (I) an amino acid sequence having at least 95% homology to a sequence of a framework region other than each of the CDR sequences in the sequences of (f) to (h); and (j) between (f) to ( i) An amino acid sequence in which one or more amino acids are deleted, substituted, or added in a sequence of a framework region other than each CDR sequence in the sequence.         The antibody or functional fragment of the antibody according to claim 13, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of any one of the following (a) to (i): ( a) A heavy chain variable region comprising an amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16 and an amino acid sequence described in amino acid number 21 to 133 in sequence identification number 23 Light chain variable region; (b) a heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16, and an amino acid number 21 to 21 in sequence identification number 25 The light chain variable region of the amino acid sequence described in 133; (c) the heavy chain variable region including the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16, and the sequence identification number The light chain variable region of the amino acid sequence described in amino acid numbers 21 to 133 in 27; (d) The heavy chain containing the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 18 may be A variable region, and a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 23; (e) including the amino acid sequence in SEQ ID NO: 18 The heavy chain variable region of the amino acid sequence described in Nos. 20 to 137 and the light chain variable region including the amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 25; (f) includes The heavy chain variable region of the amino acid sequence described in amino acid numbers 20 to 137 in SEQ ID NO: 18 and the light chain containing the amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 27 Variable region; (g) a heavy chain variable region comprising an amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 20 and an amino acid number 21 to 133 included in sequence identification number 23 The light chain variable region of the amino acid sequence of the amino acid sequence; (h) the heavy chain variable region including the amino acid sequence of amino acid numbers 20 to 137 in SEQ ID NO: 20 and the amine of sequence ID 25 The light chain variable region of the amino acid sequence described in amino acid numbers 21 to 133; (i) the heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 20, And a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 27.         The antibody or functional fragment of the antibody according to claim 13 or 14, comprising: a heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16; and sequence identification The light chain variable region of the amino acid sequence described in No. 23 of the amino acid Nos. 21 to 133.         The antibody or functional fragment of the antibody according to claim 13 or 14, comprising: a heavy chain variable region comprising the amino acid sequence described in the amino acid numbers 20 to 137 in the sequence identification number sequence identification number 20, and A light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 23.         The antibody or functional fragment of the antibody according to claim 13 or 14, comprising: a heavy chain variable region comprising the amino acid sequence described in the amino acid numbers 20 to 137 in the sequence identification number sequence identification number 20, and A light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 27.         The antibody or functional fragment of an antibody according to any one of claims 13 to 17, comprising a heavy chain and a light chain selected from the group consisting of any one of the following groups (a) to (i): (a ) A heavy chain comprising the amino acid sequences described in amino acid numbers 20 to 467 in sequence identification number 16 and a light chain comprising the amino acid sequences described in amino acid numbers 21 to 238 in sequence identification number 23; (b) A heavy chain containing the amino acid sequences described in amino acid numbers 20 to 467 in sequence identification number 16 and a light chain containing the amino acid sequences described in amino acid numbers 21 to 238 in sequence identification number 25 Chain; (c) a heavy chain comprising an amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 16 and an amino acid sequence described in amino acid numbers 21 to 238 in sequence identification number 27 (D) a heavy chain comprising the amino acid sequences described in amino acid numbers 20 to 467 in sequence identification number 18, and an amino group described in amino acid numbers 21 to 238 in sequence identification number 23 Light chain of an acid sequence; (e) a heavy chain including the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 18, and a sequence identification number The light chain of the amino acid sequence described in amino acid numbers 21 to 238 in 25; (f) the heavy chain including the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 18, and the containing sequence The light chain of the amino acid sequence described in amino acid numbers 21 to 238 in identification number 27; (g) the heavy chain including the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 20, and A light chain comprising an amino acid sequence described in amino acid numbers 21 to 238 in sequence identification number 23; (h) A heavy chain comprising an amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 20 And a light chain comprising the amino acid sequence described in amino acid numbers 21 to 238 in sequence identification number 25; (i) a light chain including the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 20 A heavy chain and a light chain including the amino acid sequences described in amino acid numbers 21 to 238 in SEQ ID NO: 27.         The antibody or the functional fragment of the antibody according to any one of claims 13 to 18, comprising: a heavy chain comprising the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 16; The light chain of the amino acid sequence described in the amino acid number 21 to 238 in the identification number 23.         The antibody or the functional fragment of the antibody according to any one of claims 13 to 18, comprising: a heavy chain comprising the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 20, and a containing sequence The light chain of the amino acid sequence described in the amino acid number 21 to 238 in the identification number 23.         The antibody or the functional fragment of the antibody according to any one of claims 13 to 18, comprising: a heavy chain comprising the amino acid sequence described in amino acid numbers 20 to 467 in sequence identification number 20, and a containing sequence The light chain of the amino acid sequence described in the amino acid number 21 to 238 in the identification number 27.         The functional fragment of the antibody of 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.         A polynucleotide encoding an antibody according to any one of claims 1 to 22 or a functional fragment of the antibody.         The polynucleotide of claim 23, comprising: a CDRH1 encoding an amino acid sequence described in SEQ ID NO: 7; a CDRH2 including an amino acid sequence described in SEQ ID NO: 8 or 21; and a sequence ID: 9 A polynucleotide comprising CDRH3 of the amino acid sequence described therein; and CDRL1 comprising the amino acid sequence of SEQ ID NO: 12; CDRL2 including the amino acid sequence of SEQ ID NO: 13; and a sequence identification number A polynucleotide of CDRL3 of the amino acid sequence according to 14.         The polynucleotide according to claim 23 or 24, which comprises a polynucleotide selected from any one of the group consisting of the following (a) to (j): (a) encodes the nucleotide sequence described in SEQ ID NO: 6; Polynucleotide of the variable region of the heavy chain of the amino acid sequence, and polynucleotide encoding the variable region of the light chain including the amino acid sequence described in SEQ ID NO: 11; (b) encoding of the sequence including the sequence identification number Polynucleotide of the heavy chain variable region of the amino acid sequence described in the amino acid numbers 20 to 137 in 16, and the nucleotide sequence encoding the amino acid sequence described in the amino acid numbers 21 to 133 in the sequence identification number 23 Polynucleotide of light chain variable region; (c) Polynucleotide encoding heavy chain variable region comprising amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 16, and encoding sequence identification Polynucleotide of the light chain variable region of the amino acid sequence described in amino acid numbers 21 to 133 in No. 25; (d) encoding the amino group described in amino acid numbers 20 to 137 in sequence identification number 16 Polynucleotide of the heavy chain variable region of the acid sequence, and encoding the amino group described in amino acid numbers 21 to 133 in amino acid sequence number 27 A polynucleotide of the light chain variable region of the sequence; (e) a polynucleotide encoding a heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in SEQ ID NO: 18, and encoding comprising Polynucleotide of the light chain variable region of the amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 23; (f) encoding a gene comprising the amino acid sequences described in amino acid numbers 20 to 137 in sequence identification number 18 Polynucleotide of a heavy chain variable region of an amino acid sequence, and a polynucleotide encoding a light chain variable region comprising the amino acid sequence described in amino acid numbers 21 to 133 in sequence identification number 25; (g ) A polynucleotide encoding a heavy chain variable region comprising an amino acid sequence described in amino acid numbers 20 to 137 in SEQ ID NO: 18, and encoding a sequence including amino acid numbers 21 to 133 in sequence identification number 27 Polynucleotide of the light chain variable region of the amino acid sequence; (h) Polynucleotide encoding the heavy chain variable region comprising the amino acid sequence of amino acid numbers 20 to 137 in SEQ ID NO: 20 And a large number of light chain variable regions encoding amino acid sequences described in amino acid numbers 21 to 133 in sequence identification number 23 (I) a polynucleotide encoding a heavy chain variable region comprising the amino acid sequence described in amino acid numbers 20 to 137 in sequence identification number 20, and encoding an amino acid sequence including the amino acid number in sequence identification number 25 Polynucleotide of the light chain variable region of the amino acid sequence according to 21 to 133; (j) A heavy chain variable encoding the amino acid sequence according to amino acid number 20 to 137 in sequence identification number 20 And a polynucleotide encoding a light chain variable region comprising an amino acid sequence described in amino acid numbers 21 to 133 in SEQ ID NO: 27.         A performance vector comprising a polynucleotide according to any one of claims 23 to 25.         A host cell transformed with a performance vector as claimed in claim 26.         The host cell of claim 26, wherein the host cell is a eukaryotic cell.         A method for producing an antibody or a functional fragment of the antibody, comprising a step of culturing a host cell as claimed in claim 27 or 28, and taking the objective or the functional fragment of the antibody from the culture obtained in the step A step of.         An antibody or a functional fragment of the antibody, which is characterized in that it is obtained by the production method according to claim 29.         The functional fragment of the antibody of claim 30, wherein the functional fragment is selected from the group consisting of Fab, F (ab) 2, Fab ', and Fv.         The antibody or functional fragment of any one of claims 1 to 22, 30, and 31 comprising one or more modifications selected from the group consisting of: glycosylation of the N-bond, O-bond glycosylation, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, N-terminal methionine residues Addition, amination of proline residues, and deletion of one or two amino acids at the carboxy terminus of the heavy chain.         The antibody of claim 32, wherein one or two amino acids are deleted from the carboxy terminus of the heavy chain.         The antibody of claim 33, wherein one amino acid is deleted at the carboxy terminus on both sides of the two heavy chains.         The antibody of any one of claims 32 to 34, wherein the proline residue at the carboxy terminus of the heavy chain is further amidated.         A medicinal composition, characterized in that it comprises, as an active ingredient, any one selected from the group consisting of the antibody of any one of claims 1 to 22, 30 to 35, or a functional fragment of the antibody, a salt thereof Such hydrates, such as the polynucleotide of any one of claims 23 to 25, such as the expression vector of claim 26, such as the host cell of claim 27 or 28.         The pharmaceutical composition according to claim 36, which is a therapeutic agent for bleeding disorders.         The pharmaceutical composition according to claim 37, wherein the bleeding disorder is at least any one selected from hemophilia A, hemophilia B, acquired hemophilia, and Von Willebrand's disease.         The pharmaceutical composition according to claim 38, wherein the bleeding disorder is hemophilia A and / or hemophilia B.         A method for treating a bleeding disorder, comprising administering any one of an antibody selected from claims 1 to 22 and 30 to 35 or a functional fragment of the antibody, a salt thereof, or a hydrate thereof To the individual.         The method according to claim 40, wherein the bleeding disorder is at least any one selected from hemophilia A, hemophilia B, acquired hemophilia, and Fon Willi disease.         The method of claim 41, wherein the bleeding disorder is hemophilia A and / or hemophilia B.         A method for treating a bleeding disorder, characterized by comprising at least one selected from the group consisting of antibodies such as claims 1 to 22 and 30 to 35 or functional fragments of the antibodies, salts thereof, or hydrates thereof The pharmaceutical composition and at least one medicine for treating a bleeding disorder are simultaneously, separately or continuously administered to an individual.         The method of claim 43, wherein the bleeding disorder is at least any one selected from the group consisting of hemophilia A, hemophilia B, acquired hemophilia, and Fon Willi disease.         The method of claim 44, wherein the bleeding disorder is hemophilia A and / or hemophilia B.