JP2018123055A - Pharmaceutical composition for use in prevention and/or treatment of blood coagulation factor xi (fxi) abnormalities comprising multispecific antigen binding molecule replacing function of blood coagulation factor viii (fviii) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for use in the prevention and/or treatment of blood coagulation factor XI (FXI) abnormalities.SOLUTION: A pharmaceutical composition for use in the prevention and/or treatment of blood coagulation factor XI abnormalities comprising a multispecific antigen binding molecule replacing the function of blood coagulation factor VIII. The multispecific antigen binding molecule replacing the function of blood coagulation factor VIII is a bispecific antibody that binds to blood coagulation factor IX and/or active coagulation factor IX and blood coagulation factor X.EFFECT: The multispecific antigen binding molecule replacing the function of blood coagulation factor VIII, can be used not only for preventing and/or treating bleeding resulting from FVIII dysfunction in hemophilia A, acquired Hemophilia A, and von Willebrand disease, but can also be used for preventing and/or treating bleeding due to diseases related to an abnormality in blood coagulation factor XI by the coagulation promoting action thereof.SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition used for the prevention and / or treatment of blood coagulation factor XI (FXI) abnormality, comprising a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII (FVIII). .

  An FXI abnormality is a rare bleeding disorder caused by a congenital defect or dysfunction of FXI (Non-Patent Document 1 [Haemophilia 2006; 12 (suppl. 3): 137-142]). FXI abnormalities are sometimes referred to as hemophilia C. FXI is a precursor of an enzyme, and when the blood coagulation reaction starts, the precursor changes to an active blood coagulation factor XI (FXIa) having an enzyme activity. The coagulation factors that activate FXI are thrombin, activated factor XII, and FXIa itself, and this FXI-mediated reaction plays an important role in the maintenance mechanism of blood coagulation reaction. In FXI abnormalities, activated partial thromboplastin time (APTT) is abnormally prolonged, as is hemophilia A and hemophilia B. Differentiation from hemophilia A and hemophilia B is performed by blood coagulation factor quantification and FXI quantification. Although bleeding with FXI abnormalities is treated with plasma supplementation or the like, since it is a rare bleeding disorder, effective prevention and / or treatment for bleeding has not been established.

  Hemophilia A is a hemorrhagic disease caused by a congenital defect or dysfunction of blood coagulation factor VIII (FVIII) among blood coagulation factors. The severity of hemophilia A is well correlated with FVIII activity in the blood. Patients with an activity of less than 1% are classified as severe, patients with an activity of 1% or more but less than 5% are moderate, and those with an activity of 5% or more but less than 40% are classified as mild. Severe patients, accounting for about half of hemophilia A patients, have several bleeding episodes per month, which is significantly more frequent than moderate and mild patients. Therefore, in severe hemophilia A patients, maintaining FVIII activity in blood at 1% or higher by FVIII replacement therapy is considered effective in preventing bleeding symptoms (Non-Patent Documents). 2 [Haemophilia 2003; 9 (suppl.1): 32]).

  For the prevention and / or treatment of hemorrhage in hemophilia A patients, FVIII preparations mainly purified from plasma or produced by genetic engineering techniques are used. Using these preparations, on-demand administration for the purpose of hemostasis at the time of bleeding, or preventive administration for preventing the occurrence of a bleeding event is performed (Non-Patent Document 3 [Blood 1981; 58: 1-13], Non-Patent Document 4 [Nature 1984; 312: 330-337]). However, since the blood half-life of the FVIII preparation is about 12 hours, frequent administration of about 3 times a week is required for preventive administration (Non-Patent Document 5 [Nature 1984; 312: 337-342], Non-Patent Document 6 [Biochim Biophys Acta 1986; 871: 268-278]). In on-demand administration, additional administration is necessary at regular intervals as necessary to prevent rebleeding. In addition, administration of the FVIII preparation is performed intravenously. Therefore, there is a strong demand for a drug that has a lower burden of administration (less administration frequency and does not require intravenous injection) compared to existing FVIII preparations.

  In addition, antibodies (inhibitors) against FVIII preparations may occur in hemophilia A patients (Non-patent Document 7 [Blood 2007; 109 (2): 546-551]). Inhibitors counteract the effects of FVIII preparations, making subsequent prevention / treatment with FVIII preparations difficult. By-pass preparations (active FVII preparations and APCC preparations) are administered for bleeding in patients with inhibitors (inhibitor patients) (Non-patent Document 7 [Blood 2007; 109 (2): 546-551]) . Their mechanism of action is less dependent on the function of FVIII and is hemostatic in hemophilia A. However, since its blood half-life is as short as about 2 to 8 hours, frequent intravenous injection is required, and there are cases where bleeding cannot be stopped sufficiently as the degree of hemostatic activity. Therefore, there is a strong demand for drugs that are not affected by the presence of inhibitors and have a low administration burden.

  As a related bleeding disorder, acquired hemophilia A in which blood coagulation factor is impaired due to the generation of a self-neutralizing antibody against blood coagulation factor acquired (Non-Patent Document 8 [Semin Thromb Hemost 2012; 38: 433-446], Non-Patent Document 9 [Thromb Haemost 2013; 110: 1114-1120]) and acquired FXI abnormalities. For the bleeding of these patients with acquired bleeding abnormalities, bypass preparations and the like are administered, but the problem is that bleeding cannot be stopped sufficiently due to frequent intravenous injection and the degree of hemostatic activity.

  In addition, von Willebrand disease caused by dysfunction or deficiency of von Willebrand factor (vWF) is known as a bleeding disorder involving FVIII. vWF is not only necessary for platelets to normally adhere to the subendothelial tissue at the site of vascular wall injury, but is also required to form a complex with FVIII and maintain normal FVIII levels in the blood It is. In patients with von Willebrand disease, these functions are reduced and hemostasis is abnormal (Non-Patent Document 10 [Blood 2013; 122: 3735-3740]). For bleeding in patients with von Willebrand disease, FVIII preparations containing desmopressin (DDAVP) and plasma-derived vWF are administered, but the bleeding cannot be stopped sufficiently due to the frequent intravenous injection and the degree of hemostatic activity. Case is a challenge.

  In recent years, it has multiple specificity that binds to both activated blood coagulation factor IX (FIXa) and blood factor X (FX) and replaces the cofactor function of FVIII, that is, the function of promoting FX activation by FIXa Sex antigen-binding molecules were found (Patent Documents 1 to 3). Among multispecific antigen-binding molecules, bispecific antibodies are being developed as pharmaceutical compositions for the prevention and / or treatment of hemophilia A bleeding (Patent Documents 1 to 3, Non-Patent Documents). Document 11 [Nature Medicine 2012; 18 (10): 1570-1574], Non-Patent Document 12 [PLOS ONE 2013; 8 (2): e57479], Non-Patent Document 13 [J Thromb Haemost 2014; 12 (2): 206 -213], Non-Patent Document 14 [Blood 2014; 124 (20): 3165-3171]). In addition, multispecific antigen-binding molecules that have a function to substitute for FVIII function may be applied to the prevention and / or treatment of bleeding in acquired hemophilia A and von Willebrand disease, in which FVIII dysfunction is involved. (Patent Documents 1 to 3).

Haemophilia 2006; 12 (suppl. 3): 137-142 Haemophilia 2003; 9 (suppl.1): 32 Blood 1981; 58: 1-13 Nature 1984; 312: 330-337 Nature 1984; 312: 337-342 Biochim Biophys Acta 1986; 871: 268-278] Blood 2007; 109 (2): 546-551 Semin Thromb Hemost 2012; 38: 433-446 Thromb Haemost 2013; 110: 1114-1120 Blood 2013; 122: 3735-3740 Nature Medicine 2012; 18 (10): 1570-1574 PLOS ONE 2013; 8 (2): e57479 J Thromb Haemost 2014; 12 (2): 206-213 Blood 2014; 124 (20): 3165-3171

WO2005 / 035756 WO2006 / 109592 WO2012 / 067176

  An object of the present invention is to provide a pharmaceutical composition for use in the prevention and / or treatment of FXI abnormalities, which contains a multispecific antigen-binding molecule that substitutes for the function of FVIII.

  In order to solve the above-mentioned problems, the present inventors have examined the plasma coagulation promoting action of a multispecific antigen-binding molecule that substitutes for the function of FVIII using commercially available plasma derived from a patient with an FXI abnormality. Examples include commercial plasma derived from patients with FXI deficiency or dysfunction (referred to as FXI-deficient plasma), or plasma obtained by adding anti-FXI neutralizing antibody to commercially available normal plasma to reduce FXI activity (referred to as FXI neutralized plasma). ) To promote plasma clotting by an antibody having a function of FVIII alternative activity in each plasma clotting evaluation method of activated partial thromboplastin time (APTT), clotting waveform analysis (CWA) and thrombin generation test (TGA) Examined.

As a result, the present inventors have found that in commercially available plasma derived from patients with FXI deficiency or dysfunction, antibodies having a function of FVIII alternative activity exhibit plasma coagulation promoting effects in all of the above-mentioned plasma coagulation evaluation methods. It was. Furthermore, the present inventors have found that an antibody having a function of FVIII substitute activity exhibits plasma coagulation promoting action even in plasma obtained by neutralizing FXI function. Therefore, a multispecific antigen-binding molecule that substitutes for the function of FVIII is a prophylactic and / or therapeutic agent for bleeding in hemophilia A, acquired hemophilia A and von Willebrand disease caused by FVIII dysfunction. It was shown that it can be used as a prophylactic and / or therapeutic agent for bleeding in abnormal FXI due to its procoagulant activity.
Based on such findings, the present invention contains FXI abnormalities other than hemophilia A, acquired hemophilia A, and von Willebrand disease, which contain a multispecific antigen-binding molecule that substitutes for the function of FVIII. The present invention relates to a pharmaceutical composition used for treatment, and more specifically to the following.
[1] A pharmaceutical composition used for the prevention and / or treatment of blood coagulation factor XI abnormality, comprising a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII.
[2] Bispecific antibody in which a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII binds to blood coagulation factor IX and / or active blood coagulation factor IX and blood coagulation factor X The pharmaceutical composition according to [1], wherein
[3] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The pharmaceutical composition according to [1] or [2], which is a specific antibody;
The first polypeptide is an H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in SEQ ID NO: 1, 2, 3 (H chain CDR of Q499), the second polypeptide is SEQ ID NO: 4, H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in 5, 6 (H chain CDR of J327), the third polypeptide and the fourth polypeptide are SEQ ID NO: 7, 8, 9 (L404 A bispecific antibody (Q499-z121 / J327-z119 / L404-k) consisting of a common L chain comprising the amino acid sequences of the L chain CDR1, 2, and 3 described in the above (L chain CDR).
[4] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The pharmaceutical composition according to any one of [1] to [3], which is a specific antibody;
The first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is the H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the third polypeptide and the fourth poly A bispecific antibody (Q499-z121 / J327-z119 / L404-k), wherein the peptide consists of a common L chain described in SEQ ID NO: 12.
[5] Blood coagulation factor XI abnormality is a disease that develops and / or progresses due to decreased activity, functional abnormality and / or deficiency of blood coagulation factor XI and / or active blood coagulation factor XI, [ [1] The pharmaceutical composition according to any one of [4].
[6] The pharmaceutical composition according to any one of [1] to [5], wherein the blood coagulation factor XI abnormality is a congenital or acquired disease.
[7] The pharmaceutical composition according to any one of [1] to [6], wherein the blood coagulation factor XI abnormality is hemophilia C.
[A1] A method for preventing and / or treating blood coagulation factor XI abnormality, comprising a step of administering an effective amount of a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII to a subject.
[A2] A bispecific antibody in which a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII binds to blood coagulation factor IX and / or active blood coagulation factor IX and blood coagulation factor X The method according to [A1].
[A3] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The method according to [A1] or [A2], which is a specific antibody;
The first polypeptide is an H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in SEQ ID NO: 1, 2, 3 (H chain CDR of Q499), the second polypeptide is SEQ ID NO: 4, H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in 5, 6 (H chain CDR of J327), the third polypeptide and the fourth polypeptide are SEQ ID NO: 7, 8, 9 (L404 A bispecific antibody (Q499-z121 / J327-z119 / L404-k) consisting of a common L chain comprising the amino acid sequences of the L chain CDR1, 2, and 3 described in the above (L chain CDR).
[A4] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The method according to any one of [A1] to [A3], which is a specific antibody;
The first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is the H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the third polypeptide and the fourth poly A bispecific antibody (Q499-z121 / J327-z119 / L404-k), wherein the peptide consists of a common L chain described in SEQ ID NO: 12.
[A5] Blood coagulation factor XI abnormality is a disease that develops and / or progresses due to decreased activity, functional abnormality and / or deficiency of blood coagulation factor XI and / or active blood coagulation factor XI. The method according to any one of [A1] to [A4].
[A6] The method according to any one of [A1] to [A5], wherein the blood coagulation factor XI abnormality is a congenital or acquired disease.
[A7] The method according to any one of [A1] to [A6], wherein the blood coagulation factor XI abnormality is hemophilia C.
[B1] A multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII for use in the prevention and / or treatment of blood coagulation factor XI abnormality.
[B2] A bispecific antibody in which a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII binds to blood coagulation factor IX and / or active blood coagulation factor IX and blood coagulation factor X The antigen-binding molecule according to [B1].
[B3] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The antigen-binding molecule according to [B1] or [B2], which is a specific antibody;
The first polypeptide is an H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in SEQ ID NO: 1, 2, 3 (H chain CDR of Q499), the second polypeptide is SEQ ID NO: 4, H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in 5, 6 (H chain CDR of J327), the third polypeptide and the fourth polypeptide are SEQ ID NO: 7, 8, 9 (L404 A bispecific antibody (Q499-z121 / J327-z119 / L404-k) consisting of a common L chain comprising the amino acid sequences of the L chain CDR1, 2, and 3 described in the above (L chain CDR).
[B4] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The antigen-binding molecule according to any one of [B1] to [B3], which is a specific antibody;
The first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is the H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the third polypeptide and the fourth poly A bispecific antibody (Q499-z121 / J327-z119 / L404-k), wherein the peptide consists of a common L chain described in SEQ ID NO: 12.
[B5] Blood coagulation factor XI abnormality is a disease that develops and / or progresses due to decreased activity, abnormal function and / or deficiency of blood coagulation factor XI and / or active blood coagulation factor XI. The antigen-binding molecule according to any one of [B1] to [B4].
[B6] The antigen-binding molecule according to any one of [B1] to [B5], wherein the blood coagulation factor XI abnormality is a congenital or acquired disease.
[B7] The antigen-binding molecule according to any one of [B1] to [B6], wherein the blood coagulation factor XI abnormality is hemophilia C.
[C1] Use of a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII in the manufacture of a medicament used for the prevention and / or treatment of blood coagulation factor XI abnormality.
[C2] A bispecific antibody in which a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII binds to blood coagulation factor IX and / or active blood coagulation factor IX and blood coagulation factor X The use according to [C1].
[C3] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. Use according to [C1] or [C2], which is a specific antibody;
The first polypeptide is an H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in SEQ ID NO: 1, 2, 3 (H chain CDR of Q499), the second polypeptide is SEQ ID NO: 4, H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in 5, 6 (H chain CDR of J327), the third polypeptide and the fourth polypeptide are SEQ ID NO: 7, 8, 9 (L404 A bispecific antibody (Q499-z121 / J327-z119 / L404-k) consisting of a common L chain comprising the amino acid sequences of the L chain CDR1, 2, and 3 described in the above (L chain CDR).
[C4] The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The use according to any one of [C1] to [C3], which is a specific antibody;
The first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is the H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the third polypeptide and the fourth poly A bispecific antibody (Q499-z121 / J327-z119 / L404-k), wherein the peptide consists of a common L chain described in SEQ ID NO: 12.
[C5] Blood coagulation factor XI abnormality is a disease that develops and / or progresses due to decreased activity, functional abnormality and / or deficiency of blood coagulation factor XI and / or active blood coagulation factor XI. Use in any one of C1]-[C4].
[C6] The use according to any one of [C1] to [C5], wherein the blood coagulation factor XI abnormality is a congenital or acquired disease.
[C7] The use according to any one of [C1] to [C6], wherein the blood coagulation factor XI abnormality is hemophilia C.

  According to the present invention, it is used for the prevention and / or treatment of FXI abnormalities other than hemophilia A, acquired hemophilia A and von Willebrand disease, which contains a multispecific antigen-binding molecule that substitutes for the function of FVIII. A pharmaceutical composition was provided. Multispecific antigen-binding molecules that replace FVIII function can be used as preventive and / or therapeutic agents for hemorrhage in hemophilia A, acquired hemophilia A and von Willebrand disease caused by FVIII dysfunction It can be used as a prophylactic and / or therapeutic agent for FXI abnormality due to its procoagulant activity. Since FXI abnormality is a rare bleeding disorder, no effective preventive and / or therapeutic agent for bleeding has been found, and the present invention is promising as a prophylactic and / or therapeutic agent for FXI abnormalities. it is conceivable that.

In APTT measurement using FXI-deficient plasma, ACE910, one of the bispecific antibodies having a function of FVIII alternative activity, shows plasma coagulation promoting action (individual data of 5 lots out of 10 lots). ACE910 showed a shortened clotting time in a concentration-dependent manner in any lot of plasma. In APTT measurement using FXI-deficient plasma, ACE910, one of the bispecific antibodies having a function of FVIII alternative activity, shows plasma coagulation promoting action (individual data of 5 lots out of 10 lots). ACE910 showed a shortened clotting time in a concentration-dependent manner in any lot of plasma. In APTT measurement using FXI-deficient plasma, ACE910 shows plasma coagulation promoting action (FXI def: mean value and standard deviation of 9-10 lots). Further, in APTT measurement using FXI neutralized plasma, ACE910 shows plasma coagulation promoting action (Normal + FXI Ab: individual data of 1 lot). ACE910 showed shortened clotting time in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma. In the CWA measurement by APTT reagent induction using FXI deficient plasma, ACE910 is a figure which shows a plasma coagulation promoting action (individual data of all 10 lots). ACE910 showed an increase in maximum clotting rate in a concentration-dependent manner in any lot of plasma. In the CWA measurement by APTT reagent induction using FXI deficient plasma, ACE910 shows the plasma coagulation promoting action (FXI def: average value and standard deviation of 9-10 lots). ACE910 showed an increase in maximum clotting rate in a concentration-dependent manner in FXI-deficient plasma. In CWA measurement by APTT reagent induction using FXI deficient plasma, ACE910 is a figure which shows a plasma coagulation promoting action (individual data of 5 lots out of all 10 lots). ACE910 showed an increase in maximum clotting acceleration in a concentration-dependent manner in any lot of plasma. In CWA measurement by APTT reagent induction using FXI deficient plasma, ACE910 is a figure which shows a plasma coagulation promoting action (individual data of 5 lots out of all 10 lots). ACE910 showed an increase in maximum clotting acceleration in a concentration-dependent manner in any lot of plasma. In the CWA measurement by APTT reagent induction using FXI deficient plasma, ACE910 shows the plasma coagulation promoting action (FXI def: average value and standard deviation of 9-10 lots). In addition, ACE910 shows plasma coagulation promoting action in CWA measurement by APTT reagent induction using FXI neutralized plasma (Normal + FXI Ab: individual data of 1 lot). ACE910 showed an increase in maximum clotting acceleration in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma. FIG. 4 is a diagram showing ACE910 having a plasma coagulation promoting action in measurement of coagulation time by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (individual data of all 5 lots). ACE910 showed a shortened clotting time in a concentration-dependent manner in any lot of plasma. ACE910 shows plasma coagulation-promoting effect in the measurement of coagulation time by induction of mixed reagents (including tissue factor and ellagic acid) using FXI-deficient plasma (FXI def: mean value and standard deviation of 4-5 lots) . In addition, ACE910 shows plasma coagulation-promoting action in the measurement of coagulation time by inducing mixed reagent (including tissue factor and ellagic acid) using FXI neutralized plasma (Normal + FXI Ab: Individual data of 1 lot) . ACE910 showed shortened clotting time in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma. FIG. 3 is a diagram showing ACE910 having a plasma coagulation promoting action in CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (individual data of all 5 lots). ACE910 showed an increase in maximum clotting rate in a concentration-dependent manner in any lot of plasma. FIG. 4 is a view showing that ACE910 has a plasma coagulation promoting action in a CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (FXI def: mean value and standard deviation of 4-5 lots). Further, ACE910 shows a plasma coagulation promoting action in CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI neutralized plasma (Normal + FXI Ab: individual data of 1 lot). ACE910 showed an increase in maximum clotting rate in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma. FIG. 3 is a diagram showing ACE910 having a plasma coagulation promoting action in CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (individual data of all 5 lots). ACE910 showed an increase in maximum clotting acceleration in a concentration-dependent manner in any lot of plasma. FIG. 4 is a view showing that ACE910 has a plasma coagulation promoting action in a CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (FXI def: mean value and standard deviation of 4-5 lots). Further, ACE910 shows a plasma coagulation promoting action in CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI neutralized plasma (Normal + FXI Ab: individual data of 1 lot). ACE910 showed an increase in maximum clotting acceleration in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma. FIG. 3 is a diagram showing plasma coagulation promoting action of ACE910 in TGA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (individual data of all 10 lots). ACE910 showed an increase in thrombin generation peak height in a concentration-dependent manner.

  The multispecific antigen-binding molecule that substitutes for the function of FVIII of the present invention can also be referred to as a multispecific antigen-binding molecule having FVIII-like activity. In the present invention, “substitute the function of FVIII” means promoting the activation of FX by FIXa (facilitating the production of FXa by FIXa). More specifically, in the present invention, “substitute FVIII function” recognizes FIX and / or FIXa and FX and promotes the activation of FX by FIXa (facilitates the production of FXa by FIXa) ) Means. FXa production promoting activity can be carried out by a method well known to those skilled in the art, and can be evaluated, for example, by a measurement system comprising FIXa, FX, synthetic substrate S-2222 (FXa synthetic substrate), and phospholipid. .

  The multispecific antigen-binding molecule of the present invention includes a first antigen-binding site and a second antigen-binding site that can specifically bind to at least two different antigens or epitopes. The first antigen-binding site and the second antigen-binding site are not particularly limited as long as they have binding activity to FIX and / or FIXa and FX, respectively. For example, antibodies, Scaffold molecules (antibody-like molecules) ), A site necessary for binding to an antigen such as a peptide, or a fragment containing the site. A Scaffold molecule is a molecule that performs its function by binding to a target molecule, and any polypeptide that is a three-dimensionally stable polypeptide that can bind to at least one target antigen. Can be used. Examples of such polypeptides include, for example, antibody variable regions, fibronectin (WO2002 / 032925), Protein A domain (WO1995 / 001937), LDL receptor A domain (WO2004 / 044011, WO2005 / 040229), ankyrin (WO2002). Nygren et al. (Current Opinion in Structural Biology 1997; 7: 463-469, Journal of Immunol Methods 2004; 290: 3-28), Binz et al. (Nature Biotech 2005; 23: 1257-1266), Hosse (Protein Science 2006; 15: 14-27). Alternatively, peptide molecules that can bind to the target antigen can be used as described in Curr Opin Mol Ther 2010; 12 (4): 487-95 and Drugs 2008; 68 (7): 901-12 .

  In the present invention, the multispecific antigen-binding molecule is not particularly limited as long as it is a molecule that can bind to at least two different antigens or epitopes. For example, the above antigens such as antibodies, Scaffold molecules, and fragments thereof Examples thereof include a polypeptide containing a binding site, an aptamer composed of a nucleic acid molecule or a peptide, and may be a single molecule or a multimer thereof. Preferred multispecific antigen binding molecules include multispecific antibodies that can specifically bind to at least two different antigens. In the antibody having activity substituting the function of FVIII of the present invention, as a particularly preferred antibody, a bispecific antibody (BsAb) (two types) capable of specifically binding to two different antigens is used. (Sometimes called specific antibodies).

  In the present invention, the “common L chain” is an L chain that can associate with two or more different H chains and exhibit binding ability to each antigen. Here, “different H chains” preferably refer to H chains of antibodies against different antigens, but are not limited thereto and mean H chains having different amino acid sequences. The common L chain can be obtained, for example, according to the method described in WO2006 / 109592.

  A multispecific antigen-binding molecule (preferably a bispecific antibody) in the present invention is a molecule comprising an antibody or antibody fragment having specificity for two or more different antigens. The antibody of the present invention is not particularly limited, but is preferably monoclonal.

  In addition, the L chains of the antibodies that serve as the multispecific antigen-binding molecules of the present invention may be different, but preferably have a common L chain.

  In one embodiment of the present invention, the multispecific antigen-binding molecule is a multispecific antigen-binding molecule that has a function of recognizing FIX and / or FIXa and FX and substituting the function of FVIII. The antibody of the present invention usually has a structure including a variable region in an anti-FIXa antibody and a variable region in an anti-FX antibody.

  In one embodiment of the present invention, the multispecific antigen-binding molecule comprises a first polypeptide and a third polypeptide that contain an antigen-binding site that recognizes FIX and / or FIXa, and an antigen-binding site that recognizes FX. A second polypeptide and a fourth polypeptide. The first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide include the antigen binding site of the antibody H chain and the antigen binding site of the antibody L chain.

  For example, in the multispecific antigen-binding molecule of the present invention, the first polypeptide and the third polypeptide each contain an antigen-binding site of an antibody H chain or L chain against FIX or FIXa, and the second polypeptide and the second polypeptide. Each of the four polypeptides contains an antigen binding site of an antibody H chain or L chain against FX. At this time, the antigen binding sites of the L chains of the antibodies contained in the first polypeptide and the third polypeptide, and the second polypeptide and the fourth polypeptide may be a common L chain.

  The polypeptide containing the antigen binding site of the antibody L chain in the present invention preferably contains the whole or a part of the sequence of the antibody L chain that binds to FIX, FIXa and / or FX.

  As preferred embodiments of the substance having an activity that substitutes for the function of FVIII described in the present invention, FIX and / or FIXa and bispecific antibodies that bind to FX and / or FXa can be exemplified. Such an antibody can be obtained, for example, according to the method described in WO2005 / 035756, WO2006 / 109592, WO2012 / 067176, and the like. The bispecific antibodies of the present invention include the antibodies described in these documents.

As a preferable bispecific antibody, the following antibody (ACE910) which is a bispecific antibody described in the patent document (WO 2012/067176) can be mentioned.
A bispecific antibody in which a first polypeptide and a third polypeptide are associated, and a second polypeptide and a fourth polypeptide are associated, wherein the first polypeptide is SEQ ID NO: 1, 2 H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in 3 (Q499 H chain CDR), the second polypeptide is described in SEQ ID NOs: 4, 5, 6 (H chain CDR of J327) H chain comprising the amino acid sequence of H1, CDR2, 2, and 3, L chain CDR1, as described in SEQ ID NO: 7, 8, 9 (L404 CDR of L404), the third polypeptide and the fourth polypeptide, A bispecific antibody consisting of a common L chain containing a few amino acid sequences (Q499-z121 / J327-z119 / L404-k).
More specifically, a bispecific antibody in which a first polypeptide and a third polypeptide are associated, and a second polypeptide and a fourth polypeptide are associated, wherein the first polypeptide has The H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is the H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, and the third and fourth polypeptides are SEQ ID NO: 12. Bispecific antibody (Q499-z121 / J327-z119 / L404-k) comprising the common L chain described in 1.

  The polypeptide in the present invention usually refers to peptides and proteins having a length of about 10 amino acids or more. Moreover, although it is normally a polypeptide derived from a living organism | raw_food, it will not specifically limit, For example, the polypeptide which consists of a sequence designed artificially may be sufficient. Moreover, any of natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. may be sufficient. Furthermore, fragments of the above polypeptides are also included in the polypeptides of the present invention.

  The term “antibody” is used in the broadest sense and is monoclonal, polyclonal, dimeric, multimeric, multispecific (eg, bispecific) as long as it exhibits the desired biological activity. Alternatively, antibody derivatives and modified antibodies may be used (Miller K et al. J Immunol. 2003, 170 (9), 4854-61). Antibodies may be mouse, human, humanized, chimeric, or derived from other species or artificially synthesized. The antibodies disclosed herein can be any type of immunoglobulin molecule (eg, IgG, IgE, IgM, IgD and IgA), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. It can be. The immunoglobulin can be from any species (eg, human, mouse or rabbit). The terms “antibody”, “immunoglobulin” and “immunoglobulin” are used interchangeably in a broad sense.

  “Antibody derivatives” comprise a portion of an antibody, preferably the variable domain of an antibody, or at least the antigen binding region of an antibody. Antibody derivatives include, for example, Fab, Fab ′, F (ab ′) 2, Fv fragment, linear antibody, single chain antibody (scFv), sc (Fv) 2, Fab3, domain antibody (dAb) (International Publication No. 2004/058821, International Publication No. WO 2003/002609), including but not limited to multispecific antibodies formed from diabodies, triabodies, tetrabodies, minibodies and antibody derivatives. Here, “Fab” is composed of one light chain and the CH1 region and variable region of one heavy chain. “Fv” is the smallest antibody derivative and includes a complete antigen recognition region and an antigen binding region. The antibody derivative may be, for example, a fusion of IgG antibody with Fc. For example, US Pat. No. 5,461,870, Example 2; Zapata G et al. Protein Eng. 1995, 8 (10), 1057-1062; Olafsen T et al. Protein Eng. Design & Sel. 2004, 17 (4 ): 315-323; Holliger P et al. Nat. Biotechnol. 2005, 23 (9); 1126-36; Fischer N et al. Pathobiology. 2007, 74 (1): 3-14; Shen J et al. J Immunol Methods. 2007, 318, 65-74; Wu et al. Nat Biotechnol. 2007, 25 (11), 1290-7.

  Diabodies refer to bivalent, low molecular weight antibodies constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448, EP 404,097 , WO93 / 11161 etc.). A diabody is a dimer composed of two polypeptide chains, each of which is in a position where the L chain variable region (VL) and H chain variable region (VH) cannot bind to each other in the same chain. Short, for example, 2 to 12 residues are preferable, 3 to 10 residues are more preferable, and the residues are particularly connected by a linker of about 5 residues. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain variable region fragment because the linker between them is short, a diabody forms two antigen-binding sites. Will have.

  Single chain antibodies or scFv antibody fragments include the VH and VL regions of an antibody, and these regions are present in a single polypeptide chain. In general, Fv polypeptides further contain a polypeptide linker between the VH and VL regions, which allows the scFv to form the necessary structure for antigen binding (for a review of scFv, see Pluckthun The The Pharmacology of Monoclonal Antibodies, Vol. 113 (see Rosenburg and Mooreed (Springer Verlag, New York) pp. 269-315, 1994)). The linker in the present invention is not particularly limited as long as it does not inhibit the expression of the antibody variable region linked to both ends thereof.

  Bispecific antibodies can also be produced by chemically cross-linking Fab ′. For example, Fab ′ prepared from one antibody is maleimidized with o-PDM (ortho-phenylenedi-maleimide) and reacted with Fab ′ prepared from the other antibody to crosslink Fab ′s derived from different antibodies. Bispecific F (ab ′) 2 can be generated (Keler T et al. Cancer Research 1997; 57: 4008-4014). Also known is a method of chemically binding Fab'-thionitrobenzoic acid (TNB) derivatives and antibody fragments such as Fab'-thiol (SH) (Brennan M et al. Science 1985; 229: 81-83). .

  A leucine zipper derived from Fos, Jun or the like can be used instead of chemical crosslinking. Fos, Jun also forms homodimers, but utilizes the preferential formation of heterodimers. Expression and preparation of Fab ′ added with Fos leucine zipper and the other Fab ′ added with Jun. Bispecific F (ab ') 2 can be formed by mixing and reacting with the reduced monomers Fab'-Fos and Fab'-Jun under mild conditions (Kostelny SA et al. J of Immunology, 1992; 148: 1547-53). This method is not limited to Fab ′, and can also be applied to scFv, Fv, and the like.

  In addition, IgG-scFv (Protein Eng Des Sel. 2010; 23 (4): 221-8), sc (Fv) 2 such as BiTE (Drug Discov Today 2005; 15; 10 (18): 1237-44), DVD -Ig (Nat Biotechnol 2007; 25 (11): 1290-7. Epub 2007 Oct 14, MAbs 2009; 1 (4): 339-47. Epub 2009 Jul 10) and others (IDrugs 2010; 13: 698-700 ), Two-in-one antibody (Science 2009; 20; 323 (5921): 1610-4, Immunotherapy 2009; 1 (5): 749-51), bispecific such as Tri-Fab, tandem scFv, and diabody Sexual antibodies are also known (MAbs 2009; 1 (6): 539-547.). Furthermore, even when molecular forms such as scFv-Fc and scaffold-Fc are used, by secreting a heterogeneous combination of Fc preferentially (Ridgway JB et al. Protein Engineering 1996; 9: 617-621, Merchant AM et al Nature Biotechnology 1998; 16: 677-681, WO2006 / 106905, Davis JH et al. Protein Eng Des Sel 2010; 4: 195-202), bispecific antibodies can be made efficiently.

  Bispecific antibodies can also be made in diabodies. A bispecific diabody is a heterodimer of two cross-over scFv fragments. In other words, VH (A) -VL (B), VH (B) -VL (A) prepared by linking VH and VL from two antibodies A and B with a relatively short linker of about 5 residues Can be used to construct heterodimers (Holliger P et al. Proc of the National Academy of Sciences of the USA 1993; 90: 6444-6448).

  At this time, two kinds of scFvs are connected by a flexible, relatively long linker of about 15 residues (single-stranded diabody: Kipriyanov SM et al. J of Molecular Biology 1999; 293: 41-56), and appropriate amino acid substitution (knobs-into-holes: Zhu Z et al. Protein Science 1997; 6: 781-788, VH / VL interface engineering: Igawa T et al. Protein Eng Des Sel 2010; 8: 667-77) The composition of can be promoted.

  Sc (Fv) 2, which can be produced by linking two kinds of scFvs with a flexible relatively long linker of about 15 residues, can also be a bispecific antibody (Mallender WD et al. J of Biological Chemistry 1994; 269: 199 -206).

  Examples of the modified antibody include antibodies bound to various molecules such as polyethylene glycol (PEG). The antibody of the present invention includes these modified antibodies. In the modified antibody of the present invention, the substance to be bound is not limited. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody. These methods are already established in this field.

  A “bispecific” antibody refers to an antibody having variable regions that recognize different epitopes within the same antibody molecule. The bispecific antibody may be an antibody that recognizes two or more different antigens, or an antibody that recognizes two or more different epitopes on the same antigen. Bispecific antibodies may include whole antibodies as well as antibody derivatives. The antibodies of the present invention also include bispecific antibodies. In the present specification, the anti-FIXa / FX bispecific antibody is used synonymously with a bispecific antibody that binds to FIXa and FX.

Method for producing gene recombinant antibody As an antibody, a recombinant antibody produced using gene recombination technology can be used. Recombinant antibodies are produced by cloning DNA encoding them from hybridomas or antibody-producing cells such as sensitized lymphocytes that produce antibodies, incorporating them into vectors, and introducing them into hosts (host cells). Can be obtained.

  The origin of the antibody is not limited, such as a human antibody, a mouse antibody, or a rat antibody. Further, it may be a genetically modified antibody such as a chimeric antibody or a humanized antibody.

  Methods for obtaining human antibodies are already known. For example, a target human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a target antigen (International Patent Application Publication) No. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

  Genetically modified antibodies can be produced using known methods. Specifically, for example, a chimeric antibody is an antibody comprising the H chain and L chain variable regions of an immunized animal antibody, and the H chain and L chain constant regions of a human antibody. A chimeric antibody can be obtained by ligating a DNA encoding the variable region of an antibody derived from an immunized animal with a DNA encoding the constant region of a human antibody, incorporating it into an expression vector, introducing it into a host, and producing it. .

  A humanized antibody is a modified antibody also referred to as a reshaped human antibody. Humanized antibodies are constructed by grafting the CDRs of antibodies from immunized animals to the complementarity determining regions of human antibodies. Its general genetic recombination techniques are also known (European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 96/02576, Sato K et al, Cancer Research 1993, 53: 851-856, International Patent Application Publication number WO 99/51743).

  Bispecific antibodies are antibodies that have specificity for two different antigens.

  Bispecific antibodies are not limited to those of IgG type, but for example, IgG type bispecific antibodies can be secreted by hybrid hybridoma (quadroma) generated by fusing two hybridomas producing IgG antibodies (Milstein C et al. Nature 1983, 305: 537-540). Moreover, it can be secreted by co-expressing the L chain and H chain genes constituting the two types of IgG of interest, a total of four genes, by introducing them into cells.

  In this case, it is also possible to preferentially secrete IgG with a heterogeneous combination with respect to the H chain by making an appropriate amino acid substitution in the CH3 region of the H chain (Ridgway JB et al. Protein Engineering 1996, 9: 617-621, Merchant AM et al. Nature Biotechnology 1998, 16: 677-681, WO2006 / 106905, Davis JH et al. Protein Eng Des Sel. 2010, 4: 195-202.).

  As for the L chain, since the diversity of the L chain variable region is lower than that of the H chain variable region, it is expected that a common L chain capable of giving binding ability to both H chains can be obtained. The antibody may be an antibody having a common L chain. By introducing the common L chain and both H chain genes into the cells, the expression of IgG enables efficient expression of the bispecific IgG.

Antibody Production Method The antibody of the present invention can be produced by methods known to those skilled in the art. Specifically, DNA encoding the target antibody is incorporated into an expression vector. In that case, it integrates into an expression vector so that it may express under the control of an expression control region, for example, an enhancer and a promoter. Next, host cells are transformed with this expression vector to express the antibody. In that case, a combination of an appropriate host and an expression vector can be used.

  Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, and the like. For the purpose of subcloning and excision of cDNA, for example, pGEM-T, pDIRECT, pT7 and the like can be used in addition to the above vector.

  An expression vector is particularly useful when a vector is used for the purpose of producing an antibody. As an expression vector, for example, when the host is E. coli such as JM109, DH5α, HB101, XL1-Blue, a promoter that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341). , 544-546; FASEB J. (1992) 6, 2422-2427), araB promoter (Better et al., Science (1988) 240, 1041-1043), or T7 promoter is essential. Examples of such vectors include pGEX-5X-1 (manufactured by Pharmacia), “QIAexpress system” (manufactured by QIAGEN), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase). BL21 is preferred).

  The vector may contain a signal sequence for polypeptide secretion. As a signal sequence for polypeptide secretion, for example, a pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4397) may be used when it is produced in the periplasm of E. coli. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

  In addition to E. coli expression vectors, vectors for producing the antibody of the present invention include, for example, mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS (Nucleic Acids. Res. 1990, 18). (17), p5322), pEF, pCDM8), insect cell-derived expression vectors (eg “Bac-to-BAC baculovairus expression system” (GIBCO BRL), pBacPAK8), plant-derived expression vectors (eg pMH1, pMH2 ), Animal virus-derived expression vectors (for example, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (for example, pZIPneo), yeast-derived expression vectors (for example, “Pichia Expression Kit” (manufactured by Invitrogen), pNV11 , SP-Q01), and an expression vector derived from Bacillus subtilis (for example, pPL608, pKTH50).

  When intended for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., a promoter necessary for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMTV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CAG promoter (Gene. (1991) 108, 193), CMV promoter, etc. More preferably, it has a gene for selecting transformed cells. Examples of genes for selecting transformed cells include drug resistance genes that can be distinguished by drugs (neomycin, G418, etc.). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

  Furthermore, when the gene is stably expressed and the purpose is to amplify the copy number of the gene in the cell, a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway (for example, , PCHOI, etc.) and amplifying with methotrexate (MTX), and for the purpose of transient expression of the gene, COS having a gene expressing SV40 T antigen on the chromosome An example is a method of transforming with a vector (such as pcD) having an SV40 replication origin using cells. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, for gene copy number amplification in host cell systems, the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and the like.

  The antibody of the present invention thus obtained can be isolated from the inside of the host cell or outside the cell (such as a medium) and purified as a substantially pure and homogeneous antibody. Separation and purification of antibodies may be carried out using separation and purification methods used in normal antibody purification, and are not limited in any way. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected, When combined, antibodies can be separated and purified.

  Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel). R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC. Examples of columns used for affinity chromatography include Protein A columns and Protein G columns. For example, columns using Protein A include Hyper D, POROS, Sepharose FF (GE Amersham Biosciences) and the like. The present invention also encompasses antibodies highly purified using these purification methods.

  The obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited thereto. Examples of columns used for affinity chromatography include Protein A columns and Protein G columns.

  The pharmaceutical composition of the present invention to be used for therapeutic or prophylactic purposes may be prepared by mixing with an appropriate pharmaceutically acceptable carrier, vehicle, etc., if necessary, to obtain a lyophilized preparation or a solution preparation. it can. Suitable pharmaceutically acceptable carriers and media include, for example, sterilized water, physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, histidine, Other organic acids), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders and the like can be mentioned. In addition, other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulin, glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine and lysine and other amino acids, polysaccharides and monosaccharides such as saccharides and carbohydrates In addition, sugar alcohols such as mannitol and sorbitol may be contained. In the case of an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, You may use together with adjuvants, such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, polysorbate 20, poloxamer 188, HCO-50) etc. It is also possible to administer a larger liquid volume subcutaneously by mixing hyaluronidase in the preparation (Expert Opin Drug Deliv. 2007 Jul; 4 (4): 427-40.). Moreover, the pharmaceutical composition of the present invention may be previously placed in a syringe. The solution preparation can be prepared according to the method described in WO2011 / 090088.

  Administration of the pharmaceutical composition of the present invention can be administered to a patient via any suitable route. For example, by intravenous, intramuscular, intraperitoneal, intracerebral spinal, transdermal, subcutaneous, intraarticular, sublingual, intrasynovial, oral, inhalation, topical or topical route as a bolus or by continuous infusion over a period of time To be administered. Intravenous or subcutaneous administration is preferred.

  For example, in the case of the bispecific antibody, the dosage is 0.001 to 1000 mg / kg. In the case of the bispecific antibody, the dosing interval is at least 1 day.

  APTT, CWA, TGA and the like are widely known as methods for monitoring the efficacy of blood coagulation factors such as FVIII, and those skilled in the art can use these methods with appropriate modifications and corrections. These methods may be used alone or in combination, and the drug efficacy can be determined based on the result of at least one method.

APTT has been widely used for a long time as a method for monitoring the efficacy of FVIII preparations. APTT after addition of APTT reagent test plasma, the addition of CaCl _2, time fibrinogen is converted to insoluble fibrin, i.e. a method of measuring the time until coagulation is initiated.

  CWA is a test in which the amount of fibrin produced while the coagulation reaction is enhanced is measured over time as an optical (for example, absorbance) change amount. In CWA, a series of coagulation reactions from the start stage of fibrin generation of the coagulation reaction to the amplification stage can be evaluated over time. Furthermore, as for the coagulation initiation reagent of CWA, APTT reagent (Thromb Haemost 2002; 87 (3): 436-41, J Thromb Haemost 2006; 4 (2): 377-84), low concentration tissue factor (TF) and A reagent combined with a mixed solution of blood coagulation factor XII (FXII) activator (ellagic acid) (J Thromb Haemost 2014; 12 (3): 355-62) has been reported. The coagulation waveform is a waveform that represents a change over time in optical information (absorbance) regarding the amount of light. The coagulation waveform is differentiated (first derivative) to calculate the coagulation rate, and the maximum coagulation rate is used as a parameter. The coagulation rate is differentiated (second derivative) to calculate the coagulation acceleration, and the maximum coagulation acceleration is used as a parameter (Haemophilia 2008; 14: 83-92, J Thromb Haemost 2014; 12 (3): 355-62) .

  TGA is a test that measures the amount of thrombin generated while the coagulation reaction is enhanced using a fluorescent substrate for thrombin as enzyme activity over time (Haemophilia 2008; 14 (suppl. 3): 83-92) .

The present invention, containing the multispecific antigen-binding molecules to replace the function of FVIII, used in the prevention and / or treatment of hemophilia A, acquired hemophilia A and other than a von Willebrand's disease F XI disorders Pharmaceutical compositions are provided. An FXI abnormality is a rare hemorrhagic disease caused by a congenital defect or dysfunction of FXI, and examples thereof include hemophilia C. In addition, the decrease or deficiency of FXI activity can be exemplified by congenital and / or acquired causes, but is not limited thereto. The degree of FXI activity decrease is preferably less than 40% (eg, less than 40%, less than 30%, less than 20%, less than 10%), more preferably less than 10% (eg, 10%) compared to healthy subjects. Less than 9%, less than 8%, less than 7%, less than 6%), more preferably less than 5% (eg less than 5%, less than 4%, less than 3%, less than 2%), particularly preferably less than 1% Include, but are not limited to: Methods for measuring the activity of FXI are well known to those skilled in the art (for example, “Basics and Clinics of Hemophilia Useful for Everyone”, Hakuho, Pharmaceutical Journal, 2009, etc.).

As used herein, an aspect represented by the expression “comprising” is an aspect represented by an expression “essentially consisting of”. And embodiments represented by the expression “consisting of”.
The contents of all patents and references explicitly cited herein are hereby incorporated by reference.
The present invention is further illustrated by the following examples, but is not limited to the following examples.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited to these Examples.
[Example 1]
In the present invention, using plasma obtained from a patient with FXI abnormality other than hemophilia A, acquired hemophilia A and von Willebrand disease, plasma with a multispecific antigen-binding molecule having a function of FVIII alternative activity Attempts were made to verify the procoagulant action. As an example, a patent document (WO) that is one of the antibodies having a function of FVIII substitute activity in each of the APTT, CWA and TGA plasma coagulation evaluation methods using commercially available FXI-deficient plasma or FXI-neutralized plasma The plasma coagulation promoting action of ACE910, a bispecific antibody described in 2012/067176) was examined.

[Example 2]
Preparation of anti-FIXa / FX bispecific antibody with FVIII function alternative activity
ACE910, which is one of the anti-FIXa / FX bispecific antibodies having FVIII function alternative activity, was obtained by the method described in WO2005 / 035756, WO2006 / 109592, and WO2012 / 067176. The bispecific antibody was expressed by incorporating the antibody gene into an animal cell expression vector and transfecting it into CHO cells. Subsequently, the bispecific antibody contained in the cell culture supernatant was purified.
Measurement of the FVIII function-substituting activity of the bispecific antibody thus purified was performed by the enzyme assay shown below. At room temperature, 1 nM human FIXa (Enzyme Research Laboratories), 140 nM human FX (Enzyme Research Laboratories), 20 μM phospholipids (10% phosphatidylserine, 60% phosphatidylcholine, 30% phosphatidylethanolamine), and bispecific antibodies After mixing with Tris-buffered saline solution containing 5 mM CaCl ₂ and 0.1% bovine serum albumin, the mixture was incubated for 2 minutes to promote the FX activation reaction with FIXa. The reaction was stopped by adding EDTA. Next, FXa-specific chromogenic substrate solution S-2222 (CHROMOGENIX) was added, and the absorbance change at 405 nm was measured using SpectraMax 340PC384 (Molecular Devices). A calibration curve was prepared from changes in absorbance with known concentrations of human FXa (Enzyme Research Laboratories), and the FXa production promoting activity by the bispecific antibody was evaluated.

[Example 3]
APTT measurement and CWA measurement
Thrombocheck APTT-SLA (Sysmex) was used as the APTT reagent. Human plasma supplemented with 50 μL FXI-deficient human plasma (George King Bio-Medical) containing the bispecific antibody ACE910 or anti-FXI neutralizing antibody (XI-5108, J Thromb Haemost2006; 4: 1496-1501) 50 μL of APTT reagent was added to 50 μL of a commercial product (George King Bio-Medical). After incubation for 3 minutes, 50 μL of a 0.02 mol / L calcium chloride solution was added to start the coagulation reaction, and APTT was measured with a blood coagulation automatic measuring device (CS-2000i, Sysmex) according to a conventional method. The amount of fibrin produced while the coagulation reaction was promoted was measured over time as the amount of change in absorbance, and the maximum coagulation rate and the maximum coagulation acceleration were calculated by CWA. A total of 10 lots of FXI-deficient human plasma were evaluated.

[Example 4]
Coagulation time measurement and CWA measurement by inducing mixed reagent As a reagent for inducing the coagulation reaction, mixed reagent (including tissue factor inobin 0.1 pM [Sysmex], synthetic phospholipid 10 μM [Sysmex] and ellagic acid 0.3 μM [Sysmex]: Measurement concentration) was used. 50μL of FXI-deficient human plasma (commercial product, George King Bio-Medical) containing bispecific antibody ACE910 or human plasma (commercial product, George King Bio-Medical) supplemented with anti-FXI neutralizing antibody (XI-5108) Was added 50 μL of the mixed reagent. After incubation for 3 minutes, 50 μL of a 0.02 mol / L calcium chloride solution was added to start the coagulation reaction, and the coagulation time was measured with an automatic blood coagulation analyzer (CS-2000i, Sysmex). The amount of fibrin produced while the coagulation reaction was promoted was measured over time as the amount of change in absorbance, and the maximum coagulation rate and the maximum coagulation acceleration were calculated by CWA. All 5 lots of FXI-deficient human plasma were evaluated.

[Example 5]
96-well fluorescence plate fluorometer (Thermo Fisher Scientific Instruments) equipped with TGA measurement excitation wavelength 390 nm / fluorescence wavelength 460 nm filter set, dispenser, analysis software (Thrombinoscope software version 3.0.0.29, Thrombinoscope BV) Then, TGA measurement was performed according to a conventional method by calibrated automated thrombography. 80μL of FXI-deficient human plasma (commercial product, George King Bio-Medical) containing bispecific antibody ACE910 or 80μL of human plasma (commercial product, George King Bio-Medical) supplemented with anti-FXI neutralizing antibody (XI-5108) Was added to each well of a 96 well plate. Next, 20 μL of mixed reagents (including tissue factor inobin 0.5 pM [Sysmex], synthetic phospholipid 4 μM [Sysmex] and ellagic acid 0.3 μM [Sysmex]: concentration at the time of measurement) Added to each well. In order to convert the measured fluorescence intensity into the thrombin amount, a well to which 20 μL of Thrombin Calibrator (Thrombinoscope BV) was added instead of the mixed reagent was also provided. In order to start the reaction, 20 μL of FluCa reagent prepared from FluCa kit (Thrombinoscope BV) was automatically dispensed by a programmed apparatus. The thrombogram and peak height (maximum thrombin amount) were calculated by the instrument software. All 5 lots of FXI-deficient human plasma were evaluated.

[Example 6]
APTT measurement results
In the APTT measurement using FXI-deficient plasma, ACE910, one of the bispecific antibodies having a function of FVIII substitute activity, showed a shortening action on clotting time (FIG. 1, individual data of all 10 lots). ACE910 showed a shortened clotting time in a concentration-dependent manner in any lot of plasma. In the results of all 10 lots of FXI-deficient plasma, ACE910 also confirmed the effect of shortening the clotting time (Figure 2, FXI def: mean and standard deviation of 9-10 lots). Moreover, in APTT measurement using FXI neutralized plasma, ACE910 showed a coagulation time shortening action (FIG. 2, Normal + FXI Ab: individual data of 1 lot). As described above, ACE910 showed shortening of the coagulation time in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasmas.

[Example 7]
Results of CWA measurement using APTT reagent
In CWA measurement by APTT reagent induction using FXI-deficient plasma, ACE910 showed an effect of increasing the maximum coagulation rate (Fig. 3, individual data of all 10 lots). ACE910 showed an increase in maximum clotting rate in a concentration-dependent manner in any lot of plasma. In the results of all 10 lots of FXI-deficient plasma, the effect of increasing the maximum clotting rate of ACE910 was also confirmed (Fig. 4, FXI def: mean value and standard deviation of 9-10 lots).
In CWA measurement by APTT reagent induction using FXI-deficient plasma, ACE910 showed the maximum coagulation acceleration action (Fig. 5, individual data of all 10 lots). ACE910 showed an increase in maximum clotting acceleration in a concentration-dependent manner in any lot of plasma. In the result aggregation of all 10 lots of FXI-deficient plasma, the effect of increasing the maximum clotting acceleration of ACE910 was also confirmed (FIG. 6, FXI def: mean value and standard deviation of 9-10 lots). Moreover, in CWA measurement by APTT reagent induction using FXI neutralized plasma, ACE910 showed a shortening action of the maximum clotting acceleration time (FIG. 6, Normal + FXI Ab: individual data of one lot). As described above, ACE910 showed an increase in the maximum coagulation acceleration in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma.

[Example 8]
Results of coagulation time measurement by mixing reagent induction
In measurement of clotting time by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma, ACE910 showed a shortening action of clotting time (FIG. 7, individual data of all 5 lots). ACE910 showed a shortened clotting time in a concentration-dependent manner in any lot of plasma. In the results of all five lots of FXI-deficient plasma, ACE910 also confirmed the effect of shortening the clotting time (Fig. 8, FXI def: mean and standard deviation of 4-5 lots). In addition, ACE910 showed the effect of shortening the clotting time in the measurement of clotting time by inducing mixed reagent (including tissue factor and ellagic acid) using FXI neutralized plasma (Figure 8, Normal + FXI Ab: Individual data of 1 lot) ). As described above, ACE910 showed shortening of the coagulation time in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasmas.

[Example 9]
Result of CWA measurement by inducing mixed reagent
ACE910 showed the effect of increasing the maximum clotting rate in the CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (Fig. 9, individual data of all 5 lots). ACE910 showed an increase in maximum clotting rate in a concentration-dependent manner in any lot of plasma. In the result summation of all 5 lots of FXI-deficient plasma, the effect of increasing the maximum clotting rate of ACE910 was also confirmed (FIG. 10, FXI def: mean value and standard deviation of 4-5 lots). In addition, ACE910 showed an effect of increasing the maximum clotting rate in the CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI neutralized plasma (Figure 10, Normal + FXI Ab: Individual data of 1 lot ). As described above, ACE910 showed an increase in the maximum coagulation rate in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma.
ACE910 showed an effect of increasing the maximum clotting acceleration in CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI-deficient plasma (Fig. 11, individual data of all 5 lots). ACE910 showed an increase in maximum clotting acceleration in a concentration-dependent manner in any lot of plasma. In the results of all five lots of FXI-deficient plasma, ACE910 also confirmed the effect of increasing the maximum clotting acceleration (Fig. 12, FXI def: mean value and standard deviation of 4-5 lots). In addition, ACE910 showed the effect of increasing the maximum coagulation acceleration in the CWA measurement by inducing mixed reagent (including tissue factor and ellagic acid) using FXI neutralized plasma (Figure 12, Normal + FXI Ab: Individual data of 1 lot) ). As described above, ACE910 showed an increase in the maximum coagulation acceleration in a concentration-dependent manner in both FXI-deficient and FXI-neutralized plasma.

[Example 10]
Results of TGA measurement by inducing mixed reagent
ACE910 showed plasma coagulation-promoting action in TGA measurement induced by mixed reagents (including tissue factor and ellagic acid) using FXI-deficient plasma (FIG. 13, individual data for all 10 lots). ACE910 showed an increase in thrombin generation peak height in a concentration-dependent manner.

  According to the present invention, in FXI abnormalities other than hemophilia A, acquired hemophilia A and von Willebrand disease, a method for preventing the onset and / or progression of bleeding, bleeding-related diseases, or diseases caused by bleeding And / or methods of using multispecific antigen binding molecules to replace the function of FVIII as therapeutics. Multispecific antigen-binding molecules that replace FVIII function can be used as a prophylactic and / or therapeutic approach for hemorrhage in hemophilia A, acquired hemophilia A, and von Willebrand disease caused by FVIII dysfunction It can be used as a preventive and / or therapeutic method against bleeding in other FXI abnormalities because of its procoagulant activity. Since FXI abnormalities are rare hemorrhagic diseases, effective prevention methods and / or treatment methods for bleeding have not been established, and the present invention prevents prophylaxis against the onset and / or progression of bleeding in FXI abnormal diseases. Promising as a method and / or treatment.

Claims (7)

A pharmaceutical composition used for the prevention and / or treatment of blood coagulation factor XI abnormality, comprising a multispecific antigen-binding molecule that substitutes for the function of blood coagulation factor VIII. A multispecific antigen binding molecule that replaces the function of blood coagulation factor VIII is a bispecific antibody that binds to blood coagulation factor IX and / or active blood coagulation factor IX and blood coagulation factor X, The pharmaceutical composition according to claim 1. The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The pharmaceutical composition according to claim 1 or 2.
The first polypeptide is an H chain comprising the amino acid sequence of H chain CDR1, 2, 3 described in SEQ ID NO: 1, 2, 3 (H chain CDR of Q499), the second polypeptide is SEQ ID NO: 4, H chain containing the amino acid sequence of H chain CDR1, 2, 3 described in 5, 6 (H chain CDR of J327), the third polypeptide and the fourth polypeptide are SEQ ID NO: 7, 8, 9 (L404 A bispecific antibody (Q499-z121 / J327-z119 / L404-k) consisting of a common L chain comprising the amino acid sequences of the L chain CDR1, 2, and 3 described in the above (L chain CDR). The bispecific antibody is an antibody described below, wherein the first polypeptide and the third polypeptide are associated, and the second polypeptide and the fourth polypeptide are associated. The pharmaceutical composition according to any one of claims 1 to 3.
The first polypeptide is an H chain consisting of the amino acid sequence set forth in SEQ ID NO: 10, the second polypeptide is the H chain consisting of the amino acid sequence set forth in SEQ ID NO: 11, the third polypeptide and the fourth poly A bispecific antibody (Q499-z121 / J327-z119 / L404-k), wherein the peptide consists of a common L chain described in SEQ ID NO: 12. The blood coagulation factor XI abnormality is a disease that develops and / or progresses due to decreased activity, functional abnormality and / or deficiency of blood coagulation factor XI and / or active blood coagulation factor XI. 5. The pharmaceutical composition according to any one of 4. The pharmaceutical composition according to any one of claims 1 to 5, wherein the blood coagulation factor XI abnormality is a congenital or acquired disease. The pharmaceutical composition according to any one of claims 1 to 6, wherein the blood coagulation factor XI abnormality is hemophilia C.

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