WO2021210667A1 - Bispecific antigen-binding molecule, composition associated therewith, and use, kit, and method for producing composition - Google Patents

Abstract

The present invention provides a bispecific antigen-binding molecule that recognizes both of two specific factors out of various coagulation-related factors, as well as a composition and a kit containing the bispecific antigen-binding molecule. In one non-limiting embodiment, the bispecific antigen-binding molecule, composition, and kit of the present invention are used to prevent and/or treat hemorrhaging, diseases accompanied by hemorrhaging, or diseases caused by hemorrhaging. Additionally, the present invention provides a method for using the bispecific antigen-binding molecule to promote coagulation. Furthermore, the present invention provides a screening method for substances that are effective for preventing and/or treating hemorrhaging, diseases accompanied by hemorrhaging, or diseases caused by hemorrhaging.

Description

Bispecific antigen-binding molecules and their associated compositions, uses, kits, and methods for the manufacture of compositions.

The present disclosure relates to bispecific antigen-binding molecules that recognize both of two specific blood coagulation-related factors, as well as compositions containing them, their use in the manufacture of compositions, and kits. .. The present disclosure also relates to a method for promoting blood coagulation using the bispecific antigen-binding molecule. Furthermore, the present disclosure relates to methods of screening for substances that are effective in the prevention and / or treatment of bleeding, bleeding-related diseases, or diseases caused by bleeding.

Blood coagulation consists of a series of components, especially fibrinogen, blood coagulation factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII (F.II, respectively). FV, F.VII, F.VIII, F.IX, FX, F.XI, F.XII) and their active forms (F.IIa, F.Va, F.VIIa, F.VIIIa, F.IXa, respectively) , F.Xa, F.XIa, F.XIIa), as well as a complex process involving the continuous interaction of von Villebrand factors.

Hemophilia is a bleeding disease in which blood coagulation is caused by deficiency of these components or inhibition of their functionality. If it is caused by F.VIII, it is called hemophilia A, and if it is caused by F.IX, it is called hemophilia B. In patients with hemophilia, bleeding symptoms are seen in deep tissues such as joints and muscles, and in severe cases, intracranial hemorrhage also occurs.

The severity of hemophilia is classified based on F.VIII activity or F.IX activity in the blood. Specifically, assuming that the F.VIII activity or F.IX activity of a healthy person is 100%, the activity is severe in patients with an activity of less than 1%, and moderate in patients with an activity of 1% or more and less than 5%. Patients with 5% or more and less than 40% are classified as mild. Patients with severe hemophilia present significantly more frequent bleeding symptoms than patients with moderate and mild illness. However, F.VIII or F.IX replacement therapy can dramatically reduce the frequency of bleeding by maintaining F.VIII or F.IX activity in the patient's blood at 1% or higher. .. For replacement therapy, coagulation factor preparations purified from plasma or prepared by genetic recombination technology are mainly used.

For bleeding in hemophilia A patients, F.VIII preparation is usually administered (on-demand administration). In recent years, in order to prevent bleeding events, F.VIII preparations are prophylactically administered (Non-Patent Documents 1 and 2) (preventive administration). The half-life of the F.VIII product in blood is about 12 to 16 hours. Therefore, for continuous prophylaxis, the F.VIII preparation is administered to the patient three times a week (Non-Patent Documents 3 and 4). In addition, in on-demand administration, in order to prevent rebleeding, the F.VIII preparation is additionally administered at regular intervals as needed. In addition, administration of F.VIII preparation is performed intravenously. Occasionally, antibodies (inhibitors) to F.VIII occur in patients with hemophilia. The inhibitor counteracts the effect of the F.VIII preparation. Therefore, there has been a strong demand for a drug that is less burdensome to administer than the F.VIII preparation and is not affected by the presence of the inhibitor.

Bispecific antibodies that substitute for the function of F.VIII and their use have been reported as means for solving these problems (Patent Documents 1, 2, 3 and 4). Bispecific antibodies against F.IXa and FX exert F.VIII cofactor function substitution activity and can substitute F.VIII function by positioning both factors in the vicinity (non-F.VIII cofactor function substitution activity). Patent Document 5). One of the antibodies, ACE910 (Emicizumab), which has high F.VIII cofactor function substitution activity, has been confirmed to have excellent pharmacokinetics (long half-life) and tolerability in clinical trials in healthy subjects. (Non-Patent Document 6), in clinical trials in patients with hemophilia A who do not have or carry inhibitors, the number of bleedings significantly increased by administration of ACE910 (Emicizumab) compared to before administration of ACE910 (Emicizumab). Suppression was observed (Non-Patent Document 7).

For bleeding in hemophilia B patients, F.IX preparation is regularly administered. However, the conventional F.IX preparation having a standard half-life requires intravenous administration twice a week, and frequent administration is a burden on patients and their families. In addition, anti-F.IX antibodies (inhibitors) may develop during hemostasis management. As a result, hemostasis management is often difficult. It is well known that patients with F.IX inhibitors exhibit anaphylactic symptoms as a characteristic side reaction not seen in F.VIII inhibitors, which is a problem (Non-Patent Document 8). Therefore, there has been a strong demand for a drug that is less burdensome to administer than the F.IX preparation and is not affected by the presence of the inhibitor.

F.XI is a protease precursor of F.XIa and contributes to hemostasis through activation of F.IX. In recent years, it has been reported that the activation peptide of F.X can be cleaved by adding a high concentration of F.XIa to F.X, and that F.Va is produced by adding it to F.V (Non-Patent Document 9). In addition, it was reported that the duration of bleeding was reduced by intravenous administration of an excessive amount of F.XIa (estimated plasma concentration of 60 nM) in hemophilia B model mice genetically deficient in F.IX. (Non-Patent Document 10). Therefore, administration of excess F.XI or F.XIa to patients with hemophilia B may reduce the number of bleedings with or without inhibitors. However, it has been reported that the F.XI preparation causes thrombosis when administered to F.XI-deficient patients, and has not been established as a safe treatment method (Non-Patent Document 11). This may be due to the fact that the F.XIa preparation contains F.XIa, and administration of the F.XIa preparation is not a safe treatment method. Therefore, there has been a demand for a therapeutic method for safely preventing bleeding in hemophiliacs by a method other than overdose of the F.XI preparation or the F.XIa preparation.

WO 2005/035754 WO 2005/035756 WO 2006/109592 WO 2012/067176

Blood 58, 1-13 (1981) Nature 312, 330-337 (1984) Nature 312, 337-342 (1984) Biochim.Biophys.Acta 871, 268-278 (1986) Nat Med. 2012 Oct; 18 (10): 1570-4. Blood. 2016, Vol.127, 13: 1633-1641. New Eng J Med 2016, 374; 21, 2044-2053 Semin Thromb Hemost. 2018; 44 (6): 578-589. J Thromb Haemost. 2013; 11 (12): 2118-2127. J Thromb Haemost. 2018; 16 (10): 2044-2049. Haemophilia. 2015; 21 (4): 477-480.

The invention in the present disclosure has been made in view of the above circumstances and is used in a non-limiting aspect for the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding. The purpose is to provide new molecules.

In one non-limiting aspect, the present inventors recognize two blood coagulation-related factors in which cofactors that are close to each other in vivo have not been confirmed as a result of diligent research. The production was successful. We also found that the bispecific antigen-binding molecule contributes to the promotion of blood coagulation.

The present disclosure is based on such findings and specifically includes embodiments exemplified below.
[A] A bispecific antigen-binding molecule that recognizes both an enzyme and a substrate that can undergo a catalytic reaction of the enzyme, but a bispecific antigen-binding molecule that substitutes for the function of a peptidic cofactor or heparin expressed in a living body. Excludes molecules.
[B] The bispecific antigen-binding molecule according to [A], which is a bispecific antigen-binding molecule that recognizes both an enzyme and a substrate capable of undergoing a catalytic reaction of the enzyme, and promotes hydrolysis of the substrate by the enzyme. Sex antigen binding molecule.
[C] The bispecific antigen-binding molecule according to [A], which is a bispecific antigen-binding molecule that recognizes both an enzyme and a substrate capable of undergoing a catalytic reaction of the enzyme, and promotes activation of the substrate by the enzyme. Sex antigen binding molecule.
[D] The bispecific antigen-binding molecule according to any one of [A] to [C], wherein the enzyme is a protease.
[E] The bispecific antigen-binding molecule according to any one of [A] to [C], wherein the enzyme is a serine protease.
[F] The bispecific antigen according to any one of [A] to [E], wherein the enzyme is a blood coagulation enzyme and the substrate capable of undergoing a catalytic reaction of the enzyme is a blood coagulation substrate. Binding molecule.
[G] Blood coagulation factor V, blood coagulation factor VIII, tissue factor (TF), thrombomodulin (TM), protein S (PS), protein Z (PZ), which are peptide complements expressed in the living body. A dual alternative to the function of at least one complement or heparin selected from the group consisting of complement C4b, complement regulatory factor H, membrane cofactor protein (MCP), and complement receptor 1 (CR1). The bispecific antigen-binding molecule according to any one of [A] to [F], excluding the specific antigen-binding molecule.
[H] The bispecific antigen-binding molecule according to any one of [A] to [G], which is a bispecific antibody.
[1] The bispecific antigen-binding molecule according to any one of (a) to (h) below:
(a) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X;
(b) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor X and blood coagulation factor X;
(c) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor VII and blood coagulation factor X;
(d) Activated blood coagulation factor VII-A bispecific antigen-binding molecule that recognizes both Tissue Factor complex and blood coagulation factor X;
(e) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor X;
(f) A bispecific antigen-binding molecule that recognizes both Thrombin and blood coagulation factor X;
(g) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor IX; or
(h) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor XI.
[1a] A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X.
[2] The bispecific antigen-binding molecule according to [1] or [1a], which promotes activation of the substrate by the enzyme.
[2a] The bispecific antigen-binding molecule according to [1] or [1a], which exhibits an absorbance of 0.1 or more 30 minutes after the addition of the color-developing substrate solution in an in vitro enzyme reaction measurement system. Sex antigen binding molecule.
[3] The bispecific antigen-binding molecule according to [1] or [1a], [2] or [2a], which is a bispecific antibody.
[4] A composition comprising the antigen-binding molecule according to any one of [1] to [3] and a pharmaceutically acceptable carrier.
[5] The composition according to [4], which is a pharmaceutical composition used for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
[6] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [5].
[7] The composition according to [6], wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activation of blood coagulation factor IX is hemophilia B.
[8] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor IX and / or activated blood coagulation factor IX are associated with blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [6], which is a disease in which an inhibitor appears.
[9] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [5].
[10] Blood coagulation factor VIII and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII are hemophilia A, acquired hemophilia, or Fonville brand disease. The composition according to [9].
[11] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII are associated with blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [9], which is a disease in which an inhibitor appears.
[12] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [5].
[13] The composition according to [12], wherein the disease that develops and / or progresses due to a decrease or deficiency of the activity of blood coagulation factor XI and / or activation of blood coagulation factor XI is hemophilia C.
[14] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor XI and / or activation with respect to blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [12], which is a disease in which an inhibitor appears.
[15] Use of the bispecific antigen-binding molecule according to any one of [1] to [3] for producing the composition according to any one of [4] to [14].
[16] Bleeding, a disease associated with bleeding, or a disease caused by bleeding, which comprises at least the bispecific antigen-binding molecule according to any one of [1] to [3] or the composition according to [4]. A kit for use in methods of preventing and / or treating.
[17] Bleeding or bleeding containing at least the bispecific antigen-binding molecule according to any one of [1] to [3] or the composition according to [4] and containing blood coagulation factor IX. A kit for use in methods of preventing and / or treating associated diseases or diseases caused by bleeding in combination with blood coagulation factor IX.
[18] Bleeding, bleeding containing at least the bispecific antigen-binding molecule according to any one of [1] to [3] or the composition according to [4] and containing blood coagulation factor VIII. A kit for use in methods of preventing and / or treating associated diseases or diseases caused by bleeding in combination with blood coagulation factor VIII.
[19] Bleeding or bleeding containing at least the bispecific antigen-binding molecule according to any one of [1] to [3] or the composition according to [4] and containing blood coagulation factor XI. A kit for use in a method of preventing and / or treating a concomitant disease or a disease caused by bleeding in combination with blood coagulation factor XI.
[20] Containing at least the bispecific antigen-binding molecule according to any one of [1] to [3] or the composition according to [4], and caused by bleeding, a disease accompanied by bleeding, or bleeding. A kit for use in methods of preventing and / or treating a disease in combination with a bypass formulation.
[21] A method for promoting blood coagulation using a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X.
[22] A method for screening a substance effective for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A screening method comprising (1) evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) evaluating the binding between the test substance and blood coagulation factor X.
[23] A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using the screening method (3) test substance according to [22], which further comprises the following steps.
[24] A quality test method for a substance or composition effective for the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A quality test method including (1) a step of evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) a step of evaluating the binding between the test substance and blood coagulation factor X.
[25] The quality test method according to [24], which further comprises the following steps:
(3) A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance.
[26] A nucleic acid encoding the bispecific antigen-binding molecule according to any one of [1] to [3].
[27] A vector into which the nucleic acid according to [26] has been inserted.
[28] A cell containing the nucleic acid according to [26] or the vector according to [27].
[29] A method for producing the bispecific antigen-binding molecule according to any one of [1] to [3] by culturing the cells according to [28].
[30] The bispecific antigen-binding molecule according to any one of (i) to (o) below:
(i) Activated A bispecific antigen-binding molecule that recognizes both blood coagulation factor XI and blood coagulation factor X, and is a bispecific antigen-binding molecule that activates blood coagulation factor X;
(j) Activated A bispecific antigen-binding molecule that recognizes both blood coagulation factor X and blood coagulation factor X, and is a bispecific antigen-binding molecule that activates blood coagulation factor X;
(k) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor VII and blood coagulation factor X, and is a bispecific antigen-binding molecule that activates blood coagulation factor X;
(l) Activated blood coagulation factor VII-A bispecific antigen-binding molecule that recognizes both Tissue Factor complex and blood coagulation factor X, and is a bispecific antigen that activates blood coagulation factor X. Binding molecule;
(m) Activated A bispecific antigen-binding molecule that recognizes both blood coagulation factor XII and blood coagulation factor X, and is a bispecific antigen-binding molecule that activates blood coagulation factor X;
(n) A bispecific antigen-binding molecule that recognizes both Thrombin and blood coagulation factor X and activates blood coagulation factor X; or
(o) Activated A bispecific antigen-binding molecule that recognizes both blood coagulation factor XII and blood coagulation factor IX, and is a bispecific antigen-binding molecule that activates blood coagulation factor IX.
[30a] A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X, and is a bispecific antigen-binding molecule that activates blood coagulation factor X.
[31] The bispecific antigen-binding molecule according to [30] or [30a], which is a bispecific antibody.
[32] A composition comprising the antigen-binding molecule according to [30], [30a], or [31] and a pharmaceutically acceptable carrier.
[33] The composition according to [32], which is a pharmaceutical composition used for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
[34] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [33].
[35] The composition according to [34], wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activation of blood coagulation factor IX is hemophilia B.
[36] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor IX and / or activated blood coagulation factor IX are associated with blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [34], which is a disease in which an inhibitor appears.
[37] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [33].
[38] Blood coagulation factor VIII and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII are hemophilia A, acquired hemophilia, or Fonville brand disease. The composition according to [37].
[39] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII are associated with blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [37], which is a disease in which an inhibitor appears.
[40] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [33].
[41] The composition according to [40], wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activation of blood coagulation factor XI is hemophilia C.
[42] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor XI and / or activation are associated with blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [40], which is a disease in which an inhibitor appears.
[43] Use of the bispecific antigen-binding molecule of [30] or [31] for producing the composition according to any one of [32] to [42].
[44] Due to bleeding, a disease associated with bleeding, or bleeding, which comprises at least the bispecific antigen binding molecule according to [30], [30a], or [31], or the composition according to [32]. A kit for use in methods of preventing and / or treating bleeding disorders.
[45] Bleeding, which comprises at least the bispecific antigen-binding molecule according to [30], [30a], or [31], or the composition according to [32], and contains blood coagulation factor IX. A kit for use in methods of preventing and / or treating bleeding-related or bleeding-related disorders in combination with blood coagulation factor IX.
[46] Bleeding, bleeding, comprising at least the bispecific antigen binding molecule according to [30], [30a] or [31], or the composition according to [32], and containing blood coagulation factor VIII. A kit for use in a method of preventing and / or treating a disease associated with blood coagulation or a disease caused by bleeding in combination with blood coagulation factor VIII.
[47] Bleeding, bleeding, comprising at least the bispecific antigen binding molecule according to [30], [30a] or [31], or the composition according to [32], and containing blood coagulation factor XI. A kit for use in a method of preventing and / or treating a disease associated with or caused by bleeding in combination with blood coagulation factor XI.
[48] Containing at least the bispecific antigen-binding molecule according to [30], [30a] or [31], or the composition according to [32], and caused by bleeding, a disease associated with bleeding, or bleeding. A kit for use in methods of preventing and / or treating bleeding disorders in combination with bypass formulations.
[49] A method for promoting blood coagulation using a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X.
[50] A method for screening a substance effective for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A screening method comprising (1) evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) evaluating the binding between the test substance and blood coagulation factor X.
[51] A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using the screening method (3) test substance according to [50], which further comprises the following steps.
[52] A quality test method for a substance or composition effective for the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A quality test method including (1) a step of evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) a step of evaluating the binding between the test substance and blood coagulation factor X.
[53] The quality test method according to [52], further comprising the following steps:
(3) A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance.
[54] The nucleic acid encoding the bispecific antigen-binding molecule according to [30], [30a] or [31].
[55] A vector into which the nucleic acid according to [54] has been inserted.
[56] A cell containing the nucleic acid according to [54] or the vector according to [55].
[57] The method for producing the bispecific antigen-binding molecule according to [30], [30a], or [31] by culturing the cells according to [56].
[58] The bispecific antigen-binding molecule according to any one of [A] to [H], [1] to [3], and [30] to [31], or [4] to [14] and A method for preventing and / or treating bleeding, a disease associated with bleeding, or a disease caused by bleeding, which comprises the step of administering the composition according to any one of [32] to [42].
[59] [A]-[H], [1]-[3], and [30]-[31] for use in the prevention and / or treatment of bleeding, diseases associated with bleeding, or diseases caused by bleeding. ], Or the composition according to any one of [4] to [14] and [32] to [42].
[60] [A]-[H], [1]-[3], and [30]-[in the manufacture of prophylactic and / or therapeutic agents for bleeding, diseases associated with bleeding, or diseases caused by bleeding. Use of the bispecific antigen-binding molecule according to any one of 31] or the composition according to any one of [4] to [14] and [32] to [42].
[61] The bispecific antigen-binding molecule according to any one of [A] to [H], [1] to [3], and [30] to [31], or [4] to [14] and A prophylactic and / or therapeutic agent for bleeding, a disease associated with bleeding, or a disease caused by bleeding, which comprises the composition according to any one of [32] to [42].
[62] The bispecific antigen-binding molecule according to any one of [A] to [H], [1] to [3], and [30] to [31], or [4] to [14] and A method for promoting blood coagulation, which comprises the step of administering the composition according to any one of [32] to [42].
[63] The bispecific antigen-binding molecule according to any one of [A] to [H], [1] to [3], and [30] to [31] for use in promoting blood coagulation. Alternatively, the composition according to any one of [4] to [14] and [32] to [42].
[64] The bispecific antigen-binding molecule according to any one of [A] to [H], [1] to [3], and [30] to [31] in the production of a blood coagulation promoter, or Use of the composition according to any one of [4] to [14] and [32] to [42].
[65] The bispecific antigen-binding molecule according to any one of [A] to [H], [1] to [3], and [30] to [31], or [4] to [14] and A blood coagulation promoter comprising the composition according to any one of [32] to [42].

[1b] The antigen-binding molecule according to any one of (a) to (h) below:
(a) Antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X;
(b) Antigen-binding molecule that recognizes both activated blood coagulation factor X and blood coagulation factor X;
(c) Antigen-binding molecule that recognizes both activated blood coagulation factor VII and blood coagulation factor X;
(d) Activated blood coagulation factor VII-An antigen-binding molecule that recognizes both Tissue Factor complex and blood coagulation factor X;
(e) Antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor X;
(f) Antigen-binding molecule that recognizes both Thrombin and blood coagulation factor X;
(g) Antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor IX; or
(h) An antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor XI.
[2b] The antigen-binding molecule according to [1b], which promotes activation of the substrate by the enzyme.
[2c] The antigen-binding molecule according to [1b], which has an absorbance of 0.1 or more 30 minutes after the addition of the color-developing substrate solution in an in vitro enzyme reaction measurement system.
[3b] The antigen-binding molecule according to [1b], [2b] or [2c], which is an antibody.
[4b] A composition comprising the antigen-binding molecule according to any one of [1b] to [3b] and a pharmaceutically acceptable carrier.
[5b] The composition according to [4b], which is a pharmaceutical composition used for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
[6b] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [5b].
[7b] The composition according to [6b], wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activation of blood coagulation factor IX is hemophilia B.
[8b] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor IX and / or activated blood coagulation factor IX are associated with blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [6b], which is a disease in which an inhibitor appears.
[9b] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [5b].
[10b] Blood coagulation factor VIII and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII are hemophilia A, acquired hemophilia, or Fonville brand disease. The composition according to [9b].
[11b] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII are associated with blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [9b], which is a disease in which an inhibitor appears.
[12b] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [5b].
[13b] The composition according to [12b], wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activation of blood coagulation factor XI is hemophilia C.
[14b] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor XI and / or activation are associated with blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [12b], which is a disease in which an inhibitor appears.
[15b] Use of any of the antigen-binding molecules of [1b] to [3b] for producing the composition according to any one of [4b] to [14b].
[16b] Preventing and / or bleeding, bleeding-related diseases, or diseases caused by bleeding, which contain at least the antigen-binding molecule according to any one of [1b] to [3b] or the composition according to [4b]. Or a kit for use in therapeutic methods.
[17b] Bleeding, a disease associated with bleeding, or a disease containing blood coagulation factor IX and containing at least the antigen-binding molecule according to any one of [1b] to [3b] or the composition according to [4b]. A kit for use in methods of preventing and / or treating diseases caused by bleeding in combination with blood coagulation factor IX.
[18b] Bleeding, a disease associated with bleeding, or a disease containing at least the antigen-binding molecule according to any one of [1b] to [3b], or the composition according to [4b], and containing blood coagulation factor VIII. A kit for use in methods of preventing and / or treating diseases caused by bleeding in combination with blood coagulation factor VIII.
[19b] Bleeding, a disease associated with bleeding, or a disease containing blood coagulation factor XI and containing at least the antigen-binding molecule according to any one of [1b] to [3b] or the composition according to [4b]. A kit for use in methods of preventing and / or treating diseases caused by bleeding in combination with blood coagulation factor XI.
[20b] Bypasses bleeding, diseases associated with bleeding, or diseases caused by bleeding, which contain at least the antigen-binding molecule according to any one of [1b] to [3b] or the composition according to [4b]. A kit for use in prophylactic and / or therapeutic methods in combination with formulations.
[21b] A method for promoting blood coagulation using an antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X.
[22b] A method for screening a substance effective for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A screening method comprising (1) evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) evaluating the binding between the test substance and blood coagulation factor X.
[23b] A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using the screening method (3) test substance according to [22b], which further comprises the following steps.
[24b] A quality test method for a substance or composition effective for the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A quality test method including (1) a step of evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) a step of evaluating the binding between the test substance and blood coagulation factor X.
[25b] The quality test method according to [24b], further comprising the following steps:
(3) A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance.
[26b] A nucleic acid encoding the antigen-binding molecule according to any one of [1b] to [3b].
[27b] A vector into which the nucleic acid according to [26b] has been inserted.
[28b] A cell containing the nucleic acid according to [26b] or the vector according to [27b].
[29b] A method for producing the antigen-binding molecule according to any one of [1b] to [3b] by culturing the cells according to [28b].
[30b] The antigen-binding molecule according to any one of (i) to (o) below:
(i) Activated antigen-binding molecule that recognizes both blood coagulation factor XI and blood coagulation factor X, and is an antigen-binding molecule that activates blood coagulation factor X;
(j) Activated antigen-binding molecule that recognizes both blood coagulation factor X and blood coagulation factor X, and is an antigen-binding molecule that activates blood coagulation factor X;
(k) Activated antigen-binding molecule that recognizes both blood coagulation factor VII and blood coagulation factor X, and is an antigen-binding molecule that activates blood coagulation factor X;
(l) Activated blood coagulation factor VII-An antigen-binding molecule that recognizes both Tissue Factor complex and blood coagulation factor X, and is an antigen-binding molecule that activates blood coagulation factor X;
(m) Activated antigen-binding molecule that recognizes both blood coagulation factor XII and blood coagulation factor X, and activates blood coagulation factor X;
(n) An antigen-binding molecule that recognizes both Thrombin and blood coagulation factor X and activates blood coagulation factor X; or
(o) Activated antigen-binding molecule that recognizes both blood coagulation factor XII and blood coagulation factor IX, and is an antigen-binding molecule that activates blood coagulation factor IX.
[31b] The antigen-binding molecule according to [30b], which is an antibody.
[32b] A composition comprising the antigen-binding molecule according to [30b] or [31b] and a pharmaceutically acceptable carrier.
[33b] The composition according to [32b], which is a pharmaceutical composition used for the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
[34b] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [33b].
[35b] The composition according to [34b], wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activation of blood coagulation factor IX is hemophilia B.
[36b] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor IX and / or activated blood coagulation factor IX are associated with blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to [34b], which is a disease in which an inhibitor appears.
[37b] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [33b].
[38b] Blood coagulation factor VIII and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII are hemophilia A, acquired hemophilia, or Fonville brand disease. The composition according to [37b].
[39b] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII are associated with blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to [37b], which is a disease in which an inhibitor appears.
[40b] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [33b].
[41b] The composition according to [40b], wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activation of blood coagulation factor XI is hemophilia C.
[42b] Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor XI and / or activation are associated with blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to [40b], which is a disease in which an inhibitor appears.
[43b] Use of the antigen-binding molecule of [30b] or [31b] for producing the composition according to any one of [32b] to [42b].
[44b] Prevents and / or treats bleeding, bleeding-related diseases, or diseases caused by bleeding, which comprises at least the antigen-binding molecule according to [30b] or [31b], or the composition according to [32b]. Kit for use in the method.
[45b] Due to bleeding, bleeding-related disease, or bleeding, comprising at least the antigen-binding molecule according to [30b] or [31b], or the composition according to [32b], and containing blood coagulation factor IX. A kit for use in methods of preventing and / or treating bleeding disorders in combination with blood coagulation factor IX.
[46b] Due to bleeding, bleeding-related disease, or bleeding, comprising at least the antigen-binding molecule according to [30b] or [31b], or the composition according to [32b], and containing blood coagulation factor VIII. A kit for use in methods of preventing and / or treating bleeding disorders in combination with blood coagulation factor VIII.
[47b] Caused by bleeding, a disease associated with bleeding, or bleeding, which comprises at least the antigen-binding molecule according to [30b] or [31b], or the composition according to [32b] and contains blood coagulation factor XI. A kit for use in methods of preventing and / or treating bleeding disorders in combination with factor XI for blood coagulation.
[48b] A bleeding, a disease associated with bleeding, or a disease caused by bleeding, which comprises at least the antigen-binding molecule described in [30b] or [31b] or the composition described in [32b], is used in combination with a bypass preparation. And a kit for use in prophylactic and / or therapeutic methods.
[49b] A method for promoting blood coagulation using an antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X.
[50b] A method for screening a substance effective for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A screening method comprising (1) evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) evaluating the binding between the test substance and blood coagulation factor X.
[51b] A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using the screening method (3) test substance according to [50b], which further comprises the following steps.
[52b] A quality test method for a substance or composition effective for the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding.
A quality test method including (1) a step of evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) a step of evaluating the binding between the test substance and blood coagulation factor X.
[53b] The quality test method according to [52b], further comprising the following steps:
(3) A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance.
[54b] A nucleic acid encoding the antigen-binding molecule according to [30b] or [31b].
[55b] A vector into which the nucleic acid according to [54b] has been inserted.
[56b] A cell containing the nucleic acid according to [54b] or the vector according to [55b].
[57b] A method for producing the antigen-binding molecule according to [30b] or [31b] by culturing the cells according to [56b].
[58b] The antigen-binding molecule according to any one of [1b] to [3b] and [30b] to [31b], or any of [4b] to [14b] and [32b] to [42b]. A method for preventing and / or treating bleeding, a disease associated with bleeding, or a disease caused by bleeding, which comprises the step of administering the composition of.
[59b] The antigen according to any one of [1b] to [3b] and [30b] to [31b] for use in the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding. The binding molecule or the composition according to any one of [4b] to [14b] and [32b] to [42b].
[60b] The description of any of [1b] to [3b] and [30b] to [31b] in the manufacture of a prophylactic and / or therapeutic agent for bleeding, a disease associated with bleeding, or a disease caused by bleeding. Use of an antigen-binding molecule or the composition according to any of [4b]-[14b] and [32b]-[42b].
[61b] The antigen-binding molecule according to any one of [1b] to [3b] and [30b] to [31b], or any of [4b] to [14b] and [32b] to [42b]. A prophylactic and / or therapeutic agent for bleeding, bleeding-related diseases, or diseases caused by bleeding, which comprises the composition of.
[62b] The antigen-binding molecule according to any one of [1b] to [3b] and [30b] to [31b], or any of [4b] to [14b] and [32b] to [42b]. A method of promoting blood coagulation, which comprises the step of administering the composition of.
[63b] The antigen-binding molecule according to any one of [1b] to [3b] and [30b] to [31b] for use in promoting blood coagulation, or [4b] to [14b] and [32b]. The composition according to any one of [42b].
[64b] The antigen-binding molecule according to any one of [1b] to [3b] and [30b] to [31b] in the production of a blood coagulation promoter, or [4b] to [14b] and [32b] to. Use of the composition according to any one of [42b].
[65b] The antigen-binding molecule according to any one of [1b] to [3b] and [30b] to [31b], or any of [4b] to [14b] and [32b] to [42b]. A blood coagulation promoter, which comprises the composition of.

In one non-limiting aspect, the bispecific antigen-binding molecules, compositions, and kits of the present disclosure contribute to the promotion of blood coagulation and thus prevent bleeding, bleeding-related diseases, or diseases caused by bleeding. And / or may be useful for treatment.

The basic concept of the present invention is illustrated. Enzyme A and substrate B, which are related so that peptide cofactors do not exist in the living body, hardly proceed with the reaction as they are (upper figure). However, by adding a bispecific antigen-binding molecule that recognizes the enzyme A and the substrate B, it is possible to promote an enzymatic reaction such as a hydrolysis reaction of the substrate B by the enzyme A (see the figure below). A schematic diagram (intrinsic system / extrinsic system) of the blood coagulation cascade is shown. The measurement results of the F.X activation promoting activity of anti-F.XIa and F.X bispecific antibodies by F.XIa in the in vitro enzyme reaction system are shown. The prepared anti-F.XIa and F.X bispecific antibodies showed F.X activation promoting activity by F.XIa. The coagulation time (APTT) in F.IX-deficient plasma is shown. The prepared anti-F.XIa and F.X bispecific antibodies showed the coagulation-promoting activity of the plasma. The measurement results of the F.X activation promoting activity of anti-F.XIa and F.X bispecific antibodies by F.XIa in the in vitro enzyme reaction system are shown. The prepared anti-F.XIa and F.X bispecific antibodies showed F.X activation promoting activity by F.XIa. The coagulation time (APTT) in F.IX-deficient plasma is shown. The prepared anti-F.XIa and F.X bispecific antibodies showed the coagulation-promoting activity of the plasma.

I. Definitions The term "antigen-binding molecule" as used herein refers to a molecule that specifically binds to an antigenic determinant (epitope) in its broadest sense. In one embodiment, the antigen binding molecule is an antibody, antibody fragment, or antibody derivative. In one embodiment, the antigen-binding molecule is a non-antibody protein, or a fragment thereof, or a derivative thereof.

The bispecific antigen-binding molecule in the present invention is an antigen-binding molecule containing two types of antigen-binding domains having specificity for different antigens or epitopes. In one aspect, the bispecific antigen-binding molecule in the present invention is a bispecific antibody. The bispecific antibody is not particularly limited, but is preferably a monoclonal antibody.

As used herein, the term "antigen binding domain" refers to a region that specifically binds to and is complementary to a part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. When the molecular weight of an antigen is large, the antigen-binding domain can bind only to a specific portion of the antigen. The specific portion is called an epitope. In one aspect, the antigen binding domain comprises an antibody fragment that binds to a particular antigen. Antigen binding domains can be provided by variable domains of one or more antibodies. In one non-limiting aspect, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding domains are "scFv (single chain Fv)", "single chain antibody", "Fv", "scFv2 (single chain Fv 2)", "Fab" or "Fab' , Etc. can be mentioned. In another embodiment, the antigen binding domain comprises a non-antibody protein or fragment thereof that binds to a particular antigen. In certain embodiments, the antigen binding domain comprises a hinge region.

As used herein, the term "specifically bound" means a state in which one molecule of a specifically bound molecule does not show any significant binding to a molecule other than the other molecule to which one or more of the specifically bound molecules are bound. It means to combine with. It is also used when the antigen-binding domain is specific for a specific epitope among a plurality of epitopes contained in a certain antigen. When the epitope to which the antigen-binding domain binds is contained in a plurality of different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens including the epitope.

As used herein, the term "antibody" is used in the broadest sense, and is not limited to, but is not limited to, a monoclonal antibody, a polyclonal antibody, and a multispecific antibody (for example, as long as it exhibits a desired antigen-binding activity). Includes a variety of antibody structures, including bispecific antibodies), antibody fragments and antibody modifications.

"Binding activity" is the sum of non-covalent interactions between one or more binding sites of a molecule (eg, an antibody) and a molecule's binding partner (eg, an antigen). It refers to the strength of. Here, the binding activity is not strictly limited to a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). For example, when the members of a binding pair reflect a 1: 1 interaction in monovalent, the binding activity refers to a unique binding affinity (“affinity”). If the members of the binding pair are capable of both monovalent and multivalent binding, the binding activity is the sum of these binding forces. The binding activity of the molecule X to its partner Y can generally be expressed by the dissociation constant (KD) or the "analyte binding amount per unit ligand amount". Binding activity can be measured by conventional methods known in the art, including those described herein.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially uniform population of antibodies. That is, the individual antibodies that make up the population are possible mutant antibodies (eg, mutant antibodies that contain naturally occurring mutations, or mutant antibodies that occur during the production of monoclonal antibody preparations, such variants are usually few. Except for the amount present), it binds to the same and / or the same epitope. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is for a single determinant on the antigen. Therefore, the modifier "monoclonal" should not be construed as requiring the production of an antibody by any particular method, indicating the characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies. For example, the monoclonal antibody used according to the present invention is not limited to these, but is a hybridoma method, a recombinant DNA method, a phage display method, and a transgenic animal containing all or a part of the human immunoglobulin locus. It may be made by a variety of methods, including methods that utilize, such methods and other exemplary methods for making monoclonal antibodies are described herein.

"Natural antibody" refers to an immunoglobulin molecule with various naturally occurring structures. For example, a native IgG antibody is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two identical disulfide-bonded light chains and two identical heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a stationary light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

The term "chimeric" antibody is one in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the rest of the heavy chain and / or light chain is derived from a different source or species. It refers to an antibody.

The "class" of an antibody refers to the type of constant domain or constant region in the heavy chain of an antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. And some of them may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

The constant region as one embodiment of the present invention is preferably an antibody constant region, more preferably an IgG1, IgG2, IgG3, IgG4 type antibody constant region, and even more preferably a human IgG1, IgG2, IgG3. , IgG4 type antibody constant region. Further, the constant region as another embodiment of the present invention is preferably a heavy chain constant region, more preferably an IgG1, IgG2, IgG3, IgG4 type heavy chain constant region, and even more preferably a human. It is a heavy chain constant region of IgG1, IgG2, IgG3, and IgG4 types. The amino acid sequences of human IgG1 constant region, human IgG2 constant region, human IgG3 constant region and human IgG4 constant region are known. As constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, a plurality of allotype sequences due to gene polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242. In any of these cases. The constant region in which the amino acid of the present invention is modified may contain other amino acid mutations and modifications as long as it contains the amino acid mutation of the present invention.

The term "hinge region" refers to linking the CH1 and CH2 domains in a wild-type antibody heavy chain, eg, from around 216 to 230 according to the EU numbering system, or around 226 according to the Kabat numbering system. It means the antibody heavy chain polypeptide portion from to the 243rd position. In a native IgG antibody, the cysteine residue at position 220 of the EU numbering in the hinge region is known to form a disulfide bond with the cysteine residue at position 214 in the antibody light chain. Furthermore, it is known that between the two antibody heavy chains, the cysteine residue at EU numbering 226 and the cysteine residue at 229 in the hinge region form a disulfide bond. The hinge region herein includes the wild-type as well as variants in which amino acid residues have been substituted, added, or deleted in the wild-type.

As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least part of the constant region. The term includes the Fc region of a native sequence and the mutant Fc region. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl end of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Follow the EU numbering system (also known as the EU index) described in MD 1991.

The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody involved in binding the antibody to an antigen. The heavy and light chain variable domains of native antibodies (VH and VL, respectively) are similar, with each domain usually containing four conserved framework regions (FR) and three hypervariable regions (HVR). Has a structure. (See, for example, Kindt al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a particular antigen may be isolated by screening complementary libraries of VL or VH domains using the VH or VL domains from antibodies that bind to that antigen, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

As used herein, the terms "hypervariable region" or "HVR" are hypervariable in sequence ("complementarity determining regions" or "CDRs" (complementarity determining regions)) and / or are structurally defined. Refers to each region of the variable domain of an antibody that forms a loop (“hypervariable loop”) and / or contains antigen contact residues (“antigen contact”). Usually, the antibody contains 6 HVRs: 3 for VH (H1, H2, H3) and 3 for VL (L1, L2, L3). Illustrative HVRs herein include:
(a) At amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) The resulting hypervariable loop (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));
(b) At amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3). The resulting CDR (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));
(c) At amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3). Antigen contact that occurs (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996));
(d) HVR Amino Acid Residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49 A combination of (a), (b), and / or (c), including -65 (H2), 93-102 (H3), and 94-102 (H3).
Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., Supra.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. A variable domain FR usually consists of four FR domains: FR1, FR2, FR3, and FR4. Correspondingly, the sequences of HVR and FR usually appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

The terms "full-length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and have a structure substantially similar to that of a native antibody, or are defined herein. An antibody having a heavy chain containing an Fc region.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which foreign nucleic acids have been introduced, including progeny of such cells. .. Host cells include "transformants" and "transformants", which include primary transformants and progeny derived from those cells regardless of the number of passages. The offspring do not have to be exactly the same in the content of the parent cell and nucleic acid and may contain mutations. Variant progeny with the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

As used herein, the term "vector" refers to a nucleic acid molecule that can augment another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. Certain vectors can result in the expression of nucleic acids to which they are operably linked. Such vectors are also referred to herein as "expression vectors."

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or an antibody derived from a non-human source using a human antibody repertoire or other human antibody coding sequence. This definition of human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

A "humanized" antibody is a chimeric antibody that contains an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In some embodiments, the humanized antibody comprises substantially all of at least one, typically two variable domains, in which all or substantially all HVRs (eg, CDRs) are non-existent. Corresponds to those of human antibodies, and all or substantially all FRs correspond to those of human antibodies. The humanized antibody may optionally include at least a portion of the antibody constant region derived from the human antibody. The "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

In one embodiment, the antigen-binding molecule is a recombinant antigen-binding molecule produced using a gene recombination technique. (See, for example, Borrebaeck CAK and Larrick JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). The recombinant antigen-binding molecule clones the DNA encoding it from an antigen-binding molecule-producing cell such as a hybridoma or a sensitized lymphocyte that produces an antigen-binding molecule, incorporates it into an appropriate vector, and introduces it into a host. It can be obtained by producing it.

Furthermore, the antigen-binding molecule is not limited to an antibody and may be an aptamer. When the antigen-binding molecule is an antibody fragment, the antibody fragment includes a diabody (Db), a linear antibody, a VHH (variable domain of heavy chain of heavy chain antibody), and a single chain antibody (hereinafter, also referred to as scFv). Includes molecules and the like. Here, the "Fv" fragment is the smallest antibody fragment and contains the complete antigen recognition and binding sites. _{The "Fv" fragment is a dimer (V H-} V _L dimer) in which one heavy (H) chain variable region (V _H ) and a light (L) chain variable region (V _L ) are strongly linked by a non-covalent bond. .. The three complementarity determining regions (CDRs) of each variable region interact to form an antigen-binding site on the surface of the _{V H} - _{VL dimer.} Six CDRs confer an antigen binding site on the antibody. However, even one variable region (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigens, albeit with lower affinity than the full binding site. ..

The Fab fragment (also referred to as F (ab)) further contains a constant region of the L chain and a constant region of the H chain (CH1). The Fab'fragment differs from the Fab fragment in that it additionally has a few residues from the carboxy terminus of the H chain CH1 region containing one or more cysteines from the hinge region of the antibody. Fab'-SH refers to Fab'in which one or more cysteine residues in the constant region have a free thiol group. The F (ab') fragment is produced by cleavage of the disulfide bond in cysteine at the hinge of the _{F (ab') 2 pepsin digest.} Other chemically bound antibody fragments are also known to those of skill in the art.

Diabody refers to a bivalent antibody fragment constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097, WO 93/11161, etc.). A diabody is a dimer composed of two polypeptide chains, in which the L chain variable region ( _VL ) and the H chain variable region (V _H ) cannot bind to each other in the same chain, respectively. It is bound by a linker that is short in position, for example, about 5 residues. _{Since V L} and V _H encoded on the same polypeptide chain cannot form a single chain variable region fragment due to the short linker between them and form a dimer, the diabody binds two antigens. Will have a site.

A single chain antibody or scFv antibody fragment contains the V _H and _VL regions of the antibody, which are located within a single polypeptide chain. In general, Fv polypeptides _{also contain a polypeptide linker between the V H} and _VL regions, which allows scFv to form the structure required for antigen binding (for a review of scFv, Pluckthun. See The Pharmacology of Monoclonal Antibodies, Vol.113 (Rosenburg and Moore ed (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.

IgG-type bispecific antibodies can be secreted by hybridomas (quadromas) produced by fusing two hybridomas that produce IgG antibodies (Milstein C et al. Nature 1983, 305: 537-540). In addition, it can be secreted by co-expressing a total of four genes, L chain and H chain genes constituting the two target IgGs, into cells.

However, there are theoretically 10 combinations of H chain and L chain of IgG produced by these methods. It is difficult to purify an IgG consisting of H chains and L chains of the desired combination from 10 types of IgG. In addition, the amount of secretion of the desired combination is theoretically significantly reduced, which requires a large culture scale and further increases the manufacturing cost.

At this time, by appropriately substituting an appropriate amino acid in the CH3 region of the H chain, IgG in a heterogeneous combination for the H chain can be preferentially secreted (Ridgway JB et al. Protein Engineering 1996, 9: 617-621, Merchant). AM et al. Nature Biotechnology 1998, 16: 677-681).

Further, regarding 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 imparting a binding ability to both H chains can be obtained. Efficient bispecific IgG can be expressed by expressing IgG by introducing this common L-chain and both H-chain genes into cells (Nature Biotechnology. 1998, 16, 677-681). However, if two types of antibodies are arbitrarily selected, it is unlikely that they will contain the same L chain, and it is difficult to carry out the above idea. A method of selection has also been proposed (WO2004 / 065611). The H chain having the above mutation (Nature Biotechnology. 1998, 16, 677-681) CH3 is hardly secreted in the absence of the other H chain. Utilizing this feature, it is possible to increase the expression ratio of IgG in the desired combination by first inducing and expressing the L and H chains of the right arm, stopping this, and then inducing and expressing the L and H chains of the left arm. Yes (PCT / JP2004 / 008585).
Bispecific antigen-binding molecules can also be made by chemically cross-linking Fab'. For example, Fab'prepared from one antibody is maleimided with o-PDM (ortho-phenylenedi-maleimide), and Fab'prepared from the other antibody is reacted with this to crosslink Fab'derived from different antibodies. Bispecific F (ab') ₂ can be produced (Keler T et al. Cancer Research 1997, 57: 4008-4014). Also known is a method of chemically binding a Fab'-thionitrobenzoic acid (TNB) derivative to an antibody fragment such as Fab'-thiol (SH) (Brennan M et al. Science 1985, 229: 81-83). ..

Leucine zippers derived from Fos, Jun, etc. can be used instead of chemical cross-linking. Fos and Jun also form homodimers, but take advantage of the preferential formation of heterodimers. The expression of Fab'with the Fos leucine zipper and the other Fab'with the addition of that of Jun are prepared. _{Bispecific F (ab') 2} can be formed by mixing and reacting 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', but can also be applied to scFv, Fv, etc.

A bispecific antigen-binding molecule can also be prepared in the diabody. The bispecific diabody is a heterodimer of two cross-over scFv fragments. _{In other words, V H} (A) -V _L (B), V _H (B) _{-produced by linking V H} and V _L derived from two types of antibodies A and B with a relatively short linker of about 5 residues. It can be done by constructing a heterodimer using V _L (A) (Holliger P et al. Proc of the National Academy of Sciences of the USA 1993, 90: 6444-6448).

At this time, two kinds of scFv are connected by a flexible and relatively long linker of about 15 residues (single-chain diabody: Kipriyanov SM et al. J of Molecular Biology. 1999, 293: 41-56), and an appropriate amino acid. Substitution (knobs-into-holes: Zhu Z et al. Protein Science. 1997, 6: 781-788) can also be used to promote the desired configuration.

_{Sc (Fv) 2,} which can be produced by linking two types of scFv with a flexible and relatively long linker of about 15 residues, can also be a bispecific antigen-binding molecule (Mallender WD et al. J of Biological Chemistry, 1994, 269: 199-206).

Examples of antibody modifications include antibodies bound to various molecules such as polyethylene glycol (PEG). In the antibody modified product of the present invention, the substance to be bound is not limited. Such antibody modifications can be obtained by chemically modifying the obtained antibody. These methods have already been established in this field.

The origin of the antigen-binding molecule of the present invention is not limited, such as human antibody, mouse antibody, and rat antibody. Further, a gene-modified antibody such as a chimeric antibody or a humanized antibody may be used.

A method for obtaining a human antibody is already known. For example, a human antibody of interest can be obtained by immunizing a transgenic animal having the entire repertoire of human antibody genes with the antigen of interest (publication of international patent application). See numbers WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

The genetically modified antibody can be produced using a known method. Specifically, for example, a chimeric antibody is an antibody consisting of a variable region of H chain and L chain of an immune animal antibody and a constant region of H chain and L chain of a human antibody. A chimeric antibody can be obtained by ligating a DNA encoding a variable region of an antibody derived from an immune animal with a DNA encoding a constant region of a human antibody, incorporating this into an expression vector, introducing it into a host, and producing it. ..

A humanized antibody is a modified antibody that is also called a reshaped human antibody. Humanized antibodies are constructed by transplanting the CDRs of antibodies from immune animals into the complementarity determining regions of human antibodies. The general gene recombination method is also known.

Specifically, several oligos prepared by preparing a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody so as to have an overlapping portion at the terminal portion. It is synthesized from nucleotides by the PCR method. It is obtained by ligating the obtained DNA with the DNA encoding the human antibody constant region, then incorporating it into an expression vector, introducing it into a host and producing it (European Patent Application Publication No. EP 239400, International Patent Application Publication). See number WO 96/02576). The FR of the human antibody linked via CDR is selected so that the complementarity determining regions form a good antigen-binding site. If desired, the amino acids in the framework regions of the variable region of the antibody may be replaced so that the complementarity determining regions of the reconstituted human antibody form the appropriate antigen binding site (Sato K et al, Cancer Research 1993, 53: 851-856). It may also be replaced with framework regions derived from various human antibodies (see International Patent Application Publication No. WO 99/51743).

In one embodiment, the enzyme hydrolyzes the substrate by recognizing both the enzyme and the substrate capable of undergoing a catalytic reaction of the enzyme, but blood coagulation factor V, blood coagulation factor VIII, tissue factor (TF). , From Thrombomodulin (TM), Protein S (PS), Protein Z (PZ) Complement C4b, Complement Regulatory Factor H, membrane cofactor Protein (MCP), complement receptor1 (CR1) A bispecific antigen-binding molecule that does not replace the function of at least one complement or heparin selected from the group is provided. In another aspect, a bispecific antigen-binding molecule that recognizes both the enzyme and the substrate that can be catalyzed by the enzyme but does not replace the function of naturally occurring coagulation cofactors is provided. .. The enzyme in these embodiments is not particularly limited as long as it is a molecule having catalytic activity for a chemical reaction occurring in a living body. Examples of the enzyme include protease, amylase, cellulase, and lipase. Examples of proteases include serine proteases of the blood coagulation system. In certain embodiments, the enzyme can be a blood coagulation enzyme. Examples of serine proteases that are blood coagulation enzymes include F.XII (a), F.XI (a), F.IX (a), FX (a), F.VII (a), and F.IIa. .. In addition, F.XII (a) means F.XII and / or F.XIIa, and the same applies to others.

The substrate capable of undergoing the catalytic reaction of the enzyme referred to in the present application means a substrate in which the catalytic reaction of the enzyme occurs when the enzyme is forcibly brought close to each other. The catalytic reaction of the enzyme means that the substrate is hydrolyzed. Hydrolysis of the substrate may be performed as long as the substrate is hydrolyzed as compared with the case where the bispecific antigen-binding molecule of the present application is absent. Substrate hydrolysis reactions can be compared by measuring the amount of protein over time after the addition of a bispecific antigen-binding molecule by the Bradford method or the like using, for example, Protein Assey Kit II (Bio-Red). Alternatively, it can be measured by SDS-PAGE analysis or Western blotting (WB) using a protein before and after the addition of the bispecific antigen-binding molecule. Alternatively, it can be measured by performing mass spectrometry (MALDI-TOFMS, etc.) of the protein before and after the addition of the bispecific antigen-binding molecule, or by performing N-terminal amino acid sequence analysis (protein sequence). Substrate hydrolysis includes, for example, activation of blood coagulation factor X or blood coagulation factor IX. The activation of the substrate can be performed by using the enzyme and the reaction system containing the substrate, and the increase in the enzyme activity (substrate resolution) by adding the antigen-binding molecule can be used as an index. For example, the enzyme, the substrate, and the activity. It is a measurement system (in vitro enzyme reaction measurement system) consisting of a synthetic substrate, phospholipids, and Ca2 + of the converted substrate, and can be evaluated by the substrate activation promoting activity by the enzyme. Based on the results, as a bispecific antigen-binding molecule having the activity, in principle, a molecule showing the substrate activation promoting activity by the enzyme of 0.1 or more only in the enzyme-added group can be selected in this measurement system. The substrate activation promoting activity by the enzyme referred to here can be measured by the value of the absorbance 30 minutes after the addition of the synthetic substrate of the antigen-binding molecular solution.

"Promoting the activation of the substrate by the enzyme" means that the substrate activation promoting activity in the in vitro enzyme reaction measurement system shows an absorbance of 0.1 or more 30 minutes after the addition of the chromogenic substrate solution. The in vitro enzyme reaction measurement system can be measured with each enzyme and each substrate. For example, as shown in Example 2, as the FX activation promoting activity by F.XIa, as the absorbance value 30 minutes after the addition of the color-developing substrate solution. When it is 0.1 or more, it can be evaluated as promoting the activation of the substrate by the enzyme.

Specific examples of the enzyme (also referred to as "blood coagulation enzyme") and the substrate (also referred to as "blood coagulation substrate") in the embodiment in which the enzyme and the substrate are blood coagulation / fibrinolysis-related factors are, for example, the combinations shown in Table 1 below. Can be mentioned.

The method for obtaining the bispecific antigen-binding molecule is not particularly limited, and any method may be used for obtaining the bispecific antigen-binding molecule. For example, when a bispecific antigen-binding molecule for enzyme A and substrate B is obtained, each of enzyme A and substrate B is immunized against an immunized animal to obtain an anti-enzyme A antibody and an anti-substrate B antibody. Then, a bispecific antigen-binding molecule containing the H-chain variable region and the L-chain variable region of the anti-enzyme A antibody and the H-chain variable region and the L-chain variable region of the anti-substrate B antibody is prepared. Here, it is desirable that a plurality of types of anti-enzyme A antibody and anti-substrate B antibody are obtained, and it is preferable to prepare as many combinations of bispecific antigen-binding molecules as possible using these. After preparing the bispecific antigen-binding molecule, the antigen-binding molecule that activates substrate B is selected.

The antigen-binding molecule to the enzyme or substrate can be obtained by a method known to those skilled in the art. For example, it can be prepared by immunizing an immune animal with an antigen. Examples of the antigen that immunizes an animal include a complete antigen having immunogenicity and an incomplete antigen (including a hapten) having no immunogenicity. In immunization, an enzyme or substrate to which an antigen-binding molecule binds, or a nucleic acid expressing them is used as the above-mentioned antigen (immunogen). As the immunized animal, for example, mice, rats, hamsters, guinea pigs, rabbits, chickens, rhesus monkeys and the like can be used. Immunization of these animals with an antigen can be performed by a method well known to those skilled in the art. Preferably, the variable regions of the L and H chains of the antibody are recovered from the immunized animal or the cells of the animal. This operation can be performed using a technique generally known to those skilled in the art. Animals immunized with an antigen express antibodies against that antigen, especially in spleen cells. Therefore, for example, mRNA can be prepared from the spleen cells of an immunized animal, and the variable regions of L and H chains can be recovered by RT-PCR using the primers corresponding to the variable regions of the animal.

Specifically, immunize animals with enzymes and substrates. The enzyme or substrate used as an immunogen may be the whole protein or a partial peptide of the protein. Further, as the immunogen used for immunizing an animal, it is possible to bind an antigen to another molecule to obtain a soluble antigen, and in some cases, fragments thereof may be used.

Spleen cells are isolated from the spleen of immunized mice and fused with mouse myeloma cells to produce hybridomas. Hybridomas that bind to the antigen can be selected, and the variable regions of the L and H chains can be recovered by RT-PCR using primers and the like corresponding to the variable regions. Primers that correspond to CDRs, frameworks that are less diverse than CDRs, or primers that correspond to the signal sequence and the CH1 or L chain constant region ( _CL ) can also be used.
Alternatively, they can be produced by B cell cloning techniques known to those of skill in the art (Proc Natl Acad Sci US A. 1996; 93 (15): 7843-7848 .; WO2008 / 045140; and WO2009 / 113742). Not limited.

Alternatively, mRNA is extracted from the splenocytes of an immunized animal, and cDNA in the L-chain and H-chain variable regions is recovered by RT-PCR using a primer corresponding to the vicinity of the variable region. It can also immunize lymphocytes in vitro. Use this to build a library that presents scFv or Fab. Antigen-binding antibody clones can be concentrated and cloned by panning to obtain variable regions. At this time, it is also possible to perform screening using a similar library using mRNA derived from peripheral blood mononuclear cells, spleen, tonsils, etc. of humans and non-immune animals.

An antigen-binding molecule expression vector is prepared using the variable region. A bispecific antigen-binding molecule can be obtained by introducing an anti-enzyme antigen-binding molecule expression vector and an anti-substrate antigen-binding molecule expression vector into the same cell and expressing the antigen-binding molecule.

The antigen-binding molecule in the present invention can be selected by, for example, the following method.
(1) A reaction system containing the enzyme / substrate is used, and an increase in the enzyme activity (substrate resolution) due to the addition of the antigen-binding molecule is used as an index for selection.
(2) Using a system that measures or mimics the biological functions associated with the enzyme, the substrate, and the cofactor (for example, a plasma coagulation measurement system), at least one of the enzyme, the substrate, and the cofactor is absent. The function recovery activity by adding the antigen-binding molecule under the conditions is used as an index for selection.

The obtained antigen-binding molecule can be purified to a uniform level. For the separation and purification of the antigen-binding molecule, the separation and purification methods used for ordinary proteins may be used. For example, an antibody can be separated and purified by appropriately selecting and combining a chromatography column such as affinity chromatography, a filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and the like. It can be done (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but it is not limited to these. Examples of the column used for affinity chromatography include a protein A column and a protein G column.

The bispecific antigen-binding molecule of the present invention preferably has a variable region in an anti-F.XIa antibody when, for example, the combination of enzyme and substrate is the blood coagulation / fibrinolysis-related factors F.XIa and FX. And a variable region in the anti-FX antibody.

The bispecific antigen-binding molecule can be prepared by the following method. For example, when the combination of enzyme and substrate was F.XIa and FX, commercially available F.XIa and FX were subcutaneously immunized in mice, respectively. Spleen cells were isolated from the spleen of immune mice with elevated antibody titers and fused with mouse myeloma cells to prepare hybridomas. Hybridomas that bind to antigens (F.XIa, FX) were selected, and the variable regions of L and H chains were recovered by RT-PCR using primers corresponding to the variable regions. L chain variable region to the L chain expression vector containing a C _L, H chain variable region incorporating the respective H chain expression vector containing an H chain constant region. In addition, mRNA was extracted from the spleen of this immune mouse, and cDNA in the L-chain and H-chain variable regions was recovered by RT-PCR using primers corresponding to the variable regions. A phage library that presents scFv was constructed using these variable regions. An antigen-binding antibody clone was concentrated and cloned by panning, and an antigen-binding molecule expression vector was prepared using the variable region. Alternatively, it can be produced by a B cell cloning technique by a method known to those skilled in the art. Bispecific antigen binding by introducing an anti-F.XIa antibody (H chain, L chain) expression vector and an FX antibody (H chain, L chain) expression vector into the same cell and expressing an antigen-binding molecule. Obtained a molecule.

Regarding the obtained bispecific antigen-binding molecule, the FX activation promoting activity by F.XIa was evaluated by a measurement system consisting of F.XIa, FX, F.Xa synthetic substrate (S-2222), and phospholipid. bottom. Based on the results, as the bispecific antigen-binding molecule to have, in principle, a molecule showing F.X activation promoting activity by F.XIa of 0.1 or more in this measurement system was selected. The F.X activation promoting activity by F.XIa referred to here is a value of the absorbance 30 minutes after the addition of the synthetic substrate of the antigen-binding molecular solution.

For the bispecific antigen-binding molecule selected above or its related bispecific antigen-binding molecule, the coagulation recovery ability was measured using a coagulation time measurement system using blood coagulation factor-deficient human plasma. As a result, a bispecific antigen-binding molecule having a shorter coagulation time than when no antibody was added was obtained. The coagulation time referred to here is a measurement of the activated partial thromboplastin time using human plasma deficient in blood coagulation factor IX. Among these bispecific antigen-binding molecules, the most preferable bispecific antigen-binding molecule had the ability to shorten the coagulation time by 60 seconds or more.

The antigen-binding molecule of the present invention is not particularly limited, and examples thereof include bispecific antigen-binding molecules that recognize both the enzyme and the substrate of any combination shown in Table 1.

The antigen-binding molecule of the present invention is a combination of activated blood coagulation factor X (F.Xa) and blood coagulation factor X (FX) shown in Table 1, and activated blood coagulation factor XI (F.XIa). In the case of the combination of blood coagulation factor XI (F.XIa), it may be a monospecific antigen-binding molecule having the same heavy chain and light chain, and more preferably an antibody.

When a full-length antibody is produced using the variable region disclosed in the present invention, the constant region is not particularly limited, and constant regions known to those skilled in the art can be used. For example, Sequences of proteins of immunological interest. , (1991), US Department of Health and Human Services. Public Health Service National Institutes of Health, An efficiency route to human bispecific IgG, (1998). Nature Biotechnology vol. 16, 677-681, etc. Constant regions can be used.

In one aspect of the antigen-binding molecule in the present invention, the antigen-binding molecule does not at least replace the function of a peptide cofactor or heparin expressed in the body, but complements the relationship between the enzyme and a substrate capable of undergoing a catalytic reaction of the enzyme. Since it fulfills factor-like functions, it is expected to be an effective drug for diseases caused by decreased activity (function) or deficiency of certain cofactors, enzymes, and substrates. When the enzyme and substrate to which the antigen-binding molecule of the present invention binds are blood coagulation / fibrinolysis-related factors, examples of the above-mentioned diseases include bleeding, diseases associated with bleeding, and diseases caused by bleeding. .. In particular, functional deterioration or deficiency of F.VIII / F.VIIIa, F.IX / F.IXa, and F.XI / F.XIa is known to cause hemorrhagic disorders called hemophilia.

Among hemophilia, bleeding dysfunction due to congenital F.VIII / F.VIIIa dysfunction or deficiency is called hemophilia A, and bleeding dysfunction due to F.IX / F.IXa dysfunction or deficiency is blood. Called friendship B. If hemophilia A patients bleed, F.VIII replacement therapy is given, and if hemophilia B patients bleed, F.IX replacement therapy is given. In addition, on the day of strenuous exercise or excursion, if frequent intra-articular bleeding occurs, or if it is classified as severe hemophilia, prophylactic administration of these preparations may be performed.

The half-life of F.VIII preparation in blood is short, about 12 to 16 hours. Therefore, for continuous prophylaxis, it is necessary to administer the F.VIII preparation about three times a week. This corresponds to maintaining approximately 1% or more of F.VIII activity. Similarly, the F.IX preparation with a standard half-life should be administered about twice a week. In addition, in replacement therapy for bleeding, it is necessary to administer additional F.VIII and F.IX products regularly for a certain period of time in order to prevent rebleeding and completely stop bleeding, except when the bleeding is mild. There is.

In addition, the F.VIII preparation and the F.IX preparation are administered intravenously. There are technical difficulties in performing intravenous administration. Especially in the case of administration to younger patients, the difficulty is further increased because the veins used for administration are thin.

In the above-mentioned prophylactic administration of F.VIII and F.IX preparations and emergency administration in the event of bleeding, home remedies and self-injection are often used. The need for frequent administration and the technical difficulty of administration not only cause pain to patients during administration, but also hinder the spread of home remedies and self-injection.
Therefore, there has been a strong demand for a drug having a wider administration interval or an easy-to-administer drug as compared with the existing blood coagulation factor VIII and factor IX preparations.

Furthermore, hemophilia A patients, especially severe hemophilia A patients, may develop antibodies against F.VIII called inhibitors. Similarly, patients with hemophilia B may develop antibodies to F.IX called inhibitors. When the inhibitor is generated, the effect of the F.VIII or F.IX preparation is hindered by the inhibitor. As a result, hemostasis management for the patient becomes very difficult.

When such hemophilia A inhibitor patients develop bleeding, neutralization therapy using a large amount of F.VIII preparation, or bypass therapy using a complex concentrate or F.VIIa preparation is usually performed. , Will be implemented. However, in neutralization therapy, administration of a large amount of the F.VIII preparation may conversely increase the titer of the inhibitor (anti-F.VIII antibody). In bypass therapy, the short half-life (about 2 to 8 hours) of complex preparations and F.VIIa preparations has become a problem. Moreover, their mechanism of action is independent of the function of F.VIII / F.VIIIa, that is, the function of catalyzing FX activation by F.IXa, which in some cases makes the hemostatic mechanism work well. There are cases where it is not possible and the response becomes unresponsive. Therefore, hemophilia A inhibitor patients often do not have a sufficient hemostatic effect as compared to non-inhibitor hemophilia A patients. The same is true for patients with hemophilia B inhibitors.

Therefore, there has been a strong demand for a drug that is independent of the presence of the inhibitor and that replaces the function of F.VIII / F.VIIIa or F.IX / F.IXa.

Now, (i) the dosing interval is wide, (ii) the dosing is easy, (iii) independent of the presence of the inhibitor, and (iv) F.VIII / F.VIIIa or F.IX / F.IXa independent. A method using an antibody can be considered for the creation of a drug that replaces the function. The half-life of an antibody in blood is generally relatively long, ranging from days to weeks. In addition, it is generally known that the antibody is transferred to the blood after subcutaneous administration. That is, in general, antibody drugs satisfy the above (i) and (ii).

The present invention provides a pharmaceutical composition containing the antigen-binding molecule of the present invention as an active ingredient. For example, when the antigen-binding molecule of the present invention is an antigen-binding molecule that recognizes both the enzymes and substrates of the combinations shown in Table 1 and promotes the blood coagulation reaction in the blood coagulation factor-deficient plasma. The antigen-binding molecule is expected to be a drug (pharmaceutical composition) or drug for preventing or treating bleeding, a disease associated with bleeding, or a disease caused by bleeding.

The pharmaceutical composition containing the antigen-binding molecule of the present invention used for therapeutic or prophylactic purposes as an active ingredient is optionally mixed with an appropriate pharmaceutically acceptable carrier, medium or the like which is inert to them. Can be formulated. For example, sterile water, physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, etc.) Tween etc.), chelating agent (EDTA etc.), binder and the like can be mentioned. In addition, other low molecular weight polypeptides, serum albumin, proteins such as gelatin and immunoglobulin, amino acids such as glycine, glutamine, asparagine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, sugars such as mannitol and sorbitol. It may contain alcohol. Examples of an aqueous solution for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannitol, D-mannitol, and sodium chloride, which are appropriately dissolved. Auxiliary agents such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc. may be used in combination.

In addition, if necessary, the antigen-binding molecule of the present invention can be encapsulated in microcapsules (microcapsules of hydroxymethyl cellulose, gelatin, poly [methyl methacrylate], etc.), or a colloidal drug delivery system (liposomes, albumin microspheres, microemulsions, etc.). Nanoparticles and nanocapsules, etc.) (see "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980), etc.). Furthermore, a method of making a drug a sustained release drug is also known and can be applied to the antigen-binding molecule of the present invention (Langer et al., J. Biomed. Mater. Res. 15: 267-277 (1981); Langer, Chemtech. 12: 98-105 (1982); US Patent No. 3,773,919; European Patent Application Publication (EP) No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP No. 133,988) ..

The antigen-binding molecule or composition of the present invention can be used in combination with blood coagulation factor VIII. Blood coagulation factor VIII may be made from human blood or genetically modified. The antigen-binding molecule or composition of the present invention may be administered at the same time as blood coagulation factor VIII, or may be administered at staggered times. Further, it may be carried out as a kit in which the antigen-binding molecule or pharmaceutical composition of the present invention is combined with blood coagulation factor VIII. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with blood coagulation factor VIII, it is possible to reduce the dose of each if desired, as compared with the case where either of them is used alone.

The antigen-binding molecule or composition of the present invention can be used in combination with blood coagulation factor IX. Blood coagulation factor IX may be made from human blood or genetically modified. The antigen-binding molecule or composition of the present invention may be administered at the same time as blood coagulation factor IX, or may be administered at staggered times. Further, it may be carried out as a kit in which the antigen-binding molecule or pharmaceutical composition of the present invention is combined with blood coagulation factor IX. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with blood coagulation factor IX, it is possible to reduce the dose of each if desired, as compared with the case where either of them is used alone.

The antigen-binding molecule or composition of the present invention can be used in combination with blood coagulation factor XI. Blood coagulation Factor XI may be made from human blood or genetically modified. The antigen-binding molecule or composition of the present invention may be administered at the same time as blood coagulation factor XI, or may be administered at staggered times. Further, it may be carried out as a kit in which the antigen-binding molecule or pharmaceutical composition of the present invention is combined with blood coagulation factor XI. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with factor XI for blood coagulation, it is possible to reduce the dose of each dose as desired, as compared with the case where either one is used alone.

The antigen-binding molecule or composition of the present invention can be used in combination with a bypass preparation. The bypass preparation may be made from human blood or by gene recombination, for example, plasma-derived active prothrombin complex preparation (APCC preparation), gene recombination type activation first. Factor VII preparation (rF.VIIa preparation) and dry concentrated human blood coagulation factor X-activated factor VII preparation (F.VIIa / FX preparation). The antigen-binding molecule or composition of the present invention may be administered at the same time as the bypass preparation, or may be administered at different times. Further, it may be carried out as a kit in which the antigen-binding molecule or pharmaceutical composition of the present invention is combined with a bypass preparation. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with the bypass preparation, it is possible to reduce the dose of each, if desired, as compared with the case where either of them is used alone.

The dose of the pharmaceutical composition of the present invention is finally determined by a doctor in consideration of the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, type of disease and degree of progression, etc. Although it is determined as appropriate, in general, for adults, 0.1 to 2000 mg can be administered in 1 to several divided doses per day. It is more preferably 1 to 1000 mg / day, even more preferably 50 to 500 mg / day, and most preferably 100 to 300 mg / day. These doses vary depending on the body weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose. It is preferable that the administration period is also appropriately determined according to the healing course of the patient and the like.

It is also conceivable to incorporate the gene encoding the antigen-binding molecule of the present invention into a gene therapy vector to perform gene therapy. As an administration method, in addition to direct administration with a naked plasmid, it is packaged in liposomes or the like, or formed as various virus vectors such as retrovirus vector, adenovirus vector, vaccinia virus vector, poxvirus vector, adenovirus-related vector, and HVJ vector. (See Adolph "Virus Genome Method", CRC Press, Florid (1996)), or it can be administered by coating it on a bead carrier such as colloidal gold particles (WO93 / 17706, etc.). Further, the nucleic acid encoding the antigen-binding molecule of the present invention may be directly administered to a living body, or may be directly administered to a living body by an electroporation method. For example, the mRNA encoding the antigen-binding molecule of the present invention is chemically modified to enhance the stability of the mRNA in vivo, and the mRNA is directly administered to humans to express the antigen-binding molecule of the present invention in vivo. The antigen-binding molecule of the present invention can be administered by the method of causing (see EP2101823B, WO2013 / 120629). However, as long as the antigen-binding molecule is expressed in vivo and exerts its action, it may be administered by any method. Preferably, injection, infusion, or gas-induced particle impact (electron) is performed via a suitable parenteral route (intravenous, intraperitoneal, subcutaneous, intradermal, intra-adipose tissue, intramammary gland tissue, inhalation or intramuscular route). A sufficient amount is administered by (with a gun, etc.), a method via a mucosal route such as a nasal spray, etc.). The present invention is produced by administering to blood cells, bone marrow-derived cells, etc. using liposome transfection, particle impact method (US Patent No. 4,945,050), or virus infection in ex vivo, and reintroducing the cells into animals. A gene encoding an antigen-binding molecule may be administered.

The present invention also provides a method for preventing and / or treating bleeding, a disease associated with bleeding, or a disease caused by bleeding, which comprises the step of administering the antigen-binding molecule or composition of the present invention. Administration of the antigen-binding molecule or composition can be carried out, for example, by the method described above.
In one aspect, bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to decreased or deficient activity of blood coagulation factor IX and / or activated blood coagulation factor IX (eg,). , Hemophilia B). In certain embodiments, the disease is a disease in which an inhibitor of blood coagulation factor IX or activated blood coagulation factor IX has emerged. In certain embodiments, the antigen-binding molecule or composition of the invention is administered to a subject (patient carrying an inhibitor) who carries an inhibitor of blood coagulation factor IX or activated blood coagulation factor IX. In certain embodiments, the method of the invention further comprises the step of administering blood coagulation factor IX.
In another embodiment, bleeding, a disease associated with bleeding, or a disease resulting from bleeding is a disease that develops and / or progresses due to decreased or deficient activity of hemocoagulation factor VIII and / or activated hemocoagulation factor VIII. For example, hemophilia A, acquired hemophilia, or Fonville brand disease). In certain embodiments, the disease is a disease in which an inhibitor of blood coagulation factor VIII or activated blood coagulation factor VIII has emerged. In certain embodiments, the antigen-binding molecule or composition of the invention is administered to a subject (patient carrying an inhibitor) who carries an inhibitor of blood coagulation factor VIII or activated blood coagulation factor VIII. In certain embodiments, the method of the invention further comprises the step of administering blood coagulation factor VIII.
In another embodiment, bleeding, a disease associated with bleeding, or a disease resulting from bleeding is a disease that develops and / or progresses due to decreased or deficient activity of blood coagulation factor XI and / or activated blood coagulation factor XI. For example, hemophilia C, or acquired hemophilia). In a particular embodiment, the disease is a disease in which an inhibitor of blood coagulation factor XI or activated blood coagulation factor XI appears. In certain embodiments, the antigen-binding molecule or composition of the invention is administered to a subject (patient carrying an inhibitor) who carries an inhibitor of blood coagulation factor XI or activated blood coagulation factor XI. In certain embodiments, the method of the invention further comprises the step of administering blood coagulation factor XI.

The present invention also relates to the use of the antigen-binding molecule of the present invention for the production of the (pharmaceutical) composition of the present invention.

Furthermore, the present invention provides a kit for use in the above method, which comprises at least the antigen-binding molecule or composition of the present invention. The kit may also be packaged with a syringe, a needle, a pharmaceutically acceptable medium, an alcohol cotton cloth, an adhesive plaster, or instructions describing how to use it. In one aspect, the kit of the present invention is for use in a method of preventing and / or treating bleeding, a disease associated with bleeding, or a disease caused by bleeding. In certain embodiments, the kit of the invention comprises a blood coagulation factor IX or factor VIII in addition to the antigen binding molecule or composition of the invention.

In another aspect, the present invention relates to a method of promoting blood coagulation using a bispecific antigen-binding molecule that recognizes both the enzyme and the substrate of any combination shown in Table 1. In one aspect of this aspect, the method for promoting blood coagulation of the present invention comprises the step of administering a bispecific antigen-binding molecule that recognizes both the enzyme and the substrate of any combination shown in Table 1. The administration of the antigen-binding molecule can be carried out by, for example, the above-mentioned administration method. In a particular aspect of the above aspect, the bispecific antigen binding molecule used in the method for promoting blood coagulation of the present invention is a bispecific antigen that recognizes both activated blood coagulation factor XI and blood coagulation factor X. It is a binding molecule.

In yet another aspect, the present invention relates to a method for screening a substance effective for the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding. In one aspect of this aspect, the screening method of the present invention comprises (1) a step of evaluating the binding of the test substance to any combination of enzymes shown in Table 1, and (2) the test substance and Table 1. Includes a step of evaluating the binding of the combination to the substrate according to. In certain embodiments, the screening methods of the invention assess (1) the step of assessing the binding of the test substance to activated blood coagulation factor XI, and (2) assessing the binding of the test substance to blood coagulation factor X. Includes the process of In certain embodiments, the screening methods of the present invention use a test substance that binds to both the enzyme and substrate of any of the combinations listed in Table 1 to prevent bleeding, bleeding-related diseases, or diseases caused by bleeding. / Or further includes the step of selecting as a candidate for a therapeutically effective substance.

The "test substance" is not particularly limited, and includes, for example, a single substance such as a natural compound, an organic compound, an inorganic compound, a nucleic acid, a protein, and a peptide, as well as a compound library, a nucleic acid library, a peptide library, and a gene live. Examples thereof include rally expression products, cell extracts, cell culture supernatants, fermented microbial products, marine organism extracts, plant extracts, prokucleic acid cell extracts, eukaryotic single cell extracts, animal cell extracts and the like. The test substance can be appropriately labeled and used as needed. Examples of the label include a radial label, a fluorescent label and the like. In addition to the above test substances, a mixture of a plurality of these test substances is also included.

In yet another aspect, the present invention relates to a quality test method for a substance or composition effective in the prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding. In one aspect of this aspect, the quality test method of the present invention comprises (1) a step of evaluating the binding of a test substance or test composition to an enzyme of any combination shown in Table 1, and (2) a test. The step of evaluating the binding between the substance or the test composition and the substrate of the combination shown in Table 1 is included. In a particular embodiment, the quality test method of the present invention comprises (1) a step of evaluating the binding of the test substance or test composition to activated blood coagulation factor XI, and (2) the test substance or test composition and blood. Includes the step of assessing binding to coagulation factor X. In certain embodiments, the quality test method of the invention results in bleeding, a disease associated with bleeding, or bleeding that binds a test substance or composition that binds to both of the enzymes and substrates of any of the combinations listed in Table 1. It further comprises the step of assessing the quality of the substance or composition as effective for the prevention and / or treatment of the disease.

The "test composition" is not particularly limited, and examples thereof include pharmaceuticals and reagents.

(1) Examples of the steps for evaluating the binding between the test substance and the activated blood coagulation factor XI include an ELISA method (Enzyme-Linked Immuno Sorbent Assay) and a surface plasmon resonance (SPR) method, for example, Biacore. It can be evaluated by measurement using a series (GE Healthcare) and biosensor interaction analysis system using biosensor technology (BLI method), for example, Octet system (Fortebio).

(2) Examples of the steps for evaluating the binding between the test substance and blood coagulation factor X include an ELISA method (Enzyme-Linked ImmunoSorbent Assay) and a surface plasmon resonance (SPR) method, for example, the Biacore series (Biacore series). It can be evaluated by measurement using GE Healthcare) and biosensor interaction analysis system using biosensor technology (BLI method), for example, Octet system (Fortebio).

(3) Activation As a step of evaluating the activation reaction of blood coagulation factor X by factor XI using a test substance, for example, the enzyme, the substrate, the synthetic substrate of the activated substrate, phosphorus It is a measurement system consisting of lipid and Ca2 +, and can be evaluated by the substrate activation promoting activity by the enzyme. Based on the results, as a bispecific antigen-binding molecule having the activity, in principle, a molecule showing the substrate activation promoting activity by the enzyme of 0.1 or more only in the enzyme-added group can be selected in this measurement system. The substrate activation promoting activity by the enzyme referred to here can be measured by the value of the absorbance 30 minutes after the addition of the synthetic substrate of the antigen-binding molecular solution.

All prior art documents cited herein are incorporated herein by reference.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Preparation of anti-F.XIa, FX bispecific antibody For the preparation of anti-F.XIa antibody, the following variable region sequences of the antibody were used: 14E11 described in WO2010080623A2 (the present specification). Internal SEQ ID NO:: Heavy Chain Variable Region SEQ ID NO: 1, Light Chain Variable Region SEQ ID NO: 2); F.XIa antibody set forth in WO2013167669 SEQ ID NOs: 165 and 166 (described herein as F11B1: heavy) Chain variable region SEQ ID NO: 11, light chain variable region SEQ ID NO: 12); F.XIa antibody 21F12 (described herein as F11B2) set forth in WO2018145533 SEQ ID NOs: 105 and 106: heavy chain variable region SEQ ID NO: : 13, light chain variable region SEQ ID NO: 14); and F.XIa antibody set forth in WO2013167669 SEQ ID NOs: 29, 30 (described herein as F11B3: heavy chain variable region SEQ ID NO: 15, light chain Variable region SEQ ID NO: 16). The following antibody variable region sequences were used for anti-FX antibody preparation: F10B1 from SB04 described in WO2005035756A1 (SEQ ID NO:: heavy chain variable region SEQ ID NO: 3, light chain variable region herein). SEQ ID NO: 4); F10B2 consisting of a sequence in which two amino acid substitutions are introduced into the heavy chain variable region sequence of J327 and the light chain variable region sequence of JNL095 described in WO2019065795A1 (SEQ ID NO:: heavy chain in the present specification). Variable region SEQ ID NO: 5, Light chain variable region SEQ ID NO: 6); FX antibody BIIB-12-917 (described herein as F10B3: heavy chain variable region) described in SEQ ID NOs: 427 and 615 of WO2018098363. SEQ ID NO: 17, light chain variable region SEQ ID NO: 18); FX antibody mAb 00916 described in SEQ ID NOs: 4 and 5 of WO2019096874 (in the present specification, the first residue Ala of the light chain is removed and described as F10B4. : Heavy chain variable region SEQ ID NO: 19, light chain variable region SEQ ID NO: 20); and FX antibody mAb 13F62 described in SEQ ID NOs: 6 and 7 of WO2019096874 (described herein as F10B5: heavy chain variable region). SEQ ID NO: 21, light chain variable region SEQ ID NO: 22). The variable region sequence of each antibody was linked to the heavy chain constant region or light chain constant region sequence to construct an expression vector containing a gene encoding the full length of the antibody sequence. The expression vector was transiently introduced into Expi293 cells (Thermo Fisher Scientific) to express the antibody. The obtained culture supernatant was purified by a method known to those skilled in the art by affinity purification using Protein A or the like. Furthermore, bispecific antibodies consisting of these anti-F.XIa antibodies and anti-FX antibodies were prepared by methods known to those skilled in the art. The correspondence between the sequence numbers of the variable region and the constant region of each antibody is shown in Table 2, and the names of the respective monospecific antibodies and bispecific antibodies are also named as shown in Table 2.

[Example 2] Measurement of FX activation promoting activity by F.XIa of F.XIa and FX bispecific antibody by in vitro enzyme reaction measurement system The presence or absence of FX activation promoting activity by F.XIa was evaluated using an in vitro enzyme reaction measurement system using a synthetic color-developing substrate. Specifically, the measurement was carried out according to the following procedure, and all the reactions were carried out at room temperature. A mixture of 4 μL of 48 ng / mL F.XIa (Enzyme Research Laboratories) and 5 μL of antibody solution at each concentration was incubated in a 384-well plate for 30 minutes. Further, 5 μL of FX (Enzyme Research Laboratories) of 22.9 μg / mL was added to the mixed solution to initiate an enzymatic reaction. After reacting for 60 minutes, the enzyme reaction was stopped by adding 5 μL of 50 μM Aprotinin (Sigma Aldrich). Subsequently, 5 μL of a synthetic color-developing substrate solution colored by F.Xa was added to each well, and the absorbance at a wavelength of 405 nm after 30 minutes was measured by SpectraMax340PC (Molecular Devices). The FX activation promoting activity of F.XIa and FX bispecific antibodies by F.XIa was expressed as the absorbance value 30 minutes after the addition of the coloring substrate solution [Figs. 3 and 5]. The antibody concentration is shown as the concentration in the solution during the enzymatic reaction.
As a result of the evaluation, in the in vitro test system containing F.XIa and FX, an antibody concentration-dependent increase in the amount of F.Xa indicated by absorbance was confirmed in the group to which the bispecific antibody was added. On the other hand, no increase in absorbance was confirmed in the group to which the monospecific antibody was added instead of the bispecific antibody. From the above, it was suggested that F.XIa and FX bispecific antibodies promote FX activation in an in vitro enzyme reaction measurement system in a binding-dependent manner to both F.XIa and FX.
As the solvent of the antibody solution, Tris-buffered physiological saline containing 0.1% bovine serum albumin (hereinafter referred to as TBSB) was used. In addition, TBSB (hereinafter referred to as TBCP) containing 1.5 mM CaCl2 and 4.0 μM phospholipid (Sysmex) was used as the solvent for F.XIa and FX. As the color-developing substrate solution, a 1.47 mg / mL color-developing substrate S-2222 (Chromogenix) solution prepared using purified water was used.

[Example 3] Measurement of plasma coagulation activity by activated partial thromboplastin time (APTT) The blood coagulation reaction is a sequential substrate activation reaction by a plurality of serine proteases. In order to clarify whether anti-F.XIa, FX bispecific antibody having FX activation promoting activity promotes the same reaction in hemophilia B plasma and corrects coagulation ability, F.IX deficient plasma is used. The effect of the antibody on activated partial thromboplastin time (APTT), which is a widely used index for clinical diagnosis of coagulation function, was investigated. APTT measurement was carried out by a method known to those skilled in the art, and specifically, the following procedure was carried out. An antibody solution prepared at each concentration using TBSB, or 15 μL of F.IX (Chris Machine, Japan Blood Products Organization) prepared at each concentration and 135 μL of F.IX-deficient plasma (Sysmex) are mixed and contains an antibody. Plasma samples were prepared. A mixture of 50 μL of plasma sample and 50 μL of APTT reagent (Sysmex) was heated at 37 ° C. for 190 seconds. The coagulation reaction was initiated by adding 50 μL of 0.02 M calcium chloride solution (Sysmex) to the mixture. The coagulation time was measured using CS-2000i (Sysmex) based on the time when the decrease in permeability was 50% of the maximum decrease, and was shown as APTT [Figs. 4 and 6]. The antibody concentration is shown as the concentration in the plasma sample containing the antibody.
As a result of the evaluation, in F.IX-deficient plasma, the APTT shortening effect was shown in the bispecific antibody-added group as in the F.IX-added group. From the above results, it was shown that the anti-F.XIa, FX bispecific antibody has a coagulation-promoting effect in F.IX-deficient plasma, and the anti-F.XIa, FX bispecific antibody is associated with hemophilia B. It was suggested that it may correct the coagulation ability of plasma of hemophilia B patients as well as F.IX used clinically as a therapeutic drug.

Applicability to other blood coagulation factors Substrate specificity of enzymes is often conferred by binding to target substrates by exosites, as represented by thrombin and F.XIa (Mol Aspects Med. 2008 Aug; 29). (4): 203-254., Thromb Res. 2018 Jan; 161: 94-105., J Thromb Haemost. 2005; 3 (1): 54-67.). Therefore, based on the results of the above-mentioned F.XIa and FX bispecific antibodies for promoting the enzyme reaction and blood coagulation ability, a cofactor, which is a high molecular weight polypeptide that promotes the reaction of the substrate B with the enzyme A, is present in the living body. It is considered that even a combination of enzyme and substrate that does not exist can promote the reaction by forcibly bringing them close to each other by a bispecific antigen-binding molecule.

Application to other coagulation factors in the blood coagulation system The blood coagulation reaction is completed by the conversion of fibrinogen to fibrin by thrombin produced by the sequential activation of the serine protease coagulation factor precursor in plasma. A schematic diagram of the major blood coagulation cascades is shown in Figure 2 (created with reference to Blood. 2010; 115 (13): 2569-2577.). F.XII (a), F.XI (a), F.IX (a), FX (a), F.VII (a), F.IIa are serine proteases, as shown in FIG. F.XIIa activates F.XI, F.XIa activates F.IX, F.IXa activates FX, F.VIIa activates FX, and F.Xa activates prothrombin to proceed with the coagulation reaction.

Hemophilia is a hemorrhagic disease caused by a congenital or acquired decrease in activity of F.VIIIa, F.IX, and F.XI in Fig. 2. On the other hand, blood coagulation factors other than the blood coagulation factor causing the disease state have a normal function. Therefore, by applying this concept to these normally active blood coagulation factors, the endogenous coagulation cascade can be normalized in hemophiliac plasma, or the reaction of the extrinsic cascade can be promoted to promote bleeding tendency. It can be suppressed.
Specifically, the bleeding tendency in hemophilia can be suppressed by preparing a bispecific antigen-binding molecule that binds to the combination of the enzymes a to h shown in Table 1 and the substrate.

The bispecific antigen-binding molecule of the present disclosure contributes to the promotion of blood coagulation and may be useful for the prevention and / or treatment of bleeding, diseases associated with bleeding, or diseases caused by bleeding.

Claims (29)

The bispecific antigen-binding molecule according to any one of (a) to (h) below:
(a) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X;
(b) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor X and blood coagulation factor X;
(c) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor VII and blood coagulation factor X;
(d) Activated blood coagulation factor VII-A bispecific antigen-binding molecule that recognizes both Tissue Factor complex and blood coagulation factor X;
(e) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor X;
(f) A bispecific antigen-binding molecule that recognizes both Thrombin and blood coagulation factor X;
(g) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor IX; or
(h) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor XI. The bispecific antigen-binding molecule according to claim 1, which promotes activation of the substrate by the enzyme. The bispecific antigen-binding molecule according to claim 1 or 2, which is a bispecific antibody. A composition comprising the antigen-binding molecule according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. The composition according to claim 4, which is a pharmaceutical composition used for prevention and / or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding. Claim 5 that bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activated blood coagulation factor IX. The composition according to. The composition according to claim 6, wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor IX and / or activation blood coagulation factor IX is hemophilia B. Blood coagulation factor IX and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor IX, and inhibitors against blood coagulation factor IX and / or activated blood coagulation factor IX appear The composition according to claim 6, which is a disease. Claim 5 that bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII. The composition according to. Blood coagulation factor VIII and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII are hemophilia A, acquired hemophilia, or von Willebrand's disease. The composition according to claim 9. Blood coagulation factor VIII and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor VIII appear as inhibitors of blood coagulation factor VIII and / or activated blood coagulation factor VIII The composition according to claim 9, which is a disease. Claim 5 that bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activated blood coagulation factor XI. The composition according to. The composition according to claim 12, wherein the disease that develops and / or progresses due to a decrease or deficiency in the activity of blood coagulation factor XI and / or activation of blood coagulation factor XI is hemophilia C. Blood coagulation factor XI and / or activation Diseases that develop and / or progress due to decreased or deficient activity of blood coagulation factor XI appear as inhibitors of blood coagulation factor XI and / or activated blood coagulation factor XI The composition according to claim 12, which is a disease. Use of the bispecific antigen-binding molecule according to any one of claims 1 to 3 for producing the composition according to any one of claims 3 to 13. Preventing and / or preventing bleeding, bleeding-related diseases, or diseases caused by bleeding, which comprises at least the bispecific antigen-binding molecule according to any one of claims 1 to 3 or the composition according to claim 3. A kit for use in therapeutic methods. Bleeding, a disease associated with bleeding, or bleeding, which comprises at least the bispecific antigen-binding molecule according to any one of claims 1 to 3 or the composition according to claim 3 and contains blood coagulation factor IX. A kit for use in methods of preventing and / or treating diseases caused by blood coagulation factor IX in combination. Bleeding, a disease associated with bleeding, or bleeding, which comprises at least the bispecific antigen-binding molecule according to any one of claims 1 to 3 or the composition according to claim 3 and contains blood coagulation factor VIII. A kit for use in methods of preventing and / or treating diseases caused by blood coagulation factor VIII in combination. Bleeding, bleeding-related disease, or bleeding containing at least the bispecific antigen-binding molecule according to any one of claims 1 to 3 or the composition according to claim 3 and containing blood coagulation factor XI. A kit for use in methods of preventing and / or treating diseases caused by blood coagulation factor XI in combination. A bypass preparation for bleeding, a disease associated with bleeding, or a disease caused by bleeding, which contains at least the bispecific antigen-binding molecule according to any one of claims 1 to 3 or the composition according to claim 3. A kit for use in prophylactic and / or therapeutic methods in combination with. A method of promoting blood coagulation using a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X. A screening method for substances that are effective in the prevention and / or treatment of bleeding, bleeding-related diseases, or diseases caused by bleeding.
A screening method comprising (1) evaluating the binding between the test substance and activated blood coagulation factor XI, and (2) evaluating the binding between the test substance and blood coagulation factor X. The screening method according to claim 22, further comprising the following steps:
(3) A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance. A quality test method for a substance or composition that is effective in the prevention and / or treatment of bleeding, bleeding-related diseases, or diseases caused by bleeding.
Includes (1) a step of evaluating the binding of the test substance or test composition to activated blood coagulation factor XI, and (2) a step of evaluating the binding of the test substance or test composition to blood coagulation factor X. , Quality test method. The quality test method according to claim 24, further comprising the following steps:
(3) A step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance or a test composition. A nucleic acid encoding the bispecific antigen-binding molecule according to any one of claims 1 to 3. A vector into which the nucleic acid according to claim 26 is inserted. A cell comprising the nucleic acid according to claim 26 or the vector according to claim 27. The method for producing a bispecific antigen-binding molecule according to any one of claims 1 to 3 by culturing the cell according to claim 28.

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