JP2023106635A - Bispecific antigen binding molecules and compositions related thereto, uses, kits and methods for producing compositions - Google Patents

Abstract

To provide therapeutic methods for safely preventing bleeding of hemophilia patients.SOLUTION: The invention provides a bispecific antigen binding molecule that recognizes specific two factors among various coagulation-related factors. The invention also provides compositions and kits containing the bispecific antigen binding molecule. In one non-limiting embodiment, the bispecific antigen binding molecule, compositions, and kits of the invention are used to prevent and/or treat bleeding, diseases accompanied by bleeding, or diseases caused by bleeding. Additionally, the invention provides a method for using the bispecific antigen binding molecule to promote coagulation. Furthermore, the invention provides a method for screening substances that are effective for preventing and/or treating bleeding, diseases accompanied by bleeding, or diseases caused by bleeding.SELECTED DRAWING: None

Description

The present disclosure relates to bispecific antigen-binding molecules that recognize both of two specific factors among various blood coagulation-related factors, as well as compositions, uses for the manufacture of compositions, and kits comprising the same. . The present disclosure also relates to methods of promoting blood coagulation using the bispecific antigen-binding molecules. Furthermore, the present disclosure relates to screening methods for substances effective in preventing and/or treating bleeding, diseases associated with bleeding, or diseases caused by bleeding.

Blood coagulation is composed of a series of components, in particular fibrinogen, blood clotting factors II, V, VII, VIII, IX, X, XI and XII (F.II, F.II, respectively). F.V, F.VII, F.VIII, F.IX, F.X, 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) and the von Willebrand factor.

Hemophilia is a bleeding disorder in which the deficiency or impairment of these components results in the inability of the blood to clot. When F.VIII is the cause, it is called hemophilia A, and when F.IX is the cause, it is called hemophilia B. In patients with hemophilia, bleeding symptoms are observed in deep tissues such as intra-articular and intra-muscular, and intracranial hemorrhage also occurs in severe cases.

Hemophilia severity is classified based on F.VIII or F.IX activity in the blood. Specifically, when the F.VIII activity or F.IX activity of healthy subjects is 100%, patients with less than 1% activity are severe, and those with activity of 1% or more and less than 5% are moderate. More than 5% and less than 40% of patients are classified as mild. Patients with severe hemophilia present with bleeding episodes significantly more frequently than those with moderate and mild hemophilia. 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 above 1%. . Replacement therapy primarily uses clotting factor preparations purified from plasma or produced by recombinant technology.

F.VIII preparations are usually administered for bleeding in hemophilia A patients (on-demand administration). Moreover, in recent years, F.VIII preparations have been prophylactically administered to prevent bleeding events (non-patent documents 1 and 2) (prophylactic administration). The blood half-life of F.VIII preparations is approximately 12 to 16 hours. Therefore, for continuous prophylaxis, F.VIII preparations are administered to patients three times a week (Non-Patent Documents 3, 4). For on-demand administration, additional F.VIII preparations are administered at regular intervals as needed to prevent rebleeding. Administration of F.VIII preparations is also performed intravenously. Also, occasionally antibodies to F.VIII (inhibitors) develop in hemophiliacs. Inhibitors counteract the effects of F.VIII preparations. Therefore, there has been a strong demand for a drug that is less burdensome to administer than F.VIII preparations and is not affected by the presence of inhibitors.

Bispecific antibodies that substitute the function of F.VIII and their use have been reported as a means of solving these problems (Patent Documents 1, 2, 3 and 4). Bispecific antibodies against F.IXa and F.X can exert F.VIII cofactor function substitution activity and substitute the function of F.VIII by positioning both factors in the vicinity (non Patent document 5). One of these 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 a clinical trial for hemophilia A patients with no inhibitors and those with hemophilia A, compared to before administration of ACE910 (Emicizumab), the number of bleeding episodes significantly decreased after administration of ACE910 (Emicizumab). Suppression was observed (Non-Patent Document 7).

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

F.XI is a pro-protease of F.XIa and contributes to hemostasis through activation of F.IX. Recently, it has been reported that the activation peptide of F.X can be cleaved by adding high-concentration F.XIa to F.X, and that addition to F.V produces F.Va (Non-Patent Document 9). In addition, it was reported that intravenous administration of an excessive amount of F.XIa (estimated plasma concentration of 60 nM) reduced the duration of bleeding in hemophilia B model mice genetically deficient in F.IX. (Non-Patent Document 10). Therefore, administration of excessive doses of F.XI or F.XIa to patients with hemophilia B may reduce the number of bleeding episodes, regardless of the presence of inhibitors. However, F.XI preparations have been reported to cause thrombosis when administered to F.XI-deficient patients, and have not been established as a safe therapeutic method (Non-Patent Document 11). This may be because the F.XI 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 that can safely prevent bleeding in hemophiliacs without overdosing F.XI preparations or F.XIa preparations.

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. 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. 2015;21(4):477-480.

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

In a non-limiting embodiment, the present inventors have, as a result of extensive research, developed a bispecific antigen-binding molecule that recognizes two types of blood coagulation-related factors for which cofactors that are brought close to each other in vivo have not been confirmed. Successfully made. They also found that the bispecific antigen-binding molecule contributes to 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 capable of undergoing the catalytic reaction of said enzyme, provided that the bispecific antigen-binding molecule substitutes for the function of a peptide cofactor or heparin expressed in vivo Excluding molecules.
[B] The bispecific antigen-binding molecule of [A], which recognizes both an enzyme and a substrate capable of undergoing catalytic reaction by said enzyme, and promotes hydrolysis of said substrate by said enzyme sexual antigen-binding molecule.
[C] The bispecific antigen-binding molecule of [A], which 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 sexual antigen-binding molecule.
[D] The bispecific antigen-binding molecule of any one of [A] to [C], wherein the enzyme is a protease.
[E] The bispecific antigen-binding molecule of any one of [A] to [C], wherein the enzyme is a serine protease.
[F] The bispecific antigen of any one of [A] to [E], wherein the enzyme is a blood coagulation enzyme, and the substrate capable of undergoing catalytic reaction by 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 cofactors expressed in the body at least one cofactor selected from the group consisting of complement C4b, complement regulatory factor H, membrane cofactor protein (MCP), complement receptor 1 (CR1) or a dual function that substitutes for heparin The bispecific antigen-binding molecule of any one of [A] to [F], excluding the specific antigen-binding molecule.
[G] the bispecific antigen-binding molecule of any one of [A] to [G], which is a bispecific antibody [1] any one of (a) to (g) below A bispecific antigen binding molecule of:
(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) a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor VII-tissue factor complex and blood coagulation factor X;
(e) bispecific antigen-binding molecules that recognize 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; or
(g) Bispecific antigen-binding molecules that recognize both activated blood coagulation factor XII and blood coagulation factor IX.
[2] The bispecific antigen-binding molecule of [1], which is a bispecific antibody.
[3] A composition comprising the antigen-binding molecule of [1] or [2] and a pharmaceutically acceptable carrier.
[4] The composition of [3], 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.
[5] 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 [4].
[6] The composition of [5], wherein the disease that develops and/or progresses due to decreased or defective activity of blood coagulation factor IX and/or activated blood coagulation factor IX is hemophilia B.
[7] 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 is associated with blood coagulation factor IX and/or activated blood coagulation factor IX; The composition of [5], which is an inhibitory disease.
[8] Bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and/or develops 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 [4].
[9] Hemophilia A, acquired hemophilia, or von Willebrand's disease, which develops and/or progresses due to decreased or defective activity of blood coagulation factor VIII and/or activated blood coagulation factor VIII The composition according to [8].
[10] a disease that develops and/or develops due to a decrease or deficiency in the activity of blood coagulation factor VIII and/or activated blood coagulation factor VIII is associated with blood coagulation factor VIII and/or activated blood coagulation factor VIII The composition of [8], which is an inhibitory disease.
[11] 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 [4].
[12] The composition of [11], wherein the disease that develops and/or progresses due to decreased or defective activity of blood coagulation factor XI and/or activated blood coagulation factor XI is hemophilia C.
[13] a disease that develops and/or develops due to a decrease or deficiency in the activity of blood coagulation factor XI and/or activated blood coagulation factor XI is associated with blood coagulation factor XI and/or activated blood coagulation factor XI The composition of [11], which is an inhibitory disease.
[14] Use of the bispecific antigen-binding molecule of [1] or [2] for producing the composition of any one of [3] to [13].
[15] Preventing and/or bleeding, a disease associated with bleeding, or a disease caused by bleeding comprising at least the bispecific antigen-binding molecule of [1] or [2] or the composition of [3] or kits for use in methods of treatment.
[16] bleeding, a disease accompanied by bleeding, which comprises at least the bispecific antigen-binding molecule of [1] or [2] or the composition of [3] and blood coagulation factor IX; Alternatively, a kit for use in a method of preventing and/or treating a disease caused by bleeding in combination with blood coagulation factor IX.
[17] bleeding, a disease accompanied by bleeding, which comprises at least the bispecific antigen-binding molecule of [1] or [2] or the composition of [3] and blood coagulation factor VIII; Alternatively, a kit for use in a method for preventing and/or treating a disease caused by bleeding in combination with blood coagulation factor VIII.
[18] bleeding, a disease accompanied by bleeding, which comprises at least the bispecific antigen-binding molecule of [1] or [2], or the composition of [3] and blood coagulation factor XI; Alternatively, a kit for use in a method for preventing and/or treating a disease caused by bleeding in combination with blood coagulation factor XI.
[19] comprising at least the bispecific antigen-binding molecule of [1] or [2] or the composition of [3] and bleeding, a disease associated with bleeding, or a disease caused by bleeding, A kit for use in a prophylactic and/or therapeutic method in combination with a bypassing agent.
[20] 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;
[21] A screening method for a substance effective for the prevention and/or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding,
A screening method comprising the steps of (1) evaluating binding between a test substance and activated blood coagulation factor XI; and (2) evaluating binding between a test substance and blood coagulation factor X.
[22] The screening method of [21], further comprising the following steps: (3) The step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance.
[23] A method for testing the quality of 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 comprising the steps of (1) assessing the binding of a test substance to activated blood coagulation factor XI, and (2) assessing the binding of the test substance to blood coagulation factor X.
[24] The quality test method according to [23], further comprising the following steps:
(3) Evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance.
[25] a nucleic acid encoding the bispecific antigen-binding molecule of [1] or the bispecific antibody of [2];
[26] A vector into which the nucleic acid of [25] has been inserted.
[27] A cell containing the nucleic acid of [25] or the vector of [26].
[28] A method for producing the bispecific antigen-binding molecule of [1] or the bispecific antibody of [2] by culturing the cell of [27].
[29] the bispecific antigen-binding molecule according to any one of the following (h) to (g):
(h) a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XI and blood coagulation factor X, wherein the bispecific antigen-binding molecule activates blood coagulation factor X;
(i) a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor X and blood coagulation factor X, wherein the bispecific antigen-binding molecule activates blood coagulation factor X;
(j) a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor VII and blood coagulation factor X, wherein the bispecific antigen-binding molecule activates blood coagulation factor X;
(k) a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor VII-tissue factor complex and blood coagulation factor X, wherein the bispecific antigen activates blood coagulation factor X binding molecule;
(l) a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor X, wherein the bispecific antigen-binding molecule activates blood coagulation factor X;
(m) a bispecific antigen binding molecule that recognizes both thrombin and blood coagulation factor X and activates blood coagulation factor X; or
(n) A bispecific antigen-binding molecule that recognizes both activated blood coagulation factor XII and blood coagulation factor IX, wherein the bispecific antigen-binding molecule activates blood coagulation factor IX.
[30] The bispecific antigen-binding molecule of [29], which is a bispecific antibody.
[31] a composition comprising the antigen-binding molecule of [29] or [30] and a pharmaceutically acceptable carrier;
[32] The composition of [31], 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.
[33] 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 of [32].
[34] The composition of [33], wherein the disease that develops and/or progresses due to decreased or defective activity of blood coagulation factor IX and/or activated blood coagulation factor IX is hemophilia B.
[35] a disease that develops and/or develops due to a decrease or deficiency in the activity of blood coagulation factor IX and/or activated blood coagulation factor IX is associated with blood coagulation factor IX and/or activated blood coagulation factor IX The composition of [33], which is an inhibitory disease.
[36] 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 of [32].
[37] Hemophilia A, acquired hemophilia, or von Willebrand's disease, which develops and/or progresses due to decreased or defective activity of blood coagulation factor VIII and/or activated blood coagulation factor VIII The composition of [36].
[38] A disease that develops and/or develops due to decreased or defective activity of blood coagulation factor VIII and/or activated blood coagulation factor VIII is associated with blood coagulation factor VIII and/or activated blood coagulation factor VIII The composition of [36], which is an inhibitory disease.
[39] 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 of [32].
[40] The composition of [39], wherein the disease that develops and/or progresses due to decreased or defective activity of blood coagulation factor XI and/or activated blood coagulation factor XI is hemophilia C.
[41] a disease that develops and/or develops due to a decrease or deficiency in the activity of blood coagulation factor XI and/or activated blood coagulation factor XI is associated with blood coagulation factor XI and/or activated blood coagulation factor XI The composition of [39], which is an inhibitory disease.
[42] Use of the bispecific antigen-binding molecule of [29] or [30] for producing the composition of any one of [31] to [41].
[43] Preventing and preventing bleeding, a disease associated with bleeding, or a disease caused by bleeding, comprising at least the bispecific antigen-binding molecule of [29] or [30], or the composition of [31] /or a kit for use in a method of treatment.
[44] bleeding, a disease accompanied by bleeding, which comprises at least the bispecific antigen-binding molecule of [29] or [30] or the composition of [31] and blood coagulation factor IX; Alternatively, a kit for use in a method of preventing and/or treating a disease caused by bleeding in combination with blood coagulation factor IX.
[45] bleeding, a disease accompanied by bleeding, which comprises at least the bispecific antigen-binding molecule of [29] or [30] or the composition of [31] and blood coagulation factor VIII; Alternatively, a kit for use in a method for preventing and/or treating a disease caused by bleeding in combination with blood coagulation factor VIII.
[46] bleeding, a disease accompanied by bleeding, which comprises at least the bispecific antigen-binding molecule of [29] or [30] or the composition of [31] and blood coagulation factor XI; Alternatively, a kit for use in a method for preventing and/or treating a disease caused by bleeding in combination with blood coagulation factor XI.
[47] comprising at least the bispecific antigen-binding molecule of [29] or [30] or the composition of [31] and bleeding, a disease associated with bleeding, or a disease caused by bleeding, A kit for use in a prophylactic and/or therapeutic method in combination with a bypassing agent.
[48] 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;
[49] A screening method for a substance effective for the prevention and/or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding,
A screening method comprising the steps of (1) evaluating binding between a test substance and activated blood coagulation factor XI; and (2) evaluating binding between a test substance and blood coagulation factor X.
[50] The screening method of [49], further comprising the following steps: (3) evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance;
[51] A method for testing the quality of 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 comprising the steps of (1) assessing the binding of a test substance to activated blood coagulation factor XI, and (2) assessing the binding of the test substance to blood coagulation factor X.
[52] The quality test method according to [51], further comprising the following steps:
(3) Evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance.
[53] a nucleic acid encoding the bispecific antigen-binding molecule of [29] or the bispecific antibody of [30];
[54] a vector into which the nucleic acid of [53] is inserted;
[55] a cell containing the nucleic acid of [53] or the vector of [54];
[56] A method for producing the bispecific antigen-binding molecule of [29] or the bispecific antibody of [30] by culturing the cell of [55].

In one non-limiting aspect, the bispecific antigen-binding molecules, compositions, and kits of the present disclosure contribute to the promotion of blood coagulation, thus preventing bleeding, diseases associated with bleeding, or diseases caused by bleeding. and/or may be therapeutically useful.

The basic concept of the invention is illustrated. Enzyme A and substrate B, which have a relationship in which no peptidic cofactor exists in the body, hardly react (upper figure). However, by adding a bispecific antigen-binding molecule that recognizes enzyme A and substrate B, enzymatic reactions such as hydrolysis of substrate B by enzyme A can be promoted (see figure below). A schematic diagram of the blood coagulation cascade (intrinsic system and extrinsic system) is shown. The results of measurement of the anti-F.XIa, F.X bispecific antibody's ability to promote F.X activation by F.XIa in an in vitro enzymatic reaction system are shown. The prepared anti-F.XIa, F.X bispecific antibody showed F.X activation promoting activity by F.XIa. Clotting time (APTT) in F.IX-deficient plasma was shown. The anti-F.XIa, F.X bispecific antibody we prepared showed the same plasma procoagulant activity.

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

A bispecific antigen-binding molecule in the present invention is an antigen-binding molecule comprising two types of antigen-binding domains with specificities for different antigens or epitopes. In one aspect, the bispecific antigen-binding molecule in the present invention is a bispecific antibody. Although the bispecific antibody is not particularly limited, it is preferably a monoclonal antibody.

As used herein, the term "antigen-binding domain" refers to a region that specifically binds and is complementary to part or all of an antigen. As used herein, an antigen-binding molecule comprises an antigen-binding domain. If the antigen has a large molecular weight, the antigen-binding domain can only bind to a specific portion of the antigen. Such specific portions are called epitopes. In one aspect, the antigen binding domain comprises an antibody fragment that binds to a specific antigen. An antigen binding domain may be provided by one or more antibody variable domains. In one non-limiting embodiment, 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 "single chain Fv (scFv)", "single chain antibody (single chain antibody)", "Fv", "single chain Fv2 (scFv2)", "Fab" or "Fab' ” and the like. In another embodiment, the antigen binding domain comprises a non-antibody protein or fragment thereof that binds to a specific antigen. In certain embodiments, the antigen binding domain comprises a hinge region.

As used herein, the term “specifically binds” refers to a state in which one of the specifically binding molecules does not show any significant binding to molecules other than one or more molecules of its binding partner. It means to combine with It is also used when the antigen-binding domain is specific for a specific epitope among multiple epitopes contained in an antigen. Moreover, when the epitope to which the antigen-binding domain binds is contained in multiple different antigens, the antigen-binding molecule having the antigen-binding domain can bind to various antigens containing the epitope.

As used herein, the term "antibody" is used in the broadest sense, and as long as it exhibits the desired antigen-binding activity, it is not limited to, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., It encompasses 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 on a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). refers to the strength of Here, avidity is not strictly limited to 1:1 interactions between members of a binding pair (eg, antibody and antigen). For example, avidity refers to the intrinsic binding affinity (“affinity”) when the members of a binding pair reflect a 1:1 interaction with monovalence. When members of a binding pair are capable of both monovalent and multivalent binding, avidity is the sum of their avidity. The binding activity of molecule X for its partner Y can generally be expressed by the dissociation constant (KD) or "amount of analyte bound per unit amount of ligand". Binding activity can be measured by routine methods known in the art, including those described herein.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up the population are mutated antibodies that may arise (e.g., mutated antibodies containing naturally occurring mutations, or mutated antibodies that arise during the manufacture of monoclonal antibody preparations. are identical and/or bind to the same epitope, except that they are present in the same amount. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention include, but are not limited to, hybridoma technology, recombinant DNA technology, phage display technology, transgenic animals containing all or part of the human immunoglobulin locus. Such and other exemplary methods for making monoclonal antibodies are described herein.

"Native antibodies" refer to immunoglobulin molecules with varying structures that occur in nature. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

The term "chimeric" antibody means that a portion of the heavy and/or light chains are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species. Refers to antibodies.

The "class" of an antibody refers to the type of constant domain or constant region provided by the heavy chains of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM. Some of these may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called 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 or IgG4 type antibody constant region, still more preferably human IgG1, IgG2 or IgG3. , the antibody constant region of the IgG4 type. In another embodiment of the present invention, the constant region is preferably a heavy chain constant region, more preferably an IgG1, IgG2, IgG3, or IgG4 type heavy chain constant region, and even more preferably a human Heavy chain constant regions 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 the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies, multiple allotypic sequences due to genetic polymorphisms are described in Sequences of proteins of immunological interest, NIH Publication No. 91-3242. Any of them may be used. The amino acid-modified constant region of the present invention 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" connects the CH1 and CH2 domains in a wild type antibody heavy chain, e.g. to about position 243 of the antibody heavy chain polypeptide. In native IgG antibodies, the cysteine residue at EU numbering position 220 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 the cysteine residues at EU numbering position 226 and the cysteine residues at position 229 in the hinge region form disulfide bonds between the two antibody heavy chains. The hinge region herein includes not only the wild type but also variants in which amino acid residues are substituted, added, or deleted in the wild type.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain, including at least part of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of 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 according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Follows the EU Numbering System (also known as the EU Index), as described in MD 1991.

The terms "variable region" or "variable domain" refer to the domains of the heavy or light chains of an antibody that are responsible for binding the antibody to the antigen. The heavy and light chain variable domains (VH and VL, respectively) of native antibodies are usually similar, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). have a structure. (See, eg, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind a particular antigen may be isolated using the VH or VL domains from the antibody that binds the antigen to screen a complementary library of VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, the term "hypervariable region" or "HVR" is hypervariable in sequence ("complementarity determining region" or "CDR") and/or is structurally defined Refers to the regions of the variable domain of an antibody that form loops (“hypervariable loops”) and/or contain antigen-contacting residues (“antigen-contacting”). Antibodies typically contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary 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); resulting hypervariable loops (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 CDRs (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 Combinations 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. The FRs of variable domains usually consist of four FR domains: FR1, FR2, FR3 and FR4. Accordingly, HVR and FR sequences 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," "whole antibody," and "whole antibody" are used interchangeably herein and have a structure substantially similar to that of a naturally occurring antibody, or as defined herein. It refers to an antibody having a heavy chain containing an Fc region that

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell (including progeny of such a cell) into which exogenous nucleic acid has been introduced. . A host cell includes "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom at any passage number. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as with which the originally transformed cell was screened or selected are also included herein.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked. The term includes vectors as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing 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 with an amino acid sequence that corresponds to that of an antibody produced by a human or human cells or derived from a human antibody repertoire or other non-human source using human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains in which all or substantially all HVRs (e.g., CDRs) are non- Corresponds to that of a human antibody, and all or substantially all FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.

In one aspect, the antigen-binding molecule is a recombinant antigen-binding molecule produced using genetic recombination technology. (See, eg, Borrebaeck CAK and Larrick JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Recombinant antigen-binding molecules are obtained by cloning DNA encoding the same from hybridomas or antigen-binding molecule-producing cells such as sensitized lymphocytes that produce the antigen-binding molecule, inserting it into an appropriate vector, and introducing it into a host. It can be obtained by producing

Furthermore, antigen-binding molecules are not limited to antibodies, and may be aptamers. Antibody fragments when the antigen-binding molecule is an antibody fragment include diabody (Db), linear antibody, VHH (variable domain of heavy chain of heavy chain antibody), single-chain antibody (hereinafter also referred to as scFv). ) molecules are included. As used herein, an "Fv" fragment is the minimum antibody fragment, containing a complete antigen-recognition and binding site. An "Fv" fragment is a dimer in which one heavy (H) chain variable region ( _VH ) and a light (L) chain variable region ( _VL ) are tightly linked non-covalently ( _VH - _VL dimer). . The three complementarity determining regions (CDRs) of each variable region interact to form an antigen-binding site on the surface of the V _H -V _L dimer. Six CDRs confer the antigen-binding site on the antibody. However, even a single variable region (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site. .

In addition, the Fab fragment (also called F(ab)) further contains the constant region of the L chain and the constant region of the H chain (CH1). Fab' fragments differ from Fab fragments by having additionally a few residues from the carboxy terminus of the H chain CH1 region including one or more cysteines from the antibody hinge region. Fab'-SH refers to Fab' in which one or more cysteine residues in the constant region have free thiol groups. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of an F(ab') ₂ pepsin digest. Other chemically coupled antibody fragments are known to those skilled in the art.

Diabodies refer to bivalent antibody fragments constructed by gene fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO93/11161, etc.). A diabody is a dimer composed of two polypeptide chains, each of which in the same chain has a light chain variable region (V _L ) and a heavy chain variable region (V _H ) that are incapable of binding to each other. are linked by short linkers of, for example, about 5 residues. 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. It will have parts.

Single-chain antibodies or scFv antibody fragments comprise the _VH and _VL regions of an antibody, wherein these regions are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL regions, which allows the scFv to form the necessary structure for antigen binding (for a review of scFv, see Pluckthun The Pharmacology of Monoclonal Antibodies, Vol. 113 (see 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 regions linked at both ends.

IgG-type bispecific antibodies can be secreted by a hybrid hybridoma (quadroma) produced by fusing two IgG antibody-producing hybridomas (Milstein C et al. Nature 1983, 305: 537-540). In addition, a total of four types of genes, the L chain and H chain genes that constitute the two types of IgG of interest, can be co-expressed and secreted by introducing them into cells.

However, there are theoretically 10 combinations of IgG H and L chains produced by these methods. It is difficult to purify an IgG consisting of a desired combination of H and L chains from 10 types of IgG. Furthermore, since the amount of secretion of the target combination is theoretically significantly reduced, a large culture scale is required, further increasing production costs.

In this case, by subjecting the CH3 region of the H chain to an appropriate amino acid substitution, it is also possible to preferentially secrete an IgG with a heterogeneous combination of H chains (Ridgway JB et al. Protein Engineering 1996, 9: 617-621, Merchant AM et al. Nature Biotechnology 1998, 16: 677-681).

As for the L chain, since the diversity of the L chain variable region is lower than that of the H chain variable region, it is expected that a common L chain capable of imparting binding ability to both H chains can be obtained. Efficient expression of bispecific IgG becomes possible by expressing IgG by introducing the 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 contain the same L chain and it is difficult to implement the above idea. A selection method has also been proposed (WO2004/065611). The above mutation (Nature Biotechnology. 1998, 16, 677-681) The H chain having CH3 is hardly secreted in the absence of the counterpart H chain. Using this feature, the L and H chains of the right arm are first induced to express, and after stopping this, the L and H chains of the left arm are induced to express, thereby increasing the expression ratio of the desired combination of IgG. Yes (PCT/JP2004/008585).
Bispecific antigen binding molecules can also be made by chemically cross-linking Fab's. For example, Fab' prepared from one antibody is maleimidated with o-PDM (ortho-phenylenedi-maleimide), and reacted with Fab' prepared from the other antibody to cross-link Fab' derived from different antibodies. However, bispecific F(ab') ₂ can be generated (Keler T et al. Cancer Research 1997, 57: 4008-4014). A method of chemically binding antibody fragments such as Fab'-thionitrobenzoic acid (TNB) derivatives and Fab'-thiol (SH) is also known (Brennan M et al. Science 1985, 229: 81-83). .

Leucine zippers derived from Fos, Jun, etc. can also be used instead of chemical cross-linking. Fos and Jun also form homodimers, but take advantage of the fact that they preferentially form heterodimers. Fab' with a Fos leucine zipper and the other Fab' with a Jun zipper are prepared for expression. Bispecific F(ab') ₂ can be formed by mixing and reacting reduced monomeric Fab'-Fos and Fab'-Jun under mild conditions (Kostelny SA et al. J of Immunology, 1992, 148:1547-53). This method is not limited to Fab', and can also be applied to scFv, Fv, and the like.

Bispecific antigen binding molecules may also be generated in diabodies. Bispecific diabodies are heterodimers of two cross-over scFv fragments. In other words, V _H (A)-VL (B), V _H (B)-, which were created by connecting V _H and V _L derived _from two antibodies A and B with a relatively short linker of about 5 residues. This 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, the two types of scFv are connected by a flexible, 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 Substitutions (knobs-into-holes: Zhu Z et al. Protein Science. 1997, 6: 781-788) can also be made to facilitate the desired construction.

sc(Fv) ₂ , which can be produced by linking two scFvs with a relatively long flexible 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).

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

The antigen-binding molecules of the present invention are human antibodies, mouse antibodies, rat antibodies, and the like, and are not limited in origin. Genetically modified antibodies such as chimeric antibodies and humanized antibodies may also be used.

A method for obtaining a human antibody is already known. For example, a transgenic animal having an entire repertoire of human antibody genes can be immunized with an antigen of interest to obtain a human antibody of interest (International Patent Application Publication Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

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

Humanized antibodies are modified antibodies, also called reshaped human antibodies. Humanized antibodies are constructed by grafting the CDRs of an antibody from an immunized animal into the complementarity determining regions of a human antibody. The general gene recombination method is also known.

Specifically, several oligos were prepared with overlapping portions at the ends of a DNA sequence designed to connect the mouse antibody CDRs and the human antibody framework regions (FRs). It is synthesized from nucleotides by the PCR method. The obtained DNA is ligated with a DNA encoding a human antibody constant region, then incorporated into an expression vector, and introduced into a host for production (European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 96/02576). Human antibody FRs linked via CDRs are selected so that their complementarity determining regions form good antigen-binding sites. If necessary, amino acids in the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms a suitable antigen binding site (Sato K et al, Cancer Research 1993, 53:851-856). Alternatively, framework regions from various human antibodies may be substituted (see International Patent Application Publication No. WO 99/51743).

In one embodiment, the enzyme recognizes both an enzyme and a substrate that can be catalyzed by the enzyme, and the enzyme hydrolyzes the substrate, including blood coagulation factor V, blood coagulation factor VIII, and 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) At least one cofactor selected from the group consisting of or a bispecific antigen-binding molecule that does not replace the function of heparin is provided. In another aspect, bispecific antigen-binding molecules that recognize both an enzyme and a substrate that can be catalyzed by the enzyme, but do not replace the function of naturally occurring blood coagulation cofactors are provided. . Enzymes in these aspects are not particularly limited as long as they are molecules having catalytic activity for chemical reactions that occur in living organisms. Enzymes are exemplified by protease, amylase, cellulase and lipase. Proteases are exemplified by serine proteases of the blood coagulation system. In certain embodiments, the enzyme can be a blood clotting enzyme. Examples of serine proteases that are blood coagulation enzymes include F.XII(a), F.XI(a), F.IX(a), F.X(a), F.VII(a), and F.IIa. . Note that F.XII(a) means F.XII and/or F.XIIa, and so on.

The term "substrate capable of undergoing the catalytic reaction of the enzyme" as used herein refers to a substrate that undergoes the catalytic reaction of the enzyme when forced to approach. Enzyme catalysis means that the substrate is hydrolyzed. Hydrolysis of the substrate may be as long as the substrate is hydrolyzed as compared with the case where the bispecific antigen-binding molecule of the present application is not present. The substrate hydrolysis reaction can be compared by measuring the amount of protein over time after addition of the bispecific antigen-binding molecule by, for example, the Bradford method using Protein Assay Kit II (Bio-Red). Alternatively, it can be measured by SDS-PAGE analysis or Western blotting (WB) using proteins before and after addition of the bispecific antigen-binding molecule. Alternatively, it can be measured by mass spectrometry (MALDI-TOFMS, etc.) or N-terminal amino acid sequence analysis (protein sequencing) of the protein before and after addition of the bispecific antigen-binding molecule. Substrate hydrolysis includes, for example, activation of blood coagulation factor X or blood coagulation factor IX. Substrate activation can be achieved by using a reaction system containing the enzyme and the substrate, and adding the antigen-binding molecule to increase the activity of the enzyme (substrate resolution). A measurement system consisting of a synthetic substrate, phospholipids, and Ca 2+ , which is a synthesized substrate, can be evaluated by the substrate activation-promoting activity of the enzyme. Based on the results, bispecific antigen-binding molecules having this activity can be selected, in principle, from those that show a substrate activation-promoting activity of 0.1 or more by the enzyme only in the enzyme-added group in this assay system. As used herein, the substrate activation-enhancing activity of the enzyme can be measured by the absorbance value of the antigen-binding molecule solution 30 minutes after addition of the synthetic substrate.

Specific examples of enzymes (also referred to as "blood coagulation enzymes") and substrates (also referred to as "blood coagulation substrates") in embodiments in which the enzymes and substrates are blood coagulation fibrinolysis-related factors include, 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 may be obtained by any method. For example, when obtaining a bispecific antigen-binding molecule against enzyme A and substrate B, an immunized animal is immunized with enzyme A and substrate B to obtain anti-enzyme A antibody and anti-substrate B antibody. Thereafter, a bispecific antigen-binding molecule comprising the H and L chain variable regions of the anti-enzyme A antibody and the H and L chain variable regions of the anti-substrate B antibody is prepared. Here, it is desirable to obtain a plurality of anti-enzyme A antibodies and anti-substrate B antibodies, respectively, and it is preferable to use them to prepare as many combinations of bispecific antigen-binding molecules as possible. After preparing the bispecific antigen-binding molecule, an antigen-binding molecule that activates substrate B is selected.

Antigen-binding molecules for enzymes or substrates can be obtained by methods known to those skilled in the art. For example, it can be prepared by immunizing an animal to be immunized with an antigen. Antigens with which animals are immunized include immunogenic complete antigens and non-immunogenic incomplete antigens (including haptens). For immunization, an enzyme or substrate to which an antigen-binding molecule binds, or a nucleic acid expressing them is used as the antigen (immunogen). Animals to be immunized include, for example, mice, rats, hamsters, guinea pigs, rabbits, chickens, rhesus monkeys and the like. Those skilled in the art can immunize these animals with antigens by methods well known to those skilled in the art. Preferably, antibody L and H chain variable regions are recovered from the immunized animal or cells of the animal. This operation can be performed using techniques generally known to those skilled in the art. Animals immunized with an antigen develop antibodies to the antigen, particularly in spleen cells. Therefore, for example, mRNA can be prepared from spleen cells of an immunized animal, and the L and H chain variable regions can be recovered by RT-PCR using primers corresponding to the variable regions of the animal.

Specifically, animals are immunized with an enzyme and a substrate, respectively. Enzymes and substrates used as immunogens may be whole proteins or partial peptides of the proteins. In addition, as an immunogen used for immunizing animals, it is optionally possible to bind antigens to other molecules to form soluble antigens, and optionally to use fragments thereof.

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

Alternatively, mRNA is extracted from the splenocytes of the immunized animal, and cDNAs of the L and H chain variable regions are recovered by RT-PCR using primers corresponding to the vicinity of the variable region. It is also possible to immunize lymphocytes in vitro. This is used to construct a library displaying scFv or Fab. Antigen-binding antibody clones can be enriched and cloned by panning to obtain variable regions. In this case, it is also possible to perform screening using a similar library of mRNA derived from peripheral blood mononuclear cells, spleen, tonsil, etc. of humans or non-immunized animals.

An antigen-binding molecule expression vector is constructed 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.

Selection of antigen-binding molecules in the present invention can be performed, for example, by the following methods.
(1) A reaction system containing the enzyme and the substrate is used, and the increase in enzyme activity (substrate resolution) due to the addition of the antigen-binding molecule is used as an indicator for selection.
(2) Absence of at least one of the enzyme, the substrate and the cofactor using a system that measures or mimics biological functions involving the enzyme, the substrate and the cofactor (e.g., plasma coagulation measurement system) Selection is made using the function restoration activity by adding the antigen-binding molecule under the conditions as an indicator.

The resulting antigen-binding molecule can be purified to homogeneity. Separation and purification of antigen-binding molecules can be carried out using separation and purification methods commonly used for proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, and the like. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but not limited to these. Columns used for affinity chromatography include protein A columns and protein G columns.

In the bispecific antigen-binding molecule of the present invention, for example, when the combination of enzyme and substrate is blood coagulation and fibrinolysis-related factor F.XIa and F.X, the variable region of anti-F.XIa antibody is preferably and a variable region in the anti-F.X antibody.

A bispecific antigen-binding molecule can be produced by the following method. For example, when the combination of enzyme and substrate was F.XIa and FX, mice were subcutaneously immunized with commercially available F.XIa and FX, respectively. Splenocytes were isolated from the spleens of immunized mice with elevated antibody titers and fused with mouse myeloma cells to produce hybridomas. Hybridomas that bind to antigens (F.XIa and FX) were selected, and the variable regions of the L and H chains were recovered by RT-PCR using primers corresponding to the variable regions. The L chain variable region was incorporated into an L chain expression vector containing _CL , and the H chain variable region was incorporated into an H chain expression vector containing an H chain constant region. In addition, mRNA was extracted from the spleens of these immunized mice, and cDNAs of the L and H chain variable regions were recovered by RT-PCR using primers corresponding to the variable regions. A phage library displaying scFv was constructed using these variable regions. Antigen-binding antibody clones were enriched and cloned by panning, and antigen-binding molecule expression vectors were constructed using the variable regions thereof. Alternatively, it can be generated by B cell cloning techniques by methods known to those skilled in the art. Anti-F.XIa antibody (H chain, L chain) expression vector and FX antibody (H chain, L chain) expression vector are introduced into the same cell, and the antigen-binding molecule is expressed to achieve bispecific antigen binding. I got the molecule.

For the obtained bispecific antigen-binding molecules, the F.X activation-promoting activity of F.XIa was evaluated using an assay system consisting of F.XIa, F.X, a synthetic F.Xa substrate (S-2222), and phospholipids. bottom. Based on the results, those bispecific antigen-binding molecules that showed an F.X activation promotion activity by F.XIa of 0.1 or more in this measurement system were selected as principle. The F.X activation-promoting activity of F.XIa as used herein is the absorbance value 30 minutes after the addition of the synthetic substrate to the antigen-binding molecule solution.

For the bispecific antigen-binding molecules selected above or related bispecific antigen-binding molecules, the ability to restore coagulation was measured using a coagulation time measurement system using blood coagulation factor-deficient human plasma. As a result, a bispecific antigen-binding molecule was obtained that shortened the clotting time compared to when no antibody was added. The clotting time referred to herein is the value obtained by measuring the activated partial thromboplastin time using blood coagulation factor IX-deficient human plasma. Among these bispecific antigen-binding molecules, the most preferred bispecific antigen-binding molecule had the ability to shorten clotting time by 60 seconds or more.

Antigen-binding molecules of the present invention are not particularly limited, and include bispecific antigen-binding molecules that recognize both the enzyme and substrate of any combination listed in Table 1.

When producing full-length antibodies using the variable regions disclosed in the present invention, the constant regions are not particularly limited, and constant regions known to those skilled in the art can be used. , (1991), U.S. Department of Health and Human Services. Public Health Service National Institutes of Health, An efficient route to human bispecific IgG, (1998). Nature Biotechnology vol. 16, 677-681, etc. A constant region can be used.

In one aspect of the antigen-binding molecule of the present invention, the antigen-binding molecule does not replace at least the function of a peptidic cofactor or heparin expressed in vivo, but complements the enzyme in relation to the substrate capable of undergoing the catalytic reaction of the enzyme. Since it has a factor-like function, it is expected to be an effective drug against diseases caused by reduced activity (function) or deficiency of certain cofactors, enzymes, or substrates. When the enzyme and substrate to which the antigen-binding molecule of the present invention binds are blood coagulation and fibrinolysis-related factors, examples of the disease include bleeding, diseases accompanied by bleeding, and diseases caused by bleeding. . In particular, dysfunction or deficiency of F.VIII/F.VIIIa, F.IX/F.IXa, and F.XI/F.XIa is known to cause a bleeding disorder called hemophilia.

In hemophilia, bleeding disorders due to congenital hypofunction or deficiency of F.VIII/F.VIIIa are called hemophilia A, and bleeding disorders due to hypofunction or deficiency of F.IX/F.IXa are called hemophilia A. Called Friendship B. Bleeding in hemophilia A patients is treated with F.VIII replacement therapy, and hemophilia B patients with bleeding are treated with F.IX replacement therapy. In addition, prophylactic administration of these preparations may be performed on the day of strenuous exercise or excursion, in cases of frequent joint bleeding, or in cases classified as severe hemophilia.

F.VIII preparations have a short blood half-life of approximately 12 to 16 hours. Therefore, for continuous prophylaxis, it is necessary to administer F.VIII preparations about three times a week. This corresponds to maintaining approximately 1% or more of F.VIII activity. Similarly, a F.IX formulation with a standard half-life would need to be dosed about twice a week. In replacement therapy during bleeding, except for mild bleeding, additional F.VIII or F.IX preparations must be administered regularly for a certain period of time to prevent rebleeding and achieve complete hemostasis. There is

F.VIII and F.IX formulations are also administered intravenously. Technical difficulties exist in the practice of intravenous administration. Especially in administration to young patients, the difficulty is further compounded by the narrow veins used for administration.

In many cases, home remedies and self-injection are used for preventive administration of F.VIII and F.IX preparations and for emergency administration in the event of bleeding. The need for frequent administration and the technical difficulties involved in administration not only make patients feel pain during administration, but also hinder the spread of home remedies and self-injection.
Therefore, there has been a strong demand for a drug with a wider administration interval or a drug that is easier to administer than the existing blood coagulation factor VIII preparations and blood coagulation factor IX preparations.

In addition, hemophilia A patients, especially severe hemophilia A patients, may develop antibodies against F.VIII called inhibitors. Similarly, hemophilia B patients may develop antibodies to F.IX called inhibitors. When inhibitors develop, they prevent the effects of F.VIII or F.IX formulations. As a result, hemostasis management for the patient becomes very difficult.

Bleeding in such patients with hemophilia A inhibitors is usually treated with reversal therapy with large doses of F.VIII or by-pass therapy with complex concentrates or F.VIIa. , is carried out. However, in neutralizing therapy, administration of large doses of F.VIII preparations may conversely increase the inhibitor (anti-F.VIII antibody) titer. In bypass therapy, complex preparations and F.VIIa preparations have a short blood half-life (approximately 2 to 8 hours), which is a problem. Moreover, because their mechanism of action is independent of the function of F.VIII/F.VIIIa, i.e., the ability to catalyze F.X activation by F.IXa, they may, in some cases, favor hemostatic mechanisms. In some cases, it is not possible to receive a response, resulting in no response. Therefore, in patients with hemophilia A inhibitors, there are many cases in which a sufficient hemostatic effect cannot be obtained compared to non-inhibitor hemophilia A patients. The same is true for hemophilia B inhibitor patients.

Therefore, there is a strong need for agents that are independent of the presence of inhibitors and that replace the function of F.VIII/F.VIIIa or F.IX/F.IXa.

Now, (i) the dosing interval is wide, (ii) the dosing is simple, (iii) it is independent of the presence of inhibitors, and (iv) it is F.VIII/F.VIIIa or F.IX/F.IXa independent. Antibody-based methods are conceivable for the creation of pharmaceuticals that can substitute for the function of the original. The serum half-life of antibodies is generally relatively long, ranging from days to weeks. Also, antibodies are generally known to migrate into the blood after subcutaneous administration. That is, antibody drugs generally satisfy the above (i) and (ii).

The present invention provides pharmaceutical compositions containing the antigen-binding molecules of the present invention as active ingredients. For example, when the antigen-binding molecule of the present invention is an antigen-binding molecule that recognizes both the enzyme and the substrate in the combination shown in Table 1 and promotes blood coagulation in the blood coagulation factor-deficient plasma , the antigen-binding molecule is expected to serve as a pharmaceutical (pharmaceutical composition) or drug for the prevention or treatment of bleeding, diseases associated with bleeding, or diseases caused by bleeding.

A 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 inert pharmaceutically acceptable carrier, medium, etc. can be formulated as For example, sterile water or saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binding agents and the like. In addition, other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulins, amino acids such as glycine, glutamine, asparagine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, sugars such as mannitol and sorbitol May contain alcohol. In the case of an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable dissolution. Adjuvants such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. may be used in combination.

In addition, if necessary, the antigen-binding molecules of the present invention are encapsulated in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, poly[methyl methacrylic acid], etc.) or colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, microcapsules, etc.). nanoparticles, nanocapsules, etc.) (see "Remington's Pharmaceutical Science 16th edition", Oslo Ed. (1980), etc.). Furthermore, methods of making drugs into sustained-release drugs are also known and can be applied to the antigen-binding molecules of the present invention (Langer et al., J. Biomed. Mater. Res. 15: 267-277 (1981); Langer, Chemtech. 12: 98-105 (1982); U.S. Pat. No. 3,773,919; European Patent Application Publication (EP) 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); .

Antigen-binding molecules or compositions 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 simultaneously with blood coagulation factor VIII, or may be administered at different times. Alternatively, the antigen-binding molecule or pharmaceutical composition of the present invention and blood coagulation factor VIII may be combined as a kit. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with blood coagulation factor VIII, the dosage of each can be reduced as desired compared to when either is used alone.

Antigen-binding molecules or compositions of the present invention can be used in combination with blood coagulation factor IX. The 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 simultaneously with blood coagulation factor IX, or may be administered at different times. Alternatively, the antigen-binding molecule or pharmaceutical composition of the present invention and blood coagulation factor IX may be combined to form a kit. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with blood coagulation factor IX, the dosage of each can be reduced as desired compared to when either of them is used alone.

Antigen-binding molecules or compositions 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 simultaneously with blood coagulation factor XI, or may be administered at different times. Alternatively, the antigen-binding molecule or pharmaceutical composition of the present invention and blood coagulation factor XI may be combined to form a kit. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with blood coagulation factor XI, the dosage of each can be reduced as desired compared to when either of them is used alone.

Antigen-binding molecules or compositions of the present invention can be used in combination with bypassing agents. Bypassing agents may be those made from human blood or those made by genetic recombination. Factor VII preparation (rFVIIa preparation) and dried concentrated human blood coagulation factor X-activated Factor VII preparation (FVIIa/FX preparation). The antigen-binding molecule or composition of the present invention may be administered simultaneously with the bypassing preparation, or may be administered at different times. Alternatively, the antigen-binding molecule or pharmaceutical composition of the present invention and a bypass preparation may be combined as a kit. Furthermore, when the antigen-binding molecule or pharmaceutical composition of the present invention is used in combination with a bypassing preparation, the dosage of each can be reduced as desired compared to when either of them is used alone.

The dosage of the pharmaceutical composition of the present invention is ultimately determined by the doctor's judgment, taking into account the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, type and degree of progression of the disease, etc. Although it is determined appropriately, in general, for adults, 0.1 to 2000 mg can be administered in 1 to several divided doses per day. More preferably 1-1000 mg/day, even more preferably 50-500 mg/day, most preferably 100-300 mg/day. These doses vary depending on the patient's body weight, age, administration method, etc., but those skilled in the art can appropriately select appropriate doses. The administration period is also preferably determined as appropriate according to the patient's progress of healing and the like.

Gene therapy may also be performed by incorporating a gene encoding the antigen-binding molecule of the present invention into a gene therapy vector. As for the administration method, in addition to direct administration by naked plasmid, packaging in liposomes, etc., or formation as various viral vectors such as retrovirus vector, adenovirus vector, vaccinia virus vector, poxvirus vector, adenovirus-related vector, HVJ vector, etc. (see Adolph, Virus Genome Method, CRC Press, Florid (1996)), or coated on a bead carrier such as colloidal gold particles (WO93/17706, etc.). In addition, 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 increase 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 a method that induces the expression (see EP2101823B, WO2013/120629). However, any method of administration may be used as long as the antigen-binding molecule can be expressed in vivo and can exert its action. Preferably, injection, infusion, or gas-induced particle bombardment (electron A sufficient amount is administered by a gun, etc.), a method via a mucosal route such as nasal drops, etc.). Ex vivo liposome transfection, particle bombardment method (U.S. Pat. No. 4,945,050), or viral infection is used to administer blood cells, bone marrow-derived cells, etc., and reintroduce the cells into animals. A gene encoding an antigen-binding molecule may be administered.

The present invention also provides methods for preventing and/or treating bleeding, diseases associated with bleeding, or diseases caused by bleeding, comprising administering an antigen-binding molecule or composition of the present invention. Administration of antigen-binding molecules or compositions can be performed, for example, by the methods described above.
In one embodiment, bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and/or progresses due to decreased or absent activity of blood coagulation factor IX and/or activated blood coagulation factor IX (e.g. , hemophilia B). In certain embodiments, the disease is a disease in which inhibitors to blood coagulation factor IX or activated blood coagulation factor IX are present. In certain embodiments, the antigen-binding molecules or compositions of the present invention are administered to subjects with inhibitors of blood coagulation factor IX or activated blood coagulation factor IX (inhibitor-bearing patients). In certain embodiments, the methods of the invention further comprise administering blood coagulation factor IX.
In another embodiment, bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and/or develops due to decreased or absent activity of blood coagulation factor VIII and/or activated blood coagulation factor VIII ( For example, hemophilia A, acquired hemophilia, or von Willebrand disease). In certain embodiments, the disease is a disease in which inhibitors to blood coagulation factor VIII or activated blood coagulation factor VIII are present. In certain embodiments, the antigen-binding molecules or compositions of the invention are administered to a subject with an inhibitor of blood coagulation factor VIII or activated blood coagulation factor VIII (inhibitor-bearing patient). In certain embodiments, the methods of the invention further comprise administering blood coagulation factor VIII.
In another embodiment, bleeding, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and/or progresses due to decreased or absent activity of blood coagulation factor XI and/or activated blood coagulation factor XI ( For example, hemophilia C, or acquired hemophilia). In certain embodiments, the disease is a disease in which inhibitors to blood coagulation factor XI or activated blood coagulation factor XI appear. In certain embodiments, the antigen-binding molecule or composition of the invention is administered to a subject with an inhibitor of blood coagulation factor XI or activated blood coagulation factor XI (inhibitor-bearing patient). In certain embodiments, the methods of the invention further comprise administering blood coagulation factor XI.

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

Furthermore, the present invention provides kits for use in the above methods, which contain at least the antigen-binding molecules or compositions of the present invention. The kit may also be packaged with syringes, needles, pharmaceutically acceptable vehicles, alcohol swabs, bandages, or instructions for use. In one embodiment, 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, kits of the invention comprise blood coagulation factor IX or factor VIII in addition to an antigen-binding molecule or composition of the invention.

In another aspect, the invention relates to a method of promoting blood coagulation using a bispecific antigen-binding molecule that recognizes both the enzyme and substrate of any combination set forth in Table 1. In one embodiment of this aspect, the method of promoting blood coagulation of the present invention comprises administering a bispecific antigen-binding molecule that recognizes both the enzyme and substrate of any combination listed in Table 1. Administration of antigen-binding molecules can be performed, for example, by the administration methods described above. In a specific embodiment of the above aspect, the bispecific antigen-binding molecule used in the method of promoting blood coagulation of the present invention is a bispecific antigen that recognizes both activated blood coagulation factor XI and blood coagulation factor X. is a binding molecule.

In yet another aspect, the present invention relates to a screening method for substances effective in preventing and/or treating bleeding, diseases associated with bleeding, or diseases caused by bleeding. In one embodiment of this aspect, the screening method of the present invention comprises the steps of: (1) assessing binding between a test substance and any combination of enzymes listed in Table 1; evaluating the binding of the combination described in 1. to a substrate. In certain embodiments, the screening methods of the present invention comprise the steps of (1) assessing binding between a test substance and activated blood coagulation factor XI; and (2) assessing binding between a test substance and blood coagulation factor X. including the step of In certain embodiments, the screening methods of the present invention employ a test substance that binds to both the enzyme and substrate of any combination described in Table 1 for the prevention and treatment of bleeding, disorders associated with bleeding, or disorders caused by bleeding. /or further comprising the step of selecting as a candidate therapeutically effective substance.

"Test substance" is not particularly limited, for example, natural compounds, organic compounds, inorganic compounds, nucleic acids, proteins, single substances such as peptides, compound libraries, nucleic acid libraries, peptide libraries, gene live Larry expression products, cell extracts, cell culture supernatants, fermented microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts or animal cell extracts and the like can be mentioned. The test substance can be appropriately labeled and used as necessary. Labels include, for example, radioactive labels, fluorescent labels, and the like. In addition to the test substances described above, mixtures in which multiple types of these test substances are mixed are also included.

In yet another aspect, the present invention relates to a method for testing the quality of a substance or composition effective in the prevention and/or treatment of bleeding, diseases associated with bleeding, or diseases caused by bleeding. In one embodiment of this aspect, the quality testing method of the present invention comprises the steps of: (1) assessing the binding of a test substance or test composition to any combination of enzymes listed in Table 1; Evaluating the binding of the substance or test composition to the substrates of the combination described in Table 1. In certain embodiments, the quality testing methods of the present invention comprise the steps of (1) assessing the binding of a test substance or test composition to activated blood coagulation factor XI; A step of evaluating binding to coagulation factor X is included. In certain embodiments, the quality testing method of the present invention tests a test substance or composition that binds both the enzyme and substrate of any combination described in Table 1 as a test substance or composition that causes bleeding, bleeding disorders, or bleeding. It further includes a step of evaluating quality as a substance or composition effective for disease prevention and/or treatment.

A "test composition" is not particularly limited, and examples thereof include pharmaceuticals, reagents, and the like.

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

(2) The step of evaluating the binding between the test substance and blood coagulation factor X includes, for example, ELISA (Enzyme-Linked Immuno Sorbent Assay), surface plasmon resonance (SPR) method, Biacore series ( GE Healthcare) and biomolecular interaction analysis system using biosensor technology (BLI method), for example, Octet system (Fortebio).

(3) The step of evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance includes, for example, the enzyme, the substrate, a synthetic substrate of the activated substrate, phosphorus An assay system consisting of lipids and Ca2+ can be used to evaluate the substrate activation-promoting activity of the enzyme. Based on the results, bispecific antigen-binding molecules having this activity can be selected, in principle, from those that show a substrate activation-promoting activity of 0.1 or more by the enzyme only in the enzyme-added group in this assay system. As used herein, the substrate activation-enhancing activity of the enzyme can be measured by the absorbance value of the antigen-binding molecule solution 30 minutes after addition of the synthetic substrate.

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

EXAMPLES The present invention will be specifically described below by way of examples, but the present invention is not limited to these examples.

[Example 1] Preparation of anti-F.XIa, FX bispecific antibody chain variable region SEQ ID NO: 2) was used. F10B1 (heavy chain variable region SEQ ID NO: 3, light chain variable region SEQ ID NO: 4) from SB04 described in WO2005035756A1 and heavy chain variable region of J327 described in WO2019065795A1 for the preparation of anti-FX antibodies F10B2 (heavy chain variable region SEQ ID NO: 5, light chain variable region SEQ ID NO: 6) consisting of a sequence and a sequence obtained by introducing two amino acid substitutions into the light chain variable region sequence of JNL095 was used. Each antibody variable region sequence was linked to a heavy chain constant region or light chain constant region sequence to construct an expression vector containing a gene encoding the full-length 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 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 shown in Table 2.

[Example 2] Measurement of FX activation promoting activity of F.XIa and FX bispecific antibody by in vitro enzyme reaction assay system The presence or absence of FX activation-promoting activity by XIa was evaluated using an in vitro enzyme reaction measurement system using a synthetic chromogenic substrate. Specifically, measurements were carried out according to the following procedure, and all reactions were carried out at room temperature. A mixed solution of 5 µL of 48 ng/mL F.XIa (Enzyme Research Laboratories) and 5 µL of antibody solution of each concentration was incubated in a 384-well plate for 30 minutes. Further, 5 μL of 22.9 μg/mL FX (Enzyme Research Laboratories) was added to the mixture to initiate the enzymatic reaction. After reacting for 60 minutes, the enzymatic reaction was stopped by adding 5 μL of 50 μM aprotinin (Sigma Aldrich). Subsequently, 5 μL of a synthetic chromogenic substrate solution that develops color with F.Xa was added to each well, and absorbance at a wavelength of 405 nm after 30 minutes was measured using SpectraMax340PC (Molecular Devices). The activity of F.XIa and FX bispecific antibody to promote FX activation by FXIa was expressed as the absorbance value 30 minutes after addition of the chromogenic substrate solution [Fig. 3]. In addition, the concentration of the antibody was shown as the concentration in the solution during the enzymatic reaction.
As a result of the evaluation, in an 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 the F.XIa, FX bispecific antibody promotes FX activation in a binding-dependent manner to both F.XIa and FX in an in vitro enzyme reaction assay system.
Tris-buffered saline containing 0.1% bovine serum albumin (hereinafter referred to as TBSB) was used as the solvent for the antibody solution. 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. A 1.47 mg/mL chromogenic substrate S-2222 (Chromogenix) solution prepared using purified water was used as the chromogenic substrate solution.

Example 3 Measurement of Plasma Coagulation Activity by Activated Partial Thromboplastin Time (APTT) Blood coagulation is a sequential substrate activation reaction by multiple serine proteases. To clarify whether the anti-F.XIa, FX bispecific antibody with FX activation-promoting activity promotes the same reaction in hemophilia B plasma and corrects coagulability, F.IX-deficient plasma We investigated the effect of this antibody on activated partial thromboplastin time (APTT), an index widely used as a clinical diagnostic of coagulation function. APTT measurement was carried out by a method known to those skilled in the art, specifically by the following procedure. Antibody solution prepared to each concentration using TBSB, or F.IX (Krismachine, Japan Blood Products Organization) 15 μL and F.IX-deficient plasma (Sysmex) 135 μL prepared to each concentration, mixed, including 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 same mixture. The clotting time was measured using CS-2000i (Sysmex) based on the time when the decrease in permeability reached 50% of the maximum decrease, and was shown as APTT [Fig. 4]. In addition, the antibody concentration was shown as the concentration in the plasma sample containing the antibody.
As a result of the evaluation, in F.IX-deficient plasma, the bispecific antibody-added group showed an antibody concentration-dependent APTT-shortening effect, similar to the F.IX-added group. These results indicate that the anti-F.XIa, FX bispecific antibody has a procoagulant effect in F.IX-deficient plasma. Similar to F.IX, which is used clinically as a therapeutic drug, it was suggested that it may correct the coagulability of plasma from hemophilia B patients.

Applicability to other blood coagulation factors Substrate specificity of many enzymes, typified by thrombin and F.XIa, is given by exosite binding to target substrates (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 above-mentioned FXIa and FX bispecific antibody-induced enzyme reaction promotion and blood coagulation results, cofactors, which are macromolecular polypeptides that promote the reaction of substrate B by enzyme A, do not exist in living organisms. Even with such a combination of enzyme and substrate, the reaction can be promoted by forcing them into close proximity with a bispecific antigen-binding molecule.

Application to Other Coagulation Factors in the Blood Coagulation System Coagulation is completed by the conversion of fibrinogen to fibrin by thrombin generated by the sequential activation of plasma serine proteases of clotting factor precursors. A schematic diagram of the major blood coagulation cascades is shown in FIG. 2 (prepared 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 described 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 advance the coagulation reaction.

Hemophilia is a bleeding disorder caused by congenital or acquired decreased activity of F.VIIIa, F.IX, and F.XI in FIG. On the other hand, blood clotting factors other than those causing disease states have normal function. Therefore, by applying this concept to these blood coagulation factors with normal activity, the intrinsic coagulation cascade can be normalized in hemophilic plasma, or the reaction of the extrinsic cascade can be promoted to reduce bleeding tendency. can be suppressed.
Specifically, the bleeding tendency in hemophilia can be suppressed by preparing bispecific antigen-binding molecules that bind to combinations of enzymes and substrates a to g listed in Table 1.

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

Claims (28)

The bispecific antigen-binding molecule according to any one of (a) to (g) 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) a bispecific antigen-binding molecule that recognizes both activated blood coagulation factor VII-tissue factor complex and blood coagulation factor X;
(e) bispecific antigen-binding molecules that recognize 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; or
(g) Bispecific antigen-binding molecules that recognize both activated blood coagulation factor XII and blood coagulation factor IX. 2. The bispecific antigen binding molecule of claim 1, which is a bispecific antibody. 3. A composition comprising the antigen-binding molecule of claim 1 or 2 and a pharmaceutically acceptable carrier. 4. The composition according to claim 3, which is a pharmaceutical composition used for the prevention and/or treatment of bleeding, diseases associated with bleeding, or diseases caused by bleeding. Claim 4, wherein the hemorrhage, 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 . 6. The composition according to claim 5, wherein the disease developed and/or progressed by decreased activity or deficiency of blood coagulation factor IX and/or activated blood coagulation factor IX is hemophilia B. A disease that develops and/or progresses due to decreased or defective activity of blood coagulation factor IX and/or activated blood coagulation factor IX, and an inhibitor of blood coagulation factor IX and/or activated blood coagulation factor IX appears. 6. The composition of claim 5, wherein the disease is Claim 4, wherein the hemorrhage, a disease associated with bleeding, or a disease caused by bleeding is a disease that develops and/or develops 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 . Hemophilia A, acquired hemophilia, or von Willebrand's disease is a disease that develops and/or progresses due to decreased or defective activity of blood coagulation factor VIII and/or activated blood coagulation factor VIII. A composition according to claim 8 . 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, and an inhibitor of blood coagulation factor VIII and/or activated blood coagulation factor VIII appears. 9. The composition of claim 8, wherein the disease is Claim 4, wherein the hemorrhage, 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 . 12. The composition according to claim 11, wherein the disease that develops and/or develops due to decreased or defective activity of blood coagulation factor XI and/or activated blood coagulation factor XI is hemophilia C. A disease that develops and/or progresses due to decreased or defective activity of blood coagulation factor XI and/or activated blood coagulation factor XI, and an inhibitor of blood coagulation factor XI and/or activated blood coagulation factor XI appears. 12. The composition of claim 11, which is a disease affecting Use of the bispecific antigen-binding molecule according to claim 1 or 2 for the production of the composition according to any one of claims 3-13. A method for preventing and/or treating bleeding, a disease associated with bleeding, or a disease caused by bleeding, comprising at least the bispecific antigen-binding molecule of claim 1 or 2, or the composition of claim 3. Kit for use in Bleeding, a disease associated with bleeding, or caused by bleeding comprising at least the bispecific antigen-binding molecule of claim 1 or 2 or the composition of claim 3 and comprising blood coagulation factor IX A kit for use in a method for preventing and/or treating a disease in combination with blood coagulation factor IX. Bleeding, a disease associated with bleeding, or caused by bleeding comprising at least the bispecific antigen-binding molecule of claim 1 or 2 or the composition of claim 3 and comprising blood coagulation factor VIII A kit for use in a method of preventing and/or treating a disease in combination with blood coagulation factor VIII. Bleeding, a disease associated with bleeding, or caused by bleeding comprising at least the bispecific antigen-binding molecule of claim 1 or 2 or the composition of claim 3 and comprising blood coagulation factor XI A kit for use in a method of preventing and/or treating a disease in combination with blood coagulation factor XI. comprising at least the bispecific antigen-binding molecule of claim 1 or 2, or the composition of claim 3, and bleeding, a disease associated with bleeding, or a disease caused by bleeding, in combination with a bypassing preparation; A kit for use in a method of prophylaxis and/or treatment of . 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 a substance effective for the prevention and/or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding, comprising:
A screening method comprising the steps of (1) evaluating binding between a test substance and activated blood coagulation factor XI; and (2) evaluating binding between a test substance and blood coagulation factor X. 22. The screening method of claim 21, further comprising the steps of:
(3) Evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance. A method for testing the quality of a substance or composition effective for the prevention and/or treatment of bleeding, a disease associated with bleeding, or a disease caused by bleeding,
(1) assessing the binding of the test substance or test composition to activated blood coagulation factor XI; and (2) assessing the binding of the test substance or test composition to blood coagulation factor X. , quality test methods. 24. The quality testing method of claim 23, further comprising the steps of:
(3) Evaluating the activation reaction of blood coagulation factor X by activated blood coagulation factor XI using a test substance or test composition. A nucleic acid encoding the bispecific antigen-binding molecule of claim 1 or the bispecific antibody of claim 2. A vector into which the nucleic acid according to claim 25 is inserted. A cell comprising the nucleic acid of claim 25 or the vector of claim 26. A method for producing the bispecific antigen-binding molecule of claim 1 or the bispecific antibody of claim 2 by culturing the cell of claim 27.

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