Bispecific Factor VIII Mimicking Antibody

Abstract

The present invention relates to bispecific antibodies that can bind to coagulation factor IX (FIX) and / or its activated form factor IXa (FIXa), as well as factor X (FX) and / or its activated form factor Xa (FXa), and promote FX activity by FIXa, and methods and compositions for treating subjects with blood clotting disorders, such as hemophilia A, with or without inhibitors.

Description

Technical Field

[0001] Incorporation by reference of a sequence listing This application is filed with an electronic sequence listing. The entire contents of the sequence listing are hereby incorporated by reference into this specification.

Background Art

[0002] In patients with blood coagulation disorders such as humans having hemophilia A and B, various steps of the coagulation cascade become dysfunctional due to, for example, the absence or insufficient presence of functional coagulation factors. Some dysfunctions of such a coagulation cascade result in insufficient blood coagulation and potentially life-threatening bleeding, or damage to internal organs such as joints.

[0003] Factor VIII (FVIII) deficiency, generally called hemophilia A, is a congenital bleeding disorder that affects approximately 420,000 people worldwide, of which approximately 105,000 are currently diagnosed.

[0004] Patients with hemophilia A may receive coagulation factor replacement therapy such as exogenous FVIII. Conventional treatment consists of replacement therapy provided as a prophylaxis or on-demand treatment for bleeding episodes. Until recently, prophylactic treatment for patients with severe hemophilia A included intravenous injection up to three times a week using either plasma-derived FVIII or recombinant FVIII, or a long-acting variant thereof.

[0005] However, such patients are at risk of developing neutralizing antibodies, so-called inhibitors, to such exogenous factors, rendering previously effective therapies ineffective. Patients with or without hemophilia A are non-limiting examples of blood coagulation disorders that are partially congenital and partially acquired. Patients who have developed inhibitors to FVIII cannot be treated with conventional replacement therapy. Exogenous coagulation factors may only be administered intravenously, which is quite inconvenient and unpleasant for patients.

[0006] The inappropriate reduction of FXa formation and thrombin generation caused by the decrease or absence of FVIII activity is the reason underlying the bleeding tendency in patients with hemophilia A.

[0007] Proteolytic conversion of FX to its enzymatically active form, FXa, can be achieved by a specific FX activation complex that includes FIXa and its cofactor active form FVIII (FVIIIa). Cofactor binding increases the enzymatic activity of FIXa by approximately five orders of magnitude and is thought to occur through multiple mechanisms as outlined in Non-Patent Document 1. In particular, FVIIIa has been shown to stabilize the structure of FIXa with increased proteolytic activity against FX (Non-Patent Document 2). Based on this observation, recognizing that antibodies are versatile binding proteins that can mimic various protein-protein interactions, Scheiflinger et al. screened 5,280 hybridoma supernatants and conducted a screening for agonist anti-FIX(a) antibodies that are characterized by the ability to promote FX activation by FIXa in the presence of a phospholipid surface and calcium but in the absence of native cofactor FVIIIa. It was found that 88 of them produced antibodies that exhibited FIXa agonist activity to varying degrees (see Patent Document 1 and Patent Document 2). Recently, the new drug, emicizumab (HEMLIBRA®) (also known as ACE910), was approved for subcutaneous prophylaxis of hemophilia A with or without inhibitors to the factors of conventional replacement therapy. Emicizumab is a humanized bispecific anti-FIX(a) / anti-FX(a) monoclonal antibody developed by Chugai Pharmaceuticals / Roche Pharmaceuticals for the treatment of hemophilia A. Emicizumab is designed to mimic the FVIII cofactor function (see Non-Patent Document 3 and Patent Document 3). Treatment with 30 - 50 μg of emicizumab per milliliter of plasma is estimated to be equivalent to at least 10 - 15 IU of factor VIII activity per deciliter of plasma (Non-Patent Document 4). However, some patients developed inhibitors (anti-drug antibodies) to emicizumab, and treatment with this compound was ineffective.

[0008] In addition to the generation of inhibitors exemplified by emicizumab, other antibody properties are also important for achieving an effective antibody-based therapy in patients. In particular, it has been shown how antibodies with a high tendency for non-specific binding can lead to safety issues in a clinical setting. Some reports have shown that high levels of non-specific binding reduced the circulating half-life of the antibody several-fold, resulting in an ineffective and cumbersome dosing regimen for the patient (see Non-Patent Document 5 and Non-Patent Document 6).

[0009] Patent Documents 4 and 5 also disclose anti-FIX(a) anti-FX(a) bispecific antibodies and their use as blood coagulation promoters for the treatment of hemophilia.

[0010] There remain many unmet medical needs for the hemophilia community, particularly for subjects with blood coagulation disorders. The present invention relates to improved compounds that can substitute for FVIII and are thus useful for the treatment of blood coagulation disorders such as hemophilia A.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

[0013] The present invention relates to a compound that functions as a substitute for coagulation factor VIII (FVIII) in patients suffering from blood coagulation disorders, particularly in patients lacking functional FVIII such as patients with hemophilia A, including patients with hemophilia A having inhibitors.

[0014] One aspect of the present invention relates to a compound that enhances the generation of FXa and can thus partially or completely restore coagulation in patients lacking functional FVIII.

[0015] In one aspect, the compound is an antibody or an antigen-binding fragment thereof. In such an aspect, the compound is a multispecific antibody or an antigen-binding fragment thereof, such as a bispecific antibody or an antigen-binding fragment thereof.

[0016] In a particular aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that functions as a substitute for FVIII in patients lacking functional FVIII such as patients with hemophilia A.

[0017] In one such embodiment, the antibody or antigen-binding fragment thereof can bind FIX(a), increase the enzymatic activity of FIXa towards FX, and optionally bind FX.

[0018] In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that can bind FIX(a) and FX(a), comprising a bispecific antibody or antigen-binding fragment thereof that increases the enzymatic activity of FIXa towards FX.

[0019] In one aspect, the invention relates to an antibody or antigen-binding fragment thereof that can bind FIX(a) and FX(a), which has improved properties compared to antibodies disclosed in the art. In one such embodiment, the antibody or antigen-binding fragment thereof has improved blood coagulation promoting properties compared to bispecific antibodies in the art including emicizumab, and / or a reduced tendency for non-specific binding to, for example, DNA and / or insulin, and / or a reduced tendency for self-association.

[0020] A further aspect of the invention relates to individual component (intermediate) antibodies or antigen-binding fragments thereof that are part of a bispecific antibody, such as a specific anti-FIX(a) antibody or antigen-binding fragment thereof or a specific anti-FX(a) antibody or antigen-binding fragment thereof.

[0021] A further aspect of the invention is directed to the antibodies or antigen-binding fragments thereof disclosed herein for the prevention and / or treatment of blood coagulation disorders, diseases associated with blood coagulation disorders, or diseases caused by blood coagulation disorders. In one embodiment, the blood coagulation disorder is hemophilia, with or without an inhibitor, such as hemophilia A.

[0022] A still further aspect of the invention relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof disclosed herein formulated for delivery of the antibody for the prevention and / or treatment of blood coagulation disorders such as hemophilia A, with or without an inhibitor, and an injection device having the same.

[0023] A further aspect of the present invention is directed to a kit comprising (i) an antibody or an antigen-binding fragment thereof disclosed herein, such as a bispecific antibody, and (ii) instructions for use.

[0024] The present invention can also solve further problems that will become apparent from the disclosure of the exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

Figure 1

[0026] Brief Description of the Sequences SEQ ID NOs: 1 - 8 and 17 - 88 represent the sequences of the heavy chain variable domains (VH) and light chain variable domains (VL) and complementarity determining regions (CDR) of the anti-FIX(a) and anti-FX(a) monoclonal antibodies (mAbs) described herein.

[0027] SEQ ID NO: 89 represents the amino acid sequence of human coagulation factor IX.

[0028] SEQ ID NO: 90 represents the amino acid sequence of human coagulation factor X.

[0029] SEQ ID NO: 91 represents the human IgG4 heavy chain constant region including S228P and C-terminal lysine cleavage.

[0030] SEQ ID NO: 92 represents the human IgG4 heavy chain constant region including S228P, F405L, R409K, and C-terminal lysine cleavage.

[0031] Sequence number 93 represents the human kappa light chain constant region.

[0032] Sequence number 94 represents the human IgG1 heavy chain constant region including F405L and C-terminal lysine cleavage.

[0033] Sequence number 95 represents the human IgG1 heavy chain constant region including K409R and C-terminal lysine cleavage.

[0034] Sequence numbers 9 to 16 are intentionally omitted.

[0035] The table in Example 6 is linked to the individual (component) anti-FIX(a) antibody and anti-FX(a) antibody as well as the bispecific antibody of the present invention.

[0036] Description In subjects with a blood coagulation disorder such as a human having hemophilia A, the coagulation cascade becomes dysfunctional due to the absence or insufficient presence of functional FVIII. Some dysfunctions of such a coagulation cascade result in insufficient blood coagulation and potentially life-threatening bleeding, or damage to internal organs such as joints. The present invention relates to compounds that function as substitutes for coagulation factor VIII (FVIII) in patients lacking functional FVIII, such as patients with hemophilia A, including patients with hemophilia A who have inhibitors. In one aspect, such compounds are antibodies.

[0037] In particular, the inventors of the present invention have surprisingly identified antibodies that mimic FVIII cofactor activity with high potency and efficacy. In a particular aspect, the present invention relates to antibodies that function as substitutes for FVIII in patients lacking functional FVIII, such as patients with hemophilia A. In such an aspect, the antibody binds to factor IXa (FIXa) relative to factor X (FX) of coagulation, increases its enzymatic activity, and optionally also binds to FX. In such an aspect, the antibody of the present invention is a bispecific antibody capable of binding to FIX / FIXa and FX.

[0038] A further aspect of the present invention relates to individual constituent (intermediate) antibodies or antigen-binding fragments thereof that are part of a multispecific antibody, such as specific anti-FIX(a) antibodies or antigen-binding fragments thereof or specific anti-FX(a) antibodies or antigen-binding fragments thereof.

[0039] A further aspect of the present invention relates to the production of the antibodies or antigen-binding fragments thereof disclosed herein and their constituent (intermediate) components.

[0040] A further aspect of the present invention relates to antibodies that compete with the antibodies or antigen-binding fragments thereof disclosed herein for binding to FIX(a) and / or FX(a).

[0041] A further aspect of the present invention relates to antibodies or antigen-binding fragments thereof that share epitope residues with the antibodies or antigen-binding fragments thereof in the presence of FIX(a) and / or FX(a), as disclosed herein.

[0042] In one aspect, the antibody is a human antibody or humanized antibody, such as a human bispecific antibody or humanized bispecific antibody.

[0043] A further aspect of the present invention is directed to the antibodies or antigen-binding fragments thereof disclosed herein for the prevention and / or treatment of blood coagulation disorders, diseases associated with blood coagulation disorders, or diseases caused by blood coagulation disorders. In one aspect, the blood coagulation disorder is hemophilia A with or without an inhibitor.

[0044] An even further aspect of the present invention relates to a pharmaceutical composition comprising the antibodies or antigen-binding fragments thereof disclosed herein formulated for delivery of the antibody for the prevention and / or treatment of blood coagulation disorders such as hemophilia A with or without an inhibitor, and an injection device having the same.

[0045] A further aspect of the invention pertains to a kit comprising (i) an antibody or an antigen-binding fragment thereof disclosed herein, such as a bispecific antibody, and (ii) instructions for use.

[0046] Factor IX Factor IX (FIX) is a vitamin K-dependent coagulation factor that has structural similarities to factor VII, prothrombin, factor X, and protein C. FIX circulates in plasma as a single-chain enzyme precursor (SEQ ID NO: 89). The circulating zymogen form is composed of 415 amino acids divided into four distinct domains, including an N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. Activation of FIX occurs by limited proteolysis at Arg145 and Arg180, releasing an activation peptide (residues 146 - 180 of SEQ ID NO: 89). Thus, activated FIX (FIXa) is composed of residues 1 - 145 (light chain) of SEQ ID NO: 89 and residues 181 - 415 (heavy chain) of SEQ ID NO: 89.

[0047] Thus, the circulating FIX molecule includes the FIX zymogen and the active form of FIX, which are generally referred to herein as FIX and FIXa with respect to SEQ ID NO: 1.

[0048] The active form of factor IX is called factor IXa or FIXa. The term "FIX (SEQ ID NO: 1) and / or its active form (FIXa)" may also be referred to as "FIX / FIXa" or simply "FIX(a)".

[0049] FIXa is a trypsin-like serine protease that plays an important role in hemostasis by generating most of the factor Xa required to support appropriate thrombin formation during coagulation as part of the tenase complex.

[0050] In the present specification, FIX is represented by SEQ ID NO: 1 corresponding to the Ala148 genotype of human FIX (Anson et al., EMBO J. 1984 3:1053 - 1060, McGraw et al., Proc Natl Acad Sci USA. 1985 82:2847 - 2851, Graham et al., Am. J. Hum. Genet. 1988 42:573 - 580). In the present invention, FIX is intended to cover all natural variants of FIX, such as the T148 variant (Uniprot ID P00740).

[0051] Factor X FX is a vitamin K - dependent coagulation factor that has structural similarity to factor VII, prothrombin, FIX, and protein C. FX circulates in plasma as a double - chain zymogen precursor containing residues 1 - 139 (light chain) of SEQ ID NO: 2 and residues 143 - 448 (heavy chain) of SEQ ID NO: 2. The human FX zymogen precursor contains four distinct domains: an N - terminal gamma - carboxyglutamic acid - rich (Gla) domain (residues 1 - 45), two EGF domains, EGF1 (residues 46 - 82) and EGF2 (residues 85 - 125) respectively, and a C - terminal trypsin - like serine protease domain (residues 195 - 448). Activation of FX occurs by limited proteolysis at Arg194, resulting in the release of the activation peptide (residues 143 - 194). Thus, activated FX (FXa) is composed of residues 1 - 139 (light chain) of SEQ ID NO: 2 and residues 195 - 448 (activated heavy chain) of SEQ ID NO: 2. Thus, circulating factor X molecules include the FX zymogen and the active form of FX, which are referred to herein as FX and FXa respectively with respect to SEQ ID NO: 2. In the present invention, FX is intended to cover all natural variants of FX. The term "FX (SEQ ID NO: 90) and / or its active form (FXa)" may also be referred to as "FX / FXa" or "FX(a)".

[0052] antibody As used herein, the term "antibody" refers to a protein derived from an immunoglobulin sequence that can bind to an antigen or a portion thereof. The term antibody includes, but is not limited to, full-length antibodies of any class (or isotype), i.e., IgA, IgD, IgE, IgG, IgM, and / or IgY. The term antibody includes, but is not limited to, bivalent antibodies such as bispecific antibodies.

[0053] Native full-length antibodies include at least four polypeptide chains: two heavy chains (HC) and two light chains (LC) that are joined by disulfide bonds. In some cases, native antibodies include fewer than four chains, as in the case of IgNAR found in cartilaginous fish. One class of immunoglobulins with particular pharmaceutical benefits is IgG. In humans, the IgG class may be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequence of their heavy chain constant regions. Light chains can be divided into two types, kappa chains and lambda chains, based on differences in their sequence compositions. An IgG molecule is composed of two heavy chains linked by two or more disulfide bonds, and two light chains each attached to a heavy chain by a disulfide bond. An IgG heavy chain includes a heavy chain variable domain (V H ) and up to three heavy chain constant (C H ) domains: C H 1, C H 2, and C H 3. A light chain may include a light chain variable domain (V L ) and a light chain constant domain (C L ). The V H and V L regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR) or hypervariable regions (HvR) interspersed with more conserved regions called framework regions (FR). The V H and V LA domain typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable domain and the light chain variable domain containing the hypervariable regions (CDRs) form a structure capable of interacting with an antigen, while the constant regions of the antibody can mediate binding to host tissues or factors of the immunoglobulin. Such host tissues or factors include, but are not limited to, various cells of the immune system (effector cells), Fc receptors, and C1q of the C1 complex of the classical complement system, which is the first component.

[0054] The antibodies of the present invention can be monoclonal antibodies (mAbs) in the sense that they represent a series of unique heavy chain variable domain sequences and light chain variable domain sequences expressed from a single B cell or a clonal population of B cells. The antibodies of the present invention may be generated and purified using various methods known to those skilled in the art. For example, the antibodies may be generated from hybridoma cells. The antibodies may be generated by B cell proliferation. The antibodies or fragments thereof may be recombinantly expressed in mammalian or microbial expression systems or by in vitro translation. The antibodies or fragments thereof may also be recombinantly expressed as cell surface binding molecules, for example, by phage display, bacterial display, yeast display, mammalian cell display, or ribosome or mRNA display.

[0055] The antibodies of the present invention may be isolated. The term "isolated antibody" refers to an antibody that has been separated and / or recovered from other (different) components in the environment in which it was generated and / or purified from a mixture of components present in the environment in which it was generated.

[0056] Certain antigen-binding fragments of an antibody may be suitable in the context of the present invention since it has been shown that the antigen-binding function of an antibody can be carried out by fragments of the full-length antibody. The term "antigen-binding fragment" of an antibody, as described herein, refers to one or more fragments of an antibody that specifically bind to, or retain the ability to recognize, an antigen such as FIX / FIXa, FX / FXa or another target molecule. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab’, Fab2, Fab’2, Fv (typically, the V L domain and combination of V H domains), single-chain Fv (scFv), see, for example, Bird et al. Science 1988;242:423-426, and Huston et al. PNAS 1988;85:5879-5883), dsFv, Fd (typically, V H and C H 1 domain), monovalent molecules containing both a single V H domain and a single V L domain; minibodies, diabodies, triabodies, tetrabodies, kappa bodies (see, for example, Ill et al (1997) Protein Eng 10:949-57); and those containing one or more isolated CDRs or functional paratopes, where the isolated CDRs or antigen-binding residues or polypeptides can associate or link together to form a functional antibody fragment. These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

[0057] The "Fab fragment" of an antibody, including "Fab" and "Fab’2" fragments, can be derived from an antibody by cleavage of the heavy chain in the hinge region on the N-terminal or C-terminal side of the hinge cysteine residues that connect the heavy chains of the antibody. The "Fab" fragment consists of the variable and constant domains of the light chain, as well as the variable domain and C H1 contains a domain. The "Fab’2" fragment contains a pair of "Fab" fragments generally covalently linked by their hinge cysteines. Fab’ is formally derived from the Fab’2 fragment by cleavage of the hinge disulfide bond connecting the heavy chains in Fab’2. Other chemical bonds other than the disulfide bonds of antibody fragments are also known in the art. The Fab fragment retains the ability of the parental antibody to bind its antigen with potentially low affinity. The Fab’2 fragment can bind bivalently, while the Fab fragment and the Fab’ fragment can only bind monovalently. Generally, the Fab fragment lacks the constant C H 2 domain and C H 3 domain, that is, the Fc portion where the interaction between the Fc receptor and C1q will occur is lacking. Thus, the Fab fragment generally lacks effector functions. The Fab fragment can be generated by well-known methods in the art, for example, by enzymatic cleavage of an antibody using papain to obtain Fab or pepsin to obtain Fab’2. Fab fragments including Fab, Fab’, and Fab’2 can be recombinantly generated using techniques well known to those skilled in the art.

[0058] The "Fv" (fragment variable) fragment is an antibody fragment containing the complete antigen recognition and binding site, and generally contains one heavy chain and one light chain variable domain in relation to the ability to covalently bond naturally, for example, in a single-chain variable domain fragment (scFv). In this configuration, the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six hypervariable regions or subsets thereof confer antigen-binding specificity to the antibody.

[0059] The "single-chain Fv" or "scFv" antibody contains the V H domain and the V L domain, and these domains are present in a single polypeptide chain. Generally, the Fv polypeptide has a V H domain and a V LIt further contains a polypeptide linker between the domains, enabling the scFv to form the desired structure for antigen binding. For an overview of scFv, see Pluckthun, 1994, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269 - 315.

[0060] "Single-chain Fab" or "scFab" antibodies contain the V H , C H 1, V L , and C L domains, and these domains are present in a single polypeptide chain. Generally, the Fab polypeptide further contains a polypeptide linker either between the V H domain and the C L domain, or between the V L domain and the C H 1 domain, enabling the scFab to form the desired structure for antigen binding (Koerber et al. (2015) J Mol Biol. 427:576 - 86).

[0061] The term "diabody" refers to a small antibody fragment with two antigen - binding sites, where the fragment contains a heavy - chain variable domain (V H and V L ) connected to a light - chain variable domain (V L ) within the same polypeptide chain (V H ). By using a linker that is too short to allow pairing between the two variable domains on the same chain, the variable domains are forced to pair with the complementary domains on another chain, creating two antigen - binding sites.

[0062] The expression "linear antibody" refers to the antibodies described in Zapata et al. (1995) Protein Eng. 8: 1057-1062. Briefly stated, these antibodies contain a pair of tandem Fd segments (V H -C H 1-V H -C H 1) that form a pair of antigen-binding regions together with the complementary light chain polypeptides. Linear antibodies can be bispecific or monospecific.

[0063] Antibody fragments may be obtained using conventional recombinant or protein engineering techniques, and the fragments can be screened for binding to FIX and its active form, FX, or another function in the same manner as intact antibodies.

[0064] Antibody fragments of the invention can be made by truncation, for example, by removal of one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. Also, the fragments can be generated by one or more internal deletions.

[0065] Antibodies of the invention can be, or can contain, fragments of an antibody or variants of any of the antibodies disclosed herein. Antibodies of the invention can be, or can contain, an antigen-binding portion of one of these antibodies or a variant thereof. For example, an antibody of the invention can be a Fab fragment of one of these antibodies, or a variant thereof, or a single-chain antibody derived from one of these antibodies, or a variant thereof. Also, an antibody of the invention can be a combination of a full-length antibody and fragments thereof.

[0066] As used herein, the term "monospecific" antibody refers to an antibody (including but not limited to bispecific antibodies) that can bind to one particular epitope.

[0067] As used herein, the term "bispecific" antibody refers to an antibody that is capable of binding to two different antigens or two different epitopes on the same antigen.

[0068] As used herein, the term "trispecific" antibody refers to an antibody that is capable of binding to three different antigens, or three different epitopes on the same antigen, or three different epitopes present on two different antigens.

[0069] As used herein, the term "multispecific" antibody refers to an antibody that is capable of binding to two or more different antigens or two or more different epitopes on the same antigen. Thus, multispecific antibodies include bispecific and trispecific antibodies.

[0070] Full-length IgG-type bispecific antibodies can form hybrid quadromas that are generated by the fusion of two individual hybridomas and produce a mixture of antibodies containing fragments of bispecific heterodimerizing antibodies (Chelius D. et al.; MAbs. 2010 May-Jun;2(3):309-319). As another method, bispecific heterodimerizing antibodies can be generated by using recombinant techniques. Heterodimerization can also be achieved by manipulating the dimerization interface of the Fc region to promote heterodimerization. An example of this is the so-called knob-in-hole mutation, where a sterically bulky side chain (knob) is introduced into one Fc that matches the sterically small side chain (hole) on the opposing Fc, thereby creating the steric complementarity that promotes heterodimerization. Other methods of engineered heterodimerizing Fc interfaces are electrostatic complementarity, fusion to non-IgG heterodimerizing domains, or the utilization of the natural Fab arm exchange phenomenon of human IgG4 to control heterodimerization. Examples of heterodimeric bispecific antibodies are well described in the literature, for example, (Klein C, et al.; MAbs. 2012 Nov-Dec;4(6):653-663). Special attention needs to be paid to the light chains of heterodimeric antibodies. Correct pairing of LC and HC can be achieved by using a common light chain. Again, manipulation of the LC / HC interface can be used to facilitate heterodimerization or light chain crossover operations, as in the case of CrossMab. In vitro reconstitution of antibodies under mild reducing conditions from two individual IgGs containing appropriate mutations can also be used for the generation of bispecific antibodies (e.g., Labrijn et al., PNAS, 110, 5145-5150 (2013)). Also, a natural Fab arm exchange method for ensuring correct light chain pairing has been reported.

[0071] Multispecific antibody system molecules can also be recombinantly expressed as fusion proteins that combine natural modules of IgG to form multispecific antibody derivatives and multivalent antibody derivatives, as described in the literature. Examples of fusion antibodies include DVD-Igs, IgG-scFv, diabodies, DART, etc. Specific detection tags or purification tags, half-life extension moieties, or other components can be incorporated into the fusion protein. Additional non-IgG modalities can also be incorporated into the fusion protein. Bispecific full-length antibodies based on Fc heterodimerization are generally referred to as asymmetric IgG, regardless of the LC pairing method.

[0072] Generally, bispecific antibodies can be generated in various molecular formats outlined by Brinkmann et al. (Brinkmann et al. The making of bispecific antibodies. Mabs 9, 182-212 (2017)).

[0073] Multispecific antibody system molecules can also be generated by chemical bonding or coupling of individual full-length IgG, or by coupling of IgG fragments, as described in the literature, and can form multispecific antibody derivatives and multivalent antibody derivatives. Examples include chemically coupled Fab fragments, IgG-dimers, etc. Specific detection tags or purification tags, half-life extension molecules, or other components can be incorporated into the complex protein. Additional non-IgG polypeptides can also be incorporated into the fusion protein. Multispecific molecules can also be generated by combining recombinant and chemical methods, including those described above.

[0074] In one aspect, the antibody of the present invention is a chimeric antibody, a human antibody, or a humanized antibody. Such antibodies can be generated, for example, by using an appropriate antibody display or immunization platform, or other appropriate platforms or methods known in the art. As used herein, the term "human antibody" is intended to include antibodies having variable domains in which at least a portion of the framework region and / or at least a portion of the CDR region are derived from human germline immunoglobulin sequences. For example, a human antibody may have variable domains in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Further, when the antibody contains a constant region, the constant region or a portion thereof is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0075] Such human antibodies can be human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas containing B cells obtained from transgenic animals (e.g., transgenic mice having a genome containing a repertoire of human immunoglobulin heavy and light chain gene segments fused to immortalized cells).

[0076] Human antibodies can be isolated from sequence libraries constructed based on the selection of human germline sequences and further diversified with natural and synthetic sequence diversity.

[0077] Human antibodies can be prepared by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.

[0078] Human antibodies can be produced by recombinant methods known in the art.

[0079] The term "human antibody derivative" refers to any modified form of a human antibody, such as an antibody conjugated with another agent or a complex of an antibody.

[0080] As used herein, the term "humanized antibody" refers to a human / non-human antibody that contains sequences (CDR regions or portions thereof) derived from non-human immunoglobulins. Thus, a humanized antibody is a human immunoglobulin (recipient antibody), and residues from at least the hypervariable regions of the recipient are replaced with residues from the hypervariable regions of an antibody from a non-human species such as a mouse, rat, rabbit, or non-human primate (donor antibody) that have the desired specificity, affinity, sequence composition, and functionality. In some cases, framework (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues. Examples of such modifications are the introduction of one or more so-called back mutations, which are typically amino acid residues derived from the donor antibody. Humanization of an antibody can be carried out using recombinant techniques known to those skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. Lo). Appropriate human recipient frameworks for both the light chain variable domain and the heavy chain variable domain can be identified, for example, by sequence or structural homology. Alternatively, for example, based on knowledge of structure, biophysical properties, and biochemical properties, a fixed recipient framework may be used. The recipient framework can be derived from germline or from mature antibody sequences. The CDR regions from the donor antibody can be transferred by CDR grafting. CDR-grafted humanized antibodies can be further optimized, for example, in terms of affinity, functionality, and biophysical properties by the identification of important framework positions where reintroduction of amino acid residues (back mutations) from the donor antibody has a beneficial effect on the properties of the humanized antibody. In addition to back mutations from the donor antibody, humanized antibodies can be engineered by introduction of germline residues into the CDR or framework regions, removal of immunogenic epitopes, affinity maturation, etc.

[0081] Furthermore, a humanized antibody can contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody contains at least one, typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and in which all or substantially all of the FR residues are of a human immunoglobulin sequence. A humanized antibody can also optionally include at least a portion of the immunoglobulin constant region (Fc), typically that portion of a human immunoglobulin.

[0082] The term "humanized antibody derivative" refers to any modified form of a humanized antibody, such as a complex of an antibody with a chemical agent or a complex of an antibody with another antibody.

[0083] The term "chimeric antibody", as used herein, refers to an antibody that includes portions of antibodies derived from two or more species. For example, a gene encoding such an antibody includes genes encoding variable domains and genes encoding constant domains that originated from two different species. For example, a gene encoding the variable domain of a mouse monoclonal antibody can be combined with a gene encoding the constant domain of an antibody of human origin.

[0084] The crystallizable fragment region of an antibody ("Fc region" / "Fc domain") is the C-terminal region of the antibody, which is the hinge and constant C H 2 and C HIt contains 3 domains. The Fc domain can interact with cell surface receptors called Fc receptors, as well as several proteins of the complement system. The Fc region enables the antibody to interact with the immune system. In one aspect of the invention, the antibody can be engineered to contain modifications within the Fc region, typically to alter one or more of its functional properties, such as serum half-life, complement fixation, Fc receptor binding, protein stability, and / or antibody-dependent cell-mediated cytotoxic activity, or the lack thereof. Furthermore, the antibodies of the invention can also be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation to change one or more of the functional properties of the antibody. IgG1 antibodies can carry a modified Fc domain that contains one or more and perhaps all of the following mutations that result in a reduced affinity for certain Fc gamma receptors (L234A, L235E, and G237A) and a decrease in C1q-mediated complement fixation (A330S and P331S) (residue numbering according to EU indices). Alternatively, other amino acid substitutions, and combinations thereof, known in the art that result in changes (decreases or increases) in Fc gamma receptor binding, and combinations with the above can be used.

[0085] The isotype of the antibody of the present invention can be IgG, such as IgG1, such as IgG2, such as IgG4. If desired, the class of the antibody can be "switched" by known techniques. For example, an antibody originally produced as an IgM molecule can be class-switched to an IgG antibody. Class-switching techniques can also be used, for example, to convert one IgG subclass to another, such as from IgG1 to IgG2 or IgG4, from IgG2 to IgG1 or IgG4, or from IgG4 to IgG1 or IgG2. It is also possible to perform engineering of antibodies to generate constant region chimeric molecules by combining regions from different IgG subclasses.

[0086] In one embodiment, the hinge region of the antibody is modified such that the number of cysteine residues within the hinge region varies, e.g., increases or decreases. This approach is further described, for example, in U.S. Patent No. 5,677,425 by Bodmer et al.

[0087] The constant region can be modified to stabilize the antibody, e.g., to reduce the risk of a divalent antibody dissociating into half-antibodies. For example, in the IgG4 constant region, residue S228 (by EU numbering index and S241 according to Kabat) can be mutated to a proline (P) residue to stabilize the formation of the inter-heavy chain disulfide bridge in the hinge (see, e.g., Angal et al. Mol Immunol. 1993;30:105-8).

[0088] Antibodies or fragments thereof can be defined in terms of their complementarity determining regions (CDRs). The term "complementarity determining region" or "hypervariable region", as used herein, refers to the region of an antibody in which amino acid residues involved in antigen binding are located. The hypervariable regions or CDRs can be identified as the regions having the highest variability in an amino acid alignment of the antibody variable domains. Databases such as the Kabat database can be used for CDR identification, and this CDR is defined, for example, as including amino acid residues 24 - 34 (L1), 50 - 56 (L2) and 89 - 97 (L3) of the light chain variable domain, and amino acid residues 31 - 35 (H1), 50 - 65 (H2) and 95 - 102 (H3) of the heavy chain variable domain (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 - 3242). Alternatively, the CDR can be defined as residues from "hypervariable loops" (residues 26 - 33 (L1), 50 - 52 (L2), and 91 - 96 (L3) of the light chain variable domain, and residues 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) of the heavy chain variable domain, Chothia and Lesk, J. Mol. Biol. 1987; 196:901 - 917). Typically, the numbering of amino acid residues in this region is carried out by the method described by Kabat et al. above. In this specification, phrases such as "Kabat position", "Kabat residue", and "according to Kabat" refer to this numbering system for the heavy chain variable domain or the light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to shortening or insertion into the framework (FR) or CDRs of the variable domain.For example, a heavy chain variable domain may include amino acid insertions after residue 52 of CDR H2 (residues 52a, 52b, and 52c according to Kabat), and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues can be determined for a given antibody by alignment in the region of homology between the antibody's sequence and the "standard" Kabat numbering sequence.

[0089] The term "framework region" or "FR" residue refers to these V amino acid residues that are not within a CDR, as defined herein. H or V L amino acid residues.

[0090] The term "blood coagulation-promoting antibody" refers to an antibody that enhances blood coagulation, for example, by accelerating the process of blood coagulation and / or by increasing the enzymatic activity of one or more coagulation factors.

[0091] The term "blood coagulation-promoting activity" refers to the ability of a compound such as an antibody that enhances blood coagulation, for example, by accelerating the process of blood coagulation and / or by increasing the enzymatic activity of one or more coagulation factors.

[0092] The activity of blood coagulation-promoting antibodies, including bispecific, trispecific, and multispecific antibodies, can be determined by methods known in the art. Standard assays include the whole blood - thrombin generation test (TGT), and thromboelastography (TEG) and FXa generation assays (see, for example, WO2018 / 141863) to measure the clotting time.

[0093] The term "stimulating the enzymatic activity of FIXa" refers to stimulation as determined using the methodology of Example 9.

[0094] The term "antigen" (Ag) refers to a molecular entity used for the immunization of an immunocompetent vertebrate to generate an antibody (Ab) that recognizes the Ag. As used herein, Ag is more broadly referred to and is generally intended to include a target molecule that is specifically recognized by an Ab, and thus includes fragments or mimetics of molecules used in an immunization process or in other processes such as, for example, phage display, to generate an Ab.

[0095] The present invention encompasses variants of the antibodies of the present invention or antigen-binding fragments thereof that may include one, two, or three amino acid substitutions and / or deletions and / or insertions in the specific sequences disclosed herein.

[0096] In one aspect, a "substituted" variant involves the substitution of one or more amino acids having the same number of amino acids. The substitutions can be, but are not limited to, conservative substitutions. For example, an amino acid may be substituted with an amino acid having similar biochemical properties; for example, a basic amino acid may be substituted with another basic amino acid (e.g., lysine to arginine), an acidic amino acid may be substituted with another acidic amino acid (e.g., glutamate to aspartate), a neutral amino acid may be substituted with another neutral amino acid (e.g., threonine to serine), a charged amino acid may be substituted with another charged amino acid (e.g., glutamate to aspartate), a hydrophilic amino acid may be substituted with another hydrophilic amino acid (e.g., asparagine to glutamine), a hydrophobic amino acid may be substituted with another hydrophobic amino acid (e.g., alanine to valine), a polar amino acid may be substituted with another polar amino acid (e.g., serine to threonine), an aromatic amino acid may be substituted with another aromatic amino acid (e.g., phenylalanine to tryptophan), and an aliphatic amino acid may be substituted with another aliphatic amino acid (e.g., leucine to isoleucine).

[0097] In another aspect, the variant includes a structural analog of an amino acid present in the sequence of the antibody, or an antigen-binding fragment thereof of the present invention.

[0098] As used herein, the term "binding affinity" is used as a measure of the strength of non-covalent interactions between two molecules, e.g., between an antibody or a fragment thereof and an antigen. The term "binding affinity" is used to describe a monovalent interaction.

[0099] The binding affinity between two molecules, e.g., between an antibody or a fragment thereof and an antigen by a monovalent interaction, can be quantified by determining the equilibrium dissociation constant (K D ). K D can be determined by measuring the kinetics of complex formation and dissociation, e.g., by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). The rate constants corresponding to the association and dissociation of a monovalent complex are the association rate constant k a (or k on ), and the dissociation rate constant k d (or k off ), respectively. K D is related to k D and k d via the equation K a =k a / k d .

[0100] According to the above definitions, binding affinities associated with different molecular interactions, such as comparison of the binding affinities of different antibodies for a given antigen, can be compared by comparison of the K D values of the individual antibody / antigen complexes.

[0101] The value of the dissociation constant can be directly determined by well-known methods. Standard assays for evaluating the binding ability of ligands such as antibodies directed to a target are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of an antibody can also be evaluated by standard assays known in the art such as SPR. However, it is preferred that isothermal titration calorimetry (ITC) can be used not only to measure the affinity for the antibody / target interaction but also to derive the thermodynamic parameters for the interaction.

[0102] A competitive binding assay can be performed in which the binding of the antibody to the target is compared to the binding of the target by another ligand of the target such as another antibody.

[0103] The K of the antibody of the present invention for its target D can be less than 100 μM, such as less than 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM.

[0104] In such an embodiment, the antibody has a K D for FX of less than 100 μM, such as less than 9 μM, less than 8 μM, less than 7 μM, less than 6 μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, less than 0.09 μM, less than 0.08 μM, less than 0.07 μM, less than 0.06 μM, less than 0.05 μM, less than 0.04 μM, less than 0.03 μM, less than 0.02 μM, less than 0.01 μM, less than 9 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, etc., and contains an anti-FX arm having

[0105] As described herein, the antibodies and antibody fragments thereof may be combined with other antibodies and antibody fragments known in the art to create bispecific, trispecific, or multispecific antibody molecules. Compounds that mimic FVIII cofactor function have been previously made using antibodies that target FIX(a) and FX(a), and in some embodiments, this may potentially replace each of the FIX(a) or FX(a) antibodies described herein. Thus, it is clear that the antibodies targeting FIX(a) and FX(a) of the present invention, particularly antigen-binding fragments thereof, are of separate interest as individual constituent (intermediate) molecules as part of a bispecific, trispecific, or multispecific antibody containing at least one FIX(a) and / or FX(a) binding domain.

[0106] Pharmaceutical formulations In another aspect, the present invention provides compositions and formulations comprising a compound of the present invention, such as an antibody of the present invention described herein. For example, the present invention provides a pharmaceutical composition comprising one or more antibodies of the present invention formulated with a pharmaceutically acceptable carrier.

[0107] Accordingly, one object of the present invention is to provide a pharmaceutical preparation containing such an antibody present at a concentration of 0.25 mg / ml to 250 mg / ml, and the preparation has a pH of 2.0 to 10.0. The preparation may further contain one or more of a buffer system, a preservative, an isotonic agent, a chelating agent, a stabilizer, or a surfactant, and various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Remington: The Science and Practice of Pharmacy, 19 th edition, 1995 may be referred to.

[0108] In one embodiment, the pharmaceutical preparation is an aqueous preparation. Such preparations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous preparation" is defined as a preparation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0109] In another embodiment, the pharmaceutical preparation is a lyophilized preparation to which a solvent and / or diluent is added before use.

[0110] In a further aspect, the pharmaceutical preparation contains an aqueous solution of such an antibody and a buffer in which the antibody is present at a concentration of 1 mg / ml or more, and the preparation has a pH of about 2.0 to about 10.0.

[0111] In one embodiment, the present invention relates to an injection device having the contents of the composition. In some embodiments, the pharmaceutical composition of the present invention is intended for use with an injection device and / or for accommodation within an infusion device. In some embodiments, the injection device is a disposable, pre-filled, multiple-dose pen of the FlexTouch® type (supplier: Novo Nordisk A / S, Denmark). In some embodiments, the injection device is a single-shot device.

[0112] In some embodiments, the injection device is a fixed-dose device, such as one configured to deliver a plurality of predetermined doses of a medicament, and is sometimes referred to as a plurality of fixed-dose devices or a fixed-dose, multi-shot device.

[0113] In one embodiment, the pharmaceutical composition of the present invention is administered using an injection device comprising a tube having a needle of 20 gauge or more.

[0114] In one embodiment, the bispecific antibody according to Table 1 herein is administered using an injection device comprising a tube having a needle of 20 gauge or more.

[0115] In one embodiment, the bispecific antibody according to Table 1 herein is administered using an injection device comprising a tube having a needle of 20 to 36 gauge. In such an embodiment, the bispecific antibody is selected from the list consisting of bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A, bimAb7A, bimAb8A, bimAb1B, bimAb2B, bimAb3B, bimAb4B, bimAb5B, bimAb6B, bimAb7B, and bimAb8B.

[0116] Administration and Dosage The compounds of the present invention, such as antibodies, can be administered parenterally, such as intravenously, intramuscularly, subcutaneously, etc. Alternatively, the antibodies of the present invention can be administered via non-injection routes, such as orally or topically. The antibodies of the present invention can be administered prophylactically. The antibodies of the present invention can be administered therapeutically (upon request).

[0117] The dosage of the compound to be administered may be about 0.01 mg to 500 mg of the compound per day, preferably about 0.1 mg to 250 mg per day, more preferably about 0.5 mg to about 250 mg per day, and depending on the severity of the condition, may be once a day, once a week, once every two weeks, or once a month as a loading dose and a maintenance dose. Also, the suitable dosage can be adjusted based on the properties of the specific compound, including its in vivo half-life or mean residence time and its biological activity. For example, the compound to be administered may be once a week, or once every other week, or once a month in one embodiment, and in any of these embodiments, for example, at a dosage of 0.005, 0.0075, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg per kg of body weight.

[0118] The composition containing the compound disclosed herein can be administered for prophylactic treatment and / or in some embodiments for therapeutic treatment. In therapeutic use, the composition is administered in an amount sufficient to cure, alleviate, or partially prevent the disease and its complications in a subject suffering from a disease such as any bleeding disorder as described above. The amount sufficient to achieve this is defined as a "therapeutically effective amount". As will be understood by those skilled in the art, the amount effective for this purpose depends on the severity of the disease or injury, as well as the body weight and general condition of the subject.

[0119] Embodiment The present invention is further illustrated by the following embodiments. 1. An antibody or an antigen-binding fragment thereof that can bind to factor IX (FIX) according to SEQ ID NO: 89 and / or its active form (FIXa). 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antibody is a Fab. 3. The antibody or antigen-binding fragment thereof has the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 25 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 29, or has the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 33 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 37, or has the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 41 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 45, or has the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 49 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 53, or has the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 57 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 61, or has the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 65 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 69, or has the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 73 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 77, or The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, which comprises the CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 81 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 85. 4. The antibody or antigen-binding fragment thereof has the heavy chain variable domain specified by SEQ ID NO: 25 and the light chain variable domain specified by SEQ ID NO: 29, or has the heavy chain variable domain specified by SEQ ID NO: 33 and the light chain variable domain specified by SEQ ID NO: 37, or has the heavy chain variable domain specified by SEQ ID NO: 41 and the light chain variable domain specified by SEQ ID NO: 45, or The heavy chain variable domain specified by SEQ ID NO: 49, and the light chain variable domain specified by SEQ ID NO: 53, or The heavy chain variable domain specified by SEQ ID NO: 57, and the light chain variable domain specified by SEQ ID NO: 61, or The heavy chain variable domain specified by SEQ ID NO: 65, and the light chain variable domain specified by SEQ ID NO: 69, or The heavy chain variable domain specified by SEQ ID NO: 73, and the light chain variable domain specified by SEQ ID NO: 77, or The antibody or antigen-binding fragment thereof according to any of the preceding embodiments, comprising the heavy chain variable domain specified by SEQ ID NO: 81 and the light chain variable domain specified by SEQ ID NO: 85. 5. An antibody or antigen-binding fragment thereof that can bind to 5.FX (SEQ ID NO: 90) and / or its active form (FXa). 6. The antibody according to embodiment 5, wherein the antibody is Fab. 7. The antibody or antigen-binding fragment thereof The CDR sequences of the heavy chain variable domain specified by SEQ ID NO: 1 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 5, or The antibody or antigen-binding fragment thereof according to any of embodiments 5 or 6, comprising the heavy chain variable domain specified by SEQ ID NO: 17 and the CDR sequences of the light chain variable domain specified by SEQ ID NO: 21. 8. The antibody or antigen-binding fragment thereof The heavy chain variable domain specified by SEQ ID NO: 1 and the light chain variable domain specified by SEQ ID NO: 5, or The heavy chain variable domain specified by SEQ ID NO: 17 and the light chain variable domain specified by SEQ ID NO: 21, The multispecific antibody or antigen-binding fragment thereof that can bind to FIX according to SEQ ID NO: 89 or its active form (FIXa), and FX (SEQ ID NO: 90) or its active form (FXa). 10. The multispecific antibody or antigen-binding fragment thereof according to embodiment 9, wherein the antibody comprises the antibody or antigen-binding fragment thereof according to any one of the preceding embodiments 1 to 4. 11. The multispecific antibody or antigen-binding fragment thereof according to embodiment 9, wherein the antibody comprises the antibody or antigen-binding fragment thereof according to any one of the preceding embodiments 5 to 8. 12. The multispecific antibody or antigen-binding fragment thereof according to embodiment 9, wherein the antibody comprises the antibody or antigen-binding fragment thereof according to any one of the preceding embodiments 1 to 4 and the antigen-binding fragment according to any one of the preceding embodiments 5 to 8. 13. Optionally, the anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 28, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. Optionally, the anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 32, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. Optionally, the anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. Optionally, the anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, the anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 28, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. Optionally, the anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 32, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. Optionally, the anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. Optionally, the anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 36, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 40, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 36, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 40, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 44, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 48, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 44, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 48, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 52, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 56, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 52, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 56, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 60, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 64, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 60, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 64, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 68, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 72, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 68, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 72, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 76, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 80, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 76, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 80, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 84, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 88, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 20, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 24, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 84, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 88, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FX(a) antibody heavy chain CDR3 sequence specified by SEQ ID NO: 4, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9 to 12, comprising an anti-FX(a) antibody light chain CDR3 sequence specified by SEQ ID NO: 8, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. 14. The anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 26, 27, and 28, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. The anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 30, 31, and 32, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. The anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. The anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 26, 27, and 28, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. The anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 30, 31, and 32, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. The anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions. The anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, respectively, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, each of the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 34, 35, and 36, containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 38, 39, and 40, containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, or containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 34, 35, and 36, containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 38, 39, and 40, containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, or containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each of the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 42, 43, and 44, containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 46, 47, and 48, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 42, 43, and 44, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 46, 47, and 48, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 50, 51, and 52, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 54, 55, and 56, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) heavy chain CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) light chain CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, the anti-FIX(a) heavy chain CDR1-3 sequences specified by SEQ ID NOs: 50, 51, and 52, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FIX(a) light chain CDR1-3 sequences specified by SEQ ID NOs: 54, 55, and 56, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) heavy chain CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) light chain CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, the anti-FIX(a) heavy chain CDR1-3 sequences specified by SEQ ID NOs: 58, 59, and 60, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FIX(a) light chain CDR1-3 sequences specified by SEQ ID NOs: 62, 63, and 64, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) heavy chain CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, respectively, or Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 58, 59, and 60, respectively, Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 62, 63, and 64, respectively, Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, respectively, Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, respectively, or Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 66, 67, and 68, respectively, Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 70, 71, and 72, respectively, Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, respectively, Optionally, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, respectively, or Optionally, the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 66, 67, and 68, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 70, 71, and 72, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 74, 75, and 76, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FIX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 78, 79, and 80, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, Optionally, the anti-FX(a) antibody light chain CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or Optionally, the anti-FIX(a) antibody heavy chain CDR1-3 sequences specified by SEQ ID NOs: 74, 75, and 76, respectively, each containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, The anti-FIX(a) antibody light chain CDR1-3 sequences, each specified by SEQ ID NO: 78, 79, and 80, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions The anti-FX(a) antibody heavy chain CDR1-3 sequences, each specified by SEQ ID NO: 2, 3, and 4, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions The anti-FX(a) antibody light chain CDR1-3 sequences, each specified by SEQ ID NO: 6, 7, and 8, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR1-3 sequences, each specified by SEQ ID NO: 82, 83, and 84, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions The anti-FIX(a) antibody light chain CDR1-3 sequences, each specified by SEQ ID NO: 86, 87, and 88, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions The anti-FX(a) antibody heavy chain CDR1-3 sequences, each specified by SEQ ID NO: 18, 19, and 20, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions The anti-FX(a) antibody light chain CDR1-3 sequences, each specified by SEQ ID NO: 22, 23, and 24, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, or The anti-FIX(a) antibody heavy chain CDR1-3 sequences, each specified by SEQ ID NO: 82, 83, and 84, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions The anti-FIX(a) antibody light chain CDR1-3 sequences, each specified by SEQ ID NO: 86, 87, and 88, optionally containing 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions Optionally, each of the anti-FX(a) heavy chain CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, including substitution and / or deletion and / or insertion of 1, 2, or 3 amino acids. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9 to 13, optionally comprising the anti-FX(a) light chain CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, each including substitution and / or deletion and / or insertion of 1, 2, or 3 amino acids. 15. The antibody is a bispecific antibody capable of specifically binding to FIX(a) and FX(a), and the binding domains are The multispecific antibody or antigen-binding fragment thereof according to embodiment 14, which is of the mAb pair consisting of mAb1 / mAbA, mAb2 / mAbA, mAb3 / mAbA, mAb4 / mAbA, mAb5 / mAbA, mAb6 / mAbA, mAb7 / mAbA, mAb8 / mAbA, mAb1 / mAbB, mAb2 / mAbB, mAb3 / mAbB, mAb4 / mAbB, mAb5 / mAbB, mAb6 / mAbB, mAb7 / mAbB, or mAb8 / mAbB. 16. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9 to 15, wherein the antibody or antigen-binding fragment thereof is a blood coagulation promoting antibody. 17. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9 to 16, wherein the antibody or antigen-binding fragment thereof can increase the enzymatic activity of FIXa against FX. 18. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9 to 17, wherein the antibody or antigen-binding fragment thereof can functionally substitute for FVIII and / or FVIIIa. 19. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9 to 18, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody. 20. The antibody isotype is IgG1, IgG2, IgG3, or IgG4, or a combination thereof, and such an antibody has an IgG1 F C region and an IgG4 F Cincluding the region and optionally C H An antibody according to any of the preceding embodiments, comprising one or two substitutions in the three domains. 21. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any of the preceding embodiments, and optionally one or more pharmaceutically acceptable carriers. 22. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to embodiment 21, for use in the treatment of a blood coagulation disorder such as hemophilia A with or without an inhibitor, or a blood coagulation disorder. 23. An antibody or an antigen-binding fragment thereof or a composition according to any of the preceding embodiments, for use in a method of treating a blood coagulation disorder or a blood coagulation disorder, such as on-demand or prophylactic treatment. 24. An antibody or an antigen-binding fragment thereof, or a composition according to any of the preceding embodiments, for use in the treatment of hemophilia A with or without an inhibitor, such as on-demand or prophylactic treatment. 25. A method of treating a subject suffering from a blood coagulation disorder or a blood coagulation disorder, the method comprising administering to the subject an antibody or an antigen-binding fragment thereof, or a composition according to any of the preceding embodiments. 26. The method according to embodiment 25, wherein the blood coagulation disorder or the blood coagulation disorder is hemophilia A or hemophilia A with an inhibitor. 27. Use of an antibody or an antigen-binding fragment thereof, or a composition according to any of embodiments 1 to 20, for the manufacture of a medicament for use in the treatment of a subject in need of a medicament, such as on-demand or prophylactic treatment. 28. Use of an antibody or an antigen-binding fragment thereof, or a composition according to any of embodiments 1 to 21, for the manufacture of a medicament for use in the treatment of hemophilia A with or without an inhibitor, such as on-demand or prophylactic treatment. 29. A eukaryotic cell expressing an antibody or an antigen-binding fragment thereof according to any of embodiments 1 to 20. 30. A kit comprising an antibody or an antigen-binding fragment thereof, or a composition according to any of embodiments 1 to 21, and instructions for use. 31. The antibody or its antigen-binding fragment according to any one of Embodiments 1 to 8, which is a component (intermediate) for use in a multispecific antibody such as a blood coagulation-promoting bispecific antibody. 32. The antibody or its antigen-binding fragment according to any one of Embodiments 1 to 8, which is a component (intermediate) for use in the production of a multispecific antibody such as a blood coagulation-promoting bispecific antibody. 33. The multispecific antibody or its antigen-binding fragment according to any one of Embodiments 9 to 20, which is a blood coagulation-promoting bispecific antibody or the like, and the blood coagulation-promoting activity of the antibody is improved compared to the multispecific antibodies disclosed in WO2018 / 141863 and WO2019 / 065795. 34. The multispecific antibody or its antigen-binding fragment according to Embodiment 33, wherein the improvement is determined using an assay disclosed herein, such as the HA-PPP TGT assay (described in Example 7 of this specification). 35. The antibody or its antigen-binding fragment, at a compound concentration of 700 nM in the TGT assay (in HA-PPP) according to Example 7 of this specification, a) when using tissue factor as a trigger, at least 80, 81, 82, 83, 84, 95, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110, or b) when using FXIa as a trigger, can provide an average peak thrombin (nM) of at least 350, 355, 360, 365, 370, 375, 380, 385, or 390. The multispecific antibody or its antigen-binding fragment according to any one of Embodiments 9 to 20. 36. The antibody or antigen-binding fragment thereof has equivalent FVIII activity as determined according to Example 8 herein, and is improved over emicizumab and the bispecific antibodies disclosed in WO2018 / 141863 and WO2019 / 065795 (both of which are incorporated herein by reference), the multispecific antibody or antigen-binding fragment thereof according to any of embodiments 9-20. 37. The antibody isotype is IgG4 and optionally contains one or two substitutions in one C H domain. The antibody according to any of embodiments 9-20. 38. An injection device comprising the antibody or antigen-binding fragment thereof, or composition, according to any of embodiments 9-20. 39. The injection device according to embodiment 38, wherein the device is a disposable and / or pre-filled and / or multi-dose device, such as a pen, etc. 40. The injection device according to embodiment 39, wherein the device is a pre-filled pen. 41. The injection device according to embodiments 39-40, wherein the device is a multi-dose pen. 42. The injection device according to embodiments 38-41, wherein the injection device comprises a tube having a 20-36 gauge needle. 43. The substitution is a conservative substitution. The multispecific antibody or antigen-binding fragment thereof according to embodiment 13 or 14.

[0120] In one embodiment, a multispecific antibody such as a bispecific antibody of the invention does not interfere with the effect of FVIII administered to a patient with hemophilia A, such as recombinant FVIII, when the antibody is used at a clinically relevant dose in the treatment of hemophilia A.

[0121] In one embodiment, in a patient suffering from hemophilia A, when present in plasma at 43.64 μg / mL, the antibody of the present invention or an antigen-binding fragment thereof corresponds to at least 20 to 50, for example, 20 to 40, for example, 25 to 35, for example, at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 IU of equivalent factor VIII activity per deciliter of plasma.

[0122] In one embodiment, in a patient suffering from hemophilia A, when present in plasma at 30 μg / mL, the antibody of the present invention or an antigen-binding fragment thereof corresponds to at least 10 to 50, for example, 15 to 40, for example, 15 to 30, for example, 15 to 20, for example, at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 IU of equivalent factor VIII activity per deciliter of plasma.

[0123] In one embodiment, in a patient suffering from hemophilia A, when present in plasma at 15 μg / mL, the antibody of the present invention or an antigen-binding fragment thereof corresponds to at least 10 to 50, for example, 15 to 40, for example, 15 to 30, for example, 15 to 20, for example, at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 IU of equivalent factor VIII activity per deciliter of plasma.

[0124] In one embodiment, the antibody of the present invention or an antigen-binding fragment thereof has reduced immunogenicity compared to blood coagulation-promoting antibodies in the art.

[0125] In one embodiment, the antibody of the present invention or an antigen-binding fragment thereof is used for the prophylactic treatment of hemophilia A with or without an inhibitor.

[0126] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention can stimulate the enzymatic activity of FIXa against FX.

[0127] In one embodiment, the anti-FIX(a) antibody or antigen-binding fragment thereof listed in Table 2 of Example 6 can stimulate the enzymatic activity of FIXa against FX.

[0128] In a preferred embodiment of the present invention, the antibody has an IgG4 / kappa form and optionally contains one or more substitutions in the Fc constant region.

[0129] The heavy chain constant domain region (C H 1-C H 2-C H 3) has an S228P (EU numbering) substitution and a cleavage of the C-terminal lysine, against anti-FIX(a) arm human IgG4:

Chemical formula

[0130] In one embodiment, the heavy chain constant domain region (C H 1-C H 2-C H 3) has an S228P substitution and two additional substitutions (F405L and R409K (EU numbering)), and promotes heterodimerization of the heavy chain within the C H 3 domain (described in Example 4) and has a cleavage of the C-terminal lysine, against anti-FX(a) arm human IgG4:

Chemical formula

[0131] In another embodiment, the antibody has a heavy chain constant domain region (C H 1-C H 2-C H 3) for the anti-FIX(a) arm carrying the S228P, F405L, and R409K substitutions, and has a heavy chain constant domain region for the anti-FX(a) arm carrying the S228P substitution, and can also be expressed in IgG4 format with or without a C-terminal lysine deletion.

[0132] In one embodiment, the antibody can also be expressed in IgG1 / kappa format. In that case, the heavy chain constant domain region of the anti-FIX(a) arm was human IgG1 F405L with cleavage of the C-terminal lysine:

Chemical formula

Chemical formula

[0133] The antibody has a heavy chain constant domain region (C H 1-C H 2-C H 3) for the anti-FIX(a) arm carrying the K409R substitution, and has a heavy chain constant domain region for the anti-FX(a) arm carrying the F405L substitution, and can also be expressed in IgG1 format with or without a C-terminal lysine deletion.

[0134] The constant domain region may further include additional substitutions or other modifications to modulate effector function, half-life or other properties.

[0135] In one embodiment, the potency of a bispecific antibody capable of binding to FIX(a) and FX(a), such as those disclosed herein but not limited thereto, can be determined in a chromogenic potency assay comprising: a) human factor X lyophilized in the presence of a fibrin polymerization inhibitor; b) human factor IXa; c) human thrombin; d) calcium; e) synthetic phospholipids; f) factor Xa (SXa-11) (Hyphen Biomed); and a chromogenic substrate specific for the anti-FIX / anti-FX bispecific antibody being evaluated. A suitable buffer such as Tris-BSA can be used for any dilution. In such assays, the level of FX activation depends on the potency of the bispecific antibody. Active FX (FXa) hydrolyzes the chromogenic substrate, thereby releasing pNA and enabling optical spectrophotometric readings at 405 nm (e.g., using a Tecan Sunrise ELISA reader). The readings are dependent on the FXa concentration and thus are proportional to the potency of the bispecific antibody being tested. The assay should include a bispecific reference antibody with a predetermined potency.

[0136] The present disclosure also provides kits comprising an antibody or an antigen-binding fragment thereof disclosed herein suitable for treatment as described herein. In some embodiments, the kit comprises: (i) an antibody disclosed herein, such as a bispecific antibody or an antigen-binding fragment thereof, or a pharmaceutical composition, or an antibody encoded by a nucleic acid or vector, or a combination thereof; and (ii) instructions for use. One of ordinary skill in the art will readily recognize that the antibodies, bispecific molecules (e.g., bispecific antibodies), pharmaceutical compositions, nucleic acids or vectors encoding them, or combinations thereof disclosed herein can be readily incorporated into one of the established kit formats well known in the art.

[0137] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Examples

[0138]

Table A

[0139] [Example 1]: Development of anti-FIX(a) antibody and anti-FX(a) antibody The FIX(a) and FX(a) binding antibodies disclosed herein were identified using various antibody development methods. To generate a diverse set of antibodies, immunizations of mice and rabbits were performed, and phage display and the Adimab yeast antibody expression platform were also utilized.

[0140] Adimab yeast antibody platform The Adimab platform is a yeast antibody expression system that includes a fully human naïve-type IgG1 / kappa library with a diversity of 10 10 . The antibody selection process was targeted at using MACS and FACS-based methods that can monitor the applied selection criteria in real time. Since the selection was by MACS and FACS systems, a labeled antigen (e.g., biotin) was required. Selection campaigns were carried out using biotinylated active site-inhibited hFIXa (FIXa-EGR-biotin) or antibody-mediated immobilization of hFIXa. Binding hits were evaluated using the biolayer interferometry (Octet fortebio systems).

[0141] Phage display The antibody phage display platform utilized was a proprietary fully human Fab display library. The library has a diversity of 10 10It has a size and is constructed by a combinatorial approach that utilizes complemented heavy chain CDR1 and CDR2 using PCR amplification of heavy chain CDR3 from human peripheral blood mononuclear cells in addition to chemical synthesis of the light chain. To maximize epitope diversity, different panning strategies were implemented, including panning using antigen capture with biotinylated FIXa-EGR, FX, active site-inhibited FXa, or anti-FIXa antibody. Initial hits were identified by phage ELISA. After sequence analysis, unique hits were cloned, recombinantly expressed as IgG1 antibodies, and ranked using SPR (Biacore) or biolayer interferometry (Octet fortebio systems).

[0142] In vivo platform Mice and rabbits were used for antibody production using the in vivo platform. For the production of anti-FIX / FIXa antibodies, mice or rabbits were immunized with human FIXa, FIXa-EGR, or FIX using standard protocols. Mouse-derived spleen cells were fused with myeloma cells using standard techniques, and the resulting hybridoma supernatants containing antibodies were screened for binding to FIXa using ELISA. FIXa-binding rabbit B cells were sorted single cells using FACS by gating on cells that bind randomly biotinylated FIXa-EGR (detected by streptavidin-conjugated fluorophore). Sorted rabbit B cells were cultured for 7 days in 384w plates using feeder cells and conditioned media from spleen cells prior to screening for binding to FIXa by ELISA. Rabbit B cells and mouse hybridoma clones expressing FIXa-binding antibody hits were either used for recombinant expression (for rabbit or hybridoma mAbs) after VH / VL sequencing or further propagated (for mouse hybridomas) for mAb production.

[0143] For the generation of anti-FX antibodies, mice and rabbits were immunized with FX using a standard protocol. Rabbit B cells were isolated using FACS-based single cell sorting and randomly biotinylated FX (detected by streptavidin-conjugated fluorophore), while spleen cells from immunized mice were used for standard hybridoma generation. The resulting antibody-producing B cells or mouse hybridoma clones were screened for FX binding using ELISA and Octet fortebio systems. Rabbit B cells or mouse hybridoma clones expressing antibody hits were either used for recombinant expression (for rabbit or hybridoma mAbs) after VH / VL sequencing or further propagated (for mouse hybridomas) for mAb production.

[0144] Sequencing of Hybridoma-Derived Antibodies Anti-FIXa and anti-FX antibodies generating hybridomas were sequenced and expressed in HEK293 cells using standard techniques. The expressed antibodies were evaluated for antigen binding using Octet fortebio systems.

[0145] Total RNA was extracted from antibody-producing clones, and the variable domains (V H and V L ) encoding the DNA sequences were amplified using RT-PCR. The V H and V L sequences were determined and inserted into the pTT-based mammalian expression vector (Durocher et al (2002) Nucleic Acid Res. 30:E9) or the pcDNA3.4 mammalian expression vector (Invitrogen) containing the antibody constant region encoding the DNA sequence. For the pTT / pcDNA3.4 mAb expression vectors, the V H and V L DNA sequences were respectively the human IgG1 or IgG4 S228P (C H 1C H 2C H3. Optionally, additional amino acid substitutions and deletions, such as C-terminal lysine C H 3 domain substitutions and deletions) or human C encoding the DNA sequence L kappa constant region was inserted in-frame using. For the corresponding pTT / pcDNA3.4Fab expression vector, V H DNA sequence was inserted in-frame using human IgG4 C encoding the DNA sequence H 1.

[0146] [Example 2]: Recombinant Expression of Antibodies and Antibody Fab Fragments Antibodies and antibody Fab fragments were expressed using transient transfection of HEK293 suspension cells (293Expi, Invitrogen) essentially according to the manufacturer's instructions. 293Expi cells were typically passaged every 3 - 4 days in Expi293F expression medium (Invitrogen, catalog number A1435104) supplemented with 1% P / S (GIBCO catalog number 15140 - 122). Expi293F cells were transfected at a cell density of 2.5 - 3 mill / mL using Expifectamine. For each liter of Expi293F cells, a total of 1 mg of plasmid DNA (V H -C H 1 (for Fab) or V H -C H 1-C H 2-C H 3 (for mAb) and the LC plasmid in a 1:1 ratio) was introduced by diluting into 50 mL of Optimem (GIBCO, catalog number 51985 - 026, dilution A) and introducing by diluting 2.7 mL of Expifectamine into 50 mL of Optimem (dilution B). For Fab and mAb producing co-transfection, V H -C H 1 and the LC plasmid (Fab) and V H -C H 1-C H 2-C H3 and LC plasmid (mAb) were each used at a ratio of 1:1. Dilutions A and B were mixed and incubated at room temperature for 10 - 20 minutes. Thereafter, the transfection mixture was added to Expi293F cells, and the cells were incubated at 37 °C in a humidified incubator equipped with orbital rotation (85 - 125 rpm). One day after transfection, the transfected cells were supplemented with 5 ml of ExpiFectamine 293 Transfection Enhancer 1 and 50 ml of ExpiFectamine 293 Transfection Enhancer 2. The cell culture supernatant was typically harvested 4 - 5 days after transfection by centrifugation and then filtered.

[0147] [Example 3]: Purification and Characterization of Fab and Antibody All purification steps were carried out at 4 °C. For the laboratory scale, Milli-Q water was used for buffer preparation. The HPLC system used for SE-HPLC analysis was an Aglient 1100. Aggregation and LC / MS were evaluated for QC.

[0148] Capture of Fab was carried out using HiTrap Protein G HP affinity chromatography with a binding buffer that was 1×PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. One-step elution was carried out using 0.1 M glycine, pH 2.8. The final product was desalted into formulation buffer (25 mM HEPES, 150 mM NaCl) pH 7.4 via a 52 mL GE Hiprep 16 desalting column and concentrated using a centrifugal ultrafiltration device (30KD C.O.) for storage at approximately -80 °C.

[0149] To evaluate the quality of the purified Fab, SDS-PAGE and high performance size exclusion chromatography (SE-HPLC) analyses were performed. Batches that did not meet the quality criteria (e.g., <95% monomer by SE-HPLC) were further purified by size exclusion chromatography. LC / MS was performed to verify the identity of the Fab protein. The molecular weight (MW) of all Fabs was shown to match the theoretical MW of the heavy and light chains, respectively.

[0150] Purification and Characterization of Antibodies Antibody purification was carried out by affinity chromatography using Protein A MabSelect SuRe resin (GE Healthcare, catalog number 17-5438-01). For small-scale antibody production, Protein A-based purification was performed in 96-well plates, while for larger production, an AktaExplorer chromatography system (GE Healthcare, catalog number 18-1112-41) was used. The buffer systems used for the affinity purification step were 1) an equilibration buffer composed of 20 mM sodium phosphate pH 7.2, 150 mM NaCl, 2) an elution buffer composed of 10 mM formic acid pH 3.5, and 3) a pH adjustment buffer composed of 0.4 M sodium phosphate pH 9.0. The cell supernatant was applied directly to the pre-equilibrated MabSelect SuRe column without any adjustment. The column was washed with approximately 10 column volumes of the equilibration buffer, and the antibody was eluted at a uniform concentration with approximately 2 - 5 column volumes of the elution buffer. The pH of the pooled fractions was adjusted to neutral immediately after elution using the described pH adjustment buffer.

[0151] The purified antibodies were characterized using different methods such as SDS-PAGE / Coomassie, size exclusion high performance liquid-chromatography (SE-HPLC) and liquid-chromatography mass spectrometry (LC-MS) analysis. SDS-PAGE / Coomassie analysis was performed using NuPage 4-12% Bis-Tris gels (Invitrogen, catalog number NP0321BOX). Here, all antibodies showed the expected light and heavy chain components. Intact molecular mass determination was performed using a setup of liquid chromatography electrospray ionization time-of-flight mass spectrometry on an Agilent 6210 instrument and a desalting column MassPREP (Waters, catalog number USRM10008656). The buffer systems used were an equilibration buffer composed of 0.1% formic acid in LC-MS grade-H2O and an elution buffer composed of 0.1% formic acid in LC-MS grade-ACN. Analysis was performed with and without N-glycosidase F (Roche Diagnostics, catalog number 11365177001) and a reducing agent (i.e., mercaptoethanol or DTT). All antibodies showed the expected intact molecular mass based on the sequence and one heavy chain N-glycan. Purity was determined based on SE-HPLC. The final protein purity was analyzed based on a setup of an SE-HPLC method on an Agilent LC 1100 / 1200 system using a BIOSep-SEC-S3000 300×7.8 mm column (Phenomenex, catalog number 00H-2146-K0) and a running buffer composed of 200 mM sodium phosphate pH 6.9, 300 mM Nacl, and 10% isopropanol. UV280 and fluorescence (Ex 280 nm / Em 354 nm) detectors were used for detection. The antibodies eluted as a single symmetric peak using the retention time reflecting the size of the antibody. All purity estimates were between 95-99% for the different antibodies. A NanoDrop spectrophotometer (Thermo Scientific) was used with the specific extinction coefficient for each of the antibodies to measure the final protein concentration.

[0152] [Example 4]: Bispecific antibody prepared by in vitro assembly The bispecific antibody was generated using a slightly modified variant of the bispecific human IgG4 antibody detailed below by in vitro assembly of a primary and a secondary antibody by the Duobody® method (Genmab), which was described for bispecific human IgG1 antibodies (Labrijn et al. PNAS 2013, vol. 110, pp. 5145 - 5150).

[0153] In the case of IgG1, the heavy chain constant region of the primary antibody is human IgG1 K409R (anti - FIX / FIXa) and that of the secondary antibody is human IgG1 F405L (anti - FX / FXa). IgG1 can be an IgG1 variant with reduced effector function as described above.

[0154] In the case of human IgG4, the heavy chain constant region of the primary antibody is IgG4 S228P (anti - FIX / FIXa) and that of the secondary antibody is IgG4 S228P F405L + R409K (anti - FX). The two parental antibodies were produced as described in Examples 1 - 3. The Fab - arm exchange reaction was carried out in HEPES buffer (pH 7.4) under reducing conditions using 75 mM 2 - mercaptoethylamine (2 - MEA) with an incubation at 30 °C for 4 hours.

[0155] [Example 5]: Preparation of monovalent (one - armed) antibodies To avoid potential avidity effects associated with conventional monospecific and bispecific antibodies, for example, in the FXa generation assay (Example 9), a monovalent one-armed (OA) antibody format was used as described in Martens et al.: A Novel One-Armed Anti-c-Met Antibody Inhibits Glioblastoma Growth In vivo. Clin. Cancer Res. 12, 6144-6152 (2006), where the full heavy chain, truncated heavy chain (lacking the Fab region), and light chain were co-expressed. Instead of the co-expression of the three chains described by Martens et al., the monovalent antibodies of the present invention were prepared using the Duobody® principle as described for bispecific antibodies (Example 4). Thus, the monovalent antibodies were prepared by mixing a complete monospecific antibody and a bispecific antibody with a truncated heavy chain dimer (formally derived from removing the Fab region from a complete antibody), and the chains could be exchanged by proceeding under the same experimental conditions as described in Example 4. For the formation of monovalent antibodies, as described in Example 4, the antibody and the truncated heavy chain dimer are required to carry appropriate complementary mutations to promote heterodimerization, namely F405L / K409R for human IgG1 and F405L+R409K / WT for human IgG4.

[0156] For monovalent antibodies of the IgG1 subtype, the heavy chain cleavage can be made from the N-terminus to a position between Cys220 and the upper hinge Cys226 (EU numbering). A specific example of a truncated human IgG1 heavy chain is one in which residues 1-220 are cleaved.

[0157] For monovalent antibodies of the human IgG4 subtype, the heavy chain cleavage can be made from the N-terminus to a position between Cys200 and the upper hinge Cys226 (EU numbering). A specific example of a truncated human IgG4 heavy chain is one in which residues 1-214 are cleaved.

[0158] [Example 6]: Summary of bispecific antibody (component) IDs and SEQ IDs

[0159]

Table 1

[0160]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0161]

Table 3

[0162] [Example 7]: Activity of bispecific anti-FIX(a) / FX(a) antibodies in the thrombin generation test (TGT) of human hemophilia A-like platelet-poor plasma The blood coagulation-promoting activities of the bispecific antibodies bimAb6B, bimAb5B, bimAb4B, bimAb3B, bimAb2B, bimAb1B, bimAb8B, bimAb7B, bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A, bimAb7A, and bimAb8A were determined based on their ability to promote thrombin generation in the presence of a blood coagulation-promoting synthetic phospholipid membrane according to the principle described by Hemker et al. (Pathophysiol Haemost Thromb, 2002;32:249-253). For comparison, an emicizumab sequence identity analog (SIA) was included. Each bispecific antibody (test compound) was tested in a thrombin generation test (TGT) using normal human platelet-poor plasma (NHP) supplemented with a neutralizing anti-FVIII polyclonal antibody (hereinafter referred to as HA-PPP).

[0163] Materials and methods: Thrombin generation test Thrombin generation tests (TGTs) in NHPs (from healthy volunteers) supplemented with ovine anti-human FVIII polyclonal antibody (pAb, Haematologic Technologies Inc., VT, USA) were performed by automated thrombiography calibrated standardly using a 96-well plate fluorometer (Fluoroscan Ascent FL, Thermolabsystems, Helsinki, Finland). The reaction mixture contained 0.1 μg / ml anti-FVIII pAb, 4 μl of test compound diluent (diluted with 20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA), either 10 μl of 1 pM tissue factor (TF, pppLow, from Thrombinoscope BV, Maastricht, The Netherlands), or 8.3 U / ml human factor XIa (Enzyme Research Laboratories, IN, USA), and 36 μl of NHP pre-incubated with 10 μl of FluCa substrate (Thrombinoscope BV, Maastricht, The Netherlands). TGTs were calibrated using a thrombin calibrator (Thrombinoscope BV, Maastricht, The Netherlands), and 10 μl of the thrombin calibrator was mixed with 36 μl of NHP pre-incubated with 0.1 μg / ml anti-FVIII pAb and 4 μl buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA). TGTs were performed with eight concentrations of the test compound (0.32, 0.96, 2.88, 8.64, 25.9, 77, 233, and 700 nM, final plasma concentration) or with additional buffer only (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) (representing the HA control). The concentration range was tested in at least three independent experiments with HA-PPP from the same stock. Normal control levels during TGTs were measured using NHP-supplemented buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) only.The TGT was run for a total of 60 minutes, and the peak thrombin height (nM) of the TGT parameters was analyzed using Thrombinoscope software (Thrombinoscope BV).

[0164] Results and Discussion Tables 4 and 5 show the measured peak thrombin generation rates of each bispecific antibody at the concentrations tested with HA-PPP triggers using tissue factor and human FXIa, respectively. The data show that all test compounds increase the peak of thrombin formation beyond the levels observed in the absence of the antibody, i.e., exhibit blood coagulation promoting activity. In addition, bimAb6B, bimAb5B, bimAb4B, bimAb3B, bimAb2B, bimAb1B, bimAb8B, bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A, and bimAb8A all show a higher concentration-dependent thrombin generation rate than that observed with emicizumab SIA when the bimAb concentration exceeds 8.64 nM and a 1 pM tissue factor trigger is used, indicating excellent efficacy. bimAb7B was superior to emicizumab SIA between 2.88 and 233 nM of bimAb.

[0165] Furthermore, bimAb6B, bimAb5B, bimAb4B, bimAb3B, bimAb2B, bimAb1B, bimAb8B, bimAb7B, bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A, bimAb7A, and bimAb8A all show a higher thrombin generating ability than that observed with emicizumab SIA at all concentrations tested when coagulation was induced with 8.3 mU / lL of human FXIa.

[0166]

Table 4-1

Table 4-2

[0167]

Table 5

[0168] [Example 8]: Equivalent FVIII Activity of Bispecific Antibody To estimate the in vitro equivalent FVIII activity of bimAb, a plasma-based thrombin generation assay was established using peak thrombin levels to enable a wide dose response of recombinant B-domain-deleted FVIII from 1 to 100 IU / dL. The dose-response curve for recombinant B-domain-deleted FVIII was used as a standard curve to analyze thrombin generation from bimAb to estimate equivalent FVIII activity.

[0169] Method: Thrombin generation was measured as described in Example 7, but 1 U / ml of human factor XIa (Enzyme Research Laboratories, IN, USA) was used as a trigger. In addition, an 8-point dilution series of recombinant B-domain-deleted FVIII (NovoEight, Novo Nordisk A / S) from 100 IU / dL to two-fold lower was included as the FVIII standard curve.

[0170] Determination of FVIII Equivalent Activity of Bispecific Antibody: Using the non-linear analysis function of GraphPad Prism version 8.0.2, the dose-response data of recombinant B-domain-deleted FVIII was fitted to "[Agonist] vs Response (3 parameters)" (Equation 1): Equation 1. Peak thrombin = Bottom + [FVIII] * (Top - Bottom) / (EC50 + [FVIII]) Where [FVIII] is the FVIII concentration in IU / mL, EC50 is the concentration of FVIII that gives 50% activity, and Bottom and Top are the plateaus of the fit.

[0171] By solving for [FVIII], the modified equation (Equation 2) can be used to estimate the concentration of FVIII that generates the same thrombin peak as a specific bispecific antibody:

Number

[0172] Result: Nonlinear fitting of the FVIII dose-response curve yields the following constants: EC50 = 20.3 ± 2.73, top = 476 ± 18.7, and bottom = -22.6 ± 10.1. Use Equation 2 to estimate the equivalent FVIII activity for the bispecific antibody described in Example 6; see Table 6 below.

[0173]

Table 6

[0174] [Example 9]: Activity of Monovalent Anti-FIX(a) Antibodies in the FXa Generation Assay To avoid any potential avidity effects resulting from the divalency of the conventional IgG antibody format, the stimulatory activity of anti-FIX(a) antibodies against the FIXa enzymatic activity toward FX was determined after reformatting to the monovalent one-armed (OA) antibody format (see Example 5). The antibodies tested are listed in Table 7 below. The monovalent OA version of the anti-FIXa antibody ACE910 was included for comparison.

[0175] The stimulatory activity of the OA antibodies was measured in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w / v) PEG8000, pH 7.3 + 1 mg / ml BSA) at fixed concentrations of phosphatidylserine (PS):phosphatidylcholine (PC) lipid vesicles (final concentration 500 μM, Haematologic Technologies Inc, USA) and plasma-derived FIXa (final concentration 0.025 or 0.1 nM; Haematologic Technologies Inc, USA). The FIXa concentration was chosen to ensure that less than 15% of the substrate FX was converted to FXa. After pre-incubation in the presence of the monovalent OA antibodies (final concentrations listed in Table 7), 100 nM of plasma-derived FX (Haematologic Technologies Inc, USA) was added to obtain a final reaction volume of 50 μl and activation was allowed to proceed for 20 min at room temperature. The reaction was then quenched by adding 25 μl of quench buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% PEG8000, pH 7.3 + 1 mg / ml BSA), and 25 μl of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) was further added and the amount of FXa generated was determined by measuring chromogenic substrate conversion by absorbance measurements at 405 nm (ΔOD / min) in a microplate reader. The measured activity was corrected for background activity by subtracting the signal measured within the same assay, but FIXa and the antibody were replaced with assay buffer and then normalized according to the FIXa concentration ([FIXa] total ) present in the assay. This number was divided by the similarly normalized rate of FXa generation in the absence of the antibody (A FIXa,norm) An antibody stimulation index was calculated that provides the fold stimulation of FIXa activity by the antibody concentration used. Since the rate of FXa production by free FIXa is slow, the activation reaction was performed in the absence of antibody as described above, but in the presence of 5, 10, or 20 nM FIXa. The measured activity was then subtracted by the background and normalized according to the FIXa concentration in the assay. For the calculation of the stimulation index, the average of the three normalized activities of free FIXa was used.

[0176] Determination of stimulation index In summary, the calculation of the stimulation index can be described as follows Equation 3 Stimulation index = ((A FIXa+OA - A bckg ) / [FIXa] total ) / A FIXa、norm Wherein, A FIXa+OA is the activity measured in the presence of the OA antibody, A bckg is the background activity measured in the absence of FIXa and the OA antibody, [FIXa] total is the FIXa concentration in the assay, and A FIXa、norm is the average normalized activity of free FIXa.

[0177] Determination of FIXa saturation The fraction of FIXa saturated with the OA antibody in the assay was determined by the FIXa and OA antibody concentrations and the equilibrium dissociation constant (K d ) that governs their interaction. The latter can be measured by techniques known in the art such as isothermal titration calorimetry (ITC).

[0178] Since the stimulation index will increase as the concentration of the OA antibody increases until FIXa saturation is reached, the concentration of the OA antibody in the assay should be selected to ensure at least 80% saturation of the FIXa in the assay in order to provide an appropriate determination of the stimulation index at complete FIXa saturation.

[0179] Fragment of FIXa bound to the OA antibody at equilibrium (f FIXa+OA) is the total concentration of FIXa in the assay ([FIXa] total ) and the total concentration of the OA antibody ([OA] total ), and can be calculated from their equilibrium dissociation constant (K d ) for their interaction using the quadratic binding equation, which is described in Krishnaswamy et al. (1992) J. Biol. Chem., 267:23696 - 23706 and detailed in Equations 4 and 5 below, [FIXa + OA] assay represents the calculated concentration of the FIXa - OA antibody complex at equilibrium in the assay, f FIXa+OA represents the calculated percentage of FIXa bound to the OA antibody at equilibrium in the assay.

Number

Number

[0180] The stimulation index for each OA antibody was provided in Table 7. For the OA emicizumab antibody, FIXa stimulation was determined at eight different antibody concentrations, which allowed the estimation of the stimulation index at full FIXa saturation using the quadratic binding equation outlined above. This also provided the estimated equilibrium dissociation constant (K d ) for the interaction between 1.1 μM emicizumab and FIXa, which is in good agreement with the value of 1.52 μM reported by Kitazawa et al. (2017) Thromb Haemost, 117:1348 - 1357. For the remaining antibodies, the FIXa saturation was unknown, and thus the listed stimulation indices represent a conservative estimate of the stimulation that would be obtained at FIXa saturations of 80% or greater. For the antibodies tested, the measured stimulation indices were found to be higher than those measured for the OA emicizumab antibody.

[0181]

Table 7

[0182] [Example 10]: SEC-HPLC Analysis of Non-Specific Binding Low non-specific binding is an important feature of therapeutic antibodies. A high tendency for non-specific binding can lead to problems such as impaired in vivo half-life (Hotzel, I., et al., mAbs, 2012 & Avery, L. B., mAbs, 2018), and reduced solubility (Kohli, N., mAbs, 2015, and Wolf Perez, A. M., mAbs, 2019). To evaluate the non-specific binding of the anti-FIX(a) mAbs listed in Table 8 below, an SEC-HPLC method in which the mAb-column interaction results in a delay in elution was used. Thus, a long retention time is a measure of non-specific binding. A method very similar to the previously described method (Wolf Perez, A. M., mAbs, 2019)) and the method described in (Dobson, C. L., Sci Rep, 2016; 6:38644) was used. For BimAbs 1-8, there was a decrease in non-specific binding compared to the emicizumab VH / VL SIA prepared as described in Example 4, see Table 8.

[0183] Materials and Methods Size-exclusion chromatography HPLC (SEC-HPLC) analysis was performed using an HPLC system (model 1200, Agilent Technologies) and a TSK G3000 SWXL SEC column (5 μm, 7.8 × 300 mm; Tosoh Bioscience). The mobile phase consisted of 122 mM Na2HPO4, 78 mM NaH2PO4, 300 mM NaCl, and 4% 2-propanol at pH 6.8. Each analysis was run for 24 minutes at a flow rate of 0.8 mL / min and a column temperature of 28°C. A 15 μl protein solution was injected into the column and eluted, and measured at an absorbance of 280 nm. Data processing was performed using Astra v.7 (Wyatt technology).

[0184] [Table 8]

Claims

Claim 1 A bispecific antibody or an antigen-binding fragment thereof, which can bind to FIX (SEQ ID NO: 89) and / or its active form (FIXa), and can bind to FX (SEQ ID NO: 90) and / or its active form (FXa), wherein the bispecific antibody comprises an anti-FIX(a) antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, and an anti-FX(a) antibody or an antigen-binding fragment thereof comprising a heavy chain and a light chain, and a) the heavy chains of the anti-FIX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 26, 27, and 28, the light chains of the anti-FIX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 30, 31, and 32, the heavy chains of the anti-FX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, the light chains of the anti-FX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 22, 23, and 24, or b) the heavy chains of the anti-FIX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 26, 27, and 28, the light chains of the anti-FIX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 30, 31, and 32, the heavy chains of the anti-FX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 2, 3, and 4, the light chains of the anti-FX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 6, 7, and 8, or c) the heavy chains of the anti-FIX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 34, 35, and 36, the light chains of the anti-FIX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 38, 39, and 40, the heavy chains of the anti-FX(a) antibody or its antigen-binding fragment respectively comprise CDR1-3 sequences specified by SEQ ID NOs: 18, 19, and 20, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 22, 23, and 24, or d) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 34, 35, and 36, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 38, 39, and 40, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 2, 3, and 4, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 6, 7, and 8, or e) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 42, 43, and 44, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 46, 47, and 48, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 18, 19, and 20, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 22, 23, and 24, or f) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 42, 43, and 44, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 46, 47, and 48, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 2, 3, and 4, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 6, 7, and 8, or g) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 50, 51, and 52, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 54, 55, and 56, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 18, 19, and 20, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 22, 23, and 24, or h) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 50, 51, and 52, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 54, 55, and 56, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 2, 3, and 4, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 6, 7, and 8, or i) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 58, 59, and 60, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 62, 63, and 64, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 18, 19, and 20, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 22, 23, and 24, or j) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 58, 59, and 60, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 62, 63, and 64, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 2, 3, and 4, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 6, 7, and 8, or k) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 66, 67, and 68, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 70, 71, and 72, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 18, 19, and 20, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 22, 23, and 24, or l) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 66, 67, and 68, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 70, 71, and 72, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 2, 3, and 4, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 6, 7, and 8, or m) the heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 74, 75, and 76, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 78, 79, and 80, The heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 18, 19, and 20, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 22, 23, and 24, or n) The heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 74, 75, and 76, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 78, 79, and 80, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 2, 3, and 4, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 6, 7, and 8, or o) The heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 82, 83, and 84, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 86, 87, and 88, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 18, 19, and 20, the light chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 22, 23, and 24, or p) The heavy chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 82, 83, and 84, the light chain of the anti-FIX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 86, 87, and 88, the heavy chain of the anti-FX(a) antibody or its antigen-binding fragment contains CDR1-3 sequences respectively specified by SEQ ID NOs: 2, 3, and 4, The light chain of the anti-FX(a) antibody or its antigen-binding fragment contains the CDR1-3 sequences respectively specified by SEQ ID NOs: 6, 7, and 8, which is a bispecific antibody or its antigen-binding fragment. **Claim 2** The anti-FIX(a) antibody or its antigen-binding fragment a. The heavy chain variable domain specified by SEQ ID NO: 25 and the light chain variable domain specified by SEQ ID NO: 29, or b. The heavy chain variable domain specified by SEQ ID NO: 33 and the light chain variable domain specified by SEQ ID NO: 37, or c. The heavy chain variable domain specified by SEQ ID NO: 41 and the light chain variable domain specified by SEQ ID NO: 45, or d. The heavy chain variable domain specified by SEQ ID NO: 49 and the light chain variable domain specified by SEQ ID NO: 53, or e. The heavy chain variable domain specified by SEQ ID NO: 57 and the light chain variable domain specified by SEQ ID NO: 61, or f. The heavy chain variable domain specified by SEQ ID NO: 65 and the light chain variable domain specified by SEQ ID NO: 69, or g. The heavy chain variable domain specified by SEQ ID NO: 73 and the light chain variable domain specified by SEQ ID NO: 77, or h. Contains the heavy chain variable domain specified by SEQ ID NO: 81 and the light chain variable domain specified by SEQ ID NO: 85, and the anti-FX(a) antibody or its antigen-binding fragment i. The heavy chain variable domain specified by SEQ ID NO: 17 and the light chain variable domain specified by SEQ ID NO: 21, or ii. The heavy chain variable domain specified by SEQ ID NO: 1 and the light chain variable domain specified by SEQ ID NO: 5, which is the bispecific antibody or its antigen-binding fragment according to Claim 1. **Claim 3** The isotype of the bispecific antibody is IgG1 or IgG4, which is the bispecific antibody or its antigen-binding fragment according to Claim 1 or 2. **Claim 4** The antibody or its antigen-binding fragment is a blood coagulation promoting antibody or its antigen-binding fragment, which is the bispecific antibody or its antigen-binding fragment according to any one of Claims 1 to 3. **Claim 5** The antibody or its antigen-binding fragment can stimulate the enzymatic activity of FIXa, which is the bispecific antibody or its antigen-binding fragment according to any one of Claims 1 to 4. **Claim 6** A pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5 and one or more pharmaceutically acceptable carriers.

7. A medicament for use in the treatment of hemophilia, comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, or the composition.

8. The medicament according to claim 7, for use in the treatment of hemophilia A with or without an inhibitor.

9. The medicament according to claim 7, for use in the prophylactic treatment of hemophilia A with or without an inhibitor.

10. An antibody or an antigen-binding fragment thereof, which can bind to FIX (SEQ ID NO: 89) and / or its active form (FIXa), and comprises a heavy chain and a light chain, a) the heavy chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 26, 27, and 28, and the light chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 30, 31, and 32, or b) the heavy chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 34, 35, and 36, and the light chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 38, 39, and 40, or c) the heavy chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 42, 43, and 44, and the light chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 46, 47, and 48, or d) the heavy chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 50, 51, and 52, and the light chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 54, 55, and 56, or e) the heavy chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 58, 59, and 60, and the light chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 62, 63, and 64, or f) the heavy chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 66, 67, and 68, and the light chain comprises CDR1-3 sequences respectively specified by SEQ ID NOs: 70, 71, and 72, or g) each of the heavy chains comprises CDR1-3 sequences specified by SEQ ID NOs: 74, 75, and 76, and each of the light chains comprises CDR1-3 sequences specified by SEQ ID NOs: 78, 79, and 80, or h) an antibody or an antigen-binding fragment thereof, wherein each of the heavy chains comprises CDR1-3 sequences specified by SEQ ID NOs: 82, 83, and 84, and each of the light chains comprises CDR1-3 sequences specified by SEQ ID NOs: 86, 87, and 88. **Claim 11** The antibody or an antigen-binding fragment thereof according to claim 10, which is an intermediate for use in the manufacture of a bispecific antibody that can bind to FIX (SEQ ID NO: 89) and / or its active form (FIXa) and can bind to FX (SEQ ID NO: 90) and / or its active form (FXa). **Claim 12** The antibody or an antigen-binding fragment thereof according to claim 10 or 11, which is a blood coagulation promoting antibody or an antigen-binding fragment thereof. **Claim 13** The antibody or an antigen-binding fragment thereof according to any one of claims 10 to 12, which can stimulate the enzymatic activity of FIXa. **Claim 14** A kit comprising: (i) optionally, a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, or a composition, contained in an injection device, and (ii) instructions for use.

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