Bispecific Factor VIII mimetic 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) and factor X (FX) and / or its activated form factor Xa (FXa) and promote the activation of FX by FIXa, as well as methods and compositions for treating subjects suffering from coagulopathy such as hemophilia A with or without inhibitors.

Description

[0001] Incorporation by Reference of Sequence Listing

[0002] This application is submitted with a sequence listing in electronic form. The entire contents of this sequence listing are incorporated herein by reference.

[0003] background

[0004] In patients with coagulopathy, such as those with hemophilia A and B, various steps of the coagulation cascade become dysfunctional due to, for example, a lack of or insufficient presence of functional coagulation factors. This dysfunction of one part of the coagulation cascade results in inadequate blood clotting and potentially life-threatening bleeding, or damage to internal organs such as joints.

[0005] Factor VIII (FVIII) deficiency, commonly known as hemophilia A, is a congenital bleeding disorder affecting approximately 420,000 people worldwide, of whom approximately 105,000 are currently diagnosed.

[0006] Patients with hemophilia A can receive coagulation factor replacement therapy, such as exogenous FVIII. Conventional treatment consists of replacement therapy, which is provided as prophylaxis or on-demand treatment of bleeding episodes. Until recently, prophylactic treatment for patients with severe hemophilia A consisted of up to three intravenous injections of plasma-derived FVIII or recombinant FVIII or its long-acting variants weekly.

[0007] In some embodiments, the present invention relates to the treatment of hemophilia A. However, such patients have the risk of generating neutralizing antibodies (so-called inhibitors) against such exogenous factors, making previously effective therapy invalid. The hemophilia A patient with inhibitors is a non-limiting example of a partially congenital, partially acquired coagulopathy. Patients who have generated FVIII inhibitors cannot be treated with conventional alternative therapy. Exogenous coagulation factors can only be administered intravenously, which is quite inconvenient and uncomfortable for the patient.

[0008] Insufficient FXa formation and decreased thrombin generation caused by reduced or absent FVIII activity are the underlying causes of the bleeding diathesis in patients with hemophilia A.

[0009] The proteolytic conversion of FX to its enzymatically active form, FXa, can be achieved by an intrinsic FX activation complex comprising FIXa and its cofactor-activated FVIII (FVIIIa). Cofactor binding increases the enzymatic activity of FIXa by approximately five orders of magnitude and is believed to occur through a variety of mechanisms as outlined by Scheiflinger et al. (2008) J Thromb Haemost, 6: 315-322. Notably, FVIIIa has been found to stabilize the conformation of FIXa, which has increased proteolytic activity against FX (Kolkman JA, Mertens K (2000) Biochemistry, 39: 7398-7405, T, Brandstetter H (2009) Biol Chem, 390: 391-400). Based on this observation and realizing that antibodies are universal binding proteins that can mimic a variety of protein-protein interactions, Scheiflinger et al. screened for agonistic anti-FIX(a) antibodies characterized by their ability to enhance the activation of FX by FIXa in the presence of phospholipid surfaces and calcium, but in the absence of the natural cofactor FVIIIa. 88 hybridoma supernatants were found to produce antibodies exhibiting varying degrees of FIXa agonist activity, as shown in EP1220923 B1 and EP1660536 B1. Recently, a new drug, emicizumab, has been Also known as ACE910, it is approved for subcutaneous prophylaxis in hemophilia A with or without inhibitors to conventional replacement therapy factors. 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 function of the FVIII cofactor (see Sampei et al. (2013) PLoS One, 8, e57479 and WO2012 / 067176). Treatment with 30-50 μg of emicizumab per milliliter of plasma is estimated to correspond to an equivalent factor VIII activity of at least 10 to 15 IU per deciliter of plasma (Shima et al., N Engl J Med 2016; 374: 2044-53). However, some patients have developed inhibitors (anti-drug antibodies) to emicizumab, rendering treatment with this compound ineffective.

[0010] In addition to generating inhibitors using emicizumab as an example, other antibody properties are also crucial for achieving effective antibody-based treatments for patients. In particular, it has been demonstrated how antibodies with high nonspecific binding tendencies may lead to clinical safety issues. In some reports, high levels of nonspecific binding result in a shortened circulating half-life of antibodies by several times, and lead to ineffective and cumbersome dosing regimens for patients (see Dobson et al., Nature, volume 6, art. no.: 38644 (2016) and Avery et al., MAbs 2018, Vol. 10, No. 2, 244-255).

[0011] WO2018 / 141863 and WO2019 / 065795 also disclose anti-FIX(a) anti-FX(a) bispecific antibodies and their use as coagulants for the treatment of hemophilia.

[0012] There remain many unmet medical needs in the hemophilia population, particularly in subjects suffering from coagulopathy.The present invention relates to improved compounds that are capable of replacing FVIII and are therefore useful in the treatment of coagulopathy such as hemophilia A. Summary of the Invention

[0013] The present invention relates to compounds that act as replacements for coagulation factor VIII (FVIII) in patients with coagulopathy, particularly patients lacking functional FVIII, such as hemophilia A patients, including hemophilia A patients with inhibitors.

[0014] One aspect of the present invention relates to compounds that are able to enhance the production of FXa and thereby partially or fully restore coagulation in patients lacking functional FVIII.

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

[0016] In a specific aspect, the invention relates to antibodies or antigen-binding fragments thereof that serve as replacements for FVIII in patients lacking functional FVIII, such as hemophilia A patients.

[0017] In one such aspect, the antibody or antigen-binding fragment thereof is capable of binding to FIX(a) and increasing the enzymatic activity of FIXa towards FX, and optionally is also capable of binding to FX.

[0018] In one aspect, the present invention relates to antibodies or antigen-binding fragments thereof, including bispecific antibodies or antigen-binding fragments thereof, that are capable of binding to FIX(a) and FX(a), which increase the enzymatic activity of FIXa on FX.

[0019] In one aspect, the present invention relates to antibodies or antigen-binding fragments thereof that can bind to FIX(a) and FX(a) that have improved properties compared to antibodies disclosed in the art. In one such aspect, the antibodies or antigen-binding fragments thereof have improved procoagulant properties and / or reduced tendency to nonspecifically bind, for example, to DNA and / or insulin, and / or reduced tendency to self-associate, compared to bispecific antibodies in the art including emicizumab.

[0020] Another aspect of the invention relates to a separate component (intermediate) antibody or antigen-binding fragment thereof that is 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] Another aspect of the present invention relates to an antibody or antigen-binding fragment thereof disclosed herein for use in preventing and / or treating a coagulopathy, a disease associated with a coagulopathy, or a disease caused by a coagulopathy. In one aspect, the coagulopathy is a hemophilia, such as hemophilia A with or without inhibitors.

[0022] Yet another aspect of the present invention relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, formulated for delivery of the antibodies for the prevention and / or treatment of coagulopathy, such as hemophilia A with or without inhibitors, and injection devices containing the contents thereof.

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

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

[0025] Figure 1A-D Shown is an alignment of sequences representing the heavy and light chain variable domains of the anti-FIX(a) ( FIG. 1A and FIG. 1B ) and anti-FX(a) ( FIG. 1C and FIG. 1D ) IgG antibodies disclosed herein. The CDR1, 2, and 3 sequences are highlighted in bold and underlined in the top sequence and represent the remaining sequences in the corresponding figures.

[0026] Sequence Description

[0027] 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 (CDRs) of the anti-FIX(a) and anti-FX(a) monoclonal antibodies (mAbs) described herein.

[0028] SEQ ID NO: 89 represents the amino acid sequence of human coagulation factor IX.

[0029] SEQ ID NO: 90 represents the amino acid sequence of human coagulation factor X.

[0030] SEQ ID NO: 91 represents the human IgG4 heavy chain constant region with S228P and C-terminal lysine truncation.

[0031] SEQ ID NO: 92 represents a human IgG4 heavy chain constant region with S228P, F405L, R409K and a C-terminal lysine truncation.

[0032] SEQ ID NO: 93 represents the human kappa light chain constant region.

[0033] SEQ ID NO: 94 represents the human IgG1 heavy chain constant region with F405L and C-terminal lysine truncation.

[0034] SEQ ID NO: 95 represents the human IgG1 heavy chain constant region with K409R and C-terminal lysine truncation.

[0035] SEQ ID NOs: 9-16 are intentionally omitted.

[0036] The table in Example 6 correlates SEQ ID NOs to the individual (component) anti-FIX(a) and anti-FX(a) antibodies and bispecific antibodies of the invention.

[0037] describe

[0038] In subjects suffering from coagulopathy, such as those suffering from hemophilia A, due to the lack or absence of functional FVIII, the coagulation cascade becomes dysfunctional. This dysfunction of a part of the coagulation cascade leads to insufficient blood coagulation and potentially life-threatening bleeding, or damage to internal organs such as joints. The present invention relates to compounds that serve as a substitute for coagulation factor VIII (FVIII) in patients suffering from coagulopathy, particularly patients lacking functional FVIII, such as hemophilia A patients, including hemophilia A patients with inhibitors. In one aspect, such compounds are antibodies.

[0039] In particular, the inventors of the present invention have surprisingly identified antibodies that mimic the activity of FVIII cofactors with high potency and efficacy. In a specific aspect, the present invention relates to antibodies that serve as substitutes for FVIII in patients lacking functional FVIII, such as hemophilia A patients. In one such aspect, the antibodies bind to and increase the enzymatic activity of coagulation factor IXa (FIXa) on coagulation factor X (FX), optionally also in conjunction with FX. In one such aspect, the antibodies of the present invention are bispecific antibodies that can bind to FIX / FIXa and FX.

[0040] Another aspect of the invention relates to individual component (intermediate) antibodies or antigen-binding fragments thereof that are part of a multispecific 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.

[0041] Another aspect of the present invention relates to the preparation of the antibodies or antigen-binding fragments thereof as disclosed herein and components (intermediates) thereof.

[0042] Another aspect of the invention relates to antibodies that compete for binding to FIX(a) and / or FX(a) with the antibodies or antigen-binding fragments thereof disclosed herein.

[0043] Another aspect of the present invention relates to antibodies or antigen-binding fragments thereof that share epitopes on FIX(a) and / or FX(a) with the antibodies or antigen-binding fragments thereof disclosed herein.

[0044] In one aspect, the antibody is a human or humanized antibody, such as a human or humanized bispecific antibody.

[0045] Another aspect of the present invention relates to an antibody or antigen-binding fragment thereof disclosed herein for use in preventing and / or treating a coagulopathy, a disease associated with a coagulopathy, or a disease caused by a coagulopathy. In one aspect, the coagulopathy is hemophilia A with or without inhibitors.

[0046] Yet another aspect of the present invention relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, formulated for delivery of the antibodies for the prevention and / or treatment of coagulopathy, such as hemophilia A with or without inhibitors, and injection devices containing the contents thereof.

[0047] Another aspect of the present invention relates to a kit comprising (i) an antibody or antigen-binding fragment thereof, such as a bispecific antibody, disclosed herein, and (ii) instructions for use.

[0048] Coagulation factor IX

[0049] Coagulation factor IX (FIX) is a vitamin K-dependent coagulation factor with structural similarities to factor VII, prothrombin, factor X, and protein C. FIX circulates in plasma as a single-chain zymogen (SEQ ID NO: 89). The circulating zymogen form consists of 415 amino acids divided into four distinct domains, including an N-terminal γ-carboxyglutamate (Gla)-rich domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. Activation of FIX occurs by limited proteolysis at Arg145 and Arg180 to release the activation peptide (residues 146 to 180 of SEQ ID NO: 89). Thus, activated FIX (FIXa) consists of residues 1-145 of SEQ ID NO: 89 (light chain) and residues 181-415 of SEQ ID NO: 89 (heavy chain).

[0050] Thus, circulating FIX molecules comprise the FIX zymogen and the activated form of FIX, which are generally referred to herein as FIX and FIXa, with reference to SEQ ID NO: 1.

[0051] Activated Factor IX is referred to as Factor IXa or FIXa. The term "FIX (SEQ ID NO: 1) and / or its activated form (FIXa)" may also be referred to as "FIX / FIXa", or simply "FIX(a)".

[0052] FIXa is a trypsin-like serine protease that plays a key role in hemostasis by generating the majority of factor Xa necessary to support the formation of proper thrombin during coagulation as part of the tenase complex.

[0053] FIX is represented herein by SEQ ID NO: 1, which corresponds to the Ala148 allele of human FIX (Anson et al. EMBO J. 19843: 1053-1060; McGraw et al. Proc Natl Acad Sci USA. 198582: 2847-2851; Graham et al. Am. J. Hum. Genet. 1988 42: 573-580). In the present invention, FIX is intended to encompass all natural variants of FIX, such as the T148 variant (Uniprot ID P00740).

[0054] Coagulation factor X

[0055] FX is a vitamin K-dependent coagulation factor with structural similarities to factor VII, prothrombin, FIX, and protein C. FX circulates in plasma as a two-chain zymogen comprising residues 1-139 of SEQ ID NO: 2 (light chain) and residues 143-448 of SEQ ID NO: 2 (heavy chain). The human FX zymogen contains four distinct domains, including an N-terminal γ-carboxyglutamic acid (Gla)-rich domain (residues 1-45), two EGF domains: EGF1 (residues 46-82) and EGF2 (residues 85-125), and a C-terminal trypsin-like serine protease domain (residues 195-448). FX activation occurs through limited proteolysis at Arg194, which results in the release of an activation peptide (residues 143-194). Thus, activated FX (FXa) consists of residues 1-139 of SEQ ID NO: 2 (light chain) and residues 195-448 of SEQ ID NO: 2 (activated heavy chain). Thus, circulating Factor X molecules comprise the FX zymogen and the activated form of FX, which are referred to herein as FX and FXa, respectively, with reference to SEQ ID NO: 2. In the present invention, FX is intended to encompass all naturally occurring variants of FX. The term "FX (SEQ ID NO: 90) and / or its activated form (FXa)" may also be referred to as "FX / FXa" or "FX(a)."

[0056] Antibody

[0057] The term "antibody" as used herein refers to a protein derived from an immunoglobulin sequence that is capable of binding 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.

[0058] A natural full-length antibody comprises at least four polypeptide chains: two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. In some cases, natural antibodies comprise fewer than four chains, as in the case of IgNARs found in cartilaginous fish (Chondrichthyes). One class of immunoglobulins of particular pharmaceutical interest is IgG. In humans, the IgG class can be divided into four subclasses: IgG1, IgG2, IgG3 and IgG4, based on the sequence of the constant region of its heavy chain. Light chains can be divided into two types: kappa chains and lambda chains, based on differences in their sequence composition. An IgG molecule consists of two heavy chains linked to each other by two or more disulfide bonds and two light chains, each linked to a heavy chain by a disulfide bond. An IgG heavy chain may comprise one heavy chain variable domain (V H ) and up to three heavy chain constant (C H ) Domain: C H 1. CH 2 and C H 3. The light chain may comprise a light chain variable domain (V L ) and the light chain constant domain (C L ). V H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) or hypervariable regions (HvR), interspersed with regions that are more conserved, termed framework regions (FR). H and V L The domain is usually composed of three CDRs and four FRs, which are arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable domains containing the hypervariable regions (CDRs) constitute the structure capable of interacting with the antigen, while the constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including but not limited to various cells of the immune system (effector cells), Fc receptors, and the first component of the C1 complex of the classical complement system, C1q.

[0059] The antibodies of the present invention can be monoclonal antibodies (mAbs), which are in the sense that they represent a unique set of heavy and light chain variable domain sequences expressed by a single B cell or by a clonal population of B cells. The antibodies of the present invention can be produced and purified using various methods known to those skilled in the art. For example, antibodies can be produced from hybridoma cells. Antibodies can be produced by B cell expansion. Antibodies or their fragments can be recombinantly expressed in mammalian or microbial expression systems or by in vitro translation. Antibodies or their fragments can also be recombinantly expressed as cell surface-bound molecules by, for example, phage display, bacterial display, yeast display, mammalian cell display, or ribosome or mRNA display.

[0060] 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 components of the environment in which it is produced, and / or an antibody that has been purified from a mixture of components present in the environment in which it is produced.

[0061] Certain antigen-binding fragments of antibodies may be suitable in the context of the present invention, as it has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The term "antigen-binding fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to or recognize an antigen, such as FIX / FIXa, FX / FXa, or another target molecule, as described herein. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', Fab2, Fab'2, Fv (typically the V fragment of a single arm of an antibody), L and V Hdomains), single-chain Fv (scFv); see, e.g., Bird et al., Science 1988 242:423-426; and Huston et al., PNAS 1988 85:5879-5883), dsFv, Fd (generally V H and C H 1 domain); contains a single V H and a single V L The present invention also provides the monovalent molecules of both the antibody and the domain; minibodies, diabodies, triabodies, tetrabodies and kappa bodies (see, for example, 111 et al. (1997) Protein Eng 10: 949-57); and one or more isolated CDRs or functional paratopes, wherein the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together to form functional antibody fragments. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments can be screened for utility in the same manner as intact antibodies.

[0062] "Fab fragments" of antibodies, including "Fab" and "Fab'2" fragments, can be obtained from antibodies by cleaving the heavy chain in the hinge region on the N-terminal or C-terminal side of the hinge cysteine ​​residues connecting the heavy chain of the antibody. "Fab" fragments include the variable and constant domains of the light chain and the variable and C-terminal domains of the heavy chain. H 1 domain. The "Fab'2" fragment contains a pair of "Fab" fragments that are usually covalently linked by their hinge cysteines. Fab' is formally derived from the Fab'2 fragment by cleaving the hinge disulfide bond connecting the heavy chains in Fab'2. Other chemical couplings besides disulfide bonding of antibody fragments are also known in the art. The Fab fragment retains the ability of the parent antibody to bind to its antigen, potentially with lower affinity. The Fab'2 fragment is capable of divalent binding, while the Fab and Fab' fragments are only capable of monovalent binding. Typically, the Fab fragment lacks a constant C H 2 and C H The Fab fragments are composed of three domains, namely the Fc portion where interactions with Fc receptors and C1q occur. Therefore, Fab fragments generally lack effector functions. Fab fragments can be produced by enzymatic cleavage of antibodies using methods known in the art, such as using papain to obtain Fab or pepsin to obtain Fab'2. Fab fragments, including Fab, Fab', and Fab'2, can be recombinantly produced using techniques well known to those skilled in the art.

[0063] "Fv" (fragment variable) fragments are antibody fragments that contain complete antigen recognition and binding sites and generally comprise one heavy chain variable domain and one light chain variable domain associated (which may be covalent in nature), such as in a single-chain variable domain fragment (scFv). In this configuration, the three hypervariable regions of each variable domain interact to form a single heavy chain variable domain. H -V L The antigen-binding site is defined on the surface of the dimer. Collectively, these six hypervariable regions, or a subset thereof, confer antigen-binding specificity to the antibody.

[0064] A "single-chain Fv" or "scFv" antibody comprises the V H and V L domains, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide is further H and V L A polypeptide linker is included between the domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, 1994, In: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

[0065] A "single-chain Fab" or "scFab" antibody comprises the V H 、C H 1. V L and C L domains, wherein these domains are present in a single polypeptide chain. Typically, the Fab polypeptide is also H and C L or V L and C H A polypeptide linker is contained between the 1 domains, which enables the scFab to form the desired structure for antigen binding (Koerber et al. (2015) J Mol Biol. 427:576-86).

[0066] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, wherein the fragments are contained on the same polypeptide chain (V H and V L ) is connected to the light chain variable domain (V L ) of the heavy chain variable domain (V H By using a linker that is too short to allow pairing between two variable domains on the same chain, the variable domains are forced to pair with the complementary domains of another chain, creating two antigen-binding sites.

[0067] The expression "linear antibodies" refers to antibodies as described in Zapata et al. (1995) Protein Eng. 8: 1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (V H -C H 1-V H -C H 1), which together with the complementary light chain polypeptide form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[0068] Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and these fragments can be screened for binding to FIX and its activated form, FX, or another function in the same manner as intact antibodies.

[0069] Antibody fragments of the present invention can be prepared by truncation, for example by removing one or more amino acids from the N-terminus and / or C-terminus of the polypeptide. Fragments can also be generated by one or more internal deletions.

[0070] The antibodies of the present invention may be or may comprise fragments of antibodies or variants of any of the antibodies disclosed herein. The antibodies of the present invention may be or may comprise an antigen-binding portion of one of these antibodies or a variant thereof. For example, the antibodies of the present invention may be a Fab fragment of one of these antibodies or a variant thereof, or it may be a single-chain antibody derived from one of these antibodies or a variant thereof. Furthermore, the antibodies of the present invention may be a combination of full-length antibodies and fragments thereof.

[0071] As used herein, the term "monospecific" antibody refers to an antibody (including but not limited to bivalent antibodies) that is capable of binding to one specific epitope.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] Bispecific antibodies in full-length IgG format can be generated by fusing two separate hybridomas to form a hybrid tetravalent tumor that produces a mixture of antibodies that includes a portion of the bispecific heterodimeric antibody (Chelius D. et al.; MAbs. 2010 May-June; 2(3): 309-319). Alternatively, bispecific heterodimeric antibodies can be produced using recombinant techniques. Heterodimerization can also be achieved by engineering the dimerization interface of the Fc region to promote heterodimerization. One example is the so-called knob-in-hole mutation, in which a sterically bulky side chain (knob) is introduced into one Fc that is matched by a sterically smaller side chain (hole) on the opposite Fc, thereby generating steric complementarity that promotes heterodimerization. Other approaches for engineering heterodimeric Fc interfaces are electrostatic complementation, fusion with non-IgG heterodimerization domains, or exploiting 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 must be paid to the light chains in heterodimeric antibodies. Correct pairing of LC with HC can be achieved by using a common light chain. Again, engineering of the LC / HC interface can be used to promote heterodimerization or light chain cross-engineering, such as CrossMabs. Reassembly of antibodies from two separate IgG containing appropriate mutations in vitro under mild reducing conditions can also be used to generate bispecific antibodies (e.g., Labrijn et al., PNAS, 110, 5145-5150 (2013)). Natural Fab-arm exchange methods have also been reported to ensure correct light chain pairing.

[0076] The molecule based on multispecific antibody can also be recombinantly expressed as fusion protein, and this fusion protein combines the natural module of IgG to form the multispecific and multivalent antibody derivatives as described in the literature.Examples of fusion antibodies are DVD-Ig, IgG-scFV, double antibody, DART etc.Specific detection or purification tag, half-life extension part or other components can be incorporated into the fusion protein.Other non-IgG mode can also be incorporated into the fusion protein.The bispecific full-length antibody based on Fc heterodimerization is commonly referred to as asymmetric IgG, and has nothing to do with LC pairing method.

[0077] Generally, as reviewed by Brinkmann et al. (Brinkmann et al. The making of bispecific antibodies. Mabs 9, 182-212 (2017)), bispecific antibodies can be produced in a variety of molecular formats.

[0078] Molecules based on multispecific antibodies can also be produced by chemically conjugating or coupling individual full-length IgG or IgG-coupled fragments to form multispecific and multivalent antibody derivatives as described in the literature. Examples are chemically coupled Fab fragments, IgG-dimers, etc. Specific detection or purification tags, half-life extension molecules or other components can be incorporated into the conjugated protein. Additional non-IgG polypeptides can also be incorporated into the fusion protein. Multispecific molecules can also be produced by combining recombinant methods and chemical methods, including those described above.

[0079] In one aspect, the antibody of the present invention is a chimeric antibody, a human antibody or a humanized antibody. Such an antibody can be produced by using, for example, a suitable antibody display or immune platform or other suitable platforms or methods known in the art. As used herein, the term "human antibody" is intended to include antibodies with 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, human antibodies can have variable domains in which the framework region and the CDR region are both derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region or its part also derives from human germline immunoglobulin sequences. Human antibodies of the present invention can include amino acid residues that are not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo).

[0080] Such human antibodies can be human monoclonal antibodies. Such human monoclonal antibodies can be produced by hybridomas comprising B cells fused with immortalized cells, the B cells being obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a repertoire of human immunoglobulin heavy and light chain gene segments.

[0081] Human antibodies can be isolated from sequence libraries created based on selections of human germline sequences, further diversified with natural and synthetic sequence diversity.

[0082] Human antibodies can be prepared by in vitro immunization of human lymphocytes and subsequent transformation of the lymphocytes with Epstein-Barr virus.

[0083] Human antibodies can be produced by recombinant methods known in the art.

[0084] The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of an antibody with another substance or antibody.

[0085] As used herein, the term "humanized antibody" refers to a human / non-human antibody containing a sequence (CDR region or portion thereof) derived from a non-human immunoglobulin. Therefore, a humanized antibody is such a human immunoglobulin (acceptor antibody), wherein at least the residues from the hypervariable region of the acceptor are replaced by residues from the hypervariable region of an antibody (donor antibody) of a non-human species such as a mouse, rat, rabbit or non-human primate, having the desired specificity, affinity, sequence composition and functionality. In some cases, the framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. An example of this type of modification is the introduction of one or more so-called back mutations, which are generally amino acid residues derived from donor antibodies. The 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). Suitable human acceptor frameworks for both light and heavy chain variable domains can be identified by, for example, sequence or structural homology. In some embodiments, the present invention provides the humanized antibody of the present invention.For example, the humanized antibody of the present invention can be ...

[0086] In addition, humanized antibodies may be included in residues not found in acceptor antibody or donor antibody. These modifications are carried out to further improve antibody performance. Usually, humanized antibodies will comprise at least one - generally two - variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and wherein all or substantially all of the FR residues are the FR residues of human immunoglobulin sequences. Humanized antibodies may also optionally comprise at least a portion (Fc) of an immunoglobulin constant region, generally the corresponding portion of a human immunoglobulin.

[0087] The term "humanized antibody derivative" refers to any modified form of a humanized antibody, such as a conjugate of an antibody with a chemical agent or a conjugate of an antibody with another antibody.

[0088] As used herein, the term "chimeric antibody" refers to an antibody that comprises portions of antibodies derived from two or more species. For example, the genes encoding such antibodies include genes encoding variable domains derived from two different species and genes encoding constant domains derived from two different species. For example, genes encoding the variable domains of a mouse monoclonal antibody can be linked to genes encoding the constant domains of a human antibody.

[0089] The fragment crystallizable region of an antibody ("Fc region" / "Fc domain") is the region containing the hinge and constant C H 2 and C H The Fc domain is an antibody C-terminal region containing a 3-domain. The Fc domain can interact with cell surface receptors known as Fc receptors and certain proteins of the complement system. The Fc region enables antibodies to interact with the immune system. In one aspect of the invention, antibodies can be engineered to include modifications within the Fc region, generally to alter one or more functional properties, such as serum half-life, complement fixation, Fc receptor binding, protein stability, and / or antigen-dependent cellular cytotoxicity, or the lack of these properties, etc. In addition, the antibodies of the invention can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation, thereby further altering one or more functional properties of the antibody. IgG1 antibodies can carry a modified Fc domain comprising one or more, and possibly all, of the following mutations, which, respectively, result in reduced affinity for certain Fc-γ receptors (L234A, L235E, and G237A) and reduced C1q-mediated complement fixation (A330S and P331S) (residues are numbered according to the EU index). Alternatively, other amino acid substitutions and combinations thereof known in the art and in combination with the modifications described above may be used to result in altered (decreased or increased) Fc-gamma receptor binding.

[0090] The isotype of the antibodies of the present invention can be IgG, such as IgG1, IgG2, or IgG4. If desired, the class of the antibody can be "switch" using known techniques. For example, an antibody initially generated as an IgM molecule can be class-switched to an IgG antibody. Class-switch techniques can also be used to convert one IgG subclass to another, for example: from IgG1 to IgG2 or IgG4; from IgG2 to IgG1 or IgG4; or from IgG4 to IgG1 or IgG2. Antibody engineering can also be performed to generate constant region chimeric molecules by combining regions from different IgG subclasses.

[0091] In one embodiment, the hinge region of an antibody is modified such that the number of cysteine ​​residues in the hinge region is altered, eg, increased or decreased. This approach is further described in, for example, US Pat. No. 5,677,425 to Bodmer et al.

[0092] The constant region can be modified to stabilize the antibody, for example, to reduce the risk of bivalent antibodies separating into half antibodies. For example, in the IgG4 constant region, residue S228 (according to the EU numbering index, S241 according to Kabat) can be mutated to a proline (P) residue to stabilize the inter-heavy chain disulfide bond formation at the hinge (see, e.g., Angal et al. Mol Immunol. 1993; 30: 105-8).

[0093] Antibodies or their fragments can be defined according to their complementary determining regions (CDRs). The terms "complementarity determining region" or "hypervariable region" as used herein refer to regions of antibodies in which the amino acid residues involved in antigen binding are located. Hypervariable regions or CDRs can be identified as regions with the highest variability in the amino acid alignment of the antibody variable domains. Databases such as the Kabat database can be used for CDR identification, for example, CDRs are defined as comprising amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and 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, US Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, the CDRs may be defined as those residues from the "hypervariable loops" (residues 26-33 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987; 196: 901-917). Generally, the numbering of amino acid residues in this region is by the method described by Kabat et al. (supra). Phrases such as "Kabat position," "Kabat residue," and "according to Kabat" herein refer to this numbering system for the heavy chain variable domain or the light chain variable domain. By using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the framework (FR) or CDRs of the variable domain. For example, the heavy chain variable domain may comprise an amino acid insertion after residue 52 of CDRH2 (residues 52a, 52b, and 52c according to Kabat) and an insertion of residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat, etc.). The Kabat numbering of residues for a given antibody can be determined by aligning regions of homology of the antibody sequence with sequences using "standard" Kabat numbering.

[0094] The term "framework region" or "FR" residues refers to those V residues not within the CDRs as defined herein. H or V L Amino acid residues.

[0095] The term "procoagulant antibody" refers to an antibody that enhances blood coagulation, for example, by accelerating the blood coagulation process and / or increasing the enzymatic activity of one or more coagulation factors.

[0096] The term "procoagulant activity" refers to the ability of a compound, such as an antibody, to enhance blood coagulation, for example by accelerating the blood coagulation process and / or increasing the enzymatic activity of one or more coagulation factors.

[0097] The activity of procoagulant antibodies including bispecific, trispecific and multispecific antibodies can be determined by methods known in the art. Standard assays include whole blood-thrombin-generation assay (TGT), measuring clotting time and FXa generation assay by thromboelastography (TEG) (see, e.g., WO2018 / 141863).

[0098] The term "stimulating the enzymatic activity of FIXa" refers to stimulation determined using the method of Example 9.

[0099] The term "antigen" (Ag) refers to a molecular entity that is used to immunize an immunocompetent vertebrate to produce antibodies (Ab) that recognize the Ag. In this article, Ag has a broader meaning and is generally intended to include target molecules that are specifically recognized by Ab, and therefore includes molecular fragments or mimics used in the immune process or other processes such as phage display for generating Ab.

[0100] The present invention includes variants of the antibodies or antigen-binding fragments thereof of the present invention, which may comprise 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions in the respective sequences disclosed herein.

[0101] On the one hand, " substitution " variants relate to one or more amino acids being replaced with the same number of amino acids.Displacement can be, but is not limited to conservative substitutions.For example, amino acid can be replaced with an amino acid with similar biochemical properties, for example, a basic amino acid can be replaced with another basic amino acid (for example, lysine is replaced with arginine), an acidic amino acid can be replaced with another acidic amino acid (for example, glutamic acid is replaced with aspartic acid), a neutral amino acid can be replaced with another neutral amino acid (for example, threonine is replaced with serine), a charged amino acid can be replaced with another charged amino acid (for example, glutamic acid is replaced with aspartic acid), a hydrophilic amino acid can be replaced with another hydrophilic amino acid (for example, asparagine is replaced with glutamine), a hydrophobic amino acid can be replaced with another hydrophobic amino acid (for example, alanine is replaced with valine), a polar amino acid can be replaced with another polar amino acid (for example, serine is replaced with threonine), an aromatic amino acid can be replaced with another aromatic amino acid (for example, phenylalanine is replaced with tryptophan), and an aliphatic amino acid can be replaced with another aliphatic amino acid (for example, leucine is replaced with isoleucine).

[0102] In another aspect, variants comprise structural analogs of amino acids that occur in the sequence of an antibody or antigen-binding fragment thereof of the invention.

[0103] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or fragment thereof and an antigen). The term "binding affinity" is used to describe a monovalent interaction.

[0104] By determining the equilibrium dissociation constant (K D ), the binding affinity between two molecules (e.g., an antibody or fragment thereof and an antigen) through a monovalent interaction can be quantified. K can be determined by measuring the kinetics of complex formation and dissociation, for example, by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constants k and k, respectively. a (or k 结合 ) and the dissociation rate constant k d (or k 解离 ). Through equation K D =k d / k a , K D With k a and k d associated.

[0105] According to the above definition, by comparing the K DThe values ​​can be used to compare the binding affinities associated with different molecular interactions, such as comparing the binding affinities of different antibodies to a given antigen.

[0106] The value of the dissociation constant can be directly measured by well-known methods. Standard tests for evaluating the binding ability of ligands such as antibodies to targets are known in the art and include, for example, ELISA, Western blotting, RIA, and flow cytometry analysis. By standard tests known in the art, such as SPR, the binding kinetics and binding affinity of antibodies can also be assessed. However, preferably, isothermal titration calorimetry (ITC) can be used to measure the affinity of antibody / target interactions and to obtain the thermodynamic parameters of the interaction.

[0107] Competitive binding assays can be performed in which binding of an antibody to a target is compared to binding of another ligand for the target, such as another antibody.

[0108] The K of the antibody of the present invention to its target D It can be less than 100μM, such as less than 10μM, such as less than 9μM, such as less than 8μM, such as less than 7μM, such as less than 6μM, such as less than 5μM, such as less than 4μM, such as less than 3μM, such as less than 2μM, such as less than 1μM, such as less than 0.9μM, such as less than 0.8μM, such as less than 0.7μM, such as less than 0.6μM, such as less than 0.5μM, such as less than 0.4μM, such as less than 0.3μM, such as less than 0.2μM, such as less than 0.1μM.

[0109] In one such embodiment, the antibody is a bispecific antibody comprising an anti-FX arm that is resistant to the K of FX. D Less than 100 μM, such as less than 10 μM, such as less than 9 μM, such as less than 8 μM, such as less than 7 μM, such as less than 6 μM, such as less than 5 μM, such as less than 4 μM, such as less than 3 μM, such as less than 2 μM, such as less than 1 μM, such as less than 0.9 μM, such as less than 0.8 μM, such as less than 0.7 μM, such as less than 0.6 μM, such as less than 0.5 μM, such as less than 0.4 μM, such as less than 0.3 μM, such as less than 0.2 μM, such as less than 0.1 μM, such as Less than 0.09μM, such as less than 0.08μM, such as less than 0.07μM, such as less than 0.06μM, such as less than 0.05μM, such as less than 0.04μM, such as less than 0.03μM, such as less than 0.02μM, such as less than 0.01μM, such as less than 9nM, such as less than 8nM, such as less than 7nM, such as less than 6nM, such as less than 5nM, such as less than 4nM, such as less than 3nM, such as less than 2nM, such as less than 1nM, such as less than 0.5nM.

[0110] Antibodies and antibody fragments thereof as described herein can be combined with other antibodies and antibody fragments known in the art to produce bispecific, trispecific or multispecific antibody molecules. Antibodies targeting FIX(a) and FX(a) have previously been used to produce compounds that mimic the function of FVIII cofactors, and in some embodiments, these antibodies can potentially replace the FIX(a) or FX(a) antibodies described herein, respectively. It can be seen that the antibodies targeting FIX(a) and FX(a) of the present invention, particularly antigen-binding fragments thereof, have independent significance as separate component (intermediate) molecules, as part of a bispecific, trispecific or multispecific antibody comprising at least one FIX(a) and / or FX(a) binding domain.

[0111] pharmaceutical preparations

[0112] In another aspect, the present invention provides compositions and formulations comprising compounds of the invention, such as antibodies described herein. For example, the present invention provides pharmaceutical compositions comprising one or more antibodies of the invention formulated together with a pharmaceutically acceptable carrier.

[0113] Accordingly, one object of the present invention is to provide a pharmaceutical formulation comprising such an antibody at a concentration of 0.25 mg / ml to 250 mg / ml, wherein the formulation has a pH of 2.0 to 10.0. The formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, and various combinations thereof. The use of preservatives, isotonicity agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th ed., 1995.

[0114] In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such formulations are generally solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water.

[0115] In another embodiment, the pharmaceutical formulation is a freeze-dried formulation to which a solvent and / or diluent is added prior to use.

[0116] In a further aspect, the pharmaceutical formulation comprises an aqueous solution of an antibody and a buffer, wherein the antibody is present at a concentration of 1 mg / ml or greater, and wherein the pH of the formulation is from about 2.0 to about 10.0.

[0117] In one embodiment, the present invention relates to an injection device having the composition as an inclusion. In some embodiments, the pharmaceutical composition of the present invention is intended for use in and / or contained in an injection device. In some embodiments, the injection device is A disposable, pre-filled, multi-dose pen of the type ANTI-INFLAME® (supplier Novo Nordisk A / S, Denmark). In some embodiments, the injection device is a single shot device.

[0118] In some embodiments, the injection device is a fixed dose device, such as a device configured to deliver multiple predetermined doses of a drug, sometimes referred to as a multiple fixed dose device or a fixed dose, multi-shot device.

[0119] In one embodiment, the pharmaceutical compositions of the present invention are administered using an injection device comprising a cannula having a needle gauge of 20 or greater.

[0120] In one embodiment, a bispecific antibody according to Table 1 herein is administered using an injection device comprising a cannula with a needle gauge of 20 or greater.

[0121] In one embodiment, the bispecific antibody according to Table 1 herein is administered using an injection device comprising a tubing with a needle gauge of 20 to 36. In one such 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.

[0122] Administration and dosage

[0123] The compounds of the present invention, such as antibodies, can be administered parenterally, such as intravenously, intramuscularly, or subcutaneously. Alternatively, the antibodies of the present invention can be administered non-parenterally, 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 (as needed).

[0124] The dose of the compound to be delivered can 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, weekly, biweekly or monthly as loading and maintenance doses, depending on the severity of the condition. The appropriate dose can also be adjusted for a particular compound based on the properties of the compound, including its half-life or mean residence time in the body and its biological activity. For example, in one embodiment, the compound to be delivered can be administered once a week, or in another embodiment, once every other week, or in another embodiment, once a month, and in any of the embodiments, at a dose of, for example, 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 / kg body weight.

[0125] Compositions containing the compounds disclosed herein can be administered for prophylactic treatment and / or in some embodiments for therapeutic treatment. In therapeutic applications, the composition is administered to a subject already suffering from a disease, such as any of the bleeding disorders described above, in an amount sufficient to cure, alleviate, or partially prevent the disease and its complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount." As will be understood by those skilled in the art, the amount effective for this purpose will depend on the severity of the disease or injury and the weight and general condition of the subject.

[0126] Implementation Plan

[0127] The present invention is further described by the following embodiments:

[0128] 1. An antibody or antigen-binding fragment thereof capable of binding to Factor IX (FIX) according to SEQ ID NO: 89 and / or its activated form (FIXa).

[0129] 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antibody is a Fab.

[0130] 3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises

[0131] the CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 25 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 29; or

[0132] the CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 33 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 37; or

[0133] the CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 41 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 45; or

[0134] The CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 49 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 53; or

[0135] the CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 57 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 61; or

[0136] the CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 65 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 69; or

[0137] the CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 73 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 77; or

[0138] The CDR sequence of the heavy chain variable domain is represented by SEQ ID NO: 81 and the CDR sequence of the light chain variable domain is represented by SEQ ID NO: 85.

[0139] 4. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises

[0140] The heavy chain variable domain represented by SEQ ID NO: 25 and the light chain variable domain represented by SEQ ID NO: 29; or

[0141] The heavy chain variable domain represented by SEQ ID NO: 33 and the light chain variable domain represented by SEQ ID NO: 37; or

[0142] The heavy chain variable domain represented by SEQ ID NO: 41 and the light chain variable domain represented by SEQ ID NO: 45; or

[0143] The heavy chain variable domain represented by SEQ ID NO: 49 and the light chain variable domain represented by SEQ ID NO: 53; or

[0144] The heavy chain variable domain represented by SEQ ID NO: 57 and the light chain variable domain represented by SEQ ID NO: 61; or

[0145] The heavy chain variable domain represented by SEQ ID NO: 65 and the light chain variable domain represented by SEQ ID NO: 69; or

[0146] The heavy chain variable domain represented by SEQ ID NO: 73 and the light chain variable domain represented by SEQ ID NO: 77; or

[0147] The heavy chain variable domain is represented by SEQ ID NO: 81 and the light chain variable domain is represented by SEQ ID NO: 85.

[0148] 5. An antibody or antigen-binding fragment thereof that is capable of binding to FX (SEQ ID NO: 90) and / or its activated form (FXa).

[0149] 6. The antibody of embodiment 5, wherein the antibody is a Fab.

[0150] 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 5 or 6, wherein the antibody or antigen-binding fragment thereof comprises

[0151] the CDR sequence of the heavy chain variable domain represented by SEQ ID NO: 1 and the CDR sequence of the light chain variable domain represented by SEQ ID NO: 5;

[0152] or

[0153] The CDR sequence of the heavy chain variable domain is represented by SEQ ID NO: 17, and the CDR sequence of the light chain variable domain is represented by SEQ ID NO: 21.

[0154] 8. The antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof comprises

[0155] The heavy chain variable domain represented by SEQ ID NO: 1 and the light chain variable domain represented by SEQ ID NO: 5; or

[0156] The heavy chain variable domain is represented by SEQ ID NO: 17 and the light chain variable domain is represented by SEQ ID NO: 21.

[0157] 9. A multispecific antibody or antigen-binding fragment thereof capable of binding to FIX according to SEQ ID NO: 89 or its activated form (FIXa) and FX (SEQ ID NO: 90) or its activated form (FXa).

[0158] 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 1-4.

[0159] 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 5-8.

[0160] 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 1-4, and the antigen-binding fragment according to any one of the preceding embodiments 5-8.

[0161] 13. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-12, comprising

[0162] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 28, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0163] The anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 32, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0164] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0165] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0166] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 28, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0167] The anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 32, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0168] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0169] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0170] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 36, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0171] The anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 40, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0172] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0173] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0174] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 36, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0175] The anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 40, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0176] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0177] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0178] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 44, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0179] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 48, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0180] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0181] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0182] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 44, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0183] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 48, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0184] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0185] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0186] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 52, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0187] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 56, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0188] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0189] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0190] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 52, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0191] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 56, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0192] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0193] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0194] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 60, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0195] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 64, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0196] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0197] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0198] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 60, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0199] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 64, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0200] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0201] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0202] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 68, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0203] The anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 72, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0204] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0205] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0206] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 68, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0207] The anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 72, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0208] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0209] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0210] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 76, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0211] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 80, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0212] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0213] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0214] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 76, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0215] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 80, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0216] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0217] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0218] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 84, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0219] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 88, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0220] an anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 20, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0221] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 24, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions; or

[0222] an anti-FIX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 84, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0223] an anti-FIX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 88, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0224] The anti-FX(a) antibody heavy chain CDR3 sequence represented by SEQ ID NO: 4, which optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0225] The anti-FX(a) antibody light chain CDR3 sequence represented by SEQ ID NO: 8 optionally comprises 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions.

[0226] 14. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-13, comprising

[0227] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 26, 27, and 28, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0228] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 30, 31 and 32, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0229] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0230] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0231] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 26, 27, and 28, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0232] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 30, 31 and 32, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0233] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0234] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0235] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 34, 35, and 36, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0236] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 38, 39, and 40, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0237] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0238] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0239] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 34, 35, and 36, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0240] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 38, 39, and 40, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0241] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0242] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0243] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 42, 43, and 44, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0244] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 46, 47, and 48, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0245] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0246] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0247] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 42, 43, and 44, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0248] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 46, 47, and 48, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0249] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0250] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0251] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 50, 51 and 52, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0252] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 54, 55, and 56, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0253] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0254] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0255] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 50, 51 and 52, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0256] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 54, 55, and 56, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0257] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0258] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0259] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 58, 59 and 60, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0260] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 62, 63, and 64, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0261] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0262] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0263] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 58, 59 and 60, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and

[0264] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 62, 63, and 64, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0265] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0266] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0267] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 66, 67, and 68, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0268] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 70, 71, and 72, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0269] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0270] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0271] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 66, 67, and 68, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0272] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 70, 71, and 72, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0273] The anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0274] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0275] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 74, 75, and 76, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0276] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 78, 79, and 80, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0277] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0278] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0279] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 74, 75, and 76, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0280] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 78, 79, and 80, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0281] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0282] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0283] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 82, 83, and 84, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0284] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 86, 87, and 88, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0285] Anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 18, 19, and 20, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0286] The anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions; or

[0287] The anti-FIX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 82, 83, and 84, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0288] The anti-FIX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 86, 87, and 88, respectively, optionally comprising 1, 2, or 3 amino acid substitutions and / or deletions and / or insertions, and

[0289] The anti-FX(a) antibody heavy chain CDR1-3 sequences represented by SEQ ID NOs: 2, 3 and 4, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions, and the anti-FX(a) antibody light chain CDR1-3 sequences represented by SEQ ID NOs: 6, 7 and 8, respectively, optionally comprising 1, 2 or 3 amino acid substitutions and / or deletions and / or insertions.

[0290] 15. The multispecific antibody or antigen-binding fragment thereof according to embodiment 14, wherein the antibody is a bispecific antibody capable of specifically binding to FIX(a) and FX(a), wherein the binding domain is a binding domain of a mAb pair consisting of:

[0291] 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.

[0292] 16. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-15, wherein the antibody or antigen-binding fragment thereof is a procoagulant antibody.

[0293] 17. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-16, wherein the antibody or antigen-binding fragment thereof is capable of increasing the enzymatic activity of FIXa on FX.

[0294] 18. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-17, wherein the antibody or antigen-binding fragment thereof is capable of functionally replacing FVIII and / or FVIIIa.

[0295] 19. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-18, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody.

[0296] 20. The antibody according to any one of the preceding embodiments, wherein the antibody isotype is IgG1, IgG2, IgG3 or IgG4 or a combination thereof, such as comprising IgG1 F C IgG4 F C region of the antibody, and optionally in C H 3 domains contain 1 or 2 substitutions.

[0297] 21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, and optionally one or more pharmaceutically acceptable carriers.

[0298] 22. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to embodiment 21 for use in the treatment of a coagulopathy or coagulation disorder, such as hemophilia A with or without inhibitors.

[0299] 23. The antibody or antigen-binding fragment thereof or composition according to any one of the preceding embodiments, for use in a method of treating a coagulopathy or coagulation disorder, such as on-demand treatment or prophylactic treatment.

[0300] 24. The antibody or antigen-binding fragment thereof or composition according to any one of the preceding embodiments, for use in the treatment of hemophilia A with or without inhibitors, such as on-demand treatment or prophylactic treatment.

[0301] 25. A method of treating a subject having a coagulopathy or coagulation disorder, comprising administering to the subject the antibody or antigen-binding fragment thereof or composition according to any one of the preceding embodiments.

[0302] 26. The method of embodiment 25, wherein the coagulopathy or coagulopathy is A

[0303] Hemophilia A or hemophilia B with inhibitors.

[0304] 27. Use of the antibody or antigen-binding fragment thereof or composition according to any one of embodiments 1-20 in the preparation of a medicament for treating a subject in need thereof, such as on-demand treatment or prophylactic treatment.

[0305] 28. Use of the antibody or antigen-binding fragment thereof or composition according to any one of embodiments 1-21 in the preparation of a medicament for treating hemophilia A with or without inhibitors, such as on-demand treatment or prophylactic treatment.

[0306] 29. A eukaryotic cell expressing the antibody or antigen-binding fragment thereof according to any one of embodiments 1-20.

[0307] 30. A kit comprising the antibody or antigen-binding fragment thereof or composition according to any one of embodiments 1-21 and instructions for use.

[0308] 31. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-8, wherein the antibody or antigen-binding fragment thereof is a component (intermediate) for use in a multispecific antibody such as a procoagulant bispecific antibody.

[0309] 32. The antibody or antigen-binding fragment thereof according to any one of embodiments 1-8, wherein the antibody or antigen-binding fragment thereof is a component (intermediate) for preparing a multispecific antibody such as a procoagulant bispecific antibody.

[0310] 33. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-20, such as a procoagulant bispecific antibody, wherein the procoagulant activity of the antibody is improved compared to the multispecific antibodies disclosed in WO2018 / 141863 and WO2019 / 065795.

[0311] 34. The multispecific antibody or antigen-binding fragment thereof according to embodiment 33, wherein the improvement is determined using an assay as disclosed herein, such as the HA-PPP TGT assay (as described in Example 7 herein).

[0312] 35. The multispecific antibody or antigen-binding fragment thereof according to any one of embodiments 9-20, wherein the antibody or antigen-binding fragment thereof is capable of providing the following mean peak thrombin (in nM) at a compound concentration of 700 nM in a TGT assay (in HA-PPP) according to Example 7 herein:

[0313] a) when tissue factor is used 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

[0314] b) at least 350, 355, 360, 365, 370, 375, 380, 385, or 390 when FXIa is used as the trigger.

[0315] 36. The multispecific antibody or antigen-binding fragment thereof of any one of embodiments 9-20, wherein the antibody or antigen-binding fragment thereof has equivalent FVIII activity as determined according to Example 8 herein, which is improved relative to emicizumab and the multispecific antibodies disclosed in WO2018 / 141863 and WO2019 / 065795 (each incorporated herein by reference).

[0316] 37. The antibody of any one of embodiments 9-20, wherein the antibody isotype is IgG4, optionally in one C H 3 domains contain 1 or 2 substitutions.

[0317] 38. An injection device comprising the antibody or antigen-binding fragment thereof or composition according to any one of embodiments 9-20.

[0318] 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.

[0319] 40. The injection device of embodiment 39, wherein the device is a prefilled pen.

[0320] 41. The injection device of embodiments 39-40, wherein the device is a multi-dose pen.

[0321] 42. The injection device of any one of embodiments 38-41, wherein the injection device comprises a tube having a needle gauge of 20 to 36.

[0322] 43. The multispecific antibody or antigen-binding fragment thereof of embodiment 13 or 14, wherein the substitution is a conservative substitution.

[0323] In one embodiment, when the multispecific antibodies, such as bispecific antibodies, of the invention are used to treat hemophilia A at clinically relevant doses, the antibodies do not interfere with the action of FVIII, such as recombinant FVIII, administered to hemophilia A patients.

[0324] In one embodiment, the antibodies or antigen-binding fragments thereof of the invention, when present at 43.64 μg / mL plasma, correspond to at least 20-50, such as 20-40, such as 25-35, such as 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 in patients suffering from hemophilia A.

[0325] In one embodiment, the antibodies or antigen-binding fragments thereof of the invention, when present at 30 μg / mL of plasma, are equivalent to at least 10-50, such as 15-40, such as 15-30, such as 15-20, such as 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 in patients suffering from hemophilia A.

[0326] In one embodiment, the antibodies or antigen-binding fragments thereof of the invention, when present at 15 μg / mL of plasma, are equivalent to at least 10-50, such as 15-40, such as 15-30, such as 15-20, such as 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 in patients suffering from hemophilia A.

[0327] In one embodiment, the antibodies or antigen-binding fragments thereof of the invention have reduced immunogenicity compared to procoagulant antibodies in the art.

[0328] In one embodiment, the antibodies or antigen-binding fragments thereof of the invention are used for the prophylactic treatment of hemophilia A with or without inhibitors.

[0329] In one embodiment, the antibodies or antigen-binding fragments thereof of the present invention are capable of stimulating the enzymatic activity of FIXa towards FX.

[0330] In one embodiment, the anti-FIX(a) antibodies or antigen-binding fragments thereof listed in Table 2 of Example 6 are capable of stimulating the enzymatic activity of FIXa towards FX.

[0331] In a preferred embodiment, the antibodies of the invention have an IgG4 / κ format, optionally comprising one or more substitutions in the Fc constant region.

[0332] Heavy chain constant domain region (C H 1-C H 2-C H 3) for anti-FIX(a) arm human IgG4 with S228P (EU-numbering) substitution and with C-terminal lysine truncation:

[0333]

[0334] In one embodiment, the heavy chain constant domain region (C H 1-C H 2-C H 3) for anti-FX(a) arm human IgG4 with S228P substitution and in C H The 3 domain has two additional substitutions: F405L and R409K (EU numbering) to promote heterodimerization of the heavy chain (described in Example 4), and has a truncation of the C-terminal lysine:

[0335]

[0336] And, the light chain constant region (CL) is human kappa:

[0337]

[0338] In another embodiment, the antibody can also be expressed in an IgG4 format having the heavy chain constant domain region (C) for the anti-FIX(a) arm with S228P, F405L, and R409K substitutions. H 1-C H 2-C H 3), and the heavy chain constant domain region for an anti-FX(a) arm with an S228P substitution, with or without a C-terminal lysine deletion.

[0339] In one embodiment, the antibody can also be expressed in IgG1 / κ format. In this case, the heavy chain constant domain region of the anti-FIX(a) arm is human IgG1 F405L, which has a truncation of the C-terminal lysine:

[0340]

[0341] And the heavy chain constant domain region of the anti-FX(a) arm is human IgG1 K409R, which has a truncation of the C-terminal lysine:

[0342]

[0343]

[0344] The antibody can also be expressed in IgG1 format, which has the heavy chain constant domain region for the anti-FIX(a) arm with a K409R substitution (C H 1-C H 2-C H 3), and the heavy chain constant domain region for an anti-FX(a) arm with an F405L substitution, with or without a C-terminal lysine deletion.

[0345] The constant domain regions may further contain additional substitutions or other modifications, for example, to modulate effector function, half-life, or other properties.

[0346] In one embodiment, the potency of bispecific antibodies capable of binding to FIX(a) and FX(a), such as, but not limited to, those disclosed herein, can be determined in a chromogenic potency assay comprising the following components: a) lyophilized human factor X in the presence of a fibrin polymerization inhibitor, b) human factor IXa, c) human thrombin, d) calcium, and e) synthetic phospholipids, f) a chromogenic substrate specific for factor Xa (SXa-11) (Hyphen Biomed) and the anti-FIX / anti-FX bispecific antibody to be evaluated. For any dilution, a suitable buffer, such as Tris-BSA, can be used. In such an assay, the level of FX activation depends on the potency of the bispecific antibody. Activated FX (FXa) hydrolyzes the chromogenic substrate, thereby releasing pNA, thereby allowing a spectrophotometric readout at 405 nm to be obtained (e.g., using a Tecan Sunrise ELISA plate reader). This readout depends on the FXa concentration and is therefore proportional to the potency of the bispecific antibody being tested. A bispecific reference antibody with a predetermined potency should be included.

[0347] The present disclosure also provides a kit comprising an antibody or antigen-binding fragment thereof as disclosed herein that is suitable for treatment as described herein. In some embodiments, the kit comprises (i) an antibody as disclosed herein, such as a bispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition, or an encoding nucleic acid or vector, or a combination thereof, and (ii) instructions for use. It will be readily appreciated by those skilled in the art that antibodies, bispecific molecules (e.g., bispecific antibodies) and pharmaceutical compositions, or encoding nucleic acids or vectors, or a combination thereof, as disclosed herein, can be easily incorporated into an established kit format well known in the art.

[0348] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Example

[0349] List of abbreviations

[0350] ACN: Acetonitrile

[0351] bimAb: bispecific monoclonal antibody

[0352] CDR: Complementarity determining region

[0353] LC-MS liquid chromatography-mass spectrometry

[0354] FACS: Fluorescence-activated cell sorting

[0355] FIX: Coagulation factor IX

[0356] FIXa: Coagulation factor IXa

[0357] FX: Coagulation factor X

[0358] FXa: Coagulation factor Xa

[0359] HA: Hemophilia A

[0360] HA-PPP: HA-induced human platelet-poor plasma

[0361] hFIXa: human coagulation factor IXa

[0362] HPLC: High Performance Liquid Chromatography

[0363] mAb: monoclonal antibody

[0364] MACS: Magnetic-activated cell sorting

[0365] PCR: polymerase chain reaction

[0366] SEC: Size Exclusion Chromatography

[0367] SIA: sequence identical analogs

[0368] SPR: Surface Plasmon Resonance

[0369] Example 1: Development of anti-FIX(a) and anti-FX(a) antibodies

[0370] Various antibody development methods were used to identify FIX(a) and FX(a) binding antibodies as disclosed herein. To generate a diverse panel of antibodies, mice and rabbits were immunized, and phage display and Adimab yeast antibody expression platforms were also employed.

[0371] Adimab yeast antibody platform

[0372] The Adimab platform is a yeast antibody expression system that includes a fully human naive IgG1 / κ library with a diversity of 10 10 The antibody selection process was guided by a MACS and FACS-based method that allowed real-time monitoring of the applied selection criteria. Since the selection was based on MACS and FACS, a labeled antigen (e.g., biotin) was required. Selection activities were performed using biotin-labeled active site-inhibited hFIXa (FIXa-EGR-biotin) or antibody-mediated hFIXa immobilization. The binding of hits was evaluated using Bio-layer interferometry (Octet fortebio system).

[0373] Phage display

[0374] The antibody phage display platform used is a proprietary fully human Fab display library. The size of the library is 10 10 , and was constructed by a combinatorial approach utilizing chemical synthesis of light and heavy chain CDR1 and CDR2 supplemented by PCR amplification of heavy chain CDR3 from human peripheral blood mononuclear cells. In order to maximize epitope diversity, different panning strategies were explored, including panning with biotinylated FIXa-EGR, FX, active site-inhibited FXa, or antigen capture using anti-FIXa antibodies. Initial hits were identified by phage ELISA. After sequence analysis, unique hits were cloned and recombinantly expressed as IgG1 antibodies and ranked using SPR (Biacore) or Bio-layer interferometry (Octet fortebio system).

[0375] In vivo platform

[0376] Using in vivo platform, mice and rabbits are used for the generation of antibodies. In order to generate anti-FIX / FIXa antibodies, standard protocols are used to immunize mice or rabbits with human FIXa, FIXa-EGR or FIX. Standard techniques are used to fuse splenocytes from mice with myeloma cells, and ELISA is used for the resulting antibodies contained in the hybridoma supernatant for the combination screening with FIXa. By gating the cells (detected by a streptavidin-conjugated fluorophore) of random binding biotinylated FIXa-EGR, FACS is used to carry out single cell sorting of the rabbit B cells in conjunction with FIXa. Using the conditioned medium of feeder cells and splenocytes, the rabbit B cells sorted are cultivated for 7 days in 384w plates, then screened for FIXa in ELISA. Rabbit B cells and mouse hybridoma clones expressing FIXa binding antibody hits are used for VH / VL sequencing, subsequently recombinant expression (for rabbit or hybridoma mAb), or further propagated to produce mAb (mouse hybridoma).

[0377] In order to generate anti-FX antibodies, mice and rabbits were immunized with FX using a standard protocol. Rabbit B cells were isolated by single cell sorting based on FACS and using random biotinylated FX (detected by a streptavidin-conjugated fluorophore), while splenocytes from immunized mice were used for standard hybridoma development. Using ELISA and Octet fortebio systems, the resulting B cells or mouse hybridoma clones that produced antibodies were screened for FX binding. Rabbit B cells or mouse hybridoma clones expressing antibody hits were used for VH / VL sequencing, followed by recombinant expression (for rabbit or hybridoma mAb), or further propagated to produce mAb (mouse hybridoma).

[0378] Sequencing of hybridoma-derived antibodies

[0379] Hybridomas producing anti-FIXa and anti-FX antibodies were sequenced and expressed in HEK293 cells using standard techniques. Antigen binding of the expressed antibodies was evaluated using the Octet fortebio system.

[0380] Total RNA was extracted from antibody-producing clones and the variable domains (V H and V L ) encoding DNA sequence. H and V L The sequence was inserted into a pTT-based mammalian expression vector (Durocher et al. (2002) Nucleic Acid Res. 30: E9) or into a pcDNA3.4 mammalian expression vector (Invitrogen) containing the antibody constant region encoding DNA sequence. For the pTT / pcDNA3.4 mAb expression vector, V Hand V L DNA sequences were compared with human IgG1 or IgG4 S228P (C H 1C H 2C H 3, optionally with additional amino acid substitutions and deletions, such as in C H 3 domains and deletion of the C-terminal lysine) or human C L The kappa constant region encoding DNA sequence was inserted in frame. H DNA sequence and human IgG4 C H 1 The coding DNA sequence is inserted in frame.

[0381] Example 2: Recombinant expression of antibodies and antibody Fab fragments

[0382] Essentially following the manufacturer's instructions, transient transfection of HEK293 suspension cells (293Expi, Invitrogen) was used to express antibodies and antibody Fab fragments. 293Expi cells were typically subcultured 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 using Expifectamine at a cell density of 2.5-3 million / mL. For each liter of Expi293F cells, a total of 1 mg of plasmid DNA (1:1 ratio of V H -C H 1 (for Fab) or V H -C H 1-C H 2-C H 3 (for mAb) and LC plasmid) were diluted into 50 mL Optimem (GIBCO, catalog number 51985-026, diluent A) and 2.7 mL Expifectamine was diluted into 50 mL Optimem (diluent B) for transfection. For co-transfection of Fab and mAb production, V was used at a ratio of 1:1, respectively. H -C H 1 and LC plasmids (Fab) and V H -C H 1-C H 2-C H3 and LC plasmid (mAb). Diluents A and B were mixed and incubated at room temperature for 10-20 minutes. The transfection mixture was then added to Expi293F cells and the cells were incubated at 37°C in a humidified incubator 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. Cell culture supernatant was generally harvested 4-5 days after transfection by centrifugation and subsequent filtration.

[0383] Example 3: Purification and characterization of Fab and antibodies

[0384] All purification steps were performed at 4°C. For 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.

[0385] Fab was captured using HiTrap Protein G HP affinity chromatography using binding buffer in 1× PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl) (pH 7.4). Elution was performed in one step using 0.1 M glycine (pH 2.8). The final product was desalted to a pH 7.4 formulation buffer (25 mM HEPES, 150 mM NaCl) using a 52 mL GE Hiprep 16 desalting column and concentrated using a centrifugal ultrafilter (30 KD CO) and stored at -80°C.

[0386] To assess the quality of the purified Fab, SDS-PAGE and high performance size exclusion chromatography (SE-HPLC) analysis were performed. Batches that did not meet quality standards (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 weights (MW) of all Fabs were consistent with the theoretical MW of the heavy and light chains, respectively.

[0387] Antibody purification and characterization

[0388] Antibody purification was performed 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-scale production, Chromatography system (GE Healthcare, catalog number 18-1112-41). The buffer system used for the affinity purification step was 1) an equilibration buffer consisting of 20 mM sodium phosphate pH 7.2, 150 mM NaCl, and 2) an elution buffer consisting of 10 mM formic acid pH 3.5, and 3) a pH adjustment buffer consisting of 0.4 M sodium phosphate pH 9.0. The cell supernatant was directly loaded onto a pre-equilibrated MabSelect SuRe column without any adjustment. The column was washed with approximately 10 column volumes of equilibration buffer, and the antibody was isocratically eluted with approximately 2-5 column volumes of elution buffer. Immediately after elution, the pH of the combined fractions was adjusted to neutral using the pH adjustment buffer.

[0389] The purified antibodies were characterized using different methods such as SDS-PAGE / Coomassie, size exclusion high pressure 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). All antibodies showed the expected light and heavy chain components. Intact molecular weight determination was performed using a liquid chromatography electrospray ionization time-of-flight mass spectrometry setup on an Agilent 6210 instrument and a desalting column MassPREP (Waters, catalog number USRM10008656). The buffer system used was an equilibrium buffer consisting of 0.1% formic acid in LC-MS grade H2O and an elution buffer consisting 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 reducing agents (i.e., mercaptoethanol or DTT). All antibodies showed intact molecular weight consistent with the sequence and one heavy chain N-glycan. Purity was determined based on SE-HPLC. Final protein purity was analyzed based on the SE-HPLC method setup 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 consisting of 200 mM sodium phosphate pH 6.9, 300 mM NaCl, and 10% isopropanol. UV280 and fluorescence (excitation 280 nm / emission 354 nm) detectors were used for detection. The antibodies eluted as a single symmetrical peak, and the retention time reflected the size of the antibody. The purity estimates for the different antibodies were between 95-99%. To determine the final protein concentration, a NanoDrop spectrophotometer (Thermo Scientific) and the specific extinction coefficient for each antibody were used.

[0390] Example 4: Bispecific Antibodies Prepared by In Vitro Assembly

[0391] For bispecific human IgG1 antibodies described by (Labrijn et al. PNAS 2013, vol. 110, pp. 5145-5150) Methods (Genmab), while using slightly modified variants for bispecific human IgG4 antibodies, bispecific antibodies were generated by in vitro assembly of the first and second antibodies, as described in detail below.

[0392] For IgG1, the heavy chain constant region of the first antibody is human IgG1 K409R (anti-FIX / FIXa), and the heavy chain constant region of the second antibody is human IgG1 F405L (anti-FX / FXa). As previously described, the IgG1 may be an IgG1 variant with reduced effector function.

[0393] For human IgG4, the heavy chain constant region of the first antibody was IgG4 S228P (anti-FIX / FIXa), and the heavy chain constant region of the second antibody was IgG4 S228P F405L+R409K (anti-FX). These two parent antibodies were produced as described in Examples 1-3. Fab arm exchange reactions were performed under reducing conditions using 75 mM 2-mercaptoethylamine (2-MEA) in HEPES buffer (pH 7.4) and incubated at 30°C for 4 hours.

[0394] Example 5: Preparation of monovalent (one-arm) antibodies

[0395] To avoid any potential avidity effects associated with conventional monospecific and bivalent antibodies, for example, in the FXa production 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), in which a complete heavy chain, a truncated heavy chain (lacking the Fab region), and a light chain were co-expressed. Instead of co-expressing the three chains described by Martens et al., a single heavy chain (100 μg / mL) was used, as described for bispecific antibodies (Example 4). Principle Preparation of the monovalent antibodies of the present invention. Therefore, monovalent antibodies are prepared by mixing intact monospecific and bivalent antibodies with truncated heavy chain dimers (formally derived from intact antibodies by removing the Fab region) and allowing chain exchange to proceed under the same experimental conditions as described in Example 4. The formation of monovalent antibodies requires that the antibody and the truncated heavy chain dimer carry appropriate complementary mutations to promote heterodimerization, i.e., F405L / K409R for human IgG1 and F405L+R409K / WT for human IgG4, as described in Example 4.

[0396] In the case of a monovalent antibody of the IgG1 subtype, the heavy chain may be truncated from the N-terminus to a position between Cys 220 and the upper hinge Cys 226 (EU numbering). A specific example of a truncated human IgG1 heavy chain is an IgG1 heavy chain in which residues 1-220 are truncated.

[0397] In the case of a monovalent antibody of the human IgG4 subtype, the heavy chain may be truncated from the N-terminus to a position between Cys 200 and the upper hinge Cys 226 (EU numbering). A specific example of a truncated human IgG4 heavy chain is an IgG4 heavy chain in which residues 1-214 are truncated.

[0398] Example 6: Summary of Bispecific Antibody (Component) IDs and SEQ ID NOs

[0399] Table 1: Overview of bispecific antibody components and corresponding VH / VL SEQ ID NOs

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406] Example 7: Activity of bispecific anti-FIX(a) / FX(a) antibodies in a thrombin generation assay (TGT) in human hemophilia A platelet-poor plasma

[0407] The procoagulant activity of bispecific antibodies bimAb6B, bimAb5B, bimAb4B, bimAb3B, bimAb2B, bimAb1B, bimAb8B, bimAb7B, bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A, bimAb7A and bimAb8A was determined based on their ability to promote thrombin generation in the presence of procoagulant synthetic phospholipid membranes according to the principles described by Hemker et al. (Pathophysiol Haemost Thromb, 2002; 32: 249-253). An emicizumab sequence identity analog (SIA) was included for comparison. Each bispecific antibody (test compound) was tested in a thrombin generation assay (TGT) using normal human platelet-poor plasma (NHP) supplemented with neutralizing anti-FVIII polyclonal antibodies (hereinafter referred to as HA-PPP).

[0408] Materials and Methods

[0409] Thrombin generation test

[0410] Thrombin generation assay (TGT) in NHP (from healthy volunteers) supplemented with sheep anti-human FVIII polyclonal antibody (pAb, Haematologic Technologies Inc., VT, USA) was performed using a 96-well plate fluorimeter (Fluoroscan Ascent FL, Thermolabsystems, Helsinki, Finland) by standard-calibrated automated coagulation curve method (thrombography). The reaction mixture contained 36 μl NHP pre-incubated with 0.1 μg / ml anti-FVIII pAb, 4 μl test compound dilution (diluted in 20 mM HEPES, 140 mM NaCl (pH 7.4), 2% BSA), 10 μl of 1 pM tissue factor (TF, pppLow, from Thrombinoscope BV, Maastricht, the Netherlands) or 8.3 U / ml human factor XIa (Enzyme Reseach Laboratories, IN, USA) and 10 μl FluCa substrate (Thrombinoscope BV, Maastricht, the Netherlands). Use thrombin calibrator (Thrombinoscope BV, Maastricht, the Netherlands) to calibrate TGT, wherein 10 μ l thrombin calibrator and 36 μ l NHP, 4 μ l buffer (20mM HEPES, 140mM NaCl, pH 7.4, 2% BSA) mixed with 0.1 μ g / ml anti-FVIII pAb pre-incubated. TGT is carried out under the test compound (0.32, 0.96, 2.88, 8.64, 25.9, 77, 233 and 700nM, final plasma concentration) of 8 kinds of concentration or only adds buffer (20mM HEPES, 140mM NaCl, pH 7.4, 2% BSA) (representative HA controls). Test concentration range in at least three independent experiments in the HA-PPP from identical storing solution. Only use the buffer (20mM HEPES, 140mM NaCl, pH 7.4, 2% BSA) that has added NHP to measure the normal control level in TGT. TGT was allowed to proceed for a total of 60 minutes and the TGT parameter peak thrombin height (nM) was analyzed by Thrombinoscope software (Thrombinoscope BV).

[0411] Results and Discussion

[0412] Table 4 and Table 5 show the peak thrombin generation rate that every kind of bispecific antibody is recorded under the concentration tested in the HA-PPP that triggers with tissue factor and people FXIa respectively.Data display, all test compounds all make peak thrombin formation be increased to the level that is higher than observing when not having antibody, i.e. show procoagulant activity.In addition, when bimAb concentration is higher than 8.64nM and uses 1pM tissue factor triggering thing, bimAb6B, bimAb5B, bimAb4B, bimAb3B, bimAb2B, bimAb1B, bimAb8B, bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A and bimAb8A all show than the higher concentration-dependent thrombin generation rate observed for emicizumab SIA, thereby demonstrate better effectiveness.BimAb7B is better than emicizumab SIA when 2.88 to 233nM bimAb.

[0413] Furthermore, when coagulation was triggered with 8.3 mU / mL human FXIa, bimAb6B, bimAb5B, bimAb4B, bimAb3B, bimAb2B, bimAb1B, bimAb8B, bimAb7B, bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A, bimAb7A, and bimAb8A exhibited higher thrombin generation potential than that observed for emicizumab SIA at all concentrations tested.

[0414] Table 4: Thrombin generation test (TGT) in HA-PPP triggered with tissue factor

[0415] The thrombin generation test (TGT) of bispecific antibody bimAb6B, bimAb5B, bimAb4B, bimAb3B, bimAb2B, bimAb1B, bimAb8B, bimAb7B, bimAb1A, bimAb2A, bimAb3A, bimAb4A, bimAb5A, bimAb6A, bimAb7A and bimAb8A was tested in induced hemophilia A plasma (HA-PPP) triggered by human tissue factor (pppLow). The average peak thrombin generation level ± standard deviation measured at each test compound concentration in at least three independent experiments in HA-PPP was obtained. If measurement was abandoned due to poor data quality, standard deviation was omitted.

[0416]

[0417]

[0418]

[0419]

[0420]

[0421] Example 8: Equivalent FVIII activity of bispecific antibodies

[0422] To estimate the equivalent FVIII activity of bimAbs in vitro, a plasma-based thrombin generation assay was established to allow for a wide range of dose responses using peak thrombin levels for recombinant B-domain-truncated FVIII ranging from 1 to 100 IU / dL. The dose-response curve for recombinant B-domain-truncated FVIII was used as a standard curve for analyzing thrombin generation from bimAbs to estimate equivalent FVIII activity.

[0423] method:

[0424] Thrombin generation was measured as described in Example 7, however, 1 U / ml human factor XIa (Enzyme Research Laboratories, IN, USA) was used as a trigger. In addition, an 8-point dilution series of recombinant B-domain truncated FVIII (NovoEight, Novo Nordisk A / S) diluted two-fold downward from 100 IU / dL was used as a FVIII standard curve.

[0425] Determination of FVIII equivalent activity of bispecific antibodies:

[0426] The dose response data of recombinant B-domain truncated FVIII were fit to “[agonist] vs. response (three parameters)” (Equation 1) using the nonlinear analysis function of GraphPad Prism version 8.0.2:

[0427] Equation 1. Peak Thrombin = Bottom + [FVIII] * (Top - Bottom) / (EC50 + [FVIII])

[0428] where [FVIII] is the concentration of FVIII in IU / mL, EC50 is the concentration of FVIII that produces 50% activity, and bottom and top are the plateaus of the fit.

[0429] By solving for [FVIII], a modified equation (Equation 2) can be used to estimate the FVIII concentration that generates the same thrombin peak as a particular bispecific antibody:

[0430] Equation 2.

[0431] where Y is the measured peak thrombin.

[0432] result:

[0433] Non-linear fitting of the FVIII dose-response curves yielded the following constants: EC50 = 20.3 ± 2.73, Top = 476 ± 18.7 and Bottom = -22.6 ± 10.1. Using Equation 2, equivalent FVIII activity was estimated for the bispecific antibodies described in Example 6, see Table 6 below.

[0434] Table 6: Equivalent FVIII activity of bispecific antibodies.

[0435] Estimated FVIII equivalent activity of bimAbs tested at four different bimAb concentrations (10, 30, 100 and 300 nM, corresponding to 1.45, 4.36, 14.55 and 43.64 μg / mL, respectively). Results for FVIII equivalents (IU / dL) are expressed as mean ± SD (n=3).

[0436]

[0437]

[0438] Example 9: Activity of Monovalent Anti-FIX(a) Antibodies in FXa Generation Assay

[0439] In order to avoid any potential avidity effects due to the bivalence of conventional IgG antibody formats, after reformatting into a monovalent one-arm (OA) antibody format, the stimulatory activity of anti-FIX(a) antibodies for the enzymatic activity of FIXa against FX was determined (Example 5). The antibodies tested are listed in Table 7 below. The monovalent OA format of anti-FIXa antibody ACE910 was included for comparison.

[0440] The stimulatory activity of OA antibodies was determined 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) phospholipid vesicles (final concentration of 500 μM; Haematologic Technologies Inc, USA) and plasma-derived FIXa (final concentration of 0.025 or 0.1 nM; Haematologic Technologies Inc, USA). The concentration of FIXa was selected to ensure that less than 15% of the substrate FX was converted to FXa. After preincubation in the presence of monovalent OA antibodies (final concentrations listed in Table 7), 100 nM plasma-derived FX (Haematologic Technologies Inc, USA) was added to 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 quenching buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% PEG8000, pH 7.3 + 1 mg / ml BSA), and the amount of FXa generated was determined by further adding 25 μl of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) and measuring the conversion of the chromogenic substrate in a microplate reader by absorbance measurement at 405 nm (ΔOD / min). The measured activity was corrected for background activity by subtracting the signal measured in an identical assay except that FIXa and antibody were replaced with assay buffer, and then the activity was adjusted according to the concentration of FIXa present in the assay ([FIXa] 总 This value was divided by the similarly normalized FXa production rate in the absence of antibody (A FIXa，归-化 ), calculate the antibody stimulation index, and provide the stimulation multiple of the antibody to FIXa activity at the concentration used. Because free FIXa produces FXa slowly, as described above, the activation reaction in the absence of antibody is performed in the presence of 5, 10 or 20 nM FIXa. The measured activity is then background-subtracted and normalized according to the FIXa concentration in the test. To calculate the stimulation index, the average value of the three normalized activities of free FIXa is used.

[0441] Determination of stimulation index

[0442] In summary, the calculation of the stimulation index can be described as follows

[0443] Equation 3 Stimulation Index = ((A FIXa+OA -A 背景 ) / [FIXa]总 ) / A FIXa，归-化

[0444] Among them A FIXa+OA is the activity measured in the presence of OA antibodies, A 背景 is the background activity measured in the absence of FIXa and OA antibodies, [FIXa] 总 is the FIXa concentration in this assay, and A FIXa，归一化 is the mean normalized activity of free FIXa.

[0445] Determination of FIXa saturation

[0446] The fraction of FIXa that is saturated with OA antibody in this assay is determined by the concentrations of FIXa and OA antibody and the equilibrium dissociation constant (K) that governs their interaction. d The latter can be measured by techniques known in the art such as isothermal titration calorimetry (ITC).

[0447] Because the stimulation index will increase with increasing OA antibody concentration until FIXa saturation is reached, the concentration of OA antibody in the assay should be selected to ensure at least 80% saturation of FIXa in the assay to provide an appropriate measure of the stimulation index at complete FIXa saturation.

[0448] The fraction of FIXa bound to OA antibody at equilibrium (f FIXa+OA ) can be obtained from this assay using the quadratic binding equation as described by Krishnaswamy et al. (1992) J. Biol. Chem., 267: 23696-23706 and detailed in Equations 4 and 5 below. 总 ) and OA antibodies ([OA] 总 ) and the equilibrium dissociation constant (K d ) is calculated, in the equation

[0449] [FIXa+OA] 试验 represents the calculated concentration of FIXa-OA antibody complex at equilibrium in the assay

[0450] f FIXa+OA represents the calculated fraction of FIXa bound to the OA antibody at equilibrium in the assay (expressed as a percentage)

[0451] Equation 4

[0452]

[0453] Equation 5

[0454] The stimulation index for each OA antibody is provided in Table 7. In the case of the OA emicizumab antibody, FIXa stimulation was determined at eight different antibody concentrations, which allowed estimation of the stimulation index at full FIXa saturation using the quadratic binding equation as outlined above. This also provided an estimated equilibrium dissociation constant (K) of 1.1 μM for the interaction of emicizumab with FIXa. d ), 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, FIXa saturation is unknown, so the stimulation indices listed represent conservative estimates of stimulation obtained at FIXa saturation of 80% or greater. For the antibodies tested, the stimulation indices measured were found to be higher than those measured for OAemicizumab.

[0455] Table 7: Stimulation of FIXa activity by monovalent one-armed (OA) anti-FIXa antibodies

[0456] The anti-FIX mAb ID refers to the ID of the antibody used to reformat the OA form. The columns labeled "OA Antibody Concentration (nM)" and "Stimulation Index" list the concentration (nM) of the OA antibody used in the assay and the corresponding stimulation of FIXa activity measured relative to free FIXa. In the case of emicizumab, an estimated stimulation index at full FIXa saturation is provided.

[0457] Anti-FIX mAb ID OA antibody concentration (nM) Stimulation Index emicizumab saturation 1372 mAb1 1599 9841 mAb2 5640 14228 mAb3 749 8649 mAb4 1217 10537 mAb5 1463 11241 mAb6 3547 13510 mAb7 851 8727 mAb8 1276 10089

[0458] Example 10: SEC-HPLC analysis of nonspecific binding

[0459] Low nonspecific binding is an important characteristic of drug antibodies. A high tendency for nonspecific binding can lead to, for example, a shortened half-life in vivo ( I. et al., mAbs, 2012 and Avery, LB, mAbs, 2018) and reduced solubility (Kohli, N., mAbs, 2015 and Wolf Perez, AM, mAbs, 2019). To evaluate the nonspecific binding of the anti-FIX(a) mAbs listed in Table 8 below, we used an SEC-HPLC method, in which mAb-column interactions result in delayed elution. Therefore, prolonged retention time is a measure of nonspecific binding. We used a method previously described (Wolf Perez, AM, mAbs, 2019) and highly similar to the method described in (Dobson, CL, Sci Rep, 2016; 6: 38644). For BimAb1-8, nonspecific binding was reduced relative to the emicizumab VH / VL SIA prepared as described in Example 4, see Table 8.

[0460] Materials and Methods

[0461] 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 NaHPO, 78 mM NaHPO, 300 mM NaCl, and 4% 2-propanol, pH 6.8. Each analysis was run for 24 min at a flow rate of 0.8 mL / min and a column temperature of 28°C. 15 μl of protein solution was injected onto the column, and elution was followed by absorbance at 280 nm. Data were processed using Astra v.7 (Wyatt Technology).

[0462] Table 8: Changes in retention time

[0463]

Claims

1. A bispecific antibody or antigen-binding fragment thereof that is capable of binding to FIX and / or its activated form FIXa as shown in SEQ ID NO: 89 and is capable of binding to FX and / or its activated form FXa as shown in SEQ ID NO: 90, wherein the bispecific antibody comprises an anti-FIX(a) antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, and an anti-FX(a) antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, in a) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 26, 27 and 28, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 30, 31 and 32, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or b) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 26, 27 and 28, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 30, 31 and 32, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or c) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 34, 35 and 36, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 38, 39 and 40, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or d) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 34, 35 and 36, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 38, 39 and 40, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or e) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 42, 43 and 44, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 46, 47 and 48, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or f) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 42, 43 and 44, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 46, 47 and 48, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or g) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 50, 51 and 52, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 54, 55 and 56, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or h) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 50, 51 and 52, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 54, 55 and 56, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or i) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 58, 59 and 60, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 62, 63 and 64, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or j) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 58, 59 and 60, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 62, 63 and 64, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or k) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 66, 67 and 68, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 70, 71 and 72, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or 1) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 66, 67 and 68, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 70, 71 and 72, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or m) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 74, 75 and 76, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 78, 79 and 80, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or n) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 74, 75 and 76, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 78, 79 and 80, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 6, 7, and 8, respectively; or o) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 82, 83, and 84, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 86, 87, and 88, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 18, 19 and 20, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; or p) the heavy chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 82, 83, and 84, respectively, and The light chain of the anti-FIX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 86, 87, and 88, respectively, and The heavy chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises the CDR1-3 sequences represented by SEQ ID NOs: 2, 3, and 4, respectively, and The light chain of the anti-FX(a) antibody or antigen-binding fragment thereof comprises CDR1-3 sequences represented by SEQ ID NOs: 6, 7 and 8, respectively.

2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-FIX(a) antibody or antigen-binding fragment thereof comprises a. a heavy chain variable domain represented by SEQ ID NO: 25 and a light chain variable domain represented by SEQ ID NO: 29; or b. a heavy chain variable domain represented by SEQ ID NO: 33 and a light chain variable domain represented by SEQ ID NO: 37; or c. a heavy chain variable domain represented by SEQ ID NO: 41 and a light chain variable domain represented by SEQ ID NO: 45; or d. a heavy chain variable domain represented by SEQ ID NO: 49 and a light chain variable domain represented by SEQ ID NO: 53; or e. a heavy chain variable domain represented by SEQ ID NO: 57 and a light chain variable domain represented by SEQ ID NO: 61; or f. a heavy chain variable domain represented by SEQ ID NO: 65 and a light chain variable domain represented by SEQ ID NO: 69; or g. a heavy chain variable domain represented by SEQ ID NO: 73 and a light chain variable domain represented by SEQ ID NO: 77; or h. a heavy chain variable domain represented by SEQ ID NO: 81 and a light chain variable domain represented by SEQ ID NO: 85; and wherein the anti-FX(a) antibody or antigen-binding fragment thereof comprises i. a heavy chain variable domain represented by SEQ ID NO: 17 and a light chain variable domain represented by SEQ ID NO: 21; or ii. The heavy chain variable domain represented by SEQ ID NO: 1 and the light chain variable domain represented by SEQ ID NO:

5. The bispecific antibody or antigen-binding fragment thereof according to claim 1 , wherein the isotype of the bispecific antibody is IgG1 or IgG4. 4 . The bispecific antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody or antigen-binding fragment thereof is a procoagulant antibody or antigen-binding fragment thereof. 5 . The bispecific antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody or antigen-binding fragment thereof is capable of stimulating the enzymatic activity of FIXa.

6. A pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and one or more pharmaceutically acceptable carriers.

7. The bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that It is used to treat hemophilia.

8. The composition according to claim 6, characterized in that It is used to treat hemophilia.

9. The bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that It is used to treat hemophilia A with or without inhibitors.

10. The composition according to claim 6, characterized in that It is used to treat hemophilia A with or without inhibitors.

11. The bispecific antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that It is used for the prophylactic treatment of hemophilia A with or without inhibitors.

12. The composition according to claim 6, characterized in that It is used for the prophylactic treatment of hemophilia A with or without inhibitors. 13 . Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a medicament for treating hemophilia A with or without inhibitors.

14. Use of the composition according to claim 6 in the preparation of a medicament for treating hemophilia A with or without inhibitors.

15. Use according to claim 13 or 14, wherein the treatment is a prophylactic treatment.

16. An antibody or antigen-binding fragment thereof capable of binding to FIX and / or its activated form FIXa as shown in SEQ ID NO: 89, comprising a heavy chain and a light chain, wherein a) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 26, 27 and 28, respectively, and the light chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 30, 31, and 32, respectively; or b) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 34, 35 and 36, respectively, and the light chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 38, 39, and 40, respectively; or c) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 42, 43 and 44, respectively, and the light chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 46, 47, and 48, respectively; or d) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 50, 51 and 52, respectively, and the light chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 54, 55, and 56, respectively; or e) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 58, 59 and 60, respectively, and the light chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 62, 63, and 64, respectively; or f) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 66, 67 and 68, respectively, and the light chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 70, 71, and 72, respectively; or g) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 74, 75 and 76, respectively, and the light chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 78, 79, and 80, respectively; or h) the heavy chain comprises the CDR1-3 sequences represented by SEQ ID NOs: 82, 83 and 84, respectively, And the light chain comprises CDR1-3 sequences represented by SEQ ID NOs: 86, 87 and 88, respectively.

17. The antibody or antigen-binding fragment according to claim 16, wherein the antibody or antigen-binding fragment thereof is an intermediate for preparing a bispecific antibody that can bind to FIX and / or its activated form FIXa as shown in SEQ ID NO: 89, and can bind to FX and / or its activated form FXa as shown in SEQ ID NO:

90.

18. The antibody or antigen-binding fragment thereof according to claim 16 or 17, wherein the antibody or antigen-binding fragment thereof is a procoagulant antibody or antigen-binding fragment thereof.

19. The antibody or antigen-binding fragment thereof according to claim 16 or 17, wherein the antibody or antigen-binding fragment thereof is capable of stimulating the enzymatic activity of FIXa.

20. A kit comprising (i) the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, optionally contained in an injection device, and (ii) instructions for use.

21. A kit comprising (i) the composition of claim 6, optionally contained in an injection device, and (ii) instructions for use.

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