Blood coagulation antibodies

Abstract

To provide compounds which serve as a substitute for coagulation Factor VIII (FVIII) in patients suffering from a coagulopathy and in particular in patients lacking functional FVIII, such as haemophilia A patients, including haemophilia A patients with inhibitors.SOLUTION: The present invention relates to: improved procoagulant antibodies including bispecific antibodies capable of binding to coagulation Factor IX (FIX) or the activated form thereof Factor IXa (FIXa), and optionally Factor X (FX) and the activated form thereof Factor Xa (FXa), and promoting FX activation by FIXa; antibodies binding to their epitopes; and methods and compositions for treating subjects suffering from a coagulopathy such as haemophilia A.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Inclusion by referencing the sequence list The sequence listing titled "160163WO02_ST25" is 198 kilobytes in size, was created on January 30, 2018, and is incorporated herein by reference. [Background technology]

[0002] In patients with coagulation disorders, such as those with hemophilia A and B, various steps in the coagulation cascade become dysfunctional, for example, due to the absence or insufficient presence of functional coagulation factors. This dysfunction of certain parts of the coagulation cascade can lead to inadequate blood clotting and potentially life-threatening bleeding or damage to internal organs such as joints.

[0003] Hemophilia A, commonly known as factor VIII (FVIII) deficiency, is a congenital bleeding disorder that affects approximately 420,000 people worldwide, of which about 105,000 have been diagnosed.

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

[0005] However, such patients are at risk of developing neutralizing antibodies against these exogenous factors, so-called inhibitors, rendering previously effective treatments ineffective. Hemophilia A patients with inhibitors represent a non-limited example of coagulation disorders that are partly congenital and partly acquired. Patients who develop inhibitors against FVIII cannot be treated with conventional replacement therapy. Recently, a new drug called Hemlibra has been approved as a subcutaneous prophylactic treatment for hemophilia A with inhibitors. Exogenous coagulation factors can only be administered intravenously, which is considerably inconvenient and uncomfortable for patients. For example, infants and young children may require surgical insertion of an intravenous catheter into the thoracic vein to ensure access to the vein. This increases the risk of developing bacterial infections. Therefore, even with the introduction of Hemlibra, hemophilia with inhibitors still requires alternative subcutaneous treatment.

[0006] In bleeding individuals, when extravascular tissue factor (TF) is exposed to activated FVII (FVIIa) in the blood, coagulation is initiated by the formation of the tissue factor / factor VIIa (TF / FVIIa) complex. The formation of the TF / FVIIa complex involves the activation of coagulation factor X (FX) to activated coagulation factor Xa (FXa), which, together with activated coagulation factor V (FVa), produces a limited amount of thrombin, which then activates platelets. The activated platelets support the assembly of the tenase complex, which consists of activated factor VIII (FVIIIa) and activated coagulation factor IX (FIXa). The tenase complex is a highly efficient catalyst for FX activation, and the FXa produced in this second step functions as an active protease in the FVa / FXa prothrombinase complex, which is involved in the final thrombin burst. Thrombin cleaves fibrinogen to produce fibrin monomers, which polymerize to form a fibrin network, sealing the leaking vessel and stopping the bleeding. Rapid and widespread thrombin bursts are necessary conditions for solidified and stable fibrin thrombus formation.

[0007] Reduced or absent FVIII activity leads to improper FXa formation and decreased thrombin production, which underlie the bleeding diathesis in hemophilia A patients.

[0008] As mentioned, the proteolytic conversion of FX to its enzymatically active form, FXa, can be achieved by a unique FX activation complex containing FIXa and its cofactor FVIIIa. Cofactor binding increases the enzymatic activity of FIXa by approximately five orders of magnitude and is thought to occur through multiple mechanisms, as outlined by Scheiflinger et al. in (2008) J Thromb Haemost, 6:315-322. In particular, FVIIIa has been shown to stabilize the structure of FIXa with increased proteolytic activity against FX (Kolkman JA, Mertens K (2000) Biochemistry, 39:7398-7405, Zogg T, Brandstetter H (2009) Biol Chem, 390:391-400). Based on this observation, recognizing that antibodies are versatile binding proteins capable of mimicking various protein-protein interactions, Scheiflinger et al. screened for agonist anti-FIXa antibodies characterized by their ability to enhance FIXa-mediated FX activation in the presence of phospholipid surfaces and calcium, but in the absence of the intrinsic cofactor FVIIIa. Screening of hybridoma supernatants of 5280 revealed that 88 produces antibodies exhibiting varying degrees of FIXa agonist activity. See EP1220923 B1 and EP1660536 B1. Regarding the dynamics of FX activation and the ability to stimulate thrombinogenesis in FVIII-deficient human plasma, EP1660536 B1 consistently identifies 224F3 as the most efficient antibody (see, e.g., sections 0060 and 0062).

[0009] ACE910, or EmicizumAb (trade name Hemlibra®), is a humanized bispecific anti-FIX(a) / anti-FX(a) monoclonal antibody developed by Chugai Pharmaceutical for the treatment of hemophilia A. ACE910 is designed to mimic the function of FVIII cofactor (see Sampei et al.: (2013) PloS One, 8, e57479, and WO2012067176).

[0010] There are still many unmet medical needs, particularly in the hemophilia community and in subjects with coagulation disorders in general. The present invention relates to an improved compound that can replace FVIII and is therefore useful in treating coagulation disorders such as hemophilia A. [Overview of the project]

[0011] The present invention relates to a compound that can serve as a substitute for coagulation factor VIII (FVIII) in patients suffering from coagulation disorders, particularly in patients lacking functional FVIII, such as hemophilia A patients, including those with inhibitory substances.

[0012] Accordingly, one aspect of the present invention relates to a compound that enhances the production of FXa and thus can partially or completely restore coagulation in patients lacking FVIII.

[0013] In one embodiment, the compound is an antibody. In this embodiment, the compound is a multispecific antibody, such as a bispecific antibody.

[0014] In a particular embodiment, the present invention relates to a blood coagulation antibody that can serve as a substitute for FVIII in patients lacking FVIII, such as patients with hemophilia A.

[0015] In one such embodiment, the antibody binds to and increases the enzymatic activity of FIXa against FX, and optionally also binds to FX.

[0016] In one embodiment, the present invention relates to a blood coagulation antibody that binds to FX, comprising a bispecific blood coagulation antibody that increases the enzymatic activity of FIXa against FX and bound FX.

[0017] In one embodiment, the present invention relates to a blood coagulation bispecific antibody that can bind to coagulation FIX / FIXa and FX / FXa.

[0018] In one embodiment, the antibody is human or humanized.

[0019] Further aspects of the present invention relate to individual antibodies or antigen-binding fragments thereof that are part of a blood coagulation antibody, such as a specific anti-FIX or anti-FIXa antibody or its antigen-binding fragment. Further aspects of the present invention relate to individual antibodies or antigen-binding fragments thereof that are part of a blood coagulation antibody, such as a specific anti-FX or anti-FXa antibody or its antigen-binding fragment.

[0020] Further aspects of the present invention relate to the production of antibodies and intermediates thereof disclosed herein.

[0021] Further aspects of the present invention relate to antibodies that compete with the blood coagulation antibodies or antigen-binding fragments disclosed herein for binding to FIX / FIXa.

[0022] Further aspects of the present invention relate to blood coagulation antibodies that compete with the antibodies disclosed herein or this antigen-binding fragment for binding to FX / FXa.

[0023] Further aspects of the present invention relate to pharmaceutical compositions comprising a blood coagulation antibody disclosed herein, formulated for delivery of the antibody for the prevention and / or treatment of coagulation disorders.

[0024] Further aspects of the present invention relate to blood coagulation antibodies disclosed herein for the prevention and / or treatment of coagulation disorders, diseases involving coagulation disorders, or diseases caused by coagulation disorders.

[0025] The present invention may also solve further problems that become apparent from the disclosure of exemplary embodiments. [Brief explanation of the drawing]

[0026] [Figure 1-1] Figure 1 shows aligned sequences of sequence numbers 3-188, with complementarity-determining regions 1, 2, and 3 (CDR1, CDR2, and CDR3) of the heavy chain variable domain and light chain variable domain successively highlighted within the boxes. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above. [Figure 1-6] Same as above. [Figure 1-7] Same as above. [Figure 1-8] Same as above. [Figure 2] Figure 2 shows thrombin generation test (TGT) data from bispecific antibodies mAb4-7761, mAb4-7762, mAb4-7789, mAb5-0057, mAb5-1409, and ACE910 in human tissue factor-activated hemophilia A platelet-poor plasma (HA-PPP). The experiment was performed as described in Example 17. Dotted and dotted lines indicate the peak thrombin levels (nM) observed in the absence of anti-FVIII antibodies in HA-PPP and conventional PPP, respectively, and their standard deviations are shown by dotted lines. The profiles for mAb4-7761, mAb4-7762, mAb4-7789, mAb5-0057, and mAb5-1409 are indicated by upward-pointing triangles, while that of ACE910 is indicated by a downward-pointing triangle. Experiments A through D are independent experiments, and within each of these experiments, the peak thrombin level at each antibody concentration represents the mean ± standard deviation of at least three independent experiments. [Figure 3]Figure 3 shows thrombin generation assay (TGT) data from bispecific antibodies mAb5-0057, mAb5-1409, and ACE910 in human tissue factor-activated hemophilia A platelet-rich plasma (HA-PRP). Experiments were performed as described in Example 17. Dotted and dotted lines indicate peak thrombin levels (nM) observed in the absence of anti-FVIII antibodies in HA-PRP and conventional PRP, respectively, and their standard deviations are shown by dotted lines. The profiles for mAb5-0057 and mAb5-1409 are shown by upward-pointing triangles, while that of ACE910 is shown by a downward-pointing triangle. Results are shown as mean ± standard deviation from four independent experiments. [Figure 4] Figure 4 shows thrombin generation (TGT) data from monovalent one-armed (OA) antibodies mAb1-9016 and 224F3 in human factor XIa-activated platelet-poor plasma. The experiment was performed as described in Example 18. The dotted line shows the mean peak thrombin level (nM) observed in the absence of the antibody, and its standard deviation (±1SD) is shown by the dotted line. The profiles of the OA versions of mAb1-9016 and 224F3 (mAb1-1582) are shown by upward and downward triangles, respectively.

[0027] A brief explanation of arrays Sequence ID 1 is the amino acid sequence of human coagulation factor IX. Sequence ID 2 is the amino acid sequence of human coagulation factor X. Sequence IDs 3-188 are the heavy chain variable domains (V) of the anti-FIX and anti-FX monoclonal antibodies (mAbs) described herein. H ) and light chain variable domain (V L This is the sequence. The IDs of the corresponding one-armed (OA) antibody and specific bispecific antibodies are also shown in the table. The CDR1-3 sequence is highlighted in the box in Figure 1.

[0028] Antibody abbreviations, targets, and corresponding V H and V L Overview of array arrays: [Table 1-1] [Table 1-2]

[0029] The first column ("OA or Bispecific Antibody ID") contains abbreviations for monovalent one-armed (OA) antibodies and / or bispecific antibodies. The second column ("mAb ID") represents the abbreviations for the corresponding component antibodies (in the case of bispecific antibodies, the first antibody listed in the second column is the anti-FIX / FIXa antibody, and the second is the anti-FX / FXa antibody). The fourth column ("Sequence ID (V H )」) and fifth column (「Sequence number (V L )」) is V H and V L Each sequence number represents an array index, with the first array index in each column representing an anti-FIX / FIXa antibody, and the second representing an anti-FX / FXa antibody. [Modes for carrying out the invention]

[0030] In subjects with coagulation disorders, such as humans with hemophilia A, the coagulation cascade is dysfunctional due to the absence or insufficient presence of functional FVIII. Dysfunction of any part of this coagulation cascade results in inadequate blood clotting and potentially life-threatening bleeding or damage to internal organs such as joints. The present invention relates to compounds that can serve as substitutes for coagulation factor VIII (FVIII) in patients with coagulation disorders, particularly in patients lacking functional FVIII, such as hemophilia A patients, including hemophilia A patients with inhibitors. In one embodiment, such compounds are antibodies.

[0031] In particular, the inventors of this invention possess a remarkably well-identified antibody that mimics FVIII cofactor activity with high potency and effectiveness.

[0032] In a particular embodiment, the present invention relates to a coagulant antibody that serves as a substitute for functional FVIII in patients lacking functional FVIII, such as patients with hemophilia A.

[0033] In one such embodiment, the blood coagulation antibody binds to the enzymatic activity of coagulation factor Xa (FIXa) against coagulation factor X (FX), increasing the enzymatic activity, and optionally also binds to FX. In this embodiment, the antibody of the present invention is a bispecific antibody capable of binding to FIX / FIXa and FX.

[0034] coagulation factor IX FIX is a vitamin K-dependent coagulation factor structurally similar to factor VII, prothrombin, factor X, and protein C. The circulating enzyme precursor consists of 415 amino acids divided into four distinct domains, including an N-terminal γ-carboxyglutamate-rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. FIX circulates in plasma as a single-chain enzyme precursor (SEQ ID NO: 1). Activation of FIX occurs by limited proteolysis at Arg145 and Arg180, releasing an activating peptide (residues 146-180 of SEQ ID NO: 1). Therefore, activated FIX (FIXa) consists of residues 1-145 of SEQ ID NO: 1 (light chain) and residues 181-415 of SEQ ID NO: 1 (heavy chain).

[0035] Therefore, the circulating FIX molecule comprises the FIX enzyme precursor and the active form of FIX, which are generally referred to herein as FIX and FIXa with respect to Sequence ID No. 1.

[0036] Activated factor IX is called factor IXa or FIXa. The term "FIX (SEQ ID NO: 1) and / or its activated form (FIXa)" is also referred to as "FIX / FIXa" or "FIX(a)".

[0037] FIXa is a trypsin-like serine protease that plays a crucial role in hemostasis by producing most of the factor Xa necessary to support proper thrombin formation during coagulation, as part of the tenase complex.

[0038] In this specification, FIX is represented by Sequence ID No. 1, which corresponds to the Ala148 allele of human FIX (Anson et al., EMBO J.1984 3:1053-1060; McGraw et al., Proc Natl Acad Sci USA.1985 82:2847-2851; Graham et al., Am.J.Hum.Genet.1988 42:573-580). In this invention, FIX is intended to encompass all natural variants of FIX, such as the T148 variant (Uniprot ID P00740).

[0039] coagulation factor FX is a vitamin K-dependent coagulation factor structurally similar to factor VII, prothrombin, FIX, and protein C. The human FX enzyme precursor contains four distinct domains, including an N-terminal gamma-carboxyglutamate-rich (Gla) domain, two EGF domains, and a C-terminal trypsin-like serine protease domain. FX circulates in plasma as a double-chain enzyme precursor, containing residues 1-139 of SEQ ID NO: 2 (light chain) and residues 143-448 of SEQ ID NO: 2 (heavy chain). Activation of FX occurs by limited proteolysis at Arg194, resulting in the release of an activating peptide (Aa143-194). 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 (activating heavy chain). Therefore, the circulating FX molecule contains the FX enzyme precursor and the active form of FX, which are referred to herein as FX and FXa, respectively, with respect to SEQ ID NO: 2. In this invention, FX is intended to encompass all natural variants of FX. The terms “FX (SEQ ID NO: 2) and / or its active form (FXa)” are also referred to as “FX / FXa” or “FX(a)”.

[0040] antibody As used herein, the term "antibody" refers to a protein derived from an immunoglobulin sequence that has the ability to bind to an antigen or a portion thereof. The term antibody includes, but is not limited to, full-length antibodies of any class (or isotype), i.e., IgA, IgD, IgE, IgG, IgM, and / or IgY. The term antibody includes, but is not limited to, bivalent antibodies such as bispecific antibodies.

[0041] Native full-length antibodies include at least four polypeptide chains, two heavy chains (HC) and two light chains (LC) that are linked by disulfide bonds. In some cases, native antibodies include fewer than four chains, as in the case of IgNAR found in cartilaginous fish. One class of immunoglobulins for certain pharmaceuticals is IgG. In humans, the IgG class may be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequences of their heavy chain constant regions. Light chains can be divided into two types, kappa chains and lambda chains, based on differences in their sequence compositions. An IgG molecule is composed of two heavy chains linked by two or more disulfide bonds, and two light chains each linked by a disulfide bond to a heavy chain. The IgG heavy chain includes a heavy chain variable domain (V H ) and up to three heavy chain constant (C H ) domains: C H 1, C H 2, and C H 3. The light chain may include a light chain variable domain (V L ) and a light chain constant domain (C L ). The V H and V L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDR) or hypervariable regions (HvR), interspersed with more conserved regions called framework regions (FR). The V H and V LThe domain generally consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy-chain and light-chain variable domains containing the hypervariable regions (CDRs) form structures that can interact with antigens, while the constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors. These host tissues or factors include, but are not limited to, various cells of the immune system (effector cells), Fc receptors, and C1q of the traditional complement system's C1 complex, which is the primary component.

[0042] The antibodies of the present invention may be monoclonal antibodies (mAbs) in the sense that they represent a set of unique heavy chain variable domain sequences and light chain variable domain sequences expressed from a single B cell or by a clonal population of B cells. The antibodies of the present invention may be produced and purified using various methods known to those skilled in the art. For example, the antibodies may be produced from hybridoma cells. The antibodies may be produced by B cell proliferation. The antibodies or fragments thereof may be recombinantly expressed in a mammalian or microbial expression system or by in vitro translation. The antibodies or fragments thereof may also be recombinantly expressed as cell surface-bound molecules, for example by phage display, bacterial display, yeast display, mammalian cell display, or ribosome or mRNA display.

[0043] 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 in the environment in which it was produced, and / or an antibody that has been purified from a mixture of components present in the environment in which it was produced.

[0044] Antigen-binding fragments of an antibody may be relevant in the context of this invention, as it has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. The term “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody 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 Fab, Fab', Fab2, Fab'2, Fv (usually the V of a single arm of an antibody). L Domain and V H Domain combinations), single-chain Fv (scFv) (see, for example, Bird et al., Science 1988;242:423-426; and Huston et al., PNAS 1988;85:5879-5883), dSfv, Fd (usually V) H and C H 1 domain), single V H and single V L The components include (but are not limited to) monovalent molecules containing both domains, minibodies, diabodies, triabodies, tetrabodies, and kappabodies (see, e.g., III (1997) Protein Eng10:949-57), as well as one or more isolated CDRs or functional paratopes, the isolated CDRs or antigen-binding residues or polypeptides being associated or ligated together to form functional antibody fragments. These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments may be screened in a manner similar to that of intact antibodies.

[0045] The "Fab" fragment of the antibody, including the "Fab" fragment and the "Fab'2" fragment, can be derived from the antibody by cleaving the heavy chain at the N-terminal or C-terminal hinge region of the hinge cysteine ​​residues that connect the antibody's heavy chain. The "Fab" fragment consists of the variable domain and constant domain of the light chain, as well as the variable domain and C of the heavy chain. HIt contains one region. The "Fab'2" fragment generally contains a pair of "Fab" fragments covalently linked by their hinged cysteine. Fab' is formally derived from the Fab'2 fragment by cleaving the hinged disulfide bond connecting the heavy chain of Fab'2. Other chemical bonds other than disulfide bonds in antibody fragments are also known in the art. Fab fragments retain the ability of the parent antibody to bind to its antigen, albeit with potentially low affinity. The Fab'2 fragment is capable of bivalent binding, while the Fab and Fab' fragments can only bind monovalently. Generally, Fab fragments contain constant C H 2 domains and C H Fab fragments lack three domains, i.e., the Fc site where the interaction between the Fc receptor and C1q occurs. Therefore, Fab fragments generally lack effector function. Fab fragments can be generated by methods known in the art, such as by enzymatic cleavage of an antibody, for example, using papain to obtain Fab or pepsin to obtain Fab'2. Fab fragments containing Fab, Fab', and Fab'2 can be recombinantly generated using methods known in the art.

[0046] A "Fv" (fragment variable) fragment is an antibody fragment containing a complete antigen recognition and binding site, and generally, for example, in a single-chain variable domain fragment (scFv), it contains one heavy chain and one light chain variable domain, relating to the spontaneous covalent binding. In this configuration, the three hypervariable regions of each variable domain interact to form V H -V L Antigen-binding sites are defined on the surface of the dimer. Collectively, six hypervariable regions or subsets of them give antigen-binding specificity to the antibody.

[0047] "Single-chain Fv" or "scFv" antibodies are antibodies with V H Domain and V L It contains domains, and these domains are present in a single polypeptide chain. Generally, Fv polypeptides are V H Domain and V LFurther inclusion of polypeptide linkers between domains allows scFv to form a desirable 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.

[0048] "Single-chain Fab" or "scFab" antibodies are antibodies with V H , C H 1. V L and C L It contains domains, and these domains are present in a single polypeptide chain. Generally, Fab polypeptides allow both V to form the desired structure for antigen binding. H and C L Between the domain, or V L and C H It further includes a polypeptide linker between either the domain or the other (Koerber et al., (2015) J Mol Biol. 427:576-86).

[0049] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, and this fragment is made up of the same polypeptide chain (V H and V L ) Light chain variable domain (V L ) connected to the heavy chain variable domain (V H ) includes. Using a linker that is too short to allow pairing between two variable domains on the same chain, the variable domain is forced to pair with a complementary domain on another chain, creating two antigen-binding sites.

[0050] The term "linear antibody" refers to the antibodies described by Zapata et al., (1995) Protein Eng. 8:1057-1062. In short, these antibodies, together with complementary light chain polypeptides, form a pair of antigen-binding domains, consisting of a pair of tandem Fd segments (V H-C H 1~V H -C H It contains (1). Linear antibodies can be bispecific or monospecific.

[0051] Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and the fragments can be screened for binding to FIX and its activated form, FX, or another function in a manner similar to that of intact antibodies.

[0052] The antibody fragments of the present invention may be prepared, for example, by cleavage, which involves the removal of one or more amino acids from the N-terminus and / or C-terminus of a polypeptide. The fragments may also be produced by one or more internal deletions.

[0053] The antibody of the present invention may be an antibody fragment, or a variant of any of the antibodies disclosed herein, or may contain such fragments. The antibody of the present invention may be an antigen-binding moiety of one of these antibodies, or a variant thereof, or may contain such a moiety. For example, the antibody of the present invention may be a Fab fragment of one of these antibodies, or a variant thereof, or a single-chain antibody derived from one of these antibodies, or a variant thereof. Alternatively, the antibody of the present invention may be a combination of a full-length antibody and its fragment.

[0054] As used herein, the term "one-armed" refers to a specific type of monovalent antibody consisting of an antibody heavy chain, a cleaved heavy chain lacking a Fab region, and a single light chain.

[0055] As used herein, the term “single-specific” antibody refers to an antibody that can bind to one specific epitope (including, but not limited to, a bivalent antibody).

[0056] As used herein, the term “bispecific” antibody refers to an antibody that can bind to two different antigens or two different epitopes on the same antigen.

[0057] As used herein, the term “triply specific” antibody refers to an antibody that can bind to three different antigens, or three different epitopes on the same antigen, or three different epitopes present on two different antigens.

[0058] As used herein, the term “multispecific” antibody refers to an antibody capable of binding to two or more different antigens or two or more different epitopes on the same antigen. Therefore, multispecific antibodies include bispecific and triplicate antibodies.

[0059] Full-length IgG bispecific antibodies can be produced by the fusion of two separate hybridomas, forming a hybrid quadroma that generates a mixture of antibodies containing fragments of bispecific heterodimerized antibodies (Chelius D. et al.; mAb. 2010 May-Jun; 2(3):309-319). Alternatively, bispecific heterodimerized antibodies can be produced using recombinant techniques. Heterodimerization can be achieved by manipulating the dimerization interface of the Fc region to promote heterodimerization. One example of this is the so-called knob-in-hole mutation, in which a sterically bulky side chain (knob) is introduced into one Fc that matches a sterically smaller side chain (hole) on the opposing Fc, thereby creating steric complementarity that promotes heterodimerization. Other methods of manipulating the heterodimerized Fc interface include electrostatic complementarity, fusion to a non-IgG heterodimerized domain, or utilization of the natural Fab-arm exchange phenomenon of human IgG4 to control heterodimerization. Examples of heterodimerized bispecific antibodies are well described in the literature, e.g., (Klein C. et al.; mAb. 2012 Nov-Dec; 4(6):653-663). Special attention must be paid to the light chains of heterodimerized antibodies. Correct pairing of LC and HC can be achieved by using a common light chain. Also, engineering of the LC / HC interface can be used to facilitate heterodimerization or light chain crossover engineering, as in the case of CrossMab. In vitro reconstruction of antibodies from two individual IgGs containing the appropriate mutations under mild reduction conditions can also be used to generate bispecific antibodies (e.g., Labrijn et al., PNS, 110, 5145-5150 (2013)). Furthermore, a natural Fab arm exchange method has been reported to ensure correct light chain pairing.

[0060] Multispecific antibody-based molecules can also be recombinantly expressed as fusion proteins that combine native modules of IgG to form multispecific and multivalent antibody derivatives, as described in the literature. Examples of fusion antibodies include DVD-Igs, IgG-scFV, Diabody, and DART. Specific detection tags, purification tags, half-life extensions, or other components can be incorporated into the fusion protein. Additional non-IgG modalities can also be incorporated into the fusion protein. Bispecific full-length antibodies based on Fc heterodimerization are generally called asymmetric IgG, regardless of the LC pairing method.

[0061] Generally, bispecific antibodies can be produced in various molecular forms outlined by Brinkmann et al. (Brinkmann et al. The making of bispecific antibodies. Mabs 9, 182-212 (2017)).

[0062] Multispecific antibody-based molecules may also be generated by chemical bonding or coupling of individual full-length IgGs, or by coupling of IgG fragments, as described in the literature, to form multispecific antibody derivatives and polyvalent antibody derivatives. Examples include chemically bonded Fab fragments and IgG dimers. Specific detection or purification tags, half-life extension molecules, or other components can be incorporated into the complex protein. Additional non-IgG polypeptides may also be incorporated into the fusion protein. Multispecific molecules can be generated by combining recombinant and chemical methods, including those described above.

[0063] In one embodiment, the antibody of the present invention is a chimeric antibody, a human antibody, or a humanized antibody. Such antibodies can be produced, for example, by using a suitable antibody representation or immunization platform, or other suitable platform or method known in the art. As used herein, the term “human antibody” is intended to include antibodies in which at least a portion of the framework region and / or at least a portion of the CDR region has a variable domain derived from a human germline immunoglobulin sequence. For example, a human antibody may have variable domains in both the framework and the CDR region derived from a human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, the constant region or a portion of it also derives from a human germline immunoglobulin sequence. The human antibody of the present invention may contain amino acid residues not encoded by a human germline immunoglobulin sequence (for example, mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutation in vivo).

[0064] Such human antibodies may be human monoclonal antibodies. Such human monoclonal antibodies may be produced by hybridomas containing B cells obtained from genetically modified non-human animals. For example, a genetically modified mouse having a genome containing a repertoire of human immunoglobulin heavy and light chain gene segments fused to immortalized cells.

[0065] Human antibodies may also be isolated from sequence libraries constructed based on the selection of human germline sequences, and are further diversified by natural and synthetic sequence diversity.

[0066] Human antibodies can be prepared by in vitro immunization of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

[0067] Human antibodies may be produced by recombinant methods known in the art.

[0068] The term "human antibody derivative" refers to any modified form of a human antibody, such as a conjugate of an antibody with another drug or antibody.

[0069] 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. Thus, a humanized antibody is a human immunoglobulin (recipient antibody) in which at least residues from the hypervariable region of the recipient are replaced with residues from the hypervariable region of an antibody from a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, sequence composition, and functionality. In some examples, framework (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Examples of such modifications include the introduction of one or more so-called reverse mutations, which are typically amino acid residues derived from the donor antibody. Antibody humanization may be carried out using recombinant techniques known to those skilled in the art (see, e.g., Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. Lo). A suitable human recipient framework for both the light-chain and heavy-chain variable domains can be identified, for example, by sequence or structural homology. Alternatively, a fixed recipient framework may be used, for example, based on knowledge of its structure, biophysical properties, and biochemical properties. The recipient framework may be germline-derived or derived from a mature antibody sequence. The CDR region from a donor antibody can be introduced by CDR transplantation. CDR-transplanted humanized antibodies can be further optimized for properties such as affinity, functionality, and biophysical properties by identifying key framework locations where the reintroduction of amino acid residues from the donor antibody (reverse mutation) has a beneficial effect on the properties of the humanized antibody. In addition to reverse mutation of the donor antibody, humanized antibodies can be manipulated by introducing germline residues into the CDR or framework region, removing immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0070] Furthermore, humanized antibodies may contain residues not found in recipient or donor antibodies. These modifications are made to further improve antibody performance. Generally, humanized antibodies contain at least one, typically two, variable domains, in which all or substantially all CDR regions correspond to those of non-human immunoglobulins, and in which all or substantially all FR residues are from human immunoglobulin sequences. Humanized antibodies may optionally also contain at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulins.

[0071] The term "humanized antibody derivative" refers to any modified form of a humanized antibody, such as a conjugate of an antibody with a chemical drug, or a conjugate of one antibody with another.

[0072] As used herein, the term "chimeric antibody" refers to an antibody that contains antibody portions derived from two or more species. For example, the gene encoding such an antibody includes a gene encoding a variable domain and a gene encoding a constant domain derived from two different species. For example, the gene encoding the variable domain of a mouse monoclonal antibody may be bound to the gene encoding the constant domain of a human-derived antibody.

[0073] The fragment crystallizable region of an antibody ("Fc region" / "Fc domain") is the C-terminal region of the antibody, which is the hinge and constant C H 2 and C HIt contains three domains. The Fc domain may interact with cell surface receptors called Fc receptors, as well as with several proteins of the complement system. The Fc region allows the antibody to interact with the immune system. In one aspect of the present invention, the antibody may be manipulated to include modifications within the Fc region to alter one or more of its functional properties, typically such as serum half-life, complement fixation, Fc receptor binding, protein stability, and / or antigen-dependent cytotoxic activity, or the absence thereof. Furthermore, the antibody of the present invention may, again, be chemically modified (e.g., one or more chemical parts may be attached to the antibody) or modified to alter its glycosylation, in order to alter one or more of the functional properties of the antibody. An IgG1 antibody may carry a modified Fc domain, including one or more, and possibly all of the following mutations (residue numbering by EU index), which result in decreased affinity to specific Fc gamma receptors (L234A, L235E, and G237A) and decreased C1q-mediated complement binding (A330S and P331S), respectively. Alternatively, other amino acid substitutions and combinations thereof, known in the art to result in alterations (decreases or increases) in Fc gamma receptor binding, and combinations thereof with those described above, may be used.

[0074] The antibody isotype of the present invention may be IgG, such as IgG1, IgG2, or IgG4. If desired, the antibody class can be "switched" by known techniques. For example, an antibody originally produced as an IgM molecule may be class-switched to an IgG antibody. Class-switching techniques can also be used to convert one IgG subclass to another, for example, from IgG1 to IgG2 or IgG4, IgG2 to IgG1 or IgG4, or IgG4 to IgG1 or IgG2. By combining regions from different IgG subclasses, antibody engineering can also be carried out to generate constant-region chimeric molecules.

[0075] In one embodiment, the hinge region of the antibody is modified such that the number of cysteine ​​residues within the hinge region changes, for example, by increasing or decreasing it. This approach is further described, for example, in U.S. Patent No. 5,677,425 by Bodmer et al.

[0076] The constant region may be modified to stabilize the antibody, for example, to reduce the risk of bivalent antibody separation into half-antibodies. For example, in the IgG4 constant region, residue S228 (according to EU numbering index and Kabat's S241) may be mutated to a proline (P) residue to stabilize heavy chain disulfide crosslinking at the hinge (see, e.g., Angal et al. Mol Immunol. 1993;30:105-8).

[0077] Antibodies or fragments thereof can be defined in terms of their complementarity-determining regions (CDRs). The terms “complementarity-determining region” or “hypervariable region,” as used herein, refer to the region of an antibody where amino acid residues involved in antigen binding are located. A hypervariable region, or CDR, can be identified as the region of the antibody variable domain that exhibits the highest variability in amino acid alignment. Databases such as the Kabat database can be used for CDR identification, and this CDR is defined, for example, as containing 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, 5th edition, USD Department of Health and Human Services, NIH Publication Nos. 91-3242). Alternatively, the CDR can be defined as these residues from the “hypervariable loop” (residues 26-33 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain; Chothia and Lesk, J.Mol.Biol.1987;196:901-917). Typically, the numbering of amino acid residues in this region is carried out by the method described by Kabat et al. above. In this specification, phrases such as “Kabat position,” “Kabat residue,” and “according to Kabat” refer to this numbering system for the heavy chain variable domain or light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of the peptide may contain fewer or additional amino acids corresponding to the framework (FR) of the variable domain or the shortening or insertion of the CDR. For example, the heavy chain variable domain may include amino acid insertions after residue 52 of the CDR H2 (e.g., residues 52a, 52b, and 52c according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat).The Kabat numbering of residues can be determined for a given antibody by aligning the antibody sequence with the "standard" Kabat numbering sequence in homology regions.

[0078] The terms “framework region” or “FR” residues are defined herein as these V not within the CDR. H or V L This refers to an amino acid residue.

[0079] The antibody of the present invention may comprise a CDR region from one or more of the specific antibodies disclosed herein.

[0080] The term "blood coagulation antibody" refers to an antibody that enhances blood coagulation, for example, by accelerating the blood coagulation process and / or by increasing the enzymatic activity of one or more coagulation factors.

[0081] The term "blood coagulation activity" refers to the ability of compounds such as antibodies to enhance blood coagulation, for example, by accelerating the blood coagulation process and / or by increasing the enzymatic activity of one or more coagulation factors.

[0082] The term “antigen” (Ag) refers to a molecular entity used in the immunization of immune vertebrates to produce an antibody (Ab) that recognizes Ag. In this specification, Ag is a broader term generally intended to include target molecules specifically recognized by Ab, and therefore includes fragments or mimicry of immunization processes or other processes such as phage display used to generate Ab.

[0083] As used herein, the term “epitope” is defined in the context of molecular interactions between an “antigen-binding polypeptide,” such as an antibody (Ab) and its corresponding antigen (Ag). Generally, an “epitope” refers to the area or region on Ag to which Ab binds, i.e., the area or region in physical contact with Ab. Physical contact can be defined using various criteria relative to the atoms of the Ab and Ag molecules (e.g., 2–6 Å spacing, such as 3 Å, 3.5 Å, 4 Å, 4.5 Å, 5 Å, or solvent exposure).

[0084] FIX / FIXa and FX / FXa may contain a number of different epitopes, including, but are not limited to, (1) linear peptide epitopes, (2) structural epitopes consisting of one or more discontinuous amino acids located in close proximity to each other in a mature FIX / FIXa or FX / FXa structure, and (3) epitopes consisting of all or part of a molecular structure covalently bonded to FIX / FIXa or FX / FXa, such as a carbohydrate group.

[0085] The epitopes of a given antibody (Ab) / antigen (Ag) pair can be described and characterized with different levels of detail using various experimental and computational epitope mapping methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and various competitive binding methods, as well as methods known in the art. Since each method relies on its own unique principles, the description of the epitope is essentially linked to the method by which it was determined. Therefore, depending on the epitope mapping method used, the epitopes of a given Ab / Ag pair can be described differently.

[0086] For example, in the context of X-ray-derived crystal structures defined by the spatial coordinates of the Fab fragment and its inter-Ab complex such as Ag, the term epitope as used herein is specifically defined as a FIX / FIXa or FX residue having a heavy atom (i.e., a non-hydrogen atom) within a distance of 3.5 Å from the heavy atom of Ab, unless otherwise specified or contradicted by the context.

[0087] For example, epitopes described at the amino acid level, determined from X-ray structures, are said to be identical if they contain the same amino acid residue. Epitopes are said to be duplicated if at least one amino acid residue is shared by the epitope. Epitopes are said to be unique if no amino acid residues are shared by the epitope.

[0088] The definition of the term "paratope" derives from the above definition of "epitope" by reversing the perspective. Therefore, the term "paratope" refers to the area or region on Ab to which Ag binds, that is, it is in physical contact with Ag.

[0089] In the context of X-ray-derived crystal structures defined by the spatial coordinates of the Fab fragment and its inter-Ab complex such as Ag, the term "paratope" as used herein is specifically defined as an Ab residue having a heavy atom (i.e., a non-hydrogen atom) within a distance of 3.5 Å from the heavy atom of FIX / FIXa or FX, unless otherwise specified or contradicted by the context.

[0090] The epitopes and paratopes of a given antibody (Ab) / antigen (Ag) pair can be identified by routine methods. For example, the general location of an epitope can be determined by evaluating the ability of the antibody to bind to different fragments or variants of FIX / FIXa or FX. Specific amino acids in FIX / FIXa or FX that contact the antibody (epitope) and specific amino acids in the antibody that contact FIX / FIXa or FX (paratope) can be determined using routine methods. For example, the antibody and target molecule may be combined, or the Ab:Ag complex may be crystallized. The crystal structure of the complex may be determined and used to identify specific sites of interaction between the antibody and its target.

[0091] Epitopes on an antigen may contain one or more hotspot residues, i.e., residues particularly important for interaction with congener antibodies, and interactions mediated by the side chains of such hotspot residues significantly contribute to the binding energy of the antibody / antigen interaction (Peng et al. (2014). PPNAS 111, E2656-E2665). Hotspot residues can be identified by test variants of the antigen (here FIX / FIXa and FX), where a single epitope residue is substituted, for example, with alanine for binding to a congener antibody. If the substitution of an epitope residue with alanine has a strong effect on binding to the antibody, the epitope residue is considered a hotspot residue and is therefore particularly important for antibody binding to the antigen.

[0092] Antibodies that bind to the same antigen can be characterized in terms of their ability to bind to that common antigen simultaneously and can undergo “competitive binding” / “binning.” In this context, the term “binning” refers to a method of grouping antibodies that bind to the same antigen. Antibody “binning” may be based on the competitive binding of two antibodies to a common antigen in an assay, based on standard techniques.

[0093] The "bin" of antibodies is defined using a reference antibody. If a secondary antibody cannot bind to the antigen simultaneously with the reference antibody, the secondary antibody is said to belong to the same "bin" as the reference antibody. In this case, the reference antibody and the secondary antibody competitively bind to the same part of the antigen and are called "competitive antibodies." If a secondary antibody can bind to the antigen simultaneously with the reference antibody, the secondary antibody is said to belong to a separate "bin." In this case, the reference antibody and the secondary antibody do not competitively bind to the same part of the antigen and are called "non-competitive antibodies."

[0094] The antibody "Binning" does not provide direct information about the epitope.

[0095] Competitive antibodies, i.e., antibodies belonging to the same "bin," may have the same epitope, overlapping epitopes, or even distinct epitopes. The latter occurs when a reference antibody bound to that epitope on the antigen occupies the space necessary for the secondary antibody to contact that epitope on the antigen ("steric hindrance"). Non-competitive antibodies generally have distinct epitopes. Therefore, in some embodiments, the antibodies of the present invention bind to the same epitope as at least one of the antibodies specifically disclosed herein.

[0096] Competition assays for determining whether an antibody competes for binding to the anti-FIX / FIXa or anti-X antibodies disclosed herein are known in the art. Exemplary competition assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance analysis (e.g., using BIAcore® instruments), biolayer interferometry (ForteBio®), and flow cytometry.

[0097] Typically, competitive assays involve the use of an antigen bound to a solid surface or expressed on a cell surface, a test FIX or FIXa-binding antibody, and a reference antibody. The reference antibody is labeled, and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cell in the presence of the test antibody. Typically, the test antibody is present in excess (e.g., 1, 5, 10, 20, 100, 1000, 10000, or 100000 times). Antibodies identified as competitive in a competitive assay (i.e., competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, or to a duplicate epitope, and antibodies that bind to an adjacent epitope sufficiently proximal to the epitope to which the reference antibody binds due to steric hindrance.

[0098] In an exemplary competitive assay, the reference anti-FIX or anti-FIXa antibody is biotinylated using a commercially available reagent. The biotinylated reference antibody is mixed with serial dilutions of the test antibody or unlabeled reference antibody (self-competitive control) to obtain mixtures of the test antibody (or unlabeled reference antibody) at various molar ratios relative to the labeled reference antibody (e.g., 1, 5, 10, 20, 100, 1000, 10000 or 100000 times). The antibody mixture is added to a FIX or FIXa polypeptide-coated ELISA plate. The plate is then washed, and horseradish peroxidase (HRP)-streptavidin is added to the plate as the detection reagent. The amount of labeled reference antibody bound to the target antigen is detected after the addition of a chromogenic substrate known in the art (e.g., TMB (3,3',5,5'-tetramethylbenzidine) or ABTS (2,2"-azino-di-(3-ethylbenzazoline-6-sulfonate)). Optical density readings (OD units) are obtained using a spectrometer (e.g., SpectraMax® M2 spectrometer (molecular instrument)). The response (OD units) corresponding to zero percent inhibition is determined from a well that does not contain any competing antibody. The response (OD units) corresponding to 100% inhibition, i.e., the assay background, is determined from a well that does not contain any labeled reference antibody or test antibody. The inhibition rate of the labeled reference antibody against FIX or FIXa by the test antibody (or unlabeled reference antibody) at each concentration is calculated as follows: % inhibition = (1 - (OD units - 100% inhibition) / (0% inhibition - 100% inhibition)) * 100.

[0099] Those skilled in the art will understand that similar assays can be performed to determine whether two or more anti-FX / FXa antibodies share a binding region, bin, and / or competitively bind to the antigen. Those skilled in the art will also understand that competitive assays can be performed using various detection systems known in the art.

[0100] As measured in competitive binding assays, if an excess of one antibody (e.g., 1, 5, 10, 20, 100, 1000, 10000, or 100000 times) inhibits the binding of other antibodies, then, for example, at least 50%, 75%, 90%, 95%, or 99%, the test antibody competes with the reference antibody for binding to the antigen.

[0101] Unless otherwise specified, competition is determined using the competitive ELISA assay described above.

[0102] The term "binding affinity" is used herein to describe a measure of the strength of a non-covalent interaction between two molecules, for example, between an antibody or a fragment of an antigen. The term "binding affinity" is used to describe a monovalent interaction.

[0103] The binding affinity between two molecules via monovalent interactions, for example, between an antibody or a fragment of an antigen, is determined by the equilibrium dissociation constant (K). D It can be quantified by determining K. D The dynamics of complex formation and dissociation can be determined by measuring them, for example, by surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). The rate constants corresponding to the association and dissociation of monovalent complexes are the association rate constant k, respectively. a (or k on ), and the dissociation rate constant k d (or k off ) is called K D is, formula K D =k d / k a Through, k a and k d It is related to this.

[0104] According to the definition above, binding affinity, which is related to different molecular interactions such as the comparison of binding affinities of different antibodies to a given antigen, is the K of individual antibody / antigen complexes. D You can also compare them by comparing their values.

[0105] The value of the dissociation constant can be directly determined by known methods. Standard assays for evaluating the binding ability of ligands such as antibodies toward a target are known in the art and include, for example, ELISA, Western blotting, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of antibodies can also be evaluated by standard assays known in the art, such as SPR. However, it is preferable to use isothermal titration calorimetry (ITC) to measure affinity for antibody / target interactions and derive thermodynamic parameters for the interaction.

[0106] A competitive binding assay can be performed in which the binding of an antibody to a target is compared to the binding of the target by another ligand of the same target, such as another antibody.

[0107] The antibody of the present invention has a target of 1 × 10 -4 M or less, 1×10 -5 M or less, 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, or 1 × 10 -9 M or less, or 1 × 10 -10 M or less, 1×10 -11 M or less, 1×10 -12 M or less, 1×10 -13 M or less or 1 × 10 -14 K below M D It holds.

[0108] K of the antibody of the present invention D However, it may also be less than 100 μM, such as less than 1 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM, less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, less than 0.1 μM, etc.

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

[0110] The antibodies and antibody fragments described herein may be combined with other antibodies and antibody fragments known in the art to produce bispecific, tripspecific, or multispecific antibody molecules. Compounds mimicking FVIII cofactor function have been previously produced using other FIX / IXa and FX / Xa binding domains, but these may potentially substitute for the FIX / IXa and / or FX / Xa binding domains described herein, respectively. Thus, it is clear that the FIX / IXa and FX / Xa binding domains of the present invention are distinct subjects as individual molecules, as well as “intermediates” as part of a bispecific, tripspecific, or multispecific antibody containing at least one FIX / IXa and / or FX / Xa binding domain.

[0111] The activity of blood coagulation antibodies, including bi-, tri-, and multi-specific antibodies, can be determined by methods known in this art. Standard assays include the whole blood thrombin production assay (TGT), and coagulation time is measured by thromboelastography (TEG) and FXa production assays.

[0112] identity The term “identity” as known in the art refers to the relationship between sequences of two or more polypeptides, determined by comparing their sequences. In the art, “identity” also means the degree of sequence relevance between polypeptides, determined by the number of matches between strings of two or more amino acid residues. “Identity” measures the proportion of identical matches between the smaller of two or more sequences, with gap adjustments (if any) addressed by a specific mathematical model or computer program (i.e., “algorithm”). The identity of related polypeptides can be readily calculated by known methods. Such methods, but not limited to, are described below. Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 1988;48:1073.

[0113] The preferred method for determining identity is designed to obtain the greatest match between the sequences being tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 1984;12:387), Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 1990;215:403-410). The BLASTX program is published by the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., see above). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0114] For example, the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.) is used to align two polypeptides whose sequence identity ratio is determined for optimal matching of their respective amino acids ("matched spans" as determined by the algorithm). A gap start penalty (calculated as 3 times the mean oblique angle, where the "mean oblique angle" is the average of the diagonals of the comparison matrix used, and the "oblique angle" is a score or numerical value assigned to each perfect amino acid match by a particular comparison matrix) and a gap stretch penalty (usually a fraction (1 / 10) of the gap start penalty), and comparison matrices such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. Standard comparison matrices (see Dayhoff et al., 1978; Atlas of Protein Sequence and Structure, vol. 5, supp. 3 for the PAM 250 comparison matrix; see Henikoff et al., PNAS 1992; 89: 10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0115] Preferred parameters for peptide sequence comparison include: Algorithm: Needleman et al. J.Mol.Biol.1970;48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., PNAS 1992;89:10915-10919; Gap penalty: 12, Gap length penalty: 4, Similarity threshold: 0.

[0116] The GAP program is useful for the parameters mentioned above. The parameters described above are the default parameters for peptide comparison using the GAP algorithm (without terminal gap penalty).

[0117] The term "similarity," while related, refers to a sequence relationship that includes both identity and conserved substitution agreement, in contrast to "identity." For example, if two polypeptide sequences have (10 / 20) identical amino acids and the rest are all non-conservative substitutions, both the identity and similarity percentages are 50%. In the same example, if there are five more positions with conservative substitutions, the identity percentage becomes 25% and the similarity percentage becomes 75% (15 / 20). Therefore, when there are conservative substitutions, the degree of similarity between two polypeptides is higher than the degree of identity between them.

[0118] Pharmaceutical preparations In another embodiment, the present invention provides compositions and formulations comprising compounds of the present invention, such as antibodies described herein. For example, the present invention provides pharmaceutical compositions comprising one or more antibodies of the present invention, formulated with a pharmaceutically acceptable carrier.

[0119] Therefore, one object of the present invention is to provide a pharmaceutical formulation containing such antibodies present at concentrations 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 contain one or more of a buffer system, a preservative, an isotonic agent, a chelating agent, a stabilizer, or a surfactant, or various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Remington: The Science and Practice of Pharmacy, 19 th See edition 1995.

[0120] In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersants, emulsions, and multiphase materials. The term “aqueous formulation” is defined as a formulation containing at least 50% w / w water. Similarly, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water.

[0121] In another embodiment, the pharmaceutical formulation is a lyophilized formulation to which a physician or patient adds a solvent and / or diluent before use.

[0122] In a further embodiment, the pharmaceutical preparation comprises an aqueous solution of such antibody and a buffer containing the antibody at a concentration of 1 mg / ml or more, and the preparation has a pH of approximately 2.0 to approximately 10.0.

[0123] Administration The compounds of the present invention, such as antibodies, may be administered parenterally, such as intravenously, intramuscularly, or subcutaneously. Alternatively, the antibodies of the present invention may be administered via an oral route, such as orally or topically. The antibodies of the present invention may be administered prophylactically. The antibodies of the present invention may be administered therapeutically (as required).

[0124] Dosage The dose of the compound delivered may be about 0.01 mg to 500 mg per day, preferably about 0.1 mg to 250 mg per day, more preferably about 0.5 mg to 250 mg per day, and depending on the severity of the condition, it may be once a day, once a week, once every two weeks, or once a month as an initial and maintenance dose. The appropriate dose may be adjusted for a particular compound based on its properties, including its in vivo half-life or mean residence time and its biological activity. For example, the compound delivered may be administered once a week in one embodiment, once every week in another embodiment, or once a month in another embodiment, and in any of the above embodiments, it may be administered in doses of, for example, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg per kg of body weight.

[0125] Compositions containing the compounds disclosed herein may be administered in prophylactic and / or, in some embodiments, therapeutic settings. In therapeutic applications, the composition is administered to a subject suffering from any disease, such as a bleeding disorder as described above, in an amount sufficient to cure, alleviate, or partially prevent the disease and its complications. The amount appropriate to achieve this is defined as the “therapeutic effective dose.” As will be understood by those skilled in the art, the effective dose for this purpose depends on the severity of the disease or injury, as well as the subject’s weight and overall condition.

[0126] Embodiment In one embodiment, the antibody of the present invention can conjugate an epitope containing residue H256 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0127] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue H257 of FIX (SEQ ID NO: 1) or its active form FIXa.

[0128] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue N258 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0129] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue K293 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0130] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue K301 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0131] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue D332 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0132] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue R333 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0133] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue A334 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0134] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue T335 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0135] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue L337 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0136] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue R338 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0137] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue S339 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0138] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue T340 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0139] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue K341 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0140] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue T343 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0141] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue N346 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0142] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue R403 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0143] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue Y404 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0144] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue N406 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0145] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue W407 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0146] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue E410 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0147] In one embodiment, the antibody of the present invention can conjugate an epitope containing residue K411 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0148] In one embodiment, the antibody of the present invention can conjugate an epitope containing residues L337, R338, S339, T340, K341, and T343 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0149] In one embodiment, the antibody of the present invention can conjugate an epitope containing residues K301, D332, R333, A334, T335, R338, and N346 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0150] In one embodiment, the antibody of the present invention can conjugate an epitope containing residues H256, H257, N258, K293, R403, Y404, N406, W407, E410, and K411 of FIX (SEQ ID NO: 1) or its active form (FIXa).

[0151] In one embodiment, the antibody of the present invention can bind to FIX (SEQ ID NO: 1) or its active form (FIXa), where the antibody competes with Fab7236 for binding to FIX.

[0152] In one embodiment, the antibody of the present invention can bind to FIX (SEQ ID NO: 1) or its active form (FIXa), where the antibody competes with Fab7237 for binding to FIX.

[0153] In one embodiment, the antibody of the present invention can bind to FIX (SEQ ID NO: 1) or its active form (FIXa), where the antibody competes with Fab7238 for binding to FIX.

[0154] In one embodiment, the antibody of the present invention is conjugated to FIX (SEQ ID NO: 1) or its active form (FIXa), where the antibody belongs to the same "bin" as Fab7236.

[0155] In one embodiment, the antibody of the present invention is conjugated to FIX (SEQ ID NO: 1) or its active form (FIXa), where the antibody belongs to the same "bin" as Fab7237.

[0156] In one embodiment, the antibody of the present invention is conjugated to FIX (SEQ ID NO: 1) or its active form (FIXa), where the antibody belongs to the same "bin" as Fab7238.

[0157] In one embodiment, the antibody of the present invention can conjugate FX (SEQ ID NO: 2) or its active form FXa, where the antibody competes with the antibody containing the CDR of mAb1-6723.

[0158] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody contains the variable domain of mAb1-6723 as of SEQ ID NOs. 21 and 22.

[0159] In one embodiment, the antibody of the present invention can specifically bind to FX / FXa, where the antibody comprises the CDR of mAb1-6723 as of SEQ ID NOs. 21 and 22.

[0160] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as Fab, containing the variable domain of mAb1-6723 according to SEQ ID NOs. 21 and 22.

[0161] In one embodiment, the antibody of the present invention can specifically bind to FX / FXa, where the antibody belongs to the same "bin" as mAb1-6723 according to SEQ ID NOs. 21 and 22.

[0162] In one embodiment, the antibody of the present invention can specifically bind FX / FXa, where the antibody belongs to the same "bin" as the antibody or Fab containing the antigen-binding domain according to SEQ ID NOs. 21 and SEQ ID NOs. 22.

[0163] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody competes with an antibody containing the CDR of mAb1-1371.

[0164] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody contains the variable domain of mAb1-1371 as of SEQ ID NOs. 65 and 66.

[0165] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody comprises the CDR of mAb1-1371 according to SEQ ID NOs. 65 and 66.

[0166] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as Fab, referred to herein as "bin A," which includes the variable domain of mAb1-1371 according to SEQ ID NOs. 65 and 66.

[0167] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as mAb1-1371 according to SEQ ID NOs. 65 and 66.

[0168] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as the antibody or Fab containing the antigen-binding domain according to SEQ ID NOs. 65 and 66.

[0169] In one embodiment, the antibody of the present invention can specifically bind to FX / FXa, where the antibody competes with antibodies containing CDRs of mAb1-1376, mAb1-6705, mAb1-7388, or mAb1-7563. Such antibodies are referred to herein as belonging to bin B.

[0170] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody comprises a variable domain of mAb1-1376, mAb1-6705, mAb1-7388, or mAb1-7563, identified by SEQ ID NOs. 67 and 68, SEQ ID NOs. 23 and 24, SEQ ID NOs. 39 and 40, and SEQ ID NOs. 59 and 60, respectively.

[0171] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody comprises a CDR of mAb1-1376, mAb1-6705, mAb1-7388, or mAb1-7563, identified by SEQ ID NOs. 67 and 68, SEQ ID NOs. 23 and 24, SEQ ID NOs. 39 and 40, and SEQ ID NOs. 59 and 60, respectively.

[0172] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as the Fab containing the variable domain of mAb1-1376, mAb1-6705, mAb1-7388, or mAb1-7563, identified by SEQ ID NOs. 67 and 68, SEQ ID NOs. 23 and 24, SEQ ID NOs. 39 and 40, and SEQ ID NOs. 59 and 60, respectively.

[0173] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as mAb1-1376, mAb1-6705, mAb1-7388, or mAb1-7563, identified by SEQ ID NOs. 67 and 68, SEQ ID NOs. 23 and 24, SEQ ID NOs. 39 and 40, and SEQ ID NOs. 59 and 60, respectively.

[0174] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as the antibody or Fab containing the antigen-binding domain according to SEQ ID NOs. 67 and 68, SEQ ID NOs. 23 and 24, SEQ ID NOs. 39 and 40, or SEQ ID NOs. 59 and 60.

[0175] In one embodiment, the antibody of the present invention can conjugate FX / FXa, wherein the antibody comprises a CDR or variable domain of an antibody selected from the group consisting of mAb1-6723, 1-6716, 1-6721, 1-6730, 1-6731, 1-6737, 1-6754, 1-7378, 1-7413, 1-7424, 1-7466, 1-7481, 1-7483, and mAb1-7591.

[0176] In one embodiment, the antibody of the present invention can conjugate FX / FXa, wherein the antibody comprises a CDR or variable domain of an antibody selected from the group consisting of mAb1-6723, 1-6716, 1-6721, 1-6730, 1-6731, 1-6737, 1-6754, 1-7378, 1-7413, 1-7424, 1-7466, 1-7481, 1-7483, 1-7591, 1-7388, 1-7563, 1-7462 and mAb1-7571.

[0177] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as an mAb selected from a group of mAbs containing a variable sequence or its CDR selected from the following: SEQ ID NOs: 21 and 22, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 51 and 52, 53 and 54, and 55 and 56, 57 and 58, 59 and 60, 61 and 62, and 63 and 64. This “bin” of antibodies is referred to herein as bin C and is exemplified by numerous individual antibodies such as mAb1-6723, 1-6716, 1-6721, 1-6730, 1-6731, 1-6737, 1-6754, 1-7378, 1-7413, 1-7424, 1-7466, 1-7481, 1-7483, 1-7591, 1-7388, 1-7563, 1-7462, and mAb1-7571.

[0178] In one embodiment, the antibody of the present invention can bind to FX / FXa, where the antibody competes for binding to FX, FX enzyme precursor, or FXa with a reference antibody selected from a group of antibodies consisting of mAbs containing a variable sequence or its CDR selected from the following: SEQ ID NOs: 21 and 22, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 41 and 42, 43 and 44, 53 and 54, and 55 and 56, 57 and 58, and 63 and 64.

[0179] In one embodiment, the antibody or its antigen-binding fragment according to the present invention competes for binding to FX / FXa with antigen-binding fragments including the CDR of SEQ ID NOs. 21 and 22, SEQ ID NOs. 25 and 26, SEQ ID NOs. 27 and 28, SEQ ID NOs. 29 and 30, SEQ ID NOs. 31 and 32, SEQ ID NOs. 33 and 34, SEQ ID NOs. 35 and 36, SEQ ID NOs. 37 and 38, SEQ ID NOs. 41 and 42, SEQ ID NOs. 43 and 44, SEQ ID NOs. 53 and 54, SEQ ID NOs. 55 and 56, SEQ ID NOs. 57 and 58, or SEQ ID NOs. 63 and 64.

[0180] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody competes with antibodies containing CDRs of mAb 1-7447, 1-7441, 1-7571, or 1-7462. These are referred to herein as antibodies of bin D.

[0181] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody comprises variable domains of mAb1-7447, 1-7441, 1-7571, or 1-7462 according to SEQ ID NOs. 47 and 48, SEQ ID NOs. 45 and 46, SEQ ID NOs. 51 and 53, or SEQ ID NOs. 61 and 62, respectively.

[0182] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody contains variable domains of mAb1-7447 or 1-7441 according to SEQ ID NOs. 47 and 48, and SEQ ID NOs. 45 and 46, respectively.

[0183] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody comprises the CDR of mAb 1-7447, 1-7441, 1-7571, or 1-7462 according to SEQ ID NOs. 47 and 48, SEQ ID NOs. 45 and 46, SEQ ID NOs. 51 and 53, or SEQ ID NOs. 61 and 62, respectively.

[0184] In one embodiment, the antibody of the present invention can conjugate FX / FXa according to SEQ ID NO: 2, where the antibody belongs to the same "bin" as the Fab containing the variable domains of mAb1-7447, 1-7441, 1-7571, or mAb1-7462 according to SEQ ID NOs: 47 and 48, SEQ ID NOs: 45 and 46, SEQ ID NOs: 51 and 53, or SEQ ID NOs: 61 and 62, respectively.

[0185] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as mAb1-7447, 1-7441, 1-7571, or mAb1-7462, according to SEQ ID NOs. 47 and 48, SEQ ID NOs. 45 and 46, SEQ ID NOs. 51 and 53, or SEQ ID NOs. 61 and 62, respectively.

[0186] In one embodiment, the antibody of the present invention can conjugate FX / FXa, where the antibody belongs to the same "bin" as the antibody or Fab containing the antigen-binding domain according to SEQ ID NOs. 47 and 48, SEQ ID NOs. 45 and 46, SEQ ID NOs. 51 and 53, or SEQ ID NOs. 61 and 62.

[0187] In one embodiment, the antibody of the present invention is an antibody according to any of the embodiments described above, in which the antibody specifically binds to the FX enzyme precursor.

[0188] In this embodiment, the antibody specifically binds to the FX enzyme precursor, which consists of amino acid residues 1-139 and 143-448 of SEQ ID NO: 2.

[0189] In one embodiment, the antibody of the present invention is an antibody according to any of the embodiments described above, where the antibody is conjugated to FX.

[0190] In one embodiment, the antibody of the present invention is an antibody according to any of the embodiments described above, where the antibody is conjugated to FXa.

[0191] In this embodiment, the antibody specifically binds to FXa via amino acid residues 1-139 and 195-448 of SEQ ID NO: 2.

[0192] In one embodiment, the antibody is a monospecific antibody. In one embodiment, the antibody is a multispecific antibody. In such one embodiment, the antibody is a bispecific antibody. In such one embodiment, the bispecific antibody can bind to FIX or its active form (FIXa) and FX / FXa. In such one embodiment, the bispecific antibody can specifically bind to FIX / FIXa and FX / FXa.

[0193] In one embodiment, the antibody is a bispecific antibody capable of binding FIX / FIXa and FX / FXa, where the FIX / FIXa binding domain is derived from the antibody in bin 1 and the FX / FXa binding domain is derived from the antibody in bin A.

[0194] In one embodiment, the antibody is a bispecific antibody that binds FIX / FIXa and FX / FXa, where the FIX / FIXa binding domain is derived from the antibody in bin 2 and the FX / FXa binding domain is derived from the antibody in bin A.

[0195] In one embodiment, the antibody is a bispecific antibody that binds FIX / FIXa and FX / FXa, where the FIX / FIXa binding domain is derived from the antibody in bin 2 and the FX / FXa binding domain is derived from the antibody in bin B.

[0196] In one embodiment, the antibody is a bispecific antibody that binds FIX / FIXa and FX / FXa, where the FIX / FIXa binding domain is derived from the Bin2 antibody and the FX / FXa binding domain is derived from the BinB antibody. In one embodiment, the antibody is a bispecific antibody that binds FIX / FIXa and FX / FXa, where the FIX / FIXa binding domain is derived from the antibody in bin 1, and the FX / FXa binding domain is derived from the antibody in bin C or D.

[0197] In one embodiment, the antibody is a bispecific antibody that binds FIX / FIXa and FX / FXa, and the binding domains are mAb1-1371 / mAb1-1307, mAb1-6705 / mAb1-1307, mAb1-1371 / mAb0-1886, mAb1-7441 / mAb0-1886, mAb1-7447 / mAb0-1886, mAb1-7481 / mAb0-1886, mAb1-1371 / mAb0 -1998, mAb1-6716 / mAb0-1998, mAb1-6723 / mAb0-1998, mAb1-6730 / mAb0-1998, mAb1-6731 / mAb0-1998, mAb1-67 37 / mAb0-1998, mAb1-6754 / mAb0-1998, mAb1-7378 / mAb0-1998, mAb1-7441 / mAb0-1998, mAb1-7447 / mAb0-1998, m Ab1-7481 / mAb0-1998, mAb1-1371 / mAb1-4707, mAb1-6705 / mAb1-4707, mAb1-1371 / mAb1-4071, mAb1-7441 / mAb1 -5788, mAb1-7447 / mAb1-5788, mAb1-7481 / mAb1-5788, mAb1-1371 / mAb1-4857, mAb1-6716 / mAb1-4857, mAb1-672 It originates from mAb pairs consisting of 3 / mAb1-4857, mAb1-6730 / mAb1-4857, mAb1-6731 / mAb1-4857, mAb1-6737 / mAb1-4857, mAb1-6754 / mAb1-4857, mAb1-7378 / mAb1-4857, mAb1-7441 / mAb1-4857, mAb1-7447 / mAb1-4857, or mAb1-7481 / mAb1-4857.

[0198] In one embodiment, the bispecific antibody of the present invention comprises an antibody arm that binds to FX and an antibody arm that binds to FIX / FIXa. In such an embodiment, the antibody arm that binds to FX binds to an epitope containing one or more residues of the activating peptide of FX, and the antibody arm that binds to FIX / FIXa binds to an epitope containing one or more residues of the FIX protease domain.

[0199] In one embodiment, the antibody of the present invention is a multispecific antibody, such as a bispecific or tripspecific antibody.

[0200] In one embodiment, the antibody of the present invention is an IgG type such as full-length IgG4.

[0201] In one embodiment, the antibody of the present invention is a chemical conjugate of two antibody fragments, such as a conjugate of two Fab fragments or scFv fragments, or a combination thereof.

[0202] In one embodiment, the antibody of the present invention is a human or humanized antibody.

[0203] In one embodiment, the antibody disclosed herein is an intermediate for use in the production of a bispecific antibody.

[0204] In one embodiment, the present invention includes an antibody that competes with the antibody disclosed herein for binding to FIX / FIXa.

[0205] The antibodies of the present invention can be used to treat subjects having coagulation disorders and, in particular, hemophilia A. Accordingly, the present invention also relates to the use of monoclonal antibodies that can bind to the protease domain of FIX / FIXa for the treatment of subjects in need thereof, and to the use of said antibodies for the manufacture of agents for the treatment of subjects in need thereof. Furthermore, the present invention includes a method for treating subjects in need thereof using monoclonal antibodies that can bind to the protease domain of FIX / FIXa.

[0206] In one embodiment, the antibody of the present invention can bind FIXa with an affinity higher than the affinity for binding FIX.

[0207] In one embodiment, the antibody of the present invention can increase the enzymatic activity of FIXa against FX.

[0208] In one such embodiment, the antibody of the present invention can increase the enzymatic activity of FIXa against FX as measured in an FXa production assay using a monovalent one-armed antibody as described herein.

[0209] In one embodiment, the antibody of the present invention can increase the enzymatic activity of FIXa against FX as measured in an FXa production assay using the bivalent antibody described herein.

[0210] In one embodiment, the antibody of the present invention is not the anti-FIX antibody CLB-FIX 13 described below: Rohlena et al. (2003) J. Biol. Chem. 278(11):9394-9401. In one embodiment, the antibody of the present invention is not the anti-FIX antibody HIX-1 (IgG1 mouse) (Merck KGaA, Sigma-Aldrich). In one embodiment, the antibody of the present invention is not the anti-FIX antibody AHIX-5041 (IgG1) (Haematologic Technologies, Inc.).

[0211] In one embodiment, the antibody of the present invention exhibits reduced immunogenicity compared to blood coagulation antibodies in the art.

[0212] In one embodiment, a bispecific antibody or its antigen-binding fragment comprises a first antigen-binding site that recognizes FIX (SEQ ID NO: 1) or its active form (FIXa), and a second antigen-binding site that recognizes FX (SEQ ID NO: 2) or its active form (FXa), where a) The first antigen-binding site includes the following CDR sequence: V H -CDR1:DYAMH V H -CDR2:GISWRGDIIGYVDSVKG V H -CDR3:SYGSGSFYNAFDS V L -CDR1:RASQSISSWLA V L -CDR2:KASRLDR V L-CDR3: LEYSSYIRT and b) The second antigen-binding site comprises the following CDR sequences: V H -CDR1: TSWIV V H -CDR2: MIDPSDSFTSYSPSFQG V H -CDR3: LHYYHSEEFDV V L -CDR1: RASQSVSSSYLA V L -CDR2: GASSRAR V L -CDR3: QQFGSSRLFT

[0213] In one embodiment, the bispecific antibody or its antigen-binding fragment comprises a first antigen-binding site that recognizes FIX (SEQ ID NO: 1) or its active form (FIXa), and a second antigen-binding site that recognizes FX (SEQ ID NO: 2) or its active form (FXa), where a) The first antigen-binding site comprises the following CDR sequences: V H -CDR1: DYAMH V H -CDR2: GISWRGDIIGYVDSVKG V H -CDR3: SYGSGSFYNAFDS V L -CDR1: RASQSISSWLA V L -CDR2: KASRLDR V L -CDR3: LEYSSYIRT and b) The second antigen-binding site comprises the following CDR sequences: V H -CDR1: TSWIV V H -CDR2: MIDPSDSFTSYSPSFQG V H -CDR3: LHYYHSEEFDV V L-CDR1: RASQSVSSSYLA V L -CDR2: GASSRTR V L -CDR3: QQFGSSRLFT

[0214] The present invention will be further described by the following embodiments. 1. An antibody or an antigen-binding fragment thereof that can bind to factor IX (FIX) according to SEQ ID NO: 1 or its active form (FIXa).

[0215] 2. The antibody or an antigen-binding fragment thereof according to embodiment 1, wherein the antibody or an antigen-binding fragment thereof is part of "BIN1".

[0216] 3. The antibody or an antigen-binding fragment thereof according to embodiment 1, wherein the antibody or an antigen-binding fragment thereof competes with a reference antibody, and the reference antibody a. comprises a heavy chain variable domain identified by SEQ ID NO: 15 and a light chain variable domain identified by SEQ ID NO: 16, or b. comprises a heavy chain variable domain identified by SEQ ID NO: 19 and a light chain variable domain identified by SEQ ID NO: 20.

[0217] 4. The antibody according to the above-described embodiments, wherein the reference antibody is Fab.

[0218] 5. The antibody or an antigen-binding fragment thereof a. has a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 15 and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 16, b. has a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 19 and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 20. c. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 69, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 70. d. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 71, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 72. e. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 73, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 74. f. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 83, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 84. g. A heavy chain variable domain that is at least 90% identical to the sequence identified by Sequence ID No. 81, and a light chain variable domain that is at least 90% identical to the sequence identified by Sequence ID No. 82. h. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 75, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 76, i. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 77, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 78, or j. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 177, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 178.

[0219] 6. The antibody or antigen-binding fragment thereof according to Embodiment 5, wherein the heavy chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0220] 7. The antibody or antigen-binding fragment thereof according to Embodiment 5, wherein the light chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified sequence number.

[0221] 8. The antibody or antigen-binding fragment thereof according to Embodiments 6 and 7, wherein both the heavy chain variable domain and the light chain variable domain are at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0222] 9. The antibody or its antigen-binding fragment a. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 15, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 16, b. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 19, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 20, c. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 69, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 70, d. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 71, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 72, e. i. Three heavy-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the heavy-chain variable domain identified by SEQ ID NO: 73, and ii. Three light-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the light-chain variable domain identified by SEQ ID NO: 74, f. i. Three heavy-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the heavy-chain variable domain identified by SEQ ID NO: 83, and ii. Three light-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the light-chain variable domain identified by SEQ ID NO: 84, g. i. Three heavy-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the heavy-chain variable domain identified by SEQ ID NO: 81, and ii. Three light-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the light-chain variable domain identified by SEQ ID NO: 82, h. i. Three heavy-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the heavy-chain variable domain identified by SEQ ID NO: 75, and ii. Three light-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the light-chain variable domain identified by SEQ ID NO: 76, or i. i. Three heavy-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the heavy-chain variable domain identified by SEQ ID NO: 77, and ii. Three light-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the light-chain variable domain identified by SEQ ID NO: 78, or j. i. Three heavy-chain CDR sequences having a maximum of 10 amino acid changes as compared to the CDR sequences of the heavy-chain variable domain identified by SEQ ID NO: 177, and ii. An antibody or antigen-binding fragment according to any of the above embodiments, comprising three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 178.

[0223] 10. The antibody or antigen-binding fragment according to Embodiment 9, wherein the three heavy chain CDR sequences have up to nine amino acid changes, such as eight, seven, and six, compared to the CDR of the identified sequence number.

[0224] 11. The antibody or antigen-binding fragment according to Embodiment 9, wherein the three heavy chain CDR sequences have up to five amino acid changes, such as four, three, and two, or up to one amino acid change, compared to the CDR of the identified sequence number.

[0225] 12. The antibody or antigen-binding fragment according to Embodiment 9, wherein three light chain CDR sequences have up to nine amino acid changes, such as eight, seven, and six, compared to the CDR of the identified sequence number.

[0226] 13. The antibody or antigen-binding fragment according to Embodiment 9, wherein the three light chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0227] 14. The antibody or its antigen-binding fragment a. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 15, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 16, or b. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 19, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 20, or c. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 3, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 4, or d. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 109, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 110, or e. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 153, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 154, or f. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 171, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 172, or g. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 177, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 178, or h. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 187 and a CDR sequence of the light chain variable domain identified by SEQ ID NO: 188.

[0228] 15. The antibody or its antigen-binding fragment a. The heavy chain variable domain identified by SEQ ID NO: 15, and the light chain variable domain identified by SEQ ID NO: 16, or b. The heavy chain variable domain identified by Sequence ID No. 19, and the light chain variable domain identified by Sequence ID No. 20, or c. The heavy chain variable domain identified by Sequence ID No. 3, and the light chain variable domain identified by Sequence ID No. 4, or d. The heavy chain variable domain identified by SEQ ID NO: 109, and the light chain variable domain identified by SEQ ID NO: 110, or e. The heavy chain variable domain identified by Sequence ID No. 153, and the light chain variable domain identified by Sequence ID No. 154, or f. The heavy chain variable domain identified by Sequence ID No. 171, and the light chain variable domain identified by Sequence ID No. 172, or g. The heavy chain variable domain identified by Sequence ID No. 177, and the light chain variable domain identified by Sequence ID No. 178, or h. An antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising a heavy chain variable domain identified by SEQ ID NO: 187 and a light chain variable domain identified by SEQ ID NO: 188.

[0229] 16. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising one or more of the amino acid residues L337, R338, S339, T340, K341, and T343 of SEQ ID NO: 1.

[0230] 17. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment is capable of binding to an epitope containing amino acid residue R338 of SEQ ID NO: 1.

[0231] 18. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising amino acid residues R338 and K341 of SEQ ID NO: 1.

[0232] 19. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising two or three of the amino acid residues L337, R338, S339, T340, K341, and T343 of SEQ ID NO: 1.

[0233] 20. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising four or five of the amino acid residues L337, R338, S339, T340, K341, and T343 of SEQ ID NO: 1.

[0234] 21. The antibody or its antigen-binding fragment is of SEQ ID NO: 1 a. R338, S339, T340, K341 and T343, b.L337, S339, T340, K341 and T343, c.L337, R338, T340, K341 and T343, d.L337, R338, S339, K341 and T343, e.L337, R338, S339, T340 and T343 or f.L337, R338, S339, T340 and K341 An antibody or antigen-binding fragment thereof, as described in any of the above embodiments, capable of binding to an epitope containing the above.

[0235] 22. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising the amino acid residues L337, R338, S339, T340, K341 and T343 of SEQ ID NO: 1.

[0236] 23. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising one or more of the amino acid residues K301, D332, R333, A334, T335, R338 and N346 of SEQ ID NO: 1.

[0237] 24. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising the amino acid residues L337, R338, S339, T340, K341 and T343 of SEQ ID NO: 1.

[0238] 25. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising two or three of the amino acid residues K301, D332, R333, A334, T335, R338 and N346 of SEQ ID NO: 1.

[0239] 26. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising four or five of the amino acid residues K301, D332, R333, A334, T335, R338 and N346 of SEQ ID NO: 1.

[0240] 27. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising five or six of the amino acid residues K301, D332, R333, A334, T335, R338 and N346 of SEQ ID NO: 1.

[0241] 28. The antibody or its antigen-binding fragment is of SEQ ID NO: 1 a. D332, R333, A334, T335, R338 and N346 b.K301, R333, A334, T335, R338 and N346 c.K301, D332, A334, T335, R338 and N346 d.K301, D332, R333, T335, R338 and N346 e.K301, D332, R333, A334, R338 and N346 f.K301, D332, R333, A334, T335 and N346 or g.K301, D332, R333, A334, T335 and R338 An antibody or antigen-binding fragment thereof, as described in any of the above embodiments, capable of binding to an epitope containing the above.

[0242] 29. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising amino acid residues D332, R333, L337 and R338 of SEQ ID NO: 1.

[0243] 30. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising the amino acid residues K301, D332, R333, A334, T335, R338 and N346 of SEQ ID NO: 1.

[0244] 31. The antibody or antigen-binding fragment thereof according to Embodiment 1, wherein the antibody or antigen-binding fragment thereof belongs to "Bin 2".

[0245] 32. The antibody or antigen-binding fragment according to Embodiment 1, wherein the antibody or antigen-binding fragment competes with a reference antibody, and the reference antibody comprises a heavy chain variable domain identified by the sequence of SEQ ID NO: 17 and a light chain variable domain identified by the sequence of SEQ ID NO: 18.

[0246] 33. The antibody described in the above embodiment, wherein the reference antibody is Fab.

[0247] 34. The antibody or its antigen-binding fragment a. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 17, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 18. b. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 85, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 86, or c. An antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 79, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 80.

[0248] 35. The antibody or antigen-binding fragment thereof according to Embodiment 34, wherein the heavy chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0249] 36. The antibody or antigen-binding fragment thereof according to Embodiment 34, wherein the light chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0250] 37. The antibody or antigen-binding fragment thereof according to Embodiments 35 and 36, wherein both the heavy chain variable domain and the light chain variable domain are at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0251] 38. Antibodies or their antigen-binding fragments, a. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 17, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 18, b. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 85, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 86, or c. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 79, and ii. An antibody or antigen-binding fragment according to any of the above embodiments, comprising three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 80.

[0252] 39. The antibody or antigen-binding fragment according to Embodiment 38, wherein the three heavy chain CDR sequences have up to nine amino acid changes, such as 8, 7, and 6, compared to the CDR of the identified sequence number.

[0253] 40. The antibody or antigen-binding fragment according to Embodiment 38, wherein the three heavy chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0254] 41. The antibody or antigen-binding fragment according to Embodiment 38, wherein three light chain CDR sequences have up to nine amino acid changes, such as 8, 7, and 6, compared to the CDR of the identified sequence number.

[0255] 42. The antibody or antigen-binding fragment according to Embodiment 38, wherein the three light chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0256] 43. Antibodies or antigen-binding fragments a. The CDR sequence of the heavy chain variable domain identified by Sequence ID No. 17, An antibody or antigen-binding fragment according to any of the above embodiments, comprising a CDR sequence of a light chain variable domain identified by SEQ ID NO: 18.

[0257] 44. Antibodies or antigen-binding fragments a. An antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising a heavy chain variable domain identified by SEQ ID NO: 17 and a light chain variable domain identified by SEQ ID NO: 18.

[0258] 45. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising one or more of the amino acid residues H256, H257, N258, K293, R403, Y404, N406, W407, E410, and K411 of SEQ ID NO: 1.

[0259] 46. ​​The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising the amino acid residues H256, H257, N258, K293, R403, Y404, N406, W407, E410 and K411 of SEQ ID NO: 1.

[0260] 47. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising two, three, or four of the amino acid residues H256, H257, N258, K293, R403, Y404, N406, W407, E410, and K411 of SEQ ID NO: 1.

[0261] 48. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising five, six, or seven amino acid residues from among SEQ ID NO: 1, H256, H257, N258, K293, R403, Y404, N406, W407, E410, and K411.

[0262] 49. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising eight, nine, or ten of the amino acid residues H256, H257, N258, K293, R403, Y404, N406, W407, E410, and K411 of SEQ ID NO: 1.

[0263] 50. The antibody or its antigen-binding fragment is the amino acid residue of SEQ ID NO: 1. a. H257, N258, K293, R403, Y404, N406, W407, E410 and K411, b. H256, N258, K293, R403, Y404, N406, W407, E410 and K411, c.H256, H257, K293, R403, Y404, N406, W407, E410 and K411, d.H256, H257, N258, R403, Y404, N406, W407, E410 and K411, e.H256, H257, N258, K293, Y404, N406, W407, E410 and K411, f.H256, H257, N258, K293, R403, N406, W407, E410 and K411, g.H256, H257, N258, K293, R403, Y404, W407, E410 and K411, h.H256, H257, N258, K293, R403, Y404, N406, E410 and K411, i.H256, H257, N258, K293, R403, Y404, N406, W407 and K411 or j.H256, H257, N258, K293, R403, Y404, N406, W407 and E410 An antibody or antigen-binding fragment thereof, as described in any of the above embodiments, capable of binding to an epitope containing the above.

[0264] 51. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising the amino acid residues H256, H257, N258, K293, R403, Y404, N406, W407, E410 and K411 of SEQ ID NO: 1.

[0265] 52. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising amino acid residues H257, K293 and N406 of SEQ ID NO: 1.

[0266] 53. The antibody or its antigen-binding fragment according to any one of the above embodiments, wherein the antibody is a blood coagulation antibody.

[0267] 54. The antibody or antigen-binding fragment according to any one of the above embodiments, wherein the antibody or antigen-binding fragment can increase the blood coagulation activity of FIXa.

[0268] 55. An antibody or antigen-binding fragment according to any one of the above embodiments, wherein the antibody can increase the enzymatic activity of FIXa against FX.

[0269] 56. The antibody or its antigen-binding fragment according to any one of the above embodiments, wherein the antibody can functionally substitute for FVIII and / or FVIIIa.

[0270] 57. An antibody or its antigen-binding fragment that can bind to FX (SEQ ID NO: 2) or its active form (FIXa).

[0271] 58. The antibody or antigen-binding fragment thereof according to Embodiment 57, wherein the antibody or antigen-binding fragment thereof is part of "Bin A".

[0272] 59. The antibody or antigen-binding fragment according to Embodiment 57, wherein the antibody or antigen-binding fragment competes with a reference antibody, the reference antibody comprising a heavy chain variable domain identified by SEQ ID NO: 65 and a light chain variable domain identified by SEQ ID NO: 66.

[0273] 60. The antibody described in the above embodiment, wherein the reference antibody is Fab.

[0274] 61. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising one or more of the amino acid residues H101, E103, R113, T116, L117, A118, T127, S227, E228, F229, Y230, E266, R287, L303, P304, E305, L419, K420, D423, R424, M426, K427, and T428 of FX / FXa.

[0275] 62. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising the amino acid residues H101, E103, R113, T116, L117, A118, T127, S227, E228, F229, Y230, E266, R287, P304, L303, P304, E305, L419, K420, D423, R424, M426, K427 and T428 of FX / FXa.

[0276] 63. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising one or more of the amino acid residues R113, Y230, K420, D423, R424, and K427 of FX / FXa.

[0277] 64. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment can bind to an epitope comprising the amino acid residues R113, Y230, K420, D423, R424, and K427 of FX / FXa.

[0278] 65. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 65 and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 66.

[0279] 66. The antibody or antigen-binding fragment thereof according to Embodiment 65, wherein the heavy chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0280] 67. The antibody or antigen-binding fragment thereof according to Embodiment 65, wherein the light chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0281] 68. The antibody or antigen-binding fragment thereof according to Embodiments 66 and 67, wherein both the heavy chain variable domain and the light chain variable domain are at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0282] 69. Antibodies or antigen-binding fragments a. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 65, and b. An antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 66.

[0283] 70. The antibody or antigen-binding fragment thereof according to Embodiment 69, wherein three heavy chain CDR sequences have up to nine amino acid changes, such as eight, seven, and six, compared to the CDR of the identified sequence number.

[0284] 71. The antibody or antigen-binding fragment according to Embodiment 69, wherein the three heavy chain CDR sequences have up to five amino acid changes, such as four, three, and two, or up to one amino acid change, compared to the CDR of the identified sequence number.

[0285] 72. The antibody or antigen-binding fragment according to Embodiment 69, wherein three light chain CDR sequences have up to nine amino acid changes, such as eight, seven, and six, compared to the CDR of the identified sequence number.

[0286] 73. The antibody or antigen-binding fragment according to Embodiment 69, wherein the three light chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0287] 74. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment comprises a CDR sequence of a heavy chain variable domain identified by SEQ ID NO: 65 and a CDR sequence of a light chain variable domain identified by SEQ ID NO: 66.

[0288] 75. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain identified by SEQ ID NO: 65 and a light chain variable domain identified by SEQ ID NO: 66.

[0289] 76. The antibody or antigen-binding fragment according to Embodiment 57, wherein the antibody or antigen-binding fragment is part of "Bin B".

[0290] 77. The antibody or antigen-binding fragment according to Embodiment 57, wherein the antibody or antigen-binding fragment competes with a reference antibody, the reference antibody comprising a heavy chain variable domain identified by SEQ ID NO: 67 and a light chain variable domain identified by SEQ ID NO: 68.

[0291] 78. The antibody or antigen-binding fragment thereof according to the above embodiment, wherein the reference antibody is Fab.

[0292] 79. Antibodies or their antigen-binding fragments, a. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 67, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 68. b. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 23, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 24. c. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 39, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 40, or d. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 59, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 60.

[0293] 80. The antibody or antigen-binding fragment thereof according to Embodiment 79, wherein the heavy chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0294] 81. The antibody or antigen-binding fragment thereof according to Embodiment 79, wherein the light chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0295] 82. The antibody or antigen-binding fragment thereof according to Embodiments 80 and 81, wherein both the heavy chain variable domain and the light chain variable domain are at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0296] 83. Antibodies or their antigen-binding fragments, a. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 67, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 68, b. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 23, and ii. Three light chain CDR sequences with up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 24, c. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 39, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 40, or d. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 59, and ii. An antibody or antigen-binding fragment according to any of the above embodiments, comprising three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 60.

[0297] 84. The antibody or antigen-binding fragment according to Embodiment 83, wherein the three heavy chain CDR sequences have up to nine amino acid changes, such as 8, 7, and 6, compared to the CDR of the identified sequence number.

[0298] 85. The antibody or antigen-binding fragment according to Embodiment 83, wherein the three heavy chain CDR sequences have up to five amino acid changes, such as four, three, and two, or up to one amino acid change, compared to the CDR of the identified sequence number.

[0299] 86. The antibody or antigen-binding fragment according to Embodiment 83, wherein three light chain CDR sequences have up to nine amino acid changes, such as 8, 7, and 6, compared to the CDR of the identified sequence number.

[0300] 87. The antibody or antigen-binding fragment according to Embodiment 83, wherein the three light chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0301] 88. Antibodies or their antigen-binding fragments, a. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 67 and a CDR sequence of the light chain variable domain identified by SEQ ID NO: 68. b. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 23 and a CDR sequence of the light chain variable domain identified by SEQ ID NO: 24. c. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 39, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 40, or d. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 59 and a CDR sequence of the light chain variable domain identified by SEQ ID NO: 60.

[0302] 89. Antibodies or their antigen-binding fragments, a. Heavy chain variable domain identified by Sequence ID No. 67, and light chain variable domain identified by Sequence ID No. 68, b. Heavy chain variable domain identified by Sequence ID No. 23, and light chain variable domain identified by Sequence ID No. 24, c. The heavy chain variable domain identified by Sequence ID No. 39, and the light chain variable domain identified by Sequence ID No. 40, or d. An antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising a heavy chain variable domain identified by SEQ ID NO: 59 and a light chain variable domain identified by SEQ ID NO: 60.

[0303] 90. The antibody or antigen-binding fragment according to Embodiment 57, wherein the antibody or antigen-binding fragment is part of "Bin C".

[0304] 91. The antibody or antigen-binding fragment according to Embodiment 57, wherein the antibody or antigen-binding fragment competes with a reference antibody, the reference antibody comprising a heavy chain variable domain identified by SEQ ID NO: 21 and a light chain variable domain identified by SEQ ID NO: 22.

[0305] 92. The antibody or antigen-binding fragment thereof according to the above embodiment, wherein the reference antibody is Fab.

[0306] 93. Antibodies or their antigen-binding fragments, a. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 21, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 22. b. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 25, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 26. c. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 27, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 28. d. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 29, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 30. e. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 31, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 32. f. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 33, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 34. g. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 35, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 36. h. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 37, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 38, i. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 39, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 40. j. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 41, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 42, k. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 43, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 44, l. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 51, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 52, m. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 53, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 54, n. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 55, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 56. o. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 57, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 58. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 59, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 60. q. A heavy chain variable domain that is at least 90% identical to the sequence identified by sequence number 61, and a light chain variable domain that is at least 90% identical to the sequence identified by sequence number 62, or An antibody or antigen-binding fragment according to any of the above embodiments, comprising a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 63, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 64.

[0307] 94. The antibody or antigen-binding fragment thereof according to Embodiment 93, wherein the heavy chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0308] 95. The antibody or antigen-binding fragment thereof according to Embodiment 93, wherein the light chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0309] 96. The antibody or antigen-binding fragment thereof according to Embodiments 94 and 95, wherein both the heavy chain variable domain and the light chain variable domain are at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0310] 97. Antibodies or their antigen-binding fragments, a. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 21, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 22, b. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 25, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 26, c. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 27, and ii. A tri-light chain CDR sequence having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 28, or d. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 29, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 30, e. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 31, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 32, f. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 33, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 34, calcium. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 35, and ii. A tri-light chain CDR sequence having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 36, or h. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 37, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 38, i. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 39, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 40, j. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 41, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 42, k. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 43, and ii. A tri-light chain CDR sequence having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 44, or l. m. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 51, and ii. A tri-light chain CDR sequence having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 52, or n. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 53, and Compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 54, there are three light chain CDR sequences with up to 10 amino acid changes. o. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 55, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 56, p. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 57, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 58, q. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 59, and ii. Three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 60, r. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 61, and ii. A tri-light chain CDR sequence having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 62, or s. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 63, and ii. An antibody or antigen-binding fragment according to any of the above embodiments, comprising three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 64.

[0311] 98. The antibody or antigen-binding fragment according to Embodiment 97, wherein the three heavy chain CDR sequences have up to nine amino acid changes, such as eight, seven, and six, compared to the CDR of the identified sequence number.

[0312] 99. The antibody or antigen-binding fragment according to Embodiment 97, wherein the three heavy chain CDR sequences have up to five amino acid changes, such as four, three, and two, or up to one amino acid change, compared to the CDR of the identified sequence number.

[0313] 100. The antibody or antigen-binding fragment according to Embodiment 97, wherein three light chain CDR sequences have up to nine amino acid changes, such as eight, seven, and six, compared to the CDR of the identified sequence number.

[0314] 101. The antibody or antigen-binding fragment according to Embodiment 97, wherein the three light chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0315] 102. Antibodies or their antigen-binding fragments, a. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 21, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 22. b. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 25, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 26. c. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 27, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 28. d. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 29, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 30, e. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 31, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 32. f. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 33, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 34. g. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 35, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 36. h. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 37, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 38. i. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 39, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 40. j. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 41, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 42, k. CDR sequence of the heavy chain variable domain identified by sequence number 43, and CDR sequence of the light chain variable domain identified by sequence number 44, l. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 51, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 52, m. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 53, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 54, n. The CDR sequence of the heavy chain variable domain identified by sequence number 55, and the CDR sequence of the light chain variable domain identified by sequence number 56. o. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 57, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 58, p. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 59, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 60. q. The CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 61, and the CDR sequence of the light chain variable domain identified by SEQ ID NO: 62, or r. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 63 and a CDR sequence of the light chain variable domain identified by SEQ ID NO: 64.

[0316] 103. Antibodies or their antigen-binding fragments, a. Sequence IDs 21 and 22, b. Sequence IDs 25 and 26, c. Sequence IDs 27 and 28, d. Sequence IDs 29 and 30, e. Sequence IDs 31 and 32, f. Sequence numbers 33 and 34, g. Sequence IDs 35 and 36, h. Sequence IDs 37 and 38, i. Sequence IDs 39 and 40, j. Sequence IDs 41 and 42, k. Sequence IDs 43 and 44, l. Sequence IDs 51 and 52, m. Sequence IDs 53 and 54, n. Sequence IDs 55 and 56, o. Sequence IDs 57 and 58, p. Sequence IDs 59 and 60, q. Sequence IDs 61 and 62 or r. An antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising a heavy chain variable domain and a light chain variable domain, as identified by SEQ ID NO: 63 and SEQ ID NO: 64, respectively.

[0317] 104. The antibody or antigen-binding fragment thereof according to Embodiment 57, wherein the antibody or antigen-binding fragment thereof belongs to "Vin D".

[0318] 105. The antibody or antigen-binding fragment according to Embodiment 57, wherein the antibody or antigen-binding fragment competes with a reference antibody, the reference antibody comprising a heavy chain variable domain identified by SEQ ID NO: 47 and a light chain variable domain identified by SEQ ID NO: 48.

[0319] 106. The antibody described in the above embodiment, wherein the reference antibody is Fab.

[0320] 107. Antibodies or their antigen-binding fragments, a. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 47, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 48. b. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 45, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 46. c. A heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 51, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 52. or d. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 61, and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 62.

[0321] 108. The antibody or antigen-binding fragment thereof according to Embodiment 107, wherein the heavy chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0322] 109. The antibody or antigen-binding fragment thereof according to Embodiment 107, wherein the light chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0323] 110. The antibody or antigen-binding fragment thereof according to Embodiments 108 and 109, wherein both the heavy chain variable domain and the light chain variable domain are at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0324] 111. Antibodies or their antigen-binding fragments, a. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 47, and ii. Three light chain CDR sequences with up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 48, b. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 45, and ii. Three light chain CDR sequences with up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 46, c. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 51, and ii. A tri-light chain CDR sequence having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by Sequence ID No. 52, or d. i. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 61, and ii. An antibody or antigen-binding fragment according to any of the above embodiments, comprising three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 62.

[0325] 112. The antibody or antigen-binding fragment thereof according to Embodiment 111, wherein three heavy chain CDR sequences have up to nine amino acid changes, such as 8, 7, and 6, compared to the CDR of the identified sequence number.

[0326] 113. The antibody or antigen-binding fragment according to Embodiment 111, wherein the three heavy chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0327] 114. The antibody or antigen-binding fragment thereof according to Embodiment 111, wherein three light chain CDR sequences have up to nine amino acid changes, such as eight, seven, and six, compared to the CDR of the identified sequence number.

[0328] 115. The antibody or antigen-binding fragment according to Embodiment 111, wherein the three light chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0329] 116. Antibodies or their antigen-binding fragments, a. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 47, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 48. b. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 45, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 46. c. CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 51, and CDR sequence of the light chain variable domain identified by SEQ ID NO: 52. or d. An antibody or antigen-binding fragment according to any of the above embodiments, comprising a CDR sequence of the heavy chain variable domain identified by SEQ ID NO: 61 and a CDR sequence of the light chain variable domain identified by SEQ ID NO: 62.

[0330] 117. Antibodies or their antigen-binding fragments, a. Heavy chain variable domain identified by Sequence ID No. 47, and light chain variable domain identified by Sequence ID No. 48, b. Heavy chain variable domain identified by Sequence ID No. 45, and light chain variable domain identified by Sequence ID No. 46, c. Heavy chain variable domain identified by Sequence ID No. 51, and light chain variable domain identified by Sequence ID No. 52, or d. An antibody or antigen-binding fragment thereof according to any of the above embodiments, comprising a heavy chain variable domain identified by SEQ ID NO: 61 and a light chain variable domain identified by SEQ ID NO: 62.

[0331] 118. The antibody or antigen-binding fragment thereof according to Embodiment 57, wherein the antibody or antigen-binding fragment thereof belongs to "Bin E".

[0332] 119. The antibody or antigen-binding fragment according to Embodiment 57, wherein the antibody or antigen-binding fragment competes with a reference antibody, the reference antibody comprising a heavy chain variable domain identified by SEQ ID NO: 49 and a light chain variable domain identified by SEQ ID NO: 50.

[0333] 120. The antibody described in the above embodiment, wherein the reference antibody is Fab.

[0334] 121. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 49 and a light chain variable domain that is at least 90% identical to the sequence identified by SEQ ID NO: 50.

[0335] 122. The antibody or antigen-binding fragment thereof according to Embodiment 121, wherein the heavy chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0336] 123. The antibody or antigen-binding fragment thereof according to Embodiment 121, wherein the light chain variable domain is at least 92%, 94%, 96%, or 98% identical to the identified sequence number.

[0337] 124. The antibody or antigen-binding fragment thereof according to Embodiments 122 and 123, wherein both the heavy chain variable domain and the light chain variable domain are at least 92%, 94%, 96%, or 98% identical to the identified SEQ ID NO:

[0338] 125. Antibodies or their antigen-binding fragments, a. Three heavy chain CDR sequences with up to 10 amino acid changes compared to the heavy chain variable domain CDR sequence identified by Sequence ID No. 49, and b. An antibody or antigen-binding fragment according to any of the above embodiments, comprising three light chain CDR sequences having up to 10 amino acid changes compared to the CDR sequence of the light chain variable domain identified by SEQ ID NO: 50.

[0339] 126. The antibody or antigen-binding fragment according to Embodiment 125, wherein the three heavy chain CDR sequences have up to nine amino acid changes, such as 8, 7, and 6, compared to the CDR of the identified sequence number.

[0340] 127. The antibody or antigen-binding fragment according to Embodiment 125, wherein the three heavy chain CDR sequences have up to five amino acid changes, such as four, three, and two, or up to one amino acid change, compared to the CDR of the identified sequence number.

[0341] 128. The antibody or antigen-binding fragment according to Embodiment 125, wherein three light chain CDR sequences have up to nine amino acid changes, such as 8, 7, and 6, compared to the CDR of the identified sequence number.

[0342] 129. The antibody or antigen-binding fragment according to Embodiment 125, wherein the three light chain CDR sequences have up to 5 amino acid changes, such as 4, 3, and 2, or up to 1 amino acid change, compared to the CDR of the identified sequence number.

[0343] 130. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment comprises a CDR sequence of a heavy chain variable domain identified by SEQ ID NO: 49 and a CDR sequence of a light chain variable domain identified by SEQ ID NO: 50.

[0344] 131. The antibody or antigen-binding fragment according to any of the above embodiments, wherein the antibody or antigen-binding fragment comprises a heavy chain variable domain identified by SEQ ID NO: 49 and a light chain variable domain identified by SEQ ID NO: 50.

[0345] 132. A multispecific antibody or its antigen-binding fragment capable of binding to FIX / FIXa and FX / FXa.

[0346] 133. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 1 to 131 described above.

[0347] 134. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 2 to 30 described above.

[0348] 135. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 31 to 52 described above.

[0349] 136. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 57 to 131 described above.

[0350] 137. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 58 to 75 described above.

[0351] 138. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of the above embodiments 76 to 89.

[0352] 139. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 90 to 103 described above.

[0353] 140. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 104 to 117 described above.

[0354] 141. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of the embodiments 118 to 131 described above.

[0355] 142. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 1 to 56 and an antigen-binding fragment described in any of Embodiments 57 to 131.

[0356] 143. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 2 to 30 above and an antigen-binding fragment described in any of Embodiments 58 to 75 above.

[0357] 144. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 2 to 30 and an antigen-binding fragment described in any of Embodiments 76 to 89.

[0358] 145. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 2 to 30 above and an antigen-binding fragment described in any of Embodiments 90 to 103 above.

[0359] 146. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 31 to 52 and an antigen-binding fragment described in any of Embodiments 58 to 75.

[0360] 147. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 31 to 52 and an antigen-binding fragment described in any of Embodiments 76 to 89.

[0361] 148. The multispecific antibody or antigen-binding fragment according to Embodiment 132, wherein the antibody comprises an antigen-binding fragment described in any of Embodiments 31 to 52 and an antigen-binding fragment described in any of Embodiments 90 to 103.

[0362] 149. The antibody is a bispecific antibody capable of specifically binding FIX / FIXa and FX / FXa, and the binding domains are mAb1-1371 / mAb1-1307, mAb1-6705 / mAb1-1307, mAb1-1371 / mAb0-1886, mAb1-7441 / mAb0-1886, mAb1-7447 / mAb0-1886, mAb1-7481 / mAb0-1886, mAb1-1371 / mAb0-1998, mAb1-6716 / mAb0-1998, mAb1-6723 / mAb0-1998, mAb1-6730 / mAb0-1998, mAb1-6731 / A multispecific antibody or its antigen-binding fragment according to Embodiment 132, derived from an mAb pair consisting of mAb0-1998, mAb1-6737 / mAb0-1998, mAb1-6754 / mAb0-1998, mAb1-7378 / mAb0-1998, mAb1-7441 / mAb0-1998, mAb1-7447 / mAb0-1998, mAb / mAb0-1998, mAb1-6723 / mAb1-1307, mAb1-6723 / mAb0-1886, mAb1-6705 / mAb0-1886, mAb1-7481 / mAb0-1998, and mAb1-6705 / mAb0-1998.

[0363] 150. The antibody or its antigen-binding fragment according to any one of Embodiments 132 to 149, wherein the antibody is a blood coagulation bispecific antibody.

[0364] 151. The antibody or antigen-binding fragment thereof according to any one of Embodiments 132 to 149, wherein the antibody is a bispecific antibody capable of increasing the blood coagulation activity of FIXa.

[0365] 152. An antibody or antigen-binding fragment thereof according to any one of embodiments 132 to 149, wherein the antibody is a bispecific antibody capable of increasing the enzymatic activity of FIXa against FX.

[0366] 153. An antibody or antigen-binding fragment thereof according to any of Embodiments 132 to 149, wherein the antibody is a bispecific antibody capable of functionally substituting FVIII and / or FVIIIa.

[0367] 154. A multispecific antibody capable of stimulating the enzymatic activity of FIXa against FX, comprising a first antigen-binding site that recognizes FIX (SEQ ID NO: 1) or its active form (FIXa), and a second antigen-binding site that recognizes FX (SEQ ID NO: 2) or its active form (FXa), a) The first antigen-binding site contains a CDR of an antibody selected from the group consisting of mAb1-5743, mAb1-6584, mAb1-8768, mAb1-6037, mAb1-6081, mAb1-4857, mAb1-8780, mAb1-9016, mAb1-9015, mAb1-8467, mAb1-5783, or mAb1-5781, b) A multispecific antibody comprising a CDR of an antibody selected from the group consisting of mAb1-6738, mAb1-6463, mAb1-6723, mAb1-7503, or mAb1-6097 as the second antigen-binding site.

[0368] A multispecific antibody capable of stimulating the enzymatic activity of FIXa against 155.FX, The first polypeptide that recognizes FIX / FIXa, It contains a second polypeptide that recognizes FX / FXa, a) The first polypeptide comprises the heavy chain variable domain and light chain variable domain of mAb1-5743, mAb1-6584, mAb1-8768, mAb1-6037, mAb1-6081, mAb1-4857, mAb1-8780, mAb1-9016, mAb1-9015, mAb1-8467, mAb1-5783, or mAb1-5781, b) The second polypeptide is a multispecific antibody containing the heavy chain variable domain and light chain variable domain of mAb1-6738, mAb1-6463, mAb1-6723, mAb1-7503, or mAb1-6097.

[0369] 156. The antibody according to either embodiment 154 or 155, wherein the antibody is a bispecific antibody.

[0370] 157. The stimulation of the enzymatic activity of FIXa to FX is determined by the FXa production assay described herein using a monovalent, one-armed anti-FIX / FIXa antibody, and the stimulation index is at least 94, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 3000, 4000, or 5000 times, according to any one of Embodiments 150 to 156.

[0371] 158. A multispecific antibody capable of stimulating the enzymatic activity of FIXa against FX, A first antigen-binding site that recognizes FIX (SEQ ID NO: 1) or its active form (FIXa), and a second antigen-binding site that recognizes FX (SEQ ID NO: 2) or its active form (FXa), wherein The first antigen-binding site can specifically bind to an epitope containing amino acid residue R338 of FIX / FIXa, and A multispecific antibody in which the second antigen-binding site can be conjugated via the EGF-2 domain and / or the FX / FXa catalytic subunit.

[0372] 159. A multispecific antibody according to Embodiment 158, The primary antigen-binding site is a multispecific antibody capable of specifically binding to epitopes containing amino acid residues R338 and K341 of FIX / FIXa.

[0373] 160. A multispecific antibody according to Embodiment 159, The primary antigen-binding site is a multispecific antibody capable of specifically binding to epitopes containing amino acid residues L337, R338, S339, T340, K341, and T343 of FIX / FIXa.

[0374] 161. A multispecific antibody capable of stimulating the enzymatic activity of FIXa against FX, comprising a first antigen-binding site that recognizes FIX (SEQ ID NO: 1) or its active form FIXa, and a second antigen-binding site that recognizes FX (SEQ ID NO: 2) or its active form (FXa), Here The first antigen-binding site can specifically bind to epitopes containing amino acid residues D332, R333, L337, and R338 of FIX / FIXa, and A multispecific antibody in which the second antigen-binding site can be conjugated via the EGF-2 domain and / or the FX / FXa catalytic subunit.

[0375] 162. A multispecific antibody according to Embodiment 161, The primary antigen-binding site is a multispecific antibody capable of specifically binding to epitopes containing amino acid residues K301, D332, R333, A334, T335, R338, and N346 of FIX / FIXa.

[0376] A multispecific antibody capable of stimulating the enzymatic activity of FIXa against 163.FX, The first antigen-binding site that recognizes FIX / FIXa, It includes a second antigen-binding site that recognizes FX / FXa, Here The first antigen-binding site can specifically bind to epitopes containing amino acid residues H257, K293, and N406 of FIX / FIXa, and A multispecific antibody in which the second antigen-binding site can be conjugated via the EGF-2 domain and / or the FX / FXa catalytic subunit.

[0377] 164. The stimulation of FIXa's enzymatic activity to FX is measured by the FXa production assay described herein using a monovalent, one-armed anti-FIX / FIXa antibody, and the stimulation index is at least 94, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 3000, 4000, or 5000 times, according to any one of Embodiments 1-56 and 132-156.

[0378] 165. The stimulation of FIXa's enzymatic activity against FX was measured by the FXa production assay described herein using a monovalent, one-armed anti-FIX / FIXa antibody, and when measured using a one-armed antibody concentration that resulted in FIXa saturating at least 80%, the stimulation index was 94-2500 times, 100-2500 times, 200-2500 times, 300-2500 times, 400-2500 times, etc. An antibody according to any one of Embodiments 1 to 56 and 132 to 156, which is between 50 and 5000 times, such as 00 to 2500 times, 600 to 2500 times, 700 to 2500 times, 800 to 2500 times, 900 to 2500 times, 1000 to 2500 times, or 1500 to 2500 times.

[0379] 166. The antibody according to any one of the embodiments described above, wherein the antibody is a blood coagulation antibody.

[0380] 167. The antibody according to any one of the above embodiments, wherein the antibody functionally substitutes for FVIII and / or FVIIIa.

[0381] 168. The antibody according to any one of the embodiments described above, wherein the antibody is a bispecific antibody.

[0382] 169. The antibody according to any one of the embodiments described above, wherein the antibody isotype is IgG1, IgG2, IgG3, or IgG4 or a combination thereof.

[0383] 170. The antibody according to any one of Embodiments 57 to 131, wherein the light chain variable domain of the antibody or its antigen-binding fragment comprises amino acid residues R57 and R96 (SEQ ID NO: 22), and the heavy chain variable domain of the antibody comprises amino acid residues W33, D52, D55, H100, Y101, Y102, and H103 (SEQ ID NO: 21).

[0384] 171. An antibody or antigen-binding fragment thereof as described in any of the preceding embodiments for use in a method for treating coagulation disorders or blood coagulation disorders.

[0385] 172. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any of the above embodiments for the treatment of coagulation disorders or blood coagulation disorders.

[0386] 173. A method for treating a subject suffering from a coagulation disorder, comprising administering to the subject an antibody or an antigen-binding fragment thereof as described in any of the embodiments described above.

[0387] 174. The method according to Embodiment 173, wherein the coagulation disorder or blood coagulation disorder is hemophilia A or hemophilia A with an inhibitor. [Examples]

[0388] List of abbreviations ACN: Acetonitrile CDR: Complementary Determination Region EGR-CK: EGR-chloromethyl ketone LC-MS Liquid Chromatography-Mass Spectrometry FACS: Fluorescence-Activated Cell Sorting FIX: Coagulation factor IX FIXa: Coagulation factor IXa FX: Coagulation factor X FXa: Clotting factor Xa HA: Hemophilia A HA-PPP: HA-induced human platelet-poor plasma HA-PRP: HA-induced human platelet-rich plasma hFIXa: Human coagulation factor IXa ITC: Isothermal Titration Calorimetry MACS: Magnetically Activated Cell Sorting OA: One-Armed PCR: Polymerase Chain Reaction SPR: Surface Plasmon Resonance

[0389] Example 1: Development of Factor IX / FIXa Fab and mAb Expression Plasmids FIX / FIXa-conjugated antibodies as disclosed herein were identified using various antibody development methods. To generate a diverse set of antibodies targeting FIXa and FX, selection was performed from mouse and rabbit immunization as well as from phage display and Adimab yeast display.

[0390] Adimab Yeast Display The Adimab platform is 10 10 This yeast display system encompasses a fully human naive IgG1 / kappa library with diversity, covering 20 of the 42 VH families. The antibody-phage display platform used is a proprietary fully human Fab display library. The library contains 10 10The antibodies were constructed using a combinatorial approach that utilized the chemosynthesis of light chains, as well as heavy chains CDR1 and CDR2 complemented by PCR amplification of heavy chain CDR3 from human peripheral blood mononuclear cells. The antibody selection process was directed using MACS and FACS-based methods that allowed for real-time monitoring of applied selection criteria. Since selection is MACS and FACS-based, a labeled antigen (e.g., biotin) is required. Selection campaigns were carried out using biotin-labeled active-site inhibitor hFIXa (FIXa-EGR-biotin) or antibody-mediated immobilization of hFIXa. Binding hits were evaluated using biolayer interferometry (Octet fortebio systems).

[0391] Phage display To maximize epitope diversity coverage, different panning strategies were employed, including panning using biotinylated FIXa-EGR, FX, active-site inhibitory FXa, or antigen capture with anti-FIXa antibodies. Initial hits were identified by phage ELISA. Following sequence analysis, unique hits were cloned, expressed as IgG1, and ranked using SPR (Biacore) or biolayer interferometry (Octet fortebio systems).

[0392] InVivo Platform For the generation of fully human antibodies in mice, Kymouse® mouse HK and HL1.0 (utilizing kappa and lambda chains, respectively) were used. To maximize antibody diversity, booster immunization was performed in wild-type mice and rabbits.

[0393] Production of anti-FIXa antibodies Mice or rabbits were immunized with FIXa, FIXa-EGR, or FX using standard protocols. Antibodies generated in mice or rabbits were screened by ELISA. FIXa-conjugated rabbit B cells were FACS sorted using randomly biotinylated FIXa-EGR. Antibody hits from rabbits and mice were either expressed recombinantly (rabbit mAbs) or reproduced (mouse hybridomas), after which the antibodies were minimally purified.

[0394] Production of anti-FX antibodies Kymouse mice and rabbits were immunized with FX using standard or multi-site repeated immunization (RIMMS) protocols. Rabbit B cells were isolated by FACS sorting using randomly biotinylated FX. Anti-FX mAbs from fusion and sorting were screened using ELISA and Octet fortebio systems.

[0395] Sequencing of antibodies derived from Kymouse and wt mice Anti-FIXa and anti-FX antibodies that produce hybridomas derived from Kymouse mice or wt mice were sequenced and expressed in HEK293 cells using standard techniques. Antibody binding was evaluated using Octet fortebio systems.

[0396] The obtained variable domain (V) encodes the DNA sequence of the selected antibody. H Oyo V L The ) was inserted into a pTT-based mammalian expression vector (Durocher et al. (2002) Nucleic Acid Res. 30:E9) or a pcDNA3.4 mammalian expression vector containing an antibody constant region encoding a DNA sequence (Invitrogen). In the case of a pTT / pcDNA3.4 mAb expression vector, V H and V L The DNA sequences are, respectively, human IgG1 or IgG4S228P(C H 1C H 2C H3) or human C encoding a DNA sequence L In the kappa constant region, it was inserted within the frame. For the corresponding pTT / pcDNA3.4Fab expression vector, V H The DNA sequence is human IgG1C, which encodes the DNA sequence. H It was inserted into the frame in step 1.

[0397] For the 224F3 reference compound used in Examples 6, 8, and 18 below, 224F3V H and V L The sequences were obtained from EP1660536 B1 (sequences 1 and 2, respectively). The DNA sequence is encoded by 224F3 V. H and V L These are, respectively, human IgG1(C) H 1C H 2C H 3) or human C encoding a DNA sequence L The kappa constant region and the frame were inserted into a pTT5 / pcDNA3.4-based mammalian expression vector.

[0398] All expression vectors contained a 5'-terminal DNA sequence containing the Kozak sequence, an antibody encoding the DNA sequence, and a DNA sequence encoding a signal peptide within the frame.

[0399] Example 2 Recombinant expression of antibody and antibody Fab fragment Antibodies and antibody Fab fragments were expressed using transient transduction of HEK293 suspension cells (293Expi, Invitrogen) essentially according to the manufacturer's instructions. 293Expi cells were typically subcultured every 3–4 days in Expi293F expression medium (Invitrogen, catalog no. A1435104) supplemented with 1% P / S (GIBCO catalog no. 15140-122). Expi293F cells were transfected with Expifectamine at a cell density of 2.5–3 million / mL. A total of 1 mg of plasmid DNA (V) was added to each liter of Expi293F cells. H -C H 1 (Fab) or VH -C H 1-C H 2-C H Transfection was performed by diluting 3 (mAb) and LC plasmid (in a 1:1 ratio) in 50 mL of Optimem (GIBCO, catalog number 51985-026, dilution A), and by diluting 2.7 mL of Expifectamine in 50 mL of Optimem (dilution B). For Fab and mAb producing co-transduction, V H -C H 1 and LC plasmid (Fab) and V H -C H 1-C H 2-C H Plasmid 3 and LC plasmid (mAb) were used in a 1:1 ratio. Dilutions A and B were mixed and incubated at room temperature for 10–20 minutes. After this, the transduction mixture was 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 transduction, the transduced cells were supplemented with 5 ml of ExpiFectamine293 transfection enhancer 1 and 50 ml of ExpiFectamine293 transfection enhancer 2. The cell culture supernatant was typically collected 4–5 days after transduction by centrifugation and filtered.

[0400] Example 3 Purification and Characterization of Fab and Antibodies Fab purification and characterization Purification of Fab molecules was performed as a two-step process consisting of affinity chromatography using kappaSelect resin (GE Healthcare, catalog no. 17-5458-11) and molecular sieve chromatography using Superdex200 resin (GE Healthcare, catalog no. 17-1043-04). Purification was carried out using an AktaExplorer chromatography system (GE Healthcare, catalog no. 18-1112-41). The buffer system used in the affinity purification step consisted of an equilibrium buffer composed of 20 mM sodium phosphate pH 7.2 and 150 mM NaCl, an elution buffer composed of 10 mM formate pH 3.5, and a pH adjustment buffer composed of 0.4 M sodium phosphate pH 9.0. The cell supernatant was applied directly to a pre-equilibriumized kappaSelect SuRe column without preparation. The column was washed with 10 column volumes of equilibrium buffer, and Fab molecules were eluted at a uniform concentration with approximately 5 column volumes of elution buffer. The pH of the pooled fraction was adjusted to neutral immediately after elution using the described pH-adjusting buffer. The Fab molecules were further purified, and the buffer was replaced using the gel filtration resin pre-packaged in the column. The running buffer used in molecular sieve chromatography was 25 mM HEPES and 150 mM NaCl, pH 7.4. The Fab molecules eluted as a single peak in approximately 0.5 column volume. The fraction covering the peak was analyzed using molecular sieve high-performance liquid chromatography (SE-HPLC) on an Agilent LC 1100 / 1200 system with a BIOSep-SEC-S3000 300×7.8 mm column (Phenomenex, catalog no. 00H-2146-K0) and a running buffer consisting of 200 mM sodium phosphate, pH 6.9, 300 mM NaCl, and 10% isopropanol. Based on this analysis, the fraction was pooled to obtain a homogeneous protein preparation. The final preparation eluted as a single symmetrical peak at a flow rate of 1 ml / min with a retention time of approximately 10 minutes.

[0401] The purified Fab molecules were further characterized using SDS-PAGE / Coomassie and liquid-chromatography-mass spectrometry analysis. SDS-PAGE / Coomassie analysis was performed using NuPage 4-12% Bis-Tris gel (Invitrogen, catalog no. NP0321BOX). All Fab molecules exhibited 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 with an Agilent 6210 instrument and a desalting column MassPREP (Waters, catalog no. USRM10008656). The buffer systems used were 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. All Fab molecules exhibited the expected intact molecular mass according to their sequence. Final purity was determined based on SE-HPLC analysis. The estimated purity for all different Fab fragments was 95–99%. To determine the final protein concentration, absorbance measurements were performed at 280 nm using a NanoDrop spectrophotometer (Thermo Scientific), and the concentration was calculated using the specific extinction coefficient of each Fab molecule.

[0402] Antibody purification and characterization Antibody purification was performed by affinity chromatography using Protein A MabSelect SuRe resin (GE Healthcare, catalog no. 17-5438-01). Purification was carried out using the AktaExplorer chromatography system (GE Healthcare, catalog no. 18-1112-41). The buffer system used in the affinity purification process consisted of an equilibrium buffer composed of 20 mM sodium phosphate pH 7.2 and 150 mM NaCl, an elution buffer composed of 10 mM formate pH 3.5, and a pH adjustment buffer composed of 0.4 M sodium phosphate pH 9.0. Cell supernatant was applied directly to a pre-equilibriumized MabSelect SuRe column without preparation. The column was washed with 10 column volumes of equilibrium buffer, and the antibody was eluted at a uniform concentration with approximately 2-5 column volumes of elution buffer. The pH of the pooled fraction was adjusted to neutral immediately after elution using the described pH adjustment buffer.

[0403] The purified antibodies were characterized using SDS-PAGE / Coomassie, molecular sieve 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 gel (Invitrogen, catalog no. NP0321BOX). Here, 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 with an Agilent 6210 instrument and a desalting column MassPREP (Waters, catalog no. USRM10008656). The buffer systems used were 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 or without the use of N-glycosidase F (Roche Diagnostics, catalog no. 11365177001) and a reducing agent (i.e., mercaptoethanol or DTT). All antibodies showed the expected intact molecular weight according to the sequence and a single heavy chain N-glycan. Purity was determined based on SE-HPLC. Final protein purity was analyzed based on an SE-HPLC setup on an Agilent LC 1100 / 1200 system using a BIOSep-SEC-S3000 300×7.8 mm column (Phenomenex, catalog no. 00H-2146-K0) and running buffer consisting of 200 mM sodium phosphate pH 6.9, 300 mM NaCl, and 10% isopropanol. UV280 and fluorescence (Ex 280 nm / Em 354 nm) detectors were used for detection. Antibodies eluted as a single symmetrical peak, along with retention time reflecting antibody size. All purity estimates were 95–99% for different antibodies. A NanoDrop spectrophotometer (Thermo Scientific) was used to measure final protein concentrations, along with the specific extinction coefficient for each antibody.

[0404] Example 4: Binding of anti-FIXa stimulating antibody Antibodies selected for their potential to stimulate the enzymatic activity of FIXa against FX were analyzed by binning experiments, and the binding properties of the identified antibodies were determined using the method described below.

[0405] Antibody binning method Binning experiments were performed using the Octet fortebio system (HTX, Red384) equipped with an anti-human IgG sensor (Pall Life Sciences, Menlo Park, California, USA), and in 8 or 32-channel mode (Red384 and HTX). Binning assays were performed using a classic sandwich epitope binning setup. Briefly, if (1) the primary antibody was captured by an anti-human AHC chip (anti-human IgG Fc capture chip (AHC part number 18-5064)), (2) the unblocked IgG binding site on the AHC chip was blocked by human polyclonal IgG (I4506 SIGMA), (3) FIXa was bound to the primary antibody, and (4) the antibodies were evaluated as belonging to the same bin if a competing antibody was presented to the antibody-antigen complex on the chip and binding of the secondary antibody was not detected. The analysis identified two distinct vials, vial 1 and vial 2, defined by antibodies mAb0-1886 and mAb1-1307, respectively.

[0406] Binning of anti-FIX antibodies The selected anti-FIX antibodies were binned together, and two different bins (bin 1 and 2) were identified. The numbers refer to the mAb numbers; for example, 0-1998 indicates mAb 0-1998. [Table 2]

[0407] The summary indicates that several antibodies, including mAb0-1886 and mAb0-1998, were found to belong to bin 1. Bin 2 was found to contain four antibodies in addition to mAb1-1307. Two antibodies, mAb1-0072 and mAb1-0073, were common to bins 1 and 2.

[0408] The variants of the parental antibody (strain) disclosed herein share the vin and epitope (hotspot) residues that contain the parental antibody. Since the antibody variants provided in this embodiment do not contain amino acid substitutions at positions shown to be important for epitope recognition, based on the crystal structure of the parent antibody-FIXa complex provided in Example 5, those skilled in the art will understand that the variants as starting points belong to the same bin, compete for binding, and recognize at least the same hotspot residues within the FIX / FIXa epitope as the antibodies from which they originate, i.e., mAb0-1998, mAb0-1886, or mAb1-1307.

[0409] Example 5: Crystallization and epitope mapping of anti-FIX / FIXa antibodies using X-ray crystallization. The FIXa protein (Cambridge Protein Works, product code 10316) used for crystallization consists of a cleaved light chain (residues 85-142 of SEQ ID NO: 1) with a non-native methionine residue bound to the N-terminus as a result of bacterial expression, and a heavy chain containing residues 181-415 of SEQ ID NO: 1. The protease active site is blocked by EGR-chloromethyl ketone.

[0410] crystallization Fab0-7237:FIXa Crystals of Fab0-7237 (the Fab fragment corresponding to mAb0-1886) mixed with FIXa protein in a 1:1 molar ratio were grown by hanging-drop vapor diffusion at 18 °C. 0.8 μl of 7.5 mg / ml protein solution in 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 2.5 mM CaCl2 were mixed with an equal volume of 4 M sodium formate as a precipitant and incubated with 1 ml of precipitant.

[0411] Fab0-7238:FIXa Crystals of Fab0-7238 (the Fab fragment corresponding to mAb0-1998) mixed with FIXa protein in a 1:1 molar ratio were grown by sitting-drop vapor diffusion at 18°C. 0.1 μl of 6.2 mg / ml protein solution in 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 2.5 mM CaCl2 were mixed with 0.1 μl of 100 mM sodium cacodylate, pH 6.5, and 1 M trisodium citrate as a precipitant, and incubated with 60 μl of the precipitant.

[0412] Fab0-7236:FIXa Crystals of Fab0-7236 (the Fab fragment corresponding to mAb1-1307), mixed with FIXa protein in a 1:1 molar ratio, were grown by sitting-drop vapor diffusion at 18°C. 0.1 μl of 6.4 mg / ml protein solution in 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 2.5 mM CaCl2 were mixed with equal volumes of 0.2 M lithium sulfate, 40 (v / v)% PEG400, and 0.1 M Tris pH 8.5 as a precipitant, and incubated with 1 ml of the precipitant.

[0413] Diffraction data collection Fab0-7237:FIXa The crystals were cryoprotected in a solution consisting of 3M sodium formate, 4% glycerol, 4% ethylene glycol, 4.5% sucrose, and 1% glucose before flash cooling with liquid nitrogen. Diffraction data were collected at 100K on the Swiss Light Source beamline X06DA (1.0000 Å wavelength) using a Dectris Pilatus 2M pixel detector. Automated indexing, merging, and scaling of the data were performed programmatically from the XDS package (diffraction data statistics are summarized in Table 1).

[0414] Fab0-7238:FIXa The crystals were cryoprotected in a solution consisting of 75 mM sodium cacodylate, pH 6.5, 0.75 M trisodium citrate, 4% glycerol, 4% ethylene glycol, 4.5% sucrose, and 1% glucose, before flash cooling with liquid nitrogen. Diffraction data were collected at 100 K on the Swiss Light Source beamline X06DA (1.0000 Å wavelength) using a Dectris Pilatus 2M pixel detector. Automated indexing, merging, and scaling of the data were performed programmatically from the XDS package (diffraction data statistics are summarized in Table 1).

[0415] Fab0-7236:FIXa The three crystals were cryoprotected in a solution consisting of 0.15 M lithium sulfate, 30 (v / v)% PEG400, and 0.075 M Tris pH 8.5, 4% glycerol, 4% ethylene glycol, 4.5% sucrose, and 1% glucose, prior to flash cooling in liquid nitrogen. Diffraction data were collected at 100 K on the Swiss Light Source beamline X06DA (1.0000 Å wavelength) using a Pilatus 2M pixel detector on a Dectris. Automated indexing, merging, and scaling of the data were performed programmatically from the XDS package (diffraction data statistics are summarized in Table 1).

[0416] Structural determination and improvement Fab0-7237:FIXa The structure was determined by molecular substitution using Phaser, implemented in the Phenix program suite, which includes the H and L chains of Protein Databank entry 4NP4 and Protein Databank entry 3KCG. The asymmetric unit contains two Fab:FIXa complexes. The model was refined in COOT using the Phenix refinement and manual reconstruction steps. Refinement statistics are shown in Table 1.

[0417] Fab0-7238:FIXa The structure was determined by molecular substitution using Phaser, implemented in the Phenix program suite, which includes the H and L chains from Protein Databank entry 4PUB and Protein Databank entry 3KCG. The asymmetric unit contains two Fab:FIXa complexes. The model was refined in COOT using the Phenix refinement and manual reconstruction steps. Refinement statistics are shown in Table 1.

[0418] Fab0-7236:FIXa The structure of the complex Fab0-7238:FIXa complex described above was determined by molecular substitution using Phaser, implemented in the Phenix program suite, which included the Fab portion of the complex structure and the H and L chains from protein databank entry 3KCG. The asymmetric unit contains one Fab:FIXa complex. The model was refined in COOT using the Phenix refinement and manual reconstruction steps. Refinement statistics are shown in Table 1.

[0419] Table 1 - Data Collection and Improvement Statistics [Table 3-1] [Table 3-2]

[0420] Epitope determination Based on the above, it was found that mAb0-1998, mAb1-1307, and mAb0-1886 bind to different epitopes on FIXa, where the epitope is defined as a residue having at least one heavy atom within a distance of 3.5 Å from the heavy atom of the antibody.

[0421] The mAb0-1998 epitope is located in loop 170 and contains the following residues within the protease domain: L337, R338, S339, T340, K341, and T343.

[0422] The mAb1-1307 epitope contains the following residues: H256, H257, N258, K293, R403, Y404, N406, W407, E410, and K411.

[0423] The mAb0-1886 epitope is located at the 170th helix and contains the following residues within the protease domain: K301, D332, R333, A334, T335, R338, and N346.

[0424] The epitopes of mAb0-1998 and mAb0-1886 were found to overlap. This is well consistent with the observation that the two antibodies compete for binding to FIX / FIXa (Example 4).

[0425] The variants of the parental antibody (strain) disclosed herein share the vin and epitope (hotspot) residues that contain the parental antibody. Those skilled in the art will understand that, based on the crystal structure of the parent antibody-FIXa complex provided in this example, the antibody variants provided in Example 4 and the following specific examples do not contain amino acid substitutions at the positions shown to be important for epitope recognition. Therefore, the variants as starting points belong to the same bin, compete for binding, and recognize at least the same hotspot residues within the FIX / FIXa epitope as the antibodies from which they originate, i.e., mAb0-1998, mAb0-1886, or mAb1-1307.

[0426] Example 6: Activity of bivalent anti-FIX / FIXa antibody in FXa generation assay The stimulating effect of the bivalent anti-FIX / FIXa antibody on the enzymatic activity of FIXa in relation to FX was determined by its ability to promote FX activation by FIXa in the presence of the blood coagulation phospholipid membrane, according to the principle described by Scheiflinger et al., (2008) J Thromb Haemost, 6:315-322. Given the high activity of the anti-FIXa antibody 224F3 among the antibodies identified by Scheiflinger et al., 224F3 was selected as the reference for the following experiments (see Example 1 for information on the 224F3 structure).

[0427] The stimulating effect of anti-FIXa antibodies on FIXa-mediated activation of FX to FXa was measured using an automated high-volume biochemical assay in a 384-well plate. Briefly, FIXa was mixed with purified antibody in a 4-point 5x dose-reaction. FXa production was measured by adding an FX / phospholipid (PL) mixture and then an FXa substrate (Pephafour), and the substrate hydrolysis rate was measured by detecting fluorescence for 5 minutes using a multi-label reader (PheraSTAR). The relative stimulating activity of FIXa was calculated as the ratio of FXa production from the FIXa-antibody complex to FIXa alone.

[0428] Each antibody was tested in a concentration range of 0–200 nM in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w / v) PEG8000, pH 7.3 + 1 mg / ml BSA) with 3 nM human plasma-derived FIXa (Haematologic Technologies Inc., USA) and 10 μM 25:75 phosphatidylserine:phosphatidylcholine lipid vesicles (Haematologic Technologies Inc., USA) for 10 minutes, before adding human plasma-derived FX (Haematologic Technologies Inc., USA) to a concentration of 150 nM. After activation at room temperature for 10 minutes, the reaction mixture (50 μl) was quenched by adding 25 μl of chilled buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% PEG8000, pH 7.3 + 1 mg / ml BSA). Then, 25 μl of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) was added, and the amount of FXa produced was determined by measuring the chromogenic substrate conversion using a microplate reader at 405 nm absorbance (ΔOD / min). The FXa production rate at each antibody concentration was determined from standard curves prepared using known amounts of human plasma-derived FXa (Haematologic Technologies Inc, USA).

[0429] Table 2 shows the measured FXa production rates for each antibody at the tested concentrations. From this, the peak stimulating activity of each antibody was calculated as the maximum FXa production rate observed compared to that of 224F3. This data is shown in Table 3, and it shows that antibodies belonging to each of the three families (0-1886, 0-1998, and 1-1307, respectively) have 10 to 67 times higher activity than those observed with 224F3 (Scheiflinger et al.).

[0430] Table 2 - FXa production rate FXa production rates in pM / min (mean ± SD, n = 2) for the indicated anti-FIX / FIXa antibodies. Each antibody was tested in the concentration range of 0–200 nM as shown in the first column. [Table 4]

[0431] Table 3 - Peak Stimulus Activity Peak stimulating activity (mean ± SD, n=2) of anti-FIX / FIXa antibody against 224F3 (Scheiflinger et al.) in an FXa generation assay. [Table 5]

[0432] Example 7: Preparation of a monovalent (one-armed) antibody To avoid potential avidity effects associated with conventional monospecific and bivalent antibodies, e.g., FXa generation assays (Example 8) and certain SPR experiments (Examples 14 and 15), monovalent one-armed (OA) antibody types were 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), the full heavy chain, cleaved heavy chain (lacking the Fab region), and light chain are co-expressed. Instead of the co-expression of the three chains described by Martens et al., in this invention, a monovalent antibody was prepared using Duobody®, as described for bispecific antibodies (Example 10). Thus, the monovalent antibody was prepared by mixing fully monospecific and bivalent antibodies with a cleaved heavy chain dimer (formally derived from the complete antibody by removing the Fab region), and chain exchange could be carried out under the same experimental conditions as described in Example 10. For the formation of monovalent antibodies, as described in Example 10, the antibody and the cleaved heavy chain dimer must have appropriate complementary mutations to promote heterodimerization, namely F405L / K409R for IgG1 and F405L+R409K / WT for IgG4.

[0433] In the case of monovalent antibodies of the IgG1 subtype, heavy chain cleavage can occur from the N-terminus to a position between Cys220 and the upper hinge Cys226 (EU numbering). A specific example of cleaved IgG1 heavy chain is the cleavage of residues 1-220.

[0434] In the case of monovalent antibodies of the IgG4 subtype, heavy chain cleavage can occur from the N-terminus to a position between Cys200 and the upper hinge Cys226 (EU numbering). A specific example of cleaved IgG4 heavy chain is the cleavage of residues 1-214.

[0435] Example 8: Activity of monovalent anti-FIX / FIXa antibody in FXa generation assay To avoid the potential avidity effect resulting from the bivalent nature of conventional antibody types, the stimulating activity of anti-FIX / FIXa antibodies against FIXa enzyme activity was determined after reformatting to a monovalent, one-armed (OA) antibody type (see Example 8). The antibodies tested are shown in Table 4 below. The monovalent OA version of anti-FIXa antibody 224F3 (indicated as mAb1-1582), also referenced in Example 7, was included for comparison.

[0436] The stimulatory activity of the OA antibody was measured in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w / v) PEG8000, pH 7.3 + 1 mg / ml BSA) at fixed concentrations of phosphatidylserine (PS):phosphatidylcholine (PC) lipid vesicles (final concentration 500 μM, Haematologic Technologies Inc, USA) and plasma-derived FIXa (final concentrations 0.17, 0.5, or 1 nM; Haematologic Technologies Inc, USA). The FIXa concentration was selected to ensure that less than 15% of the substrate FX was converted to FXa. After pre-incubation in the presence of monovalent OA antibody (final concentrations shown in Table 1), 150 nM plasma-derived FX was added to obtain a final reaction volume of 50 μl, which was activated at room temperature for 20 minutes. The reaction mixture was then quenched by adding 25 μl of chilled buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% PEG8000, pH 7.3 + 1 mg / ml BSA), and 25 μl of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) was added. The amount of FIXa produced was determined by measuring the chromogenic substrate conversion by 405 nm absorbance measurement (ΔOD / min) using a microplate reader. The measured activity was corrected for background activity by subtracting the signal measured in the same assay, but FIXa and antibody were replaced with assay buffer and then normalized according to the FIXa concentration present in the assay ([FIXa] total ). Dividing this value by the similarly normalized rate of FXa production in the absence of the antibody, (A FIXa、normThe antibody stimulation index was calculated to provide a stimulation several times greater than the FIXa activity by the antibody used. Due to the slow rate of FIXa production by free FIXa, the activation reaction was performed in the absence of antibody as described above, but with 5, 10, or 20 nM of FIXa present. The measured activity was then subtracted by the background and normalized according to the FIXa concentration in the assay. The mean of three normalized activities of free FIXa was used to calculate the stimulation index.

[0437] Determination of the stimulus index In summary, the calculation of the stimulus index can be explained as follows: Stimulus index=((A FIXa +OA - A bckg ) / [FIXa] total ) / A FIXa、norm In the formula, A FIXa+OA This is an activity measured in the presence of OA antibodies, and A bckg [FIXa]total is the background activity measured in the absence of FIXa and monovalent antibody, and A is the FIXa concentration in the assay. FIXa、norm This is the mean normalized activity of free FIXa.

[0438] Determination of FIXa saturation In the assay, the percentage of FIXa saturated with OA antibody is determined by the concentrations of FIXa and OA antibody, and the equilibrium dissociation constant (K) governing their interaction. d The latter is determined by the following: The latter can be measured by techniques known in the art, such as isothermal titration calorimetry (ITC).

[0439] As the stimulating index increases with increasing OA antibody concentration until FIXa reaches saturation, the concentration of OA antibody in the assay should be selected so that FIXa is at least 80% saturated, and the stimulating index at the point of complete FIXa saturation should be appropriately estimated.

[0440] The fraction of FIXa bound to the OA antibody in equilibrium (f FIXa+OA) is the assay FIXa([FIXa] total ) and OA ([OA] total The total concentration of ) and the equilibrium dissociation constant (K) of the interaction using a quadratic bond equation. d This can be calculated from ), which is described in Krishnaswamy et al., (1992) J. Biol. Chem., 267:23696-23706 and is detailed in the following equations 1 and 2.

number

number

[0441] Formula 1.

number

[0442] Formula 2.

number

[0443] Table 4 provides the stimulation indices for each monovalent OA antibody. For all tested antibodies, the measured stimulation indices were found to be higher than those measured for the monovalent one-armed 224F3 antibody (mAb1-1582).

[0444] In the assay, the concentration of one-armed 224F3 antibody was 3260 nM, and the interaction with 0.477 nM FIXa was reported by Kerschbaumer et al. (US7297336-B2). d In the assay, over 95% of FIXa bound to the one-armed 224F3 antibody.

[0445] Table 4 - Stimulation of FIXa activity by monovalent one-armed (OA) anti-FIXa antibody The anti-FIX mAb ID refers to the ID of the antibody used for reformatting to OA format. The columns labeled "OA antibody concentration (nM)" and "Stimulation index" show the concentration of the OA antibody (nM) used in the assay and the corresponding stimulation of FIXa activity measured against free FIXa. [Table 6]

[0446] Example 9 Development of anti-FX / FXa Fab and mAb expression plasmids The anti-FX / FXa antibodies disclosed herein were developed using standard antibody development methods, and the expression plasmids were prepared as described in Example 1 for the anti-FIX / FIXa Fab and mAb expression plasmids. The expression, purification, and characterization of the anti-FX / Xa antibodies were similarly carried out as described for the anti-FIX / FIXa antibodies of Examples 2 and 3.

[0447] Example 10: Bispecific antibodies prepared by in vitro assembly Bispecific antibodies are generated using slightly modified variants of the bispecific IgG4 antibody, as detailed below, by in vitro assembly of primary and secondary antibodies using the Duobody® method (Genmab), as described for bispecific IgG1 antibodies (Labrijn et al., PNAS, 2013, vol. 110, pp. 5145-5150).

[0448] In the case of IgG1, the heavy chain constant region of the primary antibody is IgG1 K409R (anti-FIX / FIXa), and the heavy chain constant region of the secondary antibody is IgG1 F405L (anti-FX / FXa). As mentioned above, IgG1 may also be an IgG1 mutant with reduced effector function. In the case of IgG4, the heavy chain constant region of the primary antibody is IgG4 S228P (anti-FIX / FIXa), and the heavy chain constant region of the secondary antibody is IgG4 S228P F405L R409K (anti-FX). The two parent antibodies are produced as described in Examples 1-3. The Fab arm exchange reaction is carried out in HEPES buffer (pH 7.4) under reducing conditions using 75 mM 2-mercaptoethylamine (2-MEA) and incubated at 30°C for 3 hours.

[0449] Example 11: Blood coagulation activity of bispecific antibodies Pairs of anti-FIXa antibody and anti-hFX antibody were prepared as bispecific antibodies using the Duobody® technology described above (Example 10). The bispecific antibodies were tested for blood coagulation activity in various assays, including the FXa generation assay (Example 6) and thrombin generation assay (TGT), as described in the following paragraphs.

[0450] Thrombin Generation Test (TGT) Assay TGT was performed using an automated HTP 384-well setup with a kaolin trigger (Haemonetics Corporation, #6300). Briefly, antibodies were added to hemophilia A (HA) plasma (George King) at a concentration of 111 nM (except for mAb1-1371 added at 55 nM and mAb1-0021 added at 166 nM). Kaolin mixed with phospholipids (Rossix, #PL604T) was then added, followed by the addition of the FIIa substrate (FluCa, Thrombinoscope, #TS50.00). Fluorescence was measured at 1-minute intervals for 2 hours on a Perkin Elmer EnVision multi-label plate reader. Peak height was calculated as the maximum value observed in the thrombogram and then normalized to the peak height observed for reference anti-FIXa and anti-FX antibodies. References always include a binding domain from the anti-FX antibody mAb1-2375 (identified by SEQ ID NOs. 93 and 94) in combination with a FIX domain from each of the three families represented by mAb1-4707, mAb1-5788, and mAb1-4857. Antibodies are grouped according to their relative TGT activity as low (0-24%), + (24-50%), ++ (50-75%), and +++ (>75%), where + is preferred, ++ is more preferred, and +++ is most preferred.

[0451] Selection of preferred combinations of bispecific anti-FIXa / anti-FX antibodies Numerous anti-FX antibodies were tested as bispecific antibodies in combination with anti-FIXa antibody mutants belonging to three lines: mAb1-1307, mAb0-1886, and mAb0-1998. Table 5 shows the selected anti-FIXa / anti-FX pair combinations that showed significant activity in the TGT assay.

[0452] Table 5 - Blood coagulation activity of bispecific antibodies The selected anti-FIXa / anti-FX bispecific antibody pairs are shown along with their activity in the TGT assay (as described above). The bispecific antibodies (duobodies) were IgG4 subtype, with the exception of IgG1-0021, mAb1-1335, and mAb1-0985, which were IgG1. [Table 7-1] [Table 7-2] As is evident from Table 5, the activity levels exhibited by bispecific antibodies depend on the specific anti-FIXa / anti-FX combination. For example, the anti-FX antibody mAb1-6723 shows strong activity (+++) when combined with the anti-FIXa antibody mAb0-1998, while the activity of mAb1-6723 is low when combined with mAb0-1886(+) and mAb1-1307(+).

[0453] Example 12: Binning of anti-FX antibody Specific anti-FX antibodies that exhibit significant TGT activity in a bispecific antibody form when combined with an anti-hFIXa antibody (Example 11) were binned with each other using Octet fortebio systems, using the same setup as described for the anti-FIXa antibody (Example 4), except that FIXa was replaced with FX.

[0454] The analysis identified five distinct bins A–E, defined by antibodies mAb1-1371, mAb1-1376, mAb1-6723, mAb1-7447, and mAb1-7449, respectively. Two bins, bin A and bin E, are each represented by a single anti-FX antibody (see Table 6).

[0455] Numerous clones were found to be competing with mAb1-6723 in bin C.

[0456] Table 6 - Binning of anti-FX antibodies The selected anti-FX antibodies were binned together, and five different bins (bin AEs) were identified. The numbers indicate antibody IDs; for example, 1-6723 indicates mAb1-6723. [Table 8] Example 13: Crystallization and epitope mapping of anti-FX antibody using X-ray crystallization crystallization Attempts to crystallize Fab0-8954 (the Fab fragment corresponding to mAb1-6723) in complex with FX were unsuccessful, but good quality crystals containing the active site inhibitor FXa were obtained. Therefore, crystals of Fab0-8954 mixed with the active site inhibitor des-gla FXa (human EGR inhibitor Xa gla domainless (wild-type) bacterial expression, Lot# hGDFXAEGR-022, Cambridge ProteinWorks) in a 1:1 molar ratio were grown at 18°C ​​using the sitting drop vapor diffusion technique. A 150 nl solution of the 6.7 mg / ml complex protein in 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 2.5 mM CaCl2 was mixed with 50 nl of 0.2 M magnesium acetate, 0.1 M sodium cacodylate, pH 6.5, 20% (w / v) PEG 8000 as a precipitant, and incubated with 60 μl of the precipitant.

[0457] Diffraction data collection The crystals were cryoprotected by adding 1 μl of a precipitant containing 20% ​​ethylene glycol to the crystallization drop before flash cooling with liquid nitrogen. Diffraction data were collected at 100 K on the Swiss Light Source beamline X06DA (1.0000 Å wavelength) using a Dectris Pilatus 2M pixel detector. Automated indexing, merging, and scaling of the data were performed programmatically from the XDS package (diffraction data statistics are summarized in Table 7).

[0458] Structural determination and improvement The asymmetric unit contains four Fab:FXa complexes determined from Matthews coefficient analysis. The structure was determined by molecular substitution. Phaser, implemented in the Phenix program suite, was used in conjunction with the H and L chains of protein databank input 5I1K as a search model to localize the four Fabs. These were constructed using the correct amino acid sequence with COOT and then refined using Phenix refinement. The refined Fab models were modified using the A and B chains from protein databank input 1G2L as search models, applying molecular substitutions with Molrep from the CCP4 suite. Four FXa fragments were found. The models were refined in COOT using the Phenix refinement and manual reconstruction steps. Refinement statistics are shown in Table 7.

[0459] Table 7 - Data Collection and Improvement Statistics [Table 9]

[0460] Epitope determination The crystal structure of the Fab0-8954:FXa complex contained four copies of the complex within an asymmetric unit, which were analyzed individually to identify the epitopes and paratopes using a 3.5 Å cutoff distance.

[0461] If at least one of the four copies of the Fab0-8954:FXa complex within a unit cell meets the 3.5 Å distance criterion, the residue is included in the epitope of mAb1-6723. The epitope and paratope residues of mAb1-6723 are listed in Table 8.

[0462] Table 8 Epitope and paratope of mAb1-6723 The epitope residues of mAb1-6723 in the EGF-2 and protease domains of FX / FXa (SEQ ID NO: 2) are listed in the first and second columns, respectively. Antibody V H (Sequence ID 21) and V LThe paratope residues of (Sequence ID 22) are listed in columns 3 and 4, respectively. [Table 10]

[0463] The variants of the parental antibody (strain) disclosed herein share the vin and epitope (hotspot) residues that contain the parental antibody. Since the antibody variants provided in the examples herein do not contain amino acid substitutions at positions shown to be important for epitope recognition, based on the crystal structure of the parent antibody-FXa complex provided in these examples, those skilled in the art will understand that the variants as starting points belong to the same bin, compete for binding, and recognize at least the same hotspot residues within the FX / FXa epitope as the antibody from which they originate, i.e., mAb1-6723.

[0464] Example 14: Identification of hotspot residues on FX As described in Example 15, the data provided in this example determines the hotspot epitope residue on the FX of mAb1-6723, similar to the mapping of hotspot epitope residues on FIX for mAb1-1307, mAb0-1886, and mAb0-1998. The FX mutant used is a single-site alanine mutant of desGla-desEGF1-FX (wild-type alanine, except for position 118 where an alanine-to-serine substitution is introduced), corresponding to residues 86-448 of SEQ ID NO: 2, with an N-terminal His tag (HHHHHH, for affinity purification) added via a short GS linker (GGGGSGGGGS). Table 9 lists the mutants that cover the epitope residues defined in Example 13. Table 9 - List of generated desGla-desEGF1-FX mutants [Table 11]

[0465] The wild-type desGla-desEGF1-FX and mutants listed in Table 9 were expressed in the HEK293 system and purified by affinity chromatography. Expression or low purity was not observed in the L117A, L303A, P304A, and M426A mutants, and binding could not be evaluated for these four mutants.

[0466] Identification of hotspot epitope residues was performed using a Biacore T200 instrument at 25°C. Anti-hlgG Fc antibody from the Human Antibody Capture Kit (GE Healthcare, catalog no. BR100839) at 2 μg / ml was immobilized on a Series S Sensor Chip CM5 (GE Healthcare, catalog no. BR100530) using standard amine coupling chemistry. Anti-FX antibody mAb4-6934 (a monovalent mutant of mAb1-6723) was injected at a flow rate of 5 μL / min for 30 seconds and captured by the immobilized anti-hlgG Fc antibody. Next, 5 μM (2- or 3-fold serial dilutions) of the T116A, A118S, T127A, F229A, and E226A mutants, 10 μM (5-fold serial dilution) of the Y230A mutant, and 10 μM (2- or 3-fold serial dilutions) of the WT and H101A, E103A, R113A, S227A, E228A, R287A, E305A, L419A, K420A, D423A, R424A, K427A, and T428A mutants were injected at a flow rate of 5 μM / min for 90 seconds to enable binding to the capture anti-FX antibody, and then 90 seconds of buffer injection enabled the dissociation of the desGLA-desEGF1-FX mutants. The running buffer (also used for diluting the anti-FX antibody and the desGLA-desEGF1-hFX variant) contained 10 mM HEPES, 150 mM NaCl, 1 mg / mL BSA, and 5 mM CaCl2 (pH 7.4). Sensor chip regeneration was achieved using 1 M formic acid. Binding data were analyzed using steady-state fitting according to a 1:1 model in Biacore Evaluation Software 2.0 supplied by GE Healthcare. Binding data reported the binding of the FX variant to the anti-FX antibody (monovalent mAb1-6723) as a percentage compared to the binding of wild-type FX to the anti-FX antibody at an injected 5 or 10 μM, and were calculated according to the following formula: Combine(%)= 100% × [(R max_FXvar,Ab ) / (R max_Ab )] / [(R max_FXwt,Ab ) / (R max_Ab )] In the formula, R max_Abrepresents the capture level (RU) of the anti-FX antibody, and R max_FXvar、Ab and R max_FXwt、Ab The values ​​represent the binding (RU) of the FX variant and wild-type antibody to the capture anti-FX antibody at the same concentration (5 μM for all concentrations, except 10 μM for the Y230A variant). The results are shown in Table 10.

[0467] Table 10 - Results of SPR analysis Results of SPR analysis of monovalent mutants binding to mAb1-6723 in selected FX mutants covering the epitope residue of mAb1-6723. [Table 12]

[0468] Hotspot residues of mAb1-6723 The hotspot residue of mAb1-6723 is defined as the position where alanine substitution of the wild-type residue (or position 118 where alanine is substituted with serine) reduces antibody binding to wild-type FX by 30% or less compared to the binding of the antibody to wild-type FX at a concentration of 5 μM WT (or mutant) desGLA-desEGF1-FX.

[0469] Hotspot residue of mAb1-6723 (experimentally represented by its monovalent counterpart, mAb4-6934): R113, Y230, K420, D423, R424 and K427

[0470] Example 15: Identification of hotspot residues on FIX / FIXa To determine the residues crucial for the interaction (known as the hotspot) between anti-FIX / FIXa Abs, mAb0-1886, mAb0-1998, and mAb1-1307, and FIX, a set of FIX mutants were selected based on the crystal structure of FIXa when compounded with the corresponding Fab fragments (Fab7237, Fab7238, and Fab7236, respectively). As detailed below, the selected FIX mutants were transiently expressed in mammalian cells and purified and characterized for binding to monovalent mutants of mAb0-1886, mAb0-1998, and mAb1-1307 using surface plasmon resonance (SPR).

[0471] Generation of FIX mutants A DNA plasmid suitable for transient mammalian expression was constructed using an expression cassette encoding amino acid residues 1-461 of human FIX (corresponding to T148A of SEQ ID NO: 1, excluding the T194A mutation as determined by UNIPROT numbering) followed by six histidines (6×His-tag, for affinity purification). The secreted mature FIX protein chain produced using this construct is identical to the A148 allele of human FIX (Anson et al. EMBO J.1984 3:1053-1060, McGraw et al. Proc Natl Acad Sci USA.1985 82:2847-2851), except for the addition of a C-terminal His-tag.

[0472] Selected mutations were introduced by PCR using the construct as a template. For each single-point mutation listed in Table 11, forward primers containing the desired amino acid change and reverse primers without the amino acid change were designed. These primers were used as templates for amplifying the entire vector sequence in the standard PCR reaction using the vector described above. Using ligation-free cloning, the ends of the resulting amplified DNA fragments were ligated to a circular expression plasmid using the overlapping sequences introduced by the forward and reverse primers.

[0473] Circular plasmids were used to transform E. coli cells, which were then grown on selective agar plates to form colonies. Liquid E. coli cultures were then initiated using these colonies. After growing the E. coli cultures overnight, plasmid preparation was performed, and mutants were identified by DNA sequencing.

[0474] Recombinant protein production was performed by transfecting expi293F cells, grown in suspension culture in Expi293 Expression® medium (ThermoFisher Scientific, catalog number A1435101), with the ExpiFectamine® 293 Transfection Kit (ThermoFisher Scientific, catalog number A14525) and plasmid DNA encoding each of the desired mutants, as well as wild-type FIX (corresponding to C-terminal His tagged SEQ ID NO: 1). Vitamin K was added to a final concentration of 5 mg / mL during transfection. Transfection enhancers 1 and 2 of the ExpiFectamine® 293 Transfection Kit were added the day after transfection. Cell cultures were collected by centrifugation 5 days after transfection.

[0475] The C-terminal His tag of each FIX variant was used for batch protein purification in a multi-well robotic setup. Briefly, the collected cell culture supernatant was adjusted to binding conditions and mixed with Ni Sepharose 6 Fast Flow affinity purification resin (GE Healthcare, catalog no. 17-5318-02, 50 μl precipitate resin / ml cell culture medium), and incubated with shaking for 20 minutes. The resin / supernatant mixture was then transferred to a filter plate, and the liquid was aspirated through the filter plate by applying vacuum. The resin remaining in the filter plate was washed three times in high-imidazole buffer before elution.

[0476] The concentration of the purified protein solution was measured by ELISA using an anti-FIX antibody for detection and high-purity recombinant wild-type FIX for the standard curve.

[0477] Table 11 - List of generated FIX variants [Table 13-1] [Table 13-2]

[0478] Thermal stability of FIX mutant To test whether introducing amino acid substitutions into FIX mutants leads to instability and improper folding, we used the midpoint (T) of the mutant's thermal denaturation transition. m ) was decided.

[0479] Purified FIX mutants were loaded into standard capillaries (Prometheus NT.48 nanoDSF Grade Standard capillaries, Nanotemper Technologies GmbH, Munich) and inserted into Prometheus NT.48 (Nanotemper Technologies GmbH, Munich). Thermal denaturation was tracked at 20–90°C using a heating lamp with a heating intensity of 1.5°C / min at 70% excitation intensity. Tryptophan fluorescence was measured by excitation at 280 nm and recording emission at 330 nm and 350 nm. The T of the FIX mutant was measured. m This can be determined from the ratio of fluorescence measured at 350 nm and 330 nm (F350 / F330) (except when the protein concentrations of FIX N101D, H256A, L330A, S339A, G393I, Y404A, and N406Q are less than 20 μg / mL). In all cases, the program PR.ThermoControl v2.0.4 (NanoTemper Technologies GmbH, Munich) automatically determines T by determining the maximum value of the first derivative of the F350 / F330 expansion curve. mSuitable for wild-type FIX T m The temperature was 51°C, and the mutant T m The optimal temperature range was found to be 47-54°C, demonstrating that amino acid substitution did not induce significant destabilization.

[0480] SPR analysis The FIX variants were characterized for binding to mAb0-1886, mAb0-1998, and mAb1-1307 using surface plasmon resonance (SPR) by capturing the FIX variant via a C-terminal His tag. To avoid potential avidity effects associated with conventional bivalent antibodies, i.e., to ensure a 1:1 interaction, monovalent variants of mAb0-1886, mAb0-1998, and mAb1-1307 (prepared as described in Example 7), denoted as mAb4-0673, mAb4-0004, and mAb3-3279, respectively, were used as analytes.

[0481] SPR analysis was performed using a Biacore 4000 or Biacore T200 instrument (Biacore AB, Uppsala, Sweden). For experiments using the T200 instrument, the following conditions were applied, and measurements were performed at a temperature of 25°C. Anti-His antibody at 25 μg / ml (R&D Systems, catalog no. MAB050) was immobilized on a CM5 sensor chip using standard amine coupling chemistry. A 25 nM anti-FIX variant was injected at a flow rate of 10 μl / min for 1 minute and captured via the His tag by the immobilized anti-His antibody. Next, 200 nM (4-fold serial dilution), 1600 nM (3-fold serial dilution), and 2000 nM (3-fold serial dilution) of mAb4-0004, mAb3-3279, and mAb4-0673 were injected at a flow rate of 50 μl / min for 5 minutes to enable binding to the captured FIX variant, followed by injection of buffer for 10 minutes to enable dissociation of the monovalent anti-FIX antibody. The running buffer used was 20 mM Tris, 150 mM NaCl, 5 mM CaCl2, 0.05% Tween-20, 1 mg / ml BSA, pH 7.4. This was also used to dilute the anti-FIX antibody and FIX sample. Chip regeneration was achieved using 10 mM glycine pH 2.0. The combined data were analyzed according to a 1:1 model using BiaEvaluation 4.1, provided by the manufacturer (Biacore AB, Uppsala, Sweden). A similar experimental setup was used with the Biacore 4000 instrument.

[0482] First, all FIX variants listed in Table 11 were screened for binding to all three monovalent antibodies, mAb4-0004, mAb3-3279, and mAb4-0673, using a Biacore 4000 instrument. Antibody binding (defined by the distance criteria outlined in Example 6) to FIX variants containing mutations at positions corresponding to each epitope residue was, as expected, variably disrupted. No significant effect on antibody binding was observed for FIX variants containing mutations at positions not corresponding to each epitope residue. In particular, substitutions made in the EGF2 domain did not affect binding to any of the antibodies (data not shown).

[0483] Using the Biacore T200 instrument, more detailed binding analysis was performed on residues defined as epitope residues (see Example 6). The results are shown in Table 12.

[0484] Binding data reports the binding of the antibody to the FIX variant as a percentage compared to the binding of the antibody to wild-type FIX, and is calculated according to the following formula:

[0485] Combine(%)=100%×[(R max_Ab,FIX_var ) / (R max_FIXvar )] / [(R max_Ab,FIX_wt ) / (R max_FIXwt )] In the formula, R max_FIXvar and R max_FIXwt The values ​​represent the capture levels (RU) of the FIX mutant and wild-type FIX, respectively, and R max_Ab、FIX_var and R max_Ab、FIX_wt The values ​​represent the binding (RU) of the antibody to the captured FIX variant and wild-type FIX, respectively. The results are shown in Table 12.

[0486] Table 12 - Results of SPR analysis Results of SPR analysis of the binding of mAb3-3279, mAb4-0004, and mAb4-0673 (monovalent mutants of mAb1-1307, mAb0-1998, and mAb0-1886, respectively) to selected FIX mutants covering the epitope residues of mAb1-1307, mAb0-1998, and mAb0-1886. [Table 14]

[0487] Hotspot residues mAb1-1307, mAb0-1998, and mAb0-1886 The hotspot residues of mAb1-1307, mAb0-1998, and mAb0-1886 are defined as positions where alanine substitution of the wild-type residue reduces antibody binding by 30% or less compared to binding to the wild-type FIX.

[0488] Hotspot residue of mAb1-1307 (experimentally represented by mAb3-3279): H257, K293, and N406

[0489] Hotspot residues of mAb0-1998 (experimentally represented by mAb4-0004): R338 and K341

[0490] Hotspot residues of mAb0-1886 (experimentally represented by mAb4-0673): D332, R333, L337 and R338

[0491] For both mAb0-1998 and mAb0-1886, the residue R338 was the most significant contributor to binding. Substitution of R338 with alanine (R338A) in FIX showed the greatest impact on antibody binding, which was significantly reduced to 2% and 3% in mAb0-1998 and mAb0-1886, respectively, compared to antibody binding to wild-type FIX.

[0492] Example 16: Activity of anti-FIX(a) / FX(a) bispecific antibody in FXa generation assay The blood coagulation activity of anti-FIXa / FX bispecific antibodies was determined based on their ability to promote FIXa-mediated FX activation in the presence of the blood coagulation phospholipid membrane. The bispecific antibodies (BiAbs) tested are listed in Table 13, with ACE910 included for comparison. The blood coagulation activity of each bispecific antibody is reported as a multiple of the stimulation to FX activation by free FIXa at a given antibody concentration. Bispecific antibodies were tested at eight concentrations (prepared by three-fold serial dilutions in assay buffer) by 10-minute pre-incubation with 125 pM human plasma-derived FIXa (Haematologic Technologies Inc, USA) and 500 μM 25:75 phosphatidylserine:phosphatidylcholine lipid vesicles (Haematologic Technologies Inc, USA) in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% (w / v) PEG8000, pH 7.3 + 1 mg / ml BSA). Activation was then initiated by adding human plasma-derived FX (Haematologic Technologies Inc, USA) to a concentration of 25 nM. After activation at room temperature for 15 minutes, the reactant (50 μl) was quenched by adding 25 μl of chilled buffer (50 mM HEPES, 100 mM NaCl, 60 mM EDTA, 0.1% PEG8000, pH 7.3 + 1 mg / ml BSA). The amount of FXa produced was determined by adding 25 μl of 2 mM S-2765 chromogenic substrate (Chromogenix, Sweden) and measuring the chromogenic substrate conversion by 405 nm absorbance measurement (ΔOD / min) using a microplate reader. Similarly, FX activation by free FIXa was determined at a FIXa concentration of 25 nM and a reaction time of 60 minutes. The measured activity was normalized according to the concentration of FIXa present in the assay and the reaction time. The multiple stimulation by the antibody at a given concentration was calculated by dividing this number by the similarly normalized rate of FIXa production in the absence of the antibody.

[0493] In summary, the calculation of the biAb stimulus can be explained as follows: BiAb stimulation=(A FIXa+biAb / ([FIXa] assay ×t reaction )) / A FIXa、norm In the formula, A FIXa+biAb This is the activity measured in the presence of a bispecific antibody, [FIXa] assay This is the FIXa concentration in the assay, and t reaction This is the reaction time, and A FIXa、norm This is the normalization activity of free FIXa.

[0494] Table 13 shows the concentration at which maximum stimulation was observed, along with the maximum stimulation determined for each bispecific antibody among the eight antibody concentrations tested. For all tested bispecific antibodies, the maximum stimulation was found to be higher than that measured with ACE910 in the concentration range of 0–15300 nM. Table 13 - Maximum stimulation by bispecific anti-FIXa / FX antibody [Table 15]

[0495] Example 17: Activity of bispecific anti-FIX(a) / FX(a) antibodies in thrombinogenesis tests (TGT) in platelet-poor and platelet-rich mimic plasma of human hemophilia A. The blood coagulation activity of the bispecific antibodies mAb4-7761, mAb4-7762, mAb4-7789, mAb5-0057, and mAb5-1409 (see Table 14) was determined based on their ability to promote thrombinogenesis in the presence of either the blood coagulation synthesis phospholipid membrane or platelets, according to the principle described by Hemker et al. (Pathophysiol Haemost Thromb, 2002;32:249-253). ACE910 was included for comparison. Each antibody (test compound) was tested in the thrombinogenesis assay (TGT) in pooled platelet-poor plasma (HA-PPP) and / or HA-induced human platelet-rich plasma (HA-PRP) from hemophilia A (HA) patients.

[0496] Table 14 - Bispecific anti-FIX(a) / FX(a) antibodies [Table 16]

[0497] Hemophilia A-induced human platelet-rich plasma (HA-PRP) Blood was obtained from healthy, consenting donors via venipuncture. Six volumes of blood were collected in one volume of dextrose citrate (ACD; 85 mM sodium citrate, 110 mM dextrose, 62.3 mM citrate, pH 4.9) to a final pH of 6.5, and centrifuged at 220 g for 20 minutes at room temperature (RT). Platelet-rich plasma (PRP) was collected, and platelet concentration was determined using a Medonic CA 620 blood analyzer (Boule Diagnostics AB, SpÅnga, Sweden). Red blood cells containing the plasma portion were further centrifuged at 600 g for 10 minutes at room temperature. Platelet-poor plasma (PPP) was collected and used for PRP diluted to 300,000 platelets / μl. HA conditions were induced by adding FVIII-neutralizing anti-human FVIII antibody (sheep anti-human factor VIII-5 mg, Haematologic Technologies, VT, USA) to a final concentration of 0.1 mg / ml, and the mixture was gently rotated at 2 rpm for 30 minutes at room temperature.

[0498] Thrombin generation test Thrombin generation (TGT) studies of HA-PRP and HA-PPP (George King Bio-Medical Inc., KS, USA) were performed using a 96-well plate fluorometer (Fluoroscan Ascent FL, Thermolabsystems, Helsinki, Finland) with standard-calibrated automated thrombography. The reaction mixture comprises 70 μl HA-PRP (300,000 platelets / μl) or HA-PPP, 10 μl of test compound dilution (diluted in 20 mM HEPES, 140 mM NaCl, pH 7.4, and 2% BSAm), 20 μl of CAT reagent containing tissue factor (TF) (PRP reagent; TF without synthetic phospholipids, PPP reagent LOW; TF with synthetic phospholipids, 1 pM TF final, Thrombinoscope BV, Maastricht, Netherlands) or Thrombin Calibrator (Thrombinoscope BV), and 20 μl of a mixture containing fluorescently labeled thrombin substrate z-Gly-Gly-Arg-AMC (3 mM) and CaCl2 (90 mM) (Thrombinoscope BV). TGTs were performed with up to eight concentrations of the test compound (0.3, 1.0, 3, 10, 30, 100, 300, and 900 nM, final plasma concentrations) or with supplemental buffer only (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) (representing the HA control). The concentration range was tested in blood from the same stock or from four different donors in at least three independent experiments in HA-PPP. Normal control levels in TGTs were measured using only buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) supplemented with untreated human PRP or normal human PPP plasma pooled with CRYOcheck® (Precision Biologic Inc., Dartmouth, Canada). TGTs were allowed to proceed for a total of 90 minutes, and the peak thrombin height (nM) of the TGT parameter was analyzed using Thrombinoscope software (Thrombinoscope BV).

[0499] Figure 2 / Table 15 shows the measured peak thrombin generation rates for each bispecific antibody at concentrations tested with HA-PPP. The data show that all test compounds increased the peak thrombin formation beyond the levels observed in the absence of the antibody, i.e., indicating blood coagulation activity. Furthermore, thrombin generation levels between 30 and 300 nM for mAb4-7761, mAb4-7762, mAb4-7789, mAb5-0057, and mAb5-1409 were higher than those observed with ACE910, indicating superior potency. In addition, thrombin generation levels for mAb5-0057 and mAb5-1409 at 300 to 900 nM were higher than those observed with ACE910 at 900 nM, indicating higher potency and efficacy of these compounds compared to ACE910.

[0500] Figure 3 / Table 16 shows the measured peak thrombin generation levels of mAb5-0057 and mAb5-1409 at concentrations tested with HA-PRP. Under these conditions, mAb5-0057 and mAb5-1409 also exhibit better potency and efficacy compared to ACE910. Table 15 - Thrombin generation (TGT) assays for bispecific antibodies mAb4-7761, mAb4-7762, mAb4-7789, mAb5-0057, mAb5-1409, and ACE910. Thrombin generation studies (TGTs) of bispecific antibodies mAb4-7761, mAb4-7762, mAb4-7789, mAb5-0057, mAb5-1409, and ACE910 in human tissue factor-activated hemophilia A platelet-poor plasma (PPP). Mean peak thrombin generation levels ± standard deviation measured at each compound concentration tested in at least three independent experiments of HA-PPP. Experiments A-D refer to the independent experiments described in the legend of Figure 2. [Table 17]

[0501] Table 16 - Thrombin generation (TGT) of bispecific antibodies mAb5-0057, mAb5-1409, and ACE910. Thrombin generation (TGT) studies of bispecific antibodies mAb5-0057, mAb5-1409, and ACE910 in human tissue factor-activated hemophilia A platelet-rich plasma (PRP). Mean peak thrombin generation ± standard deviation at each compound concentration tested from four independent experiments of HA-PRP. [Table 18]

[0502] Example 18: Activity of monovalent one-armed (OA) anti-FIX / FIXa antibody in thrombin generation (TGT) in human hemophilia A platelet-poor plasma. The blood coagulation activity of the monovalent, one-armed (OA) version of mAb1-9016 was determined based on its ability to promote thrombinogenesis in the presence of the blood coagulation phospholipid membrane, according to the principle described by Hemker et al. (2002) Pathophysiol Haemost Thromb, 32:249-253. A one-armed version of the 224F3 antibody (mAb1-1582) was included for comparison. Each antibody (test compound) was tested in the thrombinogenesis assay (TGT) in pooled platelet-poor plasma (HA-PPP) from hemophilia A (HA) patients (George King Bio-Medical Inc, KS, USA) by standard-calibrated automated thrombography using a 96-well plate fluorometer (Fluoroscan Ascent FL, Thermolabsystems, Helsinki, Finland). The reaction mixture contains 70 μl of HA-PPP, 10 μl of the test compound (diluted in 20 mM HEPES, 140 mM NaCl, pH 7.4, and 2% BSAm), 20 μl of PRP reagent (synthetic phospholipid, Thrombinoscope BV, Maastricht, Netherlands) or Thrombin Calibrator (Thrombinoscope BV) containing activated human plasma-derived factor XI (hFXIa, 8.3 mU / mL final) (Enzyme Research Laboratories, IN, USA), and 20 μl of a mixture containing fluorescently labeled thrombin substrates Z-Gly-Gly-Arg-AMC (3 mM) and CaCl2 (90 mM) (Thrombinoscope BV). TGT was performed with five concentrations of the test compound (30, 100, 300, 600, and 900 nM, final plasma concentrations) or with only supplemental buffer (20 mM HEPES, 140 mM NaCl, pH 7.4, 2% BSA) (representing the HA control). The concentration range was tested in two independent experiments with HA-PPP from the same stock. TGT was allowed to proceed for a total of 90 minutes, and the peak thrombin height (nM) of the TGT parameter was analyzed using Thrombinoscope software (Thrombinoscope BV). Figure 4 and Table 17 show / list the measured peak thrombin generation rates for each monovalent one-armed antibody at the tested concentrations.The data show that the OA antibody version of mAb1-9016 increases the peak of thrombin formation beyond the levels observed in the absence of the antibody, i.e., it exhibits blood coagulation activity. Furthermore, thrombin generation induced by the OA antibody version of mAb1-9016 is higher than that observed with the monovalent OA version of the 224F3 antibody (mAb1-1582).

[0503] Table 17 - Measured peak thrombin generation rates for each monovalent one-armed antibody at tested concentrations. The list represents the mean peak thrombin ± standard deviation from two independent experiments of HA-PPP. [Table 19]

[0504] Example 19 Binding affinity determined by isothermal titration calorimetry (ITC) The binding affinity of anti-FIX / FIXa antibodies and anti-FX / FXa antibodies to FIX / FIXa and FX / FXa was measured by isothermal titration calorimetry (ITC) using a PEAQ-ITC calorimeter (Malvern, UK), respectively. The experiment was performed at 37°C and pH 7.4 using 25 mM Tris, 150 mM NaCl, and 5 mM CaCl2 (Tris buffer). A sample cell (200 μl) contained either FIX, FIXa, FX, or FXa, and the anti-FIX / FIXa and anti-FX / FXa antibodies were injected via syringe. To ensure consistent buffer conditions, all proteins were dialyzed extensively in Tris buffer before measurement. Following a thermal equilibrium step, there was a 60-second delay, after which the first 0.2 μl of antibody was injected, followed by 14 injections of 2.5 μl of antibody at 120-second intervals. The stirring speed was maintained at 750 rpm, and the reference power was kept constant at 5–10 μcal / sec. The heat associated with each antibody injection was integrated and plotted against the molar ratio of ligand to polymer. The resulting isotherms were fitted to a single-site binding model using software provided by the manufacturer to obtain affinity (KD), stoichiometry (n), and enthalpy of interaction (ΔH). Experiments were performed in double or triple replication.

[0505] While this specification has illustrated and described certain features of the present invention, many modifications, substitutions, alterations, and equivalents will come to mind for those skilled in the art. It should therefore be understood that the appended claims are intended to cover all such modifications and alterations that fall within the true spirit of the invention.

Claims

1. A bispecific antibody or its antigen-binding fragment comprising a first antigen-binding site capable of binding to FIX (SEQ ID NO: 1) and / or its active form (FIXa), and a second antigen-binding site capable of binding to FX (SEQ ID NO: 2) and / or its active form (FXa), a) The first antigen-binding site of the bispecific antibody or its antigen-binding fragment comprises a heavy chain variable domain which is at least 96% identical to the sequence described in SEQ ID NO: 177, and a light chain variable domain which is at least 96% identical to the sequence described in SEQ ID NO: 178, and is capable of binding to FIX and / or its active form (FIXa), b) The second antigen-binding site of the bispecific antibody or its antigen-binding fragment comprises a heavy chain variable domain which is at least 96% identical to the sequence described in SEQ ID NO: 21, and a light chain variable domain which is at least 96% identical to the sequence described in SEQ ID NO: 22, and is capable of binding to FX and / or its active form (FXa), The bispecific antibody or its antigen-binding fragment Compared to the three CDR sequences of the heavy chain variable domain of the anti-FIX / FIXa antibody having the amino acid sequence described in Sequence ID No. 177, the three CDR sequences have at most two amino acid changes in the entirety of the three CDR sequences, and Compared to the three CDR sequences of the light chain variable domain of the anti-FIX / FIXa antibody having the amino acid sequence described in Sequence ID No. 178, the three CDR sequences have at most two amino acid changes within the entire set of three CDR sequences. This includes, and the position of the CDR is determined by Kabat. The bispecific antibody or its antigen-binding fragment can stimulate the enzymatic activity of FIXa against FX. A bispecific antibody or its antigen-binding fragment.

2. The bispecific antibody or its antigen-binding fragment according to claim 1, wherein the antibody isotype is IgG4.

3. A pharmaceutical composition comprising a bispecific antibody or its antigen-binding fragment according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. A pharmaceutical composition comprising a bispecific antibody or an antigen-binding fragment thereof according to claim 1 or 2, for the treatment of hemophilia A, with or without an inhibitor.

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