Combined use of bispecific antibody that binds activated coagulation factor ix and coagulation factor x with blood coagulation factor x for the treatment of individuals with hemophilia

Description

1 / 23 “COMBINED USE OF A BIESPECTIVE ANTIBODY THAT BINDS TO ACTIVATED COAGULATION FACTOR IX AND FACTOR X Coagulation with blood clotting factor X for the treatment of individuals with hemophilia. TECHNICAL FIELD RELATED ORDERS

[0001] This application claims the benefit of priority from Japanese Patent Application No. 2023-074020, filed on April 28, 2023, with the Japan Patent Office. The priority application is incorporated herein by reference in its entirety.

[0002] The present invention relates to a pharmaceutical composition comprising blood coagulation Factor X for the treatment of an individual with hemophilia A, wherein blood coagulation Factor X is used in combination with a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X (hereinafter referred to as the bispecific antibody FIXa / FX). PREVIOUS TECHNIQUE

[0003] Hemophilia A is a congenital bleeding disorder caused by dysfunction or deficiency of coagulation Factor VIII (FVIII). The principle of hemostatic treatment for hemophilia A is regular replacement therapy with coagulation Factor VIII preparations; as a result, the onset of hemophilic arthropathy has been significantly suppressed, contributing significantly to improved quality of life. However, even with this standard therapy, problems such as the occurrence of alloantibodies (inhibitors) induced by the administration of the preparation exist. In this context, a bispecific antibody, FIXa / FX, which is a mimetic antibody to FVIII, was developed (Patent Document 1). This antibody, which exerts a prolonged effect through subcutaneous administration, exhibits remarkable hematological efficacy. Petition 870250094311, dated 10 / 15 / 2025, page 8 / 45 2 / 23 hemostatic and is used as regular prophylactic administration for patients with congenital hemophilia A, with or without an inhibitor. On the other hand, there are still many clinical questions to be resolved, such as thromboembolic events associated with combination with bypassing agents, hemostasis monitoring, perioperative hemostatic management, and hemostatic efficacy at high activity levels.

[0004] The bispecific antibody FIXa / FX has been indicated as a preparation for regular administration in patients with hemophilia A, and in cases where bleeding occurs during regular administration, a separate hemostatic agent may be administered. As such a hemostatic agent, in a patient with hemophilia A without an inhibitor, an FVIII preparation is the first choice; however, it involves the risk of inducing FVIII inhibitors. In patients with hemophilia A with an inhibitor, a recombinant activated coagulation factor VII (rFVIIa) preparation is the first choice as a hemostatic agent; however, since rFVIIa has a relatively short half-life of about 2 to 3 hours, frequent administration is necessary, which imposes a burden on the patient and the healthcare professional.A method for using a Factor IX preparation as a hemostatic agent is also known (Patent Document 2); however, compared with the use of a Factor X preparation, it presents the problem of a weaker effect in promoting thrombin generation.

[0005] Non-Patent Literature 2 describes a method of combining the bispecific antibody FIXa / FX with activated Factor VII, concentrated and dried, added to human blood coagulation Factor X in hemophilia A with an inhibitor. Activated FVII (FVIIa) is a risk factor for thrombotic complications related to FVIIa (Non-Patent Literature 3). Furthermore, as the package insert for activated Factor VII, added to human blood coagulation Factor X, concentrated and dried, includes a description in the warning section regarding the Petition 870250094311, dated 10 / 15 / 2025, page 9 / 45 3 / 23 Concomitant use with the bispecific antibody FIXa / FX also presents safety concerns regarding this combination. Prior Art Documents PATENT DOCUMENTS Patent Document 1: Japanese Patent No. 6013915 Patent Document 2: Japanese Patent No. 6698102 NON-PATENT LITERATURE Non-Patent Literature 1: Ferriere S, et al. Blood. (2020) 136:740-8 Non-Patent Literature 2: Yuto Nakajima, et al. Haemophilia (2022;28:e149-e152) Non-Patent Literature 3: Laura Downey, et al. Anesth Analg. (2017;125:1431-1436) SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] The present invention provides a pharmaceutical composition comprising blood coagulation Factor X for the treatment of an individual with hemophilia A, wherein blood coagulation Factor X is used in combination with a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X. WAYS TO SOLVE THE PROBLEMS

[0007] Consequently, the present invention includes the following:

[0008] Item 1. A pharmaceutical composition comprising blood coagulation Factor X for the treatment of an individual with hemophilia A, wherein blood coagulation Factor X is used in combination with a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X.

[0009] Item 2. The pharmaceutical composition of Item 1, in which the bispecific antibody FIXa / FX is administered beforehand, simultaneously Petition 870250094311, dated 10 / 15 / 2025, page 10 / 45 4 / 23 or later after FX administration.

[0010] Item 3. The pharmaceutical composition of Item 1 or 2, in which blood coagulation Factor X is administered at a dose of 50 to 1000 IU per kg of body weight. EFFECTS OF THE INVENTION

[0011] According to the present invention, a pharmaceutical composition comprising blood coagulation Factor X is provided for the treatment of an individual with hemophilia A, wherein blood coagulation Factor X is used in combination with a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1

[0012] Additive effect of Factor X on thrombin generation in plasma samples with coagulation Factor VIII deficiency in a disease model characterized by coagulation Factor VIII deficiency or dysfunction. X-axis: Time (minutes). Y-axis: Thrombin (nM). Figure 2

[0013] Additive effect of the bispecific antibody FIXa / FX and Factor X on thrombin generation in plasma samples with coagulation Factor VIII deficiency in a disease model characterized by coagulation Factor VIII deficiency or dysfunction. X-axis: Time (minutes). Y-axis: Thrombin (nM). Figure 3

[0014] Additive effect of bispecific antibody FIXa / FX with Factor X (A) or Factor IX (B) on thrombin generation in plasma samples with coagulation Factor VIII deficiency in a disease model characterized by coagulation Factor VIII deficiency or dysfunction. X-axis: Time (minutes). Y-axis: Thrombin (nM). Figure 4 Petition 870250094311, dated 10 / 15 / 2025, page 11 / 45 5 / 23

[0015] Additive effect of bispecific antibody FIXa / FX and Factor X on clot formation in Factor VIII deficient whole blood samples in a Factor VIII deficiency or dysfunction disease model (ROTEM). X-axis: Time (minutes). Y-axis: Amplitude (mm). The waveform opened earlier and increased in a Factor X concentration-dependent manner. Figure 5

[0016] Additive effect of the bispecific antibody FIXa / FX and Factor X on clotting time in whole blood samples from mice in which the coagulation Factor VIII gene was deactivated in a disease model characterized by coagulation Factor VIII deficiency or dysfunction. Y-axis: CT+CFT (sec). The clotting time of mice in which the coagulation Factor VIII gene was deactivated was prolonged approximately eight times compared to wild-type mice. Even when the bispecific antibody FIXa / FX was injected into mice in which the coagulation Factor VIII gene was deactivated, no change in clotting time was observed. This is because the antibody does not exhibit cross-reactivity with mice. However, when the antibody and Factor X were injected into the mice, the clotting time was significantly reduced compared to the uninjected state. Figure 6

[0017] Additive effect of the bispecific antibody FIXa / FX and Factor X on the fibrin generation rate in whole blood samples from mice in which the coagulation Factor VIII gene was deactivated in a disease model characterized by coagulation Factor VIII deficiency or dysfunction. Y-axis: α(°). The fibrin generation rate of mice in which the coagulation Factor VIII gene was Petition 870250094311, dated 10 / 15 / 2025, page 12 / 45 6 / 23 deactivated decreased approximately three-fold compared to wild-type mice. However, when antibody and Factor X were injected into mice in which the coagulation Factor VIII gene was deactivated, the rate of fibrin generation increased significantly compared to the uninjected state. Figure 7

[0018] Additive effect of bispecific antibody FIXa / FX and Factor X on the amount of tail bleeding in mice in which the coagulation Factor VIII gene was inactivated in a disease model characterized by coagulation Factor VIII deficiency or dysfunction. Y-axis: Amount of bleeding (10 minutes) (μL). The amount of bleeding in mice in which the coagulation Factor VIII gene was inactivated increased approximately 25 times compared to wild-type mice. However, when the antibody and Factor X were injected into mice in which the coagulation Factor VIII gene was inactivated, the amount of bleeding decreased significantly compared to the no-injection state. METHODS OF CARRYING OUT THE INVENTION DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereafter, the embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described below.

[0020] Hemophilia A is a disease in which the blood clotting Factor VIII is deficient or has reduced activity. For example, testing for hemophilia A can be done by examining the activity of a clotting factor. In one embodiment, when the activity of blood clotting Factor VIII, relative to the activity of 100% of Factor VIII in the plasma of a normal human being, is less than 1%, less than 5%, less than 10%, less than 15%, less Petition 870250094311, dated 10 / 15 / 2025, page 13 / 45 7 / 23 If the activity level is less than 20%, less than 25%, less than 30%, less than 35%, or less than 40%, the individual is diagnosed with hemophilia A. Factor VIII activity is measured, for example, by a one-stage coagulation method or a synthetic substrate method.

[0021] In this descriptive report, the term “treat” encompasses the relief, suppression, or prevention of a disorder, disease, or condition, or one or more symptoms associated with the disorder, disease, or condition; or the mitigation or elimination of the cause of the disorder, disease, or condition itself. For example, in this descriptive report, treat also includes improving the bleeding status in a patient who is unable to achieve hemostasis, who requires a long period for hemostasis, or who has difficulty achieving hemostasis.

[0022] In this descriptive report, the terms “blood clotting factor”, “clotting factor” or “blood clotting factor”, or the abbreviated form “F” preceding each blood clotting factor number (e.g., FVIII, FIX, FX, etc.) are used synonymously and refer to each human blood clotting factor of the human clotting system. Activated clotting factors are, for example, abbreviated as Factor VIIIa, Factor IXa, Factor Xa and the like. Non-activated clotting factors are abbreviated as Factor VIII, Factor IX, Factor X and the like.

[0023] In this descriptive report, the term “antibody” includes a monoclonal antibody, a polyclonal antibody, an antibody variant (such as a chimeric antibody, a humanized antibody or a low molecular weight antibody including an antibody fragment to which another molecule may optionally be added, a multispecific antibody and the like), provided that it exhibits the desired antigen-binding activity and biological activity. For example, as an “antibody” in this descriptive report, a molecule in Petition 870250094311, dated 10 / 15 / 2025, p. 14 / 45 8 / 23 which a HAS-binding structure is added to a Fab (only the Fab portion being derived from a normal antibody) is also included. In this descriptive report, the “antibody” may also be a polypeptide or a heteromultimer. The antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, an Fc fusion antibody, or a low molecular weight antibody, such as an antibody fragment. In this descriptive report, the term antibody refers to a binding protein including an antigen-binding site. The terms binding site or antigen-binding site, as used in this descriptive report, denote the region of an antibody molecule to which the antigen effectively binds. The term antigen-binding site includes a variable domain of the antibody heavy chain (VH) and a variable domain of the antibody light chain (VL) (a VH / VL pair).

[0024] In this descriptive report, the term antibody fragment refers to a molecule other than a full-length antibody that includes a portion of the full-length antibody that binds to an antigen to which the full-length antibody binds. Examples of antibody fragments include, but are not limited to, an Fv, a Fab, a Fab', a Fab'-SH, an F(ab%), a diabody, a linear antibody, a single-chain antibody molecule (e.g., an scFv), and a multispecific antibody formed from antibody fragments.

[0025] In this descriptive report, the term “chimeric antibody” refers to an antibody in which a portion of a heavy chain and / or a light chain is derived from a specific source or species, while the remaining portion of the heavy chain and / or light chain is derived from a different source or species.

[0026] In this descriptive report, the class of an antibody Petition 870250094311, dated 10 / 15 / 2025, page 15 / 45 9 / 23 refers to the type of constant domain or constant region present in the antibody heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these can be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to the different immunoglobulin classes are designated α, δ, ε, γ, and μ, respectively.

[0027] Factor X is one of the enzymes that make up the blood coagulation cascade. Factor X is usually activated by intrinsic Xase or extrinsic Xase to become Factor Xa. Factor Xa generally acts by cleaving prothrombin at two sites (the ArgThr bond and subsequently the Arg-Ile bond) to produce active thrombin.

[0028] In one embodiment, a pharmaceutical composition comprising blood coagulation Factor X is provided for use in the treatment of an individual with hemophilia A, wherein blood coagulation Factor X is used in combination with a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X. In another embodiment, blood coagulation Factor X is provided for use in the treatment of an individual with hemophilia A, wherein blood coagulation Factor X is used in combination with a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X. In another embodiment, a combination of blood coagulation Factor X and a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X is provided for use in the treatment of an individual with hemophilia A.In another embodiment, the use of blood clotting Factor X is provided for the manufacture of a medicine for the treatment of an individual with hemophilia A, wherein the treatment comprises the combination of blood clotting Factor X with... Petition 870250094311, dated 10 / 15 / 2025, page 16 / 45 10 / 23 A bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X. In a further embodiment, a method is provided for treating an individual with hemophilia A, comprising the use of a combination of blood coagulation Factor X and a bispecific antibody that binds to activated blood coagulation Factor IX and blood coagulation Factor X.

[0029] In one embodiment, the bispecific antibody that binds to activated coagulation Factor IX and coagulation Factor X is called the FIXa / FX bispecific antibody and includes a first antigen-binding site and a second antigen-binding site, and these sites are not particularly limited, provided they exhibit binding activity to FIXa and FX, respectively. The FIXa / FX bispecific antibody of the present invention may additionally bind to another antigen, provided it has binding activity to FIXa and FX. In this descriptive report, the term FIXa / FX-binding bispecific antibody refers to a bispecific antibody that can bind to FIXa and FX with sufficient affinity for the antibody to be useful as a therapeutic agent when targeting FIXa and FX.In one aspect, the extent of binding of the bispecific antibody that binds to FIXa and FX to a non-FIX protein, non-FIX protein, or unrelated non-FX protein is less than about 10% of the antibody binding to FIXa and FX when measured, for example, by a radioimmunoassay (RIA). In a particular aspect, an antibody that binds to FIXa and FX has a dissociation constant (Kd) of <100 μM, <10 μM, <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., less than or equal to 10⁻⁵ M, e.g., from 10⁻⁵ M to 10⁻¹⁰ M, or e.g., from 10⁻⁶ M to 10⁻¹⁰ M). In one particular aspect, the bispecific antibody that binds to FIXa and FX binds to an epitope of FIX, FIXa, and FX that are conserved. Petition 870250094311, dated 10 / 15 / 2025, p. 17 / 45 11 / 23 of the FIX, FIXa, and FX antibodies from different species, respectively. For example, ACE910 (Emicizumab) is exemplified as a bispecific FIXa / FX antibody (Patent Document 1). In one embodiment, an antibody that binds to both FIXa and FX exhibits a dissociation constant (Kd) <1 μM in a surface plasmon resonance assay.

[0030] In one embodiment, hemophilia A can be a congenital or acquired disease. In another embodiment, a patient with hemophilia A has a deficiency of coagulation factor VIII or suffers from coagulation factor VIII dysfunction.

[0031] In one embodiment of the present invention, the components of the combination, the bispecific antibody FIXa / FX and FX, can be formulated together or separately. When formulated separately, the administration of FX and the administration of the bispecific antibody FIXa / FX can be performed simultaneously or sequentially. For example, the administration of the bispecific antibody FIXa / FX can be performed before, simultaneously with, or after the administration of FX. In one aspect, the bispecific antibody component FIXa / FX of the combination can be administered independently as a single dose or as multiple doses.When administered in multiple doses, for example, the dosing interval for the bispecific antibody FIXa / FX is approximately 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or approximately 1, 2, or 3 weeks, or approximately 1, 2, 3, 4, 5, or 6 days, and the dosing interval for FX is approximately 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 hours.

[0032] The bispecific antibody FIXa / FX or FX of the present invention can be administered by any suitable means, including parenteral administration, pulmonary administration and intra-administration. Petition 870250094311, dated 10 / 15 / 2025, page 18 / 45 12 / 23 nasal and, when desired for local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be performed by any suitable route, depending in part on whether administration is short-term or long-term, for example, by injection, such as intravenous injection or subcutaneous injection. Several administration regimens are included, including, but not limited to, single administration, repeated administration at multiple points in time, bolus administration, and pulse infusion. Preferably, the bispecific antibody FIXa / FX is injected subcutaneously and FX is injected intravenously.

[0033] The bispecific antibody FIXa / FX is administered, for example, to an adult at approximately 0.5 mg / kg of body weight, approximately 1 mg / kg of body weight, approximately 1.5 mg / kg of body weight, approximately 2 mg / kg of body weight, approximately 2.5 mg / kg of body weight, approximately 3 mg / kg of body weight, approximately 3.5 mg / kg of body weight, approximately 4 mg / kg of body weight, approximately 4.5 mg / kg of body weight, approximately 5 mg / kg of body weight, approximately 5.5 mg / kg of body weight, approximately 6 mg / kg of body weight, approximately 6.5 mg / kg of body weight, approximately 7 mg / kg of body weight, approximately 7.5 mg / kg of body weight, approximately 8 mg / kg of body weight, approximately 8.5 mg / kg of body weight, approximately 9 mg / kg of body weight, approximately 9.5 mg / kg of body weight, approximately 10 mg / kg of body weight or more per administration.Preferably, the bispecific antibody FIXa / FX is administered in doses of approximately 1.5 mg / kg of body weight, approximately 3 mg / kg of body weight, or approximately 6 mg / kg of body weight.

[0034] Blood coagulation factor X is administered, for example, to an adult at approximately 50 IU / kg of body weight, approximately 75 IU / kg of body weight, approximately 100 IU / kg of body weight, approximately Petition 870250094311, dated 10 / 15 / 2025, page 19 / 45 13 / 23 of 150 IU / kg of body weight, approximately 200 IU / kg of body weight, approximately 250 IU / kg of body weight, approximately 300 IU / kg of body weight, approximately 350 IU / kg of body weight, approximately 400 IU / kg of body weight, approximately 450 IU / kg of body weight, approximately 500 IU / kg of body weight, approximately 550 IU / kg of body weight, approximately 600 IU / kg of body weight, approximately 650 IU / kg of body weight, approximately 700 IU / kg of body weight, approximately 750 IU / kg of body weight, approximately 800 IU / kg of body weight, approximately 850 IU / kg of body weight, approximately 900 IU / kg of body weight, approximately 950 IU / kg of body weight, approximately 1000 IU / kg of body weight or more per administration. Preferably, blood coagulation Factor X is administered in doses of 50 to 1000 IU / kg of body weight, and more preferably in doses of about 200 IU / kg of body weight or more.

[0035] The blood clotting factor used in the present invention may be a natural protein derived from a living body or it may be a recombinant protein. When derived from a living body, it may be derived from plasma. The plasma is preferably human plasma.

[0036] In this descriptive report, the term “pharmaceutical composition” refers to a preparation in a form such that the biological activity of the active ingredient contained therein can exert its effect. The term “pharmaceutically acceptable vehicle” refers to a component other than the active ingredient in the pharmaceutical composition that is not toxic to the individual. A pharmaceutically acceptable vehicle includes, but is not limited to, a buffer, an excipient, a stabilizer, or a preservative.

[0037] In one embodiment of the present invention, blood coagulation activity is improved by combining coagulation Factor X with a bispecific antibody FIXa / FX compared Petition 870250094311, dated 10 / 15 / 2025, p. 20 / 45 14 / 23 with a treatment using the bispecific antibody FIXa / FX without combination with Factor X. In another embodiment of the present invention, the blood coagulation activity of the treatment is improved in a dose-dependent manner with Factor X. For example, blood coagulation activity can be examined by measuring the amount of thrombin, the rate of thrombin generation, the blood coagulation time, clot formation, fibrin formation, the rate of fibrin formation, the frequency of bleeding, or the amount of bleeding. For example, when the amount of thrombin generation is increased, the rate of thrombin generation is elevated, the blood coagulation time is shortened, clot formation is increased, the frequency of bleeding is reduced, and / or the amount of bleeding is decreased, the blood coagulation activity is considered to be improved.In this descriptive report, for example, when, through treatment, a patient with hemophilia A achieves the equivalent of 1%, 2.5%, 5%, 10%, 15%, 20% or 25% or more in terms of Factor VIII activity, preferably 5% or more, blood clotting activity is considered improved (with the Factor VIII activity of a normal human being defined as 100%).

[0038] In one embodiment of the present invention, by combining coagulation Factor X with a bispecific antibody FIXa / FX, thrombin generation can be increased in a patient. Thrombin is an important enzyme in coagulation, and increased thrombin production is known to exert several effects on the coagulation system. Thrombin typically converts fibrinogen to fibrin, thus forming fibrin, and thrombin activates platelets. An increase in thrombin generation can also cause an increase in fibrin formation and platelet activation. Furthermore, regular thrombin production can also cause a decrease in activity. Petition 870250094311, dated 10 / 15 / 2025, page 21 / 45 15 / 23 fibrinolytic. For example, thrombin generation in biological plasma can be measured using the calibrated automated thrombogram (CAT) method (Thermo Fisher Scientific).

[0039] In one embodiment of the present invention, since the intention is to administer Factor X, which is a non-activated coagulation factor, the risk of thrombosis is expected to be lower compared with the application of an activated prothrombin complex concentrate comprising an activated coagulation factor, Activated Factor VII added to dry human blood coagulation Factor X concentrate or Factor VIIa. As no Factor VIII-related factor is present, it is expected that application to a patient with hemophilia A will have little or no risk of inducing an inhibitor against Factor VIII.

[0040] The combination of the present invention is used in advance when bleeding occurs or when bleeding is expected, although the period is not particularly limited. For example, the combination of the present invention can be used at the time of bleeding after trauma, during surgery, or before surgery.

[0041] In one embodiment of the present invention, since the half-life of Factor X (40 hours) is longer than the half-life of activated Factor VII (2.5 hours), the combination with the bispecific antibody FIXa / FX can reduce the risk of inhibitor development in a hemophilia A patient with an inhibitor. In a hemophilia A patient with an inhibitor, compared to activated Factor VII, the combination can also reduce the burden of frequent injections, maintaining a longer dosing interval.

[0042] In one embodiment of the present invention, unlike the Unpatented Literature 2, FVIIa is not used, and FX, which is a non-activated coagulation factor, is used in combination with a bispecific antibody that binds to coagulation Factor IX. Petition 870250094311, dated 10 / 15 / 2025, page 22 / 45 16 / 23 activated blood clotting and to blood coagulation Factor X, the invention is advantageous in terms of safety, such as reducing the risk of hypercoagulation.

[0043] In an embodiment of the present invention, such as the Factor Unactivated X has a relatively short half-life (40 hours) compared to the half-life of the bispecific antibody FIXa / FX (approximately 4 to 5 weeks after subcutaneous injection), the thrombin generation-promoting effect may be limited to a specific period, such as during hemostasis, and therefore may reduce the risk of thrombosis during long-term treatment.

[0044] In one embodiment of the present invention, the individual to be treated is not particularly limited, but is preferably a human. In another embodiment, the individual to be treated has developed an inhibitor against FVIII or FVIIIa (sometimes referred to as with an inhibitor) or has not developed such an inhibitor (sometimes referred to as without an inhibitor). According to the combination of the present invention, the administration of FVIII or FVIIIa can be reduced or avoided.

[0045] In one embodiment of the present invention, the individual to be treated may have received treatment with the bispecific antibody FIXa / FX prior to administration of the combination. Treatment with the bispecific antibody FIXa / FX prior to administration of the combination is not particularly limited, but may be received temporarily or regularly. Preferably, treatment is received regularly.

[0046] In this descriptive report, the term approximately refers to a range of ±10%, preferably ±5%, and most preferably ±2.5%.

[0047] The following Examples are provided to illustrate the present invention in a specific and detailed manner, but the Examples are used for illustrative purposes only and are not intended to limit the present invention. Petition 870250094311, dated 10 / 15 / 2025, page 23 / 45 17 / 23 EXAMPLES Experimental Procedure: Example 1 Thrombin Generation in Plasma with Factor VIII Deficiency

[0048] Factor VIII deficient plasma samples (Factor VIII deficient plasma, Affinity Biologicals Inc.) were used as a model of coagulation Factor VIII deficiency or dysfunction.

[0049] The bispecific antibody FIXa / FX (hereinafter abbreviated as Emi; the details are described in Patent Document 1) was added to the plasma sample at a concentration of 50 μg / ml. In addition, Factor X was added in vitro as part of the experiment. Plasma derived from healthy humans was used as a control in the experiment. Table 1: Tested Additive Combinations FVIII dp FVIII dp +130 nm FX FVIII dp +260 nm FX FVIII dp +520 nm FX FVIII dp +780 nm FX FVIII dp +1040 nm FX FVIII dp +130 nm FX +50 pg / ml Emi FVIII dp +260 nm FX +50 pg / ml Emi FVIII dp +520 nm FX +50 pg / ml Emi FVIII dp +780 nm FX +50 pg / ml Emi FVIII dp +1040 nm FX +50 pg / ml Emi FVIII dp +125 nm FX +50 pg / ml Emi FVIII dp +250 nm FX +50 pg / ml Emi FVIII dp +500 nm FX +50 pg / ml Emi FVIII dp +125 nm FX +50 pg / ml Emi FVIII dp +250 nm FX +50 pg / ml Emi FVIII dp +500 nm FX +50 pg / ml Emi Normal plasma FVIII dp: FVIII deficient plasma Petition 870250094311, dated 10 / 15 / 2025, p. 24 / 45 18 / 23

[0050] Thrombin generation was continuously determined using a fluorogenic substrate after activation of coagulation with a small amount of tissue factor (the device and all reagents were from Thermo Fisher Scientific).

[0051] Method for Measuring Thrombin Generation:

[0052] Thrombin generation in biological plasma was measured using the calibrated automated thrombogram (CAT) method (Thermo Fisher Scientific). Briefly, thrombin generation is triggered by the extrinsic coagulation pathway by the addition of 1 pM tissue factor (TF), phospholipid, and calcium ions (Ca2+). A low-affinity fluorogenic substrate is added for real-time analysis of thrombin generation. By calibrating the plasma sample with a known thrombin calibrator, corrections are made for substrate consumption, sample color, and internal filter effects. Fluorescence is detected using Thermo Fluoroskan. Thrombin activity is calculated from the measured fluorescence signal. The resulting curve shows free thrombin activity (y-axis, thrombin in nM) as a function of time (x-axis, seconds). Control Measurement: Normal Plasma and Plasma with Factor VIII Deficiency

[0053] Isolated analysis of Factor VIII deficient plasma, as expected, showed very weak thrombin generation. This indicates the physiological reason for the bleeding disorder in a patient with hemophilia A. X-Factor Addition

[0054] With the addition of Factor X, thrombin was generated rapidly, and a total of approximately 1.5 times more thrombin was produced compared to the Factor VIII deficient sample (Figure 1). Addition of Emi

[0055] With the addition of Emi and Factor X, an increase was obtained in Petition 870250094311, dated 10 / 15 / 2025, page 25 / 45 19 / 23 thrombin generation comparable to that of the sample with Factor VIII deficiency (Figure 2).

[0056] Comparison of the Activity of the Combination of Emi and Factor X with the Activity of Factor X in Thrombin Generation in Plasma Samples with Factor VIII Deficiency:

[0057] When thrombin generation in samples supplemented with the combination of Emi and Factor X or with Factor X alone was compared, the combination of Emi and Factor X produced more thrombin, and the time to peak thrombin generation was shorter with the combination of Emi and Factor X than with Factor X alone (Figures 1 and 2). Addition of Factor X or Factor IX

[0058] When Factor IX was added to Emi, no recovery of thrombin generation capacity to normal plasma levels was observed (Figure 3B). On the other hand, when Factor X was added to Emi, thrombin generation capacity was recovered to a level comparable to that of normal plasma (Figure 3A). Example 2 Blood Clot Formation in Whole Blood of Factor VIII Deficient Model Mice

[0059] Hemophilia A model mice, in which the gene (F8) encoding Factor VIII on the sex chromosome was deactivated (containing normal levels of Factor IX, Factor X and non-activated Factor II), were used as an animal model for Factor VIII coagulation deficiency or dysfunction.

[0060] Emi is known not to exhibit cross-reactivity with mouse-derived Factor IX and mouse-derived Factor X. To evaluate the combined effect of Emi and Factor X in mouse models for hemophilia A, human-derived Factor IX and human-derived Factor X were administered in addition to Petition 870250094311, dated 10 / 15 / 2025, page 26 / 45 20 / 23 Emi, based on Non-Patented Literature 1. Wild mice were used as controls in the experiment. Table 2: Combinations of Tested Substances Administered Model mouse HA Model mouse HA +3 mg / kg body weight Emi Model mouse HA +100 IU / kg hFIX + 100 IU / kg hFX Model mouse HA +3 mg / kg body weight Emi +100 IU / kg hFIX + 100 IU / kg hFX Model mouse HA +3 mg / kg body weight Emi +100 IU / kg hFIX +200 IU / kg hFX Model mouse HA +3 mg / kg body weight Emi +100 IU / kg hFIX +500 IU / kg hFX Normal mouse HA model mouse: Hemophilia A model mouse; hFIX: Human Factor IX; hFX: Human Factor X Rotational Thromboelastometry (ROTEM):

[0061] ROTEM was continuously measured for the clot formation process after activation of coagulation with calcium chloride (the device and all reagents were from Pentapharm, Germany).

[0062] Clot formation in whole blood was measured using a whole blood hemostasis analyzer (Pentapharm, Germany). Briefly, the device's measuring system employs a fixed cylindrical cup and a permanently oscillating vertical shaft. The shaft's movement is detected by an optical detection system and processed and analyzed by a computer equipped with dedicated software. A test solution is placed in the cylindrical cup, and the cup is positioned so that the tip of a pin attached to the lower end of the vertical shaft is immersed in the test solution. The center of the shaft is guided by a bearing, and the shaft oscillates laterally in a Petition 870250094311, dated 10 / 15 / 2025, page 27 / 45 21 / 23 constant angle via a spring connector. The shaft rotation is measured optically by means of a mirrored plate at the upper end of the shaft, a diode as a light source, and a photosensitive sensor. When coagulation does not occur, movement is not impeded, but when a clot forms and adheres to the surface of the pin and cup, movement is impeded. The resulting curve shows the clot force (y-axis, mm) as a function of time (x-axis, seconds). Control Measurement: Model Mice for Hemophilia A and Wild-type Mice

[0063] Analysis of hemophilia A model mice, as expected, revealed very weak clot formation. This indicates the physiological reason for the bleeding disorder in a patient with hemophilia A. Furthermore, analysis of wild-type mice, as expected, showed very strong clot formation compared to hemophilia A model mice (Figure 4). Emi Management, Human Factor IX and Human Factor X

[0064] Under the administration of Emi, Human Factor IX and Factor X In humans, clot formation was rapidly generated and, compared to plasma samples deficient in VIII, clot formation was generated at up to approximately 2.5 times the rate (Figures 4, 5, and 6). Furthermore, the effect was dose-dependent on Factor X (Figures 5 and 6). Example 3 Tail-Docking Assay in Factor VIII Deficient Model Mice

[0065] Hemophilia A model mice, in which the gene (F8) encoding Factor VIII on the sex chromosome was deactivated (containing normal levels of Factor IX, Factor X and non-activated Factor II), were used as an animal model for Factor VIII coagulation deficiency or dysfunction.

[0066] Emi was administered to the mouse model with hemo Petition 870250094311, dated 10 / 15 / 2025, page 28 / 45 22 / 23 filia A at a concentration of 3 mg / kg (50 μg / ml), which is similar to the clinically applied Emi concentration.

[0067] Emi is known to not exhibit cross-reactivity with mouse-derived Factor IX and mouse-derived Factor X. To evaluate the combined effect of Emi and Factor X in hemophilia A model mice, human-derived Factor IX and human-derived Factor X were administered in addition to Emi, based on Non-Patent Literature 1. Wild-type mice were used as controls in the experiment. Table 3: Combinations of Tested Substances Administered Model mouse HA Model mouse HA +3 mg / kg body weight Emi Model mouse HA +100 IU / kg hFIX + 100 IU / kg hFX Model mouse HA +3 mg / kg body weight Emi +100 IU / kg hFIX + 100 IU / kg hFX Model mouse HA +3 mg / kg body weight Emi +100 IU / kg hFIX +200 IU / kg hFX Model mouse HA +3 mg / kg body weight Emi +100 IU / kg hFIX +500 IU / kg hFX Normal mouse HA model mouse: Model mouse for hemophilia A. hFIX: Human-derived factor IX. hFX: Human-derived factor X.

[0068] Tail Cutting Test:

[0069] In the tail docking assay, 3 mg / kg (50 μg / ml) of Emi was administered to hemophilia A model mice under anesthesia 24 hours before tail docking, and human-derived Factor IX and human-derived Factor X were administered 5 minutes prior to tail docking. Petition 870250094311, dated 10 / 15 / 2025, page 29 / 45 23 / 23 tes of tail cutting. The tail was cut 5 mm from the tip, immediately immersed in a tube containing physiological saline solution, and the amount of bleeding (μL) was measured over 10 minutes. Control Measurement: Model Mice for Hemophilia A and Wild-type Mice

[0070] Analysis of hemophilia A model mice, as expected, revealed a very large amount of bleeding. This indicates the physiological reason for the bleeding disorder in a patient with hemophilia A. Furthermore, analysis of wild-type mice, as expected, showed a very small amount of bleeding compared to hemophilia A model mice (Figure 7). Emi Administration, Human-Derived Factor IX, and Human-Derived Factor X

[0071] With the administration of Emi, human-derived Factor IX and human-derived Factor X, the amount of bleeding was reduced, and the effect was dose-dependent of Factor X. Furthermore, compared with Factor VIII-deficient samples, the amount of bleeding was reduced by up to about five times (Figure 7). Industrial Applicability

[0072] A pharmaceutical composition comprising blood coagulation factor X is provided to treat an individual with hemophilia A. Petition 870250094311, dated 10 / 15 / 2025, page 30 / 45

Claims

1 / 1 CLAIMS 1. Pharmaceutical composition, characterized in that it comprises blood coagulation factor X for treating an individual with hemophilia A, wherein blood coagulation factor X is used in combination with a bispecific antibody that binds to activated blood coagulation factor IX and blood coagulation factor X.

2. Pharmaceutical composition, according to claim 1, characterized in that the bispecific antibody FIXa / FX is administered before, simultaneously with, or after the administration of FX.

3. Pharmaceutical composition, according to claim 1 or 2, characterized in that blood coagulation factor X is administered at 50 to 1000 IU per kg of body weight. Petition 870250094311, dated 10 / 15 / 2025, pp. 31 / 45

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