Methods of treating hemophilia a

Abstract

The present invention relates to methods of increasing hemoglobin in an individual suffering from severe hemophilia A by prophylactic administration of emicizumab. Also provided are pharmaceutical compositions comprising emicizumab.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 621,559, filed January 16, 2024, the disclosure of which is hereby incorporated herein by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The contents of the electronic serial number (146392068640SEQLIST.xml; size: 15,421 bytes; and creation date: January 9, 2025) are incorporated herein by reference in their entirety. Technical Field

[0005] This invention relates to a method for increasing hemoglobin in individuals with hemophilia A by prophylactic administration of emecizumab. This document further provides pharmaceutical compositions comprising emecizumab. Background Technology

[0006] Hemophilia A is a bleeding disorder caused by a hereditary reduction or deficiency of the function of clotting factor VIII (FVIII). Patients with hemophilia A are typically treated with FVIII replacement therapy, which includes both purified plasma-derived FVIII protein and recombinant FVIII protein. These FVIII preparations are administered as needed to manage bleeding (e.g., for acute episodes). FVIII preparations are also administered periodically as needed to prevent rebleeding. In recent years, FVIII preparations have also been used prophylactically to prevent bleeding events (e.g., as a prophylactic treatment; see, for example, Blood 58, 1-13 (1981) and Nature 312, 330-337 (1984), both of which are hereby incorporated herein by reference in their entirety). The half-life of FVIII preparations in the blood is approximately 12 to 16 hours. Therefore, for continued prevention, patients were given FVIII formulation three times a week (see, for example, Nature 312, 337-342 (1984) and Biochim. Biophys. Acta 871, 268-278 (1986), both of which are hereby incorporated in their entirety by reference).

[0007] Occasionally, anti-FVIII antibodies (i.e., inhibitors) are produced in hemophilia patients, which counteract the effects of FVIII agents. Treatment for patients who have developed inhibitors (inhibitor patients) includes bypass agents that are independent of FVIII function, which involve the catalysis of activation of coagulation factor X (FX) by activated coagulation factor IX (FIXa). However, in some cases, bypass agents are not sufficient to stop bleeding in patients (see, for example, Kempton and Meeks (2014), Blood 124(23): 3365-3372, which is hereby incorporated herein by reference in its entirety).

[0008] Recently, antibodies that functionally replace FVIII and their uses have been disclosed (see, for example, Blood 58, 1-13 (1981); Nature 312, 330-337 (1984); and Nature 312, 337-342 (1984)). These antibodies may be effective against acquired hemophilia in which anti-FVIII autoantibodies are present. Summary of the Invention

[0009] This invention provides a method for increasing hemoglobin levels in an individual diagnosed with hemophilia A, the method comprising: administering a bispecific antibody (e.g., emecizumab or a biosimilar thereof) to the individual diagnosed with hemophilia A in a treatment regimen, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X; and increasing the hemoglobin level in the individual compared to baseline. This invention further provides a method for treating hemophilia A, the method comprising: administering the bispecific antibody; and increasing the hemoglobin level in the individual compared to baseline.

[0010] In one aspect of the invention, a method is provided comprising: (a) administering a bispecific antibody to an individual diagnosed with hemophilia A in a treatment regimen, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) identical first antibody L chains and second antibody L chains, each L chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively; and (b) Increases hemoglobin levels in the individual compared to baseline. In some embodiments, the antibody is emecizumab.

[0011] In another aspect of the invention, a method for treating hemophilia A is provided, the method comprising: (a) administering a bispecific antibody to an individual diagnosed with hemophilia A, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) identical first antibody L chains and second antibody L chains, each L chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively; and (b) Compared to baseline, the individual's hemoglobin level increased. In some embodiments, the antibody is emecizumab.

[0012] In another aspect of the invention, a method for increasing hemoglobin levels in an individual diagnosed with hemophilia A is provided, the method comprising: administering a bispecific antibody to the individual diagnosed with hemophilia A, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody comprises (i) a first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) the same first antibody L chain and the second antibody L chain, each L chain comprising a variable region comprising CDRs comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively. 1, 2, and 3; and wherein the level of this hemoglobin in the individual is increased compared to baseline. In some embodiments, the antibody is emecizumab.

[0013] In another aspect of the invention, the use of a bispecific antibody in the manufacture of a medicament for treating hemophilia A in an individual is provided, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) the same first antibody L chain and second antibody L chain, each L chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively; and wherein the treatment increases hemoglobin levels in the individual compared to baseline levels.

[0014] In another aspect of the invention, the use of a bispecific antibody in the manufacture of a medicament for increasing hemoglobin levels in an individual diagnosed with hemophilia A is provided, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) the same first antibody L chain and second antibody L chain, each L chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively. And 3, where the drug increases the level of that hemoglobin in the individual compared to baseline levels.

[0015] In another aspect of the invention, a bispecific antibody for use in treating hemophilia A in an individual is provided, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) the same first antibody L chain and the second antibody L chain, each L chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively; and wherein the treatment increases the hemoglobin level in the individual compared to baseline levels.

[0016] In another aspect of the invention, a bispecific antibody is provided for increasing hemoglobin levels in individuals diagnosed with hemophilia A, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) the same first antibody L chain and second antibody L chain, each L chain comprising a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively. And 3, where the use increases the level of that hemoglobin in the individual compared to the baseline level.

[0017] In some embodiments of any of the bispecific antibodies described above, the heavy (H) chain variable region of the first antibody comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the H chain variable region of the second antibody comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the same first antibody light (L) chain variable region and the same second antibody light (L) chain variable region each comprise the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the amino acid sequence of the full-length H chain of the first antibody is as shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the full-length H chain of the second antibody is as shown in SEQ ID NO: 6. In some embodiments, the amino acid sequences of the same full-length L chain of the first antibody and the same full-length L chain of the second antibody are each as shown in SEQ ID NO: 11. In some embodiments, the antibody is emecizumab.

[0018] In some embodiments according to any of the methods described above, the bispecific antibody is administered at an initial dose of 3 mg / kg per week for one or more weeks. In some embodiments, the bispecific antibody is administered at an initial dose of 3 mg / kg per week for four weeks.

[0019] In some embodiments of the bispecific antibody according to any of the methods, uses, or intended use described above, the bispecific antibody is administered at a maintenance dose of 1.5 mg / kg weekly for at least 12 weeks, at least 24 weeks, or at least 52 weeks. In some embodiments, the bispecific antibody is administered at a maintenance dose of 3 mg / kg every two weeks for at least 12 weeks, at least 24 weeks, or at least 52 weeks. In some embodiments, the bispecific antibody is administered at a maintenance dose of 6 mg / kg every four weeks for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

[0020] In some embodiments of the methods, uses, or bispecific antibodies described above, the treatment is either flare-up treatment or prophylactic treatment. In some embodiments, the treatment includes both flare-up treatment and prophylactic treatment.

[0021] In some embodiments according to any of the methods described above, the individual's bloodstream contains a factor VIII inhibitor. In some embodiments, the individual's bloodstream does not contain a factor VIII inhibitor. In some embodiments, the individual is at least 12 years old.

[0022] In some embodiments according to any of the methods, uses, or bispecific antibodies provided for use described above, the baseline level of hemoglobin is between 80 g / L and 100 g / L. In some embodiments, the baseline level of hemoglobin is between 100 g / L and 120 g / L. In some embodiments, the baseline level of hemoglobin is between 120 g / L and 140 g / L. In some embodiments, the baseline level of hemoglobin is between 140 g / L and 220 g / L.

[0023] In some embodiments according to any of the methods, uses, or bispecific antibodies provided for use described above, the hemoglobin level increased by 5 g / L, 10 g / L, or 15 g / L compared to baseline. In some embodiments, the hemoglobin level increased by 20 g / L, 25 g / L, or 30 g / L compared to baseline. In some embodiments, the hemoglobin level increased by 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, or 25-30 g / L compared to baseline.

[0024] In some embodiments of the methods, uses, or bispecific antibodies provided for use described above, the hemoglobin level is 80-100 g / L, 100-120 g / L, or 120-140 g / L at least four weeks after administration of the initial dose. In some embodiments, the hemoglobin level is 100-120 g / L, 120-140 g / L, or 140-160 g / L at least twelve weeks after administration of the initial dose. In some embodiments, the hemoglobin level is 100-120 g / L, 120-140 g / L, or 140-160 g / L at least twenty-four weeks after administration of the initial dose.

[0025] In some embodiments of the bispecific antibodies according to any of the methods, uses, or intended use described above, the hematocrit level is further measured as a percentage. In some embodiments, it is further measured at 10... 12 / L measures the mean corpuscular volume (MCV) level. In some embodiments, the red blood cell count is further measured as a percentage. In some embodiments, the red blood cell distribution width level is further measured as a percentage. Attached Figure Description

[0026] The accompanying drawings illustrate certain embodiments of the features of this disclosure. These embodiments are not intended to limit the scope of the claims in any way.

[0027] Figures 1A-1D provide a schematic overview of the clinical trials HAVEN 1 (Figure 1A), HAVEN 3 (Figure 1B), HAVEN 4 (Figure 1C), and STASEY (Figure 1D).

[0028] Figure 2 shows the mean change in hemoglobin levels from baseline over 73–78 weeks after the first dose of emecizumab for the pooled participants and those with severe hemophilia A. Subgroups based on hemoglobin levels were as follows: 80 g / L < HgB < 100 g / L; 100 g / L ≤ HgB < 120 g / L; 120 g / L ≤ HgB < 140 g / L; and 140 g / L ≤ HgB < 180 g / L. Detailed Implementation

[0029] In some respects, this document provides a method for treating hemophilia A in an individual, wherein the treatment increases the individual's hemoglobin level compared to baseline hemoglobin levels. The disclosure of this application is based, at least in part, on the inventors' accidental discovery that the bispecific antibody described herein (i.e., emecizumab or a bioanalyte thereof) exhibits unexpected improvements in hemoglobin levels over time, particularly for individuals with very low baseline hemoglobin levels. Specifically, as reported in the examples, the inventors found that this bispecific antibody significantly increased hemoglobin levels in individuals with very low baseline hemoglobin levels. Emecizumab provides significant therapeutic value for patients with severe bleeding who are unable to clot normally. Such findings presented herein represent a significant advance in the field.

[0030] Therefore, in one aspect, the present invention relates to a method for treating hemophilia A, the method comprising administering a bispecific antibody (e.g., emecizumab or a bioanalyte thereof) to an individual diagnosed with hemophilia A, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the hemoglobin level in the individual is increased compared to baseline levels.

[0031] In another aspect, the present invention provides a method for increasing hemoglobin levels in an individual diagnosed with hemophilia A, the method comprising administering to the individual diagnosed with hemophilia A a bispecific antibody (e.g., emecizumab or a biosimilar thereof) that binds to coagulation factor IX and / or activated coagulation factor IX, and to coagulation factor X, wherein the hemoglobin level in the individual is increased compared to baseline levels.

[0032] This article also provides a method comprising: administering a bispecific antibody (e.g., emecizumab or a biosimilar thereof) to an individual diagnosed with hemophilia A in a treatment regimen, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X; and increasing the hemoglobin level in the individual compared to baseline levels.

[0033] I. Definition

[0034] Before describing the invention in detail, it should be understood that the invention is not limited to specific compositions or biological systems, which can, of course, vary. Furthermore, it should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless otherwise indicated. For example, “disease” includes one or more diseases.

[0036] As used herein, the phrase “comprising” is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of” is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “substantially constitutes” is semi-closed-ended, indicating that such embodiments may further include elements that do not materially alter the essential characteristics of such embodiments. It should be understood that aspects and embodiments of the invention described herein include those referred to by “comprising,” “consisting of,” and “substantially constitutes.”

[0037] As used in this article, a person’s “baseline” level (such as the baseline level of hemoglobin) refers to the level of that person before the administration of the drug described in this article (such as emecizumab).

[0038] As used in this article, the term "prevention" includes providing prevention against the onset or recurrence of a disease (e.g., hemophilia A) in an individual (e.g., a human subject).

[0039] The term "pharmaceutical formulation" refers to a preparation that is in a form in which the biological activity of the active ingredient is permitted and that does not contain any additional components that would have unacceptable toxicity to a subject to which the formulation will be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (mediators, additives) are excipients for which the active ingredient is reasonably administered to a test mammal to provide an effective dose.

[0040] As used herein, the term "treatment" refers to a clinical intervention aimed at altering the natural course of the treated individual or cells during the course of clinicopathology. Ideal treatment outcomes include slowing the rate of disease progression, alleviating or reducing the severity of the disease state, and improving or resolving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a disease (such as asthma) are reduced or eliminated, including but not limited to reducing symptoms caused by the disease, improving the quality of life of an individual suffering from the disease, reducing the dosage of other medications required to treat the disease, and / or prolonging the individual's survival.

[0041] An "effective amount" is at least the minimum amount required to achieve measurable improvement or prevention of a particular disease. The effective amount described herein can vary depending on factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit the expected response in an individual. An effective amount is also the amount in which the beneficial effects of treatment outweigh any toxic or harmful effects of treatment. For preventative use, beneficial or anticipated outcomes include, for example, elimination or reduction of risk, lessening of severity, or delay of disease onset, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that occur during disease development. For therapeutic use, beneficial or anticipated outcomes include clinical outcomes such as reduction of one or more symptoms caused by the disease, improvement of the patient's quality of life, reduction of the dosage of other medications required to treat the disease, enhancement of the effects of other medications (e.g., by targeting), delay of disease progression, and / or prolongation of survival. An effective amount may be administered once or multiple times. For the purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prevention or treatment. An effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved when combined with another drug, compound, or pharmaceutical composition, as understood in a clinical context. Therefore, an "effective amount" can be considered when administering one or more therapeutic agents, and an effective amount of a single agent can be considered if the desired result can be obtained or achieved in combination with one or more other agents.

[0042] As used in this article, "in combination with" or "in combination with" refers to the application of another treatment in addition to one treatment. Therefore, "in combination with" or "in combination with" means the application of another treatment before, during, or after the application of one treatment to an individual.

[0043] "Disease" is any condition that will benefit from treatment, including but not limited to chronic and acute diseases or illnesses, including those pathological conditions that make mammals susceptible to the disease.

[0044] "Subject," "patient," or "individual" used for therapeutic purposes means any animal classified as a mammal, including humans, livestock, and farm animals, as well as zoo animals, sporting animals, or pets such as dogs, horses, cats, cattle, etc. Preferably, the mammal is a human.

[0045] The term “antibody” is used in the broadest sense in this article and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity.

[0046] "Isolated" antibodies are antibodies that have been identified and isolated and / or recovered from components of their natural environment. Contaminant components of their natural environment are materials that could interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteins or non-protein solutes. In some embodiments, the antibody is purified to (1) greater than 95% by weight (e.g., determined by the Lowry method), and in some embodiments, greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence (e.g., using a rotary cup sequencer); or (3) homogenized (by SDS-PAGE under reducing or non-reducing conditions, using, for example, Coomassie blue or silver staining). Isolated antibodies include recombinant intracellular in situ antibodies, since at least one component of the antibody's natural environment will be absent. However, typically, isolated antibodies are prepared by at least one purification step.

[0047] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. Each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, from the N-terminus to the C-terminus, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain.

[0048] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to another portion of the immunoglobulin (i.e., the variable domain, which contains the antigen-binding site). The constant domain comprises the heavy chain CH1, CH2, and CH3 domains (collectively referred to as CH) and the light chain CL domain.

[0049] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain is called "VH," and the variable domain of the light chain is called "VL." These domains are typically the most variable parts of an antibody and contain antigen-binding sites.

[0050] The term "variability" refers to the fact that certain portions of a variable domain differ significantly in sequence between antibodies and are used for the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the variable domains of an antibody. It is concentrated in three segments called hypervariable regions (HVRs) in the variable domains of the light and heavy chains. The more conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FRs, which are predominantly β-sheet structures linked by three HVRs that form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in the binding of antibodies to antigens, but they have various effector functions, such as the role of antibodies in antibody-dependent cytotoxicity.

[0051] The “light chain” of antibodies (immunoglobulins) from any mammalian species, based on the amino acid sequence of their constant structural domains, can be classified into one of two distinct types, referred to as Kappa (“κ”) and Lambda (“λ”).

[0052] In this invention, the term "H chain" refers to the heavy chain of the antibody, and the term "L chain" refers to the light chain of the antibody.

[0053] In this invention, the term "common L-chain" refers to an L-chain capable of linking to two or more different H-chains and exhibiting binding capacity for each antigen. Preferably, the term "different H-chains" refers to, but is not limited to, the H-chains of antibodies against different antigens, and also refers to H-chains whose amino acid sequences differ from each other. For example, a common L-chain can be obtained according to the method described in WO 2006 / 109592.

[0054] Regarding the L chain, since the diversity of the variable regions of the L chain is lower than that of the variable regions of the H chain, a common L chain capable of conferring binding ability to both H chains can be obtained. The antibodies of the present invention can be different, but preferably have a common L chain.

[0055] As used herein, the term IgG “isotype” or “subclass” refers to any subclass of immunoglobulins defined by the chemical and antigenic characteristics of the immunoglobulin constant region.

[0056] Antibodies (immunoglobulins) can be classified into different categories based on the amino acid sequence of their heavy chain constant domains. Immunoglobulins are mainly divided into five classes: IgA, IgD, IgE, IgG, and IgM, and some of them can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, γ, α, γ, and µ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known and are generally described in references such as the following: Abbas et al., *Cellular and Molecular Immunology*, Vol. 4. Version (WB Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by the covalent or non-covalent association of an antibody with one or more other proteins or peptides.

[0057] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably herein and refer to antibodies in their substantially complete form rather than antibody fragments as defined below. These terms specifically refer to antibodies having a heavy chain containing an Fc region.

[0058] Furthermore, the antibodies of the present invention may include not only whole antibodies, but also antibody fragments, low molecular weight antibodies (microantibodies), and modified antibodies.

[0059] An "antibody fragment" comprises a portion of a complete antibody, preferably including its antigen-binding region. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bisomatic antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0060] Papain digests antibodies to produce two identical antigen-binding fragments, called "Fab" fragments. Each fragment has a single antigen-binding site and a residual "Fc" fragment, the name reflecting its tendency to crystallize. Treatment with pepsin or IdeS produces the F(ab')2 fragment, which has two antigen-binding sites and can still cross-link with the antigen.

[0061] An “Fv” is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, a double-stranded Fv species consists of a dimer of a tightly and non-covalently associated heavy chain and a light chain variable region. In a single-stranded Fv (scFv) species, a heavy chain variable region domain and a light chain variable region domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate into a “dimer” structure similar to that in the double-stranded Fv species. In this configuration, the three HVRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable region (or half of an Fv containing only three antigen-specific HVRs) can recognize and bind antigens, although with a lower affinity than a complete binding site.

[0062] The Fab fragment contains a variable domain in the heavy chain and a variable domain in the light chain, as well as a constant domain in the light chain and a first constant domain (CH1) in the heavy chain. The Fab' fragment differs from the Fab fragment in that it has residues added to the carboxyl terminus of the CH1 domain in the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the designation used herein for Fab' fragments in which the cysteine ​​residues in the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally conceived as a pair of Fab' fragments containing a hinge cysteine ​​residue. Other chemical conjugations of antibody fragments are also known.

[0063] A "single-chain Fv" or "scFv" antibody fragment contains the antibody's VH and VL domains, which are contained within a single polypeptide chain. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, allowing the scFv to form the desired antigen-binding structure. For a review of scFv, see, for example, Plückthun, *The Pharmacology of Monoclonal Antibodies*, Vol. 113, eds. Rosenburg and Moore (Springer-Verlag, New York, 1994), pp. 269-315.

[0064] The term "bisomatic antibody" refers to an antibody fragment having two antigen-binding sites, comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with a complementary domain of the other chain, resulting in two antigen-binding sites. Bisomatic antibodies can be bivalent or bispecific antibodies. Bisomatic antibodies are described more comprehensively in, for example: EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Hudson et al. also described trisomatic and tetrasomatic antibodies in Nat. Med. 9:129-134 (2003).

[0065] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, for example, a group of antibodies comprising identical individual antibodies except for a small number of possible mutations, such as naturally occurring mutations. Therefore, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies typically comprise antibodies containing a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a set of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence can be further modified, for example, to increase affinity for the target, humanize the target-binding sequence, increase its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target-binding sequences are also monoclonal antibodies of the present invention. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally not contaminated by other immunoglobulins.

[0066] The modifier “monoclonal” indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody used according to the invention can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Köhler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, Vol. 2). (1988); Hammerling et al., in the following literature: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, US Pat. No. 4,816,567), phage display technology (see, for example, Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004)), and techniques for generating human antibodies or human-like antibodies in animals having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, for example, WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year inImmunol. 7:33 (1993); US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol.14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0067] The monoclonal antibodies described herein specifically include “chimeric” antibodies, wherein a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, and fragments of such antibodies, provided they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies, wherein the antigen-binding region of the antibody is derived from an antibody produced, for example, by immunizing a rhesus monkey with a target antigen.

[0068] The “humanized” form of a non-human (e.g., mouse) antibody is a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (receptor antibody), wherein residues from the receptor HVR are replaced by residues from the HVR of a non-human species (donor antibody), such as mouse, rat, rabbit, or a non-human primate having the desired specificity, affinity, and / or ability. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not present in the receptor antibody or donor antibody. These modifications can be made to further improve antibody performance. Generally, the humanized antibody will substantially comprise at least one of the variable domains, typically two variable domains, wherein all or substantially all hypervariable loops correspond to hypervariable loops of the non-human immunoglobulin, and all or substantially all FRs are FRs of the human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of the immunoglobulin constant region (Fc), which is typically a human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Also see, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0069] "Human antibody" is an antibody having an amino acid sequence corresponding to antibodies produced by the human body and / or an antibody prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Methods also available for preparing human monoclonal antibodies include those described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been deactivated, for example, immunized xenogeneic mice (see, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 concerning XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) concerning human antibodies produced via human B-cell hybridoma technology.

[0070] "Species-dependent antibodies" are antibodies that have a stronger binding affinity for antigens from a first mammalian species than for homologs of that antigen from a second mammalian species. Typically, species-dependent antibodies have a binding affinity (Kd) value of no more than about 1 × 10⁻⁶ for human antigens (e.g., human antigens). -7 M, preferably not exceeding about 1×10 -8 M and preferably not exceeding about 1×10 -9 M) “Specific binding”, but having a binding affinity for homologs of antigens from a second non-human mammal species that is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than their binding affinity for human antigens. Species-dependent antibodies may be any of the various antibodies defined above, but are preferably humanized antibodies or human antibodies.

[0071] IgG bispecific antibodies can be secreted from hybridomas (tetramammals) produced by fusing two types of hybridomas that produce IgG antibodies (Milstein C et al., Nature 1983, 305: 537-540). They can also be secreted by acquiring the L-chain and H-chain genes (a total of four genes) that constitute two target IgGs and introducing them into the cell for co-expression of these genes. In this case, by introducing appropriate amino acid substitutions into the CH3 region of these H chains, heterologous combinations of IgGs with H chains can be preferentially secreted (Ridgway JB et al., Protein Engineering, 1996, 9:617-621; Merchant AM et al., Nature Biotechnology, 1998, 16:677-681; WO 2006 / 106905; Davis JH et al., Protein Eng Des Sel. 2010, 4:195-202).

[0072] As used herein, the terms “hypervariant region,” “HVR,” or “HV” refer to regions of antibody variable domains that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the greatest diversity among the six HVRs, with H3, in particular, considered to play a unique role in conferring fine specificity to antibodies. See, for example: Xu et al., Immunity 13:37–45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1–25 (Lo ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, for example: Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol.3:733-736 (1996).

[0073] Many HVR descriptions are applied and are included in this paper. Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, p. 5). Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Conversely, Chothia refers to the location of the structural ring (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and the Chothia structural ring and is used by the AbM antibody modeling software of Oxford Molecular. “Contact” HVR is based on the analysis of available complex crystal structures. The residues in each of these HVRs are described below.

[0074]

[0075] HVR may include the following “extended HVRs”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to the method described above by Kabat et al.

[0076] HVR may include the following “extended HVRs”: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to the method described above by Kabat et al.

[0077] “Frame” or “FR” residues are those variable domain residues other than the HVR residues defined in this paper.

[0078] The terms “Kabat-described variable domain residue numbering” or “Kabat-described amino acid position numbering” and their variations refer to the numbering system for heavy chain or light chain variable domains used in antibody compilation in the aforementioned Kabat et al. literature. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, corresponding to shortening or insertion of the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat numbering) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat numbering). The Kabat number of residues for a given antibody can be determined by comparing the antibody sequence with homologous regions of a “standard” Kabat-numbered sequence.

[0079] When referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al., Sequences of Immunological Interest. No. 5). (Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is usually used (e.g., the EU index reported by Kabat et al. above; Edelman et al., (1969), Proc Natl Acad Sci USA 63:78-85). The “EU index described by Kabat” refers to the residue numbering of human IgG1 EU antibodies.

[0080] As used herein, the terms “binding,” “specifically bound to,” or “specific to” refer to a measurable and reproducible interaction, such as the binding between a target and an antibody, in the presence of a heterogeneous population of molecules (including biomolecules) that determines the presence of the target. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to the target with greater affinity, affinity, ease, and / or longer duration of binding compared to binding to other targets. In one embodiment, the degree of binding of an antibody to an irrelevant target is less than about 10%, for example, as measured by radioimmunoassay (RIA). In some embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, or ≤ 0.1 nM. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

[0081] A “functional Fc region” possesses the “effective functions” of the native Fc region. Exemplary “effective functions” include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions typically require the Fc region in combination with a binding domain (e.g., antibody variable domain) and can be assessed using various assays, such as those disclosed in the definitions herein.

[0082] As used herein, “amino acid sequence identity percentage (%)” and “homology” relative to peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the specific peptide or polypeptide sequence after aligning the candidate sequence with the specific peptide or polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum sequence identity percentage, without taking into account any conserved substitutions as part of the sequence identity. Alignments used to determine the amino acid sequence identity percentage can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0083] II. Bispecific antibodies

[0084] This invention relates to a multispecific antigen-binding molecule (i.e., a bispecific antibody) that recognizes FIX and / or FIXa and FX, and functionally replaces the cofactor function of FVIII. The bispecific antibody comprises a first polypeptide (containing an antigen-binding site recognizing FIX and / or activated FIX (FIXa)) and a third polypeptide (containing an antigen-binding site recognizing FIX and / or activated FIX (FIXa), a second polypeptide (containing an antigen-binding site recognizing FX), and a fourth polypeptide (containing an antigen-binding site recognizing FX). The first and third polypeptides, as well as the second and fourth polypeptides, each contain an antigen-binding site on the antibody's H (heavy) chain and an antigen-binding site on the antibody's L (light) chain. For example, in one bispecific antibody of this invention, the first and third polypeptides respectively contain antigen-binding sites on the H (heavy) chain and L (light) chain of the antibody against FIX or FIXa; and the second and fourth polypeptides respectively contain antigen-binding sites on the H chain and L chain of the antibody against FX. In some embodiments, the antigen-binding sites of the antibody L chains contained in the first and third polypeptides, as well as the second and fourth polypeptides, may be common L chains.

[0085] The multispecific antigen-binding molecules (preferably bispecific antibodies) of the present invention are antibodies having specificity against two or more different antigens, or molecules comprising fragments of such antibodies. The antibodies of the present invention are not particularly limited, but are preferably monoclonal antibodies. The monoclonal antibodies used in the present invention include not only monoclonal antibodies derived from animals (such as humans, mice, rats, hamsters, rabbits, sheep, camels, and monkeys), but also artificially modified recombinant antibodies, such as chimeric antibodies, humanized antibodies, and bispecific antibodies.

[0086] Preferably, the antibody of the present invention is a recombinant antibody produced using gene recombination technology (see, for example, Borrebaeck CAK and Larrick JW, THERAPEUTIC MONOCLONAL ANTIBODIES, published by MACMILLAN PUBLISHERS LTD in the UK, 1990). Recombinant antibodies can be obtained by cloning antibody-encoding DNA from a hybridoma or antibody-producing cell (such as sensitized lymphocytes), inserting this DNA into a suitable vector, and then introducing this DNA into a host (host cell) to produce the antibody.

[0087] In some embodiments, the bispecific antibody is emecizumab or a biosimilar thereof. Emecizumab (also known as RO5534262 and ACE910) is a recombinant, humanized, bispecific immunoglobulin G4 (IgG4) monoclonal antibody that binds to activated factor IX (FIXa) and factor X (FX) with moderate affinity, thereby mimicking the cofactor function of factor VIII (FVIII). In patients with hemophilia A, hemostasis can be restored regardless of the presence or absence of FVIII inhibitors because emecizumab has no sequence homology with FVIII. In some embodiments, the first polypeptide of the bispecific antibody of the present invention comprises a heavy (H) chain variable region (VH1), the VH1 comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 3; CDR2 comprises the amino acid sequence of SEQ ID NO: 4; and CDR3 comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the second polypeptide of the bispecific antibody of the present invention comprises a heavy chain variable region (VH2), the VH2 comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 8; CDR2 comprises the amino acid sequence of SEQ ID NO: 9; and CDR3 comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the third and / or fourth polypeptide of the bispecific antibody of the present invention comprises a light chain variable region (VL), the VL comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 comprises the amino acid sequence of SEQ ID NO: 13; CDR2 comprises the amino acid sequence of SEQ ID NO: 14; and CDR3 comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the H chain variable region of the first polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the H chain variable region of the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the L chain of the third polypeptide and the L chain of the fourth polypeptide comprise the same amino acid sequence. In some embodiments, the L chain of the third polypeptide and the L chain of the fourth polypeptide comprise the same L chain variable region, each L chain variable region comprising the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the amino acid sequence of the full-length H chain of the first polypeptide is as shown in SEQ ID NO: 1.In some embodiments, the amino acid sequence of the full-length H chain of the second polypeptide is shown in SEQ ID NO: 6. In other embodiments, the amino acid sequences of each of the full-length L chains of the same third polypeptide and the fourth polypeptide are shown in SEQ ID NO: 11. In some embodiments, the bispecific antibody comprises a first polypeptide (containing the amino acid sequence of SEQ ID NO: 1), a second polypeptide (containing the amino acid sequence of SEQ ID NO: 6), and the same third and fourth polypeptides (each containing the amino acid sequence of SEQ ID NO: 11). Emecizumab is further described in WO2012 / 067176 and US 9,334,331, both of which are incorporated herein by reference in their entirety.

[0088] III. Pharmaceutical Composition

[0089] This document also provides pharmaceutical compositions and formulations, for example, for treating the conditions described herein (e.g., hemophilia A). In some embodiments, the pharmaceutical compositions and formulations further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions and formulations comprise an anti-FIXa× anti-FX antibody, such as emecizumab or a biosimilar thereof, as described herein.

[0090] Following the preparation of the anti-FIXa× anti-FX antibody (such as emecizumab or its biosimilar), a pharmaceutical formulation comprising said antibody is prepared. The therapeutically effective amount of the antibody present in the formulation is determined, for example, by taking into account the required dose volume and method of administration. In some embodiments, the anti-FIXa× anti-FX antibody is formulated at a concentration from about 25 mg / mL to about 400 mg / mL, for example, from about 25 mg / mL to about 400 mg / mL, or from about 50 mg / mL to about 350 mg / mL, or from about 35 mg / mL to about 300 mg / mL, or from about 40 mg / mL to about 250 mg / mL, or from about 45 mg / mL to about 200 mg / mL, or from about 25 mg / mL to about 150 mg / mL, or from about 30 mg / mL to about 140 mg / mL, or from about 35 mg / mL to about 130 mg / mL, or from about 50 mg / mL to about 125 mg / mL, or from about 50 mg / mL to about 120 mg / mL, or from about 50 mg / mL to about 120 mg / mL. The concentration is approximately 110 mg / mL, or from approximately 50 mg / mL to approximately 100 mg / mL, or from approximately 50 mg / mL to approximately 90 mg / mL, or from approximately 50 mg / mL to approximately 80 mg / mL, or from approximately 54 mg / mL to approximately 66 mg / mL. In some embodiments, the anti-FIXa× anti-FX antibody is formulated at any concentration of approximately 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, 250 mg / mL, 275 mg / mL, 300 mg / mL, 325 mg / mL, 350 mg / mL, 375 mg / mL, or 400 mg / mL.

[0091] Pharmaceutical compositions for therapeutic or preventative purposes contain the antibodies of the present invention as active ingredients. These pharmaceutical compositions can be formulated (if necessary) by mixing with suitable pharmaceutically acceptable carriers, mediators, etc., that are inactive with the antibody. For example, sterile water, physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphates, citrates, histidines, and other organic acids), preservatives, surfactants (such as PEG and Tween), chelating agents (such as EDTA), and binders can be used. They may also contain other low molecular weight peptides, proteins such as serum albumin, gelatin, and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine, and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol and sorbitol. When preparing aqueous solutions for injection, physiological saline and isotonic solutions containing glucose such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and other adjuvants, can be used, and if necessary, in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyols (such as propylene glycol and PEG), and nonionic surfactants (such as polysorbate 80, polysorbate 20, poloxamer 188, and HCO-50). Larger volumes of fluid can be administered subcutaneously by incorporating hyaluronidase into the formulation (Expert Opin Drug Deliv. July 2007; 4(4): 427-40).

[0092] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which comprises a histidine-acetate buffer. For example, Strickley and Lambert (2021), J Pharm Sci 110:2590-2608e2556, which is hereby incorporated herein by reference in its entirety for further description of suitable antibody formulations.

[0093] The compositions and formulations described herein may also contain more than one active ingredient essential for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in a combination of amounts effective for the intended purpose.

[0094] The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by, for example, cohesive techniques or interfacial polymerization; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, Issue 16. Edition, Osol, A. Edited (1980).

[0095] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, which is in the form of a molded article, such as a membrane or microcapsule. Formulations intended for in vivo administration are typically sterile. Sterility can be readily achieved, for example, through filtration via a sterile filter membrane. Methods for preparing pharmaceutical preparations as sustained-release pharmaceutical preparations are also well known, and such methods can be applied to the antibodies of this invention (Langer et al., J. Biomed. Mater. Res. 15: 267-277 (1981); Langer, Chemtech. 12: 98-105 (1982); US Pat. No. 3,773,919; European Patent Publication No. EP 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP 133,988).

[0096] IV. Treatment methods for hemophilia A

[0097] This article provides a method for treating hemophilia A, wherein a bispecific antibody (e.g., emecizumab or a biosimilar thereof) is administered to an individual diagnosed with hemophilia A (e.g., a human) that binds to coagulation factor IX and / or activated coagulation factor IX, and to coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain containing a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) the same first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively. And 3; and wherein the hemoglobin level in the individual is increased compared to baseline. In some embodiments, the variable region of the first polypeptide H chain contains the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variable region of the second polypeptide H chain contains the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the third polypeptide L chain and the fourth polypeptide L chain contain the same sequence. In some embodiments, the third polypeptide L chain and the fourth polypeptide L chain contain the same L chain variable region, and the variable region of each L chain contains the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the amino acid sequence of the full-length H chain of the first antibody is as shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the full-length H chain of the second antibody is as shown in SEQ ID NO: 6. In other embodiments, the amino acid sequence of each of the same full-length L chain of the third polypeptide and the fourth full-length L chain is as shown in SEQ ID NO: 11. In some embodiments, the bispecific antibody comprises a first polypeptide (containing the amino acid sequence of SEQ ID NO: 1), a second polypeptide (containing the amino acid sequence of SEQ ID NO: 6), and a third polypeptide and a fourth polypeptide (each containing the amino acid sequence of SEQ ID NO: 11).

[0098] In some embodiments, hemophilia A is severe. In some embodiments, hemophilia A is moderate. In some embodiments, hemophilia A is mild.

[0099] This article also provides a method for increasing hemoglobin levels in an individual diagnosed with hemophilia A (e.g., a human), comprising administering to the individual diagnosed with hemophilia A a bispecific antibody (e.g., emecizumab or a biosimilar thereof) that binds to coagulation factor IX and / or activated coagulation factor IX, and to coagulation factor X, wherein the bispecific antibody comprises: (i) a first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4, and 5, respectively; (ii) a second antibody H chain containing a variable region comprising CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and (iii) the same first antibody L chain and the second antibody L chain, each L chain comprising a variable region comprising CDRs comprising the amino acid sequences shown in SEQ ID NO: 13, 14, and 15, respectively. 1, 2, and 3; and wherein the hemoglobin level in the individual is increased compared to baseline. In some embodiments, the variable region of the first polypeptide H chain comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variable region of the second polypeptide H chain comprises the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the third polypeptide L chain and the fourth polypeptide L chain comprise the same sequence. In some embodiments, the same variable region of the first antibody L chain and the same variable region of the second antibody L chain each comprise the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the amino acid sequence of the full-length H chain of the first antibody is as shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the full-length H chain of the second antibody is as shown in SEQ ID NO: 6. The amino acid sequences of the same full-length L chain of the first antibody and the same full-length L chain of the second antibody are each as shown in SEQ ID NO: 11. In some embodiments, the bispecific antibody comprises a first polypeptide (containing the amino acid sequence of SEQ ID NO: 1), a second polypeptide (containing the amino acid sequence of SEQ ID NO: 6), and a third polypeptide and a fourth polypeptide (each containing the amino acid sequence of SEQ ID NO: 11).

[0100] In some embodiments, hemophilia A is severe. In some embodiments, hemophilia A is moderate. In some embodiments, hemophilia A is mild. In some embodiments, the human subject or patient is an adolescent or an adult.

[0101] In some embodiments, the treatment is acute exacerbation treatment. In other embodiments, the treatment is preventative treatment. In other embodiments, the treatment consists of both acute exacerbation treatment and preventative treatment. In other embodiments, the treatment includes both acute exacerbation treatment and preventative treatment. In other embodiments, the treatment further includes treatment of hemophilia with one or more therapeutic agents.

[0102] Patient Population

[0103] In some embodiments, the individual is a person. In some embodiments, the individual is a human subject or patient. In some embodiments, the individual is an adolescent or an adult. In some embodiments, the individual has been, is being, or will be diagnosed with hemophilia A. In some embodiments, hemophilia A is severe. In some embodiments, hemophilia A is moderate. In some embodiments, hemophilia A is mild.

[0104] In some embodiments, the individual's age is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 years of age or older. In other embodiments, the individual's age is between 12-15, 16-18, 19-21, 22-23, 24-26, 27-29, or older than 29 years of age.

[0105] i. Hemoglobin Levels

[0106] Hemoglobin (Hb or hgb) is a protein in red blood cells responsible for transporting oxygen to tissues. Normal hemoglobin levels vary with age, and are generally around 110-160 g / L for children, 120-150 g / L for non-pregnant adult women, and 135-180 g / L for adult men. Patients with hemophilia A may have lower than normal hemoglobin levels.

[0107] In some embodiments, individuals exhibit baseline hemoglobin levels of less than about 250 g / L. For example, in some embodiments, individuals exhibit baseline hemoglobin levels of less than any of the following: 240 g / L, 230 g / L, 220 g / L, 210 g / L, 200 g / L, 190 g / L, 180 g / L, 170 g / L, 160 g / L, 150 g / L, 140 g / L, 130 g / L, 120 g / L, 110 g / L, 100 g / L, 90 g / L, 80 g / L, 75 g / L, 70 g / L, 65 g / L, 60 g / L, 55 g / L, 50 g / L. In some embodiments, individuals exhibit baseline hemoglobin levels between about 50 g / L and about 250 g / L, for example, about 50 g / L to about 70 g / L, about 60 g / L to about 80 g / L, about 70 g / L to about 90 g / L, about 80 g / L to about 100 g / L, about 90 g / L to about 110 g / L, about 100 g / L to about 120 g / L, about 110 g / L to about 130 g / L, about 120 g / L to about 140 g / L, about 130 g / L to about 150 g / L, about 140 g / L to about 160 g / L, about 150 g / L to about 170 g / L, about 160 g / L to about 180 g / L, about 170 g / L to about 190 g / L, about 180 g / L to about 200 g / L. g / L, about 190 g / L to about 210 g / L, about 200 g / L to about 220 g / L, about 210 g / L to about 230 g / L, about 220 g / L to about 240 g / L, about 230 g / L to about 250 g / L, about 50 g / L to about 85 g / L, about 75 g / L to about 105 g / L, about 85 g / L to about 125 g / L, about 115 g / L to about 175 g / L, about 140 g / L to about 220 g / L, about 150 g / L to about 250 g / L, about 200 g / L to about 235 g / L, or about 215 g / L to about 250 g / L.

[0108] In some embodiments, the hemoglobin level in individuals treated with the bispecific antibody described herein increases by at least one or more of about 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, 45 g / L, and 50 g / L compared to baseline hemoglobin levels. In some embodiments, the hemoglobin level in individuals treated with the bispecific antibody described herein increases by about 5 g / L to about 10 g / L, about 10 g / L to about 15 g / L, about 15 g / L to about 20 g / L, about 20 g / L to about 25 g / L, about 25 g / L to about 30 g / L, about 30 g / L to about 35 g / L, about 35 g / L to about 40 g / L, about 40 g / L to about 45 g / L, about 45 g / L to about 50 g / L, or more compared to baseline hemoglobin levels.

[0109] In some embodiments, the hemoglobin level is at least about 80 g / L following administration of the initial dose of the bispecific antibody described herein. In another embodiment, the hemoglobin level is at least about 80 g / L at least two weeks, at least four weeks, at least six weeks, at least eight weeks, or at least ten weeks after administration of the initial dose of the bispecific antibody described herein. For example, at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, or at least twelve weeks after administration of the initial dose of the bispecific antibody described herein, the hemoglobin level is approximately 80 g / L to approximately 100 g / L, approximately 90 g / L to approximately 110 g / L, approximately 100 g / L to approximately 120 g / L, approximately 110 g / L to approximately 130 g / L, approximately 120 g / L to approximately 140 g / L, approximately 130 g / L to approximately 150 g / L, approximately 140 g / L to approximately 160 g / L, approximately 150 g / L to approximately 170 g / L, approximately 160 g / L to approximately 180 g / L, approximately 170 g / L to approximately 190 g / L, approximately 180 g / L to approximately 200 g / L, approximately 190 g / L to approximately 210 g / L, approximately 200 g / L to approximately 220 g / L. g / L, about 210 g / L to about 230 g / L, about 220 g / L to about 240 g / L, about 230 g / L to about 250 g / L, about 75 g / L to about 105 g / L, about 85 g / L to about 125 g / L, about 115 g / L to about 175 g / L, about 140 g / L to about 220 g / L, about 150 g / L to about 250 g / L, about 200 g / L to about 235 g / L, or about 215 g / L to about 250 g / L.

[0110] In some embodiments, the hemoglobin level is at least about 100 g / L after administration of the initial dose of the bispecific antibody described herein. In another embodiment, the hemoglobin level is at least about 100 g / L at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least twelve weeks, at least sixteen weeks, at least twenty weeks, at least twenty-four weeks, at least twenty-eight weeks, at least thirty weeks, at least forty weeks, at least fifty weeks, at least sixty weeks, or at least eighty weeks after administration of the initial dose of the bispecific antibody described herein. For example, at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least twelve weeks, at least sixteen weeks, at least twenty weeks, at least twenty-five weeks, at least thirty weeks, at least forty weeks, at least fifty weeks, at least sixty weeks, at least seventy weeks, or at least eighty weeks after administration of the initial dose of the bispecific antibody described herein, the hemoglobin level is about 100 g / L to about 120 g / L, about 110 g / L to about 130 g / L, about 120 g / L to about 140 g / L, about 130 g / L to about 150 g / L, about 140 g / L to about 160 g / L, about 150 g / L to about 170 g / L, about 160 g / L to about 180 g / L, about 170 g / L to about 190 g / L, about 180 g / L to about 200 g / L, about 190 g / L to about 210 g / L, or about 200 g / L. From about 220 g / L, from about 210 g / L to about 230 g / L, from about 220 g / L to about 240 g / L, from about 230 g / L to about 250 g / L, from about 115 g / L to about 175 g / L, from about 140 g / L to about 220 g / L, from about 150 g / L to about 250 g / L, from about 200 g / L to about 235 g / L, or from about 215 g / L to about 250 g / L.

[0111] In some embodiments, the hemoglobin level is at least about 120 g / L after administration of the initial dose of the bispecific antibody described herein. In another embodiment, the hemoglobin level is at least about 120 g / L at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least twelve weeks, at least sixteen weeks, at least twenty weeks, at least twenty-four weeks, at least twenty-eight weeks, at least thirty weeks, at least forty weeks, at least fifty weeks, at least sixty weeks, or at least eighty weeks after administration of the initial dose of the bispecific antibody described herein. For example, at least two weeks, at least four weeks, at least six weeks, at least eight weeks, at least ten weeks, at least twelve weeks, at least sixteen weeks, at least twenty weeks, at least twenty-five weeks, at least thirty weeks, at least forty weeks, at least fifty weeks, at least sixty weeks, at least seventy weeks, or at least eighty weeks after administration of the initial dose of the bispecific antibody described herein, the hemoglobin level is about 100 g / L to about 120 g / L, about 110 g / L to about 130 g / L, about 120 g / L to about 140 g / L, about 130 g / L to about 150 g / L, about 140 g / L to about 160 g / L, about 150 g / L to about 170 g / L, about 160 g / L to about 180 g / L, about 170 g / L to about 190 g / L, about 180 g / L to about 200 g / L, about 190 g / L to about 210 g / L, or about 200 g / L. From about 220 g / L, from about 210 g / L to about 230 g / L, from about 220 g / L to about 240 g / L, from about 230 g / L to about 250 g / L, from about 115 g / L to about 175 g / L, from about 140 g / L to about 220 g / L, from about 150 g / L to about 250 g / L, from about 200 g / L to about 235 g / L, or from about 215 g / L to about 250 g / L.

[0112] ii. FVIII Inhibitors

[0113] The development of neutralizing antibodies (inhibitors) against factor VIII (FVIII) or factor IX (FIX) is the most significant complication of hemophilia treatment, occurring in up to 33% of patients with severe hemophilia A and 13% of patients with non-severe hemophilia A (see, for example, Kempton and Meeks (2014), Blood 124(23):3365-3372). Inhibitors have an even higher incidence and prevalence in patients with severe hemophilia A.

[0114] In some embodiments, the individual's bloodstream contains factor VIII inhibitors. In some embodiments, the individual's bloodstream does not contain factor VIII inhibitors. In some embodiments, the presence of factor VIII inhibitors in the bloodstream does not affect the efficacy of the bispecific antibody described herein (e.g., emecizumab or its biosimilar), or has only a minimal effect on the efficacy of the bispecific antibody.

[0115] In some embodiments, individuals with factor VIII inhibitors in their bloodstream may have previously received or are currently receiving bypass therapy. In some embodiments, bypass agents may have been previously administered or are currently administered based on incidental bleeding or as a preventative measure against future bleeding episodes. Bypass agents treat bleeding by generating thrombin via a pathway that does not require FVIII or FIX. Currently available bypass agents include recombinant factor VIIa (rFVIIa, Novoseven RT; NovoNordisk) and activated prothrombin complex concentrate (aPCC, FEIBAVH; Baxter; see, for example, Kempton and Meeks (2014), Blood 124(23): 3365-3372).

[0116] iii. Additional Indicators

[0117] Hematocrit is defined as the fractional volume of red blood cells (RBCs) in whole blood, expressed as a percentage. Normal hematocrit levels vary with age, sex, and ethnicity. For example, in adult males, the normal range is approximately 41%–50%. In adult females, the normal range is approximately 36%–44%. Hematocrit levels below the normal range may indicate various conditions, such as anemia. In some embodiments, the hematocrit level at baseline is less than approximately 50%, for example, less than any or less of approximately 45%, 40%, 35%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, and 20%. In some embodiments, the hematocrit level is further measured as a percentage. In some embodiments, the hematocrit level in individuals treated with the bispecific antibody described herein increases by approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% or more compared to baseline levels.

[0118] Mean corpuscular volume (MCV) measures the average size and volume of red blood cells. It is useful in helping to determine the cause of anemia. The MCV value is calculated by multiplying the percentage of hematocrit by 10 and then dividing by the red blood cell count. In some embodiments, it is further multiplied by 10. 12 / L measures the mean corpuscular volume (MCV) level. In some embodiments, the MCV of red blood cells in individuals administered the bispecific antibody described herein increases by approximately 1 fL, 2 fL, 3 fL, 4 fL, 5 fL, 6 fL, 7 fL, 8 fL, 9 fL, 10 fL, 11 fL, 12 fL, 13 fL, 14 fL, or 15 fL compared to baseline levels.

[0119] Red blood cells carry hemoglobin, which binds to oxygen and is responsible for transporting oxygen to tissues throughout the body. For adult males, the normal range for red blood cells is typically approximately 4.35 million to 5.65 million per microliter (mL) of blood. For adult females, the normal range for red blood cells is typically approximately 3.92 million to 5.13 million per microliter (mL) of blood. In some embodiments, the red blood cell count (RBC) is further measured as a percentage. In some embodiments, the red blood cell count in individuals treated with the bispecific antibody described herein increases by approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% or more compared to baseline levels.

[0120] Red blood cell distribution width (RDW) measures the variation in the volume and size of red blood cells. A high RDW indicates increased variability in red blood cell size compared to normal. A high RDW can indicate anemia or related conditions. In some embodiments, the level of red blood cell distribution width is further measured as a percentage. In some embodiments, the level of red blood cell distribution width in individuals treated with the bispecific antibody described herein is increased by approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% or more compared to baseline levels.

[0121] iv. Other Treatments

[0122] In some embodiments, treatment includes further therapeutic agents for treating hemophilia A. Such therapeutic agents include, but are not limited to, FVIII replacement therapies, such as plasma-derived factor VIII concentrates or recombinant FVIII concentrates. Other therapeutic agents may include, for example, desmopressin acetate and ε-aminocaproic acid.

[0123] Such therapeutic agents may be administered before, during, or after treatment according to the methods described herein.

[0124] Patient Dosing

[0125] In some embodiments, the bispecific antibody described herein (e.g., emecizumab or its biosimilar) is administered as a prophylactic treatment. In some embodiments, the bispecific antibody is administered subcutaneously. In some embodiments, the bispecific antibody is administered intravenously.

[0126] In some embodiments, the bispecific antibody is administered at an initial weekly dose for at least about one week or more. In some embodiments, the initial dose is or is about 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, or more. In some embodiments, the bispecific antibody is administered at an initial weekly dose of about 1.5 mg / kg for at least about one week or more. In other embodiments, the bispecific antibody is administered at an initial weekly dose of about 3 mg / kg for at least about one week or more. In some embodiments, the bispecific antibody is administered at an initial weekly dose of about 6 mg / kg for at least about one week or more. For example, the bispecific antibody may be administered at an initial dose of approximately 1.5 mg / kg, 3 mg / kg, or 6 mg / kg per week for at least one week, two weeks, three weeks, four weeks, five weeks, six weeks, or longer. In a particular embodiment, the bispecific antibody may be administered at an initial dose of approximately 3 mg / kg per week for approximately four weeks.

[0127] In some embodiments, the bispecific antibody is further administered as part of a maintenance regimen. A maintenance dose is administered to maintain the therapeutic concentration of the therapeutic drug, i.e., the therapeutic concentration of the bispecific antibody described herein. In some embodiments, the maintenance dose is or is about 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, or more. The maintenance dose may be administered periodically, such as once weekly, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, or for longer periods. In some embodiments, the maintenance dose is administered periodically for at least about 12 weeks, including, for example, at least about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, etc. Any time period or longer, including 1 month, 16 months, 18 months, 20 months, 22 months, 24 months, 26 months, 28 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years.

[0128] In some embodiments, the bispecific antibody is administered at a maintenance dose of 1.5 mg / kg weekly for at least about 12 weeks, at least about 24 weeks, or at least about 52 weeks. In some embodiments, the bispecific antibody is administered at a maintenance dose of 3 mg / kg every two weeks for at least about 12 weeks, at least about 24 weeks, or at least about 52 weeks. In some embodiments, the bispecific antibody is administered at a maintenance dose of 6 mg / kg every four weeks for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

[0129] Alternative forms of the treatment methods are provided along with the description of the methods. For example, in some embodiments, a bispecific antibody, such as emecizumab or a biosimilar thereof, as described herein, is provided for use in a method of increasing hemoglobin levels in an individual diagnosed with hemophilia A. In some embodiments, a bispecific antibody, such as emecizumab or a biosimilar thereof, as described herein, is provided for use in manufacturing a medicament for increasing hemoglobin levels in an individual diagnosed with hemophilia A.

[0130] V. Reagent kits and products

[0131] This application further provides articles comprising the compositions described herein (such as pharmaceutical compositions) in suitable packaging. Suitable packaging for the compositions described herein (such as pharmaceutical compositions) is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, cans, flexible packaging (such as sealed polyester films or plastic bags), etc. These articles may be further sterilized and / or sealed.

[0132] This disclosure also provides a kit for use in the methods described above. In some embodiments, the kit comprises: i) a pharmaceutical composition comprising a bispecific antibody (such as emecizumab or a biosimilar thereof) as described herein; and ii) instructions for administering the pharmaceutical composition to a human subject suffering from a condition described herein (e.g., hemophilia A) by subcutaneous injection. In some embodiments, the pharmaceutical composition is an aqueous solution further comprising L-arginine hydrochloride, L-histidine, and L-histidine hydrochloride monohydrate. In some embodiments, the pharmaceutical composition further comprises sucrose. In some embodiments, the pharmaceutical composition is present in a pre-filled syringe. In some embodiments, the pharmaceutical composition is a lyophilized powder present in a vial, and the kit further includes instructions for reconstituted the lyophilized powder in water for injection.

[0133] Other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of various reagents can vary widely to provide the concentration of that reagent in solution, thereby substantially optimizing the antibody formulation. Specifically, these reagents may be provided in the form of a dry powder including excipients, typically lyophilized, which, once dissolved, provides a reagent solution with an appropriate concentration.

[0134] Exemplary embodiments

[0135] Example 1. A method comprising:

[0136] (a) Administering a bispecific antibody to an individual diagnosed with hemophilia A according to a treatment regimen, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody contains

[0137] (i) A first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively;

[0138] (ii) A second antibody H chain comprising a variable region containing CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and

[0139] (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and

[0140] (b) Increase the hemoglobin level in the individual compared to baseline.

[0141] Example 2. A method for treating hemophilia A, the method comprising:

[0142] Administering a bispecific antibody to an individual diagnosed with hemophilia A, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody contains...

[0143] (i) A first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively;

[0144] (ii) A second antibody H chain comprising a variable region containing CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and

[0145] (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and

[0146] Compared to baseline, the individual's hemoglobin level increased.

[0147] Example 3. A method for increasing hemoglobin levels in an individual diagnosed with hemophilia A, the method comprising:

[0148] Administering a bispecific antibody to an individual diagnosed with hemophilia A, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody contains...

[0149] (i) A first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively;

[0150] (ii) A second antibody H chain comprising a variable region containing CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and

[0151] (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and

[0152] Compared to baseline, the level of this hemoglobin in the individual increased.

[0153] Example 4. According to any one of Examples 1 to 3, the variable region of the first antibody H chain contains the amino acid sequence shown in SEQ ID NO: 2.

[0154] Example 5. According to any one of Examples 1 to 4, the variable region of the second antibody H chain contains the amino acid sequence shown in SEQ ID NO: 7.

[0155] Example 6. According to any one of Examples 1 to 5, the same first antibody L-chain variable region and the same second antibody L-chain variable region each contain the amino acid sequence shown in SEQ ID NO: 12.

[0156] Example 7.According to any one of Examples 1 to 6, the amino acid sequence of the full-length H chain of the first antibody is shown in SEQ ID NO: 1.

[0157] Example 8. According to any one of Examples 1 to 7, the amino acid sequence of the full-length H chain of the second antibody is as shown in SEQ ID NO: 6.

[0158] Example 9. According to any one of Examples 1 to 8, the amino acid sequences of the same full-length L chain of the first antibody and the full-length L chain of the second antibody are each shown as shown in SEQ ID NO: 11.

[0159] Example 10. According to any one of Examples 1 to 9, the bispecific antibody is administered at an initial dose of 3 mg / kg per week for one week or more weeks.

[0160] Example 11. According to the method described in Example 10, the bispecific antibody is administered at an initial dose of 3 mg / kg per week for four weeks.

[0161] Example 12. According to any one of Examples 1 to 11, the bispecific antibody is administered at a maintenance dose of 1.5 mg / kg per week for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

[0162] Example 13. According to any one of Examples 1 to 11, the bispecific antibody is administered at a maintenance dose of 3 mg / kg every two weeks for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

[0163] Example 14. According to any one of Examples 1 to 11, the bispecific antibody is administered at a maintenance dose of 6 mg / kg every 4 weeks for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

[0164] Example 15. The method according to any one of Examples 1 to 14, wherein the treatment is an acute phase treatment or a preventive treatment.

[0165] Example 16. According to any one of Examples 1 to 14, the treatment includes both acute-onset treatment and preventative treatment.

[0166] Example 17. The method according to any one of Examples 1 to 16, wherein the bloodstream of the individual contains factor VIII inhibitors.

[0167] Example 18. The method according to any one of Examples 1 to 16, wherein the bloodstream of the individual does not contain factor VIII inhibitors.

[0168] Example 19. The method according to any one of Examples 1 to 18, wherein the individual is at least 12 years old.

[0169] Example 20. According to any one of Examples 1 to 19, the baseline level of hemoglobin is between 80 g / L and 100 g / L.

[0170] Example 21. The method according to any one of Examples 1 to 19, wherein the baseline level of hemoglobin is between 100 g / L and 120 g / L.

[0171] Example 22. The method according to any one of Examples 1 to 19, wherein the baseline level of hemoglobin is between 120 g / L and 140 g / L.

[0172] Example 23. The method according to any one of Examples 1 to 19, wherein the baseline level of hemoglobin is between 140 g / L and 220 g / L.

[0173] Example 24. According to any one of Examples 1 to 23, the hemoglobin level increased by 5 g / L, 10 g / L or 15 g / L compared with the baseline level.

[0174] Example 25. According to any one of Examples 1 to 23, the hemoglobin level increased by 20 g / L, 25 g / L or 30 g / L compared with the baseline level.

[0175] Example 26. According to any one of Examples 1 to 23, the hemoglobin level increased by 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L or 25-30 g / L compared with the baseline level.

[0176] Example 27.According to any one of Examples 1 to 26, the hemoglobin level is 80-100 g / L, 100-120 g / L, or 120-140 g / L at least four weeks after the administration of the initial dose.

[0177] Example 28. According to any one of Examples 1 to 26, the hemoglobin level is 100-120 g / L, 120-140 g / L, or 140-160 g / L for at least twelve weeks after the administration of the initial dose.

[0178] Example 29. According to any one of Examples 1 to 26, the hemoglobin level is 100-120 g / L, 120-140 g / L, or 140-160 g / L at least twenty-four weeks after the administration of the initial dose.

[0179] Example 30. The method according to any one of Examples 1 to 29, wherein the hematocrit level is further measured as a percentage.

[0180] Example 31. According to any one of Examples 1 to 30, wherein 10 12 / L measures the mean corpuscular volume (MCV) of red blood cells.

[0181] Example 32. The method according to any one of Examples 1 to 31, wherein the red blood cell count is further measured as a percentage.

[0182] Example 33. According to any one of Examples 1 to 32, the red blood cell distribution width level is further measured as a percentage.

[0183] Example 34. The method according to any one of Examples 1 to 33, wherein the antibody is emecizumab.

[0184] Example 35. Use of a bispecific antibody in the manufacture of a medicament for treating hemophilia A in an individual, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises

[0185] (i) A first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively;

[0186] (ii) A second antibody H chain comprising a variable region containing CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and

[0187] (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and

[0188] Compared to baseline levels, this treatment increased the individual's hemoglobin levels.

[0189] Example 36. Use of a bispecific antibody in the manufacture of a drug for increasing hemoglobin levels in individuals diagnosed with hemophilia A, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises

[0190] (i) A first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively;

[0191] (ii) A second antibody H chain comprising a variable region containing CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and

[0192] (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15.

[0193] Compared to baseline levels, the drug increases the level of this hemoglobin in the individual.

[0194] Example 37. A bispecific antibody for use in treating hemophilia A in an individual, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises

[0195] (i) A first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively;

[0196] (ii) A second antibody H chain comprising a variable region containing CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and

[0197] (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and

[0198] Compared to baseline levels, this treatment increased the individual's hemoglobin levels.

[0199] A bispecific antibody for increasing hemoglobin levels in individuals diagnosed with hemophilia A, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises

[0200] (i) A first antibody H chain containing a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 containing the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively;

[0201] (ii) A second antibody H chain comprising a variable region containing CDRs 1, 2, and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10, respectively; and

[0202] (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15.

[0203] Compared to baseline levels, this use increases the level of that hemoglobin in the individual.

[0204] Example

[0205] The following examples are provided to provide a complete disclosure and description of how to make and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider their invention, nor to represent that the following experiments are all or only the experiments performed. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account.

[0206] Example 1: Emesizumab prophylactic treatment in patients with hemophilia A resulted in improvements in hemoglobin levels.

[0207] This case describes the assessment of changes in hemoglobin and related red blood cell (RBC) indices from baseline in participants (≥12 years old) receiving emecizumab prophylaxis for severe hemophilia A.

[0208] method

[0209] Post-hoc analyses were performed on data collected from the HAVEN 1 (NCT02622321), HAVEN 3 (NCT02847637), HAVEN 4 (NCT03020160), and STASEY (NCT03191799) clinical trials. In these trials, a total of N = 501 adolescent and adult participants (≥ 12 years of age) with severe hemophilia A initially received prophylactic subcutaneous injections of emecizumab at 3 mg / kg / week for 4 weeks, and were subsequently randomized to one of the following groups: 1.5 mg / kg emecizumab weekly, 3.0 mg / kg emecizumab every two weeks, or 6.0 mg / kg emecizumab every four weeks, administered subcutaneously as described in the clinical trial protocols. Figures 1A-1D A schematic overview of each trial is provided. Clinical trial protocols can be found at: https: / / clinicaltrials.gov / study / NCT02622321 (HAVEN 1), https: / / clinicaltrials.gov / study / NCT02847637 (HAVEN 3), https: / / clinicaltrials.gov / study / NCT03020160 (HAVEN 4) and https: / / clinicaltrials.gov / study / NCT03191799 (STASEY), all of which are hereby incorporated herein by reference in their entirety.

[0210] In this post-hoc analysis, biomarkers such as annual bleeding rate, hemoglobin (g / L), and red blood cell count (10-1) were evaluated as described in each protocol. 12 The biomarkers included hematocrit (fraction of 1), mean corpuscular volume (fL), and red blood cell distribution width (fraction of 1). Baseline levels of each biomarker were assessed prior to the administration of the first dose of emecizumab or placebo. Experimental levels of each biomarker were collected at multiple time points throughout weeks 73–78. Experimental biomarker levels were then compared to baseline to analyze changes relative to baseline, as shown in Table 1 below.

[0211] Changes in hemoglobin from baseline were assessed at two levels: 1) the pooled population (N = 497); and 2) subgroups defined by the following factors: i. prior treatment regimen, ii. inhibitor status, and iii. baseline hemoglobin level. Baseline hemoglobin levels were categorized into the following subgroups:

[0212] ●80 g / L < Hemoglobin < 100 g / L

[0213] ● 100 g / L ≤ Hemoglobin < 120 g / L

[0214] ●120 g / L ≤ Hemoglobin < 140 g / L

[0215] ● 140 g / L ≤ Hemoglobin < 180 g / L

[0216] The changes in the relevant RBC index compared to the baseline were assessed.

[0217] result

[0218] Improvements in mean hemoglobin levels were observed in the pooled population of patients with hemophilia A. In this pooled population, the mean hemoglobin (standard deviation [SD], g / L) was 145.3 at baseline [15.8] and 151.3 at weeks 73–78 [13.2] (Table 1). Mean hemoglobin levels increased from baseline to weeks 73–78 for all subgroups (baseline hemoglobin level, prior treatment regimen, and baseline inhibitor status; Table 1). These improvements were observed over time, particularly in participants who exhibited lower baseline hemoglobin levels (Figure 2).

[0219] The greatest improvement in hemoglobin was observed in the subgroup of patients with inhibitors and in patients with the lowest hemoglobin levels at baseline. The subgroup of patients with inhibitors included those who had previously been treated with bypass agents (i.e., aPCC and rFVIIa) prior to trial enrollment and treatment with emecizumab. Patients with inhibitors had lower baseline hemoglobin levels than those without inhibitors and experienced a greater increase in hemoglobin at weeks 73–78 (mean [SD], g / L) (8.1 for patients with inhibitors [15.9] vs. 2.9 for patients without inhibitors [10.9]).

[0220] In the additional subgroups of patients, mean hemoglobin levels increased from baseline to weeks 73–78 for all subgroups (baseline hemoglobin levels, prior treatment regimens, and baseline inhibitor status; Table 1).

[0221] Table 1. Changes in hemoglobin levels and other laboratory measurements in each participant subgroup from baseline to weeks 73–78.

[0222]

[0223] Red blood cell count (10) 12 Data for erythrocyte distribution width (r / L) and fractional red blood cell distribution width (fractional) are from the HAVEN 1 study only. CBU, chromogenic Bethesda units; F, factor; HgB, hemoglobin; NE, not estimable; RBC, red blood cells; SD, standard deviation.

[0224] Additional laboratory measurements are shown in Table 1 above. Further analysis of participant subgroups showed that in participants with low baseline hemoglobin (< 120 g / L), the annual bleeding rate (ABR; at weeks 27–52) was negatively correlated with hemoglobin (at week 52) (r = −0.48).

[0225] For participants with hemophilia A who received emecizumab, these data supplemented previously observed decreases in ABR; all studied subgroups experienced elevated hemoglobin levels.

[0226] This disclosure is not intended to limit its scope to the specific embodiments disclosed herein, which are provided, for example, to illustrate various aspects of this disclosure. Various modifications to the described compositions and methods will become apparent from the description and teaching herein. Such modifications may be practiced without departing from the true scope and spirit of this disclosure, and such modifications are intended to fall within the scope of this disclosure.

[0227] sequence list

[0228]

Claims

1. A method comprising: (a) Administering a bispecific antibody to an individual diagnosed with hemophilia A according to a treatment regimen, said bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein said bispecific antibody contains (i) A first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively; (ii) A second antibody H chain comprising a variable region comprising CDRs 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9 and 10, respectively; as well as (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and (b) Increase the hemoglobin level in the individuals compared to the baseline level.

2. A method for treating hemophilia A, the method comprising: Administering a bispecific antibody to an individual diagnosed with hemophilia A, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody contains... (i) A first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively; (ii) A second antibody H chain comprising a variable region comprising CDRs 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 8, 9 and 10, respectively; as well as (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and Compared to baseline levels, the individuals in these cases showed an increase in hemoglobin levels.

3. A method for increasing hemoglobin levels in an individual diagnosed with hemophilia A, the method comprising: Administering a bispecific antibody to an individual diagnosed with hemophilia A, the bispecific antibody binding to coagulation factor IX and / or activated coagulation factor IX, and binding to coagulation factor X, wherein the bispecific antibody contains... (i) A first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively; (ii) A second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10; and (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and The hemoglobin levels in the individuals were increased compared to baseline levels.

4. The method according to claim 1, 2 or 3, wherein the first antibody H chain variable region comprises the amino acid sequence shown in SEQ ID NO:

2.

5. The method according to claim 1, 2, 3 or 4, wherein the variable region of the second antibody H chain comprises the amino acid sequence shown in SEQ ID NO:

7.

6. The method according to any one of claims 1 to 5, wherein the variable regions of the same first antibody L chain and the second antibody L chain each contain the amino acid sequence shown in SEQ ID NO:

12.

7. The method according to any one of claims 1 to 6, wherein the amino acid sequence of the full-length H chain of the first antibody is as shown in SEQ ID NO:

1.

8. The method according to any one of claims 1 to 7, wherein the amino acid sequence of the full-length H chain of the second antibody is as shown in SEQ ID NO:

6.

9. The method according to any one of claims 1 to 8, wherein the amino acid sequences of the identical full-length L-chain of the first antibody and the full-length L-chain of the second antibody are each shown in SEQ ID NO:

11.

10. The method according to any one of claims 1 to 9, wherein the bispecific antibody is administered at an initial dose of 3 mg / kg per week for one or more weeks.

11. The method of claim 10, wherein the bispecific antibody is administered at an initial dose of 3 mg / kg per week for four weeks.

12. The method according to any one of claims 1 to 11, wherein the bispecific antibody is administered at a maintenance dose of 1.5 mg / kg per week for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

13. The method according to any one of claims 1 to 11, wherein the bispecific antibody is administered at a maintenance dose of 3 mg / kg every two weeks for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

14. The method according to any one of claims 1 to 11, wherein the bispecific antibody is administered at a maintenance dose of 6 mg / kg every 4 weeks for at least 12 weeks, at least 24 weeks, or at least 52 weeks.

15. The method according to any one of claims 1 to 14, wherein the treatment is an acute phase treatment or a preventive treatment.

16. The method according to any one of claims 1 to 14, wherein the treatment comprises both acute treatment and preventive treatment.

17. The method according to any one of claims 1 to 16, wherein the blood flow of the individual contains factor VIII inhibitor.

18. The method according to any one of claims 1 to 16, wherein the blood flow of said individual does not contain factor VIII inhibitors.

19. The method according to any one of claims 1 to 18, wherein the individual is at least 12 years old.

20. The method according to any one of claims 1 to 19, wherein the baseline level of hemoglobin is between 80 g / L and 100 g / L.

21. The method according to any one of claims 1 to 19, wherein the baseline level of hemoglobin is between 100 g / L and 120 g / L.

22. The method according to any one of claims 1 to 19, wherein the baseline level of hemoglobin is between 120 g / L and 140 g / L.

23. The method according to any one of claims 1 to 19, wherein the baseline level of hemoglobin is between 140 g / L and 220 g / L.

24. The method according to any one of claims 1 to 23, wherein the hemoglobin level is increased by 5 g / L, 10 g / L or 15 g / L compared to the baseline level.

25. The method according to any one of claims 1 to 23, wherein the hemoglobin level is increased by 20 g / L, 25 g / L or 30 g / L compared to a baseline level.

26. The method according to any one of claims 1 to 23, wherein the hemoglobin level is increased by 5-10 g / L, 10-15 g / L, 15-20 g / L, 20-25 g / L, or 25-30 g / L compared to a baseline level.

27. The method according to any one of claims 1 to 26, wherein the hemoglobin level is 80-100 g / L, 100-120 g / L, or 120-140 g / L for at least four weeks after the administration of the initial dose.

28. The method according to any one of claims 1 to 26, wherein the hemoglobin level is 100-120 g / L, 120-140 g / L, or 140-160 g / L for at least twelve weeks after administration of the initial dose.

29. The method according to any one of claims 1 to 26, wherein the hemoglobin level is 100-120 g / L, 120-140 g / L, or 140-160 g / L at least twenty-four weeks after the administration of the initial dose.

30. The method according to any one of claims 1 to 29, wherein the hematocrit level is further measured as a percentage.

31. The method according to any one of claims 1 to 30, wherein further comprising 10 12 / L measures the mean erythrocyte volume (MCV) level.

32. The method according to any one of claims 1 to 31, wherein the red blood cell count is further measured as a percentage.

33. The method according to any one of claims 1 to 32, wherein the red blood cell distribution width level is further measured as a percentage.

34. The method according to any one of claims 1 to 33, wherein the antibody is emecizumab.

35. Use of a bispecific antibody in the manufacture of a medicament for treating hemophilia A in an individual, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises (i) A first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively; (ii) A second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10; and (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and The treatment increases hemoglobin levels in the individual compared to baseline levels.

36. Use of a bispecific antibody in the manufacture of a medicament for increasing hemoglobin levels in an individual diagnosed with hemophilia A, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises (i) A first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively; (ii) A second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10; and (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15. The drug increases the hemoglobin level in the individual compared to baseline levels.

37. A bispecific antibody for use in treating hemophilia A in an individual, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises (i) A first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively; (ii) A second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10; and (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15; and The treatment increases hemoglobin levels in the individual compared to baseline levels.

38. A bispecific antibody for increasing hemoglobin levels in individuals diagnosed with hemophilia A, wherein the bispecific antibody binds to a) coagulation factor IX and / or activated coagulation factor IX, and b) coagulation factor X, wherein the bispecific antibody comprises (i) A first antibody H chain comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 comprising the amino acid sequences shown in SEQ ID NO: 3, 4 and 5, respectively; (ii) A second antibody H chain comprising a variable region comprising CDRs 1, 2, and 3, respectively, comprising the amino acid sequences shown in SEQ ID NO: 8, 9, and 10; and (iii) Identical first antibody L chain and second antibody L chain, each L chain containing a variable region comprising CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 13, 14, and 15. The use of this method increases the hemoglobin level in the individual compared to the baseline level.

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