TFPI antibody used in the treatment of hemophilia

Abstract

Provided is a use of a TFPI antibody in the treatment of hemophilia. Specifically, using the TFPI antibody can significantly reduce the bleeding frequency of hemophilia A and hemophilia B patients with or without inhibitors.

Description

TFPI antibodies for the treatment of hemophilia

[0001] This application claims the benefit of Chinese Patent Application No. 2023117141330 filed on December 13, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present application relates to the field of biomedicine, and specifically to a TFPI antibody for treating hemophilia. Background Art

[0003] Hemophilia is an X-linked recessive inherited bleeding disorder that can be divided into hemophilia A and hemophilia B. The former is characterized by a deficiency of coagulation factor VIII (FVIII), accounting for 80% to 85% of cases; the latter is characterized by a deficiency of coagulation factor IX (FIX), accounting for 15% to 20%. In the male population, the incidence of hemophilia A is approximately 1 in 5,000, and the incidence of hemophilia B is approximately 1 in 25,000; female hemophilia patients are extremely rare. The clinical manifestations of hemophilia are mainly bleeding in the joints, muscles, and deep tissues. There may also be bleeding in the gastrointestinal tract, urinary tract, central nervous system, bleeding after tooth extraction, and delayed bleeding after trauma or surgery. Repeated bleeding and untimely treatment can lead to joint deformities and / or pseudotumor formation, seriously affecting the patient's quality of life. Severe bleeding may directly endanger the patient's life.

[0004] To date, the most effective treatment for hemophilia is to supplement the coagulation factors that the patient lacks, that is, replacement therapy, which includes on-demand treatment and preventive treatment. On-demand treatment refers to replacement therapy given to patients when they are bleeding, with the aim of stopping bleeding in time; preventive treatment refers to regular replacement therapy given on a regular basis, with the aim of preventing patients from repeated bleeding leading to joint deformities or life-threatening bleeding. The World Federation of Hemophilia's 2020 Guidelines for the Management of Hemophilia strongly recommends that patients with frequent bleeding and / or severe hemophilia maintain adequate preventive treatment and try to avoid bleeding at all times. Even in countries with limited medical conditions, preventive treatment is advocated over on-demand treatment, and low-intensity preventive treatment is acceptable. All types of preventive treatment are more beneficial than on-demand treatment; it is recommended to start preventive treatment early, that is, primary preventive treatment.

[0005] Some hemophilia patients will develop coagulation factor inhibitors in their bodies, especially in severe patients with long-term disease and repeated use of coagulation factor replacement therapy. The appearance of inhibitors will bring difficulties to treatment: 1. In case of bleeding, large doses of FVIII / FIX preparations or bypass preparations are required: the available bypass preparations include recombinant activated coagulation factor VII (rFVIIa) and prothrombin complex (PCC). Since PCC may cause increased inhibitor responsiveness, rFVIIa is the first choice for treatment, but the high cost increases the difficulty of medication; 2. Conventional preventive treatment is difficult to carry out, especially for hemophilia B patients: there are no drugs approved in China for the prevention and treatment of hemophilia B with inhibitors (HBW); although there are drugs approved for the prevention and treatment of hemophilia A (HAW) with inhibitors, the high cost of treatment brings obstacles to its widespread and long-term use.

[0006] Prophylactic treatment for hemophilia patients with inhibitors remains an urgent clinical issue. Promoting thrombin generation by inhibiting the activity of tissue factor pathway inhibitor (TFPI) may provide a new option for prophylactic treatment.

[0007] Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor that exists primarily in two molecular forms: TFPI-α and TFPI-β. TFPI-α consists of a negatively charged acidic N-terminus, three tandemly repeated Kunitz-type protease inhibitory domains (K1, K2, and K3), and a positively charged basic C-terminus. TFPI-β contains K1, K2, and a C-terminus for a glycosylphosphatidylinositol anchor. TFPI is a key negative regulator of the extrinsic coagulation pathway: K2 binds to activated coagulation factor X (FXa) and inactivates it. Subsequently, K1 rapidly binds to the adjacent tissue factor (TF)-activated coagulation factor FVII (FVIIa) complex in an FXa-dependent manner, forming the FXa-TFPI-TF-FVIIa quaternary complex, inhibiting the catalytic activity of the TF-FVIIa complex and further preventing FX activation. Furthermore, the K3 domain can bind to protein S, anchoring it to the cell surface and enhancing its inhibitory activity against FXa. By inhibiting the activity of TFPI, its inhibition on the activity of FXa and TF-FVIIa complex can be eliminated, thereby maintaining the generation of thrombin and improving the coagulation dysfunction of hemophilia patients. Summary of the Invention

[0008] Treatments for hemophilia

[0009] In a first aspect, the present application relates to a method for treating hemophilia, comprising the step of administering to a subject in need thereof an antibody or an antigen-binding fragment thereof against tissue factor pathway inhibitor (TFPI), wherein the antibody comprises a heavy chain variable region and a light chain variable region,

[0010] The heavy chain variable region comprises: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4;

[0011] The light chain variable region includes: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8.

[0012] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:1; for example, the heavy chain variable region can comprise an amino acid sequence that has about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:1.

[0013] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:5; or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:5; for example, the heavy chain variable region can comprise an amino acid sequence that has about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:5.

[0014] In some embodiments, the heavy chain of the antibody further comprises a constant region of human IgG4 or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO: 9.

[0015] In some embodiments, the light chain of the antibody further comprises a constant region of human Igκ or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO: 10.

[0016] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:11.

[0017] In some embodiments, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:12.

[0018] In some embodiments, the treatment is prophylactic treatment.

[0019] In some embodiments, the hemophilia is hemophilia A, for example, hemophilia A with inhibitors or hemophilia A without inhibitors.

[0020] In some embodiments, the hemophilia A is severe or moderately severe hemophilia A (factor VIII activity level ≤ 2%).

[0021] In some embodiments, the hemophilia is hemophilia B, for example, hemophilia B with inhibitors or hemophilia B without inhibitors.

[0022] In some embodiments, the hemophilia B is severe or moderately severe hemophilia B (factor IX activity level ≤ 2%).

[0023] In some embodiments, the hemophilia is hemophilia A with or without inhibitors, or hemophilia B with or without inhibitors.

[0024] In some embodiments, the hemophilia is von Willebrand disease.

[0025] In some embodiments, the hemophilia is type 1, type 2, or type 3 von Willebrand disease.

[0026] In some embodiments, the subject is a male hemophilia patient aged 12 to 70 years old.

[0027] In some embodiments, the subject weighs ≥30 kg and has a body mass index (BMI) <28 kg / m 2 .

[0028] In some embodiments, the subject is a hemophilia A patient, for example, a patient with severe or moderately severe hemophilia A (coagulation factor VIII activity level ≤ 2%).

[0029] In some embodiments, the subject is a hemophilia A patient with an inhibitor or a hemophilia A patient without an inhibitor.

[0030] In some embodiments, the subject is a hemophilia B patient, for example, a patient with severe or moderately severe hemophilia B (coagulation factor IX activity level ≤ 2%).

[0031] In some embodiments, the subject is a hemophilia B patient with an inhibitor or a hemophilia B patient without an inhibitor.

[0032] In some embodiments, the subject is a von Willebrand disease patient, for example, a von Willebrand disease type 1, type 2, or type 3 patient.

[0033] In some embodiments, the subject has not previously used TFPI antibody drugs.

[0034] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.0 mg / kg to 2.0 mg / kg, such as 1.0 mg / kg, 1.05 mg / kg, 1.1 mg / kg, 1.15 mg / kg, 1.2 mg / kg, 1.25 mg / kg, 1.3 mg / kg, 1.35 mg / kg, 1.4 mg / kg, 1.45 mg / kg, 1.5 mg / kg, 1.55 mg / kg, 1.6 mg / kg, 1.65 mg / kg, 1.7 mg / kg, 1.75 mg / kg, 1.8 mg / kg, 1.85 mg / kg, 1.9 mg / kg, 1.95 mg / kg, or 2.0 mg / kg, based on the subject's body weight.

[0035] In some embodiments, the subject weighs ≥ 30 kg.

[0036] In some embodiments, the subject's body weight is ≥50 kg; alternatively, the subject's body weight is ≥40 kg and <50 kg; alternatively, the subject's body weight is ≥30 kg and <40 kg.

[0037] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg of the subject's body weight.

[0038] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg based on the subject's body weight; the subject weighs ≥ 50 kg.

[0039] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.75 mg / kg of the subject's body weight.

[0040] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.75 mg / kg based on the subject's body weight; the subject's body weight is ≥40 kg and <50 kg.

[0041] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg to 1.75 mg / kg based on the subject's body weight.

[0042] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg to 1.75 mg / kg based on the subject's body weight; the subject weighs ≥ 40 kg.

[0043] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 2.0 mg / kg of the subject's body weight.

[0044] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 2.0 mg / kg based on the subject's body weight; the subject's body weight is ≥30 kg and <40 kg.

[0045] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight.

[0046] In some embodiments, the treatment method comprises administering the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight; the subject's body weight is ≥30 kg.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof can be administered by intravenous drip or subcutaneous injection. Preferably, the antibody or antigen-binding fragment thereof is administered by subcutaneous injection.

[0048] In some embodiments, the treatment method comprises subcutaneously injecting the antibody or antigen-binding fragment thereof once a week at a dose of 1.0 mg / kg, 1.5 mg / kg, or 2.0 mg / kg of the subject's body weight.

[0049] In some embodiments, the treatment method comprises subcutaneously injecting the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight.

[0050] In some embodiments, the treatment method comprises subcutaneous injection of the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight, and the subject's body weight is ≥30 kg.

[0051] In some embodiments, the treatment method comprises subcutaneously injecting the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg based on the subject's body weight, and the subject's body weight is ≥ 50 kg.

[0052] In some embodiments, the treatment method comprises subcutaneously injecting the antibody or antigen-binding fragment thereof once a week at a dose of 1.75 mg / kg based on the subject's body weight, and the subject's body weight is ≥40 kg and <50 kg.

[0053] In some embodiments, the treatment method comprises subcutaneously injecting the antibody or antigen-binding fragment thereof once a week at a dose of 2.0 mg / kg based on the subject's body weight, and the subject's body weight is ≥30 kg and <40 kg.

[0054] In some embodiments, after administration of the antibody or antigen-binding fragment, the subject's annualized bleeding rate (ABR) is no higher than 10.0, preferably no higher than 8.0, more preferably no higher than 5.0, and even more preferably no higher than 2.0. For example, the subject's annualized bleeding rate is no higher than 9.5, no higher than 9.0, no higher than 8.5, no higher than 8.0, no higher than 7.5, no higher than 7.0, no higher than 6.5, no higher than 6.0, no higher than 5.5, no higher than 5.0, no higher than 4.5, no higher than 4.0, no higher than 3.5, no higher than 3.0, no higher than 2.5, no higher than 2.0, no higher than 1.5, or no higher than 1.0.

[0055] In some embodiments, after administration of the antibody or antigen-binding fragment, the annualized bleeding rate of the subject is reduced by no less than 60%, preferably no less than 70%, more preferably no less than 80%, and even more preferably no less than 90% compared to before administration. For example, the annualized bleeding rate of the subject is reduced by no less than 65%, no less than 70%, no less than 75%, no less than 80%, no less than 81%, no less than 82%, no less than 83%, no less than 84%, no less than 85%, no less than 86%, no less than 87%, no less than 88%, no less than 89%, no less than 90%, no less than 91%, no less than 92%, no less than 93%, no less than 94%, no less than 95%, no less than 96%, no less than 97%, no less than 98%, or no less than 99% compared to before administration.

[0056] The annualized bleeding rate (ABR) was calculated as follows: ABR = number of bleeding episodes during the follow-up period (days) ÷ number of follow-up days × 365.25.

[0057] In some embodiments, the annualized bleeding rate is calculated based on spontaneous and traumatic bleeding.

[0058] use

[0059] In a second aspect, the present application also relates to an antibody or an antigen-binding fragment thereof for treating hemophilia, wherein the antibody comprises a heavy chain variable region and a light chain variable region.

[0060] The heavy chain variable region comprises: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4;

[0061] The light chain variable region includes: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8.

[0062] In a third aspect, the present application also relates to the use of an antibody or an antigen-binding fragment thereof against tissue factor pathway inhibitor (TFPI) in the preparation of a drug for treating hemophilia, wherein the antibody comprises a heavy chain variable region and a light chain variable region.

[0063] The heavy chain variable region comprises: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4;

[0064] The light chain variable region includes: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8.

[0065] The second and third aspects are further defined as follows:

[0066] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:1; for example, the heavy chain variable region can comprise an amino acid sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:1.

[0067] In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO:5; or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:5; for example, the heavy chain variable region can comprise an amino acid sequence that has about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:5.

[0068] In some embodiments, the heavy chain of the antibody further comprises a constant region of human IgG4 or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO: 9.

[0069] In some embodiments, the light chain of the antibody further comprises a constant region of human Igκ or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO: 10.

[0070] In some embodiments, the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:11.

[0071] In some embodiments, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:12.

[0072] In some embodiments, the treatment is prophylactic treatment.

[0073] In some embodiments, the hemophilia is hemophilia A, for example, hemophilia A with inhibitors or hemophilia A without inhibitors.

[0074] In some embodiments, the hemophilia A is severe or moderately severe hemophilia A (factor VIII activity level ≤ 2%).

[0075] In some embodiments, the hemophilia is hemophilia B, for example, hemophilia B with inhibitors or hemophilia B without inhibitors.

[0076] In some embodiments, the hemophilia B is severe or moderately severe hemophilia B (factor IX activity level ≤ 2%).

[0077] In some embodiments, the hemophilia is hemophilia A with or without inhibitors, or hemophilia B with or without inhibitors.

[0078] In some embodiments, the hemophilia is von Willebrand disease.

[0079] In some embodiments, the hemophilia is type 1, type 2, or type 3 von Willebrand disease.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.0 mg / kg to 2.0 mg / kg, such as 1.0 mg / kg, 1.05 mg / kg, 1.1 mg / kg, 1.15 mg / kg, 1.2 mg / kg, 1.25 mg / kg, 1.3 mg / kg, 1.35 mg / kg, 1.4 mg / kg, 1.45 mg / kg, 1.5 mg / kg, 1.55 mg / kg, 1.6 mg / kg, 1.65 mg / kg, 1.7 mg / kg, 1.75 mg / kg, 1.8 mg / kg, 1.85 mg / kg, 1.9 mg / kg, 1.95 mg / kg, and 2.0 mg / kg, based on the subject's body weight.

[0081] In some embodiments, the subject weighs ≥ 30 kg.

[0082] In some embodiments, the subject's body weight is ≥50 kg; alternatively, the subject's body weight is ≥40 kg and <50 kg; alternatively, the subject's body weight is ≥30 kg and <40 kg.

[0083] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg of the subject's body weight.

[0084] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg based on the subject's body weight; the subject's body weight is ≥ 50 kg.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.75 mg / kg of the subject's body weight.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.75 mg / kg based on the subject's body weight; the subject's body weight is ≥40 kg and <50 kg.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg to 1.75 mg / kg based on the subject's body weight.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg to 1.75 mg / kg based on the subject's body weight; the subject's body weight is ≥ 40 kg.

[0089] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 2.0 mg / kg of the subject's body weight.

[0090] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 2.0 mg / kg based on the subject's body weight; the subject's body weight is ≥30 kg and <40 kg.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight; the subject's body weight is ≥30 kg.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof can be administered by intravenous drip or subcutaneous injection. Preferably, the antibody or antigen-binding fragment thereof is administered by subcutaneous injection.

[0094] In some embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously once a week at a dose of 1.0 mg / kg, 1.5 mg / kg, or 2.0 mg / kg of the subject's body weight.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof is injected subcutaneously once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof is injected subcutaneously once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight, and the subject's body weight is ≥30 kg.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously once a week at a dose of 1.5 mg / kg based on the subject's body weight, and the subject's body weight is ≥ 50 kg.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously once a week at a dose of 1.75 mg / kg based on the subject's body weight, and the subject's body weight is ≥40 kg and <50 kg.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof is administered subcutaneously once a week at a dose of 2.0 mg / kg based on the subject's body weight, and the subject's body weight is ≥30 kg and <40 kg.

[0100] In some embodiments, after administration of the antibody or antigen-binding fragment, the subject's annualized bleeding rate (ABR) is no higher than 10.0, preferably no higher than 8.0, more preferably no higher than 5.0, and even more preferably no higher than 2.0. For example, the subject's annualized bleeding rate is no higher than 9.5, no higher than 9.0, no higher than 8.5, no higher than 8.0, no higher than 7.5, no higher than 7.0, no higher than 6.5, no higher than 6.0, no higher than 5.5, no higher than 5.0, no higher than 4.5, no higher than 4.0, no higher than 3.5, no higher than 3.0, no higher than 2.5, no higher than 2.0, no higher than 1.5, or no higher than 1.0.

[0101] In some embodiments, after administration of the antibody or antigen-binding fragment, the annualized bleeding rate of the subject is reduced by no less than 60%, preferably no less than 70%, more preferably no less than 80%, and even more preferably no less than 90% compared to before administration. For example, the annualized bleeding rate of the subject is reduced by no less than 65%, no less than 70%, no less than 75%, no less than 80%, no less than 81%, no less than 82%, no less than 83%, no less than 84%, no less than 85%, no less than 86%, no less than 87%, no less than 88%, no less than 89%, no less than 90%, no less than 91%, no less than 92%, no less than 93%, no less than 94%, no less than 95%, no less than 96%, no less than 97%, no less than 98%, or no less than 99% compared to before administration.

[0102] Definition of terms

[0103] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0104] As used herein, the term "tissue factor pathway inhibitor" or "TFPI" refers to any variant, isoform, and species homolog of human TFPI that is naturally expressed by cells. An exemplary human TFPI comprises the amino acid sequence shown in SEQ ID NO:13.

[0105] The term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural or partially or completely synthetically (e.g., recombinantly), including any fragment thereof that retains the binding specificity of a full-length immunoglobulin comprising at least a portion of the variable region of an immunoglobulin molecule. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin antigen-binding domain (antibody binding site). Antibodies include antibody fragments, such as anti-tumor cell antibody fragments. As used herein, the term antibody includes synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies provided herein include members of any immunoglobulin class (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0106] A full-length antibody generally refers to an immunoglobulin molecule composed of two identical pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions can be connected by a "J" region of approximately 12 or more amino acids, and the heavy chain may also include a "D" region of approximately 3 or more amino acids. The heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region can be composed of three domains (CH1, CH2, and CH3). The light chain can be composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region can be composed of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). For example, VH and VL can each comprise or consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site.

[0107] The term "antibody fragment" or "antigen-binding fragment" refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antigen-binding sites) and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains immunospecific binding (i.e., exhibits at least or at least about 10 7 -10 8 M -1 Antibodies to the Ka) antigen.

[0108] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0109] The regions of CDRs defined by the Kabat, AbM, IMGT, or Chothia schemes are as follows:

[0110] Unless otherwise specified, this application uses the Kabat scheme to define CDR regions.

[0111] As used herein, the term "and / or" refers to any one of the alternatives or two or more of the alternatives.

[0112] As used herein, the term "comprises" or "comprising" means including the elements, integers or steps mentioned, but not excluding any other elements, integers or steps. In this article, when the term "comprises" or "comprising" is used, unless otherwise indicated, combinations of the elements, integers or steps mentioned are also covered.

[0113] The term "administering" generally refers to a method of administering a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administration can be performed by any suitable means, including parenteral, intrapulmonary, and intranasal, and (if desired for local treatment) intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0114] In this application, the term "about" generally refers to variation within a range of 0.5%-10% above or below a specified value, for example, variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Unless otherwise specified, the numerical values ​​mentioned in this application are considered to be modified by "about". If in doubt, or if the error range for a particular value or parameter is not commonly understood in the art, "about" means ±5% of that value or parameter.

[0115] The term "effective amount" refers to an amount or dosage of an antibody, fragment, composition, or combination of the present invention that, after administration to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. Depending on the desired effect, both a "therapeutically effective amount" and a "prophylactically effective amount" may be included.

[0116] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome, at the dosage and for the period of time required. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" is an amount effective to achieve the desired preventive outcome, at the dosage and for the period of time required. Typically, because a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0117] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] The accompanying drawings are briefly described as follows:

[0119] Figure 1 shows the average dPT results of plasma from patients with different types of hemophilia and VWD3 patients after in vitro intervention with different concentrations of KN057.

[0120] Figures 2A-2E show the comparison of dPT results after different concentrations of KN057 and PF-06741086 intervened in the plasma of hemophilia patients and VWD3 patients in vitro.

[0121] Figures 3A and 3B show the TGA test results of plasma from patients with different types of hemophilia and VWD3 after in vitro intervention with different concentrations of KN057.

[0122] Figures 4A-4E show the comparison of peak thrombin results after different concentrations of KN057 and PF-06741086 intervened in the plasma of hemophilia patients and VWD3 patients in vitro.

[0123] Figures 5A-5E show the comparison of ETP results after different concentrations of KN057 and PF-06741086 intervened in the plasma of hemophilia patients and VWD3 patients in vitro. DETAILED DESCRIPTION

[0124] List of abbreviations

[0125] Example 1. Preparation of anti-TFPI antibodies

[0126] KN057 is a humanized anti-TFPI monoclonal antibody, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 11, and the amino acid sequence of its light chain is shown in SEQ ID NO: 12. It can be prepared by referring to the method in patent application WO2021037197.

[0127] The amino acid sequence of PF-06741086 (Marstacimab) is published in WHO Drug Information, Vol. 32, No. 2, 2018; INN Proposed List 119. A vector containing a nucleic acid sequence encoding the above-mentioned antibody is transferred into cells, and the corresponding antibody is expressed and purified.

[0128] Example 2: Phase II clinical study of KN057 in the treatment of hemophilia

[0129] Purpose of the test:

[0130] To evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and efficacy of multiple subcutaneous injections of KN057 in patients with HA or HB with or without inhibitors.

[0131] Dosage regimen

[0132] The subjects received a subcutaneous injection of KN057 injection once a week, with a total of three dose groups, each receiving a dose of 1.0 mg / kg, 1.5 mg / kg and 2.0 mg / kg respectively.

[0133] Subject inclusion criteria

[0134] 1. Male, 18 to 70 years old (inclusive), weight ≥ 40 kg;

[0135] 2. Severe and moderately severe HA or HB (coagulation factor VIII activity or coagulation factor IX activity level ≤ 2%);

[0136] 3. Patients without inhibitors must meet the following criteria: a) FVIII or FIX inhibitor test is negative during the screening period; b) ≥ 6 bleeding episodes requiring treatment (spontaneous and / or traumatic, excluding bleeding episodes during surgery) in the past 6 months and are willing to receive combined on-demand treatment during the study; c) ≥ 50 exposure days using coagulation factor replacement therapy before screening.

[0137] 4. Patients with inhibitors must meet the following criteria: a) FVIII or FIX inhibitor test is positive during the screening period; b) ≥ 6 bleeding episodes requiring treatment (spontaneous and / or traumatic, excluding bleeding episodes during surgery) in the past 6 months and are willing to receive combined on-demand treatment during the study.

[0138] 5. Ability and consent to wash out the original treatment regimen before administering KN057: recombinant activated coagulation factor VII (rFVIIa) must be washed out for at least 48 hours, FVIII and prothrombin complex (PCC) for at least 72 hours, and FIX for at least 96 hours; other drugs with longer half-lives or drugs used in clinical trials, such as emicizumab, require washing out for at least 5 drug half-lives.

[0139] 6. Voluntarily accept the trial and be able to comply with the relevant management regulations of the research implementation unit, and be able to cooperate in completing the medication, follow-up and various examinations specified in the protocol during the study.

[0140] Number of subjects

[0141] The target number of subjects to be enrolled is 24.

[0142] End point indicators

[0143] Primary endpoints:

[0144] 1) Incidence and severity of TEAEs (D85);

[0145] 2) the number of patients who withdrew from the study due to TEAEs (D85);

[0146] 3) the frequency and severity of abnormalities in various laboratory safety indicators (D85);

[0147] 4) changes in safety indicators during ECG, vital signs, and physical examination (D85);

[0148] 5) Injection site reaction (D85).

[0149] Secondary endpoints:

[0150] 1) Peak plasma concentration (Cmax), time to peak (Tmax), plasma concentration after administration, and area under the time-to-dose curve (AUClast) (D8) after the first dose;

[0151] 2) peak plasma concentration (Cmax,ss), time to reach Cmax,ss (Tmax,ss), area under the concentration-time curve (AUCτ), apparent clearance (CL / F), and minimum plasma concentration (Cmin) after administration on D36 (D43);

[0152] 3) Pharmacodynamic indicators: Total TFPI (Total TFPI) and free TFPI (Free TFPI); thrombin generation assay (TGA); prothrombin fragment 1+2 (PF1+2) concentration and change from baseline (D85, D169);

[0153] 4) Immunogenicity: number and ratio of patients producing anti-KN057 antibodies (ADA); number and ratio of patients producing anti-KN057 neutralizing antibodies (NAb) (NAb testing was performed only on ADA-positive samples and corresponding baseline samples) (D85, D169);

[0154] 5) Effectiveness evaluation: Annualized bleeding rate (ABR) (D85, D169).

[0155] Note: In this example, D followed by the number n represents the nth day after the first administration of KN057.

[0156] Summary of test results

[0157] The study completed enrollment of 24 subjects, including six HA patients in Group 1 (1.0 mg / kg, once weekly), six HA patients in Group 2 (1.5 mg / kg, once weekly), six HAW patients in Group 4 (1.5 mg / kg, once weekly), and five HA and one HB patients in Group 3 (2.0 mg / kg, once weekly). Safety results showed that the only adverse event related to the trial drug in the subjects was "decreased fibrinogen" (grade 2 on the CTCAE 5.0 scale); all others were grade 1. No SAEs related to the trial drug occurred. KN057 was safe and well-tolerated at doses of 1.0 mg / kg, 1.5 mg / kg, and 2.0 mg / kg.

[0158] The efficacy results showed that the annualized bleeding rate (ABR) of subjects in the 1.0 mg / kg dose group (Group 1) had decreased, but the efficacy had not yet reached expectations. The ABR of the 1.5 mg / kg dose group showed a significant decrease during the main trial period (12 weeks of treatment): the Mean and Median values ​​of the ABR of Group 2 (HA) were 7.648 and 2.03, respectively, and further decreased to 5.083 and 1.05 during the full treatment period (24 weeks of treatment). In addition, 3 of the 6 subjects (50%) had an ABR of 0 during the full treatment period. The Mean and Median values ​​of the ABR of Group 4 (HAW) during the main trial period were 1.255 and 0.00, respectively. The drug was continuously effective and had equivalent ABR during the full treatment period. In addition, 4 of the 6 subjects (66.7%) had an ABR of 0 during the full treatment period. The ABR of the 2.0 mg / kg dose group (Group 3) also decreased compared with the baseline, but was higher than that of the 1.5 mg / kg dose group. This is mainly due to the small number of patients with rare diseases and the large differences in individual conditions and efficacy in the 2.0 mg / kg dose group.

[0159] A subject in Group 2 experienced several episodes of right hip pain and was given FVIII treatment for suspected bleeding. However, imaging examinations and the investigators' judgment revealed that this was not true bleeding. Therefore, a sensitivity analysis of the ABR was performed excluding this subject. The results showed that the mean and median values ​​of the ABR in Group 2 during the main trial period were 2.400 and 0.00, respectively, which were similar to the results in Group 4.

[0160] Analysis of the PD indicators in the trial revealed that Free TFPI was negatively correlated with drug concentration, while Total TFPI and PF1+2 were positively correlated with drug concentration. Changes in these PD indicators (Free TFPI, Total TFPI, and PF1+2) increased with increasing dose and showed a dose-dependent relationship. Changes in Free TFPI did not further increase with increasing dose from 1.5 mg / kg to 2.0 mg / kg, while PF1+2 showed a dose-dependent increase at 2.0 mg / kg. Based on the safety, efficacy, and PD results, the Phase III dose of KN057 was determined to be 1.5 mg / kg, with a once-weekly dosing frequency.

[0161] Note: Annualized bleeding rate during the main trial period = (number of bleeding episodes requiring treatment from D1 to [D85+6 / early discharge date, whichever occurs earlier if the subject does not enter the D92 visit; D92-1 if the subject enters the D92 visit]) ÷ ([D85+6 / early discharge date, whichever occurs earlier if the subject does not enter the D92 visit; D92-1 if the subject enters the D92 visit] - date of first dose + 1) × 365.25.

[0162] Annualized bleeding rate during the entire treatment period = (number of bleeding episodes requiring treatment from D1 to the last dose date + 6) ÷ (last dose date + 6 - first dose date + 1) × 365.25.

[0163] Example 3: A randomized, open-label study evaluating the efficacy and safety of KN057 injection for prophylactic treatment in patients with hemophilia A and hemophilia B with inhibitors

[0164] Purpose of the test

[0165] Main Purpose:

[0166] The effectiveness of KN057 prophylactic treatment was demonstrated in patients with hemophilia A (HAW) and hemophilia B (HBW) with inhibitors.

[0167] Secondary objectives:

[0168] In HAW and HBW patients: 1) Evaluate the effect of KN057 prophylaxis in improving quality of life; 2) Evaluate the safety of KN057; 3) Evaluate the changes in KN057 blood concentrations and pharmacodynamic indicators; 4) Evaluate the immunogenicity of KN057.

[0169] Trial population and sample size

[0170] There were 51 male patients with HAW and HBW aged 12 to 70 years (including the cut-off value).

[0171] ·Trial groups and trial plans

[0172] Successful screened subjects were randomly assigned to the experimental group or control group at a ratio of 2:1: subjects in the experimental group were given KN057 preventive treatment for 52 weeks upon enrollment; subjects in the control group were first given bypass preparations as needed for 26 weeks upon enrollment, and then switched to KN057 preventive treatment for another 26 weeks.

[0173] The KN057 prophylactic treatment regimen is subcutaneous injection once a week. The dose for subjects weighing ≥50 kg is 1.5 mg / kg, for subjects weighing 40 kg ≤ <50 kg is 1.75 mg / kg, and for subjects weighing 30 kg ≤ <40 kg is 2.0 mg / kg. If a subject experiences ≥2 spontaneous bleeding episodes within 8 weeks of KN057 administration and post-dose changes in coagulation function do not indicate a safety risk, the dose can be escalated by 0.25 mg / kg at the original dose. Each subject can receive a maximum of two KN057 dose escalations.

[0174] If bleeding occurs during the first 26 weeks after enrollment in the control group, the subjects will continue to use the commonly used bypass preparations before screening for treatment as needed, including recombinant activated coagulation factor VII (rFVIIa) or human prothrombin complex (PCC); after 26 weeks, they will be switched to KN057 prophylaxis (PCC is prohibited within at least 96 hours before switching to KN057).

[0175] If any subjects experience bleeding during KN057 prophylaxis, rFVIIa will be given priority for hemostasis, and the lowest expected hemostatic dose of PCC will only be used when other effective hemostatic drugs are not available.

[0176] Test cycle

[0177] The trial period is 59 weeks, including a 3-week screening period, a 26-week main trial, a 26-week extension period, and a 4-week follow-up period after the last medication.

[0178] After completing 26 weeks of KN057 prophylaxis (the main trial), subjects in the experimental group continued KN057 prophylaxis for another 26 weeks (the extension phase). After completing 26 weeks of non-prophylaxis (the main trial), subjects in the control group switched to KN057 prophylaxis for another 26 weeks (the extension phase). After the main trial and the extension phase were completed, the subjects entered the post-dose follow-up phase.

[0179] Trial endpoints

[0180] Primary endpoint:

[0181] Annualized bleeding rate (ABR) calculated based on spontaneous and traumatic bleeding in the experimental and control groups after 26 weeks of treatment

[0182] Secondary endpoints:

[0183] 1) After 26 weeks of treatment, different types of ABR in the experimental group and the control group (based on bleeding & ,treat # Spontaneous bleeding, treatment # Joint bleeding, treatment # target joints * Bleeding calculation);

[0184] 2) During the 27th to 52nd week of treatment, the ABR of the control group (based on the bleeding & ,treat # Bleeding & ,treat # Spontaneous bleeding, treatment # Joint bleeding, treatment # target joints * Bleeding calculation);

[0185] 3) After 52 weeks of treatment, the different types of ABR in the experimental group (based on bleeding & ,treat # Bleeding & ,treat # Spontaneous bleeding, treatment # Joint bleeding, treatment # target joints * Bleeding calculation);

[0186] 4) the proportion of subjects in the experimental group who did not experience treatment-related bleeding at 26 and 52 weeks of treatment;

[0187] 5) the proportion of subjects in the control group who experienced untreated bleeding during treatment weeks 1 to 26 and weeks 27 to 52;

[0188] 6) Changes in the Hemophilia Joint Health Score (HJHS) of the experimental and control groups compared with baseline after 26 weeks of treatment

[0189] 7) Changes in HJHS results compared to baseline in the experimental group after 52 weeks of treatment, and changes in HJHS results compared to 26 weeks of treatment in the control group

[0190] 8) Changes in the EQ-5D-5L results compared to baseline in the experimental and control groups after 26 weeks of treatment

[0191] 9) Changes in EQ-5D-5L results compared to baseline in the experimental group after 52 weeks of treatment, and changes in EQ-5D-5L results compared to 26 weeks of treatment in the control group

[0192] 10) Treatment-emergent adverse events (TEAEs), TEAEs related to the trial drug, serious adverse events (SAEs), and thromboembolic events in the experimental and control groups after 26 weeks of treatment and at the end of the study @ , thrombotic microangiopathy, disseminated intravascular coagulation, hypersensitivity reactions (excluding injection site reactions), incidence and severity of injection site reactions

[0193] 11) The incidence and severity of abnormalities in various laboratory safety indicators in the experimental and control groups, as well as changes in electrocardiograms, vital signs, and safety indicators in physical examinations after 26 weeks of treatment and at the end of the study

[0194] 12) KN057 blood concentration

[0195] 13) Total TFPI (Total TFPI), free TFPI (Free TFPI) and prothrombin fragment 1+2 (PF1+2) levels and changes compared with baseline

[0196] 14) Patient and sample positive rates of anti-KN057 antibodies (ADA) and neutralizing antibodies (Nab).

[0197] Remark: & Bleeding included spontaneous and / or traumatic bleeding, excluding surgery / procedure-related bleeding; # Treated bleeding refers to the use of bypass agents and / or coagulation factors for hemostasis of the bleeding; * Target joints were defined as joints that had spontaneously bled ≥3 times within 6 months before screening.

[0198] @ Thromboembolic events include acute coronary syndrome, acute ischemic stroke, venous thrombosis, pulmonary thromboembolism, and other clinically significant thromboembolic events (excluding superficial thrombophlebitis associated with venous access devices). They require a comprehensive assessment based on symptoms, signs, and laboratory tests, and the diagnosis must be confirmed by imaging examinations (color Doppler ultrasound, CT, magnetic resonance imaging, etc.).

[0199] Inclusion criteria

[0200] All patients must meet all of the following criteria to be included:

[0201] (1) Male, aged 12 to 70 years (including cutoff values) at the time of informed consent, weighing ≥30 kg and with a BMI <28 kg / m2 at the time of screening 2 ;

[0202] (2) The FVIII or FIX inhibitor test during the screening period was high-titer positive (≥5BU / ml); or the FVIII or FIX inhibitor test during the screening period was low-titer positive (0.6BU / ml or upper limit of normal value < inhibitor titer <5BU / ml), and bypassing agent (rFVIIa or PCC) treatment has been started;

[0203] (3) ≥6 bleeding episodes (spontaneous and / or traumatic, excluding surgery / procedure-related bleeding) treated with drugs (including bypass preparations, plasma products, FVIII or FIX products, FX activators) within 26 weeks before screening;

[0204] (4) No previous use of TFPI antibody drugs;

[0205] (5) Able to and agree to elute drugs for the treatment of hemophilia: rFVIIa is eluted for at least 48 hours, FVIII products (short-acting) are eluted for at least 72 hours, PCC, FIX products (short-acting), cryoprecipitate or fresh frozen plasma are eluted for at least 96 hours; other drugs with a longer half-life or drugs used in clinical trials need to be eluted for at least 5 drug half-lives, such as Ainocoag α for injection (recombinant human coagulation factor IX-Fc fusion protein for injection) for at least 20 days.

[0206] (6) Be able to understand and comply with the requirements of this trial and voluntarily accept the trial.

[0207] Example 4: A randomized, open-label study evaluating the efficacy and safety of KN057 injection for prophylactic treatment in patients with hemophilia A and hemophilia B without inhibitors

[0208] Purpose of the test

[0209] Main Purpose:

[0210] The effectiveness of KN057 prophylaxis was demonstrated in patients with hemophilia A (HA) without inhibitors and hemophilia B (HB) without inhibitors.

[0211] Secondary objectives:

[0212] In patients with HA and HB: 1) Evaluate the effectiveness of KN057 in preventing various types of bleeding events; 2) Evaluate the effect of KN057 preventive treatment in improving quality of life; 3) Evaluate the safety of KN057; 4) Evaluate the changes in KN057 blood concentrations and pharmacodynamic indicators; 5) Evaluate the immunogenicity of KN057.

[0213] Trial population and sample size

[0214] There were 123 male patients with HA and HB aged 12 to 70 years (including the cut-off value).

[0215] ·Trial groups and trial plans

[0216] Subjects who met the requirements of Part A were randomly assigned to the experimental group (Group 1) or the control group (Group 2) at a ratio of 2:1: the experimental group received KN057 prophylaxis for 52 weeks; the control group first received standard half-life coagulation factor VIII (FVIII) / coagulation factor IX (FIX) products as needed for 26 weeks, and then switched to KN057 prophylaxis for 26 weeks.

[0217] Subjects who met the conditions of Part B were enrolled in the prevention group (Group 3) and first received standard prophylactic treatment with coagulation factors (the regimen was: the investigators evaluated and decided to continue the original prophylactic treatment regimen; or standard half-life FVIII 25 IU / kg, three times a week [HA subjects], standard half-life FIX 40 IU / kg, twice a week [HB subjects]) for 26 weeks, and then switched to KN057 prophylactic treatment for 26 weeks.

[0218] The KN057 prophylactic treatment regimen is subcutaneous injection once a week. The dose for subjects weighing ≥50 kg is 1.5 mg / kg, for subjects weighing 40 kg ≤ <50 kg is 1.75 mg / kg, and for subjects weighing 30 kg ≤ <40 kg is 2.0 mg / kg. If a subject experiences ≥2 spontaneous bleeding episodes within 8 weeks of KN057 administration and post-dose changes in coagulation function do not indicate a safety risk, the dose can be escalated by 0.25 mg / kg at the original dose. Each subject can receive a maximum of two KN057 dose escalations.

[0219] All subjects who experience bleeding during prophylaxis can receive standard half-life FVIII / FIX products as needed.

[0220] Test cycle

[0221] The trial period is 59 weeks. Part A includes a 3-week screening period, a 26-week main trial period, a 26-week extension period, and a 4-week follow-up period; Part B includes a 3-week screening period, a 26-week factor prevention period, a 26-week KN057 prevention period, and a 4-week follow-up period.

[0222] Part A trial group subjects continued KN057 prophylaxis for 26 weeks (extension phase) after completing 26 weeks of KN057 prophylaxis (main trial phase). Part A control group subjects switched to KN057 prophylaxis for 26 weeks (extension phase) after completing 26 weeks of on-demand treatment (main trial phase). Part B subjects switched to KN057 prophylaxis for 26 weeks (KN057 prophylaxis phase) after completing 26 weeks of standard coagulation factor prophylaxis (factor prophylaxis phase). All subjects were followed up for 4 weeks after the last dose (follow-up phase).

[0223] Trial endpoints

[0224] Primary endpoint:

[0225] 1) Part A: The superiority of the experimental group compared with the control group in the annualized bleeding rate (ABR, based on spontaneous and traumatic bleeding after treatment) after 26 weeks of treatment.

[0226] 2) Part B: Non-inferiority of KN057 prophylaxis compared with factor prophylaxis for ABR (based on treatment-induced spontaneous and traumatic bleeding).

[0227] Secondary endpoints:

[0228] In Part A:

[0229] 1) After 26 weeks of treatment, the ABR of the experimental group and the control group (based on the bleeding & ,treat # Spontaneous bleeding, treatment # Joint bleeding, treatment # target joints * Bleeding)

[0230] 2) During the 27th to 52nd week of treatment, the ABR of the control group (based on the bleeding & , treat bleeding & ,treat # Spontaneous bleeding, treatment # Joint bleeding, treatment # target joints * Bleeding) and comparison with weeks 1 to 26

[0231] 3) After 52 weeks of treatment, the ABR of the experimental group (based on bleeding & , treat bleeding & ,treat # Spontaneous bleeding, treatment # Joint bleeding, treatment # target joints * Bleeding

[0232] 4) The proportion of subjects in the experimental group who experienced untreated bleeding after 26 and 52 weeks of treatment

[0233] 5) Proportion of subjects with bleeding in the control group without treatment during weeks 1 to 26 and weeks 27 to 52

[0234] 6) Annual dosage of on-demand medication (weight-adjusted) in the experimental and control groups during treatment weeks 1 to 26 and 27 to 52

[0235] 5) Annual dosage of on-demand medication in the control group (adjusted by weight) from week 27 to week 52 of treatment

[0236] 7) Changes in HJHS results compared with baseline in the experimental and control groups after 26 weeks of treatment

[0237] 8) Changes in HJHS results compared to baseline in the experimental group after 52 weeks of treatment and in the control group after 26 weeks of treatment

[0238] 9) Changes in EQ-5D-5L results compared to baseline in the experimental and control groups after 26 weeks of treatment

[0239] 10) Changes in EQ-5D-5L results compared to baseline in the experimental group after 52 weeks of treatment, and changes in EQ-5D-5L results compared to 26 weeks of treatment in the control group

[0240] 11) TEAEs, TEAEs related to the trial drug, SAEs, and thromboembolic events in the experimental and control groups after 26 weeks of treatment and at the end of the study @ , thrombotic microangiopathy, disseminated intravascular coagulation, hypersensitivity reactions (excluding injection site reactions), incidence and severity of injection site reactions

[0241] 12) The incidence and severity of abnormalities in various laboratory safety indicators in the experimental and control groups, as well as changes in electrocardiograms, vital signs, and safety indicators in physical examinations after 26 weeks of treatment and at the end of the study

[0242] 13) KN057 blood concentration

[0243] 14) Contents of free Total TFPI, Free TFPI, and PF1+2 and their changes compared with baseline

[0244] 15) Patient and sample positive rates of ADA and Nab

[0245] In Part B:

[0246] 1) ABR during KN057 prevention period and factor prevention period (based on bleeding & ,treat # Spontaneous bleeding, treatment # Joint bleeding, treatment # target joints * Bleeding)

[0247] 2) Proportion of subjects with untreated bleeding during the KN057 prophylaxis period and the factor prophylaxis period

[0248] 3) Changes in HJHS results during the factor prevention period (26 weeks) compared with the time of enrollment; Changes in HJHS results during the KN057 prevention period (52 weeks) compared with the factor prevention period (26 weeks) and the time of enrollment

[0249] 4) Changes in EQ-5D-5L results during the factor prevention period (26 weeks) compared to the time of enrollment; Changes in EQ-5D-5L results during the KN057 prevention period (52 weeks) compared to the factor prevention period (26 weeks) and the time of enrollment

[0250] 5) TEAEs, TEAEs related to the trial drug, SAEs, and thromboembolic events during the study @ , thrombotic microangiopathy, disseminated intravascular coagulation, hypersensitivity reactions (excluding injection site reactions), incidence and severity of injection site reactions

[0251] 6) The incidence and severity of abnormalities in various laboratory safety indicators during the study, as well as changes in safety indicators in electrocardiograms, vital signs, and physical examinations

[0252] 7) KN057 blood concentration

[0253] 8) Changes in Total TFPI, Free TFPI, and PF1+2 Contents

[0254] 9) Patient and sample positive rates of ADA and Nab

[0255] Inclusion criteria

[0256] All patients must meet all of the following criteria to be included:

[0257] (1) Male, aged 12 to 70 years (including cutoff values) at the time of informed consent, weighing ≥30 kg and with a BMI <28 kg / m2 at the time of screening 2 ;

[0258] (2) Severe or moderately severe hemophilia A or hemophilia B (FVIII or FIX activity level ≤ 2%; previous regular test results are acceptable for Part B);

[0259] (3) FVIII or FIX inhibitor test was negative (<0.6BU / ml) or below the lower limit of normal laboratory value during the screening period;

[0260] (4) No history of FVIII or FIX inhibitors; or had inhibitors in the past, but received successful immune tolerance induction (ITI) for at least 5 years without reappearance of inhibitors (positive inhibitors detected after successful ITI);

[0261] (5) Use of coagulation factor replacement therapy for at least 100 exposure days before screening;

[0262] (6) No previous use of TFPI antibody drugs, such as Marstacimab (Pfizer) and Concizumab (Novo Nordisk);

[0263] (7) Ability to and consent to the elution of hemophilia treatment drugs: FVIII products (standard half-life) must be eluted for at least 72 hours; FIX products (standard half-life), prothrombin complex, cryoprecipitate or fresh frozen plasma must be eluted for at least 96 hours; other drugs with longer half-lives or drugs used in clinical trials must be eluted for at least 5 half-lives, such as Ainocoag α for injection (recombinant human coagulation factor IX-Fc fusion protein for injection) must be eluted for at least 20 days;

[0264] (8) Be able to understand and comply with the requirements of this trial and voluntarily accept the trial.

[0265] Patients admitted to Part A must meet all of the following criteria:

[0266] (1) ≥6 bleeding episodes (spontaneous and / or traumatic, excluding surgery / procedure-related bleeding) treated with drugs (including plasma products, FVIII or FIX products, prothrombin complex, FX activators) within 26 weeks before screening;

[0267] (2) The patient was undergoing on-demand treatment or non-standard preventive treatment before screening, or the standard preventive treatment course did not exceed 12 weeks.

[0268] Patients admitted to Part B must also meet one of the following criteria:

[0269] (1) The patient was undergoing standardized preventive treatment for more than 12 weeks before screening (standardized preventive treatment means that the compliance with the predetermined preventive regimen was at least 80%);

[0270] (2) After the completion of enrollment in Part A of this study, the investigators will assess the subjects who are suitable for factor prophylaxis and whose FVIII or FIX activity levels during the screening period are ≤2%.

[0271] Example 5: In vitro pharmacodynamic study of KN057 in plasma of hemophilia patients

[0272] Purpose of the test

[0273] Through the results of in vitro pharmacodynamic studies, the effective concentration range of KN057 in the plasma of hemophilia patients (type A and type B, including those with and without inhibitors; von Willebrand disease) was obtained.

[0274] Experimental design

[0275] This study plans to select 31 fasting blood samples from hemophilia patients who meet the requirements for in vitro pharmacodynamics testing. The hemophilia patients include 10 patients with hemophilia A (HA), 6 patients with hemophilia B (HB), 6 patients with hemophilia A with inhibitors (HAW), 3 patients with hemophilia B with inhibitors (HBW), and 6 patients with von Willebrand disease (VWD) type 3. (Considering the special characteristics of VWD patients, the number of patients can be adjusted according to actual clinical conditions and is tentatively set at 6.) Each blood donor who meets the enrollment criteria will need to collect approximately 20ml of fasting whole blood (using a 3.2% sodium citrate anticoagulant tube), centrifuge at the research center, obtain platelet-poor plasma (PPP), and send it to a third-party laboratory for in vitro pharmacodynamics testing.

[0276] Plasma samples from hemophilia patients were treated with different concentrations of KN057 or other clinically investigated drugs with the same target in vitro. The in vitro efficacy of KN057 was evaluated by measuring the plasma diluted prothrombin time (dPT) and thrombin generation assay (TGA), and the differences in in vitro efficacy between KN057 and drugs with the same target were compared.

[0277] Sample size

[0278] A total of 29 cases were actually enrolled and 29 cases were analyzed. The specific distribution is shown in the table below.

[0279] Inclusion criteria

[0280] Blood donors must meet all of the following criteria (2 or 3 of them must be met) to be eligible:

[0281] 1) ≥18 years old;

[0282] 2) Severe HA or HB (coagulation factor VIII or IX activity level <1 IU / dL; if the result is ≥1 IU / dL, a previous test certificate of coagulation factor VIII or IX activity level <1 IU / dL is acceptable);

[0283] Blood donors without inhibitors of factor VIII or IX must also meet the following inclusion criteria: no inhibitors detected, or documentation (test results within one month are acceptable) proving that the donor has no inhibitors;

[0284] Patients donating blood with inhibitors of factor VIII or IX must also meet the following inclusion criteria: a positive inhibitor test result (≥0.6BU / mL, with documentation of the test within six months acceptable);

[0285] 3) For patients with von Willebrand disease (VWD3 type only): the plasma VWF antigen (VWF:Ag) level and coagulation factor VIII activity level of the blood donor must be obtained;

[0286] 4) Voluntarily sign the informed consent form before the trial.

[0287] In vitro efficacy results

[0288] The dPT results showed:

[0289] a) In vitro treatment of plasma from hemophilia patients (HA, HB, HAW, HBW) with KN057 showed no significant change in plasma dPT at concentrations between 0 and 0.224 nM. However, dPT was significantly shortened at 1.12 nM, reaching its maximum shortening at 5.6 nM. The dPT plateaued at concentrations between 5.6 and 140 nM, indicating that KN057 shortens dPT in hemophilia plasma in a concentration-dependent manner. Furthermore, the effect trend and effective concentration range of KN057 were largely consistent across the plasma of patients with various hemophilia types. The effect of KN057 in the plasma of patients with VWD3 was similar to that in patients with hemophilia (Figure 1).

[0290] b) The effect of KN057 on dPT in the plasma of hemophilia patients without inhibitors (HA and HB) and with inhibitors (HAW and HBW) was basically the same, and there was no significant difference in the effect of KN057 (5.6nM and 28nM) in shortening dPT.

[0291] c) Comparison of the effects of KN057 and PF-06741086 at the same molar concentration on plasma dPT in patients with different types of hemophilia showed that KN057 was more effective in shortening dPT than PF-06741086 (as shown in Figures 2A-2E).

[0292] TGA results show:

[0293] a) In the plasma of patients with various hemophilia types, KN057 significantly increased peak thrombin levels and ETP at concentrations of 1.12 to 5.6 nM. The effect became more gradual at concentrations of 5.6 to 28 nM, reaching a peak at 28 nM. The average peak thrombin levels ranged from 85 to 120 nM, and the average peak ETP levels ranged from 1400 to 1650 nM / min. This suggests that KN057 increases thrombin generation in hemophilia plasma in a concentration-dependent manner. The effect trend and effective concentration range of KN057 were generally consistent across the plasma of patients with various hemophilia types. The effect of KN057 in the plasma of patients with VWD3 was similar to that in patients with hemophilia (as shown in Figures 3A and 3B).

[0294] b) The effect of KN057 on thrombin generation in the plasma of hemophilia patients without inhibitors (HA and HB) and with inhibitors (HAW and HBW) showed a consistent trend, and there was no significant difference in the effects of KN057 (5.6nM and 28nM) on increasing the peak thrombin level and ETP.

[0295] c) Comparison of the effects of KN057 and PF-06741086 at the same molar concentration on plasma thrombin generation in patients with different types of hemophilia showed that KN057 increased thrombin peak and ETP more potently than PF-06741086 (as shown in Figures 4A-4E and 5A-5E).

[0296] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for treating hemophilia, comprising the step of administering to a subject in need thereof an antibody or an antigen-binding fragment thereof directed against tissue factor pathway inhibitor (TFPI), wherein the antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4; The light chain variable region includes: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:

8. 2 . The method according to claim 1 , wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

1. 3 . The method according to claim 1 , wherein the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

5.

4. The method according to any one of claims 1 to 3, wherein the heavy chain of the antibody further comprises a constant region of human IgG4 or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO:

9.

5. The method according to any one of claims 1 to 4, wherein the light chain of the antibody further comprises a constant region of human Igκ or a variant thereof, for example, comprising the amino acid sequence shown in SEQ ID NO:

10.

6. The method according to any one of claims 1 to 5, wherein the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

11.

7. The method according to any one of claims 1 to 6, wherein the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

12.

8. The method according to any one of claims 1 to 7, wherein the hemophilia is hemophilia A.

9. The method according to claim 8, wherein the hemophilia is hemophilia A with inhibitors or hemophilia A without inhibitors.

10. The method according to any one of claims 1 to 7, wherein the hemophilia is hemophilia B.

11. The method according to claim 10, wherein the hemophilia is hemophilia B with inhibitors or hemophilia B without inhibitors.

12. The method according to any one of claims 1 to 7, wherein the hemophilia is von Willebrand disease.

13. The method of claim 12, wherein the hemophilia is von Willebrand disease type 1, type 2, or type 3.

14. The method according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.0 mg / kg to 2.0 mg / kg based on the subject's body weight.

15. The method according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg based on the subject's body weight.

16. The method according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof is administered once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight.

17. The method of any one of claims 1-16, wherein the antibody or antigen-binding fragment thereof is administered by subcutaneous injection.

18. The method according to claim 17, wherein the treatment method comprises subcutaneous injection of the antibody or antigen-binding fragment thereof once a week at a dose of 1.5 mg / kg to 2.0 mg / kg based on the subject's body weight.

19. The method according to any one of claims 1 to 18, wherein after administration of the antibody or antigen-binding fragment thereof, the annualized bleeding rate of the subject is no higher than 10.0, preferably no higher than 8.0, more preferably no higher than 5.0, and even more preferably no higher than 2.

0.

20. The method according to any one of claims 1-19, wherein after administration of the antibody or antigen-binding fragment thereof, the annualized bleeding rate of the subject decreases by no less than 60%, preferably no less than 70%, more preferably no less than 80%, and even more preferably no less than 90% compared with that before administration.

21. An antibody or antigen-binding fragment thereof against tissue factor pathway inhibitor (TFPI) for treating hemophilia, the antibody comprising a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4; The light chain variable region includes: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:

8.

22. Use of an antibody or an antigen-binding fragment thereof against tissue factor pathway inhibitor (TFPI) in the preparation of a drug for treating hemophilia, wherein the antibody comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region comprises: a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 4; The light chain variable region includes: a VL CDR1 comprising the amino acid sequence shown in SEQ ID NO:6, a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO:7, and a VL CDR3 comprising the amino acid sequence shown in SEQ ID NO:8.

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