WO2023098637A1 - 预防和/或治疗血栓栓塞性疾病的方法 - Google Patents

预防和/或治疗血栓栓塞性疾病的方法 Download PDF

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WO2023098637A1
WO2023098637A1 PCT/CN2022/134868 CN2022134868W WO2023098637A1 WO 2023098637 A1 WO2023098637 A1 WO 2023098637A1 CN 2022134868 W CN2022134868 W CN 2022134868W WO 2023098637 A1 WO2023098637 A1 WO 2023098637A1
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seq
single variable
variable domain
fxi
immunoglobulin single
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French (fr)
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徐霆
尹群
鄢荣梅
李倩
王燕
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Suzhou Alphamab Co Ltd
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Suzhou Alphamab Co Ltd
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Priority to US18/714,678 priority patent/US20250034277A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to the field of biomedicine, in particular to a method for preventing and/or treating thromboembolic diseases.
  • the human blood coagulation pathway is divided into two kinds of blood coagulation pathways: endogenous and exogenous.
  • the extrinsic coagulation pathway starts with the release of TF after tissue injury, and TF forms a complex with FVII, thereby activating FIX and FX.
  • the intrinsic coagulation pathway is mainly initiated by the activation of FXII factor bound by negatively charged molecules.
  • FXII is activated into FXIIa, activated FXIIa activates FXI to become FXIa, FXIa then activates downstream FIX, and activates FX under the action of cofactor FVIII.
  • the activated FXa and FVa of the exogenous and intrinsic coagulation pathways jointly constitute the prothrombin complex to activate prothrombin (FII), generate active thrombin and fibrin clot, and finally cause coagulation.
  • FXI is one of the factors in the endogenous blood coagulation pathway, which exists in the form of a dimer. Its monomer consists of four Apple domains and a serine protease domain.
  • the activated FXIa has proteolytic activity and activates its main substrate.
  • the substance FIX promotes the generation of thrombin.
  • FXIa can also activate FX, FV and FVIII.
  • FXI deficiency may protect against thrombotic disease.
  • FXI deficiency may protect against thrombotic disease.
  • there is no marketed drug targeting FXI so there is an urgent need to develop anti-thrombotic disease drugs targeting FXI.
  • the present application provides a method for preventing and/or treating thromboembolic diseases.
  • Using the coagulation factor XI (FXI) binding protein described in this application can effectively prevent and/or treat thromboembolic diseases.
  • thrombus formation can be prevented in vivo; thrombus weight can be effectively reduced; APTT can be prolonged; and/or platelet aggregation rate can be reduced.
  • the present application provides a method for preventing and/or treating thromboembolic diseases, which includes the following steps: administering coagulation factor XI (FXI) binding protein to subjects in need, which may contain at least one specific An immunoglobulin single variable domain that binds FXI, the at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in any one of SEQ ID NO: 4, 10 and 14 .
  • FXI coagulation factor XI
  • the FXI binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein the first immunoglobulin single variable domain comprises SEQ ID NO CDR1, CDR2 and CDR3 in the VHH shown in NO:14, and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:17.
  • the thromboembolic disease comprises venous thromboembolism (VTE).
  • VTE venous thromboembolism
  • the venous thromboembolism is a complication of tumor, and/or a complication of joint replacement.
  • the venous thromboembolism is associated with a peripherally implanted central catheter (PICC).
  • PICC peripherally implanted central catheter
  • the subject has, or is at risk of having, venous thromboembolism (VTE).
  • VTE venous thromboembolism
  • the subject is a cancer patient.
  • the subject has a peripherally implanted central catheter (PICC).
  • PICC peripherally implanted central catheter
  • the venous thromboembolism is associated with hip and/or knee arthroplasty.
  • the subject is a joint replacement recipient.
  • the subject has had a hip and/or knee replacement.
  • the thromboembolic disease comprises deep vein thrombosis (DVT).
  • DVD deep vein thrombosis
  • the deep vein thrombosis comprises acute deep vein thrombosis and/or recurrence of deep vein thrombosis after occurrence of acute deep vein thrombosis.
  • the venous thromboembolism comprises pulmonary thromboembolism (PTE).
  • PTE pulmonary thromboembolism
  • the subject has, or is at risk of having, deep vein thrombosis (DVT).
  • DVD deep vein thrombosis
  • the subject has acute deep vein thrombosis and/or recurrence of deep vein thrombosis after occurrence of acute deep vein thrombosis.
  • the subject has pulmonary thromboembolism (PTE).
  • PTE pulmonary thromboembolism
  • the thromboembolic disease comprises systemic thromboembolism.
  • the systemic thromboembolism is a complication of tumor, a complication of atrial fibrillation, and/or a complication of dialysis.
  • the atrial fibrillation comprises non-valvular atrial fibrillation.
  • the subject has, or is at risk of having, systemic thromboembolism.
  • the subject has non-valvular atrial fibrillation.
  • the subject is a dialysis patient.
  • the subject is an adult.
  • the subject has hypertension, diabetes, congestive heart failure, atrial fibrillation, and/or has a history of stroke.
  • the subject is at least 75 years old.
  • the subject is permanently bedridden.
  • the CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs, or IMGT CDRs.
  • CDR1, CDR2, and CDR3 in the VHH set forth in SEQ ID NO:4 are selected from any of the following groups: SEQ ID NO:60-62, SEQ ID NO:63-65, SEQ ID NO :66-68, SEQ ID NO:69-71.
  • the at least one immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 4, 306-311.
  • CDR1, CDR2, and CDR3 in the VHH set forth in SEQ ID NO: 10 are selected from any of the following groups: SEQ ID NOs: 132-134, SEQ ID NOs: 135-137, SEQ ID NOs : 138-140, SEQ ID NO: 141-143.
  • the at least one immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 10, 312-317.
  • CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 14 are selected from any of the following groups: SEQ ID NO: 180-182, SEQ ID NO: 183-185, SEQ ID NO : 186-188, SEQ ID NO: 189-191.
  • the at least one immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 14, 318-328.
  • the factor XI (FXI) binding protein binds the Apple2 domain of FXI.
  • the coagulation factor XI (FXI) binding protein may further comprise an immunoglobulin Fc region.
  • amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO: 336.
  • the factor XI (FXI) binding protein is administered at a dose of about 0.5 mg/Kg to about 10 mg/kg.
  • the method further comprises administering to the subject: a nucleic acid molecule encoding the coagulation factor XI (FXI) binding protein described in the present application, which may comprise all the expression regulatory elements operably linked
  • a nucleic acid molecule encoding the coagulation factor XI (FXI) binding protein described in the present application, which may comprise all the expression regulatory elements operably linked
  • the pharmaceutical composition of protein and pharmaceutically acceptable carrier may comprise all the expression regulatory elements operably linked
  • the present application provides a use of a blood coagulation factor XI (FXI) binding protein in the preparation of a medicine for preventing and/or treating thromboembolic diseases, wherein the blood coagulation factor XI (FXI) binds
  • the protein may comprise at least one immunoglobulin single variable domain capable of specifically binding FXI, said at least one immunoglobulin single variable domain may comprise any one of SEQ ID NO:4, 10 and 14 CDR1, CDR2 and CDR3 in VHH.
  • the present application provides a blood coagulation factor XI (FXI) binding protein, which is used for preventing and/or treating thromboembolic diseases, wherein the blood coagulation factor XI (FXI) binding protein may comprise at least one specific An immunoglobulin single variable domain that binds FXI, the at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in any one of SEQ ID NO: 4, 10 and 14 .
  • Figure 1 shows the results of prolongation of human plasma APTT by coagulation factor XI (FXI) binding protein described in the present application.
  • FXI coagulation factor XI
  • Figure 2 shows the prolongation effect of coagulation factor XI (FXI) binding protein on cynomolgus monkey plasma APTT.
  • FXI coagulation factor XI
  • Fig. 3 shows the results of prolongation effect of blood coagulation factor XI (FXI) binding protein on rabbit plasma APTT.
  • Figure 4 shows the prolongation effect of coagulation factor XI (FXI) binding protein described in the present application on mouse plasma APTT.
  • FXI coagulation factor XI
  • Fig. 5 shows the inhibitory effect of coagulation factor XI (FXI) binding protein described in the present application on thrombus weight.
  • Figure 6 shows the prolongation effect of coagulation factor XI (FXI) binding protein on APTT according to the present application.
  • Fig. 7 shows the influence of coagulation factor XI (FXI) binding protein described in the present application on platelet aggregation ability.
  • Figure 8 shows the effect of coagulation factor XI (FXI) binding protein described in the present application on PT.
  • FXI coagulation factor XI
  • Figure 9 shows the effect of coagulation factor XI (FXI) binding proteins described herein on bleeding.
  • thromboembolic disease includes diseases caused by both the pathological processes of thrombosis and thromboembolism.
  • Thrombosis usually refers to the pathological process in which formed components of blood form emboli in blood vessels (mostly small blood vessels) under certain conditions, resulting in partial or complete blockage of blood vessels and obstruction of blood supply to corresponding parts.
  • Thromboembolism usually refers to the detachment of thrombus from the formation site, partially or completely blocking certain blood vessels during the process of moving with the blood flow, causing ischemia, hypoxia, necrosis (arterial thrombosis), congestion, and edema of corresponding tissues and (or) organs (venous thrombosis) pathological process.
  • thromboembolic disease can comprise (1) venous thromboembolic disease according to the blood vessel type that takes place thrombosis: i.e. venous thromboembolic disease, for example can comprise pulmonary thromboembolism, deep vein thrombosis; (2) arterial thromboembolic disease : For example, it may include acute coronary syndrome, atrial fibrillation, arterial ischemic attack, and stroke; (3) Microvascular thrombotic disease: For example, it may include disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, etc.
  • DIC disseminated intravascular coagulation
  • thrombotic thrombocytopenic purpura etc.
  • VTE venous thromboembolism
  • VTE venous thromboembolism
  • abnormal coagulation of blood in veins causing complete or incomplete blockage of blood vessels, which belongs to venous reflux disorders.
  • the venous thromboembolism can often occur in the deep veins of the lower extremities.
  • An embolus may break off from where it formed and enter the bloodstream, which is called an embolism.
  • the venous thromboembolism can be caused by disruption of blood flow (eg, prolonged bed rest with a cast or stent, especially in combination with dehydration and previous venous disease (chronic venous insufficiency)), damage to the vein wall (eg, , surgery, trauma, or inflammation), or thrombophilia (eg, an imbalance between coagulation and fibrinolysis due to procoagulants or extreme drugs).
  • disruption of blood flow eg, prolonged bed rest with a cast or stent, especially in combination with dehydration and previous venous disease (chronic venous insufficiency)
  • damage to the vein wall eg, surgery, trauma, or inflammation
  • thrombophilia eg, an imbalance between coagulation and fibrinolysis due to procoagulants or extreme drugs.
  • the term "complication” generally refers to the development of one disease that causes the occurrence of another disease or symptom, and the latter is the complication of the former. In some cases, the complication may also include the combination of one disease and another disease or several diseases related to this disease during the course of treatment.
  • peripheral inserted central catheter generally refers to a catheter inserted into a central vein via peripheral venous puncture (ie Peraipherally Inserted Central Catheter).
  • the peripheral vein implanted central venous catheter can be inserted from the vein of the elbow or upper arm, and then advance along the direction of the vein, and the catheter is finally sent to the great blood vessels close to the heart.
  • the PICC can be long-term, safe and painless, and can be applied to patients with long-term intravenous therapy and infusion of hypertonic and irritating drugs.
  • the PICC may cause complications such as infection, thrombosis, and phlebitis.
  • joint replacement generally refers to the technology of artificial joint prosthesis made according to the shape, structure and function of human joints and surgically implanted into the human body.
  • the artificial joint prosthesis can be made of artificial materials, such as metal, high molecular polyethylene and/or ceramics.
  • the term "deep vein thrombosis (DVT)” generally refers to a condition caused by abnormal clotting of blood in deep veins.
  • the deep vein thrombosis can occur in the lower extremities, and often occurs after major orthopedic surgery.
  • the detachment of the deep vein thrombosis can cause pulmonary embolism (pulmonary embolism, PE), collectively known as venous thromboembolism.
  • PE pulmonary embolism
  • the deep vein thrombosis can be caused by the following factors: venous blood flow stagnation, vein wall damage and blood hypercoagulation state.
  • pulmonary thromboembolism generally refers to a disease characterized by pulmonary circulation and respiratory dysfunction as the main clinical and pathophysiological features caused by thrombus from the venous system or right heart blocking the pulmonary artery or its branches.
  • the thrombus that causes PTE mainly comes from deep vein thrombosis (DVT), and PTE and DVT can be collectively referred to as venous thromboembolism (VTE).
  • DVT deep vein thrombosis
  • VTE venous thromboembolism
  • the causative factors of PTE may include genetic factors (for example, genetic variation) and acquired factors (for example, surgery, trauma, acute medical diseases (such as heart failure, respiratory failure, infection, etc.), certain chronic diseases (such as anti- phospholipid syndrome, nephrotic syndrome, etc.) and malignant tumors).
  • genetic factors for example, genetic variation
  • acquired factors for example, surgery, trauma, acute medical diseases (such as heart failure, respiratory failure, infection, etc.), certain chronic diseases (such as anti- phospholipid syndrome, nephrotic syndrome, etc.) and malignant tumors).
  • the term "atrial fibrillation” generally refers to atrial fibrillation, a common cardiac arrhythmia. Refers to the loss of regular and orderly atrial electrical activity, replaced by rapid and disorderly fibrillation waves, which is a serious atrial electrical activity disorder.
  • the atrial fibrillation may be associated with coronary artery disease, hypertension and/or heart failure.
  • the atrial fibrillation can be divided into first-diagnosed atrial fibrillation, paroxysmal atrial fibrillation, persistent atrial fibrillation, long-term persistent atrial fibrillation and permanent atrial fibrillation.
  • the atrial fibrillation can lead to complications such as heart failure and arterial embolism, and even induce sudden death in severe cases.
  • nonvalvular atrial fibrillation generally refers to atrial fibrillation occurring in the absence of rheumatic mitral stenosis, biological or mechanical valve replacement, or mitral valve repair.
  • Non-valvular atrial fibrillation can be treated with non-vitamin K antagonist oral anticoagulants (DOACs) for stroke prevention.
  • DOACs oral anticoagulants
  • dialysis generally refers to a separation and purification technique that separates small molecules from biological macromolecules. It is an artificial process that replaces the kidneys to remove metabolic waste and excess water from the body when the kidneys are not working properly.
  • the dialysis (therapy) may include a therapeutic method in which components in body fluids (solutes or water) are excreted through a semipermeable membrane, generally including hemodialysis, peritoneal dialysis and/or colon dialysis.
  • congestive heart failure usually refers to tissue and organ blood perfusion insufficiency, accompanied by pulmonary circulation and/or systemic circulation congestion, which is a clinical syndrome of various heart diseases developing to a severe stage disease.
  • the congestive heart failure may be caused by poor ventricular pumping or filling function, and the cardiac output cannot meet the metabolic needs of the body.
  • the congestive heart failure may be characterized by left ventricular hypertrophy or dilation, which may lead to neuroendocrine disorders and abnormal circulatory function, such as typical clinical symptoms: dyspnea, fluid retention, and fatigue.
  • hypotension generally refers to a systemic arterial blood pressure (systolic and/or diastolic) as the main feature (in the absence of antihypertensive drugs, systolic blood pressure ⁇ 140 mm Hg and/or or diastolic blood pressure ⁇ 90 mm Hg), may be accompanied by a clinical syndrome of functional or organic damage to the heart, brain, kidney and other organs.
  • the high blood pressure may be the most important risk factor for cardiovascular and cerebrovascular diseases.
  • diabetes generally refers to a group of metabolic diseases characterized by chronic hyperglycemia caused by disorders of insulin secretion and (or) utilization caused by multiple etiologies. Long-term carbohydrate, fat, and protein metabolic disorders can cause multi-system depletion, leading to chronic progressive disease, functional decline, and failure of tissues and organs such as eyes, kidneys, nerves, heart, and blood vessels.
  • the diabetes may comprise type 1, type 2, other specific types, gestational diabetes.
  • long-term bed rest generally refers to a clinical phenomenon in which the ability of daily life is reduced due to long-term illness or disability, and partial or complete assistance is required.
  • antibody or “immunoglobulin” used interchangeably in this application, whether referring to heavy chain antibodies or conventional 4 chain antibodies, are used as generic terms to include full length antibodies, their Individual chains as well as all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments such as VHH domains or VH/VL domains, respectively).
  • sequence as used in this application (for example in terms of “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) should generally refer to It is understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding said sequence, unless the application requires a more limited interpretation.
  • domain refers to a folded protein structure that is capable of maintaining its tertiary structure independently of the rest of the protein.
  • domains are responsible for individual functional properties of a protein, and in many cases can be added, removed or transferred to other proteins without loss of function of the remainder of the protein and/or domain.
  • immunoglobulin domain refers to the globular region of an antibody chain (eg, that of a conventional 4-chain antibody or that of a heavy chain antibody), or refers to a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized in that they maintain the immunoglobulin fold characteristic of antibody molecules.
  • immunoglobulin variable domain refers to the term "framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region” and 3” or “FR3”, and four “framework regions” of "framework region 4" or “FR4", wherein the framework regions are referred to in the art and hereinafter as “complementarity determining regions 1” respectively “ or “ CDR1 ", “complementarity determining region 2” or “CDR2”, and “complementarity determining region 3" or “CDR3” are separated by three “complementarity determining regions” or "CDRs".
  • an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity to an antibody for an antigen by having an antigen-binding site.
  • immunoglobulin single variable domain refers to an immunoglobulin variable domain capable of specifically binding an antigenic epitope without pairing with another immunoglobulin variable domain.
  • An example of an immunoglobulin single variable domain within the meaning of the present application is a "domain antibody”, eg an immunoglobulin single variable domain VH and VL (VH domain and VL domain).
  • Another example of an immunoglobulin single variable domain is a "VHH domain” (or simply "VHH") of Camelidae as defined below.
  • VHH domains also known as heavy chain single domain antibodies, VHH, VHH domains, VHH antibody fragments, and VHH antibodies, are antigen-binding immunoantibodies known as "heavy chain antibodies” (ie, "antibodies lacking light chains”).
  • Variable domains of globulins Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448( 1993)).
  • VHH domain is used to distinguish the variable domain from the heavy chain variable domain present in conventional 4 chain antibodies (which is referred to in this application as a "VH domain”) and the variable domain present in conventional 4 chain antibodies.
  • light chain variable domains (which are referred to in this application as "VL domains") in antibodies.
  • a VHH domain specifically binds an epitope without the need for another antigen binding domain (in contrast to the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain).
  • the VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain.
  • VHH domain In the context of this application, the terms “heavy chain single domain antibody”, “VHH domain”, “VHH”, “VHH domain”, “VHH antibody fragment”, and “VHH antibody” are used interchangeably.
  • the amino acid residues used in the VHH domain of Camelidae can be based on the VH domain given by Kabat et al.
  • the general numbering method to number (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • AbM CDRs represent a compromise between Kabat hypervariable regions and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs were then based on analysis of the available crystal structures of the complexes. The residues of the CDRs from each method are described below:
  • the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e. according to One or more positions of the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows).
  • numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence.
  • CDR may include "extended (extended) CDR", such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 ( LCDR3); 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.
  • extended CDR such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 ( LCDR3)
  • HCDR1 50-65 or 49-65
  • HCDR3 93-102
  • HCDR3 94-102 or 95-102
  • the total number of amino acid residues in the VHH domain will generally range from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domains and polypeptides containing them can be summarized as follows:
  • VHH domains (which have been naturally "designed" to functionally bind antigen in the absence and interaction of light chain variable domains) can serve a single and relatively small function Antigen-binding structural units, domains or polypeptides. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suitable for practical use as a single antigen-binding protein or as a single variable domain of an immunoglobulin, but require some kind of form or another combination to provide a functional antigen binding unit (eg in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain).
  • a functional antigen binding unit eg in the form of a conventional antibody fragment such as a Fab fragment; or in the form of a scFv consisting of a VH domain covalently linked to a VL domain.
  • VHH domains - alone or as part of a larger polypeptide - offers many advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments)
  • Significant advantage of only a single domain is required to bind antigen with high affinity and high selectivity, making it neither necessary to have two separate domains nor to ensure that the two domains exist in the proper spatial conformation and configuration (e.g.
  • VHH domains can be expressed from a single gene and do not require post-translational folding or modification; VHH domains can be easily engineered into multivalent and multispecific formats (formatting); VHH structure Domains are highly soluble and have no tendency to aggregate; VHH domains are highly stable to heat, pH, proteases, and other denaturing agents or conditions, and thus can be prepared, stored, or shipped without the use of refrigeration equipment, thereby achieving cost, time, and environmental savings ; VHH domains are easy and relatively inexpensive to prepare, even on the scale required for production; VHH domains are relatively small compared to conventional 4-chain antibodies and antigen-binding fragments thereof (approximately 15 kDa or 1/3 the size of conventional IgG 10), thus exhibiting higher tissue permeability and can be administered in higher doses compared to conventional 4-chain antibodies and antigen-binding fragments thereof; VHH domains may exhibit so-called lumen-binding properties (especially due to the similarity with conventional VH
  • a VHH domain derived from Camelidae can be modified by substituting one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in a human conventional 4-chain antibody VH domain.
  • Humanization also referred to in this application as “sequence optimization", in addition to humanization, “sequence optimization” may also cover other modifications to the sequence by one or more mutations that provide improved properties of the VHH, such as transplantation except for potential post-translational modification sites).
  • a humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be accomplished using protein surface amino acid resurfacing and/or humanized universal framework CDR grafting (CDR grafting to a universal framework), for example, as exemplified in the Examples.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be a "linear " epitope or "conformational” epitope. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed.
  • Epitopes for a given antigen can be identified using a number of epitope mapping techniques well known in the art. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • linear epitopes can be determined, for example, by simultaneously synthesizing a large number of peptides on a solid support, wherein these peptides correspond to parts of the protein molecule, and allowing these peptides, while still attached to the support, to antibody response.
  • These techniques are known in the art and are described, for example, in U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci.
  • conformational epitopes can be identified by determining the spatial configuration of amino acids such as by, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See eg Epitope Mapping Protocols (ibid).
  • Antibodies can be competitively screened for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for antigen binding. A high-throughput method to obtain antibodies binding to the same epitope based on their cross-competition is described in International Patent Application WO 03/48731. Therefore, antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present application for binding to the same epitope on FXI can be obtained using routine techniques known to those skilled in the art.
  • the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (eg, an immunoglobulin single variable domain of the application) can bind. Specificity of an antigen binding protein can be determined based on its affinity and/or avidity.
  • the affinity expressed by the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the greater the distance between the epitope and the antigen-binding protein.
  • affinity can also be expressed as an association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner, depending on the particular antigen of interest.
  • Avidity is a measure of the strength of binding between an antigen binding protein (eg, an immunoglobulin, antibody, immunoglobulin single variable domain, or a polypeptide comprising the same) and the associated antigen.
  • Avidity is related to both the affinity between its antigen binding site on the antigen binding protein and the number of associated binding sites present on the antigen binding protein.
  • coagulation factor XI (FXI) binding protein means any protein capable of specifically binding coagulation factor XI (FXI).
  • a FXI binding protein may comprise an antibody as defined herein against FXI.
  • FXI binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • a "FXI binding protein” of the present application may comprise at least one immunoglobulin single variable domain such as VHH that binds FXI.
  • the "FXI binding molecule” of the present application may comprise 2, 3, 4 or more immunoglobulin single variable domains such as VHH that bind FXI.
  • the FXI-binding proteins of the present application may also contain linkers and/or moieties with effector functions, such as half-life extending moieties (such as immunoglobulin single variable domains that bind serum albumin), in addition to FXI-binding immunoglobulin single variable domains.
  • the "FXI-binding protein" of the present application also covers bispecific antibodies, which contain immunoglobulin single variable domains that bind different antigens or different regions of the same antigen (such as different epitopes).
  • the FXI-binding proteins of the application will be in the range of from 10 ⁇ 7 to 10 ⁇ 10 moles/liter (M), from 10 ⁇ 8 to 10 ⁇ 10 moles/liter, or even as measured in a Biacore or KinExA or Fortibio assay.
  • An association constant (KA) of -1 binds the desired antigen (ie, FXI). Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding.
  • Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays and/or competitive binding assays as described herein (for example radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competitive assays.
  • SPR surface plasmon resonance
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • sandwich competitive assays sandwich competitive assays.
  • sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences.
  • sequence similarity indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.
  • sequence identity For determination of sequence identity see, for example: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., EDS., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology , Von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • a polypeptide or nucleic acid molecule is considered “isolated” when it is isolated from another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component).
  • a polypeptide or nucleic acid molecule is considered “isolated” when it has been purified at least 2-fold, especially at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more.
  • An "isolated" polypeptide or nucleic acid molecule may be substantially homogeneous as determined by a suitable technique (eg, a suitable chromatographic technique, such as polyacrylamide gel electrophoresis).
  • Effective amount means the amount of the FXI binding protein or pharmaceutical composition of the present application that can lead to a reduction in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic phase of the disease, or to prevent damage or disability caused by the suffering of the disease .
  • thrombosis refers to the formation or presence of a clot (also referred to as a "thrombus") within a blood vessel that blocks the flow of blood through the circulatory system.
  • thrombosis is usually caused by abnormalities in blood composition, quality of vessel walls, and/or properties of blood flow. Clot formation is usually caused by injury to the vessel wall, such as from trauma or infection, and a slowing or stagnation of blood flow through the point of injury. In some instances, blood clotting abnormalities cause thrombus formation.
  • the present application provides a method for preventing and/or treating thromboembolic diseases, which includes the following steps: administering coagulation factor XI (FXI) binding protein to subjects in need, which may contain at least one specific An immunoglobulin single variable domain that binds FXI, the at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in any one of SEQ ID NO: 4, 10 and 14 .
  • FXI coagulation factor XI
  • the present application provides a use of a blood coagulation factor XI (FXI) binding protein in the preparation of a medicine for preventing and/or treating thromboembolic diseases, wherein the blood coagulation factor XI (FXI) binds
  • the protein may comprise at least one immunoglobulin single variable domain capable of specifically binding FXI, said at least one immunoglobulin single variable domain may comprise any one of SEQ ID NO:4, 10 and 14 CDR1, CDR2 and CDR3 in VHH.
  • the present application provides a blood coagulation factor XI (FXI) binding protein, which is used for preventing and/or treating thromboembolic diseases, wherein the blood coagulation factor XI (FXI) binding protein may comprise at least one specific An immunoglobulin single variable domain that binds FXI, the at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in any one of SEQ ID NO: 4, 10 and 14 .
  • the FXI binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein the first immunoglobulin single variable domain comprises SEQ ID NO: 14 and the second immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:17.
  • the method may also include administering to the subject: a nucleic acid molecule encoding the coagulation factor XI (FXI) binding protein described in the present application, which may comprise the expression regulatory element operably linked
  • a nucleic acid molecule encoding the coagulation factor XI (FXI) binding protein described in the present application, which may comprise the expression regulatory element operably linked
  • the thromboembolic disease may include venous thromboembolism (VTE).
  • VTE venous thromboembolism
  • said venous thromboembolism may be a complication of a tumor, and/or a complication of a joint replacement.
  • the venous thromboembolism may be associated with a peripherally implanted central catheter (PICC).
  • PICC peripherally implanted central catheter
  • the venous thromboembolism may be associated with hip and/or knee arthroplasty.
  • muscle contraction activity in the lower extremity may be reduced, and the bleeding from the replacement incision may make the blood more likely to clot, which may lead to deep vein thrombosis in the lower extremity.
  • the thromboembolic disease can include deep vein thrombosis (DVT).
  • DVD deep vein thrombosis
  • the deep vein thrombosis may include acute deep vein thrombosis and/or recurrence of deep vein thrombosis after occurrence of acute deep vein thrombosis.
  • the venous thromboembolism can include pulmonary thromboembolism (PTE).
  • PTE pulmonary thromboembolism
  • a deep vein thrombosis can follow blood back up into the lungs, where it can get stuck in non-main blood vessels.
  • the pulmonary thromboembolism may lead to fatal pulmonary embolism.
  • the thromboembolic disease can include systemic thromboembolism.
  • the systemic thromboembolism may be a complication of a tumor, a complication of atrial fibrillation, and/or a complication of dialysis.
  • the atrial fibrillation may include non-valvular atrial fibrillation.
  • VTE venous thromboembolism
  • peripheral vein of the subject may be implanted with a central venous catheter (PICC).
  • PICC central venous catheter
  • the subject may be a joint replacement patient.
  • the subject's hip and/or knee may be replaced.
  • the subject may have deep vein thrombosis (DVT), or be at risk of having DVT.
  • DVT deep vein thrombosis
  • the subject may suffer from acute deep vein thrombosis and/or recurrence of deep vein thrombosis after occurrence of acute deep vein thrombosis.
  • the subject may suffer from pulmonary thromboembolism (PTE).
  • PTE pulmonary thromboembolism
  • the subject may suffer from systemic thromboembolism, or be at risk of suffering from systemic thromboembolism.
  • the subject may suffer from non-valvular atrial fibrillation.
  • the subject may be a dialysis patient.
  • the dialysis patient may have undergone and/or undergo hemodialysis.
  • the hemodialysis may include a step of removing various harmful and redundant metabolic wastes and excess electrolytes in the blood out of the body through diffusion using the principle of a semi-permeable membrane.
  • the hemodialysis can achieve the purpose of purifying the blood and correcting electrolyte and acid-base balance disorders.
  • the dialysis patient may have undergone and/or undergo peritoneal dialysis.
  • the peritoneal dialysis may include the steps of using the peritoneum as a semi-permeable membrane, pouring the prepared dialysate into the patient's peritoneal cavity through a catheter using gravity, and constantly replacing the peritoneal dialysate.
  • the peritoneal dialysis can achieve the purpose of removing metabolites and toxic substances in the body and correcting the disturbance of water and electrolyte balance.
  • the dialysis patients may have cardio-cerebral complications. For example, symptoms of high blood pressure, cerebral hemorrhage, and/or heart failure may develop or have been present during dialysis.
  • the subject may be an adult.
  • the age of the adult may be over eighteen years old.
  • the subject may be a tumor patient.
  • the tumor may be a malignant tumor.
  • the tumor may include solid tumors and/or non-solid tumors.
  • the subject may suffer from hypertension, diabetes, congestive heart failure, atrial fibrillation and/or have a history of stroke.
  • the stroke may include stroke.
  • the stroke may be caused by carotid stenosis, atrial fibrillation, cerebral hemorrhage and/or ischemic stroke.
  • For the treatment method of stroke please refer to the "China Stroke Series Guidelines-2015 Release Edition".
  • the subject may be at least 75 years old (eg, may be at least 75 years old, at least 80 years old, at least 85 years old or older).
  • the subject can be bedridden for a long time (eg, can be bedridden for at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years or longer).
  • the dosage of the coagulation factor XI (FXI) binding protein may be about 0.5 mg/Kg-about 10 mg/kg.
  • it can be about 0.5mg/Kg-about 8.0mg/kg, about 0.5mg/Kg-about 7.5mg/kg, about 0.5mg/Kg-about 7.0mg/kg, about 0.5mg/Kg-about 6.5mg/kg kg, about 0.5mg/Kg-about 6mg/kg, about 0.5mg/Kg-about 5.5mg/kg, about 0.5mg/Kg-about 5.0mg/kg, about 0.5mg/Kg-about 4.5mg/kg, about 0.5mg/Kg-approximately 4.0mg/kg, approximately 0.5mg/Kg-approximately 3.5mg/kg, approximately 0.5mg/Kg-approximately 3.0mg/kg, approximately 0.5mg/Kg-approx
  • the present application provides a FXI-binding protein, which may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI.
  • the at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in any one of SEQ ID NO: 1-23.
  • the CDR may be a Kabat CDR, AbM CDR, Chothia CDR or IMGT CDR.
  • the at least one immunoglobulin single variable domain may comprise a group of CDR1, CDR2 and CDR3 selected from:
  • At least one immunoglobulin single variable domain in the FXI binding proteins of the present application is VHH.
  • the VHH may comprise any amino acid sequence in SEQ ID NO: 1-23.
  • At least one immunoglobulin single variable domain in the FXI binding protein of the present application is a humanized VHH.
  • At least one immunoglobulin single variable domain in the FXI-binding protein of the present application is a humanized VHH
  • the humanized VHH may comprise any of SEQ ID NO: 1-23
  • the sequences are amino acid sequences having at least 80%, possibly at least 90%, possibly at least 95%, even possibly at least 99% sequence identity.
  • the amino acid sequence of the humanized VHH may contain one or more amino acid substitutions compared with any of SEQ ID NO: 1-23, and may be conservative amino acid substitutions. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions may be included.
  • At least one immunoglobulin single variable domain in the FXI binding protein of the present application is a humanized VHH, wherein the humanized VHH may comprise any of SEQ ID NO:300-335 amino acid sequence.
  • said at least one immunoglobulin single variable domain binds the Apple2 domain of FXI.
  • the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in any one of SEQ ID NO:4, SEQ ID NO:10 or SEQ ID NO:14.
  • the immunoglobulin single variable domain may comprise a sequence selected from SEQ ID NO:60-62, SEQ ID NO:63-65, SEQ ID NO:66-68, SEQ ID NO:69-71 , SEQ ID NO:132-134, SEQ ID NO:135-137, SEQ ID NO:138-140, SEQ ID NO:141-143, SEQ ID NO:180-182, SEQ ID NO:183-185, SEQ ID NO:183-185, SEQ ID NO:183-185 A group of CDR1, CDR2 and CDR3 of ID NO: 186-188, SEQ ID NO: 189-191.
  • the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 10 or SEQ ID NO: 14. In the present application, the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NO: 306-323.
  • said at least one immunoglobulin single variable domain binds the Apple3 domain of FXI.
  • the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 17.
  • the immunoglobulin single variable domain may comprise a sequence selected from SEQ ID NO:216-218, SEQ ID NO:219-221, SEQ ID NO:222-224, SEQ ID NO:225-227 A set of CDR1, CDR2 and CDR3.
  • the immunoglobulin single variable domain may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NO: 324-329.
  • said at least one immunoglobulin single variable domain binds the Apple4 domain of FXI.
  • the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:1.
  • the immunoglobulin single variable domain may comprise a sequence selected from SEQ ID NO:24-26, SEQ ID NO:27-29, SEQ ID NO:30-32, SEQ ID NO:33-35 A set of CDR1, CDR2 and CDR3.
  • the immunoglobulin single variable domain may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NO: 300-305.
  • said at least one immunoglobulin single variable domain binds the Apple1-2 region of FXI (the region between the Apple1 domain and the Apple2 domain).
  • said at least one immunoglobulin single variable domain binds the Apple2-3 region of FXI (the region between the Apple2 domain and the Apple3 domain).
  • the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:20.
  • the immunoglobulin single variable domain may comprise a sequence selected from SEQ ID NO:252-254, SEQ ID NO:255-257, SEQ ID NO:258-260, SEQ ID NO:261-263 A set of CDR1, CDR2 and CDR3.
  • the immunoglobulin single variable domain may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NO:330-335.
  • said at least one immunoglobulin single variable domain binds the Apple3-4 region of FXI (the region between the Apple3 domain and the Apple4 domain).
  • the FXI-binding protein may comprise an immunoglobulin single variable domain that specifically binds to FXI.
  • the FXI-binding protein may comprise at least two, such as 2, 3, 4 or more immunoglobulin single variable domains that specifically bind to FXI.
  • the at least two immunoglobulin single variable domains bind to the same region or epitope of FXI, or compete for binding or partially compete for binding to the same region or epitope of FXI, for example, the at least two immunoglobulin single variable domains
  • the globulin single variable domains are identical.
  • said at least two immunoglobulin single variable domains bind to different regions or epitopes of FXI, or do not compete for binding to the same region or epitope of FXI.
  • the at least two immunoglobulin single variable domains that specifically bind FXI are directly linked to each other.
  • the at least two immunoglobulin single variable domains that specifically bind FXI are connected to each other through a linker.
  • the linker may be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary structure.
  • the linker is a flexible linker such as GGGGS, GS, GAP, (GGGGS) 3 and the like.
  • the FXI binding protein may comprise a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein,
  • the first immunoglobulin single variable domain can comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 1
  • the second immunoglobulin single variable domain can comprise the VHH set forth in SEQ ID NO: 4 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain can comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 1
  • the second immunoglobulin single variable domain can comprise the VHH set forth in SEQ ID NO: 9 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 1
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 10 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 1
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 14 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 1
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 17 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 1
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 20 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:4, and the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO:9 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:4, and the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO:10 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:4, and the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO:14 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:4, and the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO:17 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO:4, and the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO:20 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 9
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 10 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 9
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 14 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 9
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 17 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 9
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 20 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 10
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 14 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 10
  • the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 17 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain can comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 10
  • the second immunoglobulin single variable domain can comprise the VHH set forth in SEQ ID NO: 20 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain can comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 14, and the second immunoglobulin single variable domain can comprise the VHH set forth in SEQ ID NO: 17 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 14, and the second immunoglobulin single variable domain may comprise the VHH set forth in SEQ ID NO: 20 CDR1, CDR2 and CDR3 in the VHH; or
  • the first immunoglobulin single variable domain can comprise CDR1, CDR2 and CDR3 in the VHH set forth in SEQ ID NO: 17
  • the second immunoglobulin single variable domain can comprise the VHH set forth in SEQ ID NO: 20 CDR1, CDR2 and CDR3 in VHH.
  • CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO:1, 4, 9, 10, 14, 17 or 20 are shown in the following table:
  • the FXI-binding protein may comprise a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein,
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 1, 300-305, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 4, 306- the amino acid sequence shown in one of 311; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO:1, 300-305, and the second immunoglobulin single variable domain may comprise the amino acid sequence set forth in SEQ ID NO:9. amino acid sequence; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 1, 300-305, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 10, 312- the amino acid sequence shown in one of 317; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 1, 300-305, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 14, 318- The amino acid sequence shown in one of 323; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 1, 300-305, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 17, 324- The amino acid sequence shown in one of 329; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 1, 300-305, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 20, 330- The amino acid sequence shown in one of 335; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO:4, 306-311, and the second immunoglobulin single variable domain may comprise the amino acid sequence set forth in SEQ ID NO:9. amino acid sequence; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 4, 306-311, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 10, 312- The amino acid sequence in the VHH shown in one of 317; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 4, 306-311, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 14, 318- The amino acid sequence shown in one of 323; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 4, 306-311, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 17, 324- The amino acid sequence shown in one of 329; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 4, 306-311, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 20, 330- The amino acid sequence shown in one of 335; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 10, 312-317 amino acid sequence; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 14, 318-323. amino acid sequence; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NO: 17, 324-329. amino acid sequence; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence set forth in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the amino acid sequence set forth in one of SEQ ID NOs: 20, 330-335. amino acid sequence; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 10, 312-317, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 14, 318- The amino acid sequence shown in one of 323; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 10, 312-317, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 17, 324- The amino acid sequence shown in one of 329; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 10, 312-317, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 20, 330- The amino acid sequence shown in one of 335; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 14, 318-323, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 17, 324- The amino acid sequence shown in one of 329; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 14, 318-323, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 20, 330- The amino acid sequence shown in one of 335; or
  • the first immunoglobulin single variable domain may comprise the amino acid sequence shown in one of SEQ ID NO: 17, 324-329, and the second immunoglobulin single variable domain may comprise SEQ ID NO: 20, 330- The amino acid sequence shown in one of 335.
  • the FXI-binding protein may have CDR1-CDR3 in the VHH shown in SEQ ID NO:14.
  • the FXI binding protein can have CDR1 (SEQ ID NO: 183) in the VHH shown in SEQ ID NO: 14.
  • the FXI binding protein can have CDR2 (SEQ ID NO: 181) in the VHH shown in SEQ ID NO: 14.
  • the FXI binding protein can have CDR3 (SEQ ID NO: 182) in the VHH shown in SEQ ID NO: 14.
  • the FXI-binding protein may have CDR1 set forth in SEQ ID NO:183, CDR2 set forth in SEQ ID NO:181, and CDR3 set forth in SEQ ID NO:182.
  • the FXI binding protein can have a VHH set forth in SEQ ID NO:349.
  • the FXI-binding protein may have CDR1-CDR3 in the VHH shown in SEQ ID NO:17.
  • the FXI binding protein can have CDR1 (SEQ ID NO:219) in the VHH shown in SEQ ID NO:17.
  • the FXI binding protein can have CDR2 (SEQ ID NO:217) in the VHH set forth in SEQ ID NO:17.
  • the FXI binding protein can have CDR3 (SEQ ID NO: 218) in the VHH shown in SEQ ID NO: 17.
  • the FXI-binding protein may have CDR1 set forth in SEQ ID NO:219, CDR2 set forth in SEQ ID NO:217, and CDR3 set forth in SEQ ID NO:218.
  • the FXI binding protein can have a VHH set forth in SEQ ID NO:350.
  • the FXI binding protein can have CDR1 shown in SEQ ID NO: 183, CDR2 shown in SEQ ID NO: 181 and CDR3 shown in SEQ ID NO: 182; and, the FXI binding protein can have SEQ ID CDR1 shown in NO:219, CDR2 shown in SEQ ID NO:217 and CDR3 shown in SEQ ID NO:218.
  • the FXI-binding protein can have a VHH shown in SEQ ID NO:349, and the FXI-binding protein can have a VHH shown in SEQ ID NO:350.
  • the FXI-binding protein can have the amino acid sequence shown in SEQ ID NO:344.
  • first immunoglobulin single variable domain is located at the N-terminal of the second immunoglobulin single variable domain.
  • said second immunoglobulin single variable domain is located at the N-terminus of said first immunoglobulin single variable domain.
  • the FXI-binding protein of the present application may further comprise an immunoglobulin Fc region in addition to at least one immunoglobulin single variable domain capable of specifically binding to FXI.
  • an immunoglobulin Fc region in the FXI binding proteins of the present application may allow the binding molecules to form dimers.
  • Fc regions useful in the present application may be from different subclasses of immunoglobulins, eg, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgAl, IgA2, IgD, IgE or IgM.
  • mutations can be introduced into the wild-type Fc sequence to change the related activities mediated by Fc.
  • Such mutations include, but are not limited to: a). Mutations that alter Fc-mediated CDC activity; b). Mutations that alter Fc-mediated ADCC activity; or c). Mutations that alter FcRn-mediated half-life in vivo.
  • Such mutations are described in: Leonard G Presta, Current Opinion in Immunology 2008, 20:460-470; Esohe E. Idusogie et al., J Immunol 2000, 164:4178-4184; RAPHAEL A. CLYNES et al. , Nature Medicine, 2000, Volume 6, Number 4: 443-446; Paul R.
  • mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in the CH2 region can be used to increase or remove Fc-mediated ADCC or CDC activity or enhance or weaken FcRn affinity.
  • protein stability can be increased by mutating 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the hinge region.
  • mutations can be introduced into the Fc sequence, so that the mutated Fc can more easily form homodimers or heterodimers.
  • the knob-hole model using the space interaction of amino acid side chain groups on the Fc contact interface makes it easier to form heterodimers between different Fc mutations; another example is CN 102558355A Or in CN 103388013A, by changing the charge of the amino acid on the Fc contact interface, and then changing the ionic interaction force between the Fc contact interfaces, it is easier to form heterodimers between different Fc mutant pairs (CN 102558355A), also Or it is easier to form homodimers between Fc with the same mutation (CN 103388013A).
  • the immunoglobulin Fc region may be a human immunoglobulin Fc region, or a human IgG1 Fc region.
  • the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO:336.
  • the immunoglobulin Fc region (such as the Fc region of human IgG1) is directly or indirectly connected to the immunoglobulin single variable domain through a linker (such as a peptide linker) (such as the C-terminus of VHH).
  • a linker such as a peptide linker
  • the FXI-binding protein of the present application may comprise an immunoglobulin single variable domain that specifically binds to FXI, which is connected directly or through a linker to an immunoglobulin Fc region that allows the The FXI-binding proteins form dimeric molecules that may comprise two FXI-binding domains.
  • FXI-binding proteins are also referred to as bivalent FXI-binding proteins.
  • the dimer is a homodimer.
  • the FXI-binding protein of the present application may comprise two immunoglobulin single variable domains specifically binding to FXI and an immunoglobulin Fc region connected to each other directly or through a linker, and the immunoglobulin Fc region
  • the FXI-binding protein is allowed to form a dimeric molecule that may contain four FXI-binding domains.
  • Such FXI-binding proteins are also referred to as tetravalent FXI-binding proteins.
  • the dimer is a homodimer.
  • the two immunoglobulin single variable domains specifically binding to FXI in the FXI-binding protein respectively bind to different regions or different epitopes of FXI.
  • the FXI-binding protein of the present application can inhibit the activity of FXI. In the present application, the FXI-binding protein of the present application can inhibit the coagulation function of FXI.
  • nucleic acid nucleic acid, vector, host cell
  • the present application relates to nucleic acid molecules encoding the FXI binding proteins of the present application.
  • a nucleic acid of the present application may be RNA, DNA or cDNA.
  • the nucleic acid of the present application is a substantially isolated nucleic acid.
  • a nucleic acid of the present application may also be in the form of, may be present in and/or may be part of a vector, such as a plasmid, cosmid or YAC.
  • the vector may especially be an expression vector, ie a vector providing for in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system) expression of the FXI binding protein.
  • the expression vector can generally comprise at least one nucleic acid of the present application, which is operably linked to one or more suitable expression control elements (eg, promoter, enhancer, terminator, etc.). The selection of said elements and their sequences for expression in a particular host is within the general knowledge of the person skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the FXI binding proteins of the present application, such as promoters, enhancers, terminators, integrators, selectable markers, leader sequences, reporter genes.
  • the nucleic acid of the present application can be prepared or obtained by known means (for example, by automatic DNA synthesis and/or recombinant DNA technology) based on the information about the amino acid sequence of the polypeptide of the present application given in the present application, and/or can be obtained from a suitable Isolated from natural sources.
  • the present application relates to recombinant host cells expressing or capable of expressing one or more FXI binding proteins of the present application and/or containing the nucleic acids or vectors of the present application.
  • the possible host cells of the present application are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include Gram-negative bacterial strains, such as Escherichia coli, Proteus, and Pseudomonas strains, and Gram-positive bacterial strains, such as Bacillus (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli, Proteus, and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus (Bacillus) strains, Streptomyces (Streptomyces) strains, Staphylococcus (Staphylococcus) strains and Lactococcus (Lactococcus) strains
  • Suitable fungal cells include cells of species of Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (e.g., Saccharomyces cerevisiae), fission Saccharomyces (Schizosaccharomyces (such as Schizosaccharomyces pombe)), Pichia (such as Pichia pastoris and Pichia methanolica) and Han A cell of a species of Hansenula.
  • Saccharomyces e.g., Saccharomyces cerevisiae
  • fission Saccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Han A cell of a species of Hansenula a species of Hansenula.
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins can also be used herein.
  • the present application also provides a method for producing the FXI-binding protein of the present application, and the method may generally comprise the following steps:
  • the FXI-binding proteins of the present application can be produced intracellularly (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies) in cells as described above, followed by isolation and optional further purification from the host cell; or they can be produced extracellularly Produced (eg, in the medium in which the host cells are cultivated), followed by isolation from the medium and optionally further purification.
  • the FXI-binding protein of the present application can also be obtained by other protein-producing methods known in the art, such as chemical synthesis, including solid-phase or solution-phase synthesis.
  • the present application provides a composition, such as a pharmaceutical composition, comprising one or a combination of the FXI-binding proteins of the present application formulated together with a pharmaceutically acceptable carrier.
  • a composition may comprise one or a combination (eg, two or more different) of the FXI-binding proteins of the application.
  • a pharmaceutical composition of the present application may contain a combination of antibody molecules that bind to different epitopes on the target antigen (FXI).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the active compound ie, the antibody molecule
  • compositions of the present application may also contain pharmaceutically acceptable antioxidants.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbic acid palmitate esters, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, ⁇ -tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA ), sorbitol, tartaric acid, phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbic acid palmitate esters, butylated hydroxyanisole (
  • compositions may also contain, for example, preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol or sodium oxide, may be included in the composition.
  • Prolonged absorption of injectable drugs can be brought about by including in the composition agents which delay absorption, for example, monostearate salts and gelatin.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • Conventional media or agents, to the extent any incompatible with the active compounds, are possible in the pharmaceutical compositions of the present application.
  • Supplementary active compounds can also be incorporated into the compositions.
  • compositions generally must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.
  • Sterile injectable solutions can be prepared by mixing the active compound in the required amount in an appropriate solvent and, if necessary, adding one or a combination of ingredients enumerated above, followed by sterile microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • vacuum drying and freeze-drying are possible methods of preparation which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with carrier materials to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Typically, based on 100%, this amount will range from about 0.01% to about 99% of the active ingredient, such as from about 0.1% to about 70%, or from about 1% to about 30%, with the pharmaceutically acceptable combination of carriers.
  • Dosage regimens can be adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to result in combination with the required pharmaceutical carrier. desired therapeutic effect.
  • the particular specifications for dosage unit forms herein are limited to and directly dependent upon (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such for the treatment of sensitivity in an individual. Active compound restrictions.
  • dosages range from about 0.0001 to 100 mg/kg, more typically 0.01 to 30 mg/kg of recipient body weight.
  • the dosage may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight, 20 mg/kg body weight or 30 mg/kg body weight, or within the range of 1-30 mg/kg .
  • Exemplary treatment regimens entail weekly dosing, once every two weeks, once every three weeks, once every four weeks, once monthly, once every 3 months, once every 3-6 months, or an initial dosing interval Slightly shorter (eg once a week to once every three weeks) followed by longer dosing intervals (eg monthly to once every 3-6 months).
  • the antibody molecule can also be administered as a sustained release formulation, in which case less frequent dosing is required. Dosage and frequency vary according to the half-life of the antibody molecule in the patient. In general, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. Dosage and frequency of administration will vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively lower doses are administered at infrequent intervals over a prolonged period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to administer higher doses at shorter intervals until the progression of the disease is reduced or stopped, possibly until the patient shows partial or complete improvement in disease symptoms. Thereafter, the patient can be dosed in a prophylactic regimen.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this application may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the application employed or its ester, salt or amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the therapeutic Duration, other drugs, compounds and/or materials used in conjunction with the particular composition applied, age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field
  • compositions of the present application can be administered by one or more routes of administration using one or more methods known in the art. Those skilled in the art will appreciate that the route and/or manner of administration will vary depending on the desired result.
  • the possible administration routes of the FXI-binding protein of the present application include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as injection or infusion.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intrasaccular, intraorbital , intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.
  • the FXI-binding proteins of the present application may also be administered by non-parenteral routes, such as topical, epidermal or mucosal routes, eg, intranasally, orally, vaginally, rectally, sublingually or topically.
  • non-parenteral routes such as topical, epidermal or mucosal routes, eg, intranasally, orally, vaginally, rectally, sublingually or topically.
  • KN060 is an anti-FXI bispecific single domain antibody fusion protein screened from camel immune library. It is a homodimer formed by two identical peptide chains, wherein the amino acid sequence of the peptide chain is shown in SEQ ID NO.344.
  • the control antibody 14E11 is an anti-FXI antibody developed by Aronora Company. Wherein the amino acid sequence of the light chain of 14E11 is shown in SEQ ID NO.346; the amino acid sequence of the heavy chain is shown in SEQ ID NO.345.
  • the control antibody BAY1213790 is an anti-FXI antibody developed by Bayer Company. Wherein the amino acid sequence of the light chain of BAY1213790 is shown in SEQ ID NO.348; the amino acid sequence of the heavy chain is shown in SEQ ID NO.347.
  • the vector that can contain the nucleic acid sequence encoding the fusion protein is transferred into cells, and the corresponding fusion protein is obtained by expression and purification.
  • the binding kinetics of KN060 to hFXI-CHis protein was measured by a biolayer interferometry (BLI) technique using a molecular interaction instrument.
  • the specific operations include: diluting KN060 to 10 ⁇ g/ml and immobilizing it on the Protein A biosensor, then diluting hFXI-Chis protein to 5 concentration gradients of 50nM, 25nM, 12.5nM, 6.25nM, and 3.125nM to combine with KN060.
  • the results were fitted with a 1:1 model, and the equilibrium dissociation constant (KD) value of KN060 binding to hFXI-Chis protein was calculated. 4E11 and BAY1213790 were used as controls.
  • the measured affinity of KN060 to hFXI-Chis protein was 6.74E-10KD(M).
  • the affinity of control 14E11 to hFXI-Chis protein was 3.02E-09(M).
  • the affinity of the control BAY1213790 was 4.12E-09(M). It can be seen that the affinity of KN060 to hFXI-CHis is slightly higher than that of the two control samples.
  • the measured affinity of KN060 for hApple-Chis protein was ⁇ 1.0E-12KD(M).
  • KN060 exhibited a good affinity for human FXI factor protein and human Apple domain.
  • this experiment used Bio-Layer Interferometry (BLI) technology to evaluate the affinity of KN060 to human FXIa (hFXIa).
  • BBI Bio-Layer Interferometry
  • hFXIa was diluted to 400nM, 200nM, 100nM, 50nM, 25nM, a total of 5 concentration gradients were combined with KN060.
  • KD equilibrium dissociation constant
  • KN060 The affinity of KN060 to cynomolgus monkey and rabbit FXI was evaluated by Bio-Layer Interferometry (BLI) technology. Dilute the KN060 stock solution 201113DS to 10 ⁇ g/ml and solidify it on the Protein A biosensor. Then the cynomolgus monkey and rabbit FXI-apple proteins were diluted to 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, respectively, and combined with KN060 in five concentration gradients. The results were fitted with a 1:1 model, and the equilibrium dissociation constant (KD) value of the sample was calculated.
  • KD equilibrium dissociation constant
  • KN060 on standard human plasma APTT (activated partial thromboplastin time, Activated partial thromboplastin time) was measured by an automatic hemagglutination analyzer to analyze the inhibitory activity of KN060 on FXI in vitro.
  • the KN060 sample was diluted to 9.766ng/ml-20000ng/ml with the coagulation calibrator, and then the APTT after treatment with different concentrations of KN060 was detected, and the FXI activity value was calculated by the calibration curve of the coagulation calibrator.
  • SoftMax Pro software to process the data, draw a curve with the KN060 concentration as the abscissa and the measured FXI activity mean as the ordinate, and fit the curve with 4 parameters, according to the detection data and the SoftMax Pro software’s own function Relative Potency*100 The inhibitory effect of KN060 on FXI activity was calculated and analyzed.
  • Anti-FXI antibodies can prolong plasma APTT (activated partial thromboplastin time, Activated partial thromboplastin time) by binding to FXI.
  • APTT activated partial thromboplastin time, Activated partial thromboplastin time
  • the automatic hemagglutination analyzer was used to detect the prolongation effect of KN060 on plasma APTT of different species (including the prolongation multiple and prolongation time).
  • Coagulation instrument detection APTT is a common detection method to evaluate the intrinsic coagulation pathway. The operation method is to activate endogenous blood coagulation factors through appropriate amount of phospholipids, surface activators and incubation plasma, and then add calcium ions to trigger the coagulation process.
  • KN060 prolongs human plasma APTT significantly stronger than 14E11 and BAY1213790. The results are shown in Figure 1 and Table 5.
  • KN060 and 14E11 In rabbit plasma, KN060 and 14E11 at concentrations of 10 ⁇ g/ml, 5 ⁇ g/ml and 2 ⁇ g/ml all have a certain prolongation effect on rabbit plasma APTT, and the prolongation effect is concentration-dependent. Compared with blank plasma, KN060 and 14E11 had the maximum prolongation folds of APTT of 1.96 and 2.05 times, respectively. The effect of KN060 on prolonging APTT in rabbit plasma is similar to that of 14E11. The results are shown in Figure 3 and Table 7.
  • Table 7 KN060 and 14E11 extend the multiples of rabbit plasma APTT
  • KN060 and 14E11 In rat plasma, KN060 and 14E11 at concentrations of 30 ⁇ g/ml, 10 ⁇ g/ml and 5 ⁇ g/ml all have a certain prolongation effect on rat plasma APTT, and the prolongation effect is concentration-dependent. Compared with blank plasma, KN060 and 14E11 had a maximum extension of APTT of 1.76 and 1.37 times, respectively. The effect of KN060 on prolonging APTT in rat plasma is similar to that of 14E11. The results are shown in Figure 4 and Table 8.
  • Table 8 KN060 and 14E11 extend the multiples of rat plasma APTT
  • the FXI antibody was diluted to different concentrations with standard human plasma (purchased by Sigma), and the whole blood APTT time was measured after incubation at 37 degrees for 3 minutes. 14E11 and BAY1213790 were positive controls.
  • Table 9 shows the APTT time as a function of antibody concentration. The results show that KN060 can effectively prolong the APTT clotting time of whole blood; and exhibit a better effect of inhibiting coagulation.
  • Example KN060 and standard products were diluted to different concentrations with rabbit plasma (purchased from Sigma), and the whole blood APTT time was detected. 14E11 and BAY1213790 were positive controls.
  • a total of 24 male common-grade New Zealand rabbits were randomly divided into 4 groups according to body weight (G1-vehicle control group; G2-KN060 1mpk; G3-KN060 0.4mpk; G4-KN060 0.04mpk).
  • the animals were kept under anesthesia, and the left carotid artery and the right jugular vein were connected with a PE tube, which contained a 10 cm surgical thread and was pre-filled with normal saline. Animals were dosed 15 minutes before arteriovenous connection. After connection, perfusion was performed for 30 min to allow thrombus to form.
  • APTT and PT Prothrombin time, Prothrombin time
  • the average prolongation is 1.41, 1.92 and 2.64 times (see Figure 6 and Table 15), and the drug effect is obviously dose-dependent.
  • the platelet aggregation ability induced by ADP was tested at the same time.
  • the mean platelet aggregation rates of the vehicle control group and the KN060 low, medium and high dose treatment groups were 66.29%, 60.59%, 44.08% and 33.62%, respectively.
  • KN060 treatment had no significant effect on PT and bleeding (see Figure 8, Figure 9 and Table 17, Table 18, Table 19, Table 20).
  • KN060 can dose-dependently reduce thrombus weight, prolong APTT and reduce platelet aggregation rate in rabbit AV-shunt thrombosis model, showing obvious antithrombotic effect, and has a significant effect on PT and normal hemostasis. No noticeable effect.
  • the observation index is the thrombus weight, and the experimental data are represented by X ⁇ SD, and the significance test is carried out by GraphPad Prism 5 1way ANOVA (see Table 21).
  • the KN060 obtained in the above examples was selected, and its non-specific binding to other coagulation-related proteins was investigated by biolayer interferometry (BLI) technology.
  • the antibody protein was immobilized on the AHC chip, and the proteins investigated were commercially available FVII, FIX, FV, FXII, pro-thrombin, ⁇ -kallikrein, FVIIa, FIXa, FVa, FXIIa, and Thrombin.
  • the experimental results showed that all bispecific antibodies had no binding to FVII, FIX, FV, FXII, pro-thrombin, ⁇ -kallikrein, FVIIa, FIXa, FVa, FXIIa, Thrombin.
  • Embodiment 7 the clinical study of KN060
  • Trial design A randomized, double-blind, placebo-controlled phase I clinical study to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of KN060 injection in Chinese healthy subjects
  • Number of subjects 38 healthy subjects are planned to be enrolled.
  • Test group a single intravenous infusion of KN060 injection, a total of 6 dose groups, the doses of each group were 0.1mg/kg, 0.3mg/kg, 1.0mg/kg, 2.5mg/kg, 5.0mg/kg, 10.0mg/kg;
  • Control group a single intravenous infusion of placebo.
  • the selected subjects must meet the following criteria at the same time: 1. Healthy male or postmenopausal/amenorrhea female subjects; 2. At the time of signing the informed consent, the age is between 18 and 55 years old (including the cut-off value); 3. Weight Index (BMI) between 19.0 and 26.0kg/m2 (not including cut-off value); male body weight ⁇ 50.0kg, female body weight ⁇ 45.0kg; 4. Activated partial thrombin time (APTT), prothrombin time (PT) , International Normalized Ratio (INR) and platelet values are normal; 5. Understand the procedures of this research protocol, and are willing to accept and abide by the requirements of this protocol.
  • Administration time 6-8 hours after operation (no more than 24 hours after incision closure)
  • Exclusion criteria bleeding disorders or high risk of bleeding; history of VTE or ongoing anticoagulant therapy that cannot be interrupted; severe liver and kidney insufficiency; contraindications for angiography; active malignant tumors being treated; regional anesthesia is required, Such as spinal or epidural anesthesia; preoperative planning or postoperative epidural analgesia, etc.
  • the main efficacy endpoint the incidence of venous thromboembolism 10 to 14 days after operation (including DVT confirmed by venography, symptomatic DVT and PE, fatal PE and unexplained death that cannot be ruled out by PE);
  • the main safety endpoint the incidence of clinically relevant bleeding (defined as major bleeding or clinically relevant non-major bleeding) from signing the informed consent form (ICF) to 14 days after surgery;
  • PKPD endpoint (secondary): some subjects were tested for PKPD and correlated with efficacy and safety endpoints.

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