Methods for preventing and / or treating thromboembolic disorders

Abstract

The present application relates to a method for preventing and / or treating a thromboembolic disease, comprising the step of administering to a subject in need thereof a blood coagulation factor XI (FXI) binding protein, which may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI, wherein said at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as set forth in any one of SEQ ID NOs: 4, 10 and 14.

Description

[Technical field]

[0001] The present application relates to the biopharmaceutical field, and in particular to methods for preventing and / or treating thromboembolic disorders. [Background technology]

[0002] The human blood coagulation pathway is divided into two blood coagulation pathways: intrinsic and extrinsic. The extrinsic blood coagulation pathway begins with the release of TF after tissue injury, which then forms a complex with FVII and further activates FIX and FX. The intrinsic blood coagulation pathway is mainly initiated by the activation of factor FXII, which binds to negatively charged molecules, activating FXII to FXIIa, which activates FXI to FXIa, which further activates downstream FIX, and then activates FX under the action of cofactor FVIII. FXa and FVa activated in the extrinsic and intrinsic blood coagulation pathways together form a prothrombin complex to activate prothrombin (FII), producing active thrombin and fibrin clots, and ultimately causing blood coagulation.

[0003] FXI is one of the factors in the intrinsic blood coagulation pathway, and exists in a dimeric form, with its monomer consisting of four Apple domains and one serine protease domain. Activated FXIa has protease activity and activates its main substrate FIX, promoting the production of thrombin. FXIa can activate FX, FV, and FVIII in addition to the substrate FIX.

[0004] Deficiency of FXI may play a protective role against thrombotic diseases, but there are no commercially available drugs that target FXI, making the development of FXI-targeting antithrombotic drugs urgently necessary. Summary of the Invention [Problem to be solved by the invention]

[0005] The present application provides methods for preventing and / or treating thromboembolic disorders. [Means for solving the problem]

[0006] In one aspect, the present application provides a method for preventing and / or treating a thromboembolic disease, comprising administering to a subject in need thereof a blood coagulation factor XI (FXI) binding protein, which may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI, wherein said at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as set forth in any one of SEQ ID NOs: 4, 10 and 14.

[0007] In one embodiment, the FXI binding protein comprises a first immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14, and a second immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17.

[0008] In certain embodiments, the thromboembolic disease comprises venous thromboembolism (VTE). In one embodiment, the venous thromboembolism is a complication of a tumor and / or a complication of a joint replacement surgery. In certain embodiments, the venous thromboembolism is associated with a peripherally inserted central catheter (PICC).

[0009] In certain embodiments, the subject has or is at risk for having venous thromboembolism (VTE). In certain embodiments, the subject is a tumor patient. In one embodiment, the subject has a peripheral vein inserted into a central venous catheter (PICC). In one embodiment, the venous thromboembolism is associated with hip and / or knee joint replacement surgery.

[0010] In some embodiments, the subject has had a joint replacement. In one embodiment, the subject's hip and / or knee joint is replaced. In certain embodiments, the thromboembolic disease comprises deep vein thrombosis (DVT). In certain embodiments, the deep vein thrombosis includes acute deep vein thrombosis and / or recurrent deep vein thrombosis following an episode of acute deep vein thrombosis. In certain embodiments, the venous thromboembolism comprises pulmonary thromboembolism (PTE). In certain embodiments, the subject has or is at risk for deep vein thrombosis (DVT). In certain embodiments, the subject has suffered from acute deep vein thrombosis and / or has recurrent deep vein thrombosis following an acute deep vein thrombosis episode.

[0011] In certain embodiments, the subject is suffering from pulmonary thromboembolism (PTE). In certain embodiments, the thromboembolic disorder comprises systemic thromboembolism. In certain embodiments, the systemic thromboembolism is a complication of a tumor, a complication of atrial fibrillation, and / or a complication of dialysis. In certain embodiments, the atrial fibrillation includes non-valvular atrial fibrillation.

[0012] In certain embodiments, the subject has or is at risk of having a systemic thromboembolic disorder. In certain embodiments, the subject is suffering from non-valvular atrial fibrillation.

[0013] In certain embodiments, the subject is a dialysis patient. In certain embodiments, the subject is an adult. In certain embodiments, the subject suffers from hypertension, diabetes, congestive heart failure, atrial fibrillation, and / or has had a stroke. In one embodiment, the subject is at least 75 years of age. In certain embodiments, the subject is bedridden for an extended period of time.

[0014] In certain embodiments, the CDR may be a Kabat CDR, an AbM CDR, a Chothia CDR, or an IMGT CDR.

[0015] In one embodiment, CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 4 are any one group selected from SEQ ID NOs: 60 to 62, SEQ ID NOs: 63 to 65, SEQ ID NOs: 66 to 68 and SEQ ID NOs: 69 to 71. In certain embodiments, the at least one immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 4, 306-311.

[0016] In one embodiment, CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 10 are any one group selected from SEQ ID NOs: 132 to 134, 135 to 137, 138 to 140 and 141 to 143. In certain embodiments, the at least one immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 10, 312-317.

[0017] In one embodiment, CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 14 are any one group selected from SEQ ID NOs: 180 to 182, SEQ ID NOs: 183 to 185, SEQ ID NOs: 186 to 188 and SEQ ID NOs: 189 to 191. In certain embodiments, the at least one immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 14, 318-328.

[0018] In one embodiment, the blood coagulation factor XI (FXI) binding protein binds to the Apple2 domain of FXI. In certain embodiments, the blood coagulation factor XI (FXI) binding protein may further comprise an immunoglobulin Fc region. In one embodiment, the amino acid sequence of the immunoglobulin Fc region is set forth in SEQ ID NO:336.

[0019] In one embodiment, the dose of the blood coagulation factor XI (FXI) binding protein is about 0.5 to about 10 mg / kg.

[0020] In certain embodiments, the method further comprises administering to the subject a nucleic acid molecule encoding the blood coagulation factor XI (FXI) binding protein described herein, an expression vector which may comprise the nucleic acid molecule operably linked to an expression control element, a cell which may comprise the nucleic acid molecule or be transformed by the expression vector and capable of expressing the blood coagulation factor XI (FXI) binding protein, and / or a pharmaceutical composition which may comprise the blood coagulation factor XI (FXI) binding protein and a pharma- ceutical acceptable carrier.

[0021] In another aspect, the present application provides the use of a blood coagulation factor XI (FXI) binding protein in the preparation of a medicament for preventing and / or treating a thromboembolic disease, wherein said blood coagulation factor XI (FXI) binding protein may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI, and said at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as shown in any one of SEQ ID NOs: 4, 10 and 14.

[0022] In another aspect, the present application provides a blood coagulation factor XI (FXI) binding protein for preventing and / or treating a thromboembolic disease, wherein said blood coagulation factor XI (FXI) binding protein may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI, and said at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as shown in any one of SEQ ID NOs: 4, 10 and 14. Effect of the Invention

[0023] The blood coagulation factor XI (FXI) binding protein described in the present application can effectively prevent and / or treat thromboembolic diseases, for example, can prevent thrombosis in the body, effectively reduce the weight of thrombus, prolong APTT, and / or reduce the platelet aggregation rate. [Brief description of the drawings]

[0024] [Figure 1] 1 shows the results of the effect of the blood coagulation factor XI (FXI) binding protein described in the present application on extending the APTT in human plasma. [Diagram 2] 1 shows the results of the effect of the blood coagulation factor XI (FXI) binding protein described in the present application on prolonging APTT in cynomolgus monkey plasma. [Diagram 3] 1 shows the results of the effect of the blood coagulation factor XI (FXI) binding protein described in the present application on extending the APTT in rabbit plasma. [Figure 4] 1 shows the results of the effect of the blood coagulation factor XI (FXI) binding protein described in the present application on prolonging the APTT in mouse plasma. [Diagram 5] 1 shows the inhibitory effect of the blood coagulation factor XI (FXI) binding protein described in the present application on thrombus weight. [Figure 6] 1 shows the APTT-prolonging effect of the blood coagulation factor XI (FXI) binding protein described in the present application. [Figure 7] 1 shows the effect of the blood coagulation factor XI (FXI) binding protein described in the present application on platelet aggregation. [Figure 8] 1 shows the effect of the blood coagulation factor XI (FXI) binding protein described in this application on PT. [Figure 9] 1 shows the effect of the blood coagulation factor XI (FXI) binding protein described herein on bleeding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Specific features of the present invention are as set forth in the appended claims. The features and advantages of the present invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present application. As will be apparent to those skilled in the art, the contents of the present application allow those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the present invention. Accordingly, the drawings and descriptions in the present application are merely illustrative, not limiting.

[0026] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0027] 1. Definitions In this application, the term "thromboembolic disease" includes diseases caused by two pathological processes: thrombosis and thromboembolism. Thrombosis generally refers to a pathological process in which blood particles form emboli in blood vessels (most of which are small blood vessels) under certain conditions, causing partial or complete occlusion of the blood vessel and interrupting blood supply to the corresponding site. Thromboembolism generally refers to a pathological process in which a thrombus breaks off from the formation site and travels through the bloodstream, partially or completely occludes a certain blood vessel, causing ischemia, hypoxia, necrosis (arterial thrombosis) and congestion and edema (venous thrombosis) in the corresponding tissue and / or organ. The above thromboembolic diseases may include, depending on the type of blood vessel in which thrombosis occurs, (1) venous thromboembolic diseases, which are venous thromboembolism, which may include, for example, pulmonary embolism, deep vein thrombosis; (2) arterial thromboembolic diseases, which may include, for example, acute coronary syndrome, atrial fibrillation, arterial ischemic attack, stroke; and (3) microvascular thrombotic diseases, which may include, for example, disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura, etc.

[0028] In this application, the term "venous thromboembolism (VTE)" generally refers to the formation of blood clots in veins, i.e., complete or incomplete occlusion of blood vessels due to abnormal clotting of blood in veins, and belongs to venous return disorder. The above-mentioned venous thromboembolism is commonly found in the deep veins of the lower limbs. The embolus is called an embolus because it may break off from its site of formation and enter the blood circulation. The above-mentioned venous thromboembolism may be caused by factors such as blockage of blood flow (e.g., long-term bedridden cast or stent braking, especially the combination of dehydration and existing venous disease (chronic venous insufficiency)), vein wall damage (e.g., surgery, trauma, or inflammation), or thrombosis tendency (e.g., imbalance between blood coagulation and fibrinolysis due to procoagulant factors or special drugs). In this application, the term "complication" generally refers to the occurrence of another disease or condition during the progression of one disease, the latter being a complication of the former. In some cases, the complication may include the occurrence of another disease or several diseases related to one disease during treatment due to the presence of one disease.

[0029] In this application, the term "peripherally inserted central catheter (PICC)" generally refers to a catheter placed in a central vein by peripheral vein puncture (i.e., peripherally inserted central catheter). The peripherally inserted central catheter may be placed in a vein in the elbow or upper arm, followed along the vein's course, and finally delivered close to the great vessels of the heart. The PICC may have long-term, safe, and painless characteristics, and is applicable to patients undergoing long-term intravenous therapy and infusion of hypertonic and irritant drugs. The PICC may cause complications such as infection, thrombosis, and phlebitis.

[0030] In this application, the term "joint replacement" generally refers to the technique of manufacturing and surgically implanting an artificial joint into the human body according to the form, structure and function of a human joint. For example, the artificial joint can be made of artificial materials such as metal, high molecular weight polyethylene and / or ceramic.

[0031] In this application, the term "deep vein thrombosis (DVT)" generally refers to a disease caused by abnormal clotting of blood in deep veins. DVT can occur in the lower limbs and is common after major orthopedic surgery. If the deep vein thrombus breaks off, it can cause pulmonary embolism (PE), which are collectively called venous thromboembolism. DVT can be caused by factors such as slow venous blood flow, damage to the vein wall, and a hypercoagulable state.

[0032] In this application, the term "pulmonary thromboembolism (PTE)" generally refers to a disease caused by a thrombus from the venous system or the right heart obstructing the pulmonary artery or its branches, and whose main clinical and pathophysiological features are pulmonary circulation and respiratory dysfunction. The thrombus that causes PTE is mainly derived from deep vein thrombosis (DVT), and PTE and DVT may be collectively referred to as venous thromboembolism (VTE). The factors that cause the above-mentioned PTE may include genetic factors (e.g., genetic mutations) and acquired factors (e.g., surgery, trauma, acute medical diseases (e.g., heart failure, respiratory failure, infection, etc.), certain chronic diseases (e.g., antiphospholipid syndrome, nephrotic syndrome, etc.), and malignant tumors).

[0033] In this application, the term "atrial fibrillation" generally refers to atrial fibrillation, which is one of the common cardiac arrhythmias. It is a serious disturbance of atrial electrical activity, meaning that regular atrial electrical activity is lost and replaced by fast and chaotic fibrillation waves. The atrial fibrillation may lead to coronary heart disease, hypertension and / or heart failure. The atrial fibrillation may be divided into initial atrial fibrillation, paroxysmal atrial fibrillation, persistent atrial fibrillation, long-term persistent atrial fibrillation and permanent atrial fibrillation. The atrial fibrillation can cause complications such as heart failure, arterial embolism, and even sudden death in severe cases.

[0034] In this application, the term "non-valvular atrial fibrillation" generally refers to atrial fibrillation occurring in the absence of rheumatic mitral stenosis, bioprosthetic or mechanical valve replacement, or mitral valve repair, which can be treated with non-vitamin K antagonist oral anticoagulants (DOACs) to prevent stroke.

[0035] In this application, the term "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 in removing metabolic waste and excess water from the body when the kidneys fail to function normally. The above dialysis (treatment) may include any therapeutic method in which components (solutes or water) in bodily fluids are removed from the body through a semipermeable membrane, and may generally include hemodialysis, peritoneal dialysis, and / or colon dialysis.

[0036] In this application, the term "congestive heart failure (CHF)" generally refers to the occurrence of pulmonary and / or systemic circulation congestion along with reduced tissue and organ blood flow, and is a clinical syndrome in which various cardiac diseases have progressed to a severe stage. The congestive heart failure may occur due to a decrease in the pumping or filling function of the ventricles, causing the cardiac output to be unable to meet the metabolic demands of the body. The congestive heart failure may be characterized by left ventricular hypertrophy or dilation, which may lead to neuroendocrine disorders and circulatory dysfunction, and may result in typical clinical symptoms such as dyspnea, fluid retention, and fatigue.

[0037] In this application, the term "hypertension" generally refers to a clinical syndrome characterized primarily by an increase in systemic arterial pressure (systolic and / or diastolic pressure) (systolic pressure ≥ 140 mmHg and / or diastolic pressure ≥ 90 mmHg in the absence of antihypertensive drugs) and may be accompanied by functional or organic damage to organs such as the heart, brain, and kidneys. Hypertension may be the most important risk factor for cardiovascular and cerebrovascular diseases. In this application, the term "diabetes" generally refers to a group of metabolic disorders characterized by chronic hyperglycemia caused by impaired insulin secretion and / or utilization due to multiple etiologies. Long-term metabolic disorders of carbohydrates, fats, and proteins can cause multisystem wasting, leading to chronic progressive lesions, deterioration, and failure of tissues and organs, including eyes, kidneys, nerves, heart, and blood vessels. The diabetes may include type 1, type 2, other special types, and gestational diabetes.

[0038] In this application, the term "long-term bedridden" generally refers to a clinical phenomenon in which a person's ability to perform activities of daily living is reduced due to a long-term illness or physical disability, and he or she requires partial or total assistance.

[0039] Unless otherwise specified, the terms "antibody" or "immunoglobulin", which can be used interchangeably, are used herein as a general term to include full length antibodies, single chains thereof and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, for example VHH domains or VH / VL domains, respectively), regardless of whether they refer to heavy chain antibodies or conventional four chain antibodies. Furthermore, the term "sequence" as used herein (e.g. in terms such as "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include not only the relevant amino acid sequence, but also the nucleic acid or nucleotide sequence encoding said sequence, unless a more restrictive interpretation is required in the present application.

[0040] As used herein, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of the protein, and in many cases can be added, removed, or transferred to other proteins without loss of function of the other parts and / or domains of the protein.

[0041] The term "immunoglobulin domain" as used herein refers to a globular region of an antibody chain (e.g., a chain of a conventional four-chain antibody or a chain of a heavy-chain antibody), or a polypeptide consisting essentially of such a globular region. Immunoglobulin domains are characterized by maintaining the immunoglobulin folding characteristic of antibody molecules.

[0042] The term "immunoglobulin variable domain" as used herein refers to an immunoglobulin domain that essentially consists of four "framework regions" referred to in the art and below as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, of which said framework regions are separated by three "complementarity determining regions" or "CDRs" referred to in the art and below as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain may be depicted as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain contains the antigen binding site and thus confers specificity to the antibody for the antigen.

[0043] The term "immunoglobulin single variable domain" as used herein refers to a variable domain of an immunoglobulin that is capable of specifically binding to an epitope of an antigen without pairing with another immunoglobulin variable domain. One example of an immunoglobulin single variable domain as addressed in this application is a "domain antibody", such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is the camelid "VHH domain" (or abbreviated as "VHH"), as defined below.

[0044] A "VHH domain" is also called a heavy chain single domain antibody, VHH, VHH domain, VHH antibody fragment and VHH antibody, and is a variable domain of an antigen-binding immunoglobulin called a "heavy chain antibody" (i.e., a "light chain devoid antibody") (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)). The term "VHH domain" is used to distinguish the above variable domain from the heavy chain variable domain present in a conventional four-chain antibody (referred to herein as a "VH domain") and the light chain variable domain present in a conventional four-chain antibody (referred to herein as a "VL domain"). VHH domains specifically bind to an epitope without the need for another antigen-binding domain (this is the opposite of the VH or VL domains in a conventional four-chain antibody, where the epitope is recognized by both the VL and VH domains). VHH domains are small, stable and efficient antigen recognition units formed by a single immunoglobulin domain.

[0045] In the context of this application, the terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment" and "VHH antibody" can be used interchangeably. For example, as shown in Figure 2 of Riechmann & Muyldermans, J. Immunol. Methods 231, 25-38 (1999), the amino acid residues used in Camelidae VHH domains may be numbered according to the general numbering scheme for VH domains proposed by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0046] Alternative methods of numbering the amino acid residues of VH domains are known in the art and are applicable to VHH domains as well. For example, Chothia CDRs refer to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917(1987)). AbM CDRs represent intermediate states between Kabat hypervariable regions and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residues of CDRs according to each method are explained as follows:

[0047] TIFF2024544057000001.tif48170

[0048] However, it should be noted that, as is known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions based on 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), which generally means that the Kabat numbering may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0049] For example, the CDRs may include "extended CDRs", such as 24 to 36 or 24 to 34 (LCDR1), 46-56 or 50 to 56 (LCDR2) and 89 to 97 or 89 to 96 (LCDR3) in VL, and 26 to 35 (HCDR1), 50 to 65 or 49 to 65 (HCDR2) and 93 to 102, 94 to 102 or 95 to 102 (HCDR3) in VH.

[0050] The total number of amino acid residues in a VHH domain is generally in the range of 110 to 120, and often between 112 and 115. It should be noted that both smaller and longer sequences are also applicable for the purposes described herein.

[0051] Other structural and functional properties of VHH domains and polypeptides containing them can be summarized as follows. VHH domains (which are naturally "designed" to functionally bind antigen in the absence and without interaction with a light chain variable domain) can be used as single and relatively small functional antigen-binding structural units, domains or polypeptides, which distinguish them from the VH and VL domains of conventional four-chain antibodies, which by themselves are generally not suitable for practical use as single antigen-binding proteins or immunoglobulin single variable domains, but must be combined in one form or another to provide a functional antigen-binding unit (e.g. in the form of a conventional antibody fragment such as a Fab fragment, or in the form of an scFv consisting of a VH domain covalently linked to a VL domain).

[0052] Due to their unique properties, the use of VHH domains, either alone or as part of a larger polypeptide, offers a number of significant advantages over the use of conventional VH and VL domains, scFvs or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments): by binding antigen with high affinity and high selectivity with only a single domain, there is no need to have two separate domains or to ensure that the two domains are in the correct spatial conformation and arrangement (e.g. scFvs generally require the use of specially designed linkers), VHH domains can be derived from a single gene, VHH domains can be expressed from any suitable vector and do not require post-translational folding or modification, and multivalent and multispecific formats can be easily engineered from VHH domains; VHH domains have good solubility and are not prone to aggregation; VHH domains are highly stable to heat, pH, proteases and other denaturing agents or conditions, thereby eliminating the need for refrigeration during preparation, storage or transportation, thereby saving costs, time and protecting the environment; VHH domains are easy to prepare and relatively inexpensive, even on a production scale; VHH domains are relatively small (approximately 15 kDa or 1 / 10 the size of a conventional IgG) compared to conventional four-chain antibodies and their antigen-binding fragments, thereby allowing for relatively high tissue penetration and administration at relatively high doses compared to conventional four-chain antibodies and their antigen-binding fragments; and VHH domains can exhibit so-called cavity-binding properties (especially their CDR3 loops, which are extended compared to conventional VH domains), allowing them to reach targets and epitopes that are inaccessible to conventional four-chain antibodies and their antigen-binding fragments.

[0053] Methods for obtaining VHHs that bind to specific antigens or epitopes have already been disclosed in the following documents: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J. Biol. Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8(12), pp. 2645-2652, 17 June, 2009; and WO94 / 04678.

[0054] A VHH domain derived from Camelidae can be "humanized" by replacing 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 the VH domain of a human conventional four-chain antibody (also referred to in the present application as "sequence optimization", which may also cover, besides humanization, other modifications made to the sequence by one or more mutations that provide improved properties of the VHH, such as the removal of potential post-translational modification sites). A humanized VHH domain may comprise one or more fully human framework region sequences. Humanization can be achieved, for example, by methods of protein surface amino acid resurfacing and / or CDR grafting to a universal framework, as illustrated in the examples.

[0055] As used herein, the term "epitope" or the interchangeable term "antigenic determinant" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Antigenic determinants generally include chemically active surface groups of molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural features and specific charge characteristics. For example, an epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, and may be a "linear" or "conformational" epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all of the interaction sites between a protein and an interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur across amino acid residues of the protein that are separated from each other.

[0056] Epitopes of a given antigen can be identified by many epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). For example, linear epitopes can be determined by, for example, synthesizing large amounts of peptides corresponding to each part of a protein molecule simultaneously on a solid support and reacting these peptides with an antibody while still attached to the support. These techniques are known in the art and described, for example, in U.S. Pat. No. 4,708,871; Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al., (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes can be identified by determining the spatial arrangement of amino acids, for example, by X-ray crystallography or two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (ibid.).

[0057] Antibodies can be screened for their binding competitiveness to the same epitope by conventional techniques known to those skilled in the art. For example, antibodies that compete or cross-compete with each other to bind to an antigen can be obtained by performing competition and cross-competition tests. A high-throughput method for obtaining antibodies that bind to the same epitope by cross-competition is described in International Patent Application WO03 / 48731. Thus, antibodies and antigen-binding fragments thereof that compete with the antibody molecule of the present application to bind to the same epitope in FXI can be obtained by conventional techniques known to those skilled in the art.

[0058] In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (e.g., an immunoglobulin single variable domain according to the present application) can bind. The specificity can be determined based on the affinity and / or affinity of the antigen-binding protein. The affinity, as indicated by the dissociation equilibrium constant (KD) of 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, i.e., the smaller the KD value, the stronger the binding strength between the epitope and the antigen-binding protein (alternatively, affinity may be indicated by the association constant (KA), which is 1 / KD). Depending on the specific antigen of interest, affinity may be measured in known manners, as known to those skilled in the art. Affinity is a measure of the binding strength between an antigen-binding protein (e.g., an immunoglobulin, an antibody, an immunoglobulin single variable domain or a polypeptide comprising it) and the relevant antigen. Affinity is related to the affinity for the antigen-binding site on the antigen-binding protein and the number of relevant binding sites present in the antigen-binding protein.

[0059] As used herein, the term "blood coagulation factor XI (FXI) binding protein" refers to any protein capable of specifically binding to blood coagulation factor XI (FXI). FXI binding proteins may include antibodies as defined herein against FXI. FXI binding proteins further cover immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules.

[0060] An "FXI binding protein" according to the present application may comprise at least one immunoglobulin single variable domain, e.g., a VHH, that binds to FXI. An "FXI binding molecule" according to the present application may comprise two, three, four or more immunoglobulin single variable domains, e.g., a VHH, that bind to FXI. An FXI binding protein according to the present application may comprise, in addition to an immunoglobulin single variable domain that binds to FXI, a linker and / or a moiety with effector function, e.g., a half-life extending moiety (e.g., an immunoglobulin single variable domain that binds to serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region). In the present application, an "FXI binding protein" according to the present application further covers bispecific antibodies comprising immunoglobulin single variable domains that bind to different antigens or different regions of the same antigen (e.g., different epitopes).

[0061] Generally, the FXI binding proteins according to the present application preferably have a 10 -7 ~10 -10 moles / liter (M), more preferably 10 -8 ~10 -10 moles / liter, more preferably 10 -9 ~10 -10 or less, and / or with a dissociation constant (KD) of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , and more preferably at least 10 10 M -1 It can bind to the antigen to be bound (i.e., FXI) with an association constant (KA) of 10. -4Any K value greater than M is generally considered to indicate non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any suitable method known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays) as described herein.

[0062] "Sequence identity" between two polypeptide sequences refers to the percentage of amino acids that are the same between the sequences. "Sequence similarity" refers to the percentage of amino acids that are the same amino acids or that 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, the identity of amino acid sequences is generally measured by sequence analysis software. For example, identity can be determined by the BLAST program of the NCBI database. For the determination of sequence identity, reference may be made, for example, to Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988, Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, 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.

[0063] A polypeptide or nucleic acid molecule is considered to be "isolated" if it is isolated from at least one of the other components that are typically associated with it in its origin or medium (culture medium) (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or minor component) compared to its natural organism origin and / or the reaction medium or culture medium from which the polypeptide or nucleic acid molecule was obtained. In particular, a polypeptide or nucleic acid molecule is considered to be "isolated" if it is purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and even 1000-fold or more. It has been determined by suitable techniques (e.g., suitable chromatographic techniques, e.g., polyacrylamide gel electrophoresis) that an "isolated" polypeptide or nucleic acid molecule can be essentially homogeneous.

[0064] By "effective amount" is meant an amount of the FXI binding protein or pharmaceutical composition of the present application that results in a reduction in the severity of symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of damage or disability due to pain of the disease.

[0065] As used herein, "thrombosis" refers to the formation or presence of a clot (also called a "thrombus") in a blood vessel that blocks blood flow through the circulatory system. Thrombosis is typically caused by abnormalities in blood components, vessel wall quality and / or blood flow characteristics. Clot formation is typically caused by injury to the vessel wall (e.g., injury to the vessel wall due to trauma or infection) and reduced or stagnant blood flow through the injured site. In some cases, thrombosis is caused by a clotting abnormality.

[0066] 2. Detailed Description of the Invention In one aspect, the present application provides a method for preventing and / or treating a thromboembolic disease, comprising administering to a subject in need thereof a blood coagulation factor XI (FXI) binding protein, which may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI, wherein said at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as set forth in any one of SEQ ID NOs: 4, 10 and 14. In another aspect, the present application provides the use of a blood coagulation factor XI (FXI) binding protein in the preparation of a medicament for preventing and / or treating a thromboembolic disease, wherein said blood coagulation factor XI (FXI) binding protein may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI, and said at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as shown in any one of SEQ ID NOs: 4, 10 and 14.

[0067] In another aspect, the present application provides a blood coagulation factor XI (FXI) binding protein for preventing and / or treating a thromboembolic disease, wherein said blood coagulation factor XI (FXI) binding protein may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI, and said at least one immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH as shown in any one of SEQ ID NOs: 4, 10 and 14.

[0068] For example, the FXI binding protein comprises a first immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 in a VHH as shown in SEQ ID NO: 14, and a second immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 in a VHH as shown in SEQ ID NO: 17.

[0069] In the present application, the method may further comprise administering to the subject a nucleic acid molecule encoding the blood coagulation factor XI (FXI) binding protein described herein, an expression vector which may comprise the nucleic acid molecule operably linked to an expression control element, a cell which may comprise the nucleic acid molecule or be transformed by the expression vector and capable of expressing the blood coagulation factor XI (FXI) binding protein, and / or a pharmaceutical composition which may comprise the blood coagulation factor XI (FXI) binding protein and a pharma- ceutical acceptable carrier.

[0070] In this application, the thromboembolic disease may include venous thromboembolism (VTE). For example, said venous thromboembolism may be a complication of a tumor and / or of a joint replacement surgery.

[0071] For example, the venous thromboembolism may be associated with a peripherally inserted central catheter (PICC).

[0072] For example, such venous thromboembolism may be associated with hip and / or knee joint replacement surgery, in which muscle contractile activity in the lower limbs may be reduced and bleeding from the replacement incision may make the blood more prone to clotting, potentially leading to deep vein thrombosis in the lower limbs.

[0073] For example, the thromboembolic disease may include deep vein thrombosis (DVT). For example, the deep vein thrombosis may include acute deep vein thrombosis and / or recurrent deep vein thrombosis following an episode of acute deep vein thrombosis. For example, the venous thromboembolism may include pulmonary thromboembolism (PTE), where a deep vein thrombosis, for example, can travel back through the bloodstream to the lungs and block a non-mainstream blood vessel, potentially resulting in a fatal pulmonary embolism. For example, the thromboembolic disease may include systemic thromboembolism. For example, the systemic thromboembolism may belong to a complication of a tumor, a complication of atrial fibrillation, and / or a complication of dialysis. In the present application, the atrial fibrillation may include non-valvular atrial fibrillation.

[0074] In this application, the subject may be suffering from or at risk of suffering from venous thromboembolism (VTE). In the present application, a central venous catheter (PICC) may be inserted into a peripheral vein of the subject. In the present application, the subject may be a joint replacement patient, for example, the subject's hip and / or knee joints may be replaced.

[0075] In this application, the subject may be suffering from or at risk of suffering from deep vein thrombosis (DVT). In the present application, the subject may be suffering from acute deep vein thrombosis and / or may have recurrent deep vein thrombosis after the onset of acute deep vein thrombosis. In the present application, the subject may be suffering from pulmonary thromboembolism (PTE). In this application, the subject may be suffering from or at risk of suffering from systemic thromboembolism. In the present application, the subject may be suffering from non-valvular atrial fibrillation.

[0076] In the present application, the subject may be a dialysis patient. The dialysis patient may have undergone and / or is undergoing hemodialysis. The hemodialysis may include a step of removing various harmful and excessive metabolic wastes and excess electrolytes in the blood from the body by diffusion using the semipermeable membrane principle. 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 is undergoing peritoneal dialysis. The peritoneal dialysis may include a step of using the peritoneum as a semipermeable membrane, injecting a prepared dialysis solution into the peritoneal cavity of the patient at regular intervals using gravity through a catheter, and continuously exchanging the peritoneal dialysis solution. The peritoneal dialysis can achieve the purpose of removing metabolic products and toxic substances in the body and correcting water and electrolyte balance disorders. In the present application, the dialysis patient may develop heart and brain complications. For example, symptoms of high blood pressure, cerebral hemorrhage and / or heart failure may occur or already exist during dialysis.

[0077] In the present application, the subject may be an adult. In the present application, the adult may be 18 years of age or older.

[0078] In the present application, the subject may be a tumor patient. In the present application, the tumor may be a malignant tumor. In the present application, the tumor may include a solid tumor and / or a non-solid tumor.

[0079] In the present application, the subject may suffer from hypertension, diabetes, congestive heart failure, atrial fibrillation, and / or may have a history of stroke. In the present application, the stroke may include cerebral stroke. The stroke may be caused by carotid artery stenosis, atrial fibrillation, cerebral hemorrhage, and / or ischemic stroke. For the treatment method of the stroke, the Chinese Stroke Series Guideline-2015 Edition can be referred to.

[0080] In the present application, the subject may be at least 75 years old (eg, at least 75 years old, at least 80 years old, at least 85 years old or older).

[0081] In this application, the subject may be bedridden for an extended period of time (eg, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years or longer).

[0082] In the present application, the dosage of the blood coagulation factor XI (FXI) binding protein may be about 0.5 to about 10 mg / kg. For example, it may be about 0.5 to about 8.0 mg / kg, about 0.5 to about 7.5 mg / kg, about 0.5 to about 7.0 mg / kg, about 0.5 to about 6.5 mg / kg, about 0.5 to about 6 mg / kg, about 0.5 to about 5.5 mg / kg, about 0.5 to about 5.0 mg / kg, about 0.5 to about 4.5 mg / kg, about 0.5 to about 4.0 mg / kg, about 0.5 to about 3.5 mg / kg, about 0.5 to about 3.0 mg / kg, about 0.5 to about 2.5 mg / kg, about 0.5 to about 2.0 mg / kg, about 0.5 to about 1.5 mg / kg, about 0.5 to about 1.0 mg / kg, about 1.0 to about 10 mg / kg, or about 1.0 to about 8.0 mg / kg.

[0083] FXI binding protein In one embodiment, the present application provides a FXI binding protein, which may comprise at least one immunoglobulin single variable domain capable of specifically binding to FXI.

[0084] In the present application, the at least one immunoglobulin single variable domain comprises CDR1, CDR2 and CDR3 in a VHH shown in any one of SEQ ID NOs: 1 to 23. The CDRs may be Kabat CDRs, AbM CDRs, Chothia CDRs or IMGT CDRs.

[0085] In the present application, said at least one immunoglobulin single variable domain may comprise a group of CDR1, CDR2 and CDR3 selected from the following:

[0086] TIFF2024544057000002.tif190170

[0087] In the present application, at least one immunoglobulin single variable domain in the FXI binding protein according to the present application is a VHH. In the present application, the VHH may comprise any one of the amino acid sequences of SEQ ID NOs: 1 to 23.

[0088] In the present application, at least one immunoglobulin single variable domain in the FXI binding protein according to the present application is a humanized VHH.

[0089] In the present application, at least one immunoglobulin single variable domain in the FXI binding protein according to the present application is a humanized VHH, and the humanized VHH may comprise an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity to any one of SEQ ID NOs: 1 to 23. In the present application, the amino acid sequence of the humanized VHH may comprise one or more amino acid substitutions, or may be conservative amino acid substitutions, compared to any one of SEQ ID NOs: 1 to 23. For example, it may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions.

[0090] In the present application, at least one immunoglobulin single variable domain in the FXI binding protein according to the present application is a humanized VHH, wherein said humanized VHH may comprise any one of the amino acid sequences of SEQ ID NOs: 300-335.

[0091] In the present application, the at least one immunoglobulin single variable domain binds to the Apple2 domain of FXI. For example, the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in a VHH 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 a group of CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 60 to 62, SEQ ID NOs: 63 to 65, SEQ ID NOs: 66 to 68, SEQ ID NOs: 69 to 71, SEQ ID NOs: 132 to 134, SEQ ID NOs: 135 to 137, SEQ ID NOs: 138 to 140, SEQ ID NOs: 141 to 143, SEQ ID NOs: 180 to 182, SEQ ID NOs: 183 to 185, SEQ ID NOs: 186 to 188 and SEQ ID NOs: 189 to 191. In the present application, the immunoglobulin single variable domain may comprise an 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 an amino acid sequence as shown in any one of SEQ ID NOs: 306-323.

[0092] In the present application, the at least one immunoglobulin single variable domain binds to the Apple3 domain of FXI. For example, the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 17. In the present application, the immunoglobulin single variable domain may comprise a group of CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 216 to 218, 219 to 221, 222 to 224 and 225 to 227. In the present application, the immunoglobulin single variable domain may comprise the amino acid sequence shown in SEQ ID NO: 17. In the present application, the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 324 to 329.

[0093] In the present application, the at least one immunoglobulin single variable domain binds to the Apple4 domain of FXI. For example, the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 1. In the present application, the immunoglobulin single variable domain may comprise a group of CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 24 to 26, 27 to 29, 30 to 32 and 33 to 35. In the present application, the immunoglobulin single variable domain may comprise the amino acid sequence shown in SEQ ID NO: 1. In the present application, the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 300 to 305.

[0094] In the present application, the at least one immunoglobulin single variable domain binds to the Apple1-2 region of FXI (the region between the Apple1 and Apple2 domains).

[0095] In the present application, the at least one immunoglobulin single variable domain binds to the Apple2-3 region of FXI (the region between the Apple2 domain and the Apple3 domain). For example, the immunoglobulin single variable domain may comprise CDR1, CDR2 and CDR3 in the VHH shown in SEQ ID NO: 20. In the present application, the immunoglobulin single variable domain may comprise a group of CDR1, CDR2 and CDR3 selected from SEQ ID NOs: 252 to 254, SEQ ID NOs: 255 to 257, SEQ ID NOs: 258 to 260 and SEQ ID NOs: 261 to 263. In the present application, the immunoglobulin single variable domain may comprise the amino acid sequence shown in SEQ ID NO: 20. In the present application, the immunoglobulin single variable domain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 330 to 335.

[0096] In the present application, the at least one immunoglobulin single variable domain binds to the Apple3-4 region of FXI (the region between the Apple3 and Apple4 domains).

[0097] In the present application, the FXI binding protein may comprise one immunoglobulin single variable domain that specifically binds to FXI. In the present application, the FXI binding protein may comprise at least two, for example, two, three, four or more immunoglobulin single variable domains that specifically bind to FXI. In the present application, the at least two immunoglobulin single variable domains bind to the same region or epitope of FXI or competitively or partially competitively bind to the same region or epitope of FXI, e.g., the at least two immunoglobulin single variable domains are the same. In the present application, the at least two immunoglobulin single variable domains bind to different regions or epitopes of FXI or do not competitively bind to the same region or epitope of FXI.

[0098] Whether two antibodies or immunoglobulin single variable domains bind or competitively bind to the same region or epitope can be determined by epitope binning using biolayer interferometry (BLI), e.g., as shown in the Examples herein.

[0099] In the present application, the at least two FXI-specific binding immunoglobulin single variable domains are directly linked to each other. In the present application, the at least two immunoglobulin single variable domains that specifically bind to FXI are interconnected via a linker. The linker may be a non-functional amino acid sequence with a length of 1 to 20 or more amino acids and no secondary or higher structure. For example, the linker is a flexible linker, such as GGGGS, GS, GAP, (GGGGS)3, etc.

[0100] In the present application, the FXI binding protein may comprise a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, among which: The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:4, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:9, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 1, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 10, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 1, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 1, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO:20, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 4, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 9, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 4, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 10, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 4, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 4, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 4, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 20, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 10, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 9, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 20, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 10, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 10, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 10, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 20, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 20, or The first immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17, and the second immunoglobulin single variable domain may comprise the CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 20.

[0101] In the present application, the CDR1, CDR2 and CDR3 in the VHH represented by SEQ ID NO: 1, 4, 9, 10, 14, 17 or 20 are as shown in the following table:

[0102] TIFF2024544057000003.tif58170

[0103] In the present application, the FXI binding protein may comprise a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, among which: The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 1, 300 to 305, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 4, 306 to 311, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 1, 300 to 305, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in SEQ ID NO: 9, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 1, 300 to 305, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 10, 312 to 317, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 1, 300 to 305, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 14, 318 to 323, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 1, 300 to 305, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 17, 324 to 329, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 1, 300 to 305, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 20, 330 to 335, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 4, 306 to 311, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in SEQ ID NO: 9, or The first immunoglobulin single variable domain may comprise an amino acid sequence as shown in one of SEQ ID NOs: 4, 306 to 311, and the second immunoglobulin single variable domain may comprise an amino acid sequence in a VHH as shown in one of SEQ ID NOs: 10, 312 to 317, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 4, 306 to 311, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 14, 318 to 323, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 4, 306 to 311, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 17, 324 to 329, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 4, 306 to 311, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 20, 330 to 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 NOs: 10, 312 to 317, 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: 14, 318 to 323, 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: 17, 324 to 329, 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 to 335, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 10, 312 to 317, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 14, 318 to 323, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 10, 312 to 317, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 17, 324 to 329, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 10, 312 to 317, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 20, 330 to 335, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 14, 318 to 323, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 17, 324 to 329, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 14, 318 to 323, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 20, 330 to 335, or The first immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 17, 324-329, and the second immunoglobulin single variable domain may comprise an amino acid sequence as set forth in one of SEQ ID NOs: 20, 330-335.

[0104] In the present application, the FXI binding protein may have CDR1 to CDR3 in the VHH shown in SEQ ID NO: 14. For example, the FXI binding protein may have CDR1 (SEQ ID NO: 183) in the VHH shown in SEQ ID NO: 14. For example, the FXI binding protein may have CDR2 (SEQ ID NO: 181) in the VHH shown in SEQ ID NO: 14. For example, the FXI binding protein may have CDR3 (SEQ ID NO: 182) in the VHH shown in SEQ ID NO: 14. For example, the FXI binding protein may have a CDR1 as set forth in SEQ ID NO:183, a CDR2 as set forth in SEQ ID NO:181, and a CDR3 as set forth in SEQ ID NO:182. For example, the FXI binding protein may have a VHH as shown in SEQ ID NO:349.

[0105] In the present application, the FXI binding protein may have CDR1 to CDR3 in the VHH shown in SEQ ID NO: 17. For example, the FXI binding protein may have CDR1 (SEQ ID NO: 219) in the VHH shown in SEQ ID NO: 17. For example, the FXI binding protein may have CDR2 (SEQ ID NO: 217) in the VHH shown in SEQ ID NO: 17. For example, the FXI binding protein may have CDR3 (SEQ ID NO: 218) in the VHH shown in SEQ ID NO: 17. For example, the FXI binding protein may have a CDR1 as set forth in SEQ ID NO:219, a CDR2 as set forth in SEQ ID NO:217, and a CDR3 as set forth in SEQ ID NO:218. For example, the FXI binding protein may have a VHH as shown in SEQ ID NO:350. For example, the FXI binding protein may have a CDR1 as set forth in SEQ ID NO: 183, a CDR2 as set forth in SEQ ID NO: 181, and a CDR3 as set forth in SEQ ID NO: 182, and the FXI binding protein may have a CDR1 as set forth in SEQ ID NO: 219, a CDR2 as set forth in SEQ ID NO: 217, and a CDR3 as set forth in SEQ ID NO: 218. For example, the FXI binding protein may have a VHH as shown in SEQ ID NO:349, and the FXI binding protein may have a VHH as shown in SEQ ID NO:350. For example, the FXI binding protein may have the amino acid sequence shown in SEQ ID NO:344.

[0106] In the present application, the first immunoglobulin single variable domain is N-terminal to the second immunoglobulin single variable domain. In some other embodiments, the second immunoglobulin single variable domain is N-terminal to the first immunoglobulin single variable domain.

[0107] In the present application, the FXI binding protein according to 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. The FXI binding protein according to the present application may comprise an immunoglobulin Fc region so that the binding molecule can form a dimer. The Fc regions that can be used in the present application may be derived from different subtypes of immunoglobulins, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.

[0108] In the present application, a mutation may be introduced into the wild-type Fc sequence to change the Fc-mediated related activity. The above mutations include, but are not limited to, a) a mutation that changes the Fc-mediated CDC activity, b) a mutation that changes the Fc-mediated ADCC activity, or c) a mutation that changes the FcRn-mediated in vivo half-life. Such mutations are described in the following documents: 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. Hinton et al., J Immunol, 2006, 176:346-356. For example, mutating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the CH2 region can increase or eliminate Fc-mediated ADCC or CDC activity, or enhance or decrease affinity for FcRn. Additionally, mutating 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in the hinge region can improve protein stability.

[0109] In the present application, a mutation may be introduced into the Fc sequence so that the mutated Fc is more likely to form homodimers or heterodimers. As described in Ridgway, Presta et al. 1996 and Carter 2001, the knob-hole model utilizing the spatial effect of amino acid side groups at the Fc contact interface makes it more likely to form heterodimers between different Fc mutations, and for example, in CN102558355A or CN103388013A, the charge of the amino acid at the Fc contact interface is changed to change the ionic interaction force between the Fc contact interface, making it more likely to form heterodimers between different Fc mutation pairs (CN102558355A), or making it more likely to form homodimers between Fcs with the same mutation (CN103388013A).

[0110] The immunoglobulin Fc region may be a human immunoglobulin Fc region or a human IgG1 Fc region. In the present application, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO:336.

[0111] In the present application, in the FXI binding protein according to the present application, the immunoglobulin Fc region (e.g., the Fc region of human IgG1) is linked directly or indirectly via a linker (e.g., a peptide linker) to the C-terminus of the immunoglobulin single variable domain (e.g., VHH). In the present application, the FXI binding protein of the present application may comprise one immunoglobulin single variable domain that specifically binds to FXI, linked directly or via a linker to an immunoglobulin Fc region, which allows the FXI binding protein to form a dimeric molecule that may comprise two FXI binding domains. Such FXI binding proteins are also referred to as bivalent FXI binding proteins. In the present application, the dimer is a homodimer.

[0112] In the present application, the FXI binding protein of the present application may comprise two immunoglobulin single variable domains that specifically bind to FXI and one immunoglobulin Fc region, which are interconnected directly or via a linker, and the immunoglobulin Fc region enables the FXI binding protein to form a dimeric molecule that may comprise two FXI binding domains. Such a FXI binding protein is also called a tetravalent FXI binding protein. In the present application, the dimer is a homodimer. In the present application, the two immunoglobulin single variable domains that specifically bind to FXI in the FXI binding protein each bind to a different region or different epitope of FXI.

[0113] In the present application, the FXI binding protein of the present application is capable of inhibiting the activity of FXI. In the present application, the FXI binding protein of the present application is capable of inhibiting the blood coagulation function of FXI.

[0114] Nucleic acids, vectors and host cells In another aspect, the present application relates to a nucleic acid molecule encoding the FXI binding protein of the present application. The nucleic acid of the present application may be RNA, DNA or cDNA. According to one embodiment of the present application, the nucleic acid of the present application is an essentially isolated nucleic acid.

[0115] The nucleic acid according to the present application may be in the form of, present within and / or part of a vector, for example a plasmid, cosmid or YAC. The vector may in particular be an expression vector, i.e. a vector that allows the expression of the FXI binding protein in vitro and / or in vivo (i.e. in a suitable host cell, host organism and / or expression system). The expression vector may generally comprise at least one nucleic acid according to the present application operably linked to one or more suitable expression control elements (e.g. promoters, enhancers, terminators, etc.). The selection of said elements and their sequences for expression in a particular host is within the knowledge of a person skilled in the art. Specific examples of control elements and other elements useful or essential for the expression of the FXI binding protein according to the present application are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0116] The nucleic acids of the present application may be prepared or obtained in a known manner (e.g., by automated DNA synthesis and / or recombinant DNA techniques) based on the information of the amino acid sequence of the polypeptide of the present application described herein, and / or may be isolated from a suitable natural source.

[0117] In another aspect, the present application relates to a recombinant host cell that expresses or is capable of expressing one or more FXI binding proteins according to the present application and / or that comprises a nucleic acid or vector according to the present application. The host cell of the present application may be a bacterial cell, a fungal cell or a mammalian cell.

[0118] Suitable bacterial cells include cells of gram-negative strains (e.g., Escherichia coli, Proteus and Pseudomonas strains) and gram-positive strains (e.g., Bacillus, Streptomyces, Staphylococcus and Lactococcus strains). Suitable fungal cells include cells of species of Trichoderma, Neurospora, and Aspergillus, or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0119] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0120] However, the present application may also use amphibian cells, insect cells, plant cells and any other cells in the art for expressing heterologous proteins.

[0121] The present application further provides a method for producing a FXI binding protein according to the present application, the method generally comprising: Culturing a host cell according to the present application under conditions allowing expression of a FXI binding protein according to the present application; recovering the FXI binding protein expressed by said host cells from the culture; Optionally, further purifying and / or modifying the FXI binding protein according to the present application; may include:

[0122] The FXI binding proteins of the present application may be produced in an intracellular manner in cells as described above (e.g., in the cytoplasm, periplasm or in inclusion bodies) and then isolated from the host cells and optionally further purified, or may be produced in an extracellular manner (e.g., in the medium in which the host cells are cultured) and then isolated from the medium and optionally further purified.

[0123] Methods and reagents for recombinantly producing polypeptides are known in the art, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. Similarly, protein isolation and purification techniques applied in the methods for preparing FXI binding proteins according to the present application are known to those skilled in the art.

[0124] However, the FXI binding proteins according to the present application can be obtained by other methods for producing proteins known in the art, for example chemical synthesis, including solid phase or liquid phase synthesis.

[0125] Pharmaceutical Compositions In another aspect, the present application provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of the FXI binding proteins of the present application, formulated with a pharma- ceutically acceptable carrier. Such a composition may comprise one or a combination (e.g., two or more different) of the FXI binding proteins of the present application. For example, a pharmaceutical composition of the present application may comprise a combination of antibody molecules that bind to different epitopes on the target antigen (FXI).

[0126] As used herein, a "pharmaceutical 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. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., via injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that inactivate the compound.

[0127] The pharmaceutical composition of the present application may contain a pharma- ceutically acceptable antioxidant. Examples of the pharma-ceutically acceptable antioxidant include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium hydrogen sulfate, sodium pyrosulfite, and sodium sulfite, (2) oil-soluble antioxidants such as ascorbic acid palmitate, butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol, and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid. The composition may further contain preservatives, wetting agents, emulsifying agents, dispersing agents and the like.

[0128] Prevention of the presence of microorganisms can be ensured by a sterilization process or by the inclusion of various antibacterial and antifungal agents, such as paraben esters, chlorobutanol, phenol, sorbic acid, etc. In many cases, the composition will also contain isotonic agents, such as sugars, polyols, for example, mannitol or sorbitol, or sodium oxide. Prolonged absorption of the injectable drug can be achieved by adding to the composition agents that delay absorption, for example, monostearate and gelatin.

[0129] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of these media and agents for pharmaceutical active substances is well known in the art. Any conventional media or agent may be present in the pharmaceutical compositions of the present application, except to the extent that it is incompatible with the active compound. Supplementary active compounds may also be incorporated into the compositions.

[0130] Therapeutic compositions must generally be sterile and stable under preparation and storage conditions. The compositions may be formulated as solutions, microemulsions, liposomes, or ordered structures suitable for high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0131] Sterile injections can be prepared by mixing the active compound in the required amount in a suitable solvent, adding one or a combination of the above-listed ingredients as necessary, and then performing sterilization precision filtration. In general, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other necessary ingredients listed above. The preferred method for preparing sterile powders for preparing sterile injections is vacuum drying and freeze-drying (lyophilization), which obtains the active ingredient from the previously sterilized filtered solution and adds the powder of any other necessary ingredients.

[0132] The amount of active ingredient that can be combined with carrier materials to prepare a single dosage form varies depending on the subject being treated and the specific mode of administration. The amount of active ingredient that can be combined with carrier materials to prepare a single dosage form is generally the amount of the composition that achieves a therapeutic effect. Generally, on a 100% basis, this amount ranges from about 0.01% to about 99% of active ingredient, for example, from about 0.1% to about 70%, or from about 1% to about 30% of active ingredient, combined with a pharma- ceutically acceptable carrier.

[0133] The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus can be administered, or separate doses can be administered over time, or the dosage can be proportionally reduced or increased according to the exigencies of the therapeutic situation. It is particularly advantageous for parenteral compositions to be prepared in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discontinuous unit suitable for use with subjects treated at a unit dose, containing a predetermined amount of active compound per unit, calculated to produce a desired therapeutic effect in combination with the required pharmaceutical carrier. The specific description of dosage unit form in the present application is limited by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the inherent limitations in the art for the preparation of such active compounds to treat individual susceptibility.

[0134] Administration of the antibody molecule will be in the dose range of about 0.0001 to 100 mg / kg, more typically 0.01 to 30 mg / kg of subject body weight. For example, the dose 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 to 30 mg / kg. Exemplary therapeutic regimens include weekly, biweekly, 3 weekly, 4 weekly, monthly, 3 monthly, 3 to 6 monthly dosing, or requiring slightly shorter dosing intervals in the early stages (e.g., weekly to 3 weekly) and longer dosing intervals later (e.g., monthly to 3 to 6 monthly).

[0135] Alternatively, the antibody molecule may be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will depend on the half-life of the antibody molecule in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dose and frequency will depend on whether the treatment is prophylactic or therapeutic. In prophylactic use, a relatively low dose is administered at relatively infrequent intervals over an extended period of time. Some patients will continue treatment for the rest of their lives. In therapeutic use, a relatively high dose may need to be administered at relatively short intervals until the progression of the disease is alleviated or stopped, preferably until the patient shows some or all of the improvement of the disease symptoms. Thereafter, the patient can be administered a prophylactic regimen.

[0136] The pharmaceutical compositions of the present application are capable of effectively achieving the therapeutic response required for a particular patient, composition and mode of administration, but the actual dosage level of the active ingredient may be varied to obtain an amount of the active ingredient that is not toxic to the patient. The selection of the dosage level is determined by various pharmacokinetic factors, including the activity of the particular composition of the present application or its esters, salts or amides used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors known in the medical arts.

[0137] The compositions of the present application may be administered by one or more routes of administration using one or more methods known in the art. It should be understood by those skilled in the art that the route and / or mode of administration will vary depending on the desired outcome. Possible routes of administration of the FXI binding proteins of the present application include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as injection or infusion. The term "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, typically injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0138] Alternatively, the FXI binding proteins of the present application may be administered parenterally, for example via topical, epidermal or mucosal routes, such as intranasal, oral, vaginal, rectal, sublingual or topical. Without being limited to any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods and uses of the present invention, and are not intended to limit the scope of the present invention. EXAMPLES

[0139] Example 1 Preparation of Blood Coagulation Factor XI (FXI) Binding Protein KN060 is an anti-FXI bispecific single domain antibody fusion protein screened from a camel immune library. It is a homodimer formed from two identical peptide chains, of which the amino acid sequence is shown in SEQ ID NO:344. The control antibody 14E11 is an anti-FXI antibody developed by Aronora, Inc. Here, the amino acid sequence of the light chain of 14E11 is shown in SEQ ID NO:346, and 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. Here, the amino acid sequence of the light chain of BAY1213790 is shown in SEQ ID NO:348, and the amino acid sequence of the heavy chain is shown in SEQ ID NO:347.

[0140] A vector, which may contain a nucleic acid sequence encoding a fusion protein, is transferred into cells, expressed, and purified to obtain the corresponding fusion protein.

[0141] Example 2 Detection of affinity of blood coagulation factor XI (FXI) binding protein to FXI 2.1 Affinity of KN060 to FXI (1) The binding kinetics of KN060 to hFXI-Chis protein was measured by Biolayerinterferometry (BLI) using a molecular interaction analyzer. The specific procedure included the following: KN060 was diluted to 10 μg / mL and cured on a Protein A biosensor, and then hFXI-Chis protein was diluted to five concentration gradients of 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM and bound to KN060. The results were fitted with a 1:1 model to calculate the equilibrium dissociation constant (KD) value of KN060 binding to hFXI-Chis protein. 4E11 and BAY1213790 were used as controls. The affinity of KN060 to hFXI-Chis protein was measured to be 6.74E-10 KD (M). The affinity of the control 14E11 to the hFXI-Chis protein is 3.02E-09 (M). The affinity of the control BAY1213790 is 4.12E-09 (M). KN060 was found to have a slightly higher affinity to hFXI-CHis than the two control samples.

[0142] TIFF2024544057000004.tif37170

[0143] (2) KN060 was diluted to a final concentration of 10 μg / mL and directly applied to the AHC biosensor to measure the kinetics. hApple-Chis (sequence referenced in the Uniprot database, accession number P03951, the first 387th amino acid sequence of the Apple domain was selected) was diluted to five concentrations, and the baseline was 60 s, the binding time was 120 s, and the dissociation time was 900 s. The dilution solution was kinetic buffer, the regeneration solution was glycine-HCl (pH 1.7), and the neutralization solution was the dilution solution. The biosensor was Protein A. The binding rate (kon) and dissociation rate (kdis) were calculated by a simple one-to-one Languir binding model (Octet K2 Data Analysis Software 9.0). The equilibrium dissociation constant (kD) was calculated as the ratio kdis / kon.

[0144] The affinity of KN060 for the hApple-Chis protein was measured to be <1.0E-12 KD(M). As is clear from the above results, KN060 showed excellent affinity for human FXI factor protein and the human Apple domain.

[0145] 2.2 Affinity of KN060 to FXIa To investigate whether KN060 can bind to FXIa, this experiment evaluated the affinity of KN060 to human FXIa (hFXIa) by Bio-Layer Interferometry (BLI). KN060 was diluted to 10 μg / mL and cured on a Protein A biosensor. Then, hFXIa was diluted to 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM and bound to KN060 at a total of five concentration gradients. The results were fitted with a 1:1 model to calculate the equilibrium dissociation constant (KD) value of KN060 binding to human FXIa. As a result, the KD value of KN060 binding to hFXIa was shown to be 1.88E-09M.

[0146] TIFF2024544057000005.tif29170

[0147] 2.3 Affinity of KN060 to cynomolgus monkey and rabbit FXI The affinity of KN060 to cynomolgus monkey and rabbit FXI was evaluated by Bio-Layer Interferometry (BLI). KN060 stock solution 201113DS was diluted to 10 μg / mL and cured on a Protein A biosensor. Cynomolgus monkey and rabbit FXI-apple proteins were then diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM, respectively, and bound to KN060 at a total of five concentration gradients. The results were fitted with a 1:1 model to calculate the equilibrium dissociation constant (KD) values ​​of the samples.

[0148] As a result, KN060 was capable of binding to both cynomolgus monkey and rabbit FXI, with KD values ​​of 1.520E-13M and 1.834E-10M, respectively, suggesting that KN060 has a higher affinity for cynomolgus monkey FXI than for rabbit FXI.

[0149] TIFF2024544057000006.tif30170

[0150] Example 3 Detection of the inhibitory effect of blood coagulation factor XI (FXI) binding protein on FXI activity 3.1 Inhibitory effect of KN060 on FXI activity in vitro The inhibitory activity of KN060 against FXI in vitro was analyzed by measuring the effect of KN060 on standard human plasma APTT (activated partial thromboplastin time) using a fully automated blood coagulation analyzer. After diluting KN060 samples to 9.766-20000 ng / mL with a blood coagulation test kit, APTTs treated with different concentrations of KN060 were detected, and FXI activity was calculated using the standard curve of the blood coagulation test kit. The data was processed using SoftMax Pro software, and a curve was plotted with the KN060 concentration on the horizontal axis and the average measured FXI activity on the vertical axis. Four-parameter fitting was performed on the curve, and the inhibitory effect of KN060 against FXI activity was calculated and analyzed using the detection data and the function Relative Potency*100% included in SoftMax Pro software.

[0151] As a result, it was shown that KN060 had a clear inhibitory effect on FXI activity in vitro, with an IC50 value for inhibiting FXI activity of 650 ng / mL.

[0152] TIFF2024544057000007.tif113170

[0153] Example 4 Prolongation or inhibition effect of KN060 on plasma APTT Anti-FXI antibody can prolong plasma APTT (activated partial thromboplastin time) by binding to FXI. In this experiment, the prolongation effect of KN060 on plasma APTT of different species (including prolongation ratio and prolongation time) was detected using a fully automated blood coagulation analyzer. Detection of APTT by a blood coagulation analyzer is a common detection method for evaluating the intrinsic blood coagulation pathway. The operation method was to activate the intrinsic blood coagulation factors with an appropriate amount of phospholipids, surfactant and incubated plasma, and then to trigger the blood coagulation process by adding calcium ions.

[0154] 4.1 Prolongation of APTT in human plasma In human plasma, KN060, 14E11 and BAY1213790 at concentrations of 10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL and 0.5 μg / mL all have some degree of prolongation effect on standard human plasma APTT, and the prolongation effect shows concentration dependency. Compared with blank human plasma, KN060, 14E11 and BAY1213790 have maximum APTT prolongation ratios of 3.71, 2.09 and 2.22 times, respectively. KN060 has a clearly stronger prolongation effect on human plasma APTT than 14E11 and BAY1213790. The results are shown in Figure 1 and Table 5.

[0155] TIFF2024544057000008.tif42170

[0156] 4.2 Prolongation of APTT in cynomolgus monkey plasma In cynomolgus monkey plasma, KN060 and 14E11 at concentrations of 10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL and 0.5 μg / mL all have some degree of prolongation effect on cynomolgus monkey plasma APTT, and the prolongation effect shows concentration dependency. Compared with blank plasma, KN060 and 14E11 have maximum APTT prolongation ratios of 3.38 and 2.53 times, respectively. KN060 has a clearly stronger prolongation effect on monkey plasma APTT than 14E11. The results are shown in Figure 2 and Table 6.

[0157] TIFF2024544057000009.tif37170

[0158] 4.3. Prolongation of APTT in rabbit plasma In rabbit plasma, KN060 and 14E11 at concentrations of 10 μg / mL, 5 μg / mL, and 2 μg / mL all have some degree of prolongation effect on rabbit plasma APTT, and the prolongation effect shows concentration dependency. Compared with blank plasma, KN060 and 14E11 have maximum APTT prolongation ratios of 1.96 and 2.05 times, respectively. KN060 has a prolongation effect on APTT in rabbit plasma close to that of 14E11. The results are shown in Figure 3 and Table 7.

[0159] TIFF2024544057000010.tif37170

[0160] 4.4. Prolongation of APTT in rat plasma In rat plasma, KN060 and 14E11 at concentrations of 30 μg / mL, 10 μg / mL, and 5 μg / mL all have some degree of prolongation effect on rat plasma APTT, and the prolongation effect shows concentration dependency. Compared with blank plasma, KN060 and 14E11 have maximum APTT prolongation ratios of 1.76 and 1.37 times, respectively. KN060 has a similar prolongation effect on APTT in rat plasma as 14E11. The results are shown in Figure 4 and Table 8.

[0161] TIFF2024544057000011.tif37170

[0162] 4.5 Inhibitory activity of human whole plasma against APTT FXI antibody was diluted to different concentrations in standard human plasma (purchased from Sigma) and whole blood APTT time was detected after 3 min incubation at 37° C. 14E11 and BAY1213790 were used as positive controls.

[0163] TIFF2024544057000012.tif57170

[0164] Table 9 shows the change in APTT time with antibody concentration. The results show that KN060 can effectively prolong the whole blood APTT clotting time and has a better blood coagulation inhibition effect.

[0165] 4.6 Inhibitory activity of monkey whole plasma against APTT KN060 was diluted to different concentrations with monkey plasma (purchased from Sigma) and incubated at 37°C for 3 min before detecting whole blood APTT time. 14E11 and BAY1213790 were used as positive controls. The results are shown in Table 10.

[0166] TIFF2024544057000013.tif57170

[0167] 4.7 Inhibitory activity of rabbit whole plasma against APTT Example KN060 and standard were diluted to different concentrations with rabbit plasma (purchased from Sigma) to detect whole blood APTT time. 14E11 and BAY1213790 were used as positive controls. The results are shown in Table 11.

[0168] TIFF2024544057000014.tif57170

[0169] The above experimental results suggest that KN060 can exhibit relatively good APTT inhibitory activity in human or monkey plasma, and both are superior to the control positive antibody.

[0170] Example 5: Detection of in vivo efficacy of KN060 (1) We investigated the efficacy of KN060 in preventing thrombosis by administering a single prophylactic dose of different doses in a rabbit AV-shunt thrombosis model.

[0171] A total of 24 male normal grade New Zealand rabbits were randomly divided into 4 groups according to body weight (G1-vehicle control group, G2-KN060 1 mpk, G3-KN060 0.4 mpk, G4-KN060 0.04 mpk). The animals were kept anesthetized, and the left carotid artery and right jugular vein were connected with PE tubes, which contained a 10 cm long surgical thread and were pre-filled with saline. The animals were dosed 15 min before connecting the artery and vein. After connection, the animals were perfused for 30 min to form a thrombus.

[0172] After the model was completed, the surgical suture was removed from the tube, the blood was removed with absorbent filter paper, and the wet weight of the thrombus was measured on a balance. Plasma was collected before administration, at the end of the model, and 30 min, 60 min, and 2 h after the model was completed, and APTT and PT (prothrombin time) were measured. At the same time, at the end of the model, whole blood was collected, PRP (platelet-poor plasma) and PPP (platelet-rich plasma) were separated, and ADP (adenosine diphosphate)-induced platelet aggregation was measured to evaluate platelet aggregation ability. Furthermore, a bleeding test was performed 5 min after the model was completed, and the amount of bleeding and bleeding time were measured to evaluate the effect of KN060 on normal hemostasis.

[0173] As a result, when 0.04, 0.4 and 1 mg / kg of KN060 were administered intravenously to the model animals, all showed a significant thrombus weight inhibition effect (see Figure 5 and Table 12) and APTT prolongation effect (see Figure 6 and Table 14). The average thrombus wet weight inhibition rates of the three low, medium and high dose treatment groups were 20.57%, 87.55% and 95.87%, respectively (see Figure 5 and Table 13), and the APTT was prolonged by an average of 1.41, 1.92 and 2.64 times in the low, medium and high dose treatment groups at 120 min after the end of the model (see Figure 6 and Table 15), showing a clear dose-dependency of the drug's efficacy. At the end of the model, ADP-induced platelet aggregation was simultaneously tested, and the mean platelet aggregation rates of the vehicle control group and the three KN060 low, medium and high dose treatment groups were 66.29%, 60.59%, 44.08% and 33.62%, respectively, and the results showed that the platelet aggregation rate was negatively correlated with the dose (see Figure 7 and Table 16). KN060 treatment had no significant effect on PT and bleeding (see Figure 8, Figure 9 and Tables 17, 18, 19 and 20).

[0174] As described above, different doses of KN060 can dose-dependently reduce thrombus weight, prolong APTT, and reduce platelet aggregation rate in the rabbit AV-shunt thrombosis model, clearly demonstrating its pharmacological efficacy in preventing thrombosis, and has no significant effect on PT and normal hemostasis.

[0175] TIFF2024544057000015.tif38170

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[0184] (2) Rabbits were fasted overnight, blood was collected from the marginal ear vein, and anesthetized by injecting an anesthetic into the marginal ear vein. The distal and proximal ends of the jugular vein were ligated, with the distance between the two ligatures standardized to about 3.0 cm. 15 min before modeling, 1 mg / kg of the test product or PBS negative control product was injected into the marginal ear vein. The proximal and distal ends of the jugular vein were clamped with an arterial clip, and the blood in the blood vessel was completely removed from the facial vein with a syringe. Furthermore, 0.3 mL of 5 mg / mL agonist was injected into the blood vessel at the closed part, and after incubation with the agonist for 5 minutes, the agonist was removed with a syringe, and the blood vessel was flushed twice with saline. The arterial clip was removed, blood flow was restored, and the diameter of the blood vessel was controlled to 0.8 mm to induce thrombosis. After 25 minutes of blood flow recovery, the distal and proximal ends of the occluded vein were clamped with arterial clips, and the surgical sutures at the proximal and distal ends of the vein were tightened, the occluded vein was cut, and the thrombus was removed. The wet weight of the thrombus was immediately weighed and recorded. The thrombus was then dried in a 60°C oven for 20 hours, after which the dry weight of the thrombus was weighed and recorded.

[0185] The observation index was thrombus weight, and the experimental data were shown as X±SD, and significance testing was performed using GraphPad Prism 5 1-way ANOVA (see Table 21).

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[0187] Example 6 Specificity of KN060 KN060 obtained in the above example was selected, and its non-specific binding to other blood coagulation-related proteins was examined by Biolayerinterferometry (BLI). The antibody protein was cured on an AHC chip, and the proteins examined were commercial products of FVII, FIX, FV, FXII, pro-thrombin, α-kallikrein, FVIIa, FIXa, FVa, FXIIa, and Thrombin. The experiment revealed that all bispecific antibodies did not bind to any of FVII, FIX, FV, FXII, pro-thrombin, α-kallikrein, FVIIa, FIXa, FVa, FXIIa, and Thrombin.

[0188] Example 7 Clinical Study of KN060 1. The safety and tolerability of a single intravenous infusion of KN060 in healthy subjects was evaluated.

[0189] Test Plan: Study design: A randomized, double-blind, placebo-controlled Phase I clinical study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of KN060 injection in healthy Chinese subjects. Number of subjects: 38 healthy subjects were planned to be enrolled. The specific groupings and administration methods are as follows: Test groups: A total of 6 dose groups were administered with a single intravenous infusion of KN060 injection, with the doses of 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 2.5 mg / kg, 5.0 mg / kg, and 10.0 mg / kg, respectively. Control group: Placebo was administered intravenously in a single dose.

[0190] Selected subjects must simultaneously meet the following criteria: 1. Healthy male or postmenopausal / amenorrhea female subjects. 2. Be between 18 and 55 years of age (inclusive) at the time of signing the informed consent; 3. Body mass index (BMI) between 19.0 and 26.0 kg / m2 (excluding the threshold value), males must weigh ≥ 50.0 kg and females must weigh ≥ 45.0 kg. 4. Activated partial thromboplastin time (APTT), prothrombin time (PT), international normalized ratio (INR) and platelet count are normal. 5. You understand the procedures of this research plan and are willing to accept and comply with the requirements of this plan.

[0191] Evaluation Item Indicators: 1) Adverse events (D1-D56), 2) Plasma concentration and PK-related parameters (D1-D56), 3) Results of APTT, FXI activity, total FXI and free FXI (D1-D56), 4) Immunogenicity (D1-D56).

[0192] Preliminary results indicate that KN060 has good safety, pharmacokinetic and pharmacodynamic properties in humans.

[0193] 2. We evaluated the efficacy and safety of a single intravenous infusion of KN060 in patients undergoing elective total knee arthroplasty.

[0194] Test Plan: Study design: A randomized, open, positive-controlled, multicenter study to compare the efficacy and safety of different doses of KN060 versus enoxaparin for the prevention of venous thromboembolism in patients undergoing elective unilateral knee arthroplasty. Number of subjects and group allocation: Approximately 400 subjects will be enrolled and randomly assigned to two dose groups of KN060, low and high, and to a LMWH group (low molecular weight heparin). Administration method: Single intravenous infusion Administration time: 6 to 8 hours after surgery (within 24 hours after incision closure) Main inclusion criteria: 1) Inclusion criteria: Men or women (women infertile) aged 18–75 years (inclusive) who were scheduled to undergo elective unilateral TKA surgery. 2) Exclusion criteria: bleeding disorder or high risk of bleeding, history of VTE or receiving anticoagulant therapy that cannot be interrupted, severe hepatic or renal failure, contraindication to angiography, active malignant tumor undergoing treatment, need for local anesthesia such as spinal or epidural anesthesia, and need for epidural analgesia planned preoperatively or after surgery.

[0195] Primary endpoint measures: 1) Primary outcome: incidence of venous thromboembolism 10-14 days after surgery (including confirmation of DVT by venography, symptomatic DVT and PE, fatal PE, and death due to unknown causes where PE cannot be excluded) 2) Primary safety endpoint: incidence of clinically relevant bleeding (defined as major or clinically relevant non-major bleeding) from signing of informed consent (ICF) to 14 days after surgery; 3) PKPD evaluation items (secondary): PKPD detection was performed on some subjects, and the correlation with therapeutic effect and safety evaluation items was analyzed.

[0196] The above detailed description is provided for purposes of explanation and illustration, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments presented herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

Claims

1. A blood coagulation factor XI (FXI) binding protein for use in preventing and / or treating a thromboembolic disease in a subject, comprising: a first immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 14 and a second immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of the VHH shown in SEQ ID NO: 17, The blood coagulation factor XI (FXI) binding protein.

2. 2. The blood coagulation factor XI (FXI) binding protein of claim 1, wherein the thromboembolic disease comprises venous thromboembolism (VTE).

3. The blood coagulation factor XI (FXI) binding protein according to claim 2, wherein the venous thromboembolism is a complication of tumors and / or joint replacement surgery.

4. The blood coagulation factor XI (FXI) binding protein according to any one of claims 2 to 3, wherein the venous thromboembolism is associated with a peripherally inserted central catheter (PICC).

5. The blood coagulation factor XI (FXI) binding protein of claim 2, wherein the venous thromboembolism is associated with hip and / or knee joint replacement surgery.

6. 3. The blood coagulation factor XI (FXI) binding protein of claim 2, wherein the thromboembolic disease comprises deep vein thrombosis (DVT).

7. 3. The blood coagulation factor XI (FXI) binding protein of claim 2, wherein the venous thromboembolism includes pulmonary thromboembolism (PTE).

8. 2. The blood coagulation factor XI (FXI) binding protein of claim 1, wherein the thromboembolic disease comprises systemic thromboembolism.

9. The blood coagulation factor XI (FXI) binding protein according to claim 8, wherein the systemic thromboembolism is a complication of a tumor, a complication of atrial fibrillation, and / or a complication of dialysis.

10. 10. The blood coagulation factor XI (FXI) binding protein of claim 9, wherein the atrial fibrillation includes non-valvular atrial fibrillation.

11. The blood coagulation factor XI (FXI) binding protein of claim 1, wherein the subject is a hypertensive patient, a diabetic patient, a congestive heart failure patient, atrial fibrillation patient, and / or a patient with a history of stroke.

12. The blood coagulation factor XI (FXI) binding protein according to claim 1, wherein CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO: 14 are any one group selected from SEQ ID NOs: 180 to 182, 183 to 185, 186 to 188, and 189 to 191.

13. 2. The blood coagulation factor XI (FXI) binding protein of claim 1, wherein the first immunoglobulin single variable domain comprises an amino acid sequence set forth in one of SEQ ID NOs: 14, 318-323.

14. The blood coagulation factor XI (FXI) binding protein according to claim 1, wherein CDR1, CDR2, and CDR3 in the VHH represented by SEQ ID NO: 17 are any one group selected from SEQ ID NOs: 216 to 218, 219 to 221, 222 to 224, and 225 to 227.

15. 2. The blood coagulation factor XI (FXI) binding protein of claim 1, wherein the second immunoglobulin single variable domain comprises an amino acid sequence set forth in one of SEQ ID NOs: 17, 324 to 329.

16. The blood coagulation factor XI (FXI) binding protein of claim 1, wherein the blood coagulation factor XI (FXI) binding protein further comprises an immunoglobulin Fc region.

17. 2. The blood coagulation factor XI (FXI) binding protein of claim 1, wherein the dosage of the blood coagulation factor XI (FXI) binding protein is about 0.5 mg / kg to about 10 mg / kg.

18. 1. Use of a blood coagulation factor XI (FXI) binding protein in the preparation of a medicament for preventing and / or treating a thromboembolic disease, comprising: The FXI binding protein comprises a first immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of a VHH as set forth in SEQ ID NO: 14, and a second immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of a VHH as set forth in SEQ ID NO: 17.

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