Anti-FXI / FXIa antibodies and uses thereof

Abstract

The present invention relates to the field of therapeutic monoclonal antibodies, and more particularly provides anti-FXI / FXIa antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the same, and methods for preparing the same. The anti-FXI / FXIa antibodies or antigen-binding fragments thereof described herein have specificity and high affinity for FXI / FXIa and can effectively inhibit the activity of FXI / FXIa. Accordingly, the present invention further provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, and their use in preparing medicaments used for the prevention and / or treatment of diseases or disorders related to coagulation or thromboembolism.

Description

[Technical field]

[0001] The present invention relates to the field of therapeutic monoclonal antibodies. More particularly, the present invention relates to antibodies against FXI and / or FXIa and the use of the antibodies for the prevention and / or treatment of diseases or disorders associated with coagulation or thromboembolism. [Background technology]

[0002] Thrombosis or embolism is associated with diseases of various organs throughout the body, mainly cardiac disease, cerebral disease, and peripheral vascular disease. Thrombosis or embolism is characterized by high incidence, severe physical dysfunction, and lethality, and is the first cause of death caused by cardiovascular disease. At present, prophylactic and therapeutic drugs for thrombotic diseases mainly include three types, namely, anticoagulants, antiplatelet drugs, and thrombolytic drugs. Anticoagulants are mainly used to prevent and treat venous thromboembolism caused by various reasons, and can also be used for the prevention of stroke in patients with atrial fibrillation and anticoagulant therapy in patients with acute coronary syndrome. There is a large market demand for anticoagulants, and there are drugs available for clinical use. However, the current anticoagulants used clinically mainly inhibit the general coagulation cascade pathway, and thus bleeding is the main complication. Traditional anticoagulants, such as warfarin, heparin, low molecular weight heparin, and new drugs that have recently been introduced into the market, such as FXa inhibitors (rivaroxaban, apixaban, etc.) and thrombin inhibitors (dabigatran etexilate, hirudin, etc.), all have good effects on reducing thrombosis, but they all face a common drawback, namely, they may cause bleeding complications.Therefore, there is an urgent clinical need for anticoagulants with a low risk of bleeding.

[0003] Coagulation factor XI (FXI) participates in the intrinsic coagulation cascade, and its active form is coagulation factor XIa (FXIa). FXI protein is a dimer formed by two homologous monomers with a molecular weight of about 80 kDa through disulfide bonds. The FXI monomer is composed of four apple domains and one catalytic domain, and is catalyzed by coagulation factor XIIa (FXIIa) to form FXIa with an exposed binding site for FIX, which binds to FIX and then promotes the conversion of FIX to active FIXa, thereby activating the downstream coagulation cascade. Coagulation factor XI is a glycoprotein in the form of a zymogen with a concentration of about 25-30 nM in mammalian plasma, and almost all FXI forms a complex with high molecular weight kininogen (HK) and circulates in the blood. So far, the effect of HK on FXI function remains unclear. HK may help FXI bind to the surface of platelets or endothelial cells, and HK has also been observed to inhibit FXI activation. The activation process of FXI involves the cleavage of each monomer between Arg369 and Ile370 under the action of different proteases, resulting in a protein consisting of a heavy chain of about 50 kDa containing the apple domain and a light chain of about 30 kDa containing the catalytic domain, connected by a disulfide bond formed between Cys362 and Cys482. Activated FXI, or FXIa, refers to the case where each monomer of the dimeric FXI is usually cleaved at Arg369, but only one monomer is activated. FXIa is a Ca 2+ With the participation of FIX, FIXa cleaves FIX to activated FIX (FIXa), which then converts coagulation factor X to its active form, Xa. Xa can then mediate the activation of coagulation factor II / thrombin. Thrombin, as the final protease in the coagulation cascade, can further promote the production of FXIa by directly activating FXI through a feedback mechanism. Both coagulation factor XIIa and FXIa (autoactivated) in the coagulation cascade convert FXI to FXIa.

[0004] In addition, activated FIX (FIXa) can directly bind to platelets and promote the formation of platelet aggregates in blood, forming distal microvascular occlusions. Thus, FIX / FIXa promotes thrombosis through various ways. Furthermore, animal studies and clinical observations show that in the absence of FIX / FIXa, the risk of bleeding is minimal. Therefore, FIX / FIXa is an ideal target for preventing and treating diseases / disorders associated with coagulation or thromboembolism (Zilberman-Rudenko J. et al., "Coagulation factor XI promotes distal platelet activation and single platelet connsumpation in the bloodstream under shear flow," Arterioscler Thromb Vasc Biol., March 2016, 36(3):510-517). Factors that regulate coagulation function through the intervention of FXI molecules and / or FXIa molecules include, but are not limited to, the formation of FXI and / or FXIa dimers; blocking contact between FXI and molecules that can activate FXI (e.g., coagulation factor XIIa, FXIa), thereby inhibiting or blocking activation of FXI; inhibiting or blocking the formation of a complex between FXI and HK; closing the catalytic domain of FXI and / or FXIa or inducing a conformational change in the catalytic domain, thereby inactivating FXI and / or FXIa, thereby inhibiting or preventing activation of the intrinsic coagulation pathway; and inhibiting or blocking the binding of FXI and / or FXIa to its substrates.

[0005] Currently, Bayer's FXIa antibody drug, BAY-1213790, is in Phase II clinical trials for venous thrombosis. BAY-1213790 was derived from a phage display library and binds to the FXIa catalytic domain. In addition, Aronora's AB-023 (14E11) is in Phase I clinical trials for venous thrombosis and in Phase II clinical trials for end-stage renal disease (ESRD). Summary of the Invention [Problem to be solved by the invention]

[0006] At present, there is no FXIa targeting antibody drug on the market. Therefore, there is an urgent need to develop anti-FXI and / or anti-FXIa antibodies with high specificity, low toxicity and side effects, good clinical efficacy, and more convenient administration, which will provide more medication options for patients. [Means for solving the problem]

[0007] In the present application, the present inventors first developed a mouse antibody with excellent properties that can specifically recognize FXI and / or FXIa. Based on this, the present inventors have made great creative efforts to deepen the research on the mouse antibody and carry out modifications thereon, thereby developing the humanized antibody.

[0008] The antibodies of the present invention (especially humanized antibodies) are highly advantageous because they not only retain (or improve upon) the functions and properties of the parental murine antibodies, such as binding to FXI and / or FXIa with high affinity and specificity, and thus have the potential to be used for the prevention and treatment of coagulation or thromboembolic disorders, but also are highly humanized and can be safely administered to human subjects without eliciting an immunogenic response. Thus, the antibodies of the present invention have great clinical value. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows detection of APTT activity in hybridoma supernatants. [Figure 2A] FIG. 1 shows the detection of the coagulation time of mouse anti-human FXI / FIXa monoclonal antibody by the APTT method. [Figure 2B] FIG. 1 shows the detection of the coagulation time of mouse anti-human FXI / FIXa monoclonal antibody by the APTT method. [Diagram 3]FIG. 1 shows detection of anticoagulant function of mouse anti-human FXI / FXIa monoclonal antibody by the BIOPHEN method. [Figure 4] FIG. 1 shows the biological activity of mouse anti-human FXI / FXIa monoclonal antibodies that specifically block FXI and FXIa. [Diagram 5] FIG. 1 shows an APTT anticoagulant activity test for humanized anti-human FXI / FXIa antibodies. [Figure 6] FIG. 1 shows detection of anticoagulant function of humanized anti-human FXI / FXIa monoclonal antibody by the BIOPHEN method. [Figure 7A] FIG. 1 shows the detection of the affinity of humanized anti-human FXI / FXIa antibodies for FXIa by ELISA. [Figure 7B] FIG. 1 shows the detection of the affinity of humanized anti-human FXI / FXIa antibodies for FXI by ELISA. [Figure 8] FIG. 1 shows detection of humanized anti-human FXI / FXIa antibodies competing with control antibody BAY-1213790 for binding to FXIa. [Figure 9A] FIG. 1 shows detection of anticoagulant activity of humanized anti-human FXI / FXIa antibodies in human blood by measuring activated partial thromboplastin time (APTT). [Figure 9B] FIG. 1 shows detection of anticoagulant activity of humanized anti-human FXI / FXIa antibodies in monkey blood by measuring activated partial thromboplastin time (APTT). [Figure 9C] FIG. 1 shows detection of anticoagulant activity of humanized anti-human FXI / FXIa antibodies in dog blood by measuring activated partial thromboplastin time (APTT). [Figure 9D] FIG. 1 shows detection of anticoagulant activity of humanized anti-human FXI / FXIa antibodies in rabbit blood by measuring activated partial thromboplastin time (APTT). [Figure 9E]FIG. 1 shows detection of anticoagulant activity of humanized anti-human FXI / FXIa antibodies in rat blood by measuring activated partial thromboplastin time (APTT). [Figure 10] FIG. 1 shows accelerated stability studies for humanized anti-human FXI / FXIa antibodies. [Figure 11A] FIG. 1 shows detection of APTT activity of humanized anti-human FXI / FXIa antibodies in cynomolgus monkeys by in vivo pharmacodynamics (PD). [Figure 11B] FIG. 1 shows detection of PT activity of humanized anti-human FXI / FXIa antibodies in cynomolgus monkeys by in vivo pharmacodynamics (PD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Antibodies of the Invention In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to FXI and / or FXIa, comprising: (a) three heavy chain CDRs: CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) as set forth in any one of SEQ ID NOs: 1, 15, 16, 17, 29, 31; and / or Three light chain CDRs: CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) as set forth in any one of SEQ ID NOs: 2, 18, 19, 20, 30, 32, as follows; or (b) three heavy chain CDRs as follows: CDR-H1 as described in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-H2 as described in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-H3 as described in (a) or a variant comprising an amino acid mutation as compared thereto; and / or Three light chain CDRs as follows: CDR-L1 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-L2 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-L3 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto. Including, At least one of the three heavy chain CDRs and / or the three light chain CDRs described in (b) contains an amino acid mutation compared to the corresponding CDR in (a), the amino acid mutation being a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of one, two or three amino acids); An antibody or antigen-binding fragment thereof is provided.

[0011] In certain preferred embodiments, the substitutions are conservative substitutions.

[0012] In certain preferred embodiments, the CDRs of an antibody or antigen-binding fragment thereof are defined according to the Kabat, IMGT, Chothia, or AbM numbering systems.

[0013] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises an immunoglobulin framework region (FR) of human or murine origin.

[0014] In certain preferred embodiments, the antibody or antigen-binding fragment thereof binds to human FXI and / or human FXIa.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (a) three heavy chain CDRs: CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) as set forth in any one of SEQ ID NOs: 1, 15, 16, 17; and / or Three light chain CDRs: CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) as set forth in any one of SEQ ID NOs: 2, 18, 19, 20, as follows: or (b) three heavy chain CDRs as follows: CDR-H1 as described in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-H2 as described in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-H3 as described in (a) or a variant comprising an amino acid mutation as compared thereto; and / or Three light chain CDRs as follows: CDR-L1 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-L2 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-L3 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto. Including, At least one of the three heavy chain CDRs and / or the three light chain CDRs set forth in (b) contains an amino acid mutation compared to the corresponding CDR in (a), which is a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of one, two, or three amino acids); in certain preferred embodiments, the substitution is a conservative substitution.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: (a) three heavy chain CDRs: CDR-H1, CDR-H2, and CDR-H3 contained in the heavy chain variable region (VH) as set forth in any one of SEQ ID NOs: 29, 31, as follows; and / or Three light chain CDRs: CDR-L1, CDR-L2, and CDR-L3 contained in the light chain variable region (VL) as set forth in any one of SEQ ID NOs: 30, 32, as follows: or (b) three heavy chain CDRs as follows: CDR-H1 as described in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-H2 as described in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-H3 as described in (a) or a variant comprising an amino acid mutation as compared thereto; and / or Three light chain CDRs as follows: CDR-L1 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-L2 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto, CDR-L3 as set forth in (a) or a variant comprising an amino acid mutation as compared thereto. Including, At least one of the three heavy chain CDRs and / or the three light chain CDRs set forth in (b) contains an amino acid mutation compared to the corresponding CDR in (a), which is a substitution, deletion, or addition of one or several amino acids (e.g., a substitution, deletion, or addition of one, two, or three amino acids); in certain preferred embodiments, the substitution is a conservative substitution.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0018] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL), the CDRs of which are defined by the IMGT numbering system as follows: (a) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 with the sequence of SEQ ID NO: 3, CDR-H2 with the sequence of SEQ ID NO: 4, and CDR-H3 with the sequence of SEQ ID NO: 5, as follows; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 with the sequence of SEQ ID NO:6, CDR-L2 with the sequence of SEQ ID NO:7, and CDR-L3 with the sequence of SEQ ID NO:8, as follows; or (b) a heavy chain variable region (VH) comprising three CDRs: CDR-H1 with the sequence of SEQ ID NO: 33, CDR-H2 with the sequence of SEQ ID NO: 34, and CDR-H3 with the sequence of SEQ ID NO: 35; and / or A light chain variable region (VL) comprising three CDRs: CDR-L1 with the sequence of SEQ ID NO: 36, CDR-L2 with the sequence of SEQ ID NO: 37, and CDR-L3 with the sequence of SEQ ID NO: 38, as follows: Includes.

[0019] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or the light chain variable region (VL) comprises an amino acid mutation, as defined by IMGT, compared to the heavy chain variable region and / or the light chain variable region described in (a) or (b), which is a substitution, deletion, or addition of one or several amino acids, or any combination thereof (e.g., a substitution, deletion, or addition of one, two, or three amino acids, or any combination thereof); preferably, the substitution is a conservative substitution.

[0020] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the invention comprise a heavy chain variable region (VH) and / or a light chain variable region (VL) whose CDRs are defined by the AbM numbering system as follows: (a) three CDRs: CDR-H1 with sequence SEQ ID NO: 9, SEQ ID NO: 10 , 45 or 46 and a CDR-H3 with the sequence of SEQ ID NO: 11; and / or a light chain variable region (VL) comprising three CDRs: CDR-L1 with the sequence of SEQ ID NO: 12, CDR-L2 with the sequence of SEQ ID NO: 13, and CDR-L3 with the sequence of SEQ ID NO: 14, as follows; or (b) three CDRs, as follows: CDR-H1 with sequence SEQ ID NO: 39, SEQ ID NO: 40 Or 47and a CDR-H3 with the sequence of SEQ ID NO: 41; and / or A light chain variable region (VL) comprising three CDRs: CDR-L1 with the sequence of SEQ ID NO: 42, CDR-L2 with the sequence of SEQ ID NO: 43, and CDR-L3 with the sequence of SEQ ID NO: 44, as follows: Includes.

[0021] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or the light chain variable region (VL) comprises an amino acid mutation, as defined by AbM, compared to the heavy chain variable region and / or the light chain variable region described in (a) or (b), which is a substitution, deletion, or addition of one or several amino acids, or any combination thereof (e.g., a substitution, deletion, or addition of one, two, or three amino acids, or any combination thereof); preferably, the substitution is a conservative substitution.

[0022] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), in which at least one CDR of the heavy chain variable region (VH) and / or the light chain variable region (VL) comprises an amino acid mutation, as defined by IMGT or AbM, compared to the heavy chain variable region and / or the light chain variable region described in (a) or (b), which is a substitution, deletion, or addition of one or several amino acids, or any combination thereof (e.g., a substitution, deletion, or addition of one, two, or three amino acids, or any combination thereof); preferably, the substitution is a conservative substitution.

[0023] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention has a heavy chain variable region (VH) comprising a framework region (FR) of the VH derived from a mouse immunoglobulin; and / or the antibody or antigen-binding fragment thereof has a light chain variable region (VL) comprising a framework region (FR) of the VL derived from a mouse immunoglobulin. Thus, in certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention is derived from a mouse immunoglobulin.

[0024] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention has a heavy chain variable region (VH) that comprises a framework region (FR) of the VH derived from a human immunoglobulin; and / or the antibody or antigen-binding fragment thereof has a light chain variable region (VL) that comprises a framework region (FR) of the VL derived from a human immunoglobulin. Thus, in certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention is humanized. In such embodiments, the FR of the heavy chain variable region and / or the FR of the light chain variable region of the antibody or antigen-binding fragment thereof according to the invention may comprise one or more non-human (e.g., mouse) amino acid residues, for example, the heavy chain framework region FR and / or the light chain framework region FR may comprise one or more amino acid backmutations, which include the corresponding mouse amino acid residues.

[0025] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a) a human immunoglobulin heavy chain framework region, or a variant thereof, having up to 20 conservative amino acid substitutions (e.g., up to 15, up to 10, up to 5 conservative amino acid substitutions; e.g., 1, 2, 3, 4, or 5 conservative amino acid substitutions) compared to the germline antibody gene sequence from which it is derived; and / or (b) a human immunoglobulin light chain framework region, or a variant thereof, having up to 20 conservative substitutions of amino acids (e.g., up to 15, up to 10, up to 5 conservative substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids) compared to the germline antibody gene sequence from which it is derived. Includes.

[0026] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof according to the invention have a degree of humanization of at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the following: (i) a sequence as set forth in any one of SEQ ID NOs: 1, 15, 16, and 17; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in any one of SEQ ID NOs: 1, 15, 16, 17 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence set forth in any one of SEQ ID NOs: 1, 15, 16, and 17; and / or (b) a light chain variable region (VL), comprising an amino acid sequence selected from the following: (iv) a sequence as set forth in any one of SEQ ID NOs: 2, 18, 19, and 20; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in any one of SEQ ID NOs: 2, 18, 19, 20 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the sequences set forth in SEQ ID NOs: 2, 18, 19, and 20. Includes.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the following: (i) a sequence as set forth in any one of SEQ ID NOs: 29, 31; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in any one of SEQ ID NOs: 29, 31 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence set forth in any one of SEQ ID NOs: 29, 31; and / or (b) a light chain variable region (VL), comprising an amino acid sequence selected from the following: (iv) a sequence as set forth in any one of SEQ ID NOs: 30 and 32; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in any one of SEQ ID NOs: 30, 32 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the sequences set forth in SEQ ID NOs: 30 and 32. Includes.

[0029] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention has a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows: a VH set forth in any one of SEQ ID NOs: 1, 15, 16, 17, and / or a VL set forth in any one of SEQ ID NOs: 2, 18, 19, 20.

[0030] In certain embodiments, an antibody or antigen-binding fragment thereof according to the present invention has a heavy chain variable region (VH) and / or a light chain variable region (VL) as follows: a VH set forth in any one of SEQ ID NOs: 29, 31, and / or a VL set forth in any one of SEQ ID NOs: 30, 32.

[0031] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:1 and / or a VL set forth in SEQ ID NO:2.

[0032] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO:1 and / or the VL set forth in SEQ ID NO:2.

[0033] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH set forth in SEQ ID NO: 1 and / or the VL set forth in SEQ ID NO: 2. In preferred embodiments, the substitutions are conservative substitutions.

[0034] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:15 and / or a VL set forth in SEQ ID NO:18.

[0035] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO:15 and / or the VL set forth in SEQ ID NO:18.

[0036] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH set forth in SEQ ID NO: 15 and / or the VL set forth in SEQ ID NO: 18. In preferred embodiments, the substitutions are conservative substitutions.

[0037] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:15 and / or a VL set forth in SEQ ID NO:20.

[0038] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO: 15 and / or the VL set forth in SEQ ID NO: 20.

[0039] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH set forth in SEQ ID NO: 15 and / or the VL set forth in SEQ ID NO: 20. In preferred embodiments, the substitutions are conservative substitutions.

[0040] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:16 and / or a VL set forth in SEQ ID NO:18.

[0041] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO:16 and / or the VL set forth in SEQ ID NO:18.

[0042] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VH set forth in SEQ ID NO: 16 and / or the VL set forth in SEQ ID NO: 18 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof). In preferred embodiments, the substitutions are conservative substitutions.

[0043] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:17 and / or a VL set forth in SEQ ID NO:19.

[0044] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO:17 and / or the VL set forth in SEQ ID NO:19.

[0045] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH set forth in SEQ ID NO: 17 and / or the VL set forth in SEQ ID NO: 19. In a preferred embodiment, the substitutions are conservative substitutions.

[0046] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:17 and / or a VL set forth in SEQ ID NO:18.

[0047] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO:17 and / or the VL set forth in SEQ ID NO:18.

[0048] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the VH set forth in SEQ ID NO: 17 and / or the VL set forth in SEQ ID NO: 18 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof). In a preferred embodiment, the substitutions are conservative substitutions.

[0049] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:29 and / or a VL set forth in SEQ ID NO:30.

[0050] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO:29 and / or the VL set forth in SEQ ID NO:30.

[0051] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH set forth in SEQ ID NO: 29 and / or the VL set forth in SEQ ID NO: 30. In preferred embodiments, the substitutions are conservative substitutions.

[0052] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:31 and / or a VL set forth in SEQ ID NO:32.

[0053] In certain embodiments, the VH and / or VL of an antibody or antigen-binding fragment thereof according to the invention has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity compared to the VH set forth in SEQ ID NO:31 and / or the VL set forth in SEQ ID NO:32.

[0054] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH set forth in SEQ ID NO: 31 and / or the VL set forth in SEQ ID NO: 32. In preferred embodiments, the substitutions are conservative substitutions.

[0055] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a) a VH having the sequence shown in SEQ ID NO:1, and a VL having the sequence shown in SEQ ID NO:2; (b) a VH having the sequence set forth in SEQ ID NO: 15, and a VL having the sequence set forth in SEQ ID NO: 18; (c) a VH having the sequence set forth in SEQ ID NO: 15, and a VL having the sequence set forth in SEQ ID NO: 20; (d) a VH having the sequence set forth in SEQ ID NO: 16, and a VL having the sequence set forth in SEQ ID NO: 18; (e) a VH having the sequence set forth in SEQ ID NO: 17, and a VL having the sequence set forth in SEQ ID NO: 19; (f) a VH having the sequence set forth in SEQ ID NO: 17, and a VL having the sequence set forth in SEQ ID NO: 18; (g) a VH having the sequence set forth in SEQ ID NO: 29, and a VL having the sequence set forth in SEQ ID NO: 30; or (h) a VH having the sequence shown in SEQ ID NO: 31, and a VL having the sequence shown in SEQ ID NO: 32 Includes.

[0056] In certain preferred embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a VH set forth in any one of (a)-(h); and / or a light chain variable region (VL) having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a VL set forth in any one of (a)-(h).

[0057] In certain preferred embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH in any one of (a)-(h); and / or a light chain variable region (VL) having one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VL in any one of (a)-(h); preferably, the substitutions are conservative substitutions.

[0058] In any of the above aspects, the antibody or antigen-binding fragment thereof according to the invention may further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin, or a variant thereof. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof according to the invention comprises the heavy chain constant region (CH) of a human or murine immunoglobulin, or a variant thereof, which variant has one or several amino acid substitutions, deletions, or additions, or any combination thereof (e.g., up to 20, up to 15, up to 10, up to 5 amino acid substitutions, deletions, or additions, or any combination thereof; e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions, or any combination thereof) compared to the wild-type sequence from which it is derived. and / or the light chain of the antibody or antigen-binding fragment thereof according to the invention comprises a light chain constant region (CL) of a human or mouse immunoglobulin, or a variant thereof, which variant has one or several amino acid substitutions, deletions or additions, or any combination thereof, compared to the wild-type sequence from which it is derived (e.g. up to 20, up to 15, up to 10, up to 5 amino acid substitutions, deletions or additions, or any combination thereof; e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions, or any combination thereof).

[0059] In certain preferred embodiments, the heavy chain of the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof, which variant has up to 20 conservative substitutions of amino acids (e.g. up to 15, up to 10, up to 5 conservative substitutions of amino acids; e.g. 5, 4, 3, 2, or 1 conservative substitutions of amino acids) compared to the wild-type sequence from which it is derived; and / or the light chain of the antibody or antigen-binding fragment thereof according to the invention comprises a light chain constant region (CL) of a human immunoglobulin, or a variant thereof, which variant has up to 20 conservative substitutions of amino acids (e.g. up to 15, up to 10, up to 5 conservative substitutions of amino acids; e.g. 5, 4, 3, 2, or 1 conservative substitutions of amino acids) compared to the wild-type sequence from which it is derived.

[0060] In certain preferred embodiments, the heavy chain of the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain constant region (CH) of a mouse immunoglobulin, or a variant thereof, which variant has up to 20 conservative substitutions of amino acids (e.g. up to 15, up to 10, up to 5 conservative substitutions of amino acids; e.g. 5, 4, 3, 2, or 1 conservative substitutions of amino acids) compared to the wild-type sequence from which it is derived. In certain embodiments, the light chain of the antibody or antigen-binding fragment thereof according to the invention comprises a light chain constant region (CL) of a mouse immunoglobulin, or a variant thereof, which variant has up to 20 conservative substitutions of amino acids (e.g. up to 15, up to 10, up to 5 conservative substitutions of amino acids; e.g. 5, 4, 3, 2, or 1 conservative substitutions of amino acids) compared to the wild-type sequence from which it is derived.

[0061] In some embodiments, the constant region is altered, e.g., by amino acid mutation, to modify the properties of the anti-FXI and / or FXIa antibody molecule (e.g., to change one or more of the following properties: binding to Fc receptors, glycosylation of the antibody, number of cysteine ​​residues, function on effector cells or complement). Such altered function can be achieved by replacing at least one amino acid residue in the constant region of the antibody with a different residue; for example, effector function can be altered (e.g., reduced) by changing the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, and CDC.

[0062] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the invention have a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; preferably, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4; more preferably, the heavy chain constant region of IgG1 or IgG4 (e.g., human IgG1 or human IgG4). In some embodiments, the antibodies or antigen-binding fragments thereof according to the invention have a light chain constant region selected from, for example, a κ light chain constant region or a λ light chain constant region, preferably a κ light chain constant region (e.g., human κ light chain).

[0063] In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from: (1) a human IgG1 heavy chain constant region; or (2) a human IgG4 heavy chain constant region.

[0064] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a) a heavy chain constant region (CH) comprising an amino acid sequence selected from the following: (i) the sequence set forth in SEQ ID NO: 21; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in SEQ ID NO: 21 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:21; and / or (b) a light chain constant region (CL) comprising an amino acid sequence selected from the following: (iv) the sequence set forth in SEQ ID NO: 22; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in SEQ ID NO: 22 (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in SEQ ID NO:22. Includes.

[0065] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0066] In certain preferred embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain constant region (CH) as set forth in SEQ ID NO:21 and a light chain constant region (CL) as set forth in SEQ ID NO:22.

[0067] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a)(i) a sequence comprising a VH set forth in SEQ ID NO: 15 and a CH set forth in SEQ ID NO: 21; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (i) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (i). and / or a heavy chain comprising an amino acid sequence selected from the group consisting of (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 18 and the CL sequence set forth in SEQ ID NO: 22; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (iv) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0068] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0069] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a)(i) a sequence comprising a VH set forth in SEQ ID NO: 15 and a CH set forth in SEQ ID NO: 21; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (i) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (i). and / or a heavy chain comprising an amino acid sequence selected from the group consisting of (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO:20 and the CL sequence set forth in SEQ ID NO:22; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (iv) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0070] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0071] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a)(i) a sequence comprising a VH set forth in SEQ ID NO: 16 and a CH set forth in SEQ ID NO: 21; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (i) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (i). and / or a heavy chain comprising an amino acid sequence selected from the group consisting of (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 18 and the CL sequence set forth in SEQ ID NO: 22; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (iv) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0072] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0073] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a)(i) a sequence comprising a VH set forth in SEQ ID NO: 17 and a CH set forth in SEQ ID NO: 21; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (i) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (i). and / or a heavy chain comprising an amino acid sequence selected from the group consisting of (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 19 and the CL sequence set forth in SEQ ID NO: 22; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (iv) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0074] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0075] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a)(i) a sequence comprising a VH set forth in SEQ ID NO: 17 and a CH set forth in SEQ ID NO: 21; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (i) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (i). and / or a heavy chain comprising an amino acid sequence selected from the group consisting of (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 18 and the CL sequence set forth in SEQ ID NO: 22; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (iv) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0076] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0077] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0078] In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises: (a)(i) a sequence comprising a VH set forth in SEQ ID NO: 31 and a CH set forth in SEQ ID NO: 21; (ii) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (i) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (i). and / or a heavy chain comprising an amino acid sequence selected from the group consisting of (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 32 and the CL sequence set forth in SEQ ID NO: 22; (v) a sequence having one or several amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence shown in (iv) (e.g., one, two, three, four, or five amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0079] In certain preferred embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

[0080] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO:1 and a heavy chain constant region (CH) set forth in SEQ ID NO:21, and a light chain comprising a VL set forth in SEQ ID NO:2 and a light chain constant region (CL) set forth in SEQ ID NO:22.

[0081] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 22.

[0082] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 20 and a light chain constant region (CL) set forth in SEQ ID NO: 22.

[0083] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 16 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 22.

[0084] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 19 and a light chain constant region (CL) set forth in SEQ ID NO: 22.

[0085] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 22.

[0086] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO:29 and a heavy chain constant region (CH) set forth in SEQ ID NO:21, and a light chain comprising a VL set forth in SEQ ID NO:30 and a light chain constant region (CL) set forth in SEQ ID NO:22.

[0087] In certain preferred embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO:31 and a heavy chain constant region (CH) set forth in SEQ ID NO:21, and a light chain comprising a VL set forth in SEQ ID NO:32 and a light chain constant region (CL) set forth in SEQ ID NO:22.

[0088] In certain preferred embodiments, the antibody of the invention is a chimeric or humanized antibody. In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the invention is selected from an ScFv, a Fab, a Fab', a (Fab')2, an Fv fragment, a disulfide-linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.

[0089] In certain embodiments, the antibody or antigen-binding fragment thereof has reduced ADCC activity.

[0090] In certain embodiments, the antibody or antigen-binding fragment thereof has reduced CDC activity.

[0091] In certain embodiments, the antibody or antigen-binding fragment thereof does not have CDC activity.

[0092] In certain embodiments, the antibody or antigen-binding fragment thereof has no ADCC activity.

[0093] In certain preferred embodiments, the antibody or antigen-binding fragment thereof has reduced ADCC and reduced CDC activity.

[0094] In certain preferred embodiments, the antibody or antigen-binding fragment thereof has no ADCC or CDC activity.

[0095] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof according to the invention have the following characteristics: (a) a K for FXI and / or FXIa (e.g., human FXI and / or human FXIa) of less than about 100 nM, e.g., less than about 10 nM, 1 nM, 0.1 nM, or less; D preferably, K D may be measured by techniques known in the art, for example, by Biolayer Interferometry (BLI) (e.g., ForteBio Octet®); (b) an EC for FXI and / or FXIa (e.g., human FXI and / or human FXIa) of less than about 500 nM, e.g., less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or less; 50 preferably EC 50 can be measured by techniques known in the art, for example, by flow cytometry or cell-based competitive ELISA; (c) binding to FXI and / or FXIa inhibits or blocks the binding of FXI and / or FXIa to their substrates, thereby prolonging clotting time; (d) binding to FXI and / or FXIa inhibits or blocks the catalytic action of FXI and / or FXIa on a substrate; (e) binding to FXI and / or FXIa does not affect extrinsic coagulation; (f) having reduced ADCC activity and / or CDC activity; (g) having no ADCC activity and / or CDC activity; (h) binding to FXI and / or FXIa inhibits or blocks the formation of FXI dimers and / or FXIa dimers; (i) binding to FXI and / or FXIa inhibits or blocks the formation of a complex between FXI and / or FXIa and HK; (j) binding to and / or inducing a conformational change in the catalytic domain of FXI and / or FXIa; (k) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors; or (l) Any combination of (a) to (k) The present invention has at least one of the following:

[0096] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof according to the invention, upon binding to FXI, prevent the FXI catalytic domain from assuming an active conformation. In certain preferred embodiments, the antibodies or antigen-binding fragments thereof according to the invention, upon binding to FXI, prevent the FXI catalytic domain from assuming an active conformation by inducing a conformational change in the zymogen structure, thereby further inhibiting binding to FIX.

[0097] Derived antibody The antibodies or antigen-binding fragments thereof according to the invention may be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to FXI and / or FXIa (especially human FXI and / or FXIa). Thus, the antibodies or antigen-binding fragments thereof according to the invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof according to the invention may be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment thereof to another molecule.

[0098] One type of derivatized antibody (e.g., bispecific antibody) is produced by crosslinking two or more antibodies (of the same or different types). Methods for obtaining such bispecific antibodies are well known in the art, examples of which include, but are not limited to, chemical crosslinking, cell engineering (hybridoma method), or genetic engineering.

[0099] Another type of derivatized antibody is a labeled antibody. For example, an antibody or antigen-binding fragment thereof according to the invention can be linked to a detectable label. A detectable label of the invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art and examples include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C, or 32These include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine ester compounds, colorimetric labels such as magnetic beads (e.g., Dynabeads®), colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. Patents that teach the use of these labels include, but are not limited to, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). The detection labels described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product resulting from the action of the enzyme on the substrate, and colorimetric labels are detected by simple visualization of the colored label. In some embodiments, such labels may be suitable for immunological detection (e.g., enzyme immunometric assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.) In some embodiments, the above-described detection labels may be linked to the antibodies or antigen-binding fragments thereof according to the present invention via linkers of different lengths to reduce potential steric hindrance.

[0100] In addition, the antibodies or antigen-binding fragments thereof according to the invention may also be derivatized with chemical groups, such as polyethylene glycol (PEG) groups, methyl or ethyl groups, or sugar groups, which can be used to improve the biological properties of the antibody, such as extending its serum half-life.

[0101] As one of the antibody derivatives, the present invention provides a conjugate. In some embodiments, the conjugate comprises any antibody or antigen-binding fragment thereof according to the present invention that specifically binds to FXI and / or FXIa, and a conjugate moiety, the conjugate moiety being a detection label such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substrate, or an enzyme as described above. In certain embodiments, the conjugate moiety is a therapeutic agent; optionally, the therapeutic agent is bound to one or more of the antibodies or antigen-binding fragments thereof according to the present invention via a linker. Optionally, the therapeutic agent is bound to the antibodies or antigen-binding fragments thereof according to the present invention via a linker. The therapeutic agent is selected from any one of the drugs mentioned in the uses, the therapeutic methods, and the pharmaceutical compositions in this disclosure. The linker does not significantly affect the biological activity of the antibodies or antigen-binding fragments thereof according to the present invention.

[0102] As an antibody derivative, the present invention provides a multispecific antibody comprising an antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa in accordance with the present invention and another antibody or antigen-binding fragment thereof, or an antibody mimetic. In some embodiments, the multispecific antibody is a conjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa in accordance with the present invention and another antibody or antigen-binding fragment thereof, or an antibody mimetic.

[0103] In certain embodiments, a multispecific antibody is formed by coupling an antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa in accordance with the invention with another antibody or antigen-binding fragment thereof, or antibody mimetic, where each antibody or antigen-binding fragment thereof, or antibody mimetic maintains its original binding specificity. In certain preferred embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0104] Preparation of antibodies The antibody of the present invention may be prepared by various methods known in the art, for example, it may be obtained by recombinant techniques by genetic engineering. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector, which is then transfected into a host cell. The transfected host cell is then cultured under specific conditions to express the antibody of the present invention.

[0105] The antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods, 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol., 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')2 fragments can also be directly isolated from the culture medium of recombinant host cells. Those skilled in the art are well aware of other techniques for preparing these antigen-binding fragments.

[0106] Thus, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, or one or more CDRs thereof, according to the present invention. In some embodiments, according to codon degeneracy known in the art, the nucleotide sequence may be replaced based on the degeneracy of the codons. In certain embodiments, the nucleotide sequence is codon-optimized.

[0107] In certain preferred embodiments, the isolated nucleic acid molecule comprises a first nucleic acid and a second nucleic acid encoding a heavy chain variable region and a light chain variable region, respectively, of an antibody or antigen-binding fragment thereof in accordance with the invention, or a first nucleic acid encoding a heavy chain variable region and a heavy chain constant region, respectively, of an antibody or antigen-binding fragment thereof in accordance with the invention, or a second nucleic acid encoding a light chain variable region and a light chain constant region, respectively, of an antibody or antigen-binding fragment thereof in accordance with the invention, or a first nucleic acid and a second nucleic acid encoding a heavy chain variable region and a light chain constant region, respectively, of an antibody or antigen-binding fragment thereof in accordance with the invention. In certain preferred embodiments, the antibody of the invention, as described above, is selected from any one of the following groups: 36G9.10, 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93, 7B2, 7B2-hz11. In certain preferred embodiments, the first and second nucleic acids comprise nucleic acids having substantially the same sequence as the first and second nucleic acids described above. For example, the isolated nucleic acid molecule may comprise the nucleotide sequences shown in SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28 in the sequence listing, or sequences substantially identical thereto. For example, sequences substantially identical thereto refer to sequences having at least about 85%, 90%, 95%, 99% or more identity to the sequence to which it is compared, or sequences having one or more nucleotide substitutions, or sequences that do not differ by more than 3, 6, 15, 30, or 45 nucleotides.

[0108] In certain preferred embodiments, the invention includes a nucleic acid molecule encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule encoding a light chain variable region of an antibody, wherein the nucleic acid molecule encoding the heavy chain variable region of the antibody has (a) the nucleotide sequence set forth in SEQ ID NO:23, or (b) a sequence substantially the same as the nucleotide sequence set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence set forth in (a)), or (c) a sequence having more than 3, 6, 15, 30, or 45 nucleotides identical to the nucleotide sequence set forth in (a). a nucleic acid molecule encoding a light chain variable region of the antibody has a nucleotide sequence selected from the group consisting of: (d) the nucleotide sequence set forth in SEQ ID NO:24; or (e) a sequence substantially the same as the nucleotide sequence set forth in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence set forth in (d)); or (f) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence set forth in (d).

[0109] In certain preferred embodiments, the invention includes a nucleic acid molecule encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule encoding a light chain variable region of an antibody, wherein the nucleic acid molecule encoding the heavy chain variable region of the antibody has (a) the nucleotide sequence set forth in SEQ ID NO:27, or (b) a sequence substantially the same as the nucleotide sequence set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence set forth in (a)), or (c) a sequence having more than 3, 6, 15, 30, or 45 nucleotides identical to the nucleotide sequence set forth in (a). a nucleic acid molecule encoding a light chain variable region of the antibody has a nucleotide sequence selected from the group consisting of: (d) the nucleotide sequence set forth in SEQ ID NO:28; or (e) a sequence substantially the same as the nucleotide sequence set forth in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence set forth in (d)); or (f) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence set forth in (d).

[0110] In certain preferred embodiments, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule as set forth in SEQ ID NO:23 encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule as set forth in SEQ ID NO:24 encoding a light chain variable region of an antibody.

[0111] In certain preferred embodiments, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule as set forth in SEQ ID NO:27 encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule as set forth in SEQ ID NO:28 encoding a light chain variable region of an antibody.

[0112] In certain preferred embodiments, the invention includes a nucleic acid molecule encoding an antibody heavy chain and / or a nucleic acid molecule encoding an antibody light chain, wherein the nucleic acid molecule encoding the antibody heavy chain has (a) the nucleotide sequence set forth in SEQ ID NO:25; or (b) a sequence substantially the same as the nucleotide sequence set forth in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence set forth in (a)); or (c) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence set forth in (a). and / or a nucleic acid molecule encoding an antibody light chain has a sequence selected from the group consisting of: (d) the nucleotide sequence set forth in SEQ ID NO:26, or (e) a sequence substantially the same as the nucleotide sequence set forth in (d) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or more identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence set forth in (d)), or (f) a sequence that differs by no more than 3, 6, 15, 30, or 45 nucleotides from the nucleotide sequence set forth in (d).

[0113] In certain preferred embodiments, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule encoding an antibody heavy chain as set forth in SEQ ID NO:25, and / or a nucleic acid molecule encoding an antibody light chain as set forth in SEQ ID NO:26.

[0114] Another aspect of the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule of the invention. In certain preferred embodiments, a vector of the invention is, for example, a plasmid, a cosmid, a bacteriophage, a lentivirus, etc. In certain preferred embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof in accordance with the invention in a subject (e.g., a mammal such as a human).

[0115] Another aspect of the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention. The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain preferred embodiments, the host cell of the invention is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44) cell.

[0116] Another aspect of the invention provides a method for preparing an antibody or antigen-binding fragment thereof according to the invention, comprising culturing a host cell of the invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the culture of the cultured host cells.

[0117] Uses, Treatment Methods, and Pharmaceutical Compositions Another aspect of the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector, a host cell, a multispecific antibody, or a conjugate according to the invention and a pharma- ceutically acceptable carrier and / or excipient.

[0118] In certain preferred embodiments, the pharmaceutical compositions of the invention comprise an antibody or antigen-binding fragment thereof in accordance with the invention and a pharma- ceutically acceptable carrier and / or excipient.

[0119] In certain preferred embodiments, the pharmaceutical compositions of the invention comprise an isolated nucleic acid molecule, vector, or host cell according to the invention, and a pharma- ceutically acceptable carrier and / or excipient. In such embodiments, the host cell comprises an isolated nucleic acid molecule or vector as previously described.

[0120] In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharma- ceutical active agent, hi certain preferred embodiments, the additional pharma- ceutical active agent is an antiplatelet agent, an anticoagulant agent, or a thrombolytic agent.

[0121] In certain preferred embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof according to the invention and the further pharma- ceutical active agent are provided as separate components or as components of a single composition, and thus the antibody or antigen-binding fragment thereof according to the invention and the further pharma- ceutical active agent may be administered simultaneously, separately or sequentially.

[0122] In certain preferred embodiments, the pharmaceutical composition may further comprise an additional pharma- ceutical active agent. The additional pharma-ceutical active agent is selected from the group consisting of aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, unfractionated heparin, heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, streptokinase, urokinase, alteplase, prourokinase, or any combination thereof.

[0123] In another aspect, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody, or conjugate in the pharmaceutical composition of the present invention exhibits the following biological activity in a subject: (a) binding to and / or inducing a conformational change in the catalytic domain of FXI and / or FXIa; (b) inhibiting or blocking the binding of FXI and / or FXIa to a substrate; (c) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors; (d) inhibiting or blocking the binding of FXI to coagulation factor XIIa (FXIIa), thereby inhibiting or blocking the conversion of FXI to active FXIa; (e) inhibiting or blocking the binding of FXIa to the coagulation factor FIX, thereby inhibiting or blocking the conversion of FIX to active FIXa; (f) inhibiting or blocking FXI and / or FXIa-mediated activation of the intrinsic coagulation pathway; (g) inhibiting or blocking the activity of FXI and / or FXIa in thrombosis; (h) prolonging FXI and / or FXIa-mediated clotting time; (i) inhibiting thrombosis; (j) preventing and / or treating a disease or disorder associated with coagulation or thromboembolism mediated by FXI and / or FXIa; or (k) Any combination of (a) to (j) is sufficient to result in at least one of

[0124] In another aspect, the pharmaceutical composition of the present invention further comprises a second antibody or a nucleic acid encoding a second antibody, which is another antibody that recognizes a different epitope of FXI or FXIa, or an antibody that specifically binds to a receptor or ligand selected from the group consisting of thrombin, antiplasmin, factor XII, factor VIII, factor VII, factor X, factor IX, factor II, tissue factor, P-selectin and its ligands, L-selectin and its ligands, and any combination of the above antibodies.

[0125] Another aspect of the invention provides the use of an antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition of the invention in the manufacture of a medicament, comprising: (a) binding to and / or inducing a conformational change in the catalytic domain of FXI and / or FXIa; (b) inhibiting or blocking the binding of FXI and / or FXIa to a substrate; (c) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors; (d) inhibiting or blocking the binding of FXI to coagulation factor XIIa (FXIIa), thereby inhibiting the conversion of FXI to active FXIa; (e) inhibiting or blocking the binding of FXIa to the coagulation factor FIX, thereby inhibiting the conversion of FIX to active FIXa; (f) inhibiting or blocking FXI and / or FXIa-mediated activation of the intrinsic coagulation pathway; (g) inhibiting or blocking the activity of FXI and / or FXIa in thrombosis; (h) prolonging FXI and / or FXIa-mediated clotting time; (i) inhibiting thrombosis; (j) preventing and / or treating a disease or disorder associated with coagulation or thromboembolism mediated by FXI and / or FXIa; or (k) Any combination of (a) to (j) Used for.

[0126] In certain preferred embodiments, when the isolated nucleic acid molecule, vector, or host cell of the invention is used for the manufacture of a medicament, the host cell comprises the isolated nucleic acid molecule or vector described above.

[0127] In certain preferred embodiments, when the vectors or host cells of the invention are used for the manufacture of a medicament, the medicament is used for the prevention and / or treatment of a disease or disorder associated with coagulation or thromboembolism in a subject (e.g., a human).

[0128] In certain embodiments, the subject is a mammal, hi certain preferred embodiments, the subject is a human.

[0129] In certain preferred embodiments, when the vectors or host cells of the invention are used for the manufacture of a medicament, the medicament is used to delay the onset of a disease or disorder associated with coagulation or thromboembolism in a subject (e.g., a human).

[0130] In certain preferred embodiments, when the vectors or host cells of the invention are used for the manufacture of a medicament, the medicament is used to reduce or inhibit the recurrence of a disease or disorder associated with coagulation or thromboembolism in a subject (e.g., a human).

[0131] In certain preferred embodiments, when a vector or host cell of the invention is used for the manufacture of a medicament, the medicament is used in a subject (eg, a human).

[0132] In certain preferred embodiments, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition of the invention is used to treat thrombosis, thrombotic stroke, atrial fibrillation, stroke prevention associated with atrial fibrillation (SPAF), or other conditions that may be associated with thrombosis, thrombotic stroke, atrial fibrillation, or stroke prevention associated with atrial fibrillation (SPAF). fibrillation), deep vein thrombosis, venous thromboembolism, acute coronary syndrome (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, systemic embolism, myocardial infarction (MI), acute myocardial infarction (AMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention, peripheral arterial occlusive disease (PAOD), renal vein thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, diffuse intravascular coagulation, thromboembolic disorders caused by medical devices (e.g., catheters), severe systemic inflammatory response syndrome, metastatic cancer, infectious diseases, organ failure (e.g., renal failure) , toxicity caused by administration of therapeutic proteins in the body, multiple trauma, ischemia-reperfusion injury, localized fibrin deposition, adult pulmonary alveolar proteinosis, venous thromboembolic events (VTE) before and after total knee arthroplasty (TKA), coronary heart disease, thromboembolism after myocardial infarction, stroke in patients with non-valvular atrial fibrillation, thrombosis and thromboembolism in chronic kidney disease, thrombosis and thromboembolism in patients undergoing hemodialysis and extracorporeal membrane oxygenation, deep vein thrombosis (DVT), or pulmonary embolism (PE).

[0133] In another aspect, the present invention provides a method for preventing and / or treating a disease or disorder associated with coagulation or thromboembolism in a subject. In another aspect, the present invention provides a method for delaying the onset of a disease or disorder associated with coagulation or thromboembolism in a subject. In another aspect, the present invention provides a method for reducing or inhibiting the recurrence of a disease or disorder associated with coagulation or thromboembolism in a subject. The above-described methods comprise the step of administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition of the present invention.

[0134] When the host cell of the invention is used in the methods described above, the host cell comprises an isolated nucleic acid molecule or a vector, as described above.

[0135] In another aspect, the method further comprises administering a second therapy to the subject, wherein the second therapy is selected from the group consisting of antiplatelet agents, anticoagulants, and thrombolytic agents.

[0136] In certain preferred embodiments, the second treatment is selected from aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, unfractionated heparin, heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, streptokinase, urokinase, alteplase, prourokinase, and any combination thereof.

[0137] In certain preferred embodiments, the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition of the invention is used to treat thrombosis, thrombotic stroke, atrial fibrillation, stroke prevention associated with atrial fibrillation (SPAF), or other conditions that may be associated with thrombosis, thrombotic stroke, atrial fibrillation, or stroke prevention associated with atrial fibrillation (SPAF). fibrillation), deep vein thrombosis, venous thromboembolism, acute coronary syndrome (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, systemic embolism, myocardial infarction (MI), acute myocardial infarction (AMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention, peripheral arterial occlusive disease (PAOD), renal vein thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, diffuse intravascular coagulation, thromboembolic disorders caused by medical devices (e.g., catheters), severe systemic inflammatory response syndrome, metastatic cancer, infectious diseases, organ failure (e.g., renal failure) , toxicity caused by administration of therapeutic proteins in the body, multiple trauma, ischemia-reperfusion injury, localized fibrin deposition, adult pulmonary alveolar proteinosis, venous thromboembolic events (VTE) before and after total knee arthroplasty (TKA), coronary heart disease, thromboembolism after myocardial infarction, stroke in patients with non-valvular atrial fibrillation, thrombosis and thromboembolism in chronic kidney disease, thrombosis and thromboembolism in patients undergoing hemodialysis and extracorporeal membrane oxygenation, deep vein thrombosis (DVT), or pulmonary embolism (PE).

[0138] The antibody or antigen-binding fragment thereof according to the present invention and the pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical art, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention shall be sterile and stable under the conditions of preparation and storage. The preferred dosage form is an injection. Such an injection may be a sterile injection solution. For example, a sterile injection solution may be prepared by the following method: incorporating the required amount of the recombinant protein of the present invention into a suitable solvent, optionally incorporating other desired ingredients at the same time (including but not limited to pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. Additionally, sterile injectable solutions can be prepared for storage and use as sterile lyophilized powders (e.g., via vacuum drying or freeze drying). Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, prior to use.

[0139] In addition, the antibodies or antigen-binding fragments thereof according to the invention may be present in pharmaceutical compositions in unit dosage form for easy administration.

[0140] The antibody or antigen-binding fragment thereof, and pharmaceutical composition of the present invention may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intravesical, inguinal, intravesical, topical (such as powder, ointment, or drop), or nasal. However, for many therapeutic applications, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will appreciate that the route / mode of administration will vary according to the intended purpose. In a preferred embodiment, the antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present invention is administered by intravenous infusion or injection.

[0141] The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody, or conjugate of the invention. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest a disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of an antibody or antigen-binding fragment thereof according to the invention may vary according to the following factors: the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the mode of administration of the drug, other treatments administered simultaneously, etc.

[0142] In the present invention, the administration regimen can be adjusted to obtain the best desired response (e.g., therapeutic or prophylactic response), for example, administration can be a single dose or multiple doses over a period of time, and the dose can be reduced or increased depending on the exigencies of the therapeutic situation.

[0143] A typical non-limiting range of a therapeutically or prophylactically effective amount of the recombinant protein of the present invention is 0.02-100 mg / kg, such as 0.1-100 mg / kg, 0.1-50 mg / kg, or 1-50 mg / kg. It should be noted that dosages may vary depending on the type and severity of the condition being treated. In addition, those skilled in the art will understand that for any particular patient, the detailed dosing regimen should be adjusted over time according to the needs of the patient and the professional assessment of the physician; dosage ranges given herein are for illustrative purposes only and are not intended to limit the use or scope of the pharmaceutical composition of the present invention.

[0144] In the present invention, the subject can be a mammal, such as a human.

[0145] Detection Methods and Kits The antibodies or antigen-binding fragments thereof according to the present invention are capable of specifically binding to FXI and / or FXIa (e.g., human FXI and / or human FXIa) and therefore can be used to detect the presence or level of FXI and / or FXIa in a sample.

[0146] Thus, in another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof according to the present invention. In certain preferred embodiments, the antibody or antigen-binding fragment thereof according to the present invention carries a detectable label. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to the present invention. Preferably, the second antibody further comprises a detectable label.

[0147] In the present invention, the detectable label can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are particularly preferred for immunological detection (e.g., enzyme immunometric assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). Such labels are well known in the art and include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H, 125 I, 35 S, 14 C, or 32These include, but are not limited to, fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine ester compounds, colorimetric labels such as magnetic beads (e.g., Dynabeads®), colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned labels. Patents that teach the use of these labels include, but are not limited to, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). The detection labels described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product resulting from the action of the enzyme on the substrate, and colorimetric labels are detected by simple visualization of the colored label. In some embodiments, the detectable labels described above can be linked to the antibodies or antigen-binding fragments thereof according to the invention via linkers of different lengths to reduce potential steric hindrance.

[0148] In another aspect, the invention provides a method for detecting the presence or level of FXI and / or FXIa (e.g. human FXI and / or human FXIa) in a sample, the method comprising using an antibody or antigen-binding fragment thereof according to the invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention also carries a detectable label. In another preferred embodiment, the method further comprises using a reagent carrying a detectable label to detect the antibody or antigen-binding fragment thereof according to the invention. The method may be used for diagnostic or non-diagnostic purposes (e.g. the sample is a cell sample and is not derived from a patient).

[0149] In another aspect, the present invention provides a method for detecting the presence or level of FXI and / or FXIa (e.g., human FXI and / or human FXIa) in a sample, comprising the step of contacting an antibody or antigen-binding fragment thereof according to the present invention with the sample under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof or conjugate and FXI and / or FXIa.

[0150] In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof according to the invention in the manufacture of a kit for detecting the presence or level of FXI and / or FXIa (e.g. human FXI and / or human FXIa) in a sample. In another aspect, the invention provides a diagnostic or therapeutic kit comprising one or more of the following substances that may be provided by the invention: an antibody or antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector, a host cell, a multispecific antibody, a conjugate, or a pharmaceutical composition. The diagnostic or therapeutic kit also comprises instructions for use.

[0151] The antibody or antigen-binding fragment of the present invention has high binding affinity and extremely strong specificity for FXI and / or FXIa. Therefore, the antibody or antigen-binding fragment of the present invention is suitable for the prevention and / or treatment of diseases or disorders associated with coagulation or thromboembolism. The humanized antibody of the present invention retains the functions and properties of the parent mouse antibody. Furthermore, the humanized antibody of the present invention is highly humanized so that it can be safely administered to human subjects without inducing an immunogenic response. In addition, the antibody or antigen-binding fragment of the present invention has no or little risk of bleeding. Therefore, the antibody or antigen-binding fragment of the present invention has great clinical value.

[0152] Abbreviations and definitions CDR Complementarity determining region in immunoglobulin variable region FR: framework region of an antibody: amino acid residues other than CDR residues in the antibody variable region VH Heavy chain variable region of an antibody VL antibody light chain variable region IgG Immunoglobulin G An immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991) Chothia The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, e.g., Chothia and Lesk (1987), J. Mol. Biol., 196:901-917; Chothia (1989), Nature, 342:878-883). The AbM CDR definition is derived from Martin's related work (Martin ACR, Cheetham JC, Rees AR (1989), "Modeling antibody hypervariable loops: A combined algorithm", Proc Natl Acad Sci USA 86:9268-9272), and this definition integrates the partial definition of Kabat and the partial definition of Chothia. IMGT A numbering system based on the International ImmunoGeneTics Information System® (IMGT), originated by Lefranc et al. (see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). mAb Monoclonal antibody EC50 - the concentration at which 50% efficacy or binding occurs IC50 Concentration at which 50% inhibition occurs ELISA Enzyme-linked immunosorbent assay PCR Polymerase Chain Reaction HRP Horseradish Peroxidase KD equilibrium dissociation constant Ka Association rate constant Kd dissociation rate constant ADCC antibody-dependent cytotoxicity CDC Complement-dependent cytotoxicity FACS Fluorescence Activated Cell Sorting CDR-H1 Complementarity determining region 1 in the immunoglobulin heavy chain variable region CDR-H2 Complementarity determining region 2 in the immunoglobulin heavy chain variable region CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region CDR-L1 Complementarity determining region 1 in the immunoglobulin light chain variable region CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region APTT Activated partial thromboplastin time CFA Complete Freund's Adjuvant EC50 50% effective concentration FACS Flow Cytometry Technology IC50 50% inhibitory concentration IFA Incomplete Freund's Adjuvant MFI Mean Fluorescence Intensity FXI Human coagulation factor XI FXIa Activated human coagulation factor XI RLU Relative Light Unit In the present invention, unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.In addition, all experimental procedures used herein, such as cell culture methods, biochemistry, nucleic acid chemistry, immunology, etc., are standard procedures widely used in the corresponding fields.In addition, in order to better understand the present invention, the following provides definitions and explanations of related terms.

[0153] As used herein, the terms "FXI protein", "FXI antigen" and "FXI" are used interchangeably to refer to various species of coagulation factor XI proteins. The terms "FXIa protein", "FXIa antigen" and "FXIa" are used interchangeably to refer to various species of activated factor XI proteins. The terms "FXI" and "FXIa" (and similar terms) include mutants and variants of native FXI and FXIa proteins, respectively, which mutants and variants have substantially the same amino acid sequence as the native primary structure (amino acid sequence) described herein.

[0154] In the context of the present invention, the terms "clotting and coagulation cascade", "clotting cascade model" and similar terms are well known and refer to the process in which the coagulation pathway leads to the production of thrombin through a proteolytic cascade, which then converts soluble fibrinogen into fibrin, which forms a blood clot. The various enzymes of this pathway exist in the zymogen form (inactive form) in plasma, but upon activation undergo proteolytic cleavage to release active clotting factors. The process of thrombin generation can be divided into three stages: the intrinsic and extrinsic pathways, and the final common pathway.

[0155] As used herein, the term "antibody" refers to an immunoglobulin molecule, which is typically composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). The light chains of antibodies can be classified as κ (kappa) light chains and λ (lambda) light chains. The heavy chains can be classified as μ, δ, γ, α, or ε heavy chains, and thus the antibody isotype is defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chains also contain a "D" region of about 3 or more amino acids. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in binding of the antibody to an antigen, but exhibits various effector functions, such as mediating the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or host factors, including the first component of the classical complement system (C1q). The VH and VL regions can also be subdivided into hypervariable regions (called complementarity determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and each VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form an antigen-binding site, respectively. In each region or domain, the amino acid assignments may be according to numbering systems such as IMGT, Kabat, Chothia, or AbM.

[0156] Unless the context clearly dictates otherwise, when the term "antibody" is referred to herein, this term includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0157] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody which are responsible for antigen binding. The exact boundaries of these amino acid residues can be determined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia and Lesk (1987), J. Mol. Biol., 196:901-917; Chothia et al. (1989), Nature, 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol., 27:55-77, 2003), or the AbM numbering system (Martin ACR, Cheetham JC, Rees AR, Proc Natl Acad Sci. USA, 86:9268-9272, 1989). For a given antibody, one skilled in the art can identify the CDRs defined by each numbering system. Furthermore, one skilled in the art is familiar with the correspondence between different numbering systems (see, for example, Lefranc et al., Dev.Comparat.Immunol., 27:55-77, 2003).

[0158] As used herein, the term "framework region" or "FR" residues refers to amino acid residues other than the above-defined CDR residues in antibody variable regions.

[0159] As used herein, the term "germline antibody genes" refers to non-lymphocyte-encoded immunoglobulin sequences that have not undergone the processes that can result in gene rearrangement and maturation for expression of a particular immunoglobulin. One advantage provided by various embodiments of the present invention stems from the recognition that germline antibody genes retain more of the important amino acid sequence structures that characterize individual animal species than do mature antibody genes. Thus, when applied therapeutically to the species, germline antibody genes are less likely to be recognized as foreign by the species.

[0160] The term "antibody" is not limited to any particular method for making the antibody. For example, the term "antibody" includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibodies can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0161] As used herein, the term "antigen-binding fragment" of an antibody refers to a full-length antibody or a polypeptide that is a partial fragment of an antibody, such as a polypeptide that is a fragment of a full-length antibody, also referred to as an "antigen-binding portion", that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or the ability to compete with the full-length antibody for specific binding to an antigen. See generally, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety and for all purposes. Antigen-binding fragments of antibodies may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology by Domantis), domain antibodies (technology by Ablynx), and polypeptides of sufficient size to retain the specific antigen-binding ability of a full-length antibody. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0162] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains", where "full-length heavy chains" or "heavy chains" refers to a polypeptide chain consisting of, from the N-terminus to the C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; if the full-length antibody is an antibody of the IgE isotype, it also optionally includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of, from the N-terminus to the C-terminus, VH, CH1, HR, CH2, and CH3. A "full-length light chain" or "light chain" is a polypeptide chain consisting of, from the N-terminus to the C-terminus, a light chain variable region (VL) and a light chain constant region (CL). The two pairs of full-length antibody chains are connected by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present invention may be derived from a single species, such as human; it may also be a chimeric or humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites, each formed by a pair of VH and VL, and the two antigen-binding sites specifically recognize / bind to the same antigen. As used herein, the term "Fd fragment" refers to an antibody fragment composed of the VH domain and the CH1 domain; the term "dAb fragment" refers to an antibody fragment composed of the VH domain (Ward et al., Nature, 341:544-546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of the VL domain, the VH domain, the CL domain, and the CH1 domain; the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; and the term "Fab' fragment" refers to a fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in an F(ab')2 fragment composed of an intact light chain and a heavy chain Fd fragment (composed of the VH domain and the CH1 domain).

[0163] As used herein, the term "Fv fragment" refers to an antibody fragment composed of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. In general, the six CDRs are considered to confer antigen-binding specificity to the antibody. However, even the variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can still recognize and bind to the antigen, although its affinity may be lower than that of the complete binding site.

[0164] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the second and third constant regions of a first heavy chain and the second and third constant regions of a second heavy chain of an antibody linked via disulfide bonds. The Fc fragment of an antibody has many different functions but does not participate in binding to an antigen.

[0165] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL domain and a VH domain, where the VL and VH are connected by a linker (see, for example, Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Pluckthun, Pharmacology of Monoclonal Antibodies, Vol. 113, Eds. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH, or NH2-VH-linker-VL-COOH. According to the prior art, suitable linkers consist of repeats of the amino acid sequence GGGGS, or variations thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, although variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA, 90:6444-6448). Other linkers that may be used in the present invention are described by Alfthan et al. (1995), Protein Eng., 8:725-731; Choi et al. (2001), Eur. J. Immunol., 31:94-106; Hu et al. (1996), Cancer Res., 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol., 293:41-56; and Roovers et al. (2001), Cancer Immunol. Optionally, a disulfide bond may also be present between the VH and VL of the scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by linking two scFvs.

[0166] As used herein, the term "diabody" refers to when the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, such that the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); and Poljak RJ et al., Structure, 2:1121-1123 (1994)).

[0167] Each of the foregoing antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or the ability to compete with the full-length antibody for specific binding to an antigen.

[0168] As used herein, the term "multispecific antibody" refers to an antibody with multiple different antigen-binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. A "bispecific antibody" refers to an antibody with two different antigen-binding specificities formed by conjugating a first antibody (or a fragment thereof) with a second antibody (or a fragment thereof) or an antibody mimic via a coupling arm, and coupling methods include, but are not limited to, chemical reaction, gene fusion, protein fusion, polypeptide fusion, and enzymatic reaction. A trispecific antibody is an antibody with three different antigen-binding specificities, and a tetraspecific antibody is an antibody with four different antigen-binding specificities.

[0169] As used herein, an "antibody mimic" is a substance that specifically binds to an antigen like an antibody, but does not have the structure of an antibody. Antibody mimics are usually artificial peptides or proteins with a molar mass of about 3-20 kDa, such as designed ankyrin repeat proteins (DARPins) and finomers. DARPins (designed ankyrin repeat proteins) are linked to IgG antibodies, scFv-Fc antibody fragments, or combinations thereof, as described in Chinese Patent Application No. 104341529A. Anti-IL-17a finomers bind to anti-IL-6R antibodies, as described in WO2015141862A1.

[0170] As used herein, "immunoglobulin" or "Ig" may refer to a type of protein that functions as an antibody. Antibodies expressed by B cells are sometimes called antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE, where IgG is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is the most common circulating antibody that is important in defense against bacteria and viruses.

[0171] In this context, antigen-binding fragments of an antibody (e.g., the antibody fragments described above) may be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods) and screened for specificity in the same way that intact antibodies are screened.

[0172] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and may be used interchangeably, and refer to an antibody or antibody fragment derived from a population of highly homologous antibody molecules, i.e., antibody molecules that are identical except for naturally occurring mutations that may occur spontaneously. Monoclonal antibodies have a high degree of specificity for a single epitope on an antigen. Polyclonal antibodies are referred to in contrast to monoclonal antibodies and typically contain at least two or more different antibodies, which typically recognize different epitopes on an antigen. In addition, the modifier "monoclonal" indicates only the character of the antibody as being obtained from a highly homogenous population of antibodies, and should not be construed as requiring production of the antibody by any particular method.

[0173] The monoclonal antibodies of the present invention may be prepared by a variety of techniques, including hybridoma technology (see, e.g., Kohler et al., Nature, 256:495, 1975), recombinant DNA technology (see, e.g., U.S. Pat. No. 4,816,567), or phage antibody library technology (see, e.g., Clackson et al., Nature, 352:624-628, 1991; or Marks et al. J. Mol. Biol., 222:581-597, 1991).

[0174] For example, monoclonal antibodies can be prepared as follows: First, mice or other suitable host animals are immunized with immunogens (with adjuvants added, if necessary). The injection method of immunogens or adjuvants is usually multiple-point subcutaneous injection or intraperitoneal injection. The immunogens are pre-coupled with certain known proteins, such as serum albumin or soybean trypsin inhibitor, so as to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the animals are immunized, the body will produce lymphocytes that secrete antibodies that specifically bind to the immunogens. In addition, lymphocytes can also be obtained by in vitro immunization. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusing agent, such as PEG, to obtain hybridoma cells (Goding, "Monoclonal Antibodies: Principles and Practice", pp. 59-103, Academic Press, 1996). The hybridoma cells prepared above can be inoculated into a suitable culture medium, preferably containing one or more substances capable of inhibiting the growth of unfused parental myeloma cells. For example, for parental myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), the addition of HAT (hypoxanthine, aminopterin, and thymine) medium to the culture medium will inhibit the growth of HGPRT-deficient cells. The preferred myeloma cells should have a high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among these, mouse myelomas, such as the MOP-21 or MC-11 strains (Salk Institute Cell Distribution Center, San Diego, Calif., USA) derived from mouse tumors, and the SP-2 / 0 cell line, or the X63-Ag8-653 cell line (American Type Cuture Collection, Rockville, Md., USA), are preferred for the myeloma cells.In addition, studies have also reported the use of human myeloma cell lines and human-mouse heteromyeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., "Monoclonal Antibodies Production Techniques and Applications", pp. 51-63, Marcel Dekker, Inc., New York, 1987). Culture medium for growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), etc. For example, the affinity of monoclonal antibodies can be determined using the Scatchard assay described by Munson et al., Anal. Biochem., 107:220 (1980). After determining the specificity, affinity, and reactivity of the antibodies produced by the hybridomas, the cell line of interest is subcloned by standard limiting dilution methods as described in Goding, "Monoclonal Antibodies: Principles and Practice", pp. 59-103, Academic Press, 1996. Appropriate culture media can be DMEM or RPMI-1640, etc. In addition, the hybridoma cells can also be grown in animals in the form of ascites tumors. The monoclonal antibodies secreted from the subcloned cells can be isolated from the cell culture medium, ascites, or serum using conventional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0175] Monoclonal antibodies can also be obtained by recombinant genetic engineering. DNA molecules encoding the heavy and light chain genes of a monoclonal antibody can be isolated from hybridoma cells by PCR amplification using nucleic acid primers that specifically bind to the heavy and light chain genes of the monoclonal antibody. The resulting DNA molecules are inserted into an expression vector, which is then transfected into host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) and cultured under appropriate conditions, thereby obtaining the desired recombinant antibody.

[0176] Antibodies can be purified by known techniques, such as affinity chromatography using Protein A or Protein G. A specific antigen (the target molecule recognized by the antibody) or its epitope can be immobilized on a column and the immunospecific antibody purified by immunoaffinity chromatography. For the purification of immunoglobulins, see, for example, D. Wilkinson (Scientist, Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0177] As used herein, the term "mouse antibody" refers to an antibody prepared by fusing B cells of an immunized mouse with myeloma cells and selecting mouse hybrid fusion cells that can proliferate indefinitely and secrete antibody, followed by screening, antibody preparation, and antibody purification; or an antibody secreted by plasma cells formed by B cell differentiation and proliferation after antigen infiltrates the mouse's body.

[0178] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light and / or heavy chain is derived from one antibody (which may be from a specific species and may belong to a particular specific antibody class or subclass) and another portion of its light and / or heavy chain is derived from another antibody (which may be from the same species or a different species and may belong to the same antibody class or subclass or a different antibody class or subclass), but which nevertheless retains binding activity for an antigen of interest (Cabilly et al., U.S. Pat. No. 4,816,567; Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). For example, the term "chimeric antibody" can include antibodies in which the heavy and light chain variable regions are derived from a first antibody (e.g., a murine antibody) but the heavy and light chain constant regions are derived from a second antibody (e.g., a human antibody) (e.g., a human-mouse chimeric antibody).

[0179] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies typically retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune responses, and the like. The donor antibody can be an antibody derived from a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) with the desired properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and / or ability to enhance immune responses).

[0180] Humanized antibodies are particularly advantageous because they can not only retain the properties expected of a non-human donor antibody (e.g., a mouse antibody), but also effectively reduce the immunogenicity of the non-human donor antibody (e.g., a mouse antibody) in a human subject. However, due to the problem of matching the CDRs of the donor antibody with the FRs of the acceptor antibody, the properties expected of a humanized antibody (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and / or ability to enhance immune response) are generally lower than those of a non-human donor antibody (e.g., a mouse antibody).

[0181] Thus, although researchers in the field have studied antibody humanization in depth and have made some progress (see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today, 21:397-402 (2000)), the prior art does not provide detailed guidance on how to fully humanize a donor antibody such that the humanized antibody produced is not only maximally humanized but also retains as much of the properties expected of the donor antibody as possible. To obtain a humanized antibody that is not only highly humanized (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) but also retains the properties expected of the specific donor antibody, engineers must expend a great deal of creative effort exploring, exploring and modifying the specific donor antibody.

[0182] In the present invention, in order for the humanized antibody to retain as much as possible the properties of the donor antibody (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity, and / or ability to enhance the immune response), the framework region (FR) of the humanized antibody of the present invention may contain both amino acid residues of the human acceptor antibody and amino acid residues of the corresponding non-human donor antibody.

[0183] Chimeric or humanized antibodies of the invention can be prepared based on the sequences of the mouse monoclonal antibodies prepared above. DNA encoding the heavy and light chains can be obtained from the target mouse hybridoma and engineered using standard molecular biology techniques to contain non-mouse (e.g., human) immunoglobulin sequences.

[0184] To prepare chimeric antibodies, the variable regions of mouse immunoglobulins can be linked to the constant regions of human immunoglobulins using methods known in the art (see, for example, U.S. Patent No. 4,816,567 by Cabilly et al.). For example, the DNA encoding VH is operably linked to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but generally is preferably an IgG1 or IgG4 constant region. For example, the DNA encoding the VL is operably linked to another DNA molecule encoding the light chain constant region, CL, to obtain a full-length light chain gene (as well as a light chain gene of the Fab). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region, but is generally preferably a kappa constant region.

[0185] To prepare humanized antibodies, murine CDR regions can be grafted into human framework sequences using any method known in the art (see U.S. Pat. No. 5,225,539 by Winter; U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 by Queen et al.; and Lo, Benny, KC, eds., Antibody Engineering: Methods and Protocols, Vol. 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals capable of producing fully human antibody libraries after immunization without producing endogenous immunoglobulins can also be used. For example, it has been reported that homozygous deletion of the antibody heavy chain joining (JH) region genes in chimeric germ-line mutant mice results in complete inhibition of endogenous antibody production, and introduction of human germ-line immunoglobulin gene arrays in such germ-line mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA, 90:2551; Jakobovits et al., 1993, Nature, 362:255-258; Bruggermann et al., 1993, Year in Immunology, 7:33; and Duchosal et al., 1992, Nature, 355:258). Non-limiting examples of the above-mentioned transgenic animals include the HuMAb mouse (Medarex, Inc.), which contains a minilocus of human immunoglobulin genes encoding unrearranged human heavy (μ and γ) immunoglobulin sequences and unrearranged κ light chain immunoglobulin sequences, and targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994), Nature, 368(6474):856-859); or the "KM mouse™," which carries a human heavy chain transgene and a human light chain transchromosome (see, WO 02 / 43478).Other methods for humanizing antibodies include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol., 227:381; Marks et al., J. Mol. Biol., 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech, 14:309).

[0186] As used herein, the term "degree of humanization" is an index used to assess the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be evaluated by predicting the sequence homology of the variable region to the human V domain, for example, by DomainGapAlign on the IMGT website.

[0187] As used herein, "homologous antibody" refers to an antibody variant whose amino acid sequences contained in the heavy and light chain variable regions are homologous to the amino acid sequences of an antibody or antigen-binding fragment thereof presented herein, which variant retains the desired functional properties of the antibodies against FXI and / or FXIa of the present invention.

[0188] Methods of sequence alignment for comparison are well known in the art. Various procedures and alignment algorithms are described in Smith TF and Waterman MS, Adv. Appl. Math., 2:482, 1981; Higgins DG and Sharp PM, CABIOS, 5:151, 1989. Altschul SF et al., Nature Genet., 6:119, 1994, presents a detailed discussion of sequence alignment and homology calculations.

[0189] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) or the 50% effective concentration (EC50) for the interaction.

[0190] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. Both the "association rate constant" (ka, or k) and the "dissociation rate constant" (k or k) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is the dissociation constant, K D (See Davies et al., Annual Rev Biochem, 1990, 59:439-473). The KD, k and k values ​​may be measured in any effective manner. In certain embodiments, the dissociation constant may be measured using biolayer interferometry (BLI) (e.g., Fortebio Octet). Additionally, the dissociation constant may be measured by surface plasmon resonance (e.g., Biacore) or Kinexa.

[0191] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If the vector allows for the expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may contain an origin of replication.

[0192] Expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. In general, in cloning vectors, this sequence allows the vector to replicate independently of the host chromosomal lDNA and includes an origin of replication or an autonomously replicating sequence. As used herein, the term "expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, although other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or liposomally contained plasmids), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0193] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as a Drosophila S2 cell or Sf9 cell, or an animal cell such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK293 cell, or a human cell.

[0194] As used herein, the term "identity" refers to the degree of match between two polypeptides or two nucleic acids. If two sequences for comparison have the same monomeric subunit of a base or amino acid at a particular site (e.g., two DNA molecules each have an adenine at a particular site, or two polypeptides each have a lysine at a particular site), the two molecules are identical at this site. The percent identity between two sequences is a function of the number of identical sites shared by the two sequences x 100 over the total number of sites for comparison. For example, if six sites out of ten sites of two sequences match, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six sites match). Generally, the comparison of two sequences is performed to yield the maximum identity. Such alignments can be performed using computer programs such as the Align program (DNAstar, Inc.), which is based on the method of Needleman et al. (J. Mol. Biol., 48:443-453, 1970). The percent identity between two amino acid sequences can also be determined using the algorithm by E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), incorporated into the ALIGN program (version 2.0) and using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage identity between two amino acid sequences can also be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol., 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com) and using a Blossum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0195] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence, and the resulting antibody variant retains the biological activity of its original sequence, such as specific binding to FXI or FXIa. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, valine, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng., 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA, 94:412-417 (1997), which are incorporated herein by reference).

[0196] In the present disclosure, the twenty conventional amino acids are represented in a conventional manner. See, for example, "Immunology-A Synthesis," 2nd ed., ES Golub and DR Gren, Sinauer Associates, Sunderland, Mass. (1991), incorporated herein by reference. In this disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in this disclosure, amino acids are generally represented by the one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0197] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and active ingredient, and are well known in the art (see, for example, "Remington's Pharmaceutical Sciences", edited by Gennaro AR, 19th edition, Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, osmolality maintaining agents, absorption retarding agents, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or non-ionic surfactants such as Tween-80. Ionic strength enhancing agents include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Osmolarity maintaining agents include, but are not limited to, sugars, NaCl, etc. Absorption retarding agents include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerin), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers can stabilize the desired activity of the active ingredient in the drug, and have the meanings commonly understood by those skilled in the art, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine, etc.), proteins (such as dried whey, albumin, or casein), or their decomposition products (such as lactalbumin hydrolysate).

[0198] As used herein, the term "prophylaxis" refers to a method performed to prevent or delay the occurrence of a disease or disorder or condition (e.g., a disease or disorder associated with coagulation or thromboembolism) in a subject. As used herein, the term "treatment" refers to a method performed to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., no longer worsening), delay or slowing of the onset of the disease, improvement or alleviation of the disease status, and alleviation of symptoms (partial or total), alleviation or improvement of prognosis, reduction or suppression of recurrence of the disease. In addition, the term "treatment" may also refer to an increase in survival time compared to expected survival time (if the treatment was not administered).

[0199] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from or is at risk for suffering from a disease or disorder associated with coagulation or thromboembolism.

[0200] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain a desired effect. For example, an effective amount for inhibiting a disease (e.g., a disease or disorder associated with coagulation or thromboembolism) refers to an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a disease or disorder associated with coagulation or thromboembolism); a therapeutically effective amount is an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. It is entirely within the capabilities of a person skilled in the art to determine such an effective amount. For example, an effective amount for therapeutic use will depend on the disease being treated, the general condition of the patient's own immune system, the general condition of the patient, such as age, weight, and sex, the route of administration of the drug, and other treatments administered at the same time, etc.

[0201] As used herein, the term "effector function" refers to a biological activity attributable to an antibody Fc region (native or variant amino acid sequence Fc region), which varies depending on the antibody isotype. Examples of antibody effector functions include, but are not limited to, Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance of antibodies and antigen-antibody complexes, and the like. Methods for altering antibody effector functions are known in the art, for example, by introducing mutations into the Fc region.

[0202] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which cytotoxic effector cells specifically bind to antigen-conjugated target cells via the binding of immunoglobulins to Fc receptors (FcRs) presented on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), and then secrete cytotoxins to kill the target cells. Methods for detecting the ADCC activity of an antibody are known in the art, and can be assessed, for example, by measuring the binding activity of a test antibody to an Fc receptor (e.g., CD16a).

[0203] As used herein, the term "complement-dependent cytotoxicity (CDC)" refers to a form of cytotoxicity in which the complement cascade is activated by the binding of the complement component Cq to the Fc of an antibody. Methods for detecting the CDC activity of an antibody are known in the art, and can be assessed, for example, by measuring the binding activity of a test antibody to an Fc receptor (e.g., C1q).

[0204] As used herein, the term "mediated by FXI and / or FXIa" refers to the fact that FXI and / or FXIa mediate the intrinsic coagulation pathway by directly or indirectly activating coagulation factor IX (also known as FIX), coagulation factor X (FX), and / or thrombin, and / or by binding to platelet receptors.

[0205] As used herein, the term "disease or disorder associated with coagulation or thromboembolism" or similar terms refers to a disease or disorder caused by aberrant activation or non-natural inactivation (e.g., in the absence of therapeutic measures) of the coagulation pathway. Such diseases or disorders include thrombosis, thrombotic stroke, atrial fibrillation, stroke prevention associated with atrial fibrillation (SPAF), and the like. fibrillation), deep vein thrombosis, venous thromboembolism, acute coronary syndrome (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, systemic embolism, myocardial infarction (MI), acute myocardial infarction (AMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention, peripheral arterial occlusive disease (PAOD), renal vein thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, diffuse intravascular coagulation, thromboembolic disorders caused by medical devices (e.g., catheters), severe systemic inflammatory response syndrome, metastatic cancer, infectious diseases, organs failure (e.g., renal failure), toxicity caused by administration of therapeutic proteins in the body, multiple trauma, ischemia-reperfusion injury, localized fibrin deposition, adult pulmonary alveolar proteinosis, venous thromboembolic events (VTE) before and after total knee arthroplasty (TKA), coronary heart disease, thromboembolism after myocardial infarction, stroke in patients with non-valvular atrial fibrillation, thrombosis and thromboembolism in chronic kidney disease, thrombosis and thromboembolism in patients undergoing hemodialysis and extracorporeal membrane oxygenation, deep vein thrombosis (DVT), or pulmonary embolism (PE).

[0206] Catheter-induced thromboembolic disorders include the formation of thromboembolic events in catheters (eg, the use of Hickman catheters in oncology patients and the use of extracorporeal membrane oxygenation (ECMO) can lead to blood clots).

[0207] As used herein, the expression "prophylaxis and / or treatment of a disease or disorder associated with coagulation or thromboembolism" refers to the use of an antibody against FXI and / or FXIa, or an antigen-binding fragment thereof, according to the present invention, in the treatment of one or more of the following diseases or disorders: Thromboembolism in individuals suspected or confirmed to have arrhythmias, such as sudden, persistent, or permanent atrial appendage fibrillation or atrial fibrillation; Stroke prevention in atrial fibrillation (SPAF) with a subgroup of AF patients undergoing percutaneous coronary intervention (PCI); Treatment of acute venous thromboembolic events (VTE) and prevention of secondary prolonged VTE in patients at high bleeding risk; cerebral and cardiovascular events in secondary prevention after transient ischemic attack (TIA) or non-disabling stroke, and in the prevention of thromboembolic events in heart failure extending to sinus rhythm; clot formation and thromboembolism in the left atrium of individuals undergoing defibrillation for arrhythmias; Thrombosis before ablation procedure for arrhythmia, thrombosis during ablation procedure, thrombosis after ablation procedure; Venous thrombosis, including (but not excluding) the treatment and secondary prevention of deep or superficial vein thrombosis, of the lower or upper kind, abdominal and thoracic vein thrombosis, venous sinus thrombosis, and jugular vein thrombosis; Thrombosis on any artificial surface, such as an intravenous catheter or pacemaker lead; Pulmonary embolism in patients with or without venous thrombosis; Chronic Thromboembolic Pulmonary Hypertension (CTEPH); Arterial thrombosis in ruptured atherosclerotic plaques, thrombi in intra-arterial assist devices or catheters, and thrombi in apparently normal arteries, including (but not limited to) acute coronary syndromes, ST-elevation myocardial infarction, non-ST-elevation myocardial infarction, unstable angina, stent thrombosis, thrombi on any artificial surface of the arterial system, and thrombi in the pulmonary arteries of patients with or without pulmonary hypertension; Thrombosis and thromboembolism in patients undergoing percutaneous coronary intervention (PCI); psychogenic and cryptogenic stroke; Thrombosis in patients with invasive and non-invasive cancer malignancies; thrombus in resident catheter; Thrombosis in patients with severe disease; Cardiac thrombosis and cardiothromboembolism, including (but not exclusively) cardiac thrombosis following myocardial infarction and cardiac thrombosis associated with conditions such as cardiac aneurysms, myocardial fibrosis, cardiac hypertrophy and dysfunction, myocarditis, and artificial surfaces in the heart; Thromboembolism in patients with valvular heart disease with or without atrial fibrillation; Thromboembolism in the valve mechanism or biological ancillary structures; Thromboembolism in patients with native or artificial cardiac patches, intra-arterial or intravenous catheters after cardiac repair of simple or complex cardiac anomalies; venous thrombosis and thromboembolism following knee replacement, hip replacement, and orthopedic, thoracic or abdominal surgery; Arterial or venous thrombosis after neurosurgery, including intracranial and spinal interventions; congenital or acquired thrombophilia, including (but not exclusively) clotting factor V Leiden mutation, mutations in prothrombin, deficiencies of antithrombin III, protein C, and protein S, clotting factor XIII mutations, familial afibrinogenemia, congenital plasminogen deficiency, increased clotting factor XI content, sickle cell disease, antiphospholipid syndrome, autoimmune diseases, chronic enteropathy, nephrotic syndrome, hemolytic anemia, myelodysplastic disease, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria, and heparin-induced thrombocytopenia; Thrombosis and thromboembolism in chronic kidney disease; and Thrombosis and thromboembolism in patients undergoing hemodialysis and extracorporeal membrane oxygenation The term may refer to the use of a drug for the prevention or treatment of

[0208] As used herein, the term "pharmaceutical acceptable" means that the molecule itself, a molecular fragment, or a composition does not produce an adverse, allergic, or other undesirable reaction when properly administered to an animal or human. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or components thereof include sugars such as lactose, starch, cellulose and its derivatives, vegetable oils, gelatin, polyols such as propylene glycol, alginic acid, and the like.

[0209] As used herein, combination therapy includes the use of an antibody against FXI and / or FXIa, or an antigen-binding fragment thereof, of the present invention in combination with one or more additional active therapeutic agents (e.g., chemotherapeutic agents) or other prophylactic or therapeutic modalities (e.g., antiplatelet agents, anticoagulants, thrombolytic agents) for a second treatment.

[0210] Exemplary antiplatelet agents for the second treatment are selected from aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, or any combination thereof.

[0211] Exemplary anticoagulants for the second treatment are selected from unfractionated heparin, heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, or any combination thereof.

[0212] Exemplary thrombolytic agents for the second treatment are selected from streptokinase, urokinase, alteplase, or prourokinase.

[0213] In this type of combination therapy, the various active agents often exert different complementary mechanisms of action, and the combination therapy may result in a synergistic effect.The combination therapy may allow for a reduction in the dosage of one or more of the agents, so as to reduce or eliminate the adverse effects associated with one or more of the agents.This type of combination therapy may exert a synergistic therapeutic or preventive effect on the underlying disease, disorder or condition.

[0214] As used herein, "combination" includes therapies that may be administered separately, such as separately formulated and administered separately (e.g., as may be provided in one kit), and therapies that may be administered together as a single formulation (i.e., a "co-formulation"). In certain embodiments, antibodies to FXI and / or FXIa, or antigen-binding fragments thereof, according to the present invention may be administered sequentially. In other embodiments, antibodies to FXI and / or FXIa, or antigen-binding fragments thereof, may be administered simultaneously. Antibodies to FXI and / or FXIa, or antigen-binding fragments thereof, according to the present invention may be used in any combination with at least one other (active) agent.

[0215] As used herein, the term "about" refers to the value stated in the context ±10%.

[0216] Because Chinese grammar does not have the rules of singular and plural in English, when translating this disclosure into English, "one or more" may be added before the noun.

[0217] In the following, the embodiments of the present invention will be described in detail with the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, and are not used to limit the scope of the present invention. According to the accompanying drawings and the following detailed description of the preferred embodiments, various objects and advantageous aspects of the present invention will be enabled for those skilled in the art.

[0218] Sequence information Information about the sequences involved in the present invention is given in the table below. [Table 1] EXAMPLES

[0219] The invention will now be described with reference to the following examples, which are intended to illustrate the invention rather than limit its scope.

[0220] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to those described in J. Sambrook et al., "Molecular Cloning: Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press, 1989; and FMAusubel et al., "Short Protocols in Molecular Biology", 3rd edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples are described for illustrative purposes of the present invention and are not intended to limit the scope of the protection sought by the present invention.

[0221] Example 1: Preparation of mouse anti-human FXI / FXIa monoclonal antibodies Mouse anti-human FXI / FXIa monoclonal antibodies were obtained by immunizing wild-type Balb / c mice with the proteins. For the first immunization, each Balb / c mouse was subcutaneously injected with 25 μg of human FXIa (manufacturer: Haematologic Technologies, model number: HCXI-0150) and 25 μg of human FXI (manufacturer: Haematologic Technologies, model number: HCXI-0160) emulsified in CFA (complete Freund's adjuvant, manufacturer: Sigma, model number: F5506), and booster immunizations were performed every other week using emulsions prepared in IFA (incomplete Freund's adjuvant, manufacturer: Sigma, model number: F5881). After three immunizations, serum titers were measured by ELISA. Booster immunization was performed 3-5 days before fusion, and mice with high titers were selected for intraperitoneal immunization with 10 μg FXIa and 10 μg FXI. A standard fusion process was adopted to fuse mouse spleen cells with Sp2 / 0-Ag14 (ATCC, model number: CRL-1581) mouse myeloma cells by PEG fusion, and then ELISA screening was performed 14 days later using HAT (manufacturer: Sigma, model number: H0262-10VL) for stress screening. The detailed method for ELISA screening included: ELISA plates (manufacturer: Thermo Fisher Sci., model number: 5129) were coated with 0.5 μg / mL biotin-labeled FXI (manufacturer: Haematologic Technologies, model number: HCXI-0150-B) at room temperature for 1 hour at 100 μL per well. Washing was performed three times with 200 μL of wash buffer (1×TBS containing 0.05% Tween-20), 100 μL of hybridoma supernatant was added, and incubated for 1 hour at 37° C. Washing was performed three times with 200 μL of wash buffer, 100 μL of streptavidin-HRP (Pierce, product number: 21130) diluted at a ratio of 1:8000 was added, and incubated for 1 hour at 37° C.Washing was performed three times using 200 μL of wash buffer, 100 μL of TMB (purchased from Thermo Fisher Sci., model number: TMBW-1000-01) was added, and the color was developed in the dark for 10 minutes, then 100 μL of stop solution (purchased from Thermo Fisher Sci., model number: 13361-100-10) was added, and reading was performed at 450 nm on a microplate reader.

[0222] The supernatants of 24,000 hybridoma clones were screened by ELISA to obtain 100 hybridoma clones capable of recognizing biotin-labeled FXI and transferred to 24-well plates. After 7-10 days, the hybridoma supernatants were examined by APTT assay. The best clones, 22, were subcloned by limiting dilution to obtain monoclonal hybridomas. The monoclonal hybridomas were examined for their anticoagulant activity by APTT assay via Activated Partial Thromboplastin Time (APTT) Kit (Activated Partial Thromboplastin Time Kit, Manufacturer: Thermo Fisher Sci, Model No.: 100402). The detailed steps were as follows: 100μL normal human plasma (purchased from Innovative Research, model number: IPLA-N) was added to a pre-heated test tube, then 100μL APTT reagent (purchased from Thermo Fisher Sci., model number: 100402TS) and 100μL test sample were added, mixed thoroughly, incubated at 37℃ for 5 minutes, then 100μL calcium chloride (purchased from Thermo Fisher Sci., model number: 100304, 20mM) was added, the reading at OD405 was measured, and the clotting time was calculated by curve fitting, in which Sp2 / 0-Ag14 cell supernatant was used as a negative control. 14E11 was used as a positive control antibody (prepared with reference to the patent by Aronora, US Patent US8388959B2). The fold change shown in the APTT assay results was the ratio of the clotting time measured in samples spiked with the test antibody to the clotting time measured in control samples without antibody. A fold change value of 1 or less indicated that the clotting time was neither delayed nor accelerated, whereas a fold change value of greater than 1 indicated that the clotting time was prolonged.

[0223] As shown in Figure 1, the hybridoma supernatants of a total of five subclones were examined by APTT, in which 36G9.10 showed prolonged APTT time, thus supporting significant anticoagulant function, and could be used for further analysis, whereas the other clones 6B6.9, 28A8.3, 36C3, and 40F6.9 showed no significant difference in clotting time compared with the negative control. By the same screening method, a hybridoma with prolonged APTT time, 7B2, was obtained for further analysis.

[0224] Monoclonal hybridomas 36G9.10 and 7B2 were expanded to 100-150 mL of serum-free culture. The supernatants were purified by protein G, and the purified mouse antibodies were detected by HPLC-SEC, and their purity was all above 97%. The purified mouse antibodies could be used for further functional validation.

[0225] Example 2: Functional identification of mouse anti-human FXI / FXIa monoclonal antibodies 2.1: Detection of the function of mouse anti-human FXI / FXIa antibodies in prolonging coagulation Using the APTT detection method in Example 1, 36G9.10 was compared with positive control antibodies BAY-1213790 (prepared with reference to M007-H04 in International Patent Publication No. 2013167669 by Bayer) and 14E11 under conditions in which the antibodies were diluted to have concentrations of 2.00, 1.00, 0.50, and 0.25 μg / mL, and PBS buffer was used as a negative control.

[0226] As shown in FIG. 2A, the candidate mouse antibody 36G9.10 showed a significant prolongation of the clotting time at each concentration compared to the control antibodies BAY-1213790 and 14E11. 7B2 was compared to the positive control antibody 14E11 at an antibody concentration of 5 μg / mL, and PBS buffer was used as a negative control. The results are shown in FIG. 2B. The candidate mouse antibody 7B2 showed a significant prolongation of the clotting time compared to PBS, and showed a prolongation of the clotting time equivalent to that of the positive control antibody 14E11.

[0227] 2.2: Detection of the activity of mouse anti-human FXI / FXIa antibodies that inhibit the catalysis of FXa production by FXIa and their anticoagulant function According to the kit's instruction, BIOPHEN Factor XIa Kit (HyphenBioMed, model number: 220412) was used in detection, and the release of paranitroaniline (pNA) product was measured under OD405nm to further confirm the activity of anti-human FXI / FXIa antibodies in inhibiting the catalysis of the production of FXa by FXIa. All antibodies had a detection concentration of 0.25μg / mL. The stronger the ability of anti-FXI / FXIa antibody to block FXI / FXIa activity, the smaller the signal value of pNA product at OD405nm.

[0228] The results are shown in Figure 3. The 36G9.10 mouse antibody exhibited significantly stronger anticoagulant function than the control antibodies BAY-1213790 and 14E11.

[0229] 2.3: Affinity studies on mouse anti-human FXI / FXIa monoclonal antibodies Octet ForteBio® is widely used in detecting antibody-antigen affinity kinetics, and was used to determine the affinity kinetics of candidate mouse antibody 36G9.10 and control antibodies BAY1213790 and 14E11 to FXIa. The detailed experimental steps were as follows: streptavidin biosensor was first conjugated with biotin-labeled test antibody to reach a response signal value of 0.8 nm, then conjugated with FXIa protein (3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, and 0 μg / mL) for 5 minutes, and then dissociated for 7 minutes. A bivalent analysis model was used for all fitting analyses.

[0230] As shown in Table 1, 36G9.10 exhibited a faster binding rate (as indicated by the K value) than the binding rates of the control antibodies 14E11 and BAY-1213790, a slower dissociation rate (as indicated by the K value) than the dissociation rate of BAY-1213790, and an affinity that was 1.7 and 2.5 times that of the control antibodies 14E11 and BAY-1213790, respectively. TIFF0007650807000002.tif45149

[0231] 2.4: Mouse anti-human FXI / FXIa antibodies specifically block the biological activity of FXI and FXIa To confirm that the mouse antibody, 36G9.10, specifically blocked the biological activity of FXI and FXIa, APTT clotting times were determined using human plasma lacking FXI (purchased from Innovative Research, product number: 50-643-396) by spiking with FXI (0.2, 0.4, 0.8 μg / mL), FXIa (0.2, 0.4, 0.8 μg / mL), FXI (0.4 μg / mL), and 36G9.10 (0.4 μg / mL), FXIa (0.4 μg / mL), and 36G9.10 (0.4 μg / mL).

[0232] The results are shown in Figure 4. Addition of FXI or FXIa significantly shortened the clotting time of FXI-deficient human plasma in a dose-dependent manner. Upon addition of the 36G9.10 mouse antibody, FXI and FXIa showed a significant prolongation of the clotting time, indicating that the mouse antibody 36G9.10 specifically inhibited the biological activity of FXI and FXIa.

[0233] Example 3: Identification of subtypes and amplification of variable regions of mouse anti-human FXI / FXIa antibodies To identify the antibody subtypes of the candidate hybridoma clones, the Pierce Rapid Isotyping Kit (purchased from Thermo Fisher Sci., model number: 26179) was used to identify the antibody subtypes of the candidate clones 36G9.10 and 7B2. The identification results showed that the candidate clones had heavy chains of IgG1 subtype and light chains of kappa subtype.

[0234] Hybridoma cells were cultured to a population of about 8000, lysed, and subjected to synthesis of first-strand cDNA by using a cDNA reverse transcription kit (purchased from Thermo Fisher Sci., model number: 18080-200). VH and VK genes were amplified from cDNA by PCR using custom primers, and the PCR products were purified by a DNA purification kit (purchased from Qiagen, model number: 28104), and ligated with TOPO vector (purchased from Thermo Fisher Sci., model number: K457540). Approximately 12 clones were picked for each ligation reaction and sequenced. The sequences were analyzed using a vector, NTI 11.5 (purchased from Thermo Fisher Sci.), and a 5.4.6 type sequencer (purchased from Genecodes) to obtain the variable region sequences and CDR sequences of mouse anti-FXI / FXIa antibodies shown in the sequence listing, where the 36G9.10 mouse antibody has a heavy chain variable region shown in SEQ ID NO: 1 and a light chain variable region shown in SEQ ID NO: 2; the 7B2 mouse antibody has a heavy chain variable region shown in SEQ ID NO: 29 and a light chain variable region shown in SEQ ID NO: 30. Furthermore, chimeric antibodies 36G9.10-hz00 and 7B2-hz00 were constructed for 36G9.10 and 7B2, respectively, by grafting the heavy chain variable region sequence of 36G9.10 or 7B2 onto a mutant human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21) and the light chain variable region sequence of 36G9.10 or 7B2 onto a human kappa light chain constant region (SEQ ID NO: 22).

[0235] Example 4: Humanization of anti-human FXI / FXIa mouse antibodies The mouse antibodies 36G9.10 and 7B2 were humanized by CDR grafting humanization method. Briefly, humanization involves the following steps: the amino acid sequence of mouse monoclonal antibody is compared with that of human germline antibody to find a sequence with high homology and excellent physical and chemical properties, which is used as the human germline framework sequence; the affinity of HLA-DR is analyzed and searched to select a human germline framework sequence with low affinity; and then the six CDRs of mouse antibody are grafted into the selected heavy and light chain framework sequences.

[0236] Specifically, the heavy and light chain CDR regions of the mouse antibodies 36G9.10 and 7B2 were grafted onto the corresponding humanized template FR, respectively. The heavy chain humanized template for 36G9.10 was the human germline gene sequence IGHV1-2 * 02 (see IMGT Accession No.: X62106), and IGHV1-69-2 * 01 (see IMGT Accession Number: KF698734), and the light chain humanized template is the human germline gene sequence, IGKV1-33 * 01 (see IMGT Accession No.: M64856), and IGKV1-16 * 01 (see IMGT Accession Number: J00248). The heavy chain humanized template for 7B2 was the human germline gene sequence, IGHV1-69-2 * 01 (see IMGT Accession Number: KF698734), and the light chain humanized template is the human germline gene sequence, IGKVV1-39 * 01 (see IMGT accession number: X59315).

[0237] Furthermore, the variable region and the surrounding framework amino acid sequence were analyzed by molecular docking using computer simulation technology to explore its spatial binding form. By calculating the values ​​of electrostatic force, van der Waals force, hydrophobicity, and entropy, the amino acid sequence of the mouse antibody was analyzed for key amino acids that can interact with coagulation factor XIa and maintain the spatial structure, and these mouse amino acids were retained in the grafted antibody. In other words, a series of back mutations were made to the FR region amino acid residues of the humanized template so that the humanized antibody retains the antigen-binding ability of the mouse antibody as much as possible.

[0238] According to the above method, a total of five humanized antibodies were constructed based on the CDRs of mouse antibody 36G9.10, named 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93, respectively, in which the heavy chain constant region of each antibody was human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21). Based on the CDRs of mouse antibody 7B2, humanized antibody 7B2-hz11 was constructed, in which the heavy chain constant region was mutant human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21). The light chain constant region sequences of the antibodies 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz43, 36G9.10-hz92, 36G9.10-hz93, and 7B2hz11 were sequence number 22, but none of them had ADCC or CDC effects.

[0239] The amino acid sequences of the variable and constant regions of the humanized antibodies are shown in Table 2. TIFF0007650807000003.tif72149

[0240] Example 5: Determination of affinity of anti-human FXI / FXIa antibodies Octet Fortevio® was used to determine the affinity of chimeric and humanized antibodies. The main steps for the determination were as follows: first, the chimeric and humanized antibodies (at a concentration of 0.3 μg / mL) were immobilized on an AHC (anti-human Fc) sensor, and then FXI was subjected to a 1:2 gradient dilution with an initial concentration of 3.2 μg / mL to determine the association and dissociation rates of the chimeric and humanized antibodies. The obtained data was analyzed by Octet data analysis software.

[0241] The results, shown in Table 3, indicated that the chimeric antibody 36G9.10-hz00 and the humanized antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, and 36G9.10-hz93 all had stronger affinities, KDs, than the affinity of the positive control BAY-1213790. TIFF0007650807000004.tif77149

[0242] Example 6: Determination of APTT anticoagulant activity, inhibition of FXIa activity catalyzing the production of FXa, and anticoagulant function of chimeric and humanized anti-human FXI / FXIa antibodies The anticoagulant activity of the humanized antibodies was detected using an APTT coagulation test kit, and the detailed detection method was shown in Example 1. The results were shown in Figure 5. The mean value and standard deviation were calculated by performing the determination four times. The chimeric antibody 36G9.10-hz00 and the humanized antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, and 36G9.10-hz93 all showed a greater prolongation of the clotting time than that of the positive control antibodies 14E11 and BAY-1213790.

[0243] The Biophen Factor XIa kit (purchased from Aniara; model number: 220412) was used to detect the anticoagulant activity of the humanized antibody, and the detailed method was shown in Example 2.

[0244] The detection results are shown in FIG. 6, where chimeric antibody 36G9.10-hz00, humanized antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93, antibodies 14E11 and BAY-1213790 all effectively reduced the production of FXa, and antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz92, 36G9.10-hz93 could more effectively reduce the production of FXa compared with control antibodies 14E11 and BAY-1213790, indicating that they had excellent anticoagulant activity.

[0245] Example 7: Determination of affinity of humanized anti-human FXI / FXIa antibodies to FXI / FXIa ELISA method was used to detect the affinity of 36G9.10-hz73 to human FXIa. The detailed steps were as follows: FXIa antigen (Haematologic Technologies, HCXIA-160) was diluted to 1 μg / mL with CBS coating solution (0.32 g Na2CO3, 0.59 g NaHCO3 were dissolved in demineralized water and diluted to 200 ml), and 100 μL per well was coated with FXIa antigen overnight at 4°C; the next day, the liquid in the well was discarded, and 300 μL PBS was used for washing once; 100 μL PBS (2% BSA, BOVOGEN, BSAS) was used for washing; 1.0) and blocked at 37°C for 2 hours; PBS (containing 2% BSA) was used to dilute the antibodies 36G9.10-hz73 and BAY-1213790 (4-fold dilutions, 12 concentration points starting at 10 μg / mL) and 100 μL of the dilution was added to the corresponding wells and incubated at 37°C for 2 hours; washing was performed three times with 300 μL of PBST; HRP-labeled goat anti-human secondary antibody (purchased from Jackson, 109-035-00) was diluted in PBS (containing 2% BSA) and blocked at 37°C for 2 hours. A) and 100 μL of this dilution was added to the corresponding wells and incubated at 37° C. for 1 hour; washed 5 times with 300 μL PBST; 100 μL of TMB development solution (purchased from BioPanda, TMB-S-004) was added to the corresponding wells and developed for 20 minutes at room temperature; development was stopped by adding 50 μL of 2N H2SO4 and readings were taken at OD450nm using a microplate reader (purchased from MD, SpectraMax M2) and the results were imported into Graphpad Prism for curve fitting.

[0246] The experimental results are shown in Figure 7A, in which the EC50 of 36G9.10-hz73, the affinity for FXIa, was 2.488 ng / mL, and the EC50 of BAY-1213790, the affinity for FXIa, was 6.163 ng / mL, indicating that 36G9.10-hz73 was better than BAY-1213790.

[0247] ELISA was used to detect the affinity of 36G9.10-hz73 to human FXI, where FXI antigen (PMD-F11 plasmid purchased from Sino Biological, PLVX- I The antibodies (obtained by cloning into the RES-PURO vector and transfecting into 293F cells for expression) were diluted to 1 μg / mL in CBS coating solution and coated at 100 μL per well overnight at 4° C. The remaining steps were as described in the previous section, except that the antibodies were diluted 3-fold to give 9 concentration points with an initial concentration of 0.37 μg / mL.

[0248] The experimental results are shown in Figure 7B, where the EC50, which is the affinity of 36G9.10-hz73 for FXI, was 9.68 ng / mL, and BAY-1213790 did not bind to FXI, indicating that 36G9.10-hz73 could simultaneously neutralize FXI and FXIa and perform good anticoagulant function.

[0249] Example 8: Detection of anti-human FXI / FXIa antibodies that compete with a control antibody for binding to FXIa To determine whether 36G9.10-hz73, BAY-1213790, and 14E11 recognize the same epitope of FXIa, a competitive ELISA method was used for detection. The detailed steps were as follows: FXI antigen was diluted to 1 μg / mL with CBS coating solution, and 100 μL per well was coated with FXI antigen at 4°C overnight; the next day, the liquid in the well was discarded and washed with 300 μL of PBS; 100 μL of PBS (containing 2% BSA) was added and blocked at 37°C for 2 hours; biotin-labeled 36G9.10-hz73 antibody was applied to the wells. 10ng / mL was diluted with PBS (containing 2% BSA) to 10ng / mL, which was used as the mother liquor to dilute the antibodies BAY-1213790 and 14E11 to 10μg / mL, and 100μL of this dilution was added to the corresponding wells. 10μg / mL of 36G9.10-hz73 antibody was used as a positive control, 10ng / mL of biotin-labeled 36G9.10-hz73 antibody was used as a negative control, and a blank control was also set up. All were incubated at 37°C for 2 hours; washed 3 times with 300 μL PBST; HRP-streptavidin secondary antibody (purchased from Proteintech, SA00001-0) was diluted 1:3000 with PBS (containing 2% BSA) and 100 μL of this dilution was added to the corresponding wells and incubated at 37°C for 1 hour; washed 5 times with 300 μL PBST; 100 μL of TMB development solution (purchased from BioPanda, TMB-S-004) was added to the corresponding wells and developed for 20 minutes at room temperature; development was stopped by adding 50 μL 2N H2SO4, and readings were taken at OD450nm with a microplate reader (purchased from MD, Spectramax M2), and the results were imported into GraphPad Prism for plotting.

[0250] The experimental results are shown in Figure 8, in which BAY-1213790 and 14E11 did not compete with 36G9.10-hz73 for binding to FXIa, indicating that the former two and 36G9.10-hz73 bound to different epitopes on FXIa.

[0251] Example 9: Inhibition of catalytic activity of FXIa on substrates by anti-human FXI / FXIa antibodies In 1988, S. Kawabata et al. screened several FXIa-specific fluorescently labeled substances for sensitive detection of human FIXa enzyme activity in vitro (for details, see Eur. J. Biochem., 172, 17-25 (1988)). Thus, the activity of human FXIa and the effective neutralization of FXIa by humanized anti-human FXI / FXIa antibodies were determined by detecting the catalytic cleavage of a specific fluorescent substrate (I-1575, Bachem) by human FXIa. The detailed steps were as follows: human FXIa was diluted to 1 nM with a buffer containing 50 mM Tris / HCl, 100 mM NaCl, 5 mM CaCl2, and 0.1% BSA, which was used as the mother solution to serially dilute antibodies BAY-1213790 and 14E11 to a concentration of 20 μg / mL, with 4-fold dilutions and 14 concentration points; 10 μL of the diluted antibodies were added to each well of a 384-well plate (purchased from Coring, 4514) and incubated at 37°C for 1 hour; after incubation, 10 μL of fluorescent substrate I-1575 at a concentration of 2 μM was added to each well, mixed, and immediately measured by a microplate reader to continuously read the fluorescence values ​​at 360 / 465 nm, which were imported into GraphPad Prism for curve fitting.

[0252] The experimental results are shown in Table 4, in which the antibody 36G9.10-hz73 effectively inhibited the catalytic action of FXIa on the fluorescent substrate with an IC50 of 26.215 ng / mL, and BAY-1213790 had an IC50 of 69.96 ng / mL, indicating that 36G9.10-hz73 was better than BAY-1213790. TIFF0007650807000005.tif31149

[0253] Example 10: Detection of anticoagulant activity of anti-human FXI / FXIa antibodies by measuring activated partial thromboplastin time (APTT) After adding APTT reagent to anticoagulated plasma, the intrinsic coagulation pathway was activated to activate XI to XIa, and the antibody targeting FXIa could inhibit the activity of FXIa, thereby prolonging the APTT. Therefore, the determination of APTT was used to detect the anticoagulant activity of humanized anti-human FXI / FXIa antibodies. The detailed steps were as follows: venous blood was collected from each of humans, monkeys, dogs, rabbits, and rats, and 2-3 samples were taken from each species, and 4 test tubes (1.8 ml per test tube) of blood were collected for each sample and subjected to anticoagulation treatment using 3.2% sodium citrate and centrifugation at 500×g at 4°C for 10 minutes, and then the resulting plasma was collected and stored at 4°C for further use (all plasma from each sample was combined); the test antibody was diluted with saline at 3 concentrations starting from 1500 μg / mL, and the ... The plasma was diluted in 10-fold and used as a mother solution of the test antibody with a 10-fold concentration; for the high concentration, the sample detection system was set to 150 μl by taking 135 μl of plasma and adding 15 μl of the test antibody mother solution (i.e., the mother solution of the test antibody was diluted 10-fold), and then the APTT was detected using a fully automated blood coagulation analyzer (CA1500, purchased from Sysmex); for the low concentration, the sample detection system was set to 75 μl by taking 67.5 μl of plasma and adding 7.5 μl of the test antibody mother solution (i.e., the mother solution of the test antibody was diluted 10-fold), and then the APTT was detected using a fully automated blood coagulation analyzer.

[0254] The results of the experiment are shown in FIG. 9A, where the four tested antibodies, 36G9.10-hz73, BAY-1213790, BMS-962212 (prepared with reference to J Med Chem., 2017 Dec. 14; 60(23):9703-9723; doi:10.1021 / acs.jmedchem.7b01171; Epub, 2017 Nov. 17), and 14E11, all showed APTT prolongation in human plasma, with 36G9.10-hz73 showing the best effect, with an APTT prolongation of 2.2-fold at 6 μg / mL, which was better than the effects of the other three antibodies. As shown in Figures 9B to 9E, 36G9.10-hz73 prolonged the APTT clotting time only in monkey plasma, whereas BAY-1213790 prolonged the APTT clotting time in monkey and rabbit plasma, and 14E11 prolonged the APTT clotting time in monkey, rabbit, and rat plasma, indicating that 36G9.10-hz73 differed from BAY-1213790 and 14E11 in terms of species cross-reactivity, and therefore it is speculated that 36G9.10-hz73 bound to an epitope of FXI / FXIa different from the epitopes bound by BAY-1213790 and 14E11.

[0255] The prothrombin time (PT) test mainly reflected the status of the extrinsic coagulation system. The fold change shown in the PT test result was the ratio of the clotting time measured in the sample spiked with the test antibody to the clotting time measured in the control sample without the antibody. A fold change value of 1 or less indicated that the clotting time was neither delayed nor accelerated, whereas a fold change value of more than 1 indicated that the clotting time was prolonged.

[0256] Using the same treatment as above, the effect of 36G9.10-hz73, BAY-1213790, BMS-962212 and 14E11 on PT was detected by a fully automated blood coagulation analyzer. The detection results are shown in Table 5, where all samples, even at a concentration of 150 μg / mL (1 μM), did not prolong (in terms of fold change) the PT in the plasma of five species compared with the PBS control, indicating that they did not exert an effect on extrinsic coagulation and did not increase the risk of bleeding. TIFF0007650807000006.tif47149

[0257] Example 11: Detection of accelerated stability of anti-human FXI / FXIa antibodies The antibody 36G9.10-hz73 was diluted with 20 mM His-HCl (hydrochloric acid-histidine buffer) (containing 0.03% Tween-20, pH 5.5) to a concentration of 11.5 mg / mL, and incubated at 40°C and 25°C for 14 and 28 days, respectively. Then, the APTT detection method in Example 4 was used to detect the anticoagulant activity of the sample after acceleration, and SEC was used to detect the purity of the antibody after acceleration.

[0258] The experimental results are shown in Figure 10 and Table 6, where antibody 36G9.10-hz73 incubated at 40°C and 25°C for 14 and 28 days showed no significant changes in anticoagulant activity and purity. TIFF0007650807000007.tif47149

[0259] Example 12: Detection of pharmacokinetics (PK) and pharmacodynamics (PD) of anti-human FXI / FXIa antibodies In order to detect the PK and PD of 36G9.10-hz73 in vivo, different doses of antibody 36G9.10-hz73 were administered to cynomolgus monkeys by single intravenous injection (IV) or subcutaneous injection (SC), and blood samples were taken at different time points to detect PK and APTT activity.Detailed administration groups and doses are shown in Table 7, where the volume of single intravenous injection or subcutaneous administration is 2mL / kg, the site of intravenous injection is limb vein, and the site of subcutaneous injection is back of neck. TIFF0007650807000008.tif40149

[0260] After completing blood collection at all time points, antibody 36G9.10-hz73 was used as a standard substance, and a calibration curve for antibody concentration was drawn using the ELISA method in Example 7, and the blood concentration of antibody 36G9.10-hz73 at each time point was determined by using the calibration curve under the same conditions, and finally, the measured blood concentration was imported into GraphPad Prism for curve fitting and the PK of 36G9.10-hz73 in cynomolgus monkeys was calculated.

[0261] At the same time, immediately after each blood sampling, the APTT was detected using the method in Example 10, and finally, the data at all time points were imported into GraphPad Prism for curve fitting and to calculate the PK of 36G9.10-hz73 in cynomolgus monkeys.

[0262] PK results are shown in Table 8, where intravenous injection of 3 mg / kg 36G9.10-hz73 antibody showed a half-life of 290.15 hours in cynomolgus monkeys and subcutaneous injection of 3 mg / kg 36G9.10-hz73 antibody showed a half-life of 188.02 hours in cynomolgus monkeys. Subcutaneous injection of 3 mg / kg 36G9.10-hz73 antibody showed a bioavailability of 94%±2% (n=2). TIFF0007650807000009.tif50149

[0263] The PD detection results showed that 36G9.10-hz73 can be used for intravenous injection and subcutaneous administration to achieve good effect of prolonging APTT. As shown in Figure 11A, the dose-effect relationship between the two intravenous injection dose groups was clearly shown, and the subcutaneous injection group of 3 mg / kg had the effect of prolonging APTT by about 1.3 times at 1008 hours, showing a slightly better effect on prolonging APTT than the intravenous injection group; as shown in Figure 11B, the three dose groups did not prolong PT, indicating that they had no effect on extrinsic coagulation. Therefore, the 36G9.10-hz73 antibody had the potential clinical advantage of not causing the risk of bleeding.

[0264] Although the detailed embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes may be made to the details according to all the teachings disclosed, and these changes are within the protection scope of the present invention. All of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. An antibody or an antigen-binding fragment thereof capable of specifically binding to FXI and / or FXIa, the antibody or the antigen-binding fragment thereof comprising: (1) A heavy chain variable region (VH) and a light chain variable region (VL), as follows: a heavy chain variable region (VH) comprising three CDRs: CDR-H1 consisting of the sequence of SEQ ID NO:3, CDR-H2 consisting of the sequence of SEQ ID NO:4, and CDR-H3 consisting of the sequence of SEQ ID NO:5, as follows; and a light chain variable region (VL) comprising three CDRs: CDR-L1 consisting of the sequence of SEQ ID NO:6, CDR-L2 consisting of the sequence of SEQ ID NO:7, and CDR-L3 consisting of the sequence of SEQ ID NO:8, as follows; or (2) a heavy chain variable region (VH) and a light chain variable region (VL), as follows: a heavy chain variable region (VH) comprising three CDRs: CDR-H1 consisting of the sequence of SEQ ID NO: 9, CDR-H2 consisting of the sequence of SEQ ID NO: 45, 46 or 10, and CDR-H3 consisting of the sequence of SEQ ID NO: 11, as follows; and a light chain variable region (VL) comprising three CDRs: CDR-L1 consisting of the sequence of SEQ ID NO: 12, CDR-L2 consisting of the sequence of SEQ ID NO: 13, and CDR-L3 consisting of the sequence of SEQ ID NO: 14, as follows; Including, The CDR in (1) is defined by the IMGT numbering system, and the CDR in (2) is defined by the AbM numbering system. An antibody or an antigen-binding fragment thereof.

2. (a) a VH consisting of a sequence as set forth in SEQ ID NO: 15 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a VL consisting of a sequence as set forth in SEQ ID NO: 18 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; (b) a VH consisting of a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a VL consisting of a sequence as set forth in SEQ ID NO: 2 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; (c) a VH consisting of a sequence as set forth in SEQ ID NO: 15 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a VL consisting of a sequence as set forth in SEQ ID NO: 20 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; (d) a VH consisting of a sequence set forth in SEQ ID NO: 16 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a VL consisting of a sequence set forth in SEQ ID NO: 18; (e) a VH consisting of a sequence set forth in SEQ ID NO: 17 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a VL consisting of a sequence set forth in SEQ ID NO: 19; (f) a VH consisting of a sequence as set forth in SEQ ID NO: 17 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and a VL consisting of a sequence as set forth in SEQ ID NO: 18 or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; Including, The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, which is a murine antibody, a chimeric antibody, or a humanized antibody.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising a heavy chain constant region (CH) and a light chain constant region (CL) derived from a human immunoglobulin.

5. The antibody or antigen-binding fragment thereof according to claim 4 , wherein the heavy chain constant region is an IgG heavy chain constant region and / or the light chain constant region is a κ light chain constant region.

6. the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO:21, or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO:21; and / or The antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 22, or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared to SEQ ID NO:

22. The antibody or antigen-binding fragment thereof according to claim 4.

7. The antibody, (i) a sequence comprising a VH as set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 21; or (ii) a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence shown in (i). a heavy chain comprising an amino acid sequence selected from the group consisting of: (iii) a sequence comprising the VL sequence set forth in SEQ ID NO: 18 and the light chain constant region (CL) set forth in SEQ ID NO: 22; or (iv) a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence set forth in (iii). A light chain comprising an amino acid sequence selected from The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6,

8. The antibody is selected from the group consisting of: (a) a heavy chain comprising a VH set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 22; (b) a heavy chain comprising a VH set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 20 and a light chain constant region (CL) set forth in SEQ ID NO: 22; (c) a heavy chain comprising a VH set forth in SEQ ID NO: 16 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 22; (d) a heavy chain comprising a VH set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) set forth in SEQ ID NO: 21, and a light chain comprising a VL set forth in SEQ ID NO: 19 and a light chain constant region (CL) set forth in SEQ ID NO: 22; (e) a heavy chain comprising a VH as set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) as set forth in SEQ ID NO: 21, and a light chain comprising a VL as set forth in SEQ ID NO: 18 and a light chain constant region (CL) as set forth in SEQ ID NO:

22. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, selected from the following:

9. ScFv, Fab, Fab', F(ab') ２ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is selected from an Fv fragment, a disulfide-linked Fv (dsFv), and a diabody.

10. To be labeled, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. Features: (a) a K for FXI and / or FXIa of less than about 100 nM Ｄ To combine with; (b) an EC for FXI and / or FXIa of less than about 500 nM ５０ To combine with; (c) binding to FXI and / or FXIa inhibits or blocks the binding of FXI and / or FXIa to substrates, thereby prolonging clotting time; (d) binding to FXI and / or FXIa inhibits or blocks the catalytic action of FXI and / or FXIa on a substrate; (e) binding to FXI and / or FXIa does not affect extrinsic coagulation; (f) reducing ADCC activity and / or CDC activity; (g) not having ADCC activity and / or CDC activity; (h) binding to FXI and / or FXIa inhibits or blocks the formation of FXI dimers and / or FXIa dimers; (i) binding to FXI and / or FXIa inhibits or blocks the formation of a complex between FXI and / or FXIa and high molecular weight kininogen (HK); (j) binding to and / or inducing a conformational change in the catalytic domain of FXI and / or FXIa; (k) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors; or (l) any combination of (a) to (k) The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising at least one of the following:

12. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, its heavy chain and light chain, or its heavy chain variable region and light chain variable region according to any one of claims 1 to 11.

13. (1) A nucleic acid molecule encoding a heavy chain variable region of an antibody, and / or a nucleic acid molecule encoding a light chain variable region of an antibody, the nucleic acid molecule encoding the heavy chain variable region of the antibody, (1a) a nucleotide sequence as set forth in SEQ ID NO: 23 or 27; (1b) having a nucleotide sequence selected from the group consisting of a sequence having at least about 90%, 95%, 99%, or more identity to the nucleotide sequence set forth in (1a); and / or the nucleic acid molecule encoding the light chain variable region of the antibody, (1c) a nucleotide sequence as set forth in SEQ ID NO: 24 or 28; (1d) a sequence having at least about 90%, 95%, 99%, or more identity to the nucleotide sequence set forth in (1c); having a nucleotide sequence selected from the group consisting of a nucleic acid molecule; or (2) a nucleic acid molecule encoding a heavy chain variable region of an antibody having a nucleotide sequence as set forth in SEQ ID NO:23, and / or a nucleic acid molecule encoding a light chain variable region of an antibody having a nucleotide sequence as set forth in SEQ ID NO:24; Or, (3) a nucleic acid molecule encoding a heavy chain variable region of an antibody having a nucleotide sequence as set forth in SEQ ID NO:27, and / or a nucleic acid molecule encoding a light chain variable region of an antibody having a nucleotide sequence as set forth in SEQ ID NO:28; Or, (4) A nucleic acid molecule encoding an antibody heavy chain and / or a nucleic acid molecule encoding an antibody light chain, The nucleic acid molecule encoding the antibody heavy chain is (4a) the nucleotide sequence set forth in SEQ ID NO: 25; (4b) A sequence having at least about 90%, 95%, 99%, or more identity to the nucleotide sequence described in (4a). having a nucleotide sequence selected from and / or the nucleic acid molecule encoding the antibody light chain (4c) the nucleotide sequence set forth in SEQ ID NO: 26; (4d) a sequence having at least about 90%, 95%, 99%, or more identity to the nucleotide sequence set forth in (4c); having a sequence selected from the group consisting of Nucleic acid molecules; Or, (5) A nucleic acid molecule encoding an antibody heavy chain having the nucleotide sequence shown in SEQ ID NO: 25, and / or a nucleic acid molecule encoding an antibody light chain having the nucleotide sequence shown in SEQ ID NO:

26. The isolated nucleic acid molecule of claim 12 .

14. A vector comprising the isolated nucleic acid molecule of claim 12 or 13.

15. 15. A host cell comprising the isolated nucleic acid molecule of claim 12 or 13, or the vector of claim 14.

16. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising culturing a host cell according to claim 15 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from a culture of the cultured host cells.

17. The antibody or antigen-binding fragment thereof capable of specifically binding to FXI and / or FXIa according to any one of claims 1 to 11, and another antibody or antigen-binding fragment thereof, or an antibody mimetic. Multispecific antibodies.

18. 12. A method for the treatment of a pulmonary artery disease comprising administering to a subject an antibody or an antigen-binding fragment thereof capable of specifically binding to FXI and / or FXIa according to any one of claims 1 to 11, and a conjugate moiety, the conjugate moiety being a detectable label or a therapeutic agent. Conjugate.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, the isolated nucleic acid molecule according to claim 12 or 13, the vector according to claim 14, the host cell according to claim 15, the multispecific antibody according to claim 17, or the conjugate according to claim 18, together with a pharma- ceutically acceptable carrier and / or excipient.

20. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition further comprises another pharma- ceutical active agent selected from an antiplatelet agent, an anticoagulant agent, or a thrombolytic agent.

21. The additional pharmacologic active agent is aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, unfractionated heparin, heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, streptokinase, urokinase, alteplase, prourokinase, or any combination thereof; 20. The pharmaceutical composition of claim 20.

22. The following biological activities in a subject: (a) binding to and / or inducing a conformational change in the catalytic domain of FXI and / or FXIa; (b) inhibiting or blocking the binding of FXI and / or FXIa to a substrate; (c) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors; (d) inhibiting or blocking the binding of FXI to coagulation factor XIIa (FXIIa), thereby inhibiting or blocking the conversion of FXI to active FXIa; (e) inhibiting or blocking the binding of FXIa to the coagulation factor FIX, thereby inhibiting or blocking the conversion of FIX to active FIXa; (f) inhibiting or blocking FXI and / or FXIa-mediated activation of the intrinsic coagulation pathway; (g) inhibiting or blocking the activity of FXI and / or FXIa in thrombosis; (h) prolonging FXI and / or FXIa mediated clotting time; (i) inhibiting thrombosis; (j) preventing and / or treating a disease or disorder associated with coagulation or thromboembolism mediated by FXI and / or FXIa; or (k) any combination of (a) to (j) 20. The pharmaceutical composition of claim 19, comprising the antibody or antigen-binding fragment thereof in an effective dose sufficient to induce at least one of the following:

23. 23. The pharmaceutical composition of any one of claims 19 to 22, further comprising a second antibody or a nucleic acid encoding said second antibody, wherein said second antibody is another antibody capable of recognizing a different epitope of FXI or FXIa, or an antibody capable of specifically binding to a receptor or ligand selected from the group consisting of thrombin, antiplasmin, factor XII, factor VIII, factor VII, factor X, factor IX, factor II, tissue factor, P-selectin and its ligands, L-selectin and its ligands, or any combination of the above antibodies.

24. A kit comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 11, or a vector according to claim 14, or a host cell according to claim 15, or a multispecific antibody according to claim 17, or a conjugate according to claim 18, or a pharmaceutical composition according to any one of claims 19 to 23, together with instructions for use.

25. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or the isolated nucleic acid molecule according to claim 12 or 13, or the vector according to claim 14, or the host cell according to claim 15, or the multispecific antibody according to claim 17, or the conjugate according to claim 18, or the pharmaceutical composition according to any one of claims 19 to 23, in the manufacture of a medicament for the prevention and / or treatment of a disease or disorder associated with coagulation or thromboembolism.

26. For the following purposes: (a) binding to and / or inducing a conformational change in the catalytic domain of FXI and / or FXIa; (b) inhibiting or blocking the binding of FXI and / or FXIa to a substrate; (c) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors; (d) inhibiting or blocking the binding of FXI to coagulation factor XIIa (FXIIa), thereby inhibiting the conversion of FXI to active FXIa; (e) inhibiting or blocking the binding of FXIa to the coagulation factor FIX, thereby inhibiting the conversion of FIX to active FIXa; (f) inhibiting or blocking FXI and / or FXIa-mediated activation of the intrinsic coagulation pathway; (g) inhibiting or blocking the activity of FXI and / or FXIa in thrombosis; (h) prolonging FXI and / or FXIa mediated clotting time; (i) inhibiting thrombosis; (j) preventing and / or treating a disease or disorder associated with coagulation or thromboembolism mediated by FXI and / or FXIa; or (k) any combination of (a) to (j) 25. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or an isolated nucleic acid molecule according to claim 12 or 13, or a vector according to claim 14, or a host cell according to claim 15, or a multispecific antibody according to claim 17, or a conjugate according to claim 18, or a pharmaceutical composition according to any one of claims 19 to 23, in the manufacture of a medicament for use in at least one of the following:

27. The disease or disorder associated with coagulation or thromboembolism is thrombosis, thrombotic stroke, atrial fibrillation, prevention of stroke associated with atrial fibrillation (SPAF), deep vein thrombosis, venous thromboembolism, acute coronary syndrome (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolic pulmonary hypertension, systemic embolism, myocardial infarction (MI), acute myocardial infarction (AMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention, peripheral arterial occlusive disease (PAOD), renal vein thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, diffuse intravascular coagulation, thromboembolic disorder caused by medical devices, severe systemic inflammatory response syndrome. , metastatic cancer, infectious disease, organ failure, toxicity caused by administration of therapeutic proteins in the body, multiple trauma, ischemia-reperfusion injury, localized fibrin deposition, adult pulmonary alveolar proteinosis, venous thromboembolic events (VTE) before and after total joint arthroplasty (TKA), coronary heart disease, thromboembolism after myocardial infarction, stroke in patients with non-valvular atrial fibrillation, thrombosis and thromboembolism in chronic kidney disease, thrombosis and thromboembolism in patients undergoing hemodialysis and extracorporeal membrane oxygenation, deep vein thrombosis (DVT), or pulmonary embolism (PE).

28. A method for detecting the presence or level of FXI and / or FXIa in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 11, or a conjugate described in claim 18, under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof and FXI and / or FXIa, and detecting the formation of the complex.

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