Anti-FXI / FXIa antibodies and uses thereof

Abstract

The present invention belongs to the field of therapeutic monoclonal antibodies, and specifically provides antibodies or antigen-binding fragments thereof against FXI / FXIa, nucleic acid molecules encoding them, and methods for preparing them. The anti-FXI / FXIa antibodies or antigen-binding fragments thereof of the present invention have specificity and high affinity for FXI / FXIa, and can effectively inhibit the activity of FXI / FXIa. Therefore, the present invention further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and its use in the preparation of a drug for preventing and / or treating a disease or condition associated with coagulation or thromboembolism.

Description

Technical Field

[0001] The present invention belongs to the field of therapeutic monoclonal antibodies, and more specifically, the present invention relates to an antibody against FXI and / or FXIa; and also relates to the use of the antibody in preventing and / or treating diseases or conditions associated with coagulation or thromboembolism. Technical Background

[0002] Thrombosis or embolism involves organs of all systems of the body, mainly heart, brain and peripheral vascular diseases. It is characterized by high incidence, high disability and lethality, and is the leading cause of death from cardiovascular diseases. At present, the prevention and treatment of thrombotic diseases mainly include three types of drugs: anticoagulants, antiplatelets and thrombolytics; among them, anticoagulants are mainly used in the clinic to prevent and treat venous thromboembolism caused by various reasons; in addition, they can also be used to prevent stroke in patients with atrial fibrillation and anticoagulant treatment in patients with acute coronary syndrome. The market demand for anticoagulants is large, and there are drugs available for clinical selection. However, the anticoagulants currently used in clinical practice mainly inhibit the common pathway of the coagulation cascade reaction, so bleeding is the main complication. Traditional anticoagulants, such as warfarin, heparin, low molecular weight heparin, and new drugs launched in recent years, such as FXa inhibitors (rivaroxaban, apixaban, etc.) and thrombin inhibitors (dabigatran etexilate, hirudin, etc.), have good effects on reducing thrombosis, but they all face a common deficiency - the possibility of causing bleeding complications. Therefore, there is an urgent clinical need for anticoagulant drugs with low bleeding risk.

[0003] Coagulation factor XI (FXI) participates in the intrinsic coagulation cascade, and its active form is coagulation factor XIa (FXIa). The FXI protein is a dimer formed by two homologous monomers with a molecular weight of about 80 kDa through disulfide bonds. The FXI monomer consists of four apple domains and a catalytic domain. After the formation of FXIa by coagulation factor XIIa (FXIIa), the binding site of FIX is exposed. After binding with FIX, it promotes the conversion of FIX to active FIXa, activating the downstream coagulation cascade. The concentration of coagulation factor XI in mammalian plasma is about 25-30nM. It is a glycoprotein that exists in the form of zymogen. Almost all FXI forms a complex with high molecular weight kininogen (HK) and circulates in the blood. The effect of HK on the function of FXI is still unclear. HK may assist FXI in binding to platelets or endothelial cells, and studies have also observed that HK inhibits the activation of FXI. The activation process of FXI is that under the action of different proteases, each monomer is cleaved between Arg369-Ile370 to produce a protein composed of a heavy chain of about 50kDa containing an apple domain and a light chain of about 30kDa containing a catalytic domain. The heavy chain and the light chain are connected by a disulfide bond formed by Cys362-Cys482. Activated FXI, FXIa, usually refers to each monomer of the dimer FXI cleaved at Arg369, but there are also cases where only one monomer is activated. FXIa in Ca 2+ The cleavage of FIX by the participation of FIXa turns it into activated FIX (FIXa), which then converts coagulation factor X into its active form Xa. Xa can then mediate the activation of coagulation factor II / thrombin. Thrombin, as the terminal protease in the coagulation cascade, can further promote the production of FXIa by directly activating FXI in a feedback mechanism. Both coagulation factor XIIa and FXIa (auto-activation) in the coagulation cascade can convert FXI into FXIa.

[0004] In addition, activated FIX (FIXa) can directly bind to platelets, promote the formation of platelet aggregates in the blood, and form distal microvascular occlusion. Therefore, FIX / FIXa promotes thrombosis through multiple pathways. And animal experiments and clinical observations have shown that the lack of FIX / FIXa has only a very small risk of bleeding. Therefore, FIX / FIXa is an ideal target for the prevention and treatment of diseases / disorders related to coagulation or thromboembolism. (Zilberman-Rudenko J. et. al. Coagulation factor XI promotes distal platelet activation and single platelet connversion in the bloodstream under shear flow. Arterioscler Thromb Vasc Biol. 2016 March; 36(3): 510-517.) Factors that regulate coagulation function by intervening in FXI and / or FXIa molecules include, but are not limited to: the formation of FXI and / or FXIa dimers; preventing molecules that can activate FXI, such as coagulation factor XIIa, FXIa, from contacting FXI, thereby inhibiting or blocking the activation of FXI; inhibiting or blocking the formation of a complex between FXI and HK; blocking 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 blocking the activation of the intrinsic coagulation pathway; inhibiting or blocking the binding of FXI and / or FXIa to substrates.

[0005] Currently, Bayer's FXIa antibody drug BAY-1213790 is in Phase II clinical trials for the indication of venous embolism. BAY-1213790 comes from a phage display library and binds to the catalytic domain of FXIa. In addition, Aronora's AB-023 (14E11) is in Phase I clinical trials for the indication of venous embolism, and in Phase II clinical trials for the indication of end-stage renal disease (ESRD).

[0006] Currently, there are no FXIa-targeted antibody drugs on the market. Therefore, it is urgent and necessary to develop anti-FXI and / or FXIa antibodies with higher specificity, lower toxic side effects, better clinical efficacy, and more convenient administration methods, which will provide patients with more medication options. Summary of the invention

[0007] In the present application, the inventors first developed a mouse antibody with excellent properties, which can specifically recognize FXI and / or FXIa. On this basis, the inventors have devoted a lot of creative work, conducted in-depth research and transformation on the mouse antibody, and thus developed a humanized antibody of the mouse antibody.

[0008] The antibodies (especially humanized antibodies) of the present invention are extremely advantageous, as they not only retain (or even improve) the functions and properties of the parent murine antibodies, such as binding to FXI and / or FXIa with very high affinity and specificity, thus having the potential to be used for the prevention and treatment of coagulation or thromboembolic disorders, but also have a very high degree of humanization and can be safely administered to human subjects without inducing immunogenic reactions. Therefore, the antibodies of the present invention have great clinical value.

[0009] Antibodies of the present invention

[0010] In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to FXI and / or FXIa, wherein the antibody or the antigen-binding fragment thereof comprises the following:

[0011] (a) the following three heavy chain CDRs: CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 1, 15, 16, 17, 29, 31; and / or

[0012] The following three light chain CDRs: CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 2, 18, 19, 20, 30, 32;

[0013] or,

[0014] (b) the following three heavy chain CDRs: CDR-H1 described in (a) or a variant thereof containing an amino acid mutation, CDR-H2 described in (a) or a variant thereof containing an amino acid mutation, CDR-H3 described in (a) or a variant thereof containing an amino acid mutation; and / or

[0015] The following three light chain CDRs: CDR-L1 described in (a) or a variant containing an amino acid mutation compared thereto, CDR-L2 described in (a) or a variant containing an amino acid mutation compared thereto, CDR-L3 described in (a) or a variant containing an amino acid mutation compared thereto;

[0016] 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), and the amino acid mutation is a substitution, deletion or addition of one or several amino acids (for example, a substitution, deletion or addition of 1, 2 or 3 amino acids).

[0017] In certain preferred embodiments, the substitution is a conservative substitution;

[0018] In certain preferred embodiments, the CDRs of the antibody or antigen-binding fragment thereof are defined according to the Kabat, IMGT, Chothia or AbM numbering systems.

[0019] In certain preferred embodiments, the antibody or antigen-binding fragment thereof further comprises a framework region (FRs) from human or mouse immunoglobulin.

[0020] In certain preferred embodiments, the antibody or antigen-binding fragment thereof binds to human FXI and / or human FXIa.

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0022] (a) the following three heavy chain CDRs: CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 1, 15, 16, 17; and / or

[0023] The following three light chain CDRs: CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 2, 18, 19, 20;

[0024] or,

[0025] (b) the following three heavy chain CDRs: CDR-H1 described in (a) or a variant thereof containing an amino acid mutation, CDR-H2 described in (a) or a variant thereof containing an amino acid mutation, CDR-H3 described in (a) or a variant thereof containing an amino acid mutation; and / or

[0026] The following three light chain CDRs: CDR-L1 described in (a) or a variant containing an amino acid mutation compared thereto, CDR-L2 described in (a) or a variant containing an amino acid mutation compared thereto, CDR-L3 described in (a) or a variant containing an amino acid mutation compared thereto;

[0027] 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), and the amino acid mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); in certain preferred embodiments, the substitution is a conservative substitution.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

[0029] (a) the following three heavy chain CDRs: CDR-H1, CDR-H2 and CDR-H3 contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NOs: 29 and 31; and / or

[0030] The following three light chain CDRs: CDR-L1, CDR-L2 and CDR-L3 contained in the light chain variable region (VL) as shown in any one of SEQ ID NOs: 30 and 32;

[0031] or,

[0032] (b) the following three heavy chain CDRs: CDR-H1 described in (a) or a variant thereof containing an amino acid mutation, CDR-H2 described in (a) or a variant thereof containing an amino acid mutation, CDR-H3 described in (a) or a variant thereof containing an amino acid mutation; and / or

[0033] The following three light chain CDRs: CDR-L1 described in (a) or a variant containing an amino acid mutation compared thereto, CDR-L2 described in (a) or a variant containing an amino acid mutation compared thereto, CDR-L3 described in (a) or a variant containing an amino acid mutation compared thereto;

[0034] 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), and the amino acid mutation is a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids); in certain preferred embodiments, the substitution is a conservative substitution.

[0035] 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).

[0036] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the IMGT numbering system:

[0037] (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 3, CDR-H2 with a sequence of SEQ ID NO: 4, and CDR-H3 with a sequence of SEQ ID NO: 5; and / or,

[0038] A light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 6, CDR-L2 with a sequence of SEQ ID NO: 7, and CDR-L3 with a sequence of SEQ ID NO: 8;

[0039] or

[0040] (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 33, CDR-H2 with a sequence of SEQ ID NO: 34, and CDR-H3 with a sequence of SEQ ID NO: 35; and / or,

[0041] A light chain variable region (VL) comprising the following 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.

[0042] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains an amino acid mutation compared to the heavy chain variable region and / or light chain variable region described in (a) or (b) under the aforementioned IMGT definition, and the amino acid mutation is a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof); preferably, the substitution is a conservative substitution.

[0043] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:

[0044] (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 9, CDR-H2 with a sequence of SEQ ID NO: 10, and CDR-H3 with a sequence of SEQ ID NO: 11; and / or,

[0045] a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with a sequence of SEQ ID NO: 12, CDR-L2 with a sequence of SEQ ID NO: 13, and CDR-L3 with a sequence of SEQ ID NO: 14;

[0046] or

[0047] (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 39, CDR-H2 with a sequence of SEQ ID NO: 40, and CDR-H3 with a sequence of SEQ ID NO: 41; and / or,

[0048] A light chain variable region (VL) comprising the following 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.

[0049] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains an amino acid mutation compared to the heavy chain variable region and / or light chain variable region described in (a) or (b) under the aforementioned AbM definition, and the amino acid mutation is a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof); preferably, the substitution is a conservative substitution.

[0050] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise the following heavy chain variable region (VH) and / or light chain variable region (VL), wherein at least one CDR of the heavy chain variable region (VH) and / or light chain variable region (VL) contains an amino acid mutation compared to the heavy chain variable region and / or light chain variable region described in (a) or (b) under the aforementioned IMGT or AbM definition, and the amino acid mutation is a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof); preferably, the substitution is a conservative substitution.

[0051] In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) framework region (FRs) derived from a murine immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) framework region (FRs) derived from a murine immunoglobulin. Therefore, in certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is of murine origin.

[0052] In certain preferred embodiments, the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) framework region (FRs) derived from a human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) framework region (FRs) derived from a human immunoglobulin. Therefore, in certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention is humanized. In such embodiments, the heavy chain variable region FRs and / or light chain variable region FRs of the antibody or antigen-binding fragment thereof of the present invention may comprise one or more non-human (e.g., mouse) amino acid residues, for example, the heavy chain framework region FRs and / or light chain framework region FRs may comprise one or more amino acid back mutations, in which there are corresponding mouse amino acid residues.

[0053] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0054] (a) a heavy chain framework region of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids (e.g., up to 15, up to 10, or 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; and / or

[0055] (b) a light chain framework region of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the sequence of the germline antibody gene from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 1, 2, 3, 4 or 5 conservative substitutions of amino acids).

[0056] In certain preferred embodiments, the degree of humanization of the antibodies or antigen-binding fragments thereof of the invention is 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%.

[0057] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0058] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0059] (i) a sequence shown in any one of SEQ ID NOs: 1, 15, 16, and 17;

[0060] (ii) a sequence having one or more 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 sequence shown in any one of SEQ ID NOs: 1, 15, 16, 17; or

[0061] (iii) a sequence that is 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% identical to the sequence shown in any one of SEQ ID NOs: 1, 15, 16, 17;

[0062] and / or

[0063] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0064] (iv) a sequence shown in any one of SEQ ID NOs: 2, 18, 19, and 20;

[0065] (v) a sequence having one or more 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 sequence shown in any one of SEQ ID NOs: 2, 18, 19, 20; or

[0066] (vi) a sequence that is 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% identical to the sequence shown in any one of SEQ ID NOs: 2, 18, 19, 20.

[0067] In certain embodiments, an antibody or antigen-binding fragment thereof of the invention comprises:

[0068] (a) a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of:

[0069] (i) a sequence shown in any one of SEQ ID NOs: 29 and 31;

[0070] (ii) a sequence having one or more 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 sequence shown in any one of SEQ ID NOs: 29, 31; or

[0071] (iii) a sequence that is 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% identical to the sequence shown in any one of SEQ ID NOs: 29 and 31;

[0072] and / or

[0073] (b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0074] (iv) a sequence shown in any one of SEQ ID NOs: 30 and 32;

[0075] (v) a sequence having one or more 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 sequence shown in any one of SEQ ID NOs: 30, 32; or

[0076] (vi) a sequence that is 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% identical to the sequence shown in any of SEQ ID NOs: 30, 32.

[0077] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have the following heavy chain variable region (VH) and / or light chain variable region (VL): VH shown in any one of SEQ ID NOs: 1, 15, 16, 17, and / or VL shown in any one of SEQ ID NOs: 2, 18, 19, 20.

[0078] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have the following heavy chain variable region (VH) and / or light chain variable region (VL): VH shown in any one of SEQ ID NOs: 29 and 31, and / or VL shown in any one of SEQ ID NOs: 30 and 32.

[0079] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:1, and / or the VL set forth in SEQ ID NO:2.

[0080] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO: 1 and / or the VL set forth in SEQ ID NO: 2.

[0081] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 VH shown in SEQ ID NO: 1 and / or the VL shown in SEQ ID NO: 2. In a preferred embodiment, the substitutions are conservative substitutions.

[0082] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:15, and / or the VL set forth in SEQ ID NO:18.

[0083] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO: 15 and / or the VL set forth in SEQ ID NO: 18.

[0084] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 VH shown in SEQ ID NO: 15 and / or the VL shown in SEQ ID NO: 18. In a preferred embodiment, the substitutions are conservative substitutions.

[0085] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:15, and / or the VL set forth in SEQ ID NO:20.

[0086] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO: 15 and / or the VL set forth in SEQ ID NO: 20.

[0087] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 VH shown in SEQ ID NO: 15 and / or the VL shown in SEQ ID NO: 20. In a preferred embodiment, the substitutions are conservative substitutions.

[0088] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:16, and / or the VL set forth in SEQ ID NO:18.

[0089] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO: 16 and / or the VL set forth in SEQ ID NO: 18.

[0090] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 VH shown in SEQ ID NO: 16 and / or the VL shown in SEQ ID NO: 18. In a preferred embodiment, the substitutions are conservative substitutions.

[0091] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:17, and / or the VL set forth in SEQ ID NO:19.

[0092] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO: 17 and / or the VL set forth in SEQ ID NO: 19.

[0093] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 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.

[0094] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:17, and / or the VL set forth in SEQ ID NO:18.

[0095] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO: 17 and / or the VL set forth in SEQ ID NO: 18.

[0096] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 VH shown in SEQ ID NO: 17 and / or the VL shown in SEQ ID NO: 18. In a preferred embodiment, the substitutions are conservative substitutions.

[0097] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:29, and / or the VL set forth in SEQ ID NO:30.

[0098] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO: 29 and / or the VL set forth in SEQ ID NO: 30.

[0099] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 VH shown in SEQ ID NO: 29 and / or the VL shown in SEQ ID NO: 30. In a preferred embodiment, the substitutions are conservative substitutions.

[0100] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises the VH set forth in SEQ ID NO:31, and / or the VL set forth in SEQ ID NO:32.

[0101] In certain embodiments, the VH and / or VL of the antibodies or antigen-binding fragments thereof of the invention are 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% identical to the VH set forth in SEQ ID NO:31 and / or the VL set forth in SEQ ID NO:32.

[0102] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof has one or more 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 VH shown in SEQ ID NO: 31 and / or the VL shown in SEQ ID NO: 32. In a preferred embodiment, the substitutions are conservative substitutions.

[0103] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0104] (a) a VH having the sequence shown in SEQ ID NO: 1 and a VL having the sequence shown in SEQ ID NO: 2;

[0105] (b) a VH having the sequence shown in SEQ ID NO: 15 and a VL having the sequence shown in SEQ ID NO: 18;

[0106] (c) a VH having the sequence shown in SEQ ID NO: 15 and a VL having the sequence shown in SEQ ID NO: 20;

[0107] (d) a VH having the sequence shown in SEQ ID NO: 16 and a VL having the sequence shown in SEQ ID NO: 18;

[0108] (e) a VH having the sequence shown in SEQ ID NO: 17 and a VL having the sequence shown in SEQ ID NO: 19;

[0109] (f) a VH having the sequence shown in SEQ ID NO: 17 and a VL having the sequence shown in SEQ ID NO: 18;

[0110] (g) a VH having the sequence shown in SEQ ID NO: 29 and a VL having the sequence shown in SEQ ID NO: 30; or,

[0111] (h) VH having the sequence shown in SEQ ID NO: 31 and VL having the sequence shown in SEQ ID NO: 32.

[0112] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) are compared with the VH and VL described in any one of groups (a) to (h), and the heavy chain variable region (VH) 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; and / or, the light chain variable region (VL) 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.

[0113] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) have one or several 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 VH and VL described in any one of groups (a) to (h); and / or, the light chain variable region (VL) has one or several 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); preferably, the substitutions are conservative substitutions.

[0114] In any of the above aspects, the antibody or antigen-binding fragment thereof of the present invention may further comprise a constant region sequence derived from a mammalian (eg, mouse or human) immunoglobulin or a variant thereof. In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) of a human or murine immunoglobulin or a variant thereof, which has one or more 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, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); and / or, the light chain of an antibody or antigen-binding fragment thereof of the present invention comprises a light chain constant region (CL) of a human or murine immunoglobulin or a variant thereof, which has one or more 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, or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 5, 4, 3, 2 or 1 amino acid substitutions, deletions or additions or any combination thereof).

[0115] In certain preferred embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 5, 4, 3, 2 or 1 conservative substitutions of amino acids); and / or, the light chain of the antibody or antigen-binding fragment thereof of the present invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, which has up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; for example, 5, 4, 3, 2 or 1 conservative substitutions of amino acids).

[0116] In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) of a murine immunoglobulin or a variant thereof, the variant having up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 5, 4, 3, 2, or 1 conservative substitutions of amino acids). In certain embodiments, the light chain of an antibody or antigen-binding fragment thereof of the present invention comprises a light chain constant region (CL) of a murine immunoglobulin or a variant thereof, the variant having up to 20 conservative substitutions of amino acids compared to the wild-type sequence from which it is derived (e.g., up to 15, up to 10, or up to 5 conservative substitutions of amino acids; e.g., 5, 4, 3, 2, or 1 conservative substitutions of amino acids).

[0117] In some embodiments, the constant region is altered, for example, by amino acid mutations, to modify the properties of the anti-FXI and / or FXIa antibody molecule (e.g., to change one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue, for example, changing the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby changing the effector function (e.g., reducing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0118] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention have a heavy chain constant region (Fc), which is selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; preferably selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4, more preferably selected from the heavy chain constant regions of IgG1 or IgG4 (for example, human IgG1 or IgG4). In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have a light chain constant region, which is selected from, for example, the light chain constant regions of κ or λ, preferably the κ light chain constant region (for example, human κ light chain).

[0119] 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.

[0120] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0121] (a) a heavy chain constant region (CH) comprising an amino acid sequence selected from the group consisting of:

[0122] (i) the sequence shown in SEQ ID NO:21;

[0123] (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in SEQ ID NO: 21 (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0124] (iii) a sequence that is 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% identical to the sequence shown in SEQ ID NO:21;

[0125] and / or

[0126] (b) a light chain constant region (CL) comprising an amino acid sequence selected from the group consisting of:

[0127] (iv) the sequence shown in SEQ ID NO:22;

[0128] (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in SEQ ID NO: 22 (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0129] (vi) a sequence that is 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% identical to the sequence set forth in SEQ ID NO:22.

[0130] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0131] In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) as shown in SEQ ID NO:21 and a light chain constant region (CL) as shown in SEQ ID NO:22.

[0132] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0133] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0134] (i) a sequence comprising the VH shown in SEQ ID NO: 15 and the CH shown in SEQ ID NO: 21;

[0135] (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0136] (iii) a sequence that is 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% identical to the sequence shown in (i);

[0137] and / or

[0138] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0139] (iv) a sequence comprising the VL sequence shown in SEQ ID NO: 18 and the CL sequence shown in SEQ ID NO: 22;

[0140] (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0141] (vi) a sequence that is 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% identical to the sequence shown in (iv).

[0142] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0143] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0144] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0145] (i) a sequence comprising the VH shown in SEQ ID NO: 15 and the CH shown in SEQ ID NO: 21;

[0146] (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0147] (iii) a sequence that is 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% identical to the sequence shown in (i);

[0148] and / or

[0149] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0150] (iv) a sequence comprising the VL shown in SEQ ID NO: 20 and the CL shown in SEQ ID NO: 22;

[0151] (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0152] (vi) a sequence that is 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% identical to the sequence shown in (iv).

[0153] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0154] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0155] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0156] (i) a sequence comprising the VH shown in SEQ ID NO: 16 and the CH shown in SEQ ID NO: 21;

[0157] (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0158] (iii) a sequence that is 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% identical to the sequence shown in (i);

[0159] and / or

[0160] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0161] (iv) a sequence comprising the VL shown in SEQ ID NO: 18 and the CL shown in SEQ ID NO: 22;

[0162] (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0163] (vi) a sequence that is 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% identical to the sequence shown in (iv).

[0164] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0165] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0166] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0167] (i) a sequence comprising the VH shown in SEQ ID NO: 17 and the CH shown in SEQ ID NO: 21;

[0168] (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0169] (iii) a sequence that is 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% identical to the sequence shown in (i);

[0170] and / or

[0171] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0172] (iv) a sequence comprising the VL shown in SEQ ID NO: 19 and the CL shown in SEQ ID NO: 22;

[0173] (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0174] (vi) a sequence that is 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% identical to the sequence shown in (iv).

[0175] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0176] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0177] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0178] (i) a sequence comprising the VH shown in SEQ ID NO: 17 and the CH shown in SEQ ID NO: 21;

[0179] (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0180] (iii) a sequence that is 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% identical to the sequence shown in (i);

[0181] and / or

[0182] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0183] (iv) a sequence comprising the VL shown in SEQ ID NO: 18 and the CL shown in SEQ ID NO: 22;

[0184] (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0185] (vi) a sequence that is 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% identical to the sequence shown in (iv).

[0186] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0187] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0188] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise:

[0189] (a) a heavy chain comprising an amino acid sequence selected from the group consisting of:

[0190] (i) a sequence comprising the VH shown in SEQ ID NO: 31 and the CH shown in SEQ ID NO: 21;

[0191] (ii) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (i) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0192] (iii) a sequence that is 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% identical to the sequence shown in (i);

[0193] and / or

[0194] (b) a light chain comprising an amino acid sequence selected from the group consisting of:

[0195] (iv) a sequence comprising the VL shown in SEQ ID NO: 32 and the CL shown in SEQ ID NO: 22;

[0196] (v) a sequence having one or more amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions or any combination thereof); or

[0197] (vi) a sequence that is 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% identical to the sequence shown in (iv).

[0198] In certain preferred embodiments, the substitutions described in (ii) or (v) are conservative substitutions.

[0199] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising VH shown in SEQ ID NO: 1 and a heavy chain constant region (CH) shown in SEQ ID NO: 21, and a light chain comprising VL shown in SEQ ID NO: 2 and a light chain constant region (CL) shown in SEQ ID NO: 22.

[0200] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising the VH shown in SEQ ID NO: 15 and the heavy chain constant region (CH) shown in SEQ ID NO: 21, and a light chain comprising the VL shown in SEQ ID NO: 18 and the light chain constant region (CL) shown in SEQ ID NO: 22.

[0201] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising the VH shown in SEQ ID NO: 15 and the heavy chain constant region (CH) shown in SEQ ID NO: 21, and a light chain comprising the VL shown in SEQ ID NO: 20 and the light chain constant region (CL) shown in SEQ ID NO: 22.

[0202] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising the VH shown in SEQ ID NO: 16 and the heavy chain constant region (CH) shown in SEQ ID NO: 21, and a light chain comprising the VL shown in SEQ ID NO: 18 and the light chain constant region (CL) shown in SEQ ID NO: 22.

[0203] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising VH shown in SEQ ID NO: 17 and a heavy chain constant region (CH) shown in SEQ ID NO: 21, and a light chain comprising VL shown in SEQ ID NO: 19 and a light chain constant region (CL) shown in SEQ ID NO: 22.

[0204] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising the VH shown in SEQ ID NO: 17 and the heavy chain constant region (CH) shown in SEQ ID NO: 21, and a light chain comprising the VL shown in SEQ ID NO: 18 and the light chain constant region (CL) shown in SEQ ID NO: 22.

[0205] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising the VH shown in SEQ ID NO: 29 and the heavy chain constant region (CH) shown in SEQ ID NO: 21, and a light chain comprising the VL shown in SEQ ID NO: 30 and the light chain constant region (CL) shown in SEQ ID NO: 22.

[0206] In certain preferred embodiments, the antibodies of the present invention comprise: a heavy chain comprising the VH shown in SEQ ID NO:31 and the heavy chain constant region (CH) shown in SEQ ID NO:21, and a light chain comprising the VL shown in SEQ ID NO:32 and the light chain constant region (CL) shown in SEQ ID NO:22.

[0207] In certain preferred embodiments, the antibodies of the present invention are chimeric antibodies or humanized antibodies. In certain preferred embodiments, the antibodies of the present invention or their antigen-binding fragments are selected from ScFv, Fab, Fab', (Fab')2, Fv fragments, disulfide-linked Fv (dsFv), diabodies, bispecific antibodies, and multispecific antibodies.

[0208] In certain embodiments, the antibody or antigen-binding fragment thereof has reduced ADCC activity.

[0209] In certain embodiments, the antibody or antigen-binding fragment thereof has reduced CDC activity.

[0210] In certain embodiments, the antibody or antigen-binding fragment thereof does not have CDC activity.

[0211] In certain embodiments, the antibody or antigen-binding fragment thereof does not have ADCC activity.

[0212] In certain preferred embodiments, the antibody or antigen-binding fragment thereof has reduced ADCC and reduced CDC activity.

[0213] In certain preferred embodiments, the antibody or antigen-binding fragment thereof has neither ADCC nor CDC activity.

[0214] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention possess at least one of the following characteristics:

[0215] (a) binds to FXI and / or FXIa (e.g., human FXI and / or human FXIa) with a KD of less than about 100 nM, e.g., less than about 10 nM, 1 nM, 0.1 nM or less; preferably, the KD D It can be measured by techniques known in the art, such as biofilm interferometry (BLI) (such as ForteBio ) measured;

[0216] (b) binds to FXI and / or FXIa (e.g., human FXI and / or human FXIa) with an EC50 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; preferably, the EC50 can be measured by techniques known in the art, e.g., by flow cytometry or cell competition ELISA;

[0217] (c) the binding of the antibody or antigen-binding fragment thereof to FXI and / or FXIa inhibits or blocks the binding of FXI and / or FXIa to a substrate, thereby prolonging the coagulation time;

[0218] (d) the binding of the antibody or antigen-binding fragment thereof to FXI and / or FXIa inhibits or blocks the catalytic effect of FXI and / or FXIa on the substrate;

[0219] (e) the binding of the antibody or antigen-binding fragment thereof to FXI and / or FXIa does not affect extrinsic coagulation;

[0220] (f) the antibody or antigen-binding fragment thereof has reduced ADCC and / or CDC activity;

[0221] (g) the antibody or antigen-binding fragment thereof does not have ADCC and / or CDC activity;

[0222] (h) the binding of the antibody or antigen-binding fragment thereof to FXI and / or FXIa inhibits or blocks the formation of dimers of FXI and / or FXIa;

[0223] (i) the binding of the antibody or antigen-binding fragment thereof to FXI and / or FXIa inhibits or blocks the formation of a complex between FXI and / or FXIa and HK;

[0224] (j) binding to the catalytic domain of FXI and / or FXIa, and / or inducing conformational changes thereof;

[0225] (k) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors; or

[0226] (l) Any combination of (a)-(k).

[0227] In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention prevent the FXI catalytic domain from assuming an active conformation when bound to FXI. In certain preferred embodiments, the antibodies or antigen-binding fragments thereof of the present invention prevent the FXI catalytic domain from assuming an active conformation by inducing conformational changes in the zymogen structure when bound to FXI, further inhibiting binding to FIX.

[0228] Derivatized antibodies

[0229] The antibodies or antigen-binding fragments thereof of the present invention may be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). Generally, the derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof will not adversely affect its binding to FXI and / or FXIa (particularly human FXI and / or FXIa). Therefore, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the present 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., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide (e.g., an avidin or polyhistidine tag) that is capable of mediating the binding of the antibody or antigen-binding fragment to another molecule.

[0230] One type of derivatized antibody (e.g., bispecific antibody) is produced by cross-linking 2 or more antibodies (of the same type or different types). Methods for obtaining bispecific antibodies are well known in the art, and examples thereof include, but are not limited to, chemical cross-linking, cell engineering (hybrid hybridoma method), or genetic engineering.

[0231] Another type of derivatized antibody is a labeled antibody. For example, the antibody of the invention or its antigen-binding fragment can be linked to a detectable label. The detectable label of the invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, 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 32P), 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), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as 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 labels. Patents teaching the use of such labels include, but are not limited to, U.S. Patents 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). Detectable labels such as those 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 to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of a colored label. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, detectable labels as described above can be linked to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

[0232] In addition, the antibodies or antigen-binding fragments thereof of the present invention may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups may be used to improve the biological properties of the antibodies, such as increasing serum half-life.

[0233] As one of the derivatives of the antibody, the present invention provides a conjugate. In certain embodiments, the conjugate comprises any antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa of the present invention and a coupling portion, wherein the coupling portion is a detectable label, such as the above-mentioned radioisotope, fluorescent substance, luminescent substance, colored substance or enzyme. In certain embodiments, the coupling portion is a therapeutic agent; optionally, the therapeutic agent is bound to one or more of the antibodies or antigen-binding fragments of the present invention through a linker. Alternatively, the therapeutic agent is bound to the antibodies or antigen-binding fragments of the present invention through a linker. The therapeutic agent is selected from any one of the drugs in the use, treatment methods and pharmaceutical compositions disclosed herein. The linker does not significantly affect the biological activity of the antibodies or antigen-binding fragments of the present invention.

[0234] As one of the derivatives of antibodies, the present invention provides a multispecific antibody, which comprises an antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa of the present invention, and another antibody or antigen-binding fragment thereof, or an antibody analog. In certain embodiments, the multispecific antibody is a conjugate comprising an antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa of the present invention, and another antibody or antigen-binding fragment thereof, or an antibody analog.

[0235] In certain embodiments, the multispecific antibody is formed by coupling an antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa of the present invention with another antibody or antigen-binding fragment thereof or antibody analog, and each antibody or antigen-binding fragment thereof or antibody analog retains the original binding specificity. In certain preferred embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.

[0236] Antibody preparation

[0237] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present invention.

[0238] The antigen-binding fragments of the present invention can be obtained by hydrolyzing 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 directly produced 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 directly obtained 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. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0239] Therefore, in another aspect, the present invention provides a kind of isolated nucleic acid molecule, it comprises the nucleotide sequence encoding the antibody of the present invention or its antigen-binding fragment, or its heavy chain variable region and / or light chain variable region, or its one or more CDRs.According to codon degeneracy known in the art, in certain embodiments, the nucleotide sequence can be replaced according to codon degeneracy.In certain embodiments, the nucleotide sequence is codon optimized.

[0240] In certain preferred embodiments, the isolated nucleic acid molecule comprises: a first nucleic acid and a second nucleic acid encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention, respectively, or a first nucleic acid encoding the heavy chain variable region and the heavy chain constant region of the antibody or antigen-binding fragment thereof of the present invention, respectively, and a second nucleic acid encoding the light chain variable region and the light chain constant region, or a first nucleic acid and a second nucleic acid encoding the heavy chain and the light chain of the antibody or antigen-binding fragment thereof of the present invention, respectively. In certain preferred embodiments, the antibody of the present invention 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 nucleic acid and the second nucleic acid comprise nucleic acids having substantially the same sequence as the first nucleic acid and the second nucleic acid described above. For example, the isolated nucleic acid molecule may comprise the nucleotide sequence shown in SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28 in the sequence listing, or a sequence substantially identical thereto. For example, the sequence substantially identical thereto refers to a sequence having at least about 85%, 90%, 95%, 99% or higher identity thereto, or a sequence having one or more nucleotide substitutions, or a sequence differing by no more than 3, 6, 15, 30 or 45 nucleotides).

[0241] In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region has a nucleotide sequence selected from the following: (a). A nucleotide sequence as shown in SEQ ID NO: 23, or (b). A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence described in (a), or (c). A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; the nucleic acid molecule encoding the antibody light chain variable region has a sequence selected from the following: (d). A nucleotide sequence as shown in SEQ ID NO: 23. NO:24, or (e). a sequence substantially identical to the nucleotide sequence described in (d) (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence described in (d)), or (f). a sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0242] In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid molecule encoding an antibody heavy chain variable region, and / or a nucleic acid molecule encoding an antibody light chain variable region, wherein the nucleic acid molecule encoding the antibody heavy chain variable region has a nucleotide sequence selected from the following: (a). A nucleotide sequence as shown in SEQ ID NO: 27, or (b). A sequence substantially identical to the nucleotide sequence described in (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence described in (a), or (c). A sequence that differs from the nucleotide sequence described in (a) by no more than 3, 6, 15, 30 or 45 nucleotides; the nucleic acid molecule encoding the antibody light chain variable region has a nucleotide sequence selected from the following: (d). A nucleotide sequence as shown in SEQ ID NO: NO:28, or (e). a sequence substantially identical to the nucleotide sequence described in (d) (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence described in (d)), or (f). a sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0243] In certain preferred embodiments, the isolated nucleic acid molecules of the present invention comprise a nucleic acid molecule encoding an antibody heavy chain variable region as shown in SEQ ID NO:23, and / or a nucleic acid molecule encoding an antibody light chain variable region as shown in SEQ ID NO:24.

[0244] In certain preferred embodiments, the isolated nucleic acid molecules of the present invention comprise a nucleic acid molecule encoding an antibody heavy chain variable region as shown in SEQ ID NO:27, and / or a nucleic acid molecule encoding an antibody light chain variable region as shown in SEQ ID NO:28.

[0245] In certain preferred embodiments, the present invention provides an isolated nucleic acid molecule comprising 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 nucleotide sequence selected from the following: (a). A nucleotide sequence as shown in SEQ ID NO: 25, or (b). A sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having one or more nucleotide substitutions compared to the nucleotide sequence of (a), or (c). A sequence that differs from the nucleotide sequence of (a) by no more than 3, 6, 15, 30 or 45 nucleotides, and / or, the nucleic acid molecule encoding the antibody light chain has a nucleotide sequence selected from the following: (d). A nucleotide sequence as shown in SEQ ID NO: NO:26, or (e). a sequence substantially identical to the nucleotide sequence described in (d) (for example, a sequence having at least about 85%, 90%, 95%, 99% or higher identity, or a sequence having one or more nucleotide substitutions, compared to the nucleotide sequence described in (d), or (f). a sequence that differs from the nucleotide sequence described in (d) by no more than 3, 6, 15, 30 or 45 nucleotides.

[0246] In certain preferred embodiments, the isolated nucleic acid molecules of the present invention comprise a nucleic acid molecule encoding an antibody heavy chain as shown in SEQ ID NO:25, and / or a nucleic acid molecule encoding an antibody light chain as shown in SEQ ID NO:26.

[0247] In another aspect of the present invention, a vector (e.g., a cloning vector or an expression vector) is provided, which comprises an isolated nucleic acid molecule of the present invention. In certain preferred embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain preferred embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., a mammal, such as a human).

[0248] The present invention on the other hand, provides a kind of host cell, it comprises the nucleic acid molecule of separation of the present invention or the vector of the present invention.Host cell can be eukaryotic cell (for example mammalian cell, insect cell, yeast cell) or prokaryotic cell (for example Escherichia coli).Suitable eukaryotic cell includes but is not limited to NS0 cell, Vero cell, Hela cell, COS cell, CHO cell, HEK293 cell, BHK cell and MDCKII cell.Suitable insect cell includes but is not limited to Sf9 cell.In certain preferred embodiments, host cell of the present invention is mammalian cell, for example CHO (for example CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0249] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0250] Uses, methods of treatment and pharmaceutical compositions

[0251] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody or conjugate of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0252] In certain preferred embodiments, the pharmaceutical composition of the present invention comprises the antibody or antigen-binding fragment thereof of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0253] In certain preferred embodiments, the pharmaceutical composition of the present invention comprises the isolated nucleic acid molecule of the present invention, a vector or a host cell, and a pharmaceutically acceptable carrier and / or excipient. In such embodiments, the host cell comprises an isolated nucleic acid molecule or a vector as described above.

[0254] In certain preferred embodiments, the pharmaceutical composition may further comprise another pharmaceutically active agent. In certain preferred embodiments, the other pharmaceutically active agent is an antiplatelet drug, an anticoagulant drug or a thrombolytic drug.

[0255] In certain preferred embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent are provided as separate components or as components of the same composition. Therefore, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0256] In certain preferred embodiments, the pharmaceutical composition may further comprise another pharmaceutically active agent. The other pharmaceutically active agent 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, or any combination thereof.

[0257] 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 is sufficient to produce at least one of the following biological activities in a subject:

[0258] (a) binding to the catalytic domain of FXI and / or FXIa, and / or inducing conformational changes thereof;

[0259] (b) inhibiting or blocking the binding of FXI and / or FXIa to a substrate;

[0260] (c) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors;

[0261] (d) inhibiting or blocking the binding of FXI to coagulation factor XIIa (FXIIa), thereby inhibiting or blocking the conversion of FXI to active FXIa;

[0262] (e) inhibiting or blocking the binding of FXIa and coagulation factor FIX, thereby inhibiting or blocking the conversion of FIX into active FIXa;

[0263] (f) inhibiting or blocking the activation of the intrinsic coagulation pathway mediated by FXI and / or FXIa;

[0264] (g) inhibiting or blocking the activity of FXI and / or FXIa in thrombosis;

[0265] (h) prolonging the coagulation time mediated by FXI and / or FXIa;

[0266] (i) inhibiting thrombosis;

[0267] (j) preventing and / or treating FXI and / or FXIa-mediated conditions or diseases related to coagulation or thromboembolism; or

[0268] (k) Any combination of (a)-(j).

[0269] On the other hand, the pharmaceutical composition of the present invention further comprises a second antibody or a nucleic acid encoding the second antibody, wherein the second antibody is another antibody that recognizes a different epitope of FXI or FXIa, or is an antibody that specifically binds to a receptor or ligand selected from the following: 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.

[0270] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody, conjugate or pharmaceutical composition of the present invention in the preparation of a drug, wherein the drug is used for:

[0271] (a) binding to the catalytic domain of FXI and / or FXIa, and / or inducing conformational changes thereof;

[0272] (b) inhibiting or blocking the binding of FXI and / or FXIa to a substrate;

[0273] (c) inhibiting or blocking the binding of FXI and / or FXIa to platelet receptors;

[0274] (d) inhibiting or blocking the binding of FXI to coagulation factor XIIa (FXIIa), thereby inhibiting the conversion of FXI to active FXIa;

[0275] (e) inhibiting or blocking the binding of FXIa and coagulation factor FIX, thereby inhibiting the conversion of FIX into active FIXa;

[0276] (f) inhibiting or blocking the activation of the intrinsic coagulation pathway mediated by FXI and / or FXIa;

[0277] (g) inhibiting or blocking the activity of FXI and / or FXIa in thrombosis;

[0278] (h) prolonging the coagulation time mediated by FXI and / or FXIa;

[0279] (i) inhibiting thrombosis;

[0280] (j) preventing and / or treating diseases or conditions related to coagulation or thromboembolism mediated by FXI and / or FXIa; or

[0281] (k) Any combination of (a)-(j).

[0282] In certain preferred embodiments, when the isolated nucleic acid molecule, vector or host cell of the present invention is used to prepare a medicament, the host cell comprises the isolated nucleic acid molecule or vector as described above.

[0283] In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare a medicament, the medicament is used to prevent and / or treat a disease or disorder associated with blood coagulation or thromboembolism in a subject (eg, a human).

[0284] In certain embodiments, the subject is a mammal. In certain preferred embodiments, the subject is a human.

[0285] In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare a medicament, the medicament is used to delay the occurrence of a disease or disorder associated with coagulation or thromboembolism in a subject (eg, a human).

[0286] In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare 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 (eg, a human).

[0287] In certain preferred embodiments, when the vector or host cell of the present invention is used to prepare a medicament, the medicament is used in a subject (eg, a human).

[0288] 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 present invention is directed to a disease or condition related to coagulation or thromboembolism selected from the group consisting of 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 artery intervention, peripheral arterial occlusive disease (PAOD), renal venous thrombosis ... acute limb ischemia, chronic thromboembolic pulmonary hypertension, systemic embolism, venous thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, disseminated intravascular coagulation, thromboembolic disorders induced by medical devices (e.g., catheters), severe systemic inflammatory response syndrome, metastatic cancer, infectious diseases, organ failure (e.g., renal failure), toxicity caused by in vivo administration of therapeutic proteins, multiple trauma, ischemia-reperfusion injury, localized fibrin deposition, pulmonary alveolar proteinosis in adults, thromboembolic events (VTE) before and after total knee arthroplasty (TKA) surgery, coronary artery 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 in patients subjected to adventitial membrane oxygenation, deep vein thrombosis (DVT), or pulmonary embolism (PE).

[0289] In another aspect, the present invention provides a method for preventing and / or treating a disease or condition associated with coagulation or thromboembolism in a subject. In another aspect, the present invention provides a method for delaying the occurrence of a disease or condition 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 condition associated with coagulation or thromboembolism in a subject. The above method comprises administering an effective amount of an antibody or antigen-binding fragment thereof, a vector, a host cell, a multispecific antibody, a conjugate, or a pharmaceutical composition of the present invention to a subject in need thereof.

[0290] When the host cell of the present invention is used in the above-mentioned method, the host cell comprises the isolated nucleic acid molecule or vector as described above.

[0291] In another aspect, the above method further comprises administering a second therapy to the subject, wherein the second therapy is selected from antiplatelet drugs, anticoagulant drugs, and thrombolytic drugs.

[0292] In certain preferred embodiments, the second therapy 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.

[0293] 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 present invention is directed to a disease or condition associated with coagulation or thromboembolism selected from thrombosis, thrombotic stroke, atrial fibrillation, atrial fibrillation associated stroke prevention (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 artery intervention, peripheral arterial occlusive disease (PAOD), renal venous thrombosis ... acute limb ischemia, chronic thromboembolic pulmonary hypertension, systemic embolism, venous thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, disseminated intravascular coagulation, thromboembolic disorders induced by medical devices (e.g., catheters), severe systemic inflammatory response syndrome, metastatic cancer, infectious diseases, organ failure (e.g., renal failure), toxicity caused by in vivo administration of therapeutic proteins, multiple trauma, ischemia-reperfusion injury, localized fibrin deposition, pulmonary alveolar proteinosis in adults, thromboembolic events (VTE) before and after total knee arthroplasty (TKA) surgery, coronary artery 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 in patients subjected to adventitial membrane oxygenation, deep vein thrombosis (DVT), or pulmonary embolism (PE).

[0294] The antibodies or antigen-binding fragments thereof of the present invention and the pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, 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 should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: incorporating the recombinant protein of the present invention in a suitable solvent at a required dose, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.

[0295] In addition, the antibodies or antigen-binding fragments thereof of the present invention may be present in a pharmaceutical composition in a unit dosage form for ease of administration.

[0296] The antibody or its Fab of the present invention, pharmaceutical composition can be applied by any suitable method known in the art, including but not limited to, oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The technician will understand that the route of administration and / or mode will change according to the intended purpose. In a preferred embodiment, the antibody or its Fab of the present invention, pharmaceutical composition is administered by intravenous infusion or injection.

[0297] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, multispecific antibody or conjugate of the present invention. "Prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease. "Therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment thereof of the present invention may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0298] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be administered once, multiple times over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0299] The typical non-limiting range of the therapeutic or prophylactic 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 the dosage may vary depending on the type and severity of the symptoms to be treated. In addition, those skilled in the art understand that for any particular patient, the specific dosage regimen should be adjusted over time according to the patient's needs and the doctor's professional evaluation; the dosage range given here is only for illustrative purposes and does not limit the use or scope of the pharmaceutical composition of the present invention.

[0300] In the present invention, the subject may be a mammal, such as a human.

[0301] Detection methods and kits

[0302] The antibodies or antigen-binding fragments thereof of the present invention can specifically bind to FXI and / or FXIa (eg, human FXI and / or human FXIa), and thus can be used to detect the presence or level of FXI and / or FXIa in a sample.

[0303] Therefore, in another aspect, the present invention provides a kind of test kit, it comprises the antibody of the present invention or its antigen-binding fragment.In certain preferred embodiments, the antibody of the present invention or its antigen-binding fragment carries a detectable label.In a preferred embodiment, the test kit also comprises a second antibody, which specifically recognizes the antibody of the present invention or its antigen-binding fragment.Preferably, the second antibody also comprises a detectable label.

[0304] In the present invention, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. It is particularly preferred that such labels can be used for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, 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 32P), 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)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as 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 labels. Patents teaching the use of such labels include, but are not limited to, U.S. Patents 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 labels encompassed in the present invention 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 to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of a colored label. In certain embodiments, detectable labels as described above can be attached to the antibodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

[0305] 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 using an antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention also carries a detectable label. In another preferred embodiment, the method further comprises detecting the antibody or antigen-binding fragment thereof of the present invention using a reagent with a detectable label. The method can be used for diagnostic purposes, or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0306] 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, the method comprising contacting the sample with the antibody or antigen-binding fragment thereof of the present invention under conditions allowing formation of a complex between the antibody or antigen-binding fragment thereof, or conjugate and FXI and / or FXIa, and detecting the formation of the complex.

[0307] In another aspect, the invention provides a use of an antibody or antigen-binding fragment thereof in the preparation 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 the invention can provide: the 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 includes instructions for use.

[0308] The antibody or antigen-binding fragment of the present invention has a high binding affinity to FXI and / or FXIa and has extremely strong specificity. Therefore, the antibody or antigen-binding fragment of the present invention is suitable for preventing and / or treating diseases or conditions related to coagulation or thromboembolism. The humanized antibody of the present invention retains the functions and properties of the parent mouse antibody. Moreover, the humanized antibody of the present invention has a high degree of humanization, so that it can be safely administered to human subjects without inducing immunogenic reactions. In addition, the antibody or antigen-binding fragment of the present invention has no or very little bleeding risk. Therefore, the antibody or antigen-binding fragment of the present invention has great clinical value.

[0309] Definition of Abbreviations and Terms

[0310] CDR Complementarity determining region in the variable region of an immunoglobulin

[0311] FR Antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues

[0312] VH antibody heavy chain variable region

[0313] VL Antibody light chain variable region

[0314] IgG Immunoglobulin G

[0315] Kabat The 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).

[0316] Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0317] AbM The AbM CDR definition method is derived from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268-9272). This definition method integrates part of the definitions of Kabat and Chothia.

[0318] IMGT is based on the international ImMunoGeneTics information system initiated by Lefranc et al. For the Immunoglobulin G (IMGT) numbering system, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0319] mAb monoclonal antibody

[0320] EC50 Concentration that produces 50% efficacy or binding

[0321] IC50 concentration that produces 50% inhibition

[0322] ELISA enzyme-linked immunosorbent assay

[0323] PCR polymerase chain reaction

[0324] HRP Horseradish Peroxidase

[0325] KD equilibrium dissociation constant

[0326] Ka binding rate constant

[0327] Kd dissociation rate constant

[0328] ADCC Antibody-dependent cellular cytotoxicity

[0329] CDC Complement-dependent cytotoxicity

[0330] FACS flow cytometry

[0331] CDR-H1 Complementarity determining region 1 in the variable region of the immunoglobulin heavy chain

[0332] CDR-H2 Complementarity determining region 2 in the variable region of the immunoglobulin heavy chain

[0333] CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region

[0334] CDR-L1 Complementarity determining region 1 in the immunoglobulin light chain variable region

[0335] CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region

[0336] CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region

[0337] APTT activated partial thromboplastin time

[0338] CFA Complete Freund's Adjuvant

[0339] EC50 half maximal effect concentration

[0340] FACS flow cytometry technology

[0341] IC50 half maximal inhibitory concentration

[0342] IFA Incomplete Freund's Adjuvant

[0343] MFI Mean fluorescence intensity

[0344] FXI Human coagulation factor 11

[0345] FXIa activated human coagulation factor 11

[0346] RLU Relative Light Unit

[0347] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operation steps such as cell culture, biochemistry, nucleic acid chemistry, immunology, etc. used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0348] As used herein, the terms "FXI protein", "FXI antigen" and "FXI" are used interchangeably and refer to factor XI proteins of various species. The terms "FXIa protein", "FXIa antigen" and "FXIa" are used interchangeably and refer to activated factor XI proteins of various species. The terms "FXI" and "FXIa" (and similar terms) include mutants and variants of native FXI and FXIa proteins, respectively, having an amino acid sequence substantially the same as the native primary structure (amino acid sequence) described herein.

[0349] As used herein, the terms "coagulation and coagulation cascade", "coagulation cascade model" and the like are well known and refer to the coagulation pathway that ultimately produces thrombin through a proteolytic cascade, which then converts soluble fibrinogen into fibrin that forms a clot. The various enzymes of this pathway exist in plasma in the form of zymogens (inactive forms), which undergo proteolytic cleavage of their own precursor molecules upon activation to release active coagulation factors. The process of generating thrombin can be divided into three stages: the intrinsic and extrinsic pathways, and the final common pathway.

[0350] As used herein, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists 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 the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antigen binding sites, respectively. The allocation of amino acids in each region or domain can be defined by numbering systems such as IMGT, Kabat, Chothia or AbM.

[0351] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0352] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the antibody variable region that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, as defined in 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 & 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, a person skilled in the art can easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to a person skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0353] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0354] As used herein, the term "germline antibody gene" is an immunoglobulin sequence encoded by non-lymphocytes that has not undergone genetic rearrangement and maturation leading to the expression of specific immunoglobulins. An advantage provided by various embodiments of the present invention is derived from a recognition that germline antibody genes retain more characteristic important amino acid sequence structures of individual animal species than mature antibody genes. Therefore, when applied therapeutically to the species, it is less recognized as a foreign substance by the species.

[0355] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0356] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide of a full-length or partial fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA technology 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 from Domantis), domain antibodies (technology from Ablynx), and polypeptides that are small enough to have 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.

[0357] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" or "heavy chains" and two "full-length light chains" or "light chains". Wherein, "full-length heavy chain" or "heavy chain" refers to a polypeptide chain that consists of 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 in the direction from N to C; and, when the full-length antibody is an IgE isotype, optionally also includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the direction from N to C. A "full-length light chain" or "light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N to C. Two pairs of full-length antibody chains are linked together 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 from a single species, such as human; or may be a chimeric antibody or a humanized antibody. The full-length antibody of the present invention comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0358] As used herein, the term "Fd fragment" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al., Nature 341: 544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of VH and CH1 domains).

[0359] As used herein, the term "Fv fragment" means an antibody fragment consisting 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 that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0360] As used herein, the term "Fc fragment" means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via a disulfide bond. The Fc fragment of an antibody has a variety of different functions, but does not participate in antigen binding.

[0361] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can 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. In some cases, 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.

[0362] As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of 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)).

[0363] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0364] 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. "Bispecific antibodies" refer to antibodies with two different antigen binding specificities, which are conjugates formed by coupling arms of a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or antibody analogs, and the coupling methods include, but are not limited to, chemical reactions, gene fusions, protein fusions, polypeptide fusions, and enzymatic reactions. Trispecific antibodies are antibodies with three different antigen binding specificities, and tetraspecific antibodies are antibodies with four different antigen binding specificities.

[0365] As used herein, "antibody mimetic" refers to an antibody that specifically binds to an antigen like an antibody, but does not have an antibody structure. They are typically artificial peptides or proteins with a molar mass of about 3 to 20 kDa. For example, ankyrin repeat proteins (DARPin) and fynomers. Designed ankyrin repeat proteins (DARPin) can be linked to IgG antibodies, scFv-Fc antibody fragments, or a combination thereof, as described in CN104341529A. Anti-IL-17a fynomers are bound to anti-IL-6R antibodies, as described in WO2015141862A1.

[0366] As used herein, the term "immunoglobulin" or "Ig" may refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE, of which IgG is the most common circulating antibody. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody reactions, and is important in defending against bacteria and viruses.

[0367] Herein, the technology for obtaining antibodies can use conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) to obtain an antigen-binding fragment of the antibody (e.g., the above-mentioned antibody fragment) from a given antibody (e.g., an antibody provided by the present invention), and screen the antigen-binding fragment of the antibody for specificity in the same manner as for intact antibodies.

[0368] As used herein, the terms "monoclonal antibody", "single antibody", and "mAb" have the same meaning and are used interchangeably, and refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, relative to monoclonal antibodies, generally contain at least two or more different antibodies, and these different antibodies generally recognize different epitopes on an antigen. In addition, the modifier "monoclonal" only indicates that the antibody is characterized as being obtained from a highly homologous antibody group, and it should not be understood that the antibody needs to be prepared by any specific method.

[0369] The monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256:495, 1975), recombinant DNA technology (see, for example, U.S. Patent Application 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352:624-628, 1991, or Marks et al. J. Mol. Biol. 222:581-597, 1991).

[0370] For example, monoclonal antibodies can be prepared as follows. First, mice or other suitable host animals are immunized with immunogens (adjuvants are added when necessary). The injection method of immunogens or adjuvants is usually subcutaneous multi-point injection or intraperitoneal injection. The immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitor, to enhance the immunogenicity of the antigen in the host. The adjuvant can be Freund's adjuvant or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies that specifically bind to the immunogen will be produced in the body. In addition, lymphocytes can also be obtained by in vitro immunization. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion 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 for growth, and the culture medium preferably contains one or more substances that can inhibit the growth of unfused, parent myeloma cells. For example, for maternal myeloma cells lacking hypoxanthine guanine phosphotransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin and thymidine (HAT culture medium) to the culture medium will inhibit the growth of HGPRT-deficient cells. Preferred myeloma cells should have characteristics such as high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among them, myeloma cells are preferably mouse myeloma, such as MOP-21 or MC-11 mouse tumor derivatives (THE Salk Institute Cell Distribution Center, San Diego, Calif. USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md. USA). In addition, there are also studies reporting the use of human myeloma and human-mouse heteromyeloma cell lines to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987). The culture medium of 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) and enzyme-linked immunosorbent assay (ELISA).For example, the affinity of monoclonal antibodies can be determined by the Scatchard analysis method described by Munson et al. in Anal. Biochem. 107: 220 (1980). After determining the specificity, affinity and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned by the standard limiting dilution method described in (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). Suitable culture fluids can be DMEM or RPMI-1640, etc. In addition, hybridoma cells can also grow in animals in the form of ascites tumors. Using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by subcloned cells can be separated from cell culture fluid, ascites or serum.

[0371] Monoclonal antibodies can also be obtained through genetic engineering recombination technology. Using nucleic acid primers that specifically bind to the heavy chain and light chain genes of the monoclonal antibody for PCR amplification, DNA molecules encoding the heavy chain and light chain genes of the monoclonal antibody can be isolated from hybridoma cells. The obtained DNA molecules are inserted into expression vectors, and 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 to obtain recombinantly expressed target antibodies.

[0372] Antibodies can be purified by known techniques, such as affinity chromatography using protein A or protein G. Specific antigens (target molecules recognized by the antibody) or their antigenic epitopes can be immobilized on a column and immunoaffinity chromatography can be used to purify immunospecific antibodies. Purification of immunoglobulins can be found, for example, in D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).

[0373] As used herein, the term "murine antibody" refers to an antibody obtained by fusing B cells of immunized mice with myeloma cells, followed by screening for mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, and then performing screening, antibody preparation and antibody purification; or, after antigens invade the mouse body, B cells differentiate and proliferate to form plasma cells, which can produce secreted antibodies.

[0374] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the binding activity to the target antigen (US Patent 4,816,567 to Cabilly et al.; Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy chain and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0375] As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc. The donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response).

[0376] Humanized antibodies can retain the expected properties of non-human donor antibodies (e.g., mouse antibodies) and can effectively reduce the immunogenicity of non-human donor antibodies (e.g., mouse antibodies) in human subjects, and therefore are particularly advantageous. However, due to the matching problem between the CDR of the donor antibody and the FR of the acceptor antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and / or ability to enhance immune response) are usually lower than those of non-human donor antibodies (e.g., mouse antibodies).

[0377] Therefore, although researchers in this field have conducted in-depth studies on antibody humanization and have made some progress (see, for example, 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 so that the generated humanized antibody has the highest possible degree of humanization and can retain the expected properties of the donor antibody as much as possible. Technicians need to explore, investigate and modify specific donor antibodies and put in a lot of creative work to obtain humanized antibodies that have a high degree of humanization (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% humanization) and retain the expected properties of the specific donor antibodies.

[0378] In the present invention, in order to make the humanized antibody retain the properties of the donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, the ability to increase the activity of immune cells and / or the ability to enhance the immune response), the framework region (FR) in the humanized antibody of the present invention may contain both the amino acid residues of the human acceptor antibody and the amino acid residues of the corresponding non-human donor antibody.

[0379] Chimeric antibodies or humanized antibodies of the present invention can be prepared based on the sequence of the mouse monoclonal antibody prepared above. DNA encoding heavy and light chains can be obtained from the target mouse hybridoma and engineered to contain non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques.

[0380] To prepare chimeric antibodies, methods known in the art can be used to link the murine immunoglobulin variable region to the human immunoglobulin constant region (see, e.g., U.S. Pat. No. 4,816,567 to 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 sequence of the human heavy chain constant region gene is known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, 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 heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is generally preferably an IgG1 or IgG4 constant region. For example, the DNA encoding 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 Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0381] To prepare humanized antibodies, mouse CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. No. 5,225,539; Queen et al., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be used that are capable of not producing endogenous immunoglobulins after immunization and are capable of producing a complete human antibody repertoire. For example, it has been reported that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can completely inhibit endogenous antibody production, and then transfer of the human germ-line immunoglobulin gene array into the germ-line mutant mice will result in the mice producing human antibodies when challenged with antigen (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 transgenic animals include, HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g., Lonberg et al. (1994) Nature 368 (6474): 856-859); or "KM mice TM" carrying human heavy chain transgenes and human light chain transchromosomes (see patent application WO02 / 43478). Other methods for antibody humanization 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).

[0382] As used herein, the term "humanization degree" is an indicator for evaluating the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted, for example, by using the IMGT website DomainGapAlign to predict the homology between the variable region sequence and the human V domain.

[0383] As used herein, the term "homologous antibody" refers to a variant of an antibody comprising heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the antibodies or antigen-binding fragments thereof provided herein, and wherein the variant retains the desired functional properties of the anti-FXI and / or FXIa antibodies of the present invention.

[0384] Sequence alignment methods for comparison are well known in the art. Various programs 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 provide detailed ideas for sequence alignment methods and homology calculations.

[0385] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50) of the interaction.

[0386] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the speed of formation and dissociation of the antigen binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, bioluminescence interferometry (e.g., ForteBio Octet method) can be used to measure the dissociation constant. In addition, surface plasmon resonance technology (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0387] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries 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 (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0388] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression regulatory sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0389] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0390] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0391] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of the protein / polypeptide comprising the amino acid sequence, and the variant of the antibody obtained by conservatively substituting the amino acid fully retains the biological activity of its source sequence, such as specifically 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 that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with 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, proline, 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).

[0392] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0393] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers 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. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption 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 glycerol), 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 have the meanings commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0394] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a disease or disorder associated with coagulation or thromboembolism) in a subject. As used herein, the term "treatment" refers to a method implemented in order 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, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, alleviating symptoms (whether partially or completely), alleviating or improving prognosis, reducing or inhibiting disease recurrence, etc., whether detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival period (if not treated), prolonging the survival period.

[0395] 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 a disease or disorder associated with coagulation or thromboembolism, or has a risk of suffering from the above-mentioned disease.

[0396] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a disease or condition associated with coagulation or thromboembolism) refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease (e.g., a disease or condition associated with coagulation or thromboembolism); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0397] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody (a native sequence Fc region or an amino acid sequence variant Fc region), and varies with 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), downregulation of cell surface receptors (e.g., B cell receptors), B cell activation, cytokine secretion, half-life / clearance rate of antibodies and antigen-antibody complexes, and the like. Methods for altering the effector functions of antibodies are known in the art, for example, by introducing mutations in the Fc region.

[0398] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which immunoglobulins bind to Fc receptors (FcRs) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), causing these cytotoxic effector cells to specifically bind to target cells attached to antigens, and then kill the target cells by secreting cytotoxins. Methods for detecting the ADCC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the test antibody and an Fc receptor (e.g., CD16a).

[0399] As used herein, the term "complement dependent cytotoxicity (CDC)" refers to a form of cytotoxicity that activates the complement cascade by binding complement component C1q to antibody Fc. Methods for detecting the CDC activity of antibodies are known in the art, and can be evaluated, for example, by measuring the binding activity between the test antibody and an Fc receptor (e.g., C1q).

[0400] As used herein, the term "FXI and / or FXIa-mediated" refers to the fact that FXI and / or FXIa mediate the activation of 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.

[0401] As used herein, the term "disease or condition associated with coagulation or thromboembolism" or similar terms refers to a condition or disease caused by abnormal activation of the coagulation pathway or by a non-natural inactivation (e.g., without therapeutic means) of the coagulation pathway. These conditions include, but are not limited to, thrombosis, thrombotic stroke, atrial fibrillation, atrial fibrillation associated stroke prevention (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 artery intervention, peripheral arterial occlusive disease (PAOD), renal vein thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, disseminated intravascular coagulation, medical device (e.g., catheter)-induced thromboembolic disorders, severe systemic inflammatory response syndrome, metastatic cancer, infectious diseases, organ failure (e.g., renal failure), toxicity caused by in vivo administration of therapeutic proteins, multiple trauma, ischemia-reperfusion injury, localized fibrin deposition, pulmonary alveolar proteinosis in adults, thromboembolic events (VTE) before and after total knee arthroplasty (TKA) surgery, coronary artery 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 in patients undergoing adventitial membrane oxygenation, deep vein thrombosis (DVT), or pulmonary embolism (PE).

[0402] Catheter-induced thromboembolic disorders involve thromboembolism that forms in catheters (e.g., Hickman catheters in cancer patients and clots that may form during extracorporeal membrane oxygenation (ECMO)).

[0403] Herein, the expression "preventing and / or treating a disease or disorder related to blood coagulation or thromboembolism" may refer to one or more of the following conditions that the anti-FXI and / or FXIa antibodies or antigen-binding fragments thereof of the present invention can be used to prevent or treat:

[0404] -Thromboembolism in individuals with suspected or confirmed cardiac arrhythmias, such as paroxysmal, persistent or permanent atrial fibrillation or atrial fibrillation;

[0405] - Stroke Prevention in Atrial Fibrillation (SPAF), with a subpopulation of AF patients undergoing percutaneous coronary intervention (PCI);

[0406] - Treatment of acute venous thromboembolic events (VTE) and long-term secondary VTE prevention in patients at high risk of bleeding;

[0407] - Cerebral and cardiovascular events in the secondary prevention of thromboembolic events after transient ischemic attack (TIA) or non-disabling stroke and in heart failure complicated by sinus rhythm;

[0408] - Clot formation and thromboembolism in the left atrium of individuals undergoing cardioversion for cardiac arrhythmia;

[0409] -Thrombosis before, during and after arrhythmogenic resection procedures;

[0410] - venous thrombosis, including (but not excluding) the treatment and secondary prevention of lower or upper deep or surface venous thrombosis, thrombosis of the abdominal and thoracic veins, sinus thrombosis and jugular vein thrombosis;

[0411] - thrombus on any artificial surface of a venous catheter or pacemaker lead;

[0412] - Pulmonary embolism in patients with or without venous thrombosis;

[0413] - Chronic thromboembolic pulmonary hypertension (CTEPH);

[0414] - Arterial thrombi on ruptured atherosclerotic plaques, thrombi on intra-arterial devices or catheters, and thrombi in apparently normal arteries, such conditions including (but not limited to) acute coronary syndrome, ST-elevation myocardial infarction, non-ST-elevation myocardial infarction, unstable angina, stent thrombosis, thrombi on any artificial surface in the arterial system, and thrombi in the pulmonary arteries of individuals with or without pulmonary hypertension;

[0415] - Thrombosis and thromboembolism in patients undergoing percutaneous coronary intervention (PCI);

[0416] -Psychogenic and cryptogenic stroke;

[0417] -Thrombosis in patients with invasive and non-invasive cancer malignancies;

[0418] - thrombosis on indwelling catheters;

[0419] -Thrombosis and thromboembolism in patients with serious illness;

[0420] - Cardiac thrombus and thromboembolism, including (but not exclusively) cardiac thrombus following myocardial infarction, cardiac thrombus associated with pathologies such as cardiac aneurysms, myocardial fibrosis, cardiac enlargement and dysfunction, myocarditis and artificial surfaces in the heart;

[0421] -Thromboembolism in patients with valvular heart disease with or without atrial fibrillation;

[0422] - Thromboembolism on the mechanical or biological valve device;

[0423] -Thromboembolism in patients with natural or artificial cardiac patches, arterial or venous conduits following cardiac repair for simple or complex cardiac malformations;

[0424] - Venous thrombosis and thromboembolism following knee replacement, hip replacement, orthopedic, chest or abdominal surgery;

[0425] - Arterial or venous thrombosis following neurosurgery including intracranial and spinal cord interventions;

[0426] - congenital or acquired thrombotic tendencies, including (but not exclusively) factor V Leiden, prothrombin mutation, antithrombin III, protein C and protein S deficiency, factor XIII mutation, familial dysfibrinogenemia, congenital plasminogen deficiency, increased factor XI, sickle cell disease, antiphospholipid syndrome, autoimmune diseases, chronic intestinal disease, nephrotic syndrome, hemolytic uremic disease, myeloproliferative disease, disseminated intravascular coagulation, paroxysmal nocturnal hemoglobinuria, and heparin-induced thrombocytopenia;

[0427] -Thrombosis and thromboembolism in chronic kidney disease; and

[0428] -Thrombosis and thromboembolism in patients undergoing hemodialysis and in patients subjected to adventitial membrane oxidation.

[0429] As used herein, the term "pharmaceutically acceptable" means that when the molecular entity, molecular fragment or composition is appropriately administered to an animal or a human, they do not produce adverse, allergic or other adverse reactions. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or their components include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0430] As used herein, combination therapy includes the use of an anti-FXI and / or FXIa antibody or antigen-binding fragment thereof encompassed by the invention in combination with one or more additional active therapeutic agents of a second therapy (e.g., a chemotherapeutic agent) or other prophylactic or therapeutic modality (e.g., an antiplatelet drug, an anticoagulant drug, a thrombolytic drug).

[0431] Exemplary antiplatelet drugs for the second therapy are selected from aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, or any combination thereof.

[0432] Exemplary anticoagulant drugs for the second therapy are selected from unfractionated heparin, heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, or any combination thereof.

[0433] An exemplary thrombolytic drug for the second therapy is selected from streptokinase, urokinase, alteplase or prourokinase.

[0434] In such combination therapies, the various active agents often have 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 to reduce or eliminate adverse effects associated with one or more of the agents. Such combination therapies may have a synergistic therapeutic or preventive effect on the underlying disease, disorder, or condition.

[0435] As used herein, "combination" includes therapies that can be administered separately, e.g., separately formulated for separate administration (e.g., can be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a "co-formulation"). In certain embodiments, the anti-FXI and / or FXIa antibodies or antigen-binding fragments thereof of the present invention can be administered sequentially. In other embodiments, the anti-FXI and / or FXIa antibodies or antigen-binding fragments thereof can be administered simultaneously. The anti-FXI and / or FXIa antibodies or antigen-binding fragments thereof of the present invention can be used in combination with at least one other (active) agent in any manner.

[0436] As used herein, the term "about" means plus or minus 10% of the numerical value recited herein.

[0437] Chinese grammar does not correspond to the singular and plural rules of English, so when the present disclosure is translated into English, "one or more" may be added before the noun.

[0438] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0439] Figure 1 :APTT activity detection of hybridoma supernatant

[0440] Figure 2A-2B :Detection of coagulation time of anti-human FXI / FIXa mouse monoclonal antibody by APTT method

[0441] Figure 3 :Detection of anticoagulant function of anti-human FXI / FXIa mouse monoclonal antibody by BIOPHEN method

[0442] Figure 4 :Anti-human FXI / FXIa mouse monoclonal antibody specifically blocks the biological activities of FXI and FXIa

[0443] Figure 5 :APTT anticoagulant activity test of humanized anti-human FXI / FXIa antibody

[0444] Figure 6 :Detection of anticoagulant function of humanized monoclonal antibody against human FXI / FXIa by BIOPHEN method Fig. 7A :ELISA to detect the affinity of anti-human FXI / FXIa humanized antibody to FXIa

[0445] Figure 7B :ELISA to detect the affinity of anti-human FXI / FXIa humanized antibody to FXI

[0446] Figure 8 :Anti-human FXI / FXIa humanized antibody competes with control antibody BAY-1213790 for binding to FXIa detection

[0447] Fig. 9A :Detection of anticoagulant activity of anti-human FXI / FXIa humanized antibody in human blood by measuring activated partial thromboplastin time (APTT)

[0448] Fig. 9B :Detection of anticoagulant activity of humanized anti-human FXI / FXIa antibody in monkey blood by measuring activated partial thromboplastin time (APTT)

[0449] Fig. 9C :Detection of anticoagulant activity of humanized anti-human FXI / FXIa antibody in canine blood by measuring activated partial thromboplastin time (APTT)

[0450] Fig.9D :Detection of anticoagulant activity of anti-human FXI / FXIa humanized antibody in rabbit blood by measuring activated partial thromboplastin time (APTT)

[0451] Fig.9E : Detection of the anticoagulant activity of anti-human FXI / FXIa humanized antibody in rat blood by measuring activated partial thromboplastin time (APTT)

[0452] Fig.10 :Accelerated stability testing of humanized anti-human FXI / FXIa antibodies

[0453] Fig.11A :Pharmacodynamics (PD) and APTT activity assay of humanized anti-human FXI / FXIa antibody in cynomolgus monkeys

[0454] Fig. 11B :Pharmacodynamic (PD) PT activity assay of humanized anti-human FXI / FXIa antibody in cynomolgus monkeys

[0455] Sequence information

[0456] The information of the sequences involved in the present invention is described in the following table:

[0457]

[0458] DETAILED DESCRIPTION

[0459] The present invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit the scope of the invention.

[0460] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0461] Example 1. Preparation of anti-human FXI / FXIa mouse monoclonal antibody

[0462] Wild-type Balb / c mice were immunized with protein to obtain anti-human FXI / FXIa mouse monoclonal antibodies. Each Balb / c mouse was subcutaneously injected with CFA (complete Freund's adjuvant, manufacturer: Sigma, catalog number F5506) emulsified with 25μg human FXIa (manufacturer: Haematologic Technologies, catalog number HCXI-0150) and 25μg human FXI (manufacturer: Haematologic Technologies, catalog number HCXI-0160) for the first immunization, and boosted immunization was performed every two weeks. The boosted immunization was emulsified with IFA (incomplete Freund's adjuvant, manufacturer: Sigma, catalog number: F5881). After three immunizations, the serum titer was detected by ELISA. The boosted immunization was performed 3-5 days before fusion, and mice with higher titers were selected and immunized intraperitoneally with 10μg FXIa and 10μg FXI. Mouse spleen cells and Sp2 / 0-Ag14 (ATCC, Cat#CRL-1581) mouse myeloma cells were fused by PEG fusion using the standard fusion process, followed by HAT (manufacturer: Sigma, catalog number: H0262-10VL) pressure screening, and ELISA screening was performed 14 days later. Specific ELISA screening method: 0.5μg / mL biotin-labeled FXI (manufacturer: Haematologic Technologies, catalog number: HCXI-0150-B) 100μL / well, coated with ELISA plate (manufacturer: Thermo Fisher Sci., catalog number: 5129) at room temperature for 1 hour. Wash 3 times with 200μL washing buffer (1×TBS containing 0.05% Tween-20), add 100μL hybridoma supernatant, and incubate at 37°C for 1 hour. Wash three times with 200 μL of washing buffer, add 100 μL of Streptavidin-HRP (Pierce, Cat#21130) diluted at a ratio of 1:8000 and incubate at 37°C for 1 hour. Wash three times with 200 μL of washing buffer, add 100 μL of TMB (purchased from Thermo Fisher Sci., Cat#TMBW-1000-01) to develop color in the dark for 10 minutes, then add 100 μL of stop solution (purchased from Thermo Fisher Sci., Cat#13361-100-10), and read at 450 nm on a microplate reader.

[0463] The supernatants of 24,000 hybridoma clones were screened by ELISA, and 100 hybridoma clones that could recognize biotin-labeled FXI were obtained and transferred to 24-well plates. After 7-10 days, the hybridoma supernatants were tested by APTT functional assay. The best 22 clones were subcloned by limiting dilution to obtain monoclonal hybridomas. The monoclonal hybridomas were tested for anticoagulant activity by APTT functional assay, and the activated partial thromboplastin time (APTT) kit (Activated Partial Thromboplastin Time Kit, manufacturer: Thermo Fisher Sci, catalog number 100402) was used for detection. The specific steps are as follows: add 100 μL normal human plasma (purchased from Innovative Research, Cat#IPLA-N) to the preheated test tube, then add 100 μL APTT reagent (purchased from Thermo Fisher Sci. Cat#100402TS) and 100 μL test sample to mix, incubate at 37°C for 5 minutes, then add 100 μL calcium chloride (purchased from Thermo Fisher Sci. Cat#100304, 20mM), measure OD405 readings, calculate the coagulation time by curve fitting, and use Sp2 / 0-Ag14 cell supernatant as negative control. 14E11 is the positive control antibody (prepared with reference to Aronora patent US8388959B2). The multiple value in the APTT test result is the ratio of the coagulation time measured by the sample with the experimental antibody to the coagulation time measured by the control sample without the antibody. 1 times or less indicates that the coagulation time is not delayed or accelerated, and greater than 1 times indicates that the coagulation time is prolonged.

[0464] like Figure 1 As shown, APTT tested a total of 5 subclone hybridoma supernatants, of which 36G9.10 prolonged the APTT time, had significant anticoagulant function, and could be used for further analysis, while the other clones 6B6.9, 28A8.3, 36C3, and 40F6.9 had similar prolongation of coagulation time compared to the negative control. The hybridoma 7B2 with prolonged APTT duration was obtained by the same screening for further analysis.

[0465] The monoclonal hybridomas 36G9.10 and 7B2 were expanded to 100-150 mL without serum. The supernatant was purified by Protein G filler, and the purified mouse antibodies were detected by HPLC-SEC, with a purity greater than 97%. The purified mouse antibodies can be used for further functional verification.

[0466] Example 2. Functional identification of anti-human FXI / FXIa mouse monoclonal antibodies

[0467] 2.1 Anti-human FXI / FXIa mouse antibody prolonged coagulation function test

[0468] The APTT detection method in Example 1 was used to compare 36G9.10 with the positive control antibodies BAY-1213790 (prepared with reference to M007-H04 in Bayer patent WO2013167669) and 14E11 when the antibody was diluted to concentrations of 2.00, 1.00, 0.50 and 0.25 μg / mL, and PBS buffer was used as a negative control.

[0469] like Figure 2A As shown in Figure 2, the candidate mouse antibody 36G9.10 significantly prolonged the coagulation time at all concentrations compared with the control antibodies BAY-1213790 and 14E11. At an antibody concentration of 5 μg / mL, 7B2 was compared with the positive control antibody 14E11, and PBS buffer was used as a negative control. The results are shown in Figure 2. Figure 2B As shown, the candidate mouse antibody 7B2 significantly prolonged the clotting time relative to PBS, which was comparable to the prolongation of the clotting time by the positive control antibody 14E11.

[0470] 2.2 Determination of the inhibitory activity of anti-human FXI / FXIa mouse antibodies on the production of FXa catalyzed by FXIa and its anticoagulant function

[0471] The BIOPHEN Factor XIa kit (Hyphen BioMed, catalog number 220412) was used for testing according to the kit instructions, and the release of para-nitroaniline (pNA) product was measured by OD405nm to further determine the activity of anti-FXI / FXIa antibodies in inhibiting the production of FXa catalyzed by FXIa. The detection concentration of all antibodies was 0.25 μg / mL. The stronger the anti-FXI / FXIa antibody's function in blocking FXI / FXIa activity, the lower the OD405nm signal value of the pNA product.

[0472] The results are as follows Figure 3 As shown, the 36G9.10 mouse antibody has significantly stronger anticoagulant function than the control antibodies BAY-1213790 and 14E11.

[0473] 2.3 Anti-human FXI / FXIa mouse monoclonal antibody affinity test

[0474] Octet It is widely used for the detection of dynamic affinity between antibodies and antigens. The candidate mouse antibody 36G9.10 and the control antibodies BAY1213790 and 14E11 were tested for their dynamic affinity with FXIa using this method. The specific experimental steps are as follows: the streptavidin probe first binds to the biotin-labeled antibody to be tested to a response signal value of 0.8nm, then binds to FXIa protein (3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05 and 0μg / mL) for 5 minutes, then dissociates for 7 minutes, and uses a bivalent analysis model and full fitting analysis.

[0475] As shown in Table 1, 36G9.10 has a faster binding rate than control antibodies 14E11 and BAY-1213790 (as shown by the Kon value), but a slower dissociation rate than BAY-1213790 (as shown by the Kdis value), and its affinity is 1.7 and 2.5 times stronger than that of control antibodies 14E11 and BAY-1213790, respectively.

[0476] Table 1. Dynamic affinity determination of anti-FXI / FXIa mouse antibodies to FXIa

[0477] Antibody Name KD(M) Kon(1 / Ms) Kdis(1 / s) <![CDATA[R 2 ]]> 14E11 8.03E-10 7.65E+05 6.14E-04 0.9889 BAY-1213790 1.19E-09 9.18E+05 1.09E-03 0.9786 36G9.10 4.80E-10 1.50E+06 7.20E-04 0.9831

[0478] 2.4 Anti-human FXI / FXIa mouse antibodies specifically block the biological activities of FXI and FXIa

[0479] To confirm that the mouse antibody 36G9.10 specifically blocks the biological activity of FXI and FXIa, human plasma lacking FXI (purchased from Innovative Research, catalog number: 50-643-396) was used to measure the APTT clotting time by adding 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).

[0480] The results are as follows Figure 4 As shown, the addition of FXI or FXIa significantly shortened the coagulation time of FXI-deficient human plasma in a dose-dependent manner. When the 36G9.10 mouse antibody was added, the coagulation time of FXI and FXIa was significantly prolonged, indicating that the mouse antibody 36G9.10 specifically inhibited the biological activity of FXI and FXIa.

[0481] Example 3. Identification of subtypes and variable region amplification of anti-human FXI / FXIa mouse antibodies

[0482] In order to detect the antibody subtype of the candidate hybridoma clones, the Pierce Rapid Isotyping Kit (purchased from Thermo Fisher Sci.Cat#26179) was used to identify the antibody subtype of the candidate clones 36G9.10 and 7B2. The identification results showed that the heavy chain of the candidate clones was IgG1 subtype and the light chain was Kappa subtype.

[0483] When the number of hybridoma cells reached about 8,000, the cells were lysed and the first-strand cDNA was synthesized using a cDNA reverse transcription kit (purchased from ThermoFisher Sci.Cat#18080-200). VH and VK genes were amplified from cDNA by PCR using specific primers. The PCR products were purified using a DNA purification kit (purchased from Qiagen, Cat#28104) and ligated to a TOPO vector (purchased from Thermo Fisher Sci.Cat#K457540). About 12 clones were selected for sequencing for each ligation reaction. The sequence was analyzed by Vector NTI 11.5 (purchased from Thermo Fisher Sci.) and Sequencer 5.4.6 (purchased from Genecodes), and the variable region sequence and CDR sequence of the anti-FXI / FXIa mouse antibody were obtained as shown in the sequence information table, among which the heavy chain variable region of the 36G9.10 mouse antibody was SEQ ID NO: 1, and the light chain variable region was SEQ ID NO: 2; the heavy chain variable region of the 7B2 mouse antibody was SEQ ID NO: 29, and the light chain variable region was SEQ ID NO: 30. Furthermore, chimeric antibodies 36G9.10-hz00 and 7B2-hz00 of 36G9.10 and 7B2 were constructed respectively, which were obtained by grafting the heavy chain variable region sequence of 36G9.10 or 7B2 to the mutant human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21), and grafting the light chain variable region sequence of 36G9.10 or 7B2 to the human Kappa light chain constant region (SEQ ID NO: 22).

[0484] Example 4. Humanization of anti-human FXI / FXIa murine antibodies

[0485] The mouse antibody 36G9.10, 7B2 was humanized by using the CDR transplantation antibody humanization method. In short, the humanization involves the following steps: compare the amino acid sequence of the mouse monoclonal antibody with the amino acid sequence of the human embryonic antibody to find the sequence with high homology and better physical and chemical properties as the human embryonic framework sequence; analyze and examine the HLA-DR affinity and select the human embryonic framework sequence with low affinity; and then transplant the six CDRs of the mouse antibody to the selected heavy chain and light chain framework sequences.

[0486] Specifically, the heavy chain and light chain CDR regions of the murine antibodies 36G9.10 and 7B2 were transplanted onto the FR frameworks of the corresponding humanized templates. The heavy chain humanized template of 36G9.10 was the human germline gene sequence IGHV1-2*02 (see IMGT Accession No. X62106) and IGHV1-69-2*01 (see IMGT Accession No. KF698734), and the light chain humanized template was the human germline gene sequence IGKV1-33*01 (see IMGT Accession No. M64856) and IGKV1-16*01 (see IMGT Accession No. J00248). The heavy chain humanization template of 7B2 is the human germline gene sequence IGHV1-69-2*01 (see IMGT Accession No. KF698734), and the light chain humanization template is the human germline gene sequence IGKV1-39*01 (see IMGT Accession No. X59315).

[0487] Furthermore, computer simulation technology was used to apply molecular docking to analyze the variable region and its surrounding framework amino acid sequence to examine its spatial stereo binding mode. By calculating electrostatic force, van der Waals force, hydrophilicity and entropy value, the key amino acids in the amino acid sequence of the mouse antibody that can interact with coagulation factor XIa and maintain the spatial framework were analyzed, and these mouse amino acids were retained in the transplanted antibody. That is, a series of back mutations were performed on the amino acid residues in the FR region of the above humanized template so that the humanized antibody can retain the antigen binding ability of the mouse antibody as much as possible.

[0488] According to the above method, based on the CDR of the mouse antibody 36G9.10, a total of 5 humanized antibodies were constructed, named 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, and 36G9.10-hz93, respectively; wherein the heavy chain constant region of each antibody is a human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21). Based on the CDR of the mouse antibody 7B2, the humanized antibody 7B2-hz11 was constructed, and the heavy chain constant region is a mutant human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21). The light chain constant region sequences of antibodies 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz43, 36G9.10-hz92, 36G9.10-hz93 and 7B2hz11 are SEQ ID NO: 22, and none of them have ADCC and CDC effects.

[0489] The amino acid sequences of the variable and constant regions of the above humanized antibodies are shown in Table 2.

[0490] Table 2. Amino acid sequences of variable and constant regions of humanized anti-human FXI / FXIa antibodies

[0491]

[0492] Example 5. Affinity determination of anti-human FXI / FXIa antibodies

[0493] Using Octet Determine the affinity of chimeric and humanized antibodies. The main determination steps are: first, fix the chimeric and humanized antibodies (concentration of 0.3μg / mL) to the AHC (anti-human-Fc) sensor, then dilute FXI in a 1:2 gradient with a starting concentration of 3.2μg / mL to determine the binding and dissociation rates of chimeric and humanized antibodies. The obtained data were analyzed by Octet data analysis software.

[0494] The results are shown in Table 3, which show that the affinity KD of 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 are all stronger than that of the positive control BAY-1213790.

[0495] Table 3. Dynamic affinity determination of anti-human FXI / FXIa humanized antibodies binding to human FXI

[0496]

[0497] Example 6. Determination of APTT anticoagulant activity, inhibition of FXIa-catalyzed FXa production activity and anticoagulant function of anti-human FXI / FXIa chimeric and humanized antibodies

[0498] The anticoagulant activity of the humanized antibody was detected using the APTT coagulation test kit. The specific detection method is shown in Example 1. Figure 5 As shown, the mean value and standard deviation were calculated by 4 measurements, and the coagulation prolongation time of the chimeric antibody 36G9.10-hz00, humanized antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, and 36G9.10-hz93 were all greater than those of the positive control antibodies 14E11 and BAY-1213790.

[0499] The anticoagulant activity of the humanized antibody was detected using the Biophen Factor XIa kit (purchased from Aniara Cat#220412). The specific method is shown in Example 2.

[0500] Test results such as Figure 6As shown, chimeric antibody 36G9.10-hz00, humanized antibody 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93, antibody 14E11 and BAY-1213790 can all effectively reduce the production of FXa. Antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz92 and 36G9.10-hz93 are more effective in reducing the production of FXa than control antibodies 14E11 and BAY-1213790, indicating that they have superior anticoagulant activity.

[0501] Example 7. Affinity determination of anti-human FXI / FXIa humanized antibody and FXI / FXIa

[0502] The affinity of 36G9.10-hz73 to human FXIa was detected by ELISA. The specific 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, dissolved in deionized water, and fixed to 200 ml), 100 μL of FXIa antigen was coated on each well, and incubated at 4°C overnight; the next day, the liquid in the wells was discarded, and the wells were washed once with 300 μL PBS; 100 μL PBS (containing 2% BSA, BOVOGEN, BSAS1.0) was added, and the wells were blocked at 37°C for 2 hours; PBS (containing 2% 36G9.10-hz73 and BAY-1213790 antibodies (starting at 10 μg / mL, 4-fold dilution, 12 concentration points) were diluted with PBS (containing 2% BSA), 100 μL was added to the corresponding wells, and incubated at 37°C for 2 hours; 300 μL PBST was used to wash 3 times; HRP-labeled goat anti-human secondary antibody (purchased from Jackson, 109-035-00) was diluted at a ratio of 1:10000 with PBS (containing 2% BSA), 100 μL was added to the corresponding wells, and incubated at 37°C for 1 hour; 300 μL PBST was used to wash 5 times; 100 μL TMB color development solution (purchased from BioPanda, TMB-S-004) was added to the corresponding wells, and color was developed at room temperature for 20 minutes; 50 μL 2N H2SO4 was added to terminate the reaction, and the OD450 nm readings were read by an enzyme reader (purchased from MD, SpectraMax M2) and imported into Graphpad Prism for curve fitting.

[0503] The experimental results are as follows Fig. 7AAs shown, the EC50 of affinity between 36G9.10-hz73 and FXIa is 2.488 ng / mL, and the EC50 of affinity between BAY-1213790 and FXIa is 6.163 ng / mL. 36G9.10-hz73 is superior to BAY-1213790.

[0504] The affinity of 36G9.10-hz73 to human FXI was detected by ELISA. The FXI antigen (PMD-F11 plasmid purchased from Sino Biological, cloned into PLVX-IRES-PURO vector and transfected into 293F cells for expression) was diluted to 1 μg / mL with CBS coating solution, and 100 μL was coated overnight at 4°C in each well. The remaining steps were as described above. The starting concentration of the antibody was 0.37 μg / mL, 3-fold dilution, and 9 concentration points.

[0505] The experimental results are as follows Figure 7B As shown, the affinity EC50 of 36G9.10-hz73 to FXI is 9.68 ng / mL, and BAY-1213790 does not bind to FXI. 36G9.10-hz73 neutralizes both FXI and FXIa, and can exert better anticoagulant function.

[0506] Example 8. Anti-human FXI / FXIa antibody competes with control antibody for binding to FXIa detection

[0507] In order to determine whether 36G9.10-hz73 recognized the same epitope of FXIa as BAY-1213790 and 14E11, a competitive ELISA method was used for detection. The specific steps are as follows: dilute FXIa antigen to 1 μg / mL with CBS coating solution, coat each well with 100 μL of FXIa antigen, and incubate at 4°C overnight; discard the liquid in the wells the next day, wash once with 300 μL PBS; add 100 μL PBS (containing 2% BSA) and block at 37°C for 2 hours; dilute biotin-labeled 36G9.10-hz73 antibody to 10 ng / mL with PBS (containing 2% BSA), and use this as the mother solution to dilute BAY-1213790 and 14E11 antibodies to 10 μg / mL, take 100 μL and add it to the corresponding wells, and use 10 μg / mL 36G9.10-hz73 antibody as a positive control, and use only 10 ng / mL biotin-labeled 36G9.10-hz73 antibody as a negative control, and set up a blank control, incubate at 37°C for 2 hours; wash 3 times with 300 μL PBST; use PBS (containing 2% HRP-Streptavidin secondary antibody (purchased from Proteintech, SA00001-0) was diluted with BSA at a ratio of 1:3000, and 100 μL was added to the corresponding wells and incubated at 37°C for 1 hour; 300 μL PBST was used for washing 5 times; 100 μL TMB color development solution (purchased from BioPanda, TMB-S-004) was added to the corresponding wells and color developed at room temperature for 20 minutes; 50 μL 2N H2SO4 was added to terminate the reaction, and the OD450 nm reading was performed with an enzyme reader (purchased from MD, SpectraMaxM2) and imported into Graphpad Prism for plotting.

[0508] The experimental results are as follows Figure 8 As shown, BAY-1213790 and 14E11 did not compete with 36G9.10-hz73 for binding to FXIa, indicating that the former two and 36G9.10-hz73 bind to different epitopes of FXIa.

[0509] Example 9. Anti-human FXI / FXIa antibodies inhibit the catalytic effect of FXIa on substrates

[0510] In 1988, Shun-ichiro KAWABATA et al. screened several specific fluorescent-labeled substrates for FXIa for sensitive in vitro detection of human FIXa enzyme activity (see Eur. J. Biochem. 172, 17-25 (1988)). Therefore, the activity of human FXIa and the effective neutralization of anti-human FXI / FXIa humanized antibodies on FXIa were determined by detecting the catalytic cleavage of human FXIa on specific fluorescent substrates (I-1575, Bachem). The specific steps are as follows: dilute human FXIa to 1 nM with a buffer containing 50 mM Tris / HCl, 100 mM NaCl, 5 mM CaCl2 and 0.1% BSA, and use this as the mother solution to gradiently dilute 36G9.10-hz73 and BAY-1213790 antibodies, starting at 20 μg / mL, 4-fold dilution, and 14 concentration points; take 10 μL of the diluted antibody and add it to the corresponding wells of a 384-well plate (purchased from Coring, 4514) per well, and incubate at 37°C for 1 hour; after incubation, add 10 μL of a 2 μM fluorescent substrate I-1575 to each well, mix well, and immediately put it on the machine, use an enzyme reader to continuously read the fluorescence values ​​at 360 / 465 nm, and import them into Graphpad Prism for curve fitting.

[0511] The experimental results are shown in Table 4. The 36G9.10-hz73 antibody can effectively inhibit the catalytic effect of FXIa on the fluorescent substrate, with an IC50 of 26.215 ng / mL, and the BAY-1213790 IC50 of 69.96 ng / mL. 36G9.10-hz73 is superior to BAY-1213790.

[0512] Table 4. Anti-human FXI / FXIa humanized antibodies inhibit the catalytic effect of FXIa on fluorescent substrates

[0513] Antibody Name IC50 value (ng / mL) 36G9.10-hz73 26.215±3.365(n＝2) BAY-1213790 69.96±7.44(n＝2)

[0514] Example 10. Detection of anticoagulant activity of anti-human FXI / FXIa antibodies by measuring activated partial thromboplastin time (APTT)

[0515] After adding APTT reagent to anticoagulated plasma, the intrinsic coagulation pathway is initiated and XI is activated to XIa. Antibodies targeting FXIa can inhibit the activity of FXIa, thereby prolonging APTT. Therefore, APTT is used to detect the anticoagulant activity of anti-human FXI / FXIa humanized antibodies. The specific steps are as follows: venous blood is collected from humans, monkeys, dogs, rabbits, and rats, 2 to 3 samples are collected for each species, 4 tubes of blood samples (1.8 ml per tube) are collected for each sample, anticoagulated with 3.2% sodium citrate, centrifuged at 2500×g and 4°C for 10 minutes, and plasma is collected and stored at 4°C for later use (the plasma of each sample is combined); the antibody to be tested is diluted with normal saline, starting at 1500μg / mL, and 3 concentration points are used as 10× the mother solution of the antibody to be tested; at high concentrations, the sample detection system is 150μl, 135μl of plasma is taken, and then 15μl of the mother solution of the antibody to be tested is added (that is, the mother solution of the antibody to be tested is diluted 10 times), and then the APTT is detected using a fully automatic coagulation analyzer (purchased from Sysmex CA1500). At low concentration, the sample detection system is 75 μl. Take 67.5 μl of plasma, add 7.5 μl of the antibody stock solution to be tested (ie, the antibody stock solution to be tested is diluted 10 times), and then use a fully automatic coagulation analyzer to detect APTT.

[0516] The experimental results are as follows Fig. 9A As shown in the figure, the four tested antibodies 36G9.10-hz73, BAY-1213790, BMS-962212 (prepared by reference to J Med Chem. 2017 Dec 14; 60(23):9703-9723.doi:10.1021 / acs.jmedchem.7b01171.Epub 2017 Nov 17), and 14E11 can prolong APTT in human plasma. 36G9.10-hz73 has the best efficacy, and 6 μg / mL can prolong APTT by 2.2 times, which is better than the other three. Figure 9B-9E As shown, 36G9.10-hz73 only prolonged the APTT clotting time in monkey plasma, while 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 the species cross-reactivity of 36G9.10-hz73 is different from that of BAY-1213790 and 14E11. It is speculated that the antibody epitope of 36G9.10-hz73 binding to FXI / FXIa is different from that of BAY-1213790 and 14E11.

[0517] Prothrombin time (PT) test mainly reflects the status of the exogenous coagulation system. The multiple value in the PT test result is the ratio of the coagulation time measured by the sample with the experimental antibody added to the coagulation time measured by the control sample without the antibody added. 1 times or less indicates that the coagulation time is not delayed or accelerated, and more than 1 times indicates that the coagulation time is prolonged.

[0518] The same treatment method as above was used to detect the effects of 36G9.10-hz73, BAY-1213790, BMS-962212, and 14E11 on PT using a fully automatic coagulation analyzer. The test results are shown in Table 5. All samples in the plasma of 5 species, compared with PBS, did not prolong PT (expressed in multiples) at a concentration of 150 μg / mL (1 μM), indicating that it had no effect on exogenous coagulation and did not increase the risk of bleeding.

[0519] Table 5. PT activity detection of anti-human FXI / FXIa humanized antibody

[0520] Antibody Name people monkey dog rabbit Rat 36G9.10-hz73 0.96±0.04 1.00±0.01 1.03±0.01 0.99±0.01 0.99±0.01 BAY-1213790 0.95±0.05 1.00±0.01 1.02±0.01 0.99±0.01 0.99±0.01 14E11 0.95±0.05 / / 1.00±0.01 1.00±0.01 BMS-962212 0.97±0.06 0.99±0.01 1.01±0.01 1.00±0.00 1.00±0.01

[0521] Example 11. Accelerated stability test of anti-human FXI / FXIa antibodies

[0522] The 36G9.10-hz73 antibody was diluted with 20mM His-HCl (hydrochloric acid-histidine buffer) (containing 0.03% Tween-20, pH5.5) to a concentration of 11.5mg / mL, and placed at 40°C and 25°C for 14 days and 28 days, respectively. The anticoagulant activity of the accelerated sample was detected by the APTT assay method in Example 4, and the purity of the accelerated antibody was detected by SEC.

[0523] The experimental results are as follows Fig.10 As shown in Table 6, storage at 40°C or 25°C for 14 or 28 days had no significant effect on the anticoagulant activity and purity of the 36G9.10-hz73 antibody.

[0524] Table 6. Accelerated stability test of anti-human FXI / FXIa humanized antibodies

[0525] Placement conditions Purity detected by SEC (%) APTT assay (EC50, nM) Day 0 99.4 18.36 25℃-28 days 99.8 17.36 40℃-14 days 97.9 20.33 40℃-28 days 94.4 26.89

[0526] Example 12. Pharmacokinetic (PK) and pharmacodynamic (PD) testing of anti-human FXI / FXIa antibodies

[0527] In order to detect the PK and PD of 36G9.10-hz73 in vivo, different doses of 36G9.10-hz73 antibody were given to cynomolgus monkeys by single intravenous injection (IV) or subcutaneous injection (SC), and blood was collected at different time points to measure PK and APTT activity. The specific dosing groups and doses are shown in Table 7, where the volume of a single intravenous or subcutaneous injection is 2mL / kg, the intravenous injection site is the limb vein, and the subcutaneous injection site is the back of the neck.

[0528] Table 7. Dosing regimen for PK and PD testing of anti-human FXI / FXIa humanized antibodies

[0529] Group Dosage (mg / kg) Dosage Number of animals Animal Gender 1 1 Intravenous injection 2 1 male + 1 female 2 3 Intravenous injection 3 1 male + 2 female 3 3 Subcutaneous injection 3 2 males + 1 female

[0530] After completing blood sampling at all time points, the 36G9.10-hz73 antibody was used as a standard, and the ELISA method in Example 7 was used to draw a standard curve of antibody concentration. The blood concentration of the 36G9.10-hz73 antibody at each time point was determined using this standard curve under the same conditions. Finally, the measured blood concentration was imported into Graphpad Prism for curve fitting to calculate the PK of 36G9.10-hz73 in crab-eating macaques.

[0531] At the same time, after each blood collection, APTT was immediately detected using the method in Example 10. Finally, all time point data were imported into Graphpad Prism for curve fitting to calculate the PD of 36G9.10-hz73 in cynomolgus monkeys.

[0532] The PK test results are shown in Table 8. The half-life of 36G9.10-hz73 antibody in cynomolgus monkeys after intravenous injection of 3 mg / kg was 290.15 hours, and the half-life of 36G9.10-hz73 antibody in cynomolgus monkeys after subcutaneous injection of 3 mg / kg was 188.02 hours. The bioavailability of 36G9.10-hz73 antibody in subcutaneous injection of 3 mg / kg was 94%±2% (n=2).

[0533] Table 8. Anti-human FXI / FXIa humanized antibody PK assay

[0534] Dosage Half-life (hours) AUC (0-t,h*μg / mL) IV - 1 mg / kg 355.37 3997.79 IV - 3 mg / kg 290.15 5035.86 Subcutaneous injection - 3 mg / kg 188.02 9581.64

[0535] The PD test results showed that 36G9.10-hz73 can achieve a good effect of prolonging APTT when used for intravenous injection and subcutaneous administration. Fig.11A As shown in the figure, the dose-effect relationship of the two intravenous injection dose groups is clear. The effect of subcutaneous injection of 3 mg / kg in prolonging APTT is slightly better than that of intravenous injection. It still has an APTT prolongation effect of about 1.3 times at 1008 hours. Fig. 11B As shown, none of the three dose groups prolonged PT, indicating no effect on exogenous coagulation. Therefore, the 36G9.10-hz73 antibody has the potential clinical advantage of not causing the risk of bleeding.

[0536] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa, wherein: The antibody or antigen-binding fragment thereof comprises: (1) The following heavy chain variable region (VH) and light chain variable region (VL), where the CDRs are defined according to the IMGT numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 33, CDR-H2 with a sequence of SEQ ID NO: 34, and CDR-H3 with a sequence of SEQ ID NO: 35; and, a light chain variable region (VL) comprising the following three CDRs: a CDR-L1 having a sequence of SEQ ID NO: 36, a CDR-L2 having a sequence of SEQ ID NO: 37, and a CDR-L3 having a sequence of SEQ ID NO: 38; or, (2) the following heavy chain variable region (VH) and light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system: A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with a sequence of SEQ ID NO: 39, CDR-H2 with a sequence of SEQ ID NO: 40, and CDR-H3 with a sequence of SEQ ID NO: 41; and, A light chain variable region (VL) comprising the following 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.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The VH and / or VL of the antibody or antigen-binding fragment thereof include framework regions (FRs) from human or mouse immunoglobulins.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof binds to human FXI and / or human FXIa.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: The VH shown in any one of SEQ ID NOs: 29 and 31, and the VL shown in any one of SEQ ID NOs: 30 and 32.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: (a) the VH sequence shown in SEQ ID NO: 29 and the VL sequence shown in SEQ ID NO: 30; or (b) VH of the sequence shown in SEQ ID NO:31 and VL of the sequence shown in SEQ ID NO:

32.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a murine antibody or a humanized antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a chimeric antibody.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof further comprises: (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the wild-type sequence from which it is derived; and / or (b) a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the wild-type sequence from which it is derived.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein The heavy chain constant region is an IgG heavy chain constant region.

10. The antibody or antigen-binding fragment thereof according to claim 8, wherein The heavy chain constant region is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

11. The antibody or antigen-binding fragment thereof according to claim 8, wherein The light chain constant region is a kappa light chain constant region.

12. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of: (1) human IgG1 heavy chain constant region; or (2) Human IgG4 heavy chain constant region.

13. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 21 or a variant thereof, wherein the variant has up to 20 conservative amino acid substitutions compared with SEQ ID NO:

21.

14. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 22 or a variant thereof having up to 20 conservative amino acid substitutions compared to SEQ ID NO:

22.

15. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 21 and a light chain constant region (CL) as shown in SEQ ID NO:

22.

16. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: a heavy chain comprising a sequence comprising the VH shown in SEQ ID NO:31 and a heavy chain constant region (CH) shown in SEQ ID NO:21; and A light chain comprising the sequence of the VL shown in SEQ ID NO:32 and the light chain constant region (CL) shown in SEQ ID NO:

22.

17. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is labeled.

18. The antibody or antigen-binding fragment thereof according to claim 17, wherein The antibody or antigen-binding fragment thereof is detectably labeled.

19. The antibody or antigen-binding fragment thereof according to claim 18, wherein The detectable label is selected from the group consisting of an enzyme, a radionuclide, a fluorescent dye and biotin.

20. The antibody or antigen-binding fragment thereof according to claim 18, wherein The detectable label is selected from luminescent substances.

21. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, the heavy chain and light chain thereof, or the heavy chain variable region and light chain variable region thereof according to any one of claims 1 to 20.

22. A vector comprising the isolated nucleic acid molecule of claim 21.

23. The vector according to claim 22, wherein The vector is a cloning vector or an expression vector.

24. A host cell comprising the isolated nucleic acid molecule of claim 21 or the vector of claim 22 or 23.

25. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, comprising culturing the host cell according to claim 24 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

26. A multispecific antibody comprising the antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa according to any one of claims 1 to 20, and another antibody or antigen-binding fragment thereof.

27. The multispecific antibody according to claim 26, wherein The multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

28. A conjugate comprising the antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa according to any one of claims 1 to 20 and a coupling moiety, wherein the coupling moiety is a detectable label or a therapeutic agent.

29. The conjugate of claim 28, wherein The detectable label is selected from the group consisting of: radioisotopes, fluorescent substances and enzymes.

30. The conjugate of claim 28, wherein The detectable label is selected from: a luminescent substance.

31. The conjugate of claim 28, wherein The detectable label is selected from: colored substances.

32. The conjugate of claim 28, wherein The therapeutic agent is an antiplatelet drug, an anticoagulant drug, or a thrombolytic drug.

33. The conjugate of any one of claims 28 to 32, wherein The therapeutic agent is bound to the antibody or antigen-binding fragment thereof that specifically binds to FXI and / or FXIa according to any one of claims 1 to 20 via a linker.

34. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-20, the isolated nucleic acid molecule of claim 21, the vector of claim 22 or 23, the host cell of claim 24, the multispecific antibody of claim 26 or 27, or the conjugate of any one of claims 28-33, and a pharmaceutically acceptable excipient.

35. The pharmaceutical composition of claim 34, wherein The excipient is a carrier.

36. The pharmaceutical composition of claim 34, wherein The pharmaceutical composition may also contain an additional pharmaceutically active agent.

37. The pharmaceutical composition of claim 36, wherein The additional pharmaceutically active agent is an antiplatelet drug, an anticoagulant drug or a thrombolytic drug.

38. The pharmaceutical composition of claim 36, wherein The additional pharmaceutically active agent is aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, unfractionated heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, streptokinase, urokinase, alteplase, prourokinase, or any combination thereof.

39. The pharmaceutical composition of any one of claims 34-38, further comprising a second antibody or a nucleic acid encoding the second antibody, wherein the second antibody is another antibody that recognizes a different epitope of FXI or FXIa, or is an antibody that specifically binds to a receptor or ligand selected from the following: 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.

40. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-20, or the vector of claim 22 or 23, or the host cell of claim 24, or the multispecific antibody of claim 26 or 27, the conjugate of any one of claims 28-33, or the pharmaceutical composition of any one of claims 34-39.

41. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 20, or the isolated nucleic acid molecule according to claim 21, or the vector according to claim 22 or 23, or the host cell according to claim 24, or the multispecific antibody according to claim 26 or 27, the conjugate according to any one of claims 28 to 33, or the pharmaceutical composition according to any one of claims 34 to 39 in the preparation of a medicament for preventing and / or treating coagulation or thromboembolic diseases or disorders.

42. The use according to claim 41, wherein the disease or condition is selected from: thrombosis, thrombotic stroke, atrial fibrillation-related stroke prevention, acute coronary syndrome, ischemic stroke, chronic thromboembolic pulmonary hypertension, systemic embolism, myocardial infarction, transient ischemic attack, disseminated intravascular coagulation, or stroke in patients with non-valvular atrial fibrillation.

43. The use according to claim 41, wherein the disease or condition is selected from: venous thromboembolism.

44. The use according to claim 41, wherein the disease or condition is selected from: pulmonary thromboembolism, or deep vein thrombosis.

45. The use according to claim 41, wherein the disease or condition is selected from: medical device-induced thromboembolic disorders, thromboembolic events before and after joint replacement surgery, thromboembolism after myocardial infarction, thromboembolism in chronic kidney disease, or thromboembolism in patients undergoing hemodialysis and patients subjected to adventitial membrane oxidation.

46. ​​The use according to claim 41, wherein the disease or condition is selected from: pulmonary embolism.

47. The use according to claim 41, wherein the disease or disorder is selected from: renal vein thrombosis, or thrombosis in chronic kidney disease.

48. The use according to claim 42, wherein The myocardial infarction is acute myocardial infarction.

49. A method for detecting the presence or level of FXI and / or FXIa in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 20, or the conjugate of any one of claims 28 to 33, under conditions that allow formation of a complex between the antibody or antigen-binding fragment thereof and FXI and / or FXIa, and detecting the formation of the complex; wherein: The method is for non-diagnostic purposes.

Images