Antibodies against fxi and / or fxi a and uses thereof

Abstract

This invention discloses an isolated anti-FXI and / or anti-FXIa antibody or its antigen-binding fragment thereof, which can specifically bind to human FXI or FXIa. The isolated antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:22; and (b) a light chain variable region of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. It has the advantages of reduced number of reversion mutations but unchanged function, improved stability in antibody production process, excellent APTT performance, and low risk, thereby providing a drug that targets FXI, can effectively prevent or treat thrombosis, and has minimal side effects to meet medical needs.

Description

Technical Field

[0001] This invention relates to an antibody capable of binding coagulation factor XI (FXI) and / or its activating factor XIa (FXIa) and fragments of FXI and / or FXIa, and its use therein, including its use as an anticoagulant for the preparation of a treatment for thrombosis without affecting hemostasis. Background Technology

[0002] A blood clot is a serious blood vessel blockage disease characterized by the formation of one or more blood clots inside a blood vessel. These clots can block blood flow or break off and move to other parts of the body. If a moving clot gets stuck in a critical area, it can cause fatal diseases such as stroke and coronary heart disease.

[0003] The coagulation cascade mechanism in the body includes intrinsic and extrinsic pathways. The intrinsic pathway, also known as the contact activation pathway, is triggered by surface contact, leading to the activation of FXII. The intrinsic pathway involves the activation of FXI, FIX, and FVIII. The extrinsic pathway, also known as the tissue factor (TF) pathway, is triggered by vascular injury, leading to the formation of the TF-FVIIa activation complex. These two pathways cross over and activate a common pathway, ultimately converting prothrombin to thrombin, forming a cross-linked fibrin clot.

[0004] Ideally, anticoagulants should effectively prevent thrombus formation without affecting normal hemostasis. Evidence suggests that the intrinsic coagulation pathway is crucial in the coagulation amplification phase, while the extrinsic and common pathways primarily participate in the initiation and diffusion phases of coagulation. These findings indicate that the intrinsic pathway plays a secondary role in normal hemostasis, and inhibiting it can reduce bleeding risk while exerting an antithrombotic effect. As an important component of the intrinsic pathway, FXI (fibrinolytic angiotensin-releasing enzyme) has recently emerged as an attractive pharmacological target due to its potential to provide antithrombotic effects without affecting bleeding.

[0005] The most commonly used anticoagulants in clinical practice include: non-enteric anticoagulants (such as heparin), coumarin anticoagulants (such as warfarin), antiplatelet drugs (such as aspirin), and thiophene pyridine drugs (such as clopidogrel). However, all of these drugs carry a certain risk of bleeding.

[0006] Factor XI (FXI) deficiency, first described in 1955, is a rare congenital hemorrhagic disorder, more common in Ashkenazi Jews. FXI is a member of the contact activation pathway and works in conjunction with the factor VII-tissue factor pathway to lead to thrombin formation and subsequent thrombus formation. Spontaneous bleeding is usually absent in patients with FXI deficiency; bleeding typically occurs after surgery or trauma.

[0007] Animal studies have shown that FXI deficiency or inhibition has no significant effect on experimental bleeding, suggesting that the FXI amplification pathway may play a less important role in normal hemostasis. Data from animal models of arterial, venous, and cerebral thrombosis show that FXI inhibitors exhibit antithrombotic effects. Furthermore, elevated plasma FXI levels may be associated with thromboembolic diseases in humans. Evidence suggests that patients with severe FXI deficiency are less likely to experience ischemic stroke or venous thrombosis. Therefore, FXI is an attractive target for the treatment and prevention of thromboembolism due to its unique role in thrombus formation and hemostasis, and the relatively low risk associated with FXI inhibition.

[0008] Chinese patents 202111467365.1, 202111466510.4, and 201880098606.X all disclose an isolated anti-FXI or anti-FXIa antibody that specifically binds to human FXI or FXIa. The antibodies in these patents respectively contain an immunoglobulin light chain variable domain (SEQ ID NO: 204) and an immunoglobulin heavy chain variable domain (SEQ ID NO: 205), an immunoglobulin light chain variable domain (SEQ ID NO: 208) and an immunoglobulin heavy chain variable domain (SEQ ID NO: 209), and immunoglobulin light chain variable domains (SEQ ID NO: 206 and SEQ ID NO: 207). These patents also disclose methods for preparing the antibodies and their use in preparing drugs for treating and / or preventing coagulation-related conditions (e.g., thrombosis and thrombosis-related complications or conditions).

[0009] Chinese Patent 201710073984.X discloses an isolated anti-FXI or anti-FXIa antibody that specifically binds to human FXI or FXIa, wherein: the sequence of the light chain variable domain of the antibody is SEQ ID NO: 197, and the sequence of the heavy chain variable domain of the antibody is SEQ ID NO: 198; or the sequence of the light chain variable domain of the antibody is SEQ ID NO: 199, and the sequence of the heavy chain variable domain of the antibody is SEQ ID NO: 200; or the sequence of the light chain variable domain of the antibody is SEQ ID NO: 201, and the sequence of the heavy chain variable domain of the antibody is SEQ ID NO: 202.

[0010] Chinese Patent 201780007555.0 discloses an antibody or antigen-binding fragment comprising: (a) a heavy chain (HC) variable domain comprising an HC complementarity-determining region (CDR)1 composed of the amino acid sequence shown in SEQ ID NO:1, an HC CDR2 composed of the amino acid sequence shown in SEQ ID NO:2, and an HC CDR3 composed of the amino acid sequence shown in SEQ ID NO:3; and (b) a light chain (LC) variable domain comprising an LCCDR1 composed of the amino acid sequence shown in SEQ ID NO:4, an LCCDR2 composed of the amino acid sequence shown in SEQ ID NO:5, and an LCCDR3 composed of the amino acid sequence shown in SEQ ID NO:6; wherein the antibody or antigen-binding fragment binds to the apple 2 domain of coagulation factor XI (FXI) and inhibits the activation of FXI.

[0011] Chinese Patent 201780037421.3 discloses an antibody or antigen-binding fragment that binds to the apple 3 domain of coagulation factor XI (FXI), comprising (a) HC-CDR 1 of the amino acid sequence shown in SEQ ID NO: 8, HC-CDR 2 of the amino acid sequence shown in SEQ ID NO: 9, and HC-CDR 3 of the amino acid sequence shown in SEQ ID NO: 10; and (b) light chain complementarity-determining region (LC-CDR) 1 of the amino acid sequence shown in SEQ ID NO: 11, LC-CDR 2 of the amino acid sequence shown in SEQ ID NO: 12, and LC-CDR 3 of the amino acid sequence shown in SEQ ID NO: 13.

[0012] Chinese Patent 202210232062.X discloses an isolated antibody or an isolated antigen-binding fragment comprising: a heavy chain (HC) variable domain comprising an HC complementarity-determining region (HC CDR)1 composed of the amino acid sequence shown in SEQ ID NO:7, an HC CDR2 composed of the amino acid sequence shown in SEQ ID NO:8, and an HCCDR3 composed of the amino acid sequence shown in SEQ ID NO:9 or 13; and a light chain (LC) variable domain comprising an LC CDR1 composed of the amino acid sequence shown in SEQ ID NO:10, an LC CDR2 composed of the amino acid sequence shown in SEQ ID NO:11, and an LC CDR3 composed of the amino acid sequence shown in SEQ ID NO:12; and wherein the isolated antibody or isolated antigen-binding fragment binds to the apple 2 domain of coagulation factor XI (FXI) and inhibits the activation of FXI.

[0013] Chinese Patent 201380037161.1 discloses a human monoclonal antibody capable of binding coagulation factor XIa (FXIa) and its antigen-binding fragment, comprising a variable light chain domain and a variable heavy chain domain, wherein the variable light chain domain is composed of the amino acid sequence SEQ ID NO:19 and the variable heavy chain domain is composed of the amino acid sequence SEQ ID NO:20; or a human monoclonal antibody capable of binding FXIa and its antigen-binding fragment, comprising CDRH1 SEQ ID NO:21, CDRH2 SEQ ID NO:22 and CDRH3 SEQ ID NO:23 and CDRH1 SEQ ID NO:24, CDRH2 SEQ ID NO:25 and CDRH3 SEQ ID NO:26; or a human monoclonal antibody capable of binding FXIa and its antigen-binding fragment, comprising a variable light chain domain and a variable heavy chain domain, wherein the variable light chain domain is composed of the amino acid sequence SEQ ID NO:27 and the variable heavy chain domain is composed of the amino acid sequence SEQ ID NO:26. NO:20; or a human monoclonal antibody capable of binding FXIa and its antigen-binding fragment, comprising CDRH1 SEQ ID NO:21, CDRH2 SEQ ID NO:22 and CDRH3 SEQ ID NO:23, and CDRH1 SEQ ID NO:24, CDRH2 SEQ ID NO:25 and CDRH3 SEQ ID NO:28.

[0014] Chinese Patent 201910729990.5 discloses an antigen-binding fragment of an antibody against the activated form of coagulation factor XI, factor XIa. The amino acid sequence of CDR1 in the heavy chain variable region of this antigen-binding fragment is shown in SEQ ID NO:1, the amino acid sequence of CDR2 in the heavy chain variable region is shown in SEQ ID NO:2, and the amino acid sequence of CDR3 in the heavy chain variable region is shown in SEQ ID NO:3. The amino acid sequence of CDR1 in the light chain variable region of this antigen-binding fragment is shown in SEQ ID NO:4, the amino acid sequence of CDR2 in the light chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of CDR3 in the light chain variable region is shown in SEQ ID NO:6.

[0015] Because currently approved anticoagulants can interfere with the hemostasis process and increase the risk of bleeding, there is a significant unmet medical need in the market for safer anticoagulants. Existing anticoagulants, such as heparin, warfarin, aspirin, and factor Xa inhibitors, while effective in treating or preventing thrombosis, are often accompanied by significant adverse reactions.

[0016] Therefore, there is an urgent need to develop a drug that can effectively prevent or treat thrombosis and minimize side effects. Summary of the Invention

[0017] The objective of this invention is to provide a separable, improved anti-FXI and / or anti-FXIa antibody or its antigen-binding fragment that can specifically bind to human FXI or FXIa, and has better cell productivity, better antibody stability and better duration of drug efficacy.

[0018] Certain embodiments of the present invention provide antibodies or antigen-binding fragments thereof that bind to coagulation factor XI (FXI) and / or its activated form factor XIa (FXIa) and fragments of FXI and / or FXIa, specifically binding to human FXI or FXIa. In some embodiments, the antibody may be a monoclonal antibody; in some embodiments, the antibody may be a recombinant antibody; in some embodiments, the antibody may also be a humanized antibody; in some embodiments, the antibody is an immunoglobulin molecule with immunoactive activity, such as Fab, Fvs, or scFv; in some embodiments, the antibody binds to the A3 domain of FXI and / or FXIa.

[0019] In some preferred embodiments, the isolated anti-FXI and / or anti-FXIa antibodies or their antigen-binding fragments of the present invention comprise:

[0020] (a) The heavy chain variable region shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:22 in Table 1; and

[0021] (b) The light chain variable regions shown in Table 1 as SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[0022] The separable anti-FXI and / or anti-FXIa antibodies or their antigen-binding fragments involved in this invention include a heavy chain variable domain sequence selected from SEQ ID NO:5 in Table 1 or a sequence having at least 60%, 90%, 97%, or 98% sequence identity with the sequence of SEQ ID NO:5, wherein the antibody binds to FXI and / or FXIa; furthermore, the separable anti-FXI and / or anti-FXIa antibodies or their antigen-binding fragments involved in this invention also include a light chain variable domain sequence selected from SEQ ID NO:15 in Table 1 or a sequence having at least 60%, 90%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:15, wherein the light chain variable domain combines with the heavy chain variable domain to form an antigen-binding site for FXI and / or FXIa.

[0023] Preferably, the isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region selected from SEQ ID NO:5 and having at least 80% identity with it, and a light chain variable region selected from SEQ ID NO:15 and having at least 80% identity with it; more preferably, the isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region selected from SEQ ID NO:5 and having at least 97% identity with it, and a light chain variable region selected from SEQ ID NO:15 and having at least 97% identity with it; even more preferably, the isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region selected from SEQ ID NO:5 and having at least 98% or 99% identity with it, and a light chain variable region selected from SEQ ID NO:15 and having at least 98% or 99% identity with it.

[0024] As some specific embodiments of the present invention, the separable anti-FXI and / or anti-FXIa antibody or antigen-binding fragment includes a heavy chain variable region selected from SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9 and a light chain variable region selected from SEQ ID NO:17 or SEQ ID NO:18.

[0025] As some preferred embodiments of the present invention, the separable anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:7 or SEQ ID NO:10, and a light chain variable region selected from SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:21.

[0026] As some other preferred embodiments of the present invention, the separable anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:10 or SEQ ID NO:22, and a light chain variable region selected from SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.

[0027] As some other preferred embodiments of the present invention, the separable anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises:

[0028] The heavy chain of SEQ ID NO:30 and the light chain of SEQ ID NO:31; or

[0029] The heavy chain of SEQ ID NO:32 and the light chain of SEQ ID NO:33; or

[0030] The heavy chain of SEQ ID NO:34 and the light chain of SEQ ID NO:35.

[0031] As a preferred embodiment of the present invention, the separable anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises the heavy chain shown in SEQ ID NO:32 and the light chain shown in SEQ ID NO:33.

[0032] As a specific embodiment of the present invention, the separable anti-FXI and / or anti-FXIa antibody or antigen-binding fragment is a human monoclonal antibody.

[0033] On the other hand, the present invention also provides a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described above. This pharmaceutical composition can be used to treat and / or prevent thrombosis and / or thrombosis-related complications or diseases. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants, carriers, excipients, preservatives, or combinations thereof.

[0034] In another aspect, the present invention also provides the use of the above-described separable anti-FXI and / or anti-FXIa antibodies or their antigen-binding fragments or the above-described pharmaceutical compositions in the preparation of a medicament for inhibiting blood clot formation in subjects.

[0035] In some embodiments of the present invention, the above-described use refers to its use in the preparation of a medicament for the treatment or prevention of thrombosis or thrombosis-related complications or conditions, wherein the amount of antibody used does not impair the hemostatic function of the subject.

[0036] In another aspect, the present invention also provides a method for preparing the above-mentioned separable anti-FXI and / or anti-FXIa antibodies, wherein the method includes cloning the nucleic acid encoding the antibody into an expression vector and expressing it in a host cell. That is, the method includes steps including transfecting host cells with a vector encoding antibody nucleotides and expressing the antibody in the host cells.

[0037] In some embodiments of the invention, the method described above further includes purifying the expressed antibody from host cells. Furthermore, the purified antibody may be modified, and the modified recombinant antibody retains the activity of the corresponding human antibody. Alternatively, the antibodies disclosed in this specification can be generated from hybridoma culture.

[0038] Furthermore, the present invention also provides a nucleic acid encoding the anti-FXI and / or anti-FXIa antibody, or any antibody functional fragment disclosed herein, a vector comprising the nucleic acid, and a host cell comprising the vector. In some embodiments, the vector is an expression vector capable of producing the antibody or its functional fragment encoded by the nucleic acid in the host cell.

[0039] Furthermore, the present invention provides a kit comprising one or more anti-FXI and / or anti-FXIa antibodies disclosed herein for the treatment and / or prevention of thrombosis and / or thrombosis-related complications or conditions. Alternatively, the kit comprises a pharmaceutical composition comprising one or more anti-FXI and / or anti-FXIa antibodies disclosed herein for the treatment and / or prevention of thrombosis and / or thrombosis-related complications or conditions.

[0040] Furthermore, the present invention provides a method for treating and / or preventing thrombosis and / or thrombosis-related complications or conditions, the method comprising administering to a subject in need an effective dose of one or more anti-FXI and / or anti-FXIa antibodies disclosed herein. Alternatively, the method comprises administering to a subject in need an effective dose of a pharmaceutical composition containing a functional fragment of an anti-FXI antibody, an anti-FXIa antibody, or any of the antibodies.

[0041] Unless otherwise defined, all technical terms used in this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] In this invention, the terms "FXI protein," "FXI antigen," and "FXI" are used interchangeably and can refer to coagulation factor XI proteins of different species. Coagulation factor XI is mammalian plasma coagulation factor XI, a glycoprotein present in human plasma in proenzyme form at a concentration of 25-30 nM. When converted into an active serine protease through limited proteolysis, it participates in the intrinsic pathway of blood coagulation.

[0043] In this invention, the terms "FXIa protein," "FXIa antigen," and "FXIa" are used interchangeably and can refer to activated FXI proteins of different species. Proenzyme coagulation factor XI is converted to its active form, coagulation factor Xla (FXIa), through a blood-clotting contact phase or through thrombin-mediated activation on the platelet surface. During this activation of coagulation factor XI, each of the two chains of the internal peptide bond is cleaved, resulting in the activation factor Xla, a serine protease consisting of two heavy chains and two light chains linked by disulfide bonds. This serine protease FXIa converts coagulation factor IX to IXa, which subsequently activates coagulation factor X (Xa). Xa can then mediate coagulation factor XI / thrombin activation.

[0044] In this invention, the terms "FXI" and "FXIa" (and similar terms) respectively include mutants and variants of the natural FXI and FXIa proteins, which have amino acid sequences that are substantially the same as the natural primary structure (amino acid sequence) described above.

[0045] In this invention, the term "antibody" refers to a complete antibody and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain. A complete antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three regions (CH1, CH2, and CH3). Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises a domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The variable regions of the heavy and light chains contain binding domains that interact with antigens. The antibody constant regions mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0046] In this invention, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of a complete antibody that retain the ability to specifically bind to a given antigen (e.g., coagulation factor XIa (FXIa)). The antigen-binding function of an antibody can be performed by fragments of a complete antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" or "antigen-binding fragment" of an antibody include: a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; an F(ab)2 fragment, a bivalent fragment comprising two Fab fragments connected by disulfide bridges located in the hinge region; an Fd fragment consisting of VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single arm of the antibody; a single-domain antibody (dAb) fragment consisting of either a VH domain or a VL domain; and a separated complementarity-determining region (CDR).

[0047] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. However, the following description is only a part of various embodiments of the present invention. Therefore, the specific variations discussed should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various equivalent substitutions, changes, and improvements can be made to the present invention without departing from the spirit of the invention. Therefore, the scope of protection of the present invention includes such equivalent substitutions, changes, and improvements. Attached Figure Description

[0048] Figure 1 The results of the in vitro APTT assay (human plasma) for the humanized 13F4 antibody are shown, including: A. First round of in vitro APTT test of the humanized 13F4 antibody, with antibody concentrations of 5 μg / ml and 3 μg / ml, respectively; B. Comparison of APTT between 13F4-b10 and the PC control group; C. Second round of in vitro APTT test of the humanized 13F4 antibody; D. Individual comparison of APTT between 13F4-NOV-b10 and 13F4-NOV-b21; Note: PC is an abbreviation for Positive control.

[0049] Figure 2 This section describes the expression and purification of PI-regulated humanized 13F4-b21 antibody, including: A. Antibody expression and purification data; B. SDS-PAGE analysis; C. Comparison of protein A yields after purification; Abbreviations in the figure: NOV-b21, 13F4-NOV-b21; P3, 13F4-NOV-b21-P3; P4, 13F4-NOV-b21-P4; TM-b21, 13F4-TM-b21; S4, 13F4-TM-b21-S4;

[0050] Figure 3The image shows the in vitro APTT assay of the PI-regulated humanized 13F4 antibody, including: A. APTT results; B. Schematic diagram of total mAb detection; C. Schematic diagram of active mAb detection;

[0051] Figure 4 The image shows a mouse pharmacokinetic analysis of the PI-regulated humanized 13F4-b21 antibody;

[0052] Figure 5 This paper presents the expression, purification, and preliminary analysis of P329G LALA after incorporation with humanized 13F4-b21 series antibodies, including: A. Antibody expression and purification data; B. SDS-PAGE analysis; C. Size exclusion chromatography (SEC) results; Note: NOV represents NOV1401, BAY represents BAY1213790, 13F4-b21 represents 13F4-b21 LALA, P3 represents 13F4-b21-P3 LALA, and S4 represents 13F4-b21-S4 LALA.

[0053] Figure 6 The SPR affinity analysis of humanized 13F4-b21 series antibodies with human FXI protein is shown, including: SPR experimental flowchart; BF. SPR curve; G. ka, kd, KD, Rmax values; H. experimental method parameters; NOV1401 is an abbreviation for NOV1401MAA868;

[0054] Figure 7 The SPR affinity analysis of humanized 13F4-b21 series antibodies with human FXIa protein is shown, including: SPR experimental flowchart; BF. SPR curve; G. ka, kd, KD, Rmax values; H. experimental method parameters; NOV1401 is an abbreviation for NOV1401MAA868;

[0055] Figure 8 The results of differential scanning calorimetry (DSC) are shown; NOV1401 is an abbreviation for NOV1401MAA868;

[0056] Figure 9 The results of coagulation parameters in human plasma are shown. The working concentration of all mAbs is 50 nM. APTT is the activated partial thromboplastin time, PT is the activated partial thromboplastin time, FIB represents fibrinogen, and TT is the prothrombin time.

[0057] Figure 10 The results of coagulation parameters for different antibodies in monkey plasma are shown. The working concentration of all mAbs is 50 nM. APTT is the activated partial thromboplastin time, PT is the activated partial thromboplastin time, FIB represents fibrinogen, and TT is the prothrombin time.

[0058] Figure 11 The APTT activity of the antibody in human or monkey plasma in vitro was demonstrated;

[0059] Figure 12 The affinity of different antibodies for human FcγRI (CD64) was compared.

[0060] Figure 13 The affinity of different antibodies for human FcγRIIa (CD32a) was compared.

[0061] Figure 14 The affinity of different antibodies for human FcγRIII (CD16a) was compared.

[0062] Figure 15 The comparison of ELISA binding of different antibodies to human C1q is shown;

[0063] Figure 16 The affinity of different antibodies for human FcRn (pH 6.0) was compared.

[0064] Figure 17 The results of mouse PK ELISA experiments with different antibodies are shown. In the figure, AC are the total antibody and active antibody PK ELISA curves of NOV1401, BAY1213790 and 13F4-b21-P3 LALA, respectively. D and E are the comparison of the total amount or activity of the three antibodies in PK ELISA.

[0065] Figure 18 The correlation of area under the curve (AUC) of mouse PK ELISA is shown, where: A. total mAb AUC; B. active mAb AUC. P values ​​were obtained by ordinary one-way ANOVA, ns represents no significant difference, ** represents a significant difference, and P<0.05.

[0066] Figure 19 This study shows the effect of antibodies on APTT in cynomolgus monkeys, where d represents the number of days. Detailed Implementation

[0067] The sources of the main materials used in the experiments of this invention and the models of the main instruments used are shown in the various embodiments.

[0068] Example 1: Humanization of 13F4 hybridoma antibody

[0069] Antibody humanization can reduce the immunogenicity of monoclonal antibodies and enhance the activation of the human immune system by replacing non-human antibody frameworks with human antibody frameworks. This is a very important step in the discovery of therapeutic antibodies.

[0070] The original 13F4 VH and VL sequences were derived from mouse hybridoma cells. The first round of humanization design of the hybridoma 13F4 VH and VL sequences is shown in Table 1. Table 2 lists the combinations of humanized 13F4 VH and VL plasmids and antibodies, with the heavy chain of the antibody being wild-type IgG1 and the light chain being the λ subtype.

[0071] The first round of humanization yielded antibodies 13F4-b1 to b16. The yield of most antibodies was higher than that of chimeric 13F4 antibodies, except for 13F4-b3, b4, and b16 (Table 3). The number of reversion mutations in these antibodies ranged from 3 to 16. Within the variable region of the antibody, the more human germline-derived sequences, the lower the immunogenicity in humans. In our study, we found that the 13F4 VH and VL chains could potentially be further humanized by reducing the number of amino acids in the murine reversion mutations. Therefore, we conducted a second round of humanization design, obtaining antibodies 13F4-NOV-b17 to b21. The lowest number of reversion mutations was in 13F4-NOV-b21, with 5. The number of reversion mutations for each antibody is detailed in Table 2; the numbers in parentheses below the antibody name represent the sum of VH and VL reversion mutations. Fewer reversion mutations indicate lower immunogenicity in humans. The second round of humanized antibodies used the same heavy chain Fc as the positive antibody NOV1401, hence the abbreviation NOV was added to the antibody name. The light chain constant region remained the λ chain. The expression yield of the second round of humanized antibodies was also very stable, and the purity was generally above 95% (Table 4). 13F4-NOV-b21 reduced the number of reversion mutations to 5, but its yield and purity were no less than those of 13F4-NOV-b10 with 9 reversion mutations (Table 4), suggesting that 13F4-NOV-b21 may be an ideal candidate antibody for us.

[0072] Table 1. Variable region sequences of 13F4 antibody humanization

[0073]

[0074]

[0075]

[0076] Note: VH, variable region of heavy chain; VL, variable region of light chain.

[0077] Table 2. List of plasmids and antibodies corresponding to humanized 13F4 antibodies.

[0078]

[0079] Note: Heavy chain plasmids are listed on the left side of the table, and light chain plasmids are listed at the top; recombinant humanized antibodies are purified by transfecting VH:VL plasmids in a 2:3 ratio; the number of reversion mutations in the VH and VL regions is indicated in parentheses below the antibody name.

[0080] Table 3. List of expression and purification of 13F4 antibody in the first round of humanization.

[0081]

[0082] Abbreviations: 13F4, mAb13F4 xhIgG1, chimeric 13F4 antibody; 13F4-b1, mAb13F4 zhIgG1-b1, humanized 13F4-b1 antibody.

[0083] Table 4. List of expression and purification of 13F4 antibody in the second round of humanization.

[0084]

[0085] Example 2: In vitro APTT assay of humanized 13F4 antibody

[0086] Experimental Methods: The final working concentrations of the antibodies were 3 and 5 μg / ml, respectively. 75 μl of antibody was mixed with 75 μl of human plasma and incubated at room temperature for 5 minutes. 25 μl of Dade... Activated Cephaloplastin Reagent (SIEMENS, SMN10445709, LOT 557290) was mixed with 50 μl of antibody-plasma mixture and incubated at 37°C for 3 minutes. Then, 25 μl of 0.025 M CaCl2 was added and mixed. Using 1xPBS mixed with plasma as a baseline, coagulation time (in seconds) was measured using a coagulation analyzer (Sysmex CA660).

[0087] Experimental results: These are illustrated in the accompanying figures. At an antibody concentration of 5 μg / ml, the APTT clotting time of the first round of humanized 13F4-b7, b8, b10, and b12 was relatively higher than that of other antibodies (see [reference]). Figure 1 A) The number of reversion mutations for these four antibodies were 9, 11, 9, and 13, respectively. The APTT curves of 13F4-b10 were compared with those of the three positive control antibodies NOV1401, AB023, and BAY1213790. Ranked by curve trend and EC50, the order was: 13F4-b10 > NOV1401 > AB023 > BAY1213790 (see [link]). Figure 1 B).

[0088] During the second round of humanization of the 13F4 antibody, 13F4-b10 was selected as the control antibody, and five new antibodies were designed. The heavy chain Fc of these antibodies was replaced with an Fc sequence identical to that of the positive control NOV1401. The main objective of this part is to evaluate the effects of these five antibodies on in vitro APTT in human plasma at concentrations of 2.34, 4.68, 9.37, 18.75, 37.5, 75, 150, and 300 nM, compared to 13F4-NOV-b10. Figure 1 As shown in C, the APTT activities of these five antibodies in human plasma in vitro are similar to those of 13F4-NOV-b10, and the APTT activity of 13F4-NOV-b21, which has the fewest reversion mutations, shows a high degree of overlap with the curves of 13F4-NOV-b10, with almost no difference (see [reference]). Figure 1 D). Considering the above factors, 13F4-NOV-b21 is the preferred antibody with the fewest reversion mutations and unchanged APTT function.

[0089] Example 3: PI Modification of Humanized 13F4-b21 Antibody

[0090] The sequence design for adjusting the pI values ​​of humanized 13F4-b21 antibodies VH and VL is shown in Table 5, and the combination of antibodies VH and VL into antibodies is shown in Table 6. The adjusted pI values ​​of antibodies 13F4-TM-b21-s4, 13F4-NOV-b21-p3, and 13F4-NOV-b21-p4 are 6.26, 8.19, and 8.31, respectively. The constant region of the antibody heavy chain in this batch of antibodies uses the NOV-Fc and TM-Fc sequences; detailed sequence information is shown in Table 8.

[0091] Overall, the antibody yield after pI adjustment was slightly lower than before adjustment, but the 13F4-NOV-b21-p3 antibody performed well, with a yield not significantly different from the unadjusted 13F4-NOV-b21 yield. Figure 2 A and 2C). SDS-PAGE gel analysis of the antibody bands in both the non-reduced and reduced states yielded results consistent with theoretical predictions. Figure 2 B). The in vitro APTT curve in human plasma of the pI-adjusted antibody was unaffected and resembled that of the pre-adjustment antibody, as well as the positive control NOV1401 (see [link]). Figure 3 ).

[0092] Table 5. PI-regulated variable region sequence of humanized 13F4-b21 antibody.

[0093]

[0094] Table 6. List of PI-regulated humanized 13F4-b21 antibodies

[0095] Ab Name Heavy chain Light Chain Theoretical PI 13F4-TM-b21-s4 13F4 VH.h5-s1+hIgG1-TM 13F4 VL.g6-s4+human 1amda CL 6.26 13F4-NOV-b21-P3 13F4 VH.h5+hIgG1-NOV 13F4 VL.g6-p3+human lamda CL 8.19 13F4-NOV-b21-P4 13F4 VH.h5+hIgG1-NOV 13F4 VL.g6-p4+human 1amda cL 8.31

[0096] Table 7. pI and cIEF measurements of PI-regulated humanized 13F4-b21 antibody.

[0097]

[0098] Abbreviations in the table: AP, acid peak; MP, monomer peak; BP, basic peak

[0099] Table 8 Protein Sequences

[0100]

[0101]

[0102]

[0103] Example 4: In vivo stability study of PI-modified humanized 13F4-b21 antibody in mice

[0104] This embodiment aims to evaluate the pharmacokinetic (PK) performance of the PI-modified humanized 13F4-b21 antibody in mice (approximately 8 weeks old male ICR mice). Analyzing their PK curves provides a comprehensive understanding of the antibody's stability in mice, offering crucial data for subsequent experiments. Blank plasma was collected from all mice before injection. The antibody was administered via tail vein injection at a dose of 1 mg / kg, with 3-4 male mice receiving each antibody. Plasma was collected at 0.5h, 2h, 1d, 3d, 7d, 14d, 21d, and 28d post-injection. After anticoagulation with sodium citrate, the plasma was frozen at -80℃. The plasma concentrations of total IgG and active IgG were subsequently determined using ELISA. (See schematic diagram). Figure 3 B and 3C. The PK ELISA uses a standard ELISA protocol, with the following reagents:

[0105] • Capture antibody: Sheep Anti-Human IgG(Fc) Monkey Adsorbed for total IgG detection

[0106] (Selectscience-Binding Site, AU004M); Human coagulation factor XI (ERL, HFXI 1111) for the detection of active IgG;

[0107] • Antibody detection: Goat anti-Human IgG-h+l HRP Conjugated MinX monkey

[0108] (Bethyl, A80-319P);

[0109] • Carbonate buffer (CBS): Dissolve 1.5g Na2CO3 and 2.93g NaHCO3 in 1000mL of deionized water and adjust the pH to 9.6;

[0110] • Blocking buffer: PBS containing 5% skim milk;

[0111] • Detection buffer: PBS + 0.5% BSA + 0.35M NaCl + 0.05% Tween 20 + 0.25% CHAPS

[0112] +5mM EDTA;

[0113] • Sample diluent: Analytical buffer containing 1% mouse plasma;

[0114] • Antibody diluent: 0.05% PBST containing 0.5% BSA;

[0115] • Washing buffer (0.05% PBST): Add 0.5 mL of Tween 20 to 1000 mL of 1xPBS and mix well;

[0116] •TMB: TMB-S-002 (Huzhou Yingchuang);

[0117] • ELISA stop solution: Solarbio, catalog number C1058.

[0118] Experimental results: The stability of the PI-modified antibody in mice was similar to that of the unmodified antibody or the positive control NOV1401. The ELISA curves of Total and Active IgG were almost overlapping, except that the stability of 13F4-NOV-b21-P4 showed a slightly weaker trend (see [link to results]). Figure 4 ).

[0119] Example 5: Introduction of P329G LALA mutation into the heavy chain constant region of the PI-modified humanized 13F4-b21 antibody.

[0120] Tilman et al. reported that the modified hIgG1 Fc domains (hIgG1-P329G LALA and hIgG4-P329GSPLE) contained a limited number of mutations that significantly reduced the interaction between FcγR and C1q, while FcRn interaction and Fc stability remained unaffected (Schlothauer et al., 2016). We also introduced P329G LALA into hIgG1 candidate antibodies, generating 13F4-b21 LALA, 13F4-b21-P3 LALA, and 13F4-b21-S4 LALA candidate antibodies (see Table 8). The expression results of the three candidate antibodies and two positive antibodies are shown in Table 8. Figure 5A. After two rounds of purification, most antibodies achieved a purity of 99%, except for 13F4-b21-S4 LALA, which reached 97%. All antibodies, in both their non-reduced and reduced states, showed the desired bands when run on SDS-PAGE gels. Figure 5 B). SEC results for most antibodies also showed a single main peak, except for 13F4-b21-S4 LALA, which showed a small stray peak. Figure 5 C).

[0121] Example 6: Ab kinetics detection using human FXI or FXIa protein

[0122] This embodiment uses an SPR detection method to determine the affinity of the humanized 13F4-b21 antibody with the P329G LALA mutation prepared in Example 5 for FXI or FXIa antigen. The specific detection steps are as follows: SPR interaction analysis of the captured antibodies [antibodies prepared in Example 5 (13F4-b21 LALA, 13F4-b21-P3 LALA, 13F4-b21-S4 LALA) and positive antibodies (NOV1401, BAY1213790)] and FXI or FXIa was performed on a Biacore T200 system (GE Healthcare) using a highly immobilized human antibody capture kit (Cytiva). Immobilization of the anti-human IgG (Fc) antibody was performed on a CM5 chip using a standard amine conjugation kit (Cytiva) in 10 mM sodium acetate and 0.5 mg / ml anti-human IgG (Fc) antibody solution at pH 5.0. The immobilization level of anti-human IgG (Fc) reached >9000 RU. Using running buffer HBS-EP, antibody solutions of 1 μg / ml were prepared and immobilized to a level of 50 RU at a flow rate of 10 μl / min. Subsequently, 0.005–0.33 μg / ml of FXI or FXIa were applied to HBS-EP at a flow rate of 30 μl / min for 60–180 s. The dissociation phase was monitored for 1000 seconds. A 30-second washing step with 3M magnesium chloride at a flow rate of 30 μl / min was performed to regenerate the surface. Data analysis was performed using Biacore T200 evaluation software.

[0123] In SPR experiments, the humanized 13F4-b21 candidate antibody with the P329G LALA mutation showed similar high affinity to human FXI or FXIa factor as the PC control NOV1401. In contrast, the PC control BAY1213790 showed slightly lower affinity (see [link to study]). Figure 7 and 8 ).

[0124] Example 7: Differential Scanning Calorimetry (DSC) for Identifying Antibody Stability

[0125] Figure 8 In the diagram, different Tm peaks represent different components of the antibody, as shown below: Tm1: CH2; Tm2: Fab; Tm3: CH3. A higher Tm value indicates greater antibody stability. DSC results showed that the stability of 13F4-b21-P3 LALA at the Tm1 and Tm2 peaks was slightly higher than that of NOV1401, suggesting that the stability of the 13F4-b21-P3 LALA antibody at the CH2 and Fab ends may be slightly better than that of the positive control NOV1401.

[0126] Example 8: Coagulation Four Tests

[0127] Compared to the three coagulation parameters (PT, FIB, and TT) in humans and monkeys, the candidate antibody 13F4-b21-P3 LALA had the most significant effect on APTT in both humans and monkeys. Compared to the PBS negative control, 50 nM of 13F4-b21-P3 LALA significantly delayed APTT time by approximately two times in both human and monkey plasma, similar to the 50 nM NOV1401 positive group. This result indicates that the candidate antibody only prolongs the APTT time of intrinsic coagulation in humans and monkeys, and has no significant effect on extrinsic coagulation PT, FIB, or TT (see [link to relevant documentation]). Figure 9 and 10 ).

[0128] Example 9 In vitro APTT curve

[0129] To further investigate the in vitro APTT curves of human and monkey plasma, candidate antibodies were diluted into different concentration gradients to test the effects of different antibodies on the in vitro APTT of humans and monkeys. The specific experimental methods are described in Example 2. The antibody concentrations used in human plasma were 3.125, 6.25, 12.5, 25, 50, 100, and 200 nM, and the antibody concentrations used in monkey plasma were 0, 6.25, 12.5, 25, 50, 100, and 200 nM.

[0130] from Figure 11 Results A show that in human plasma, the APTT curve of 13F4-b21-P3 LALA is similar to that of the PC control NOV1401, with EC50 values ​​of 34.22 nM and 30.44 nM, respectively. The APTT curve of BAY1213790 is relatively weaker, with an EC50 value of 40.25 nM. In monkey plasma, the effect of 13F4-b21-P3 LALA on APTT is similar to that of NOV1401, with a slightly higher maximum value. Figure 11 B).

[0131] Example 10: Determination of the binding affinity of the antibody to human Fcγ receptor (FcγR) or human C1q.

[0132] Tilman et al. found that the P329G LALA mutation at the Fc end of IgG1 of antibodies can completely eliminate the ADCC, ADCP, and CDC effects (Schlothauer et al., 2016). And selecting the Fc subtype of G1m3(CH1)+nG1m1, 2(EEM, CH3) at the Fc end of monoclonal antibodies is beneficial to reducing the immunogenicity of antibodies (Ternant et al., 2016; Webster et al., 2016). In the research of this invention, the above-mentioned mutation was introduced at the Fc end of the 13F4-b21-P3 LALA antibody, and the amino acid sequence of the constant region of the antibody heavy chain is shown in the protein sequence in Table 8. The Fc sequence of this invention has several amino acid differences compared with the Fc sequences of the positive NOV1401 and BAY1213790. [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Figure 12-14 )。The binding affinity of 13F4-b21-P3 LALA to all three human FcγR receptors is lower than that of the two positive antibodies (see Figure 12-14 ).

[0143] 2) Affinity of the antibody to human C1q

[0144] For the specific steps, refer to Thermofisher's C1q Human ELISA Kit (product number BMS2099).

[0145] The experimental results show that the binding ability of the antibody to human C1q is: BAY<1213790> 13F4-b21-P3 LALA > NOV1401. Compared with the positive BAY1213790 antibody with wild-type Fc, the binding affinity of the candidate antibody 13F4-b21-P3 LALA to hC1q is weaker and it is less likely to trigger the in vivo CDC reaction. (See Figure 15 ).

[0146] Example 11 Affinity of the antibody to human neonatal Fc receptor (hFcRn)

[0147] The binding effect of the Fc region of the antibody to hFcRn (Sinobiological, CAT#CT009-H08H) can be evaluated by SPR affinity assay. For the Fc region-hFcRn affinity data obtained through the SPR experiment, the specific experimental procedure is referred to Example 10, only the ligand is replaced with hFcRn and other steps remain the same.<000036​​​​​​​​​​​​​​The results show that, when the total and active PK ELISA curves of the three antibodies are combined, the stability of our candidate antibody 13F4-b21-P3 LALA in mice after 28 days is similar to that of the positive antibody NOV1401, with a high degree of curve overlap. However, the positive control BAY1213790 shows inferior PK stability in mice compared to the other two antibodies (see [link to relevant documentation]). Figure 17 D and 17E).

[0152] AUC analysis showed no significant difference in total mAb AUC and active mAb AUC between the positive control NOV1401 and the candidate antibody 13F4-b21-P3 LALA, implying that their total and active antibody exposures in vivo were roughly equivalent. However, the BAY1213790 antibody showed significant differences in AUC values ​​for both total and active antibody concentrations compared to the two antibodies mentioned above, possibly due to differences in antibody structure, affinity, or metabolic processes (see [link to relevant documentation]). Figure 18 ).

[0153] Table 9 Grouping Design in Mouse Stability Study

[0154]

[0155] Example 13: Stability analysis of candidate antibody 13F4-b21-P3-LALA in monkeys

[0156] The objective of this experiment was to test the in vivo stability of the 13F4-b21-P3 LALA antibody in monkeys. The experimental materials used in this experiment... The cynomolgus macaques (Macaca fascicularis) used were provided by Shanghai Pengli Biotechnology. These monkeys underwent a 6-month washout period, and all vital signs were normal before the experiment. At the beginning of the experiment, blank plasma was collected from 12 monkeys for ADA testing (performed by Shanghai Pengli). Monkeys with negative ADA results were selected, and their plasma was then subjected to an in vitro APTT sensitivity test (using a NOV1401 positive antibody test, performed by Shanghai Pengli Biotechnology). Finally, male monkeys with good linearity in APTT results were selected for the next stage of the experiment.

[0157] For both NOV1401 and 13F4-b21-P3 LALA, two male monkeys with better results were selected from the previous results. Specific administration methods are shown in Table 9. Starting on Day 0, the drug was administered once via hind limb intravenous bolus. Plasma was collected at 0h, 0.5h, 6h, 12h, 1d, 3d, 7d, 14d, 21d, and 28d. APTT / PT testing was performed rapidly within 6 hours of blood collection (performed by Shanghai Pengli Biotechnology). Other plasma samples were anticoagulated with sodium citrate and frozen at -80℃, then sent back to Renhui Biotechnology on dry ice for PK ELISA (Total / ActivemAb) and ADA testing. The PK ELISA method is described in Example 4. For ADA testing, the capture antibody was the test antibody. The sample consisted of monkey plasma injected with the test antibody at different time points. The secondary antibody was Goat anti-Monkey IgG+IgA+IgM(H+L)(HRP) (Mybiosource, catalog number MBS538736), and the procedure was similar to a conventional ELISA.

[0158] Following antibody injection, monkey plasma was collected at different time points for APTT testing. The results showed that the 13F4-b21-P3 LALA antibody could prolong the APTT time in monkeys to a certain extent, increasing it by 1.5-3.5 times after injection (see [link to relevant documentation]). Figure 19 Similar to the positive antibody NOV1401, the APTT delay effect of both antibodies only showed a significant decrease after 21 days, which is similar to the metabolic half-life of ordinary IgG antibodies.

[0159] Table 10 Group Design for Comparative Analysis of Crab-Eating Mammals

[0160]

[0161] The inventors of this invention, through a series of experiments and summarizing the results, discovered that:

[0162] 1) The antibody sequence described in this invention is novel;

[0163] 2) The expression levels of most humanized 13F4 antibodies were higher than those of chimeric 13F4 antibodies (Table 3);

[0164] 3) The number of VH+VL reversion mutations in the second-round humanized antibody 13F4-NOV-b21 was reduced to 5, while the function remained unchanged;

[0165] 4) The in vitro APTT activity of 13F4-b10, based on the curve trend, is superior to that of the positive control antibodies NOV1401, AB023, and BAY1213790. Figure 1 B);

[0166] 5) After adjusting 13F4-NOV-b21 (with a neutral pI value of around 7.15) to 13F4-NOV-b21-p3 antibody (with a pI value greater than 8.0), the proportion of single peaks climbed from 53.96% to 56.81%, indicating improved stability and decreased aggregation during the antibody production process (see Table 7). Moreover, the APTT function of the 13F4-NOV-b21-p3 antibody remained unchanged( Figure 3 ), and the stability performance of the antibody in mice was similar to that of the antibody before modification( Figure 4 );

[0167] 6) In the SPR experiment, the humanized 13F4-b21 (13F4-b21 LALA) series of candidate antibodies with the P329G LALA mutation showed high affinity for human FXI or FXIa factors similar to that of the PC control NOV1401, and was significantly higher than that of the PC control BAY1213790( Figure 6 and 7 );

[0168] 7) The DSC results showed that the stability of the humanized 13F4-b21-P3 (13F4-b21-P3 LALA) with the P329G LALA mutation introduced into hIgG1 was slightly higher than that of NOV1401 at the Tm1 and Tm2 peaks, indicating that the stability of the CH2 and Fab regions of the 13F4-b21-P3LALA antibody might be slightly better than that of the positive control NOV1401( Figure 8 );

[0169] 8) 13F4-b21-P3 LALA only caused a certain degree of delay in the APTT of humans and monkeys and had no effect on the other three coagulation parameters( Figure 9 and 10 );

[0170] 9) The affinity of 13F4-b21-P3 LALA for human FcγRI (CD64), FcγRIIa (CD32a), and FcγRIII (CD16a) was lower than that of the two positive antibodies BAY1213790 and NOV1401, suggesting that the risk of ADCC and ADCP effects of 13F4-b21-P3 LALA in humans might be lower than that of the two positive antibodies( Figure 12-14 );

[0171] 10) The binding ability of the antibody to human C1q: BAY<1213790> 13F4-b21-P3 LALA > NOV1401, suggesting that the candidate antibody 13F4-b21-P3 LALA is less likely to trigger the CDC reaction compared to the positive antibody BAY1213790( Figure 15 );

[0172] 11) The modification of Fc's P329G LALA did not significantly alter the affinity of 13F4-b21-P3 LALA for human FcRn. Figure 16 );

[0173] 12) The modified 13F4-b21-P3 LALA candidate antibody showed relatively stable PK in mice, similar to the positive antibody NOV1401, and superior to the positive antibody BAY1213790. Figure 17 and 18 );

[0174] 13) The candidate antibody 13F4-b21-P3 LALA antibody can prolong the APTT time in monkeys to a certain extent, increasing the APTT time by 1.5-3.5 times after injection. Similar to the positive antibody NOV1401, the APTT delay effect of both only shows a significant decrease after 21 days, which is similar to the half-life of ordinary IgG antibodies. Figure 19 A).

Claims

1. An isolated anti-FXI and / or anti-FXIa antibody or its antigen-binding fragment, which specifically binds to human FXI or FXIa; wherein the isolated antibody or its antigen-binding fragment comprises: (a) The heavy chain variable region of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:22; and (b) The light chain variable region of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:

25.

2. The isolated antibody or antigen-binding fragment as described in claim 1, wherein, The isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:5 and an amino acid sequence having at least 60% identity with it; preferably, the isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:5 and an amino acid sequence having at least 80% identity with it; and a light chain variable region selected from SEQ ID NO:15 and an amino acid sequence having at least 60% identity with it; preferably, a light chain variable region selected from SEQ ID NO:15 and an amino acid sequence having at least 80% identity with it.

3. The isolated antibody or antigen-binding fragment as described in claim 1, wherein, The isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:5 and an amino acid sequence having at least 97% identity with it; and a light chain variable region selected from SEQ ID NO:15 and an amino acid sequence having at least 97% identity with it.

4. The isolated antibody or antigen-binding fragment as described in claim 1, wherein, The isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; and a light chain variable region selected from SEQ ID NO:17 or SEQ ID NO:

18.

5. The isolated antibody or antigen-binding fragment as described in claim 1, wherein, The isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:7 or SEQ ID NO:10; and a light chain variable region selected from SEQ ID NO:18, SEQ ID NO:20 or SEQ ID NO:

21.

6. The isolated antibody or antigen-binding fragment as described in claim 1, wherein, The isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises a heavy chain variable region selected from SEQ ID NO:10 or SEQ ID NO:22; and a light chain variable region selected from SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:

25.

7. The isolated antibody or antigen-binding fragment as described in claim 1, wherein, The isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment comprises: The heavy chain of SEQ ID NO:30 and the light chain of SEQ ID NO:31; or The heavy chain of SEQ ID NO:32 and the light chain of SEQ ID NO:33; or The heavy chain of SEQ ID NO:34 and the light chain of SEQ ID NO:

35.

8. The isolated antibody or antigen-binding fragment of claim 1, wherein, The isolated anti-FXI and / or anti-FXIa antibodies or antigen-binding fragments are human monoclonal antibodies.

9. The isolated antibody or antigen-binding fragment of claim 1, wherein, The isolated antibody or its antigen-binding fragment comprises: the heavy chain shown in SEQ ID NO:32 and the light chain shown in SEQ ID NO:

33.

10. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

11. The pharmaceutical composition of claim 10, further comprising one or more pharmaceutically acceptable adjuvants, carriers, excipients, preservatives, or combinations thereof.

12. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-9, or the pharmaceutical composition as described in any one of claims 10-11, in the preparation of a medicament for inhibiting blood clot formation in a subject.

13. The use as described in claim 12, wherein, The stated use is in the preparation of a pharmaceutical agent for the treatment or prevention of thrombosis or thrombosis-related complications or conditions, and the amount of said antibody does not impair the hemostatic function of the subject.

14. A method for preparing the antibody according to any one of claims 1-9, wherein, The method includes cloning the nucleic acid encoding the antibody into an expression vector and expressing it in a host cell.

15. The method of claim 14, wherein, The method also includes purifying the expressed antibody from host cells.

Images