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, which specifically binds to human FXI or FXIa, and provides a drug targeting FXI that effectively prevents or treats thrombosis with minimal side effects to meet medical needs. Because currently approved anticoagulants interfere with hemostasis, leading to an increased risk of bleeding, there is a significant unmet medical need for safer anticoagulants. Although thrombosis can be treated or prevented with anticoagulants (e.g., heparin, warfarin, aspirin, or factor Xa inhibitors), these treatments have significant adverse effects. Genetic and pharmacological evidence in humans and animals suggests that lowering coagulation factor XI (FXI) can successfully prevent and manage thrombosis with minimal bleeding.

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] Thrombosis is a serious blood vessel blockage disease, where a blood clot forms one or more blood clots inside a blood vessel. These clots can obstruct blood flow and may break off and move elsewhere. If a moving clot blocks a critical area, it can lead to fatal conditions such as stroke and coronary heart disease.

[0003] Factor XI (FXI) deficiency was first reported in 1955. It is a rare congenital hemorrhagic disorder, more common in Jewish people. 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 rare in FXI deficiency, occurring only after surgery or trauma. The fact that FXI deficiency or inhibition has no significant effect on experimentally induced bleeding in animals suggests that the FXI amplification pathway may be less important for normal hemostasis. Conversely, animal models of arterial, venous, and cerebral thrombosis show that elevated plasma FXI levels may contribute to thromboembolic diseases. There is also some evidence that patients with severe FXI deficiency experience fewer ischemic strokes or venous thrombosis. Therefore, FXI is an attractive target for the treatment and prevention of thromboembolism, as it can effectively prevent thrombus formation without affecting normal hemostasis.

[0004] Chinese patent application 202311845986.8 discloses a double-stranded ribonucleic acid (BRNA), a BRNA modification, a BRNA conjugate, a pharmaceutical composition, and uses for inhibiting coagulation factor XI gene expression. The BRNA comprises a sense strand and an antisense strand, the sense strand and antisense strand being complementary and / or substantially anticomplementary to form a double-stranded region of the BRNA. The sense strand contains sequence A, which differs from the target sequence by no more than 3 nucleotides from at least 15 consecutive nucleotides. The antisense strand contains sequence B, which differs from the target sequence by no more than 3 nucleotides from the anticomplementary sequence of at least 15 consecutive nucleotides. The target sequence is selected from any one of the nucleotide sequences shown in SEQ ID NO: 1–10 and SEQ ID NO: 834–843.

[0005] 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).

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] Chinese Patent 201680048982.9 discloses an isolated anti-FXI and / or anti-FXIa antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment binds to the catalytic domain of factor XI (FXI) and / or activated FXI (FXIa), wherein the isolated antibody or its antigen-binding fragment comprises: (a) the heavy chain variable region CDR1 of SEQ ID NO:23; the heavy chain variable region CDR2 of SEQ ID NO:24; the heavy chain variable region CDR3 of SEQ ID NO:25; the light chain variable region CDR1 of SEQ ID NO:33; the light chain variable region CDR2 of SEQ ID NO:34; and the light chain variable region CDR3 of SEQ ID NO:35; (b) the heavy chain variable region CDR1 of SEQ ID NO:26; the heavy chain variable region CDR2 of SEQ ID NO:27; the heavy chain variable region CDR3 of SEQ ID NO:28; and the light chain variable region CDR1 of SEQ ID NO:36; SEQ ID NO:27; the heavy chain variable region CDR2 of SEQ ID NO:28; and the light chain variable region CDR3 of SEQ ID NO:36; SEQ ID NO:27; the heavy chain variable region CDR2 of SEQ ID NO:28; and the light chain variable region CDR3 of SEQ ID NO:27; SEQ ID NO:28; and the light chain variable region CDR2 of SEQ ID NO:29; ... (c) Light chain variable region CDR2 of SEQ ID NO:37; and light chain variable region CDR3 of SEQ ID NO:38; (d) Heavy chain variable region CDR1 of SEQ ID NO:43; Heavy chain variable region CDR2 of SEQ ID NO:44; Heavy chain variable region CDR3 of SEQ ID NO:45; Light chain variable region CDR1 of SEQ ID NO:47; Light chain variable region CDR2 of SEQ ID NO:37; and Light chain variable region CDR3 of SEQ ID NO:15; or (d) Heavy chain variable region CDR1 of SEQ ID NO:46; Heavy chain variable region CDR2 of SEQ ID NO:4; Heavy chain variable region CDR3 of SEQ ID NO:5; Light chain variable region CDR1 of SEQ ID NO:33; Light chain variable region CDR2 of SEQ ID NO:14; and Light chain variable region CDR3 of SEQ ID NO:15.

[0011] 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.

[0012] 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.

[0013] The coagulation cascade mechanism in vivo 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 can intersect and activate a common pathway, resulting in the conversion of prothrombin to thrombin, ultimately forming a cross-linked fibrin clot. Evidence suggests that the intrinsic coagulation pathway is important in the expansion phase of coagulation, while the extrinsic pathway and the common pathway are more involved in the initiation and diffusion phases. An ideal anticoagulant should effectively prevent thrombus formation without increasing the risk of bleeding.

[0014] Currently, the most commonly used clinical anticoagulants include: parenteral anticoagulants (e.g., heparin), coumarin anticoagulants (e.g., warfarin), antiplatelet drugs (e.g., aspirin), and cyclopyridine (Bolivar). However, all of these drugs carry a risk of bleeding.

[0015] Because currently approved anticoagulants can interfere with hemostasis and increase the risk of bleeding, there is a significant unmet medical need for safer anticoagulants. Summary of the Invention

[0016] 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.

[0017] 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.

[0018] In some preferred embodiments, the isolated anti-FXI and / or anti-FXIa antibodies or their antigen-binding fragments of the present invention comprise:

[0019] (a) Heavy chain variable region CDR1 shown in SEQ ID NO:2 in Table 1; heavy chain variable region CDR2 shown in SEQ ID NO:3 or SEQ ID NO:6; and heavy chain variable region CDR3 shown in SEQ ID NO:4; and

[0020] (b) Light chain variable region CDR1 shown in SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20 in Table 1; light chain variable region CDR2 shown in SEQ ID NO:9; and light chain variable region CDR3 shown in SEQ ID NO:10.

[0021] 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:1 in Table 1 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:1, 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:7 in Table 1 or a sequence having at least 90%, 95%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO:7, wherein the light chain variable domain combines with the heavy chain variable domain to form an antigen-binding site for FXI and / or FXIa.

[0022] 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:1 and having at least 95% identity with it, and a light chain variable region selected from SEQ ID NO:7 and having at least 95% 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:1 and having at least 97% identity with it, and a light chain variable region selected from SEQ ID NO:7 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:1 and having at least 98% or 99% identity with it, and a light chain variable region selected from SEQ ID NO:7 and having at least 98% or 99% identity with it.

[0023] As some specific 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:1 or SEQ ID NO:5, and a light chain variable region selected from SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19.

[0024] As some preferred 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:1 and a light chain variable region selected from SEQ ID NO:13 or SEQ ID NO:15.

[0025] As some other preferred 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:5 and a light chain variable region selected from SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:15.

[0026] As a preferred embodiment of the present invention, the separable anti-FXI and / or anti-FXIa antibody or antigen-binding fragment is an antibody or antigen-binding fragment comprising the heavy chain variable region of SEQ ID NO:5 and the light chain variable region of SEQ ID NO:7.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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

[0043] Figure 1 This is a graph showing the results of the analysis of the binding of the modified antibody to FXI or FXIa (the active form of FXI) protein by ELISA. In the graph, h0 is RH003-h0, which is RH003.

[0044] Figure 2 The results show the APTT assay of the modified monoclonal antibody, including: A. APTT assay results of human plasma with antibody concentration of 3 μg / ml; B. APTT assay results of human plasma with different concentrations of antibody; C. Comparison of human plasma APTT curves of RH003-h6 ​​and RH003-h8 with NOV1401, AB023, and BAY1213790.

[0045] Figure 3This figure shows a comparison of the stability of total IgG and active IgG of different antibodies in mouse plasma, including: A. Comparison of total IgG and active IgG pK of RH003-h0, h6, and h8; B. Comparison of total IgG pK and active IgG pK of the three antibodies; C. Comparison of total IgG pK of RH003-h6, BAY1213790, and NOV1401; D. Comparison of active IgG pK of RH003-h6, BAY1213790, and NOV1401. In the figure, BAY represents BAY1213790, and NOV represents NOV1401.

[0046] Figure 4 The diagram shows a comparison of pharmacokinetic (PK) levels in mice before and after antibody modification. Specifically: A. Comparison of total IgG and active IgG PK in RH003; B. Comparison of total IgG and active IgG PK in RH003-h6 ​​and RH003-h8; C. Schematic diagram of total IgG detection; D. Schematic diagram of active IgG detection.

[0047] Figure 5 The results show antibody expression, purification, and preliminary analysis, including: A. Protein expression and purification results; B. SDS-PAGE analysis showing the left lane for non-reducing antibody electrophoresis and the right lane for reducing antibody electrophoresis. The molecular weight of the non-reducing antibody is approximately 150 kDa, observed on the left side of the SDS-PAGE results. Under reducing conditions, two different bands are visible on the right side of the results, corresponding to molecular weights of 50 kDa and 25 kDa, respectively; C. Size exclusion chromatography (SEC) purity peak. In the figure, NOV represents NOV1401, and BAY represents BAY1213790.

[0048] Figure 6 The results show the affinity of different antibodies for human FXI, including: A. SPR flowchart; B. SPR results for different antibodies; E. SPR specificity values ​​for different antibodies; F. Parameters of the SPR experimental method.

[0049] Figure 7 The results show the affinity of different antibodies for human FXIa, including: A. SPR flowchart; B. SPR results for different antibodies; E. SPR-specific values ​​for different antibodies; F. Parameters of the SPR experimental method.

[0050] Figure 8 The experimental results of differential scanning calorimetry (DSC) are shown;

[0051] Figure 9 The results of four coagulation parameters for different antibodies in human plasma are shown. Among them, APTT is activated partial thromboplastin time, PT is partial thromboplastin time, FIB represents fibrinogen, and TT is prothrombin time.

[0052] Figure 10 The results of four coagulation parameters for different antibodies in monkey plasma are shown. Among them, APTT is activated partial thromboplastin time, PT is partial thromboplastin time, FIB represents fibrinogen, and TT is prothrombin time.

[0053] Figure 11 The APTT activity of different antibodies in human or monkey plasma in vitro was demonstrated;

[0054] Figure 12 The comparison of the affinity of different antibodies for human FcγRI (CD64) is shown, where: A. SPR results of different antibodies; D. SPR specificity values ​​of different antibodies; E. Parameters of the SPR experimental method;

[0055] Figure 13 The comparison of the affinity of different antibodies for human FcγRIIa (CD32a) is shown, where: A. SPR results of different antibodies; D. SPR specificity values ​​of different antibodies; E. Parameters of the SPR experimental method;

[0056] Figure 14 The comparison of affinity of different antibodies to human FcγRIII (CD16a) is shown, where: A. SPR results of different antibodies; D. SPR specificity values ​​of different antibodies; E. Parameters of SPR experimental method;

[0057] Figure 15 The ELISA comparison of the binding of different antibodies to human C1q is shown;

[0058] Figure 16 The affinity of different antibodies for human FcRn (pH 6.0) is compared, where: A. SPR results of different antibodies; D. SPR specificity values ​​of different antibodies; E. Parameters of the SPR experimental method;

[0059] Figure 17 The results of PK ELISA experiments with different antibodies are shown. Among them, AC shows the total IgG and active IgG PK ELISA curves of NOV1401, BAY1213790 and RH003-h6 ​​LALA, while D and E show the comparison of total IgG or active IgG PK ELISA of the three monoclonal antibodies.

[0060] Figure 18 The correlation of area under the curve (AUC) of mouse PK ELISA is shown, where: A. total IgG AUC; B. active IgG AUC. In the figure, ns represents no significant difference, ** represents a significant difference, and P<0.05.

[0061] Figure 19 The results of the Cyno APTT and PT studies in RH003-h6 ​​LALA monkeys are presented, including: A. APTT assay in plasma of RH003-h6 ​​LALA injected monkeys; B. PT assay in plasma of RH003-h6 ​​LALA injected monkeys; C. Detection of APTT and FXI activities in plasma of RH003 injected monkeys.

[0062] Figure 20 The results of cynomolgus monkey PK ELISA and ADA assays for RH003-h6 ​​LALA monkeys are shown, including: A. RH003-h6 ​​LALA injected monkey (#201); B. RH003-h6 ​​LALA injected monkey (#202);

[0063] Figure 21 The results of the PK ELISA comparison of cynomolgus monkeys with different mAbs are shown. Detailed Implementation

[0064] 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.

[0065] Example 1: RH003 Sequence Modification

[0066] The modified sequences are shown in Table 1. CDR regions are indicated by italics and underline, and mutation sites are indicated by boxes. The Kabat numbering method was used to calculate the CDR regions. The combination relationship between plasmids and antibodies is shown in Table 2. Gene synthesis, plasmid construction, cell transfection, and antibody production and purification were all performed at Biointron. The yields of the modified RH003-h2, h3, h6, h8, and h9 were significantly improved (Table 3), with the most significant improvement observed in RH003-h3 and h9. The SEC purity of the modified antibodies was higher than 95%, slightly better than that of the unmodified RH003 (Table 3).

[0067] Table 1. Variable region sequence of the modified monoclonal antibody.

[0068]

[0069]

[0070] Note: VH, variable region of heavy chain; VL, variable region of light chain.

[0071] Table 2 Sequence Combinations of Modified Monoclonal Antibodies

[0072]

[0073] Note: HC, heavy chain; LC, light chain.

[0074] Table 3. Expression level and purity of the modified antibody

[0075]

[0076] Example 2: Binding analysis of the modified antibody with FXI or FXIa

[0077] The purpose of this embodiment is to determine whether the modified antibody retains its binding affinity to the antigen.

[0078] Experimental Procedure: Coat each well of the microplate with 100 μL of coating buffer, then cap and incubate overnight (12-18 hours) at 2-8°C. Aspirate from the wells and wash once with >200 μL of washing buffer / well. Invert and pat gently on absorbent paper to remove excess liquid. Block the plate with 200 μL / well of blocking buffer (1×PBS + 4% milk) at room temperature for 1 hour. Aspirate, invert, and pat gently on absorbent paper to remove excess liquid. Prepare standard and sample diluents in the blocking buffer. Transfer 100 μL of standard (two copies) and sample to the designated wells. Incubate at room temperature for 1 hour, then gently and continuously shake (~500 rpm). Aspirate from the wells and wash three times with >200 μL of washing buffer / well. Invert and pat gently on absorbent paper to remove excess liquid.

[0079] Dilute the primary antibody solution with blocking buffer. Add 100 μL of primary antibody solution to each well and incubate at room temperature for 2 hours, gently shaking continuously (~500 rpm). Aspirate the liquid from the microwells and wash each well three times with >200 μL of washing buffer, inverting and tapping on absorbent paper to remove excess liquid. Dilute the secondary antibody 2,500 times with blocking buffer to prepare the working solution. Add 100 μL of the working solution to each well and incubate at room temperature for 30 min. Aspirate the liquid from the microwells and wash each well five times with >200 μL of washing buffer, inverting and tapping on absorbent paper to remove excess liquid. Add 100 μL of TMB substrate solution (Biopanda, TMB-S-004) to each well and incubate at room temperature for 30 min. Add 100 μL of stop solution (Solarbio, C1058) to each well. The absorbance at 450 nm was measured within 30 minutes after the addition of the stop solution. The results were calculated using log-log or four-parameter curve fitting.

[0080] The materials used are shown below:

[0081] 1) Coating solution: 1x PBS, containing 100 ng / well of human FXI or human FXIa (ERL, catalog number: HFXI 1111, HFXIa1111a);

[0082] 2) Blocking buffer: 1×PBS + 5% milk (BD, 23200);

[0083] 3) Washing buffer PBST (1×PBS containing 0.05% (v / v) Tween)

[0084] 3) Primary antibody: RH003-h0~h11 mAbs.

[0085] 4) Secondary antibody: Mouse anti-human IgG1 Fc antibody HRP (1:2,500, Invitrogen, MH1715);

[0086] Experimental Results: ELISA analysis of the binding of the modified antibodies to FXI or FXIa (the active form of FXI) proteins showed that all modified antibodies specifically bound to human FXI and FXIa, and the binding affinity was similar to that of RH003. Please refer to [link to relevant documentation]. Figure 1 This indicates that the sequence modification did not weaken the affinity of the antibody for FXI and FXIa.

[0087] Example 3 Activated Partial Thromboplastin Time (APTT) Test

[0088] Experimental procedure: Dilute the antibody to eight concentration gradients with 1xPBS. Mix 75 μL of antibody with 75 μL of human plasma and incubate at room temperature for 5 min. Add 25 μL of Dade... Activated Cephaloplastin Reagent (SIEMENS, SMN10445709, batch number 557290) was mixed with 50 μL of antibody-plasma mixture and incubated at 37°C for 3 min. Then, 25 μL of 0.025 M CaCl2 was added and mixed. 1xPBS was mixed with plasma as a baseline, and coagulation time was measured using a coagulation analyzer (Sysmex CA660).

[0089] Experimental results: Please see the appendix. Figure 2 And its accompanying diagram. In the 3 μg / ml concentration test, the APTT clotting times of RH003-h2, h3, h6, h8, and h9 were similar to those of h0. Please refer to the attached diagram. Figure 2 A. Subsequently, these antibodies were tested at concentrations of 2.34, 4.68, 9.37, 18.75, 37.5, 75, 150, and 300 (nM), and the results were consistent with the previous observations. The effects of RH003-h6 ​​and RH003-h8 on APTT were similar to those of NOV1401, and showed a better trend than AB023 and BAY1213790. Please refer to the appendix. Figure 2 C.

[0090] Example 4: PK analysis of the modified antibody in mice

[0091] Experimental Procedure: On day 0, mice (male ICR mice around 8 weeks old) were administered 1 mg / kg of different antibodies. Plasma samples of 50 μL were collected from mice at t2h, 6h, 1d, 2d, 3d, 4d, 5d, 7d, 14d, 21d, and 28d and anticoagulated using sodium citrate buffer. On day 28, the plasma concentrations of total IgG and active IgG were quantitatively determined using ELISA. (See schematic diagram). Figure 4 C and 4D. PK ELISA uses the standard ELISA protocol, with the following reagents:

[0092] Capture Antibody: Sheep Anti-Human IgG (Fc) Monkey Adsorbed for Total IgG Detection (Selectscience- BindingSite ,AU004M); Human coagulation factor XI (ERL, HFXI1111) for the detection of active IgG;

[0093] ● Antibody detection: Goat anti-Human IgG-h+l HRP Conjugated MinX monkey (Bethyl, A80-319P);

[0094] ● Carbonate buffer (CBS): Dissolve 1.5g Na2CO3 and 2.93g NaHCO3 in 1000mL of deionized water and adjust the pH to 9.6;

[0095] ●Blocking buffer: PBS containing 5% skim milk;

[0096] • Detection buffer: PBS + 0.5% BSA + 0.35M NaCl + 0.05% Tween 20 + 0.25% CHAPS + 5mM EDTA;

[0097] • Sample diluent: Analytical buffer containing 1% mouse plasma;

[0098] • Antibody diluent: 0.05% PBST containing 0.5% BSA;

[0099] • Washing buffer (0.05% PBST): Add 0.5 mL Tween 20 to 1000 mL 1xPBS and mix well; • TMB: TMB-S-002 (Huzhou Yingchuang);

[0100] • ELISA stop solution: Solarbio product number C1058.

[0101] Experimental results: Please see the appendix. Figure 3 Appendix Figure 4See attached figures for explanation. As the test time increased, the plasma concentrations of all samples gradually decreased. The slower the decrease in the PK ELISA curve, the better the stability of the antibody. Judging from the decreasing trend, RH003-h6 ​​is more stable than RH003-h8. Please refer to the attached figures. Figure 3 A. In contrast, the total IgG PK curve and the active IgG PK curve of RH003 began to separate 3 days after injection, and the plasma concentration of active IgG decreased significantly. Please refer to [link to relevant documentation]. Figure 4 A indicates that the CDR region of the antibody has been inactivated, but the antibody has not been completely degraded. Therefore, RH003-h6 ​​may have a longer duration of efficacy compared to RH003.

[0102] Example 5: Candidate antibody for introducing P329G LALA into the Fc region

[0103] P329G LALA was introduced into the RH003-h6 ​​antibody (RH003-h6 ​​LALA, protein sequence shown in Table 4 below). Plasmid construction, antibody production, and purification were all performed at Biointron. The protein expression and purification results of the candidate antibody and two positive control antibodies are attached. Figure 5 As shown in Figure A. After two rounds of purification, most antibodies achieved a purity of over 99%. All antibodies showed ideal bands on SDS-PAGE gels under both reducing and non-reducing conditions; please refer to the appendix. Figure 5 B and its accompanying figures; the SEC results for the three antibodies also showed a single main peak, please refer to the appendix. Figure 5 C.

[0104] Table 4 Protein Sequences

[0105]

[0106]

[0107] Example 6: Affinity determination with human FXI and FXIa

[0108] In this embodiment, the affinity of RH003-h6 ​​LALA for FXI and FXIa was determined using SPR technology.

[0109] Experimental Procedure: Anti-human IgG (Fc) antibody was immobilized on a CM5 chip using a standard amine conjugation kit (Cytiva) at pH 5.0, 10 mM sodium acetate, and a 0.5 mg / mL anti-human IgG (Fc) antibody solution. The anti-human IgG (Fc) antibody was immobilized to a level >9000 RU using running buffer HBS-EP. 1 μg / mL antibody solutions were prepared to capture a immobilization level of 50 RU at a flow rate of 10 μl / min. Subsequently, a series of FXI or FXIa concentrations (0.005–0.33 μg / mL) were applied to HBS-EP at a flow rate of 30 μL / min for 60–180 s, with the dissociation phase monitored for 100 s and 1000 s. The surface was regenerated by washing with 3 MM MgCl2 at a flow rate of 30 μl / min for 30 s. Data were then analyzed using Biacore T200 evaluation software; please refer to the appendix. Figure 6 A and 6F.

[0110] In the SPR experiment, RH003-h6 ​​LALA showed similar affinity to human FXI and FXIa as NOV1401, both exhibiting high affinity. Please refer to the appendix. Figure 6 C and 6D, Appendix Figure 7 C and 7D have an affinity 3-4 orders of magnitude higher than BAY1213790 (see appendix). Figure 6 (B and 7B). For detailed numerical results of KD, ka, and KD of the three antibody SPRs, please refer to the appendix. Figure 6 E and 7E.

[0111] Example 7: Differential Scanning Calorimetry (DSC)

[0112] The DSC experiments were conducted by Shanghai Chempartner Co., Ltd. The DSC results showed that the Tm1 and Tm2 peak temperatures of RH003-h6LALA were both slightly higher than those of NOV1401. Please refer to the appendix. Figure 8 This suggests that the CH2 and Fab regions of RH003-h6LALA may be more stable than the positive control NOV1401.

[0113] Example 8: Coagulation Four Tests

[0114] The experimental objectives of this embodiment include: 1. evaluating the effect of antibodies on the coagulation system; 2. evaluating the safety of antibodies.

[0115] The four coagulation tests were performed at Hkeybio.

[0116] Experimental results: The prolongation effect of 50 nM RH003-h6 ​​LALA on human plasma APTT exceeded the upper limit of 100 s for machine detection time. Please refer to the appendix. Figure 9Compared to the PBS negative control, 50 nM RH003-h6LALA significantly delayed APTT in monkey plasma by approximately 2-fold, comparable to the results observed in the 50 nM NOV1401 group. (See attached image for details.) Figure 10 The results indicate that RH003-h6 ​​LALA prolongs the intrinsic clotting time of APTT in humans and monkeys, while having little effect on extrinsic clotting time (PT), fibrinogen (FIB), and triglycerides (TT). Please refer to the appendix. Figure 9 and attached Figure 10 .

[0117] Example 9 In vitro APTT curve

[0118] To further investigate the in vitro APTT curves of human and monkey plasma, we diluted the candidate antibody into different concentration gradients to test the effect of different antibodies on human in vitro APTT. Specific experimental methods are described in Example 3.

[0119] As attached Figure 11 As shown, the APTT curve of RH003-h6 ​​LALA in human plasma is similar to that of NOV1401, with EC50 values ​​of 34.22 nM and 30.44 nM, respectively. The upper plateau of RH003-h6 ​​LALA is slightly higher (see...). Figure 11 A). The APTT curve of BAY1213790 is relatively weak, with an EC50 of 40.25 nM.

[0120] Example 10: Detection of the effect of the antibody Fc terminus

[0121] Tilman et al. found that the P329G LALA mutation at the Fc terminus of antibody IgG1 can significantly eliminate ADCC, ADCP, and CDC effects. 1 Furthermore, selecting the Fc isotype G1m3(CH1)+nG1m1,2(EEM,CH3) at the Fc end of the monoclonal antibody is beneficial in reducing the immunogenicity of the antibody. 2 In this study, the Fc terminus of the RH003-h6 ​​LALA antibody, i.e., the mutation described above, was introduced. Comparison of our Fc sequence with the Fc sequences of positive NOV1401 and BAY1213790 revealed differences at several amino acid points.

[0122] 1) Antibody binds to human Fcγ receptor (FcγR)

[0123] The affinity of the purified antibody with commercial human FcγRI (CD64) (Sinobiological, CAT#10256-H08H), human FcγRIIa (CD32a) (Sinobiological, CAT#10374-H08), and human FcγRIII (CD16a) (Sinobiological, CAT#10389-H08H1) was determined using SPR technology. The specific experimental procedures were as described in Example 6.

[0124] The experimental results showed that the affinities of each antibody for FcγRI (CD64), FcγRIIa (CD32a), and FcγRIII (CD16a) were as follows: BAY<1213790> NOV1401 > RH003-h6 LALA. Please refer to the appendix Figure 12 - Appendix Figure 14 . This result indicates that the possibility of RH003-h6 LALA stimulating in vivo ADCC and ADCP responses is relatively lower compared to the two positive control antibodies.

[0125] 2) Affinity of the antibody with human C1q (hC1q)

[0126] The experimental procedure: The affinity was detected using a human C1q ELISA kit (Thermo Fisher, BMS2099).

[0127] The experimental results showed that the binding of hC1q to RH003-h6 LALA and NOV1401 was extremely weak, and the binding to BAY1213790 was relatively strong. Please refer to the appendix Figure 15 . This result indicates that the possibility of RH003-h6 LALA and NOV1401 stimulating in vivo complement-dependent cytotoxicity (CDC) responses is quite low.

[0128] Example 11 Determination of the antibody-hFcRn affinity

[0129] The experimental procedure: The affinity of the Fc region of the antibody with human hFcRn (Sinobiological, catalog number CT009-H08H) was determined using SPR technology.

[0130] The experimental results showed that the affinity of RH003-h6 LALA for hFcRn was close to that of BAY1213790 and NOV1401. The P329G LALA mutation in the Fc region did not significantly change its affinity with hFcRn. Please refer to Figure 16 . The three antibodies showed obvious binding to hFcRn only at pH 6.0. Please refer to Figure 16 .

[0131] Example 12 PK analysis of RH003-h6 ​​LALA in mice

[0132] The purpose of this study was to evaluate the pharmacokinetic (PK) performance of RH003-h6 ​​LALA in mice.

[0133] Experimental steps:

[0134] Blank plasma was collected from all mice before injection. Antibody at 1 mg / kg was then administered via tail vein. Antibody grouping design is shown in Table 4. Plasma was collected at 0.5 h, 2 h, 1 d, 3 d, 7 d, 14 d, 21 d, and 28 d post-injection, anticoagulated with sodium citrate, and frozen at -80°C. Plasma measurements were then taken. For more detailed information on the mouse PK ELISA procedure, please refer to Example 4. P-values ​​were analyzed using standard one-way ANOVA.

[0135] Experimental results showed that the PK curves of RH003-h6 ​​LALA and NOV1401 showed no significant difference in trend, while the PK stability of BAY1213790 was somewhat weaker. Area under the curve (AUC) analysis indicated that there was no significant difference in the AUC values ​​of total IgG and active IgG between NOV1401 and RH003-h6 ​​LALA; the AUC values ​​of total IgG and active IgG concentrations of BAY1213790 were much lower than those of the two antibodies mentioned above (P<0.05). Please see the appendix. Figure 17 and attached Figure 18 .

[0136] Table 5 Experimental design of mouse PK study

[0137]

[0138] Example 13 Monkey APTT / PT Detection and PK ELISA

[0139] Experimental objective: To evaluate the stability and duration of efficacy of RH003-h6 ​​LALA and NOV1401 in monkeys.

[0140] Experimental steps:

[0141] Twelve non-naive male rhesus monkeys were provided by Shanghai PharmaLegacy, and a comprehensive physical examination was performed, revealing no abnormalities. Blood samples were collected from these animals before the experiment to detect residual ADA levels. Animals with negative results were selected for further experiments. Finally, the APTT sensitivity of the animals was tested, and positive antibodies were selected as test samples. Animals showing a good linear relationship in the monkey APTT response were selected for further study. On day 0, the monkeys were intravenously injected into their hind limbs; injection details are shown in Table 5. Plasma was collected from monkeys at 0h, 0.5h, 6h, 12h, 1d, 3d, 7d, 14d, 21d, and 28d. APTT / PT measurements were performed within 4 hours of blood collection (performed by PharmaLegacy). The remaining plasma was anticoagulated with sodium citrate and then frozen at -80°C. Blood drug concentration and ADA levels were then measured in the monkey plasma. The specific method for the PK ELISA is described in Example 4. The capture antibody for ADA is the antibody sample, which is monkey plasma after injection. The detection antibody is goat anti-monkey IgG+IgA+IgM(H+L)(HRP) (Mybiosource, catalog number: MBS538736). The specific method is similar to that of conventional ELISA.

[0142] The results showed that RH003-h6 ​​LALA prolonged the APTT time by 1.3 to 2 times, with a significant delay observed only after 21 days, consistent with the half-life of conventional IgG antibodies in humans. In historical data, the APTT prolongation effect of RH003 antibody significantly weakened after 7 days, and FXI antigen activity gradually recovered 7 days after antibody injection. Please refer to the appendix. Figure 19 C. Therefore, the modification in this invention effectively prolongs the duration of antibody activity in monkeys.

[0143] In monkey #201, the total PK ELISA levels and active PK ELISA levels of the RH003-h6 ​​LALA candidate antibody gradually decreased, and no sudden decrease was observed after 7 days. This indicates that the antibody is relatively stable, similar to conventional IgG antibodies. Please refer to the appendix. Figure 20 A.

[0144] The PK ELISA curves of the RH003-h6 ​​LALA antibody and the positive control NOV1401 antibody groups showed similar trends, indicating that the two antibodies were comparable in stability in monkeys (see Appendix). Figure 21 A and 21B). The distribution half-life (T1 / 2α) and elimination phase (T1 / 2β) are also similar (see Appendix). Figure 21 C) indicates that the two antibodies were similar in distribution, elimination trend and time in monkeys.

[0145] Table 5 Group Design for Monkey PK Analysis

[0146]

[0147] The inventors of the present invention summarized a series of experimental results and found that:

[0148] 1) Although there are other FXI antibodies currently undergoing clinical trials, the monoclonal antibody sequences described in the present invention are completely different from the antibody sequences undergoing clinical trials. The sequences in the present invention are mutated based on the publicly available sequence RH003 and new technical features are obtained;

[0149] 2) The experimental results shown in Table 3 indicate that the cell productivity of the modified RH003-h2, h3, h6, h8, and h9 is significantly improved, among which RH003-h3 and h9 show the most significant improvement;

[0150] 3) Attached Figure 2 C shows that in the human plasma APTT assay, the activity of RH003-h6 is better than that of the positive controls NOV1401, AB023, and BAY121,3790;

[0151] 4) Attached Figure 4 A and attached Figure 4 B show that compared with RH003, the total PK curves and the PK curves of the active ingredients of the modified RH003-h6 and RH003-h8 are basically the same, indicating an increase in the stability of the modified antibodies;

[0152] 5) Attached Figure 12 - Attached Figure 14 shows that after replacing the Fc region of RH003-h6 from wild-type IgG1 with the P329G LALA mutation (RH003-h6 LALA), by measuring the affinity with human FcγRI (CD64), human FcγRIIa (CD32a), and human FcγRIII (CD16a), it was found that the Fc mutation effectively reduced the binding of RH003-h6 LALA to the three human FcγR receptors. Compared with two positive antibodies (BAY121,3790 and NOV1401), the affinity of RH003-h6 LALA for the three FcγR receptors is lower;

[0153] 6) Attached Figure 15 shows that the ability of RH003-h6 LALA to bind to human C1q: BAY121,3790 > NOV1401 ≈ RH003-h6 LALA; ;

[0154] 7) Attached Figure 16 shows that the P329G LALA modification did not significantly change the binding affinity of RH003-h6LALA to human FcRn;

[0155] 8) Appendix Figure 18 The experimental results shown indicate that the Tm1 and Tm2 values ​​of RH003-h6 ​​LALA in DSC (differential scanning calorimetry) are slightly higher than those of NOV1401, suggesting that the CH2 and Fab fragments of RH003-h6 ​​LALA may be relatively more stable than the PC control.

[0156] 9) Appendix Figure 9 and attached Figure 10 The experimental results shown indicate that RH003-h6 ​​LALA has a certain degree of prolongation of APTT in humans and monkeys, with minimal impact on other coagulation indicators;

[0157] 10) Appendix Figure 17 and attached Figure 18 The experimental results shown indicate that the pharmacokinetic results in mice show that the total antibody and antibody active fraction of RH003-h6 ​​LALA are similar in stability to those of NOV1401 in mice, but superior to BAY1213790.

[0158] 11) Appendix Figure 21 The experimental results shown indicate that RH003-h6 ​​LALA exhibits PK characteristics similar to NOV1401 in monkeys; compared with RH003 (see appendix) Figure 19 C), the APTT duration of RH003-h6 ​​LALA was significantly prolonged (see Appendix). Figure 19 A).

[0159] References

[0160] The references listed below are all incorporated herein by reference in their entirety, as if fully described herein.

[0161] 1.Schlothauer,T.et al.Novel human IgG1 and IgG4 Fc-engineeredantibodies with completely abolished immune effectorfunctions.ProteinEngineering,Design andSelection 29,457–466(2016).2.Webster,C.I.etal.Acomparison ofthe abilityofthe human IgG1 allotypes G1m3 and G1m1,17to stimulate T-cell responses from allotype matched and mismatcheddonors.MAbs 8,253–263(2016)。

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) Heavy chain variable region CDR1 of SEQ ID NO:2; heavy chain variable region CDR2 of SEQ ID NO:3 or SEQ ID NO:6; and heavy chain variable region CDR3 of SEQ ID NO:4; and (b) Light chain variable region CDR1 of SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20; light chain variable region CDR2 of SEQ ID NO:9; and light chain variable region CDR3 of SEQ ID NO:

10.

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:1 and an amino acid sequence having at least 95% identity with it; and a light chain variable region selected from SEQ ID NO:7 and an amino acid sequence having at least 95% 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:1 and an amino acid sequence having at least 97% identity with it; and a light chain variable region selected from SEQ ID NO:7 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:1 or SEQ ID NO:5; and a light chain variable region selected from SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:

19.

5. The isolated antibody or antigen-binding fragment as described in claim 4, 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:1; and a light chain variable region selected from SEQ ID NO:13 or SEQ ID NO:

15.

6. The isolated antibody or antigen-binding fragment as described in claim 4, 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 a light chain variable region selected from SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:

15.

7. The isolated antibody or antigen-binding fragment as described in claim 4, wherein, The isolated anti-FXI and / or anti-FXIa antibody or antigen-binding fragment is an antibody or antigen-binding fragment containing the heavy chain variable region of SEQ ID NO:5 and the light chain variable region of SEQ ID NO:

7.

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:21 and the light chain shown in SEQ ID NO:

22.

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 9-10, 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.

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