Anti-FXI antibody and use thereof

Abstract

Provided are an antibody specifically binding to FXI or an antigen-binding fragment thereof, and a composition containing the antibody or antigen-binding fragment thereof and a bispecific antibody. In addition, further provided are a nucleic acid encoding the antibody or antigen-binding fragment thereof, a host cell containing same, and the related use. Moreover, further provided is the therapeutic and diagnostic uses of the antibody or antigen-binding fragment, bispecific antibody or composition thereof. Specifically, provided is the use of the antibody or antigen-binding fragment, bispecific antibody or composition thereof in combination with other therapies, e.g., therapeutic methods or therapeutic agents.

Description

Anti-FXI antibodies and their uses

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410949423.1, filed on July 16, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to anti-FXI antibodies, their preparation methods, compositions, and uses. The invention also provides methods for treating FXI-related diseases and / or conditions, such as venous thromboembolism. Background Technology

[0004] Thrombotic diseases are caused by thrombus formation and thromboembolism, leading to symptoms such as poor blood circulation, limb swelling and pain, and tissue ischemia and necrosis. If a blood vessel ruptures and treatment is not timely, it can be life-threatening. Statistics show that the number of deaths in my country due to thrombotic diseases far exceeds those from cancer, infectious diseases, and respiratory diseases, accounting for 51% of the global total. The "China Cardiovascular Health and Disease Report 2022" indicates that the incidence and mortality rates of cardiovascular diseases in China are continuously rising, with the current number of patients reaching 330 million, posing a significant challenge to disease prevention and treatment.

[0005] Clinically common drugs for preventing and treating thrombosis are mainly divided into three categories: antiplatelet drugs, anticoagulants, and thrombolytic drugs. Antiplatelet drugs inhibit thrombus formation by suppressing platelet aggregation; examples include aspirin, clopidogrel, edtifibatide, and tirofiban. Anticoagulants include warfarin, direct oral anticoagulants (DOACs), and heparin and its low molecular weight heparin (LMWH). Thrombolytic drugs dissolve thrombi by promoting the conversion of plasminogen to plasmin in the fibrinolytic system; these include urokinase, streptokinase, and tissue plasminogen activator. While these drugs have good clinical efficacy in reducing thrombus formation, they all carry a certain risk of bleeding and have limitations in clinical use; they cannot be used in patients with a bleeding risk. Therefore, developing drugs that effectively prevent thrombosis without increasing the risk of bleeding is an important research direction for the prevention and treatment of thrombotic diseases.

[0006] FXI plays a crucial role in the amplification process of the coagulation cascade and in the intrinsic pathway, and is closely related to thrombotic diseases. Individuals with FXI deficiency generally do not experience spontaneous bleeding or only have a mild bleeding tendency. Further findings revealed that FXI deficiency is negatively correlated with the risk of death from thrombotic diseases, while a positive correlation exists in individuals with high FXI expression. Clinical trials of existing FXI antisense nucleotide drugs have also demonstrated that reducing FXI levels can significantly reduce the incidence of thrombosis in patients without increasing the risk of bleeding. Therefore, FXI and the intrinsic coagulation pathway it pertains to may play an important role in in vivo thrombosis under pathological conditions without increasing the risk of bleeding. Thus, FXI is a more advantageous antithrombotic therapeutic target compared to other coagulation factors.

[0007] Coagulation factor XI (FXI) is a serine protease proteoproteasome that maintains thrombin production through FIX activation. FXI is a 160 kDa disulfide-linked homodimer, with each subunit consisting of a heavy chain (N-terminal catalytic domain, consisting of four AP domains (apple domains, A1–A4)) and a light chain (C-terminal catalytic domain, CD)). FXI is a circulating proenzyme in a complex with high molecular weight kininogen (HK). The FXI-A2 domain binds to HK, inducing activation of coagulation in vitro and in vivo. The remaining apple domains in FXI also mediate important physiological functions. For example, the FXI-binding exosite is located in A3, while the FXII binding site is located in A4. The FXI CD domain is the light chain of the complete FXI molecule, containing amino acids 370–607. Experiments have shown that it has comparable functional activity to the complete FXI and can replace it. The investigational drug Abelacimab targets the catalytic domain of factor XI, locking it in an inactive precursor conformation (zymogen state) and preventing its activation by factor XIIa or thrombin. This gives the drug dual activity against both factor XI and its activated form, factor XIa. While reducing coagulation risk, it protects physiological coagulation function through a decoupled coagulation mechanism, preliminarily validating the effectiveness of drugs binding to the FXI CD region.

[0008] Currently, there are three main categories of drugs under development for FXI: small molecule inhibitors, RNA-targeted drugs, and antibody drugs. Selective small molecule inhibitors have not yet achieved sufficient efficacy, selectivity, and pharmacokinetics. While injectable FXI ASOs show significant efficacy and fewer adverse reactions, reducing the concentration of FXI to the therapeutic window requires a 3-4 week injection period, which greatly limits the use of FXI ASOs for treating existing or acute thrombosis.

[0009] Therefore, there is an urgent need to develop novel antithrombotic drugs that are potent, have a low risk of bleeding, and are long-lasting to meet the existing clinical gaps.

[0010] Invention Overview

[0011] This invention provides a novel anti-FXI antibody, its encoded nucleic acid, and its applications.

[0012] In some embodiments, the antibody or its antigen-binding fragment of the present invention specifically binds to FXI, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain CDRs contained in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27, and the light chain variable region comprises three light chain CDRs contained in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28, according to the IMGT numbering scheme.

[0013] In some embodiments, the antibody or its antigen-binding fragment of the present invention specifically binds to FXI, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown above, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown above.

[0014] In some embodiments, the antibody of the present invention is a bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain bind to different domains on FXI, the first antigen-binding domain binds to the apple domain (A1-A4) of FXI, and the second antigen-binding domain binds to the catalytic domain (CD) of FXI.

[0015] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention binds to the FXI apple domain A1, A2, A3 or A4, preferably to the apple domain A2.

[0016] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein the first heavy chain variable region (VH1) comprises three heavy chain CDRs contained in SEQ ID NO: 5, 7, 15, 19 or 25; the first light chain variable region (VL1) comprises three light chain CDRs contained in SEQ ID NO: 6, 8, 16, 20 or 26, preferably, the first heavy chain variable region (VH1) comprises three heavy chain CDRs contained in SEQ ID NO: 7; and the first light chain variable region (VL1) comprises three light chain CDRs contained in SEQ ID NO: 8. In some embodiments, the second antigen-binding domain of the bispecific antibody of the present invention comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2). The second heavy chain variable region (VH2) comprises the three heavy chain CDRs contained in SEQ ID NO: 1, 3, 9, 11, 17 or 27; the second light chain variable region (VL2) comprises the three light chain CDRs contained in SEQ ID NO: 2, 4, 10, 12, 18 or 28. Preferably, the second heavy chain variable region (VH2) comprises the three heavy chain CDRs contained in SEQ ID NO: 11; the second light chain variable region (VL2) comprises the three light chain CDRs contained in SEQ ID NO: 12.

[0017] In some embodiments, the antibody of the present invention comprises the Fc region of an immunoglobulin, wherein the Fc region is an IgG Fc region, for example, the Fc domain of human IgG1, IgG2, IgG3 or IgG4, preferably the Fc domain of human IgG1 or IgG4.

[0018] In some embodiments, the antibody of the present invention is a murine antibody, a chimeric antibody, or a humanized antibody.

[0019] In some experimental protocols, the antigen-binding domain of the present invention is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody, (Fab')2 fragment, single-domain antibody, or linear antibody.

[0020] In some embodiments, the present invention provides a nucleic acid encoding an antibody or antigen-binding fragment of the present invention, a vector comprising the nucleic acid, and a host cell comprising the vector.

[0021] In some embodiments, the present invention provides a method for preparing antibodies or antigen-binding fragments thereof.

[0022] In some embodiments, the present invention provides a pharmaceutical composition comprising the above-described FXI antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier and / or excipient.

[0023] In some embodiments, the present invention also provides a method for preparing antibodies or antigen-binding fragments.

[0024] In some embodiments, the present invention also provides methods and uses for preventing or treating FXI-related diseases by applying the antibody or antigen-binding fragment and pharmaceutical composition of the present invention, including but not limited to thrombotic or thromboembolic diseases or their complications.

[0025] In some embodiments, the present invention relates to the use of the antibody or its antigen-binding fragment in the preparation of a detection reagent for detecting FXI in a sample.

[0026] In some embodiments, the present invention relates to a diagnostic test kit comprising the antibody or its antigen-binding fragment described above.

[0027] The antibodies or antigen-binding fragments thereof of the present invention, especially bispecific antibodies, 1. simultaneously target and bind to the FXI CD and FXI A2 regions, exhibiting high affinity for FXI; 2. possess good cross-species activity and high selectivity; 3. have no interaction with other exogenous coagulation factors and downstream coagulation factors in common pathways; 4. can significantly prolong activated partial thromboplastin time (APTT); 5. have significant antithrombotic effects; do not affect changes in prothrombin time (PT); 6. do not increase the risk of bleeding while having antithrombotic effects; 7. have high affinity for FcRn, a long drug half-life, low dosing frequency, and high patient acceptance.

[0028] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims. Attached Figure Description

[0029] Figure 1. FXI humanized molecule binding screening

[0030] Figure 2. Experimental results showing the binding of FXI antibody to FXI and FXII.

[0031] Figure 3. Detection of FXI antibody-activated partial thromboplastin time.

[0032] Figure 4. Inhibitory effect of FXI antibody on FXIa enzyme activity.

[0033] Figure 5. Detection of the in vivo antithrombotic activity of FXI antibody.

[0034] Figure 5-1: A. Thrombus wet weight determination; B. Thrombus inhibition rate; C. Activated partial thromboplastin time (APTT) detection; D. Plasma prothrombin time (PT);

[0035] Figure 5-2: A. Bleeding weight detection; B. Bleeding duration detection; C. Rabbit weight measurement; D. Blood flow velocity detection.

[0036] Figure 6. Schematic diagram of the molecular structure of FXI bispecific antibody

[0037] Figure 7. Binding activity of FXI bispecific antibody molecules with human FXI and FXII proteins.

[0038] Figure 8. Binding activity of FXI with family proteins FVII, FIX, and FX.

[0039] Figure 9. In vitro activity and function detection of FXI bispecific antibody molecules

[0040] Figure 10. Results of the affinity experiment between the dual-antibody molecule and FcRn.

[0041] Figure 11 Results of affinity experiments between the dual-antibody molecules and FXI, FXIa, and FXI-A2.

[0042] Figure 12 Results of APTT experiment on bispecific antibodies in monkey plasma.

[0043] Figure 13. The effects of dual anti-antibody molecules on APTT and PT in cynomolgus monkeys.

[0044] Figure 14 shows the results of the experiment on the concentration of free FXI.

[0045] Figure 15. Results of the pharmacokinetic study of the dual-antibiotic monkey.

[0046] Invention Details

[0047] definition

[0048] The terminology used herein is for descriptive purposes only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0049] In this document, unless otherwise stated, the singular form of a term also covers the plural form, or vice versa.

[0050] The terms “comprising” or “including” mean that the stated elements, integers, or steps are included, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0051] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y” and should be used to provide clear support for both meanings or either meaning.

[0052] The term "antibody" is used in the broadest sense herein and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, heavy-chain antibodies, or antibody fragments thereof exhibiting the desired antigen-binding activity. Intact antibodies will typically contain at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains; for example, naturally occurring antibodies in camels may contain only heavy chains.

[0053] The term "whole antibody" (which may be used interchangeably with "full-length antibody," "complete antibody," and "intact antibody" in this document) comprises at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region. The light chain constant region consists of one domain: CL. The variable region is a domain in the heavy or light chain of the antibody that participates in the binding of the antibody to its antigen. The constant region does not directly participate in the binding of the antibody to the antigen but exhibits various effector functions. The light chain of an antibody can be classified into one of two types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant domain. Antibody heavy chains can be classified into five main types based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. Several of these types can be further subdivided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different antibody types are called α, δ, ε, γ, and μ, respectively. The term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. See, for example, Fundamental Immunology, Ch.7 (edited by Paul, W., 2nd ed., Raven Press, NY (1989)).

[0054] The term "FXI," also known as "coagulation factor XI" or "factor XI," refers to a coagulation factor in the blood coagulation system, a protein encoded by the F11 gene. The FXI molecule consists of two identical subunits, each containing 603 amino acid residues. It is part of the prothrombin complex and closely related to coagulation factors IX and VIII, playing a crucial role in the coagulation cascade, particularly in platelet aggregation and intravascular thrombosis. The FXI structure includes an apple domain (A1-A4) and a catalytic domain (CD). The apple domain, named for its apple-like shape, consists of approximately 60 amino acid residues, forming a winery-like β-barrel structure. This unique β-sheet structure provides a platform for molecular recognition and interaction, playing a vital role in the activation and regulation of FXI. The catalytic domain, located at the C-terminus of FXI, is a key component of prothrombin activator. When FXI is activated to FXIa (coagulation factor XIa), this catalytic domain undergoes a conformational change, enabling it to serve as the enzyme's active site and participate in the blood coagulation cascade reaction. An example amino acid sequence of the FXI involved in this invention is shown in SEQ ID NO:74.

[0055] In this invention, the term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigenic epitopes. In some embodiments, such multispecific antibodies are provided herein that have binding specificity against a first antigen and a second antigen, also referred to as "bispecific antibodies."

[0056] In the context of this invention, the term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding moieties defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target; the bispecific antibodies of this invention bind to different epitopes of the same target. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

[0057] The term "antigen-binding fragment" (which may be used interchangeably herein with "antibody fragment," "antigen-binding moiety," or "antigen-binding domain") refers to a molecule that is not a complete antibody but contains the portion of the complete antibody used to bind the antigen bound by that complete antibody. As those skilled in the art will understand, the antigen-binding moiety of an antibody typically contains amino acid residues from the "complementarity-determining region" or "CDR." Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab')2, Fab'-SH, Fv, single-chain Fv, diabody, triabody, tetrabody, minibody, and sdAb.

[0058] The term "Fab" typically refers to a fragment containing both a variable domain of the heavy chain and a variable domain of the light chain, and also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain; the term "Fab'" typically refers to the addition of a small number of residues (including one or more cysteine ​​residues from the antibody hinge region) to the carboxyl terminus of the CH1 domain of the heavy chain.

[0059] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including a variable region comprising a light chain and at least one antibody fragment including a variable region comprising a heavy chain, wherein the light and heavy chain variable regions are adjacent (e.g., via a synthetic linker, such as a short, flexible peptide linker) and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, the scFv may have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0060] The term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, an antibody whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody.

[0061] The term "humanized antibody" refers to an antibody for which a CDR sequence derived from another mammalian species, such as a mouse line, is inserted onto a human framework sequence. Additional framework modifications can be made within the human framework sequence, and / or additional amino acid modifications can be made to the CDR sequence, for example, to facilitate antibody affinity maturation.

[0062] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al., (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0063] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0064] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region CDR, it means the position numbered according to the IMGT numbering system.

[0065] In some embodiments, the CDR of the antibody of the present invention is defined by the IMGT rule.

[0066] The term "isolated" antibody refers to an antibody that has been separated from its components in its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0067] The term "epitope" refers to the antigenic region to which an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of a protein.

[0068] The term “Fc region” as used herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes both native and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 of the heavy chain to the C-terminus. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0069] The term "effector function" refers to biological activities attributable to the Fc region of immunoglobulins that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0070] As used herein, the term sequence “identity” refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0071] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of the interaction. In this invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to the antigen with an affinity (KD) of less than about 10⁻⁹ M, for example, less than about 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less. In some embodiments, when KD ≤ 10 × 10⁻⁸ M, the antibody or its antigen-binding fragment of the present invention is considered to specifically bind FXI.

[0072] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured using bioluminescent interferometry (e.g., the ForteBio Octet method). Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0073] In this invention, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. These conserved modifications include conserved substitutions, additions, and deletions of amino acids. Modifications can be introduced into the chimeric antigen receptor of this invention using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conserved amino acid substitution is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphiphilic properties of the residues involved. Conservative substitution refers to the substitution of one amino acid with another amino acid within the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid.

[0074] In this invention, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA.

[0075] In this invention, the term "cell" generally refers to a plasmid or vector that may contain or already contains nucleic acid molecules described in this invention, or an individual cell, cell line, or cell culture capable of expressing the antigen-binding protein described in this invention. The cell may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they need to be capable of expressing the antibody or its antigen-binding fragment described in this invention.

[0076] As used herein, the term "pharmaceuticalally acceptable excipient" is intended to include any and all excipients compatible with drug administration, including but not limited to solvents, dispersion media, buffers, diluent coatings, antimicrobial and antifungal agents, isotonic and absorption-delaying agents, etc.

[0077] In this invention, the terms "pharmaceutical combination," "combination product," or "pharmaceutical combination product" refer to non-fixed or fixed combinations, including but not limited to cassettes and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) antibodies or bispecific antibodies of the present invention and their derivatives, fragments, analogs, and homologs, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active ingredients are selected so that the combined use of the components produces an effect greater than that achieved by using any one ingredient alone in treating a disease or condition. Each component may be in a separate formulation, and the formulations may be the same or different.

[0078] The terms "combination therapy" or "combination therapy" refer to the administration of two or more therapeutic agents or treatment modalities (such as radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids).

[0079] In this invention, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0080] In this invention, the term "therapeuticly effective amount" generally refers to the amount of a subject compound or composition that elicits a biological, physiological, clinical, or medical response in cells, tissues, organs, systems, or a subject, including amounts of a compound or composition sufficient, when applied, to prevent or to some extent treat one or more signs or symptoms of the disease or ailment being treated. Therapeuticly effective amounts can vary depending on the compound or composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0081] Various aspects of the invention will be further described in the following subsections.

[0082] 1. The anti-FXI antibody of the present invention

[0083] The first aspect of the invention provides antibodies that specifically bind to FXI, preferably human FXI protein, or antigen-binding fragments thereof, especially humanized antibodies. In some embodiments, the antigen-binding fragment of the antibody of the present invention is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibodies such as scFv, (Fab')2 fragments, single-domain antibodies, bispecific antibodies (dAbs), or linear antibodies.

[0084] In some embodiments, the anti-FXI antibody of the present invention or its antigen-binding fragment has one or more of the following characteristics: (i) exhibiting the same or similar binding affinity and / or specificity to FXI as the antibody of the present invention; (ii) inhibiting (e.g., competitively inhibiting) the binding of the antibody of the present invention to FXI; (iii) binding the same or overlapping epitopes as the antibody of the present invention; (iv) competing with the antibody of the present invention for binding to FXI; (v) having one or more biological characteristics of the antibody of the present invention.

[0085] In some embodiments, the anti-FXI antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region, wherein the heavy chain variable region comprises

[0086] (i) The three complementary determinant regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29-32, 37-40 and 45-48, or

[0087] (ii) Relative to the sequence in (i), the three CDR regions contain at least one and no more than 5, 4, 3, 2 or 1 complementarity-determining regions (CDRs) with amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions).

[0088] In some embodiments, the anti-FXI antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region, said heavy chain variable region

[0089] (i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29-32, 37-40, and 45-48; or

[0090] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29-32, 37-40, and 45-48; or

[0091] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29-32, 37-40, and 45-48, preferably, the amino acid alterations do not occur in the CDR region.

[0092] In some embodiments, the anti-FXI antibody or its antigen-binding fragment of the present invention comprises a light chain variable region, wherein the light chain variable region comprises

[0093] (i) The three complementary determining regions (CDRs) contained in the VL shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 33-36, 41-44, and 49-53, or

[0094] (ii) Relative to the sequence in (i), the three CDR regions contain at least one and no more than 5, 4, 3, 2 or 1 complementarity-determining regions (CDRs) with amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions).

[0095] In some embodiments, the anti-FXI antibody or its antigen-binding fragment of the present invention comprises a light chain variable region, said light chain variable region

[0096] (i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 33-36, 41-44, and 49-53; or

[0097] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 33-36, 41-44, and 49-53; or

[0098] (iii) An amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 33-36, 41-44, and 49-53, preferably, the amino acid alterations do not occur in the CDR region.

[0099] In some embodiments, the antibody or antigen-binding fragment of the present invention that specifically binds to FXI comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain CDRs, namely HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three light chain CDRs, namely LCDR1, LCDR2, and LCDR3.

[0100] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:87, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:88, HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:89, LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:90, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:91, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:92.

[0101] (ii) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:81, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:82, HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:83, LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:84, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:85, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:86.

[0102] In some embodiments, the anti-FXI antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region VH and / or a light chain variable region VL, wherein the VH and VL are selected from:

[0103] (i) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:1, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:2;

[0104] (ii) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:3, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:4;

[0105] (iii) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:5, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:6;

[0106] (iv) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:7, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:8;

[0107] (v) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:9, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:10;

[0108] (vi) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:11, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:12;

[0109] (vii) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:13, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:14;

[0110] (viii) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:15, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:16;

[0111] (ix) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:17, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:18;

[0112] (x) A VH sequence or a variant thereof containing the amino acid sequence shown in SEQ ID NO:19, and / or a VL sequence or a variant thereof containing the amino acid sequence shown in SEQ ID NO:20;

[0113] (xi) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:21, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:22;

[0114] (xii) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:23, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:24;

[0115] (xiii) A VH sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:25, and / or a VL sequence or a variant thereof comprising the amino acid sequence shown in SEQ ID NO:26;

[0116] (xiv) A VH sequence or a variant thereof containing the amino acid sequence shown in SEQ ID NO:27, and / or a VL sequence or a variant thereof containing the amino acid sequence shown in SEQ ID NO:28.

[0117] In some embodiments, the variant of the VH sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher amino acid sequence identity compared to the VH sequence described above (preferably, in the full length or in the CDR1, 2, and 3 regions). In one embodiment, the variant of the VH sequence contains at least one and no more than 30, 10, or 5, 4, 3, 2, 1, or 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the VH sequence described above (preferably, in the full length or in the CDR1, 2, and 3 regions). Preferably, the amino acid changes do not occur in the CDR regions.

[0118] In a preferred embodiment, the variant of the VL sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher amino acid sequence identity compared to the aforementioned VL sequence (preferably, in the full length or in the CDR1, 2 and 3 regions). In a preferred embodiment, the variant of the VL sequence contains at least one and no more than 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the aforementioned VL sequence (preferably, in the full length or in the CDR1, 2 and 3 regions). Preferably, the amino acid changes do not occur in the CDR regions.

[0119] In some embodiments, the invention has at least 95-99% identity with VH, VL, or VH and VL, or contains variants with no more than 10 amino acid changes.

[0120] In any of the above embodiments, preferably, the heavy chain variable region of the antibody variant contains no more than 10, preferably no more than 5 (e.g., 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in one or more CDR (preferably all 3 CDRs) regions relative to the antibody.

[0121] In any of the above embodiments, preferably, the light chain variable region VL of the antibody variant contains no more than 10, preferably no more than 5 (e.g., 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in one or more CDR (preferably all 3 CDRs) regions.

[0122] In a preferred embodiment, the amino acid alteration described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described in this invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the substitution is a conservative substitution.

[0123] In some embodiments, the antibody or its antigen-binding fragment of the present invention specifically binds to FXI, particularly specifically binding to the C-terminal catalytic domain (CD) of FXI, which comprises a heavy chain variable region and a light chain variable region, wherein

[0124] 1) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:12;

[0125] 2) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:33;

[0126] 3) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:33;

[0127] 4) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:33;

[0128] 5) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:33;

[0129] 6) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:34;

[0130] 7) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:34;

[0131] 8) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:34;

[0132] 9) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:34;

[0133] 10) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:35;

[0134] 11) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:35;

[0135] 12) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:35;

[0136] 13) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:35;

[0137] 14) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:36;

[0138] 15) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:36;

[0139] 16) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:36; or

[0140] 17) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:36.

[0141] In some embodiments, the antibody or its antigen-binding fragment of the present invention specifically binds to FXI, particularly specifically to the AP domain of FXI, and more particularly specifically to the FXI-A2 domain, which includes a heavy chain variable region and a light chain variable region, wherein

[0142] 1) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:7, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:8;

[0143] 2) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:41;

[0144] 3) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:41;

[0145] 4) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:41;

[0146] 5) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:40, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:41;

[0147] 6) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:42;

[0148] 7) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:42;

[0149] 8) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:42;

[0150] 9) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:40, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:42;

[0151] 10) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:43;

[0152] 11) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:43;

[0153] 12) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:43;

[0154] 13) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:40, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:43;

[0155] 14) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:44;

[0156] 15) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:38, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:44;

[0157] 16) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:44; or

[0158] 17) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:40, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:44.

[0159] In some embodiments, the anti-FXI antibody or its antigen-binding fragment of the present invention further comprises a heavy chain constant region and / or a light chain constant region.

[0160] In some embodiments, the heavy chain constant region is derived from the human IgG heavy chain constant region, such as the human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is the human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 79 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the shown sequence.

[0161] In some embodiments, the light chain constant region is derived from the light chain constant region of Kappa or Lambda, such as the human Kappa or Lambda light chain constant region. In some embodiments, the light chain constant region is the human Kappa or Lambda light chain constant region. In some embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 80 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the shown sequence.

[0162] In some embodiments, the anti-FXI antibody or its antigen-binding fragment of the present invention further comprises an Fc region, preferably the Fc region being linked to the C-terminus of the heavy chain variable region. In some embodiments, a constant region CH1 is further included between the heavy chain variable region and the Fc region of the antibody of the present invention. In some embodiments, the heavy chain constant region of the antibody of the present invention comprises both CH1 and the Fc region.

[0163] In some embodiments, the Fc region is derived from IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1 or IgG4. In some embodiments, the Fc region is derived from human IgG1 or human IgG4.

[0164] In some embodiments, the Fc region: (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of human IgG1 or human IgG4; (ii) comprises or consists of the amino acid sequence of human IgG1 or human IgG4; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of human IgG1 or human IgG4.

[0165] In one embodiment of the invention, the amino acid alteration described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid alteration described herein is an amino acid substitution, and more preferably a conservative substitution.

[0166] In some implementations, the Fc region can be mutated to have reduced effector function, such as reduced binding to ADCC, ADCP, CDC and / or Clq, FcγRI, FcγRII, or FcγRIIIA. For example, the Fc region can be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, and mutations can reduce or even eliminate its ability to mediate effector function. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol. 75(24):12161-12168 (2001). For example, compared to the wild-type sequence, the Fc region may contain an amino acid sequence with one or more of the following amino acid substitutions: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G.

[0167] In some implementations, the Fc region can be mutated to have improved binding affinity to the neonatal Fc receptor FcRn, thereby significantly prolonging the half-life of the antibody in vivo. Such mutations include, for example, the M252Y / S254T / T256E mutation (YTE mutation).

[0168] Therefore, in some embodiments, the heavy chain constant region or Fc region of the anti-FXI antibody or its antigen-binding fragment of the present invention includes, relative to the wild-type heavy chain constant region or Fc region, (i) mutations that reduce the function of the Fc region effector, such as mutations that reduce or eliminate the binding of the Fc region to FcγR (FcγRI, FcγRII, FcγRIII) and C1q, such as L234A / L235A mutations and / or P329G mutations, and / or (ii) mutations that increase the binding of the Fc region to FcRn, such as M252Y / S254T / T256E mutations.

[0169] Therefore, in some embodiments, the heavy chain constant region or Fc region of the anti-FXI antibody or its antigen-binding fragment of the present invention contains, relative to the wild-type heavy chain constant region or wild-type Fc region, the L234A / L235A / M252Y / S254T / T256E / P329G mutation.

[0170] In some embodiments, the Fc region having the L234A / L235A / M252Y / S254T / T256E / P329G mutation comprises or is composed of the amino acid sequence shown in SEQ ID NO:99, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0171] In some embodiments, the IgG1 constant region having the L234A / L235A / M252Y / S254T / T256E / P329G mutation comprises or is composed of the amino acid sequence shown in SEQ ID NO:101, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it. SEQ ID NO:101 is the sequence of the constant region having the L234A / L235A / M252Y / S254T / T256E / P329G mutation compared to the wild-type IgG1 constant region, as shown in the sequence listing.

[0172] In some embodiments, the heavy chain constant region or Fc region of the anti-FXI antibody of the present invention or its antigen-binding fragment contains, relative to the wild-type heavy chain constant region or wild-type Fc region, the L234A / L235A / M252Y / S254T / T256E mutation.

[0173] In some embodiments, the Fc region having the L234A / L235A / M252Y / S254T / T256E mutation comprises or is composed of the amino acid sequence shown in SEQ ID NO:103, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0174] In some embodiments, the anti-FXI antibody of the present invention or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the heavy chain variable region VH and the heavy chain constant region as described herein, and the light chain comprises or consists of the light chain variable region VL and the light chain constant region as described herein. In some embodiments, the anti-FXI antibody of the present invention or its antigen-binding fragment comprises two heavy chains and two light chains, preferably two identical heavy chains and two identical light chains, or consists of the latter.

[0175] In some embodiments, the number of cysteine ​​residues in the antibody can be altered to modify antibody properties. For example, modification of the hinge region of CH1 can change (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region. This method is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 can be altered to, for example, promote the assembly of light and heavy chains or increase or decrease antibody stability.

[0176] Optionally, the antibodies of the present invention include post-translational modifications to the antibody chain. Exemplary post-translational modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation with labeled components.

[0177] In some embodiments, the anti-FXI antibody of the present invention is a monoclonal antibody.

[0178] In some embodiments, the anti-FXI antibody of the present invention is a full-length antibody.

[0179] In some embodiments, the anti-FXI antibody of the present invention is a chimeric antibody.

[0180] In some embodiments, the anti-FXI antibody of the present invention is humanized. Different methods for humanizing antibodies are known to those skilled in the art, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13, 1619-1633).

[0181] In some embodiments, the anti-FXI antibody of the present invention is a human antibody. Human antibodies can be prepared using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Chem. Biol 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol 20(4):450-9 (2008).

[0182] In some embodiments, at least a portion of the framework sequence of the anti-FXI antibody is a human common framework sequence. In one embodiment, the anti-FXI antibody of the present invention also encompasses its antibody fragments, preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv) or (Fab')2, single-domain antibody, bispecific antibody (dAb), or linear antibody.

[0183] In some embodiments, the anti-FXI antibody molecules of the present invention are in the form of bispecific or multispecific antibody molecules. In some embodiments, the multispecific antibody molecule may be, for example, a trispecific antibody molecule, comprising a first antigen-binding domain against FXI and second and third antigen-binding domains against one or more molecules.

[0184] In some embodiments, the anti-FXI antibody of the present invention is a bispecific antibody comprising the above-mentioned FXI antibody, which contains a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain and the second antigen-binding domain binding to different structural domains on FXI, wherein the first antigen-binding domain binds to the apple structural domain (A1-A4) of FXI, and the second antigen-binding domain binds to the catalytic structural domain (CD) of FXI.

[0185] 2. Bispecific antibodies

[0186] In a second aspect, the present invention provides a bispecific antibody that specifically binds to FXI, comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain bind to different structural domains of FXI, for example, the first antigen-binding domain binds to the apple structural domains (A1-A4) of FXI, and the second antigen-binding domain binds to the catalytic structural domain (CD) of FXI. In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention binds to the apple structural domains A1, A2, A3, or A4 of FXI, preferably to the apple structural domain A2. In some specific embodiments, the bispecific antibody of the present invention comprises one or two first antigen-binding domains and one or two second antigen-binding domains. In some specific embodiments, the bispecific antibody of the present invention comprises two first antigen-binding domains and two second antigen-binding domains. In some specific embodiments, the bispecific antibody of the present invention comprises one first antigen-binding domain and one second antigen-binding domain.

[0187] In some embodiments, the first antigen-binding domain and the second antigen-binding domain may respectively comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the antibody that specifically binds to FXI as described in the first aspect of this document.

[0188] In some embodiments, the antigen-binding domain of the bispecific antibody of the present invention comprises a first / second heavy chain variable region (VH1 / VH2) and / or a first / second light chain variable region (VL1 / VL2), wherein the first / second heavy chain variable region (VH1 / VH2) comprises three heavy chain CDRs contained in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27 and / or the first / second light chain variable region (VL1 / VL2) comprises three light chain CDRs contained in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28.

[0189] In some embodiments, the bispecific antibody of the present invention contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alterations (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions of the heavy chain variable region or the light chain variable region.

[0190] In some embodiments, the first antigen-binding domain and the second antigen-binding domain may respectively comprise VH and VL of an antibody that specifically binds to FXI as described in the first aspect of this document, or a combination of VH and VL.

[0191] In some embodiments, the antigen-binding domain of the bispecific antibody of the present invention comprises a first / second heavy chain variable region (VH1 / VH2) and a first / second light chain variable region (VL1 / VL2), wherein the heavy chain variable region (VH1 / VH2) comprises the amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29-32, 37-40 and 45-48 or has more than 80% identity with the above sequences; the first / second light chain variable region (VL1 / VL2) comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 33-36, 41-44 and 49-53 or has more than 80% identity with the above sequences.

[0192] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein the first heavy chain variable region (VH1) comprises an amino acid sequence shown in any one of SEQ ID NO: 5, 7, 15, 19, 25, 37-40, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with any one of SEQ ID NO: 5, 7, 15, 19, 25, 37-40, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with any one of SEQ ID NO: 5, 7, 15, 19, 25, 37-40, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions compared to any one of SEQ ID NO: 5, 7, 15, 19, 25, 37-40; the first light chain variable region (VH1) comprises an amino acid sequence shown in any one of SEQ ID NO: 6, 8, 16, 20, 26, 41-44, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with any one of SEQ ID NO: 5, 7, 15, 19, 25, 37-40 ... The amino acid sequences shown in any one of SEQ ID NO: 6, 8, 16, 20, 26, 41-44 have at least 80%, 85%, 90%, 95% or 99% identity with each other, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with additions, deletions and / or substitutions compared to the amino acid sequences shown in any one of SEQ ID NO: 6, 8, 16, 20, 26, 41-44.

[0193] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1).

[0194] in,

[0195] i. The first heavy chain variable region (VH1) contains the three heavy chain CDRs contained in SEQ ID NO: 5; the first light chain variable region (VL1) contains the three light chain CDRs contained in SEQ ID NO: 6;

[0196] ii. The first heavy chain variable region (VH1) contains the three heavy chain CDRs contained in SEQ ID NO: 7; the first light chain variable region (VL1) contains the three light chain CDRs contained in SEQ ID NO: 8;

[0197] iii. The first heavy chain variable region (VH1) contains the three heavy chain CDRs contained in SEQ ID NO: 15; the first light chain variable region (VL1) contains the three light chain CDRs contained in SEQ ID NO: 16;

[0198] iv. The first heavy chain variable region (VH1) contains the three heavy chain CDRs contained in SEQ ID NO: 19; the first light chain variable region (VL1) contains the three light chain CDRs contained in SEQ ID NO: 20;

[0199] v. The first heavy chain variable region (VH1) contains the three heavy chain CDRs contained in SEQ ID NO: 25; the first light chain variable region (VL1) contains the three light chain CDRs contained in SEQ ID NO: 26.

[0200] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein the first heavy chain variable region comprises three heavy chain CDRs, namely HCDR1, HCDR2 and HCDR3, and the first light chain variable region comprises three light chain CDRs, namely LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:81, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:82, HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:83, LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:84, LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:85, and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:86.

[0201] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein 1) the VH1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:7, and the VL1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:8; or 2) the VH1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:40, and the VL1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:43.

[0202] In some embodiments, the second antigen-binding domain of the bispecific antibody of the present invention comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein the second heavy chain variable region (VH2) comprises an amino acid sequence shown in any one of SEQ ID NO: 1, 3, 9, 11, 17, 27, 29-32, or an amino acid sequence having at least 80%, 85%, 90, 95%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 1, 3, 9, 11, 17, 27, 29-32, or an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids compared to any one of SEQ ID NO: 1, 3, 9, 11, 17, 27, 29-32; the second light chain variable region (VL2) comprises SEQ ID NO: 1, 3, 9, 11, 17, 27, 29-32. The amino acid sequence shown in any one of SEQ ID NO: 2, 4, 10, 12, 18, 28, 33-36, or the amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 10, 12, 18, 28, 33-36, or the amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids compared with the amino acid sequence shown in any one of SEQ ID NO: 2, 4, 10, 12, 18, 28, 33-36.

[0203] In some embodiments, the second antigen-binding domain of the bispecific antibody of the present invention comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein the second heavy chain variable region comprises three heavy chain CDRs, namely HCDR1, HCDR2 and HCDR3, and the second light chain variable region comprises three light chain CDRs, namely LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:87, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:88, HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:89, LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:90, LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:91, and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:92.

[0204] In some embodiments, the second antigen-binding domain of the bispecific antibody of the present invention comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein 1) the VH2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11, and the VL2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; or 2) the VH2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:32, and the VL2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34.

[0205] In some embodiments, the antigen-binding domain of the bispecific antibody of the present invention may be an antibody fragment of the anti-FXI antibody described in the first aspect, preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv) or (Fab')2, single-domain antibody, bispecific antibody (dAb) or linear antibody.

[0206] In some embodiments, the first or second antigen-binding domain of the bispecific antibody of the present invention is a Fab fragment. In some embodiments, the first or second antigen-binding domain of the bispecific antibody of the present invention is an SCFV fragment.

[0207] In some embodiments, the bispecific antibody of the present invention has a first antigen-binding domain of scFv and a second antigen-binding domain of Fab fragment, or vice versa.

[0208] In some embodiments, the two antigen-binding domains of the bispecific antibody of the present invention are connected by a linker.

[0209] In some embodiments, the first antigen-binding domain of the bispecific antibody of the present invention is an scFv, the scFv comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), optionally the first light chain variable region and the first heavy chain variable region being connected via a linker. The scFv is optionally connected via a linker to the N / C terminus of VL2 or VH2 or the N / C terminus of the Fc region.

[0210] In some embodiments, the linker can be any flexible linker comprising an amino acid sequence selected from (G4)n, (G4S)n and GS(G4S)n, where n is an integer selected from 1 to 5, and preferably the linker comprises an amino acid sequence as shown in GGGGS.

[0211] In some embodiments, the bispecific antibody of the present invention is shown in Figure 6, but is not limited thereto.

[0212] In some embodiments, the bispecific antibody of the present invention may comprise an Fc region containing CH2 and CH3 of the antibody. The Fc region may be any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the Fc region is an IgG1 isotype or derived therefrom, optionally having one or more mutations or modifications. In another embodiment, the Fc region is an IgG4 isotype or derived therefrom, optionally having one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc or human IgG4 Fc.

[0213] In some embodiments, the Fc region of the bispecific antibody of the present invention is IgG1 Fc, such as human IgG1 Fc.

[0214] In some embodiments, the human IgG1 Fc comprises the amino acid sequence shown in SEQ ID NO:96, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence.

[0215] In some implementations, the Fc region has reduced effector function, such as reduced binding to ADCC, ADCP, CDC and / or Clq, FcγRI, FcγRII, or FcγRIIIA. For example, the Fc region may be an IgG1 isotype or a non-IgG1 type, such as IgG2, IgG3, or IgG4, which has been mutated to reduce or even eliminate its ability to mediate effector function. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-38 (2006) and Hezareh M et al., J Virol. 75(24):12161-8 (2001). For example, compared to the wild-type sequence, the Fc region may contain an amino acid sequence with one or more of the following amino acid substitutions: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. In a preferred experimental scheme of the present invention, the Fc region employs an L234A / L235A mutation combination. In another preferred experimental scheme of the present invention, the Fc region employs an L234A / L235A / P329G mutation combination.

[0216] In some implementations, the mutated Fc region has enhanced affinity for the neonatal Fc receptor (FcRn) compared to the wild-type Fc region; for example, by introducing the M252Y / S254T / T256E mutation into the Fc region.

[0217] Therefore, in some embodiments, the bispecific antibody of the present invention is mutated. In some embodiments, the Fc region of the bispecific antibody of the present invention comprises, relative to the wild-type Fc region, the L234A / L235A / M252Y / S254T / T256E / P329G mutation. In some embodiments, the Fc region having the L234A / L235A / M252Y / S254T / T256E / P329G mutation comprises or is composed of the amino acid sequence shown in SEQ ID NO:99, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0218] Therefore, in some embodiments, the bispecific antibody of the present invention is mutated. In some embodiments, the Fc region of the bispecific antibody of the present invention comprises, relative to the wild-type Fc region, the L234A / L235A / M252Y / S254T / T256E mutation. In some embodiments, the Fc region having the L234A / L235A / M252Y / S254T / T256E mutation comprises or is composed of the amino acid sequence shown in SEQ ID NO:103, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0219] In some embodiments, the bispecific antibody of the present invention may contain two Fc regions, which may be the same or different.

[0220] In some embodiments, the Fc region of the bispecific antibody of the present invention comprises two distinct Fc regions that heterodimerize and may each contain modifications or mutations that inhibit Fc homodimerization, such as a Knob-into-hole mutation. In some embodiments, the Fc region comprises a variant of the human IgG1 Fc wild-type sequence. Preferably, one side of the variant Fc domain is replaced with an amino acid residue with a larger side chain volume, i.e., a knob mutation, thereby forming a raised structure; the other side is replaced with an amino acid residue with a smaller side chain volume, i.e., a hole mutation, thereby forming a hollow structure; the hollow structure accommodates the raised structure, thereby forming a heterodimer.

[0221] In some embodiments, the Fc region of the bispecific antibody of the present invention comprises two different Fc regions, one of which contains a Knob mutation and the other of which contains a Hole mutation.

[0222] Preferably, the mutation schemes of the Fc domain are selected from: (a) knob mutation to T366W, and hole mutation to T366S, L368A and Y407V; (b) knob mutation to S354C and T366W, and hole mutation to Y349C, T366S, L368A and Y407V; the above numbering method is in accordance with the EU index.

[0223] Therefore, in one specific embodiment, the bispecific antibody of the present invention comprises two heterodimerized Fc regions, one Fc region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:100 and comprising the mutations S354C and T366W, and optionally the L234A / L235A / M252Y / S254T / T256E / P329G mutation, and the other Fc region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:65 and comprising the mutations Y349C, T366S, L368A, and Y407V, and optionally the L234A / L235A / M252Y / S254T / T256E / P329G mutation.

[0224] Therefore, in one specific embodiment, the bispecific antibody of the present invention comprises two heterodimerized Fc regions, one Fc region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:104 and comprising the mutations S354C and T366W, and optionally the L234A / L235A / M252Y / S254T / T256E mutation, and the other Fc region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:105 and comprising the mutations Y349C, T366S, L368A, and Y407V, and optionally the L234A / L235A / M252Y / S254T / T256E mutation.

[0225] In some specific embodiments of the present invention, the bispecific antibody of the present invention specifically binds to two domains of FXI, comprising a first antigen-binding domain A2 that specifically binds to FXI and a second antigen-binding domain CD that specifically binds to FXI, wherein the first antigen-binding domain is the scFv of the anti-FXI antibody described in the first aspect, and the second antigen-binding domain is the Fab of the anti-FXI antibody described in the first aspect, wherein the C-terminus of the Fab heavy chain of the second antigen-binding domain is directly or preferably connected to the N-terminus of the Fc region via a linker, and the C-terminus of the scFv of the first antigen-binding domain is directly or preferably connected to the N-terminus of the Fc region via a linker; optionally, the bispecific antibody comprises two first antigen-binding domains, two second antigen-binding domains, and two identical Fc regions.

[0226] In some specific embodiments of the present invention, the bispecific antibody of the present invention specifically binds to two domains of FXI, comprising a first antigen-binding domain of A2 that specifically binds to FXI and a second antigen-binding domain of CD that specifically binds to FXI, wherein the first antigen-binding domain is the scFv of the anti-FXI antibody described in the first aspect, and the second antigen-binding domain is the Fab of the anti-FXI antibody described in the first aspect, wherein the C-terminus of the Fab heavy chain of the second antigen-binding domain is directly or via a linker (preferably directly) connected to the N-terminus of the Fc, and the C-terminus of the scFv of the first antigen-binding domain is directly or preferably via a linker connected to the N-terminus of the other Fc region.

[0227] Optionally, the bispecific antibody comprises a first antigen-binding domain and a second antigen-binding domain, as well as two distinct Fc regions (e.g., the two Fc regions contain Knob mutations and Hole mutations, respectively).

[0228] In some specific embodiments of the present invention, the bispecific antibody of the present invention specifically binds to two domains of FXI, comprising a first antigen-binding domain A2 that specifically binds to FXI and a second antigen-binding domain CD that specifically binds to FXI, wherein the first antigen-binding domain comprises a first heavy chain variable region VH1 and a first light chain variable region VL1, and the second antigen-binding domain comprises a second heavy chain variable region VH2 and a second light chain variable region VL2, and the bispecific antibody comprises or is composed of a first chain, a second chain, and a third chain.

[0229] The first chain contains or consists of the following: VH2-CH1-first Fc;

[0230] The second chain includes or consists of the following: VH1-connector 1-VL1-connector 2-second Fc;

[0231] The third chain contains or consists of the following: VL2-CL;

[0232] VH1, VL1, VH2, and VL2 are defined as follows, the first Fc and the second Fc are different, and as defined above, CL is the light chain constant region; CH1 is the heavy chain constant region CH1.

[0233] In some specific embodiments of the present invention, the bispecific antibody of the present invention specifically binds to two domains of FXI, comprising a first antigen-binding domain A2 that specifically binds to FXI and a second antigen-binding domain CD that specifically binds to FXI, wherein the first antigen-binding domain comprises a first heavy chain variable region VH1 and a first light chain variable region VL1, and the second antigen-binding domain comprises a second heavy chain variable region VH2 and a second light chain variable region VL2, and the bispecific antibody comprises a first chain and a second chain, preferably two first chains and two second chains or composed of the two first chains and two second chains, wherein the first chain comprises or is composed of the following: VH2-CH1-linker 3-VH1-linker 1-VL1-linker 2-Fc;

[0234] The second chain contains or consists of the following: VL2-CL;

[0235] VH1, VL1, VH2, and VL2 are defined as described in this paper, Fc is defined as described above, CL is the light chain constant region, and CH1 is the heavy chain constant region CH1.

[0236] In some embodiments, the light chain constant region CL of the bispecific antibody of the present invention is derived from the light chain constant region of Kappa or Lambda, such as the human Kappa or Lambda light chain constant region. In some embodiments, the CL is the human Kappa or Lambda light chain constant region. In some embodiments, the CL comprises the amino acid sequence shown in SEQ ID NO: 80 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown above.

[0237] In some embodiments, the heavy chain constant region CH1 of the bispecific antibody of the present invention is or is derived from the heavy chain constant region of human IgG, such as CH1 of the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain constant region is CH1 of the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, CH1 comprises the amino acid sequence shown in SEQ ID NO: 95 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown above.

[0238] In some embodiments, linkers 1, 2, and 3 are peptide linkers, such as short amino acid sequences consisting of amino acids, for example, glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination. Preferably, the peptide linkers are of sufficient length to connect two entities in such a way that they maintain their conformation relative to each other without hindering the desired activity. In some embodiments, the linkers can be any flexible linker comprising an amino acid sequence selected from (G4)n, (G4S)n, and GS(G4S)n, where n is an integer selected from 1-5, and preferably the linker comprises an amino acid sequence such as GGGG or GGGGS.

[0239] In some embodiments, the connector 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:97, and / or the connector 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:98, and / or the connector 3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:102 (GGGGS).

[0240] In some specific embodiments of the present invention, the bispecific antibody of the present invention specifically binds to two domains of FXI, comprising a first antigen-binding domain A2 that specifically binds to FXI and a second antigen-binding domain CD that specifically binds to FXI, wherein the first antigen-binding domain comprises a first heavy chain variable region VH1 and a first light chain variable region VL1, and the second antigen-binding domain comprises a second heavy chain variable region VL2 and a second light chain variable region VL2, and the bispecific antibody comprises or is composed of a first chain, a second chain, and a third chain.

[0241] The first chain contains the amino acid sequence shown in SEQ ID NO:67, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:67;

[0242] The second chain contains the amino acid sequence shown in SEQ ID NO:68, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:68; and

[0243] The third chain contains the amino acid sequence shown in SEQ ID NO:57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:57;

[0244] Preferably, the first chain contains the amino acid sequence shown in SEQ ID NO:67, the second chain contains the amino acid sequence shown in SEQ ID NO:68, and the third chain contains the amino acid sequence shown in SEQ ID NO:57.

[0245] In some specific embodiments of the present invention, the bispecific antibody of the present invention specifically binds to two domains of FXI, comprising a first antigen-binding domain A2 that specifically binds to FXI and a second antigen-binding domain CD that specifically binds to FXI, wherein the first antigen-binding domain comprises a first heavy chain variable region VH1 and a first light chain variable region VL1, and the second antigen-binding domain comprises a second heavy chain variable region VL2 and a second light chain variable region VL2, and the bispecific antibody comprises or is composed of a first chain, a second chain, and a third chain.

[0246] The first chain contains the amino acid sequence shown in SEQ ID NO:93, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:93;

[0247] The second chain contains the amino acid sequence shown in SEQ ID NO:94, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:94; and

[0248] The third chain contains the amino acid sequence shown in SEQ ID NO:57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:57;

[0249] Preferably, the first chain contains the amino acid sequence shown in SEQ ID NO:93, the second chain contains the amino acid sequence shown in SEQ ID NO:94, and the third chain contains the amino acid sequence shown in SEQ ID NO:57.

[0250] In some specific embodiments of the present invention, the bispecific antibody of the present invention specifically binds to two domains of FXI, comprising a first antigen-binding domain A2 that specifically binds to FXI and a second antigen-binding domain CD that specifically binds to FXI, wherein the first antigen-binding domain comprises a first heavy chain variable region VH1 and a first light chain variable region VL1, and the second antigen-binding domain comprises a second heavy chain variable region VL2 and a second light chain variable region VL2, and the bispecific antibody comprises a first chain and a second chain, preferably two first chains and two second chains, or composed of the two first chains and two second chains, wherein...

[0251] The first chain contains the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:56;

[0252] The second chain contains the amino acid sequence shown in SEQ ID NO:57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:57;

[0253] Preferably, the first chain contains the amino acid sequence shown in SEQ ID NO:56, and the second chain contains the amino acid sequence shown in SEQ ID NO:57.

[0254] In some implementations, insertions, deletions, and / or substitutions may be performed in frame (FR) regions, such as FR1, FR2, FR3, and / or FR4; and / or constant regions, such as CL, CH1, CH2, and / or CH3.

[0255] In some implementations, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0256] 3. Polynucleotides, vectors, and hosts

[0257] A third aspect of the invention provides a nucleic acid encoding any of the above-described antibodies or their antigen-binding fragments (covering the anti-FXI antibody or its antigen-binding fragment of the present invention, as well as multispecific antibodies such as bispecific antibodies) or any one strand thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector, such as the pTT5 vector. In one embodiment, a host cell comprising said nucleic acid or said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells), or other cells suitable for preparing antibodies or their antigen-binding fragments. In another embodiment, the host cell is prokaryotic.

[0258] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 1-59, 61, 62, 64-68 and 93 and 94, or a nucleic acid encoding an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 1-59, 61, 62, 64-68 and 93 and 94.

[0259] The present invention also covers nucleic acids that hybridize under stringent conditions or that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions compared to the following nucleic acids: nucleic acids comprising nucleic acid sequences encoding amino acid sequences selected from any one of SEQ ID NO: 1-59, 61, 62, 64-68; or nucleic acids comprising nucleic acid sequences encoding amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acid sequences selected from any one of SEQ ID NO: 1-59, 61, 62, 64-68.

[0260] In one embodiment, one or more vectors containing the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, kinases, λ phages, or yeast artificial chromosomes (YAC). In one embodiment, the vector is a pTT5 vector.

[0261] Once the expression vector or DNA sequence for expression has been prepared, it can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells.

[0262] Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0263] In one embodiment, a host cell comprising one or more of the polynucleotides of the present invention is provided. In some embodiments, a host cell comprising the expression vector of the present invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. Suitable host cells include prokaryotic microorganisms, such as *Escherichia coli*. The host cell can also be a eukaryotic microorganism such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as a host. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK293 or 293F cells), 293 cells, young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOS cells, NSO cells, and myeloma cell lines such as Y0, NSO, P3X63, and Sp2 / 0. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a 293 cell, such as a HEK293 cell, for example, HEK293-F.

[0264] 4. Pharmaceutical compositions and pharmaceutical preparations

[0265] A fourth aspect of the invention also provides compositions (including pharmaceutical compositions or pharmaceutical formulations) comprising the anti-FXI antibody of the present invention (covering the anti-FXI antibody of the present invention or its antigen-binding fragment and multispecific antibodies such as bispecific antibodies) and compositions comprising polynucleotides encoding the anti-FXI antibody of the present invention (covering the anti-FXI antibody of the present invention or its antigen-binding fragment and multispecific antibodies such as bispecific antibodies). These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0266] The pharmaceutical carriers suitable for use in this invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations can contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, and saccharin.

[0267] Pharmaceutical formulations comprising the invention can be prepared by mixing the anti-FXI antibody of the invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0268] In the pharmaceutical compositions and formulations of the present invention, the antibodies of the present invention may be the sole active agent or may be combined with other therapeutic agents. Therapeutic agents that may be combined with the antibodies of the present invention include, but are not limited to, therapeutic agents that have beneficial therapeutic effects on the disease and / or condition to be treated. For example, the active ingredient may be required for the specific indication being treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, other pharmaceutical ingredients that can provide anticoagulant activity may be present in the pharmaceutical compositions and formulations in a suitable combination with the active ingredients in an amount effective for the intended use.

[0269] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0270] 5. Combination Products

[0271] A fifth aspect of the invention also provides a combination product or pharmaceutical combination comprising the antibody of the invention or an antigen-binding fragment thereof (covering the anti-FXI antibody of the invention or an antigen-binding fragment thereof and multispecific antibodies such as bispecific antibodies), and one or more other therapeutic agents, such as one or more other anticoagulants or anticoagulant drugs or antithrombotic drugs, preferably selected from vitamin K antagonists (e.g., warfarin), thrombin inhibitors (e.g., direct thrombin inhibitors such as gandinox, dabigatran; indirect thrombin inhibitors such as low molecular weight heparin; or special thrombin inhibitors such as FXa inhibitors, such as edoxaban, rivaroxaban or apixaban; or antiplatelet drugs (e.g., COX inhibitors, ADP receptor antagonists, GPIIb / IIIa inhibitors, PDE inhibitors); fibrinolytic drugs (e.g., propranolol).

[0272] In some embodiments, the present invention provides combination products or drug combinations, wherein the other anticoagulant is, for example, an effective blockade of intrinsic or extrinsic coagulation processes, thereby further enhancing the anticoagulant effect, such as an FXa inhibitor. The combination products or drug combinations of the present invention can be used in the treatment methods of the present invention.

[0273] In some embodiments, the combination product or drug combination is used to prevent or treat diseases or conditions associated with FXI activity or expression, such as thromboembolic diseases or their complications, including but not limited to any of the following: thrombotic or thromboembolic diseases, thrombotic or thromboembolic complications, arrhythmias, ischemic stroke, disseminated intravascular coagulation; preferably, the thrombotic or thromboembolic diseases or their complications are selected from: coronary artery disease, myocardial infarction with ST-segment elevation, myocardial infarction without ST-segment elevation, stable angina, unstable angina, and post-coronary artery intervention. Restenosis and re-stenosis can lead to peripheral artery occlusive disease, pulmonary embolism, venous thromboembolism, venous thrombosis, transient ischemic attack, thrombotic stroke and thromboembolic stroke, lung disease caused by chronic thromboembolism, pulmonary hypertension caused by CTEPH, myocardial infarction, ischemic stroke, pulmonary thromboembolism, atrial fibrillation, medical device-related thromboembolic conditions, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (such as cardiopulmonary bypass, hemodialysis and ECMO), arterial thrombosis, advanced renal disease, antiphospholipid syndrome, metastatic cancer, or infectious diseases. In some implementations, thromboembolic disease is venous thromboembolism (VTE).

[0274] The present invention also relates to a kit comprising the anti-FXI antibody or antigen-binding fragment or bispecific antibody of the present invention, or an anti-FXI antibody or its antigen-binding fragment or bispecific antibody prepared by the method of the present invention, the polynucleotide of the present invention, the vector of the present invention, the host cell of the present invention, the pharmaceutical composition of the present invention, or the pharmaceutical combination of the present invention.

[0275] 6. Treatment methods and uses

[0276] The sixth aspect of the invention is also a method or use of the antibody or antigen-binding fragment thereof of the invention (covering the anti-FXI antibody or antigen-binding fragment thereof of the invention and multispecific antibodies such as bispecific antibodies).

[0277] In this document, the terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, a subject is a human.

[0278] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0279] Because FXI plays a key role in the amplification process of the coagulation cascade and the endogenous pathway, and is closely related to the occurrence of thrombotic diseases, FXI antibodies (including the anti-FXI antibodies of the present invention or their antigen-binding fragments, as well as multispecific antibodies such as bispecific antibodies) can be used to treat thromboembolic diseases or their complications.

[0280] In some embodiments, the present invention relates to methods or uses of applying any anti-FXI antibody or fragment thereof described herein (covering the anti-FXI antibody or antigen-binding fragment thereof of the present invention and multispecific antibodies such as bispecific antibodies) for use in vivo or in vitro.

[0281] - Combine with FXI; or

[0282] -Block the activation of FXI to form FXIa; or

[0283] -Inhibits thrombus formation,

[0284] Or for use in the preparation of a drug for any of the above purposes.

[0285] In some embodiments, the present invention relates to a method for preventing or treating diseases or conditions associated with FXI activity or expression in a subject, the method comprising administering to the subject an effective amount of any anti-FXI antibody or fragment thereof described herein (covering the anti-FXI antibodies or antigen-binding fragments thereof of the present invention and multispecific antibodies such as bispecific antibodies), or a pharmaceutical composition comprising said antibody or fragment. In some embodiments, the diseases associated with FXI activity or expression include, but are not limited to, any of the following: thrombotic or thromboembolic diseases, thrombotic or thromboembolic complications, arrhythmias, ischemic stroke, or disseminated intravascular coagulation.

[0286] In this invention, the term "thromboembolic disease" or similar terms as used herein include, but are not limited to, coronary artery disease, myocardial infarction with ST-segment elevation, myocardial infarction without ST-segment elevation, stable angina, unstable angina, re-occlusion and restenosis after coronary intervention leading to peripheral artery occlusive disease, pulmonary embolism, venous thromboembolism, venous thrombosis, transient ischemic attack, thrombotic stroke and thromboembolic stroke, lung disease caused by chronic thromboembolism, pulmonary hypertension caused by CTEPH, myocardial infarction, ischemic stroke, pulmonary thromboembolism, atrial fibrillation, medical device-related thromboembolic disease, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (such as cardiopulmonary bypass, hemodialysis and ECMO), arterial thrombosis, advanced renal disease, antiphospholipid syndrome, metastatic cancer or infectious disease, preferably, venous thromboembolism.

[0287] In some embodiments, the present invention relates to a method of anticoagulation in a subject, the method comprising administering to the subject an effective amount of any antiFXI antibody or fragment thereof described herein (covering the antiFXI antibody or antigen-binding fragment thereof of the present invention and multispecific antibodies such as bispecific antibodies), or a pharmaceutical composition comprising said antibody or fragment.

[0288] In some embodiments, the present invention relates to a method for preventing or treating diseases or conditions associated with FXI activity or expression, such as thrombosis or thromboembolic diseases, in a subject, the method comprising administering to the subject an effective amount of any anti-FXI antibody or fragment thereof described herein (covering the anti-FXI antibodies or antigen-binding fragments thereof of the present invention and multispecific antibodies such as bispecific antibodies), or a pharmaceutical composition comprising said antibody or fragment.

[0289] In some embodiments, the anti-FXI antibody of the present invention (including the anti-FXI antibody of the present invention or its antigen-binding fragment and multispecific antibodies such as bispecific antibodies) is administered to thromboembolic subjects to inhibit the activation of FXI, reduce the formation of thromboembolism, and without increasing the risk of bleeding.

[0290] In some embodiments, the method described herein further includes administering one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to the subject in combination. In some embodiments, treatment modalities include surgical treatment and interventional treatment.

[0291] In some embodiments, the coagulation reaction can be inhibited by combining the anti-FXI antibody of the present invention with one or more therapeutic agents. In some embodiments, in addition to administering the antibody of the present invention, the method of the present invention further includes using at least one other therapeutic agent, such as one or more other anticoagulants or anticoagulant drugs or antithrombotic drugs, preferably selected from vitamin K antagonists (e.g., warfarin), thrombin inhibitors (e.g., direct thrombin inhibitors such as gandinox, dabigatran; indirect thrombin inhibitors such as low molecular weight heparin; or special thrombin inhibitors such as FXa inhibitors, such as edoxaban, rivaroxaban, or apixaban; or antiplatelet drugs (e.g., COX inhibitors, ADP receptor antagonists, GPIIb / IIIa inhibitors, PDE inhibitors); fibrinolytic drugs (e.g., propranolol).

[0292] The antibodies of the present invention (including the anti-FXI antibodies of the present invention or their antigen-binding fragments and multispecific antibodies such as bispecific antibodies) and pharmaceutical compositions or combinations thereof comprising the present invention, and any other therapeutic agents) may be administered by any suitable method, including oral administration, parenteral administration, intrapulmonary administration, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it may be administered by any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0293] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and progression of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient as a single treatment or after a series of treatments.

[0294] The present invention also relates to the use of the antibodies of the present invention or antigen-binding fragments thereof (covering the anti-FXI antibodies of the present invention or antigen-binding fragments thereof and multispecific antibodies such as bispecific antibodies), pharmaceutical compositions and pharmaceutical combinations comprising the present invention for anticoagulation or prevention or treatment of diseases or conditions such as thrombosis or thromboembolic diseases associated with FXI activity or expression as described herein.

[0295] This invention also relates to the use of the antibodies of the present invention or antigen-binding fragments thereof (covering the anti-FXI antibodies of the present invention or antigen-binding fragments thereof and multispecific antibodies such as bispecific antibodies), pharmaceutical compositions comprising the thereof, and pharmaceutical combinations for the preparation of medicaments for anticoagulation or prevention or treatment of diseases or conditions related to FXI activity or expression as described herein, such as thrombosis or thromboembolic diseases.

[0296] The present invention also relates to antibodies of the present invention or antigen-binding fragments thereof (covering anti-FXI antibodies of the present invention or antigen-binding fragments thereof and multispecific antibodies such as bispecific antibodies), pharmaceutical compositions and pharmaceutical combinations comprising the present invention for anticoagulation or prevention or treatment of diseases or conditions such as thrombosis or thromboembolic diseases associated with FXI activity or expression as described herein.

[0297] The present invention also relates to antibodies of the present invention or antigen-binding fragments thereof (covering anti-FXI antibodies of the present invention or antigen-binding fragments thereof and multispecific antibodies such as bispecific antibodies), pharmaceutical compositions comprising the present invention, and pharmaceutical compositions thereof for use in treating and / or preventing diseases in an individual or as a diagnostic tool for diseases, preferably, said individual being a mammal, more preferably a human.

[0298] 7. Detection Applications

[0299] The present invention also provides a method and kit for detecting FXI in a sample, wherein the method comprises: (a) contacting the sample with an antibody of the present invention or an antigen-binding fragment thereof; and (b) detecting the formation of a complex between the antibody or the antigen-binding fragment thereof and the FXI protein.

[0300] When used herein, the term "detection" includes both quantitative and qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In some embodiments, the biological sample comprises cells or tissues. In some embodiments, the FXI to be detected is a human FXI.

[0301] The following embodiments are described to aid in understanding the invention. It is not intended, and should not be construed in any way, as limiting the scope of the invention. Example

[0302] Example 1: Preparation of BMK molecules and FXI and FXII proteins

[0303] 1.1 Preparation of BMK (positive control molecule) and Isotype (negative control molecule):

[0304] Abelacimab is a fully human monoclonal antibody that targets and binds to the catalytic domain (CD region) of FXI. The molecule was prepared with reference to the sequence information provided in INN Recommended List 81 (who.int), and its heavy chain sequence (SEQ ID NO: 70) and light chain sequence (SEQ ID NO: 71).

[0305] Xisomab is a recombinant human monoclonal antibody that targets and binds to the A2 region of the FXI apple domain. The molecule was prepared with reference to the sequence information provided in INN Recommended List 91 (who.int), and its heavy chain sequence (SEQ ID NO: 72) and light chain sequence (SEQ ID NO: 73).

[0306] KN060: Recombinant humanized FCI single-domain antibody Fc fusion protein (SEQ ID NO: 78) injection.

[0307] 1.2 Preparation of FXI protein

[0308] Both FXI and FXII play important roles in the intrinsic coagulation pathway. When the blood vessel wall is damaged and the subendothelial tissue is exposed, negatively charged subendothelial collagen fibers come into contact with coagulation factors, and FXI binds to them. With the participation of HK and PK, FXI is activated to form FXIIa. Under calcium-independent conditions, FXIIa activates FXI. The human full-length FXI protein (HFXI 1111, Enzyme Research), human FXII protein (HFXI 1212, Enzyme Research), and monkey FXI protein (FXI-C52H3, Acro) used in the experiments of this invention were purchased. The FXI-A2 and FXI-CD sequences were obtained from the UniProt database and the rabbit FXI protein (19-624aa, NP_001076261.1, NCBI), and were prepared using conventional experimental methods. The specific sequence information is as follows: human FXI full-length sequence (SEQ ID NO:74); FXI A2 protein sequence (SEQ ID NO:75); FXI CD protein sequence (SEQ ID NO:76); FXII full-length sequence (SEQ ID NO:77).

[0309] Example 2: Preparation of FXI chimeric antibody

[0310] 2.1 Preparation of murine antibodies

[0311] Balb / c mice were immunized by subcutaneous injection of the full-length FXI protein (HFXI 1111, Enzyme Research). Single-cell suspensions were prepared from the spleens of animals with high titers and electrofused with myeloma cells to form hybridomas. These hybridomas were then seeded in 96-well plates and cultured continuously for about 7 days to allow the hybridomas to grow to a certain number before further screening.

[0312] First, an ELISA assay was used for preliminary screening, with BMK molecules serving as a positive control, to obtain hybridoma clones with high affinity. Further screening was conducted using activated partial thromboplastin time (APTT) and prothrombin time (PT) activity assays to select hybridomas that met the requirements. Subcloning experiments were then performed, and finally, the confirmed monoclonal cells were expanded and cultured until they reached a certain number before sequencing.

[0313] 2.2 Preparation of chimeric antibodies

[0314] The heavy / light chain sequences of the positive clones obtained above were cloned into pTT5-hIgG4.CH (containing the amino acid sequence of the constant region of the IgG4 heavy chain, SEQ ID NO: 79) and pTT5-hKappa.CL (containing the amino acid sequence of the constant region of the Kappa light chain, SEQ ID NO: 80) respectively via homologous recombination to obtain chimeric antibody heavy / light chain expression plasmids, which were then transfected into 293F cells. 293F cells in good logarithmic growth phase were seeded into 250 mL cell culture flasks and cultured in 50 mL of medium. 25 μg of each of the light and heavy chain expression plasmids were co-transfected with PEI. Cell supernatant was collected on day 7 post-transfection, centrifuged, and filtered using a 0.45 μM filter. The antibody was purified using Protein A medium and dialyzed to replace it in PBS pH 7.2 buffer. Antibody concentration and purity were determined by Nanodrop absorbance measurement, and purity was detected by sodium dodecyl sulfate gel electrophoresis and Coomassie staining. Finally, the corresponding chimeric antibody was prepared. The preferred chimeric antibody sequence information is shown below, with the underlined portion being the CDR region.

[0315] IgG4 heavy chain constant region: SEQ ID NO:79; Kappa light chain constant region: SEQ ID NO:80

[0316] Similar preparation methods were used for BMK positive control molecules and humanized antibodies.

[0317] 2.3 Data on chimeric antibody activity

[0318] APTT is an important indicator of coagulation function reflecting the intrinsic coagulation pathway. It is mainly used to detect the activity of coagulation factors (such as coagulation factors VIII, IX, XI, XII, etc.) in plasma to determine the normality of blood coagulation function. Therefore, this invention uses activated partial thromboplastin time (APTT) to evaluate the ability of anti-FXI antibody to block coagulation in vitro. Furthermore, the binding activity of the above-mentioned chimeric antibody to different domains (A1-A4, CD) of the FXI protein and the activated partial thromboplastin time (APTT) were tested.

[0319] Protein binding-ELISA assay: Plate coating: Add 100 μL of 1 μg / mL protein solution (FXI-A1, FXI-A2, FXI-A3, FXI-A4, or FXI-CD) to each well of a 96-well microplate and incubate overnight at 2-8°C; Washing: Aspirate the liquid from each well and wash twice with PBST (PBS + 0.1% Tween-20) to remove excess antigen solution. After washing, invert the plate to remove any remaining liquid; Blocking: Add 100 μL of 2% protein solution to each well at room temperature. BSA (prepared with PBS buffer), block for 1 hour; washing: aspirate the liquid from each well, wash twice with washing buffer to remove excess antigen solution, after washing, invert the plate to remove residual liquid; sample loading: add 100 μL of gradient concentration of the test antibody (starting concentration 20 μg / mL, 3-fold serial dilution) to each well, incubate at room temperature for 1 hour; washing: aspirate the liquid from each well, wash 5 times with washing buffer; add 100 μL of secondary antibody to each well, incubate at room temperature for 0.5 hours.

[0320] Washing: Aspirate the liquid from each well and wash 5 times with washing buffer; Development: Add 100 μL TMB substrate solution to each well and incubate the plate at room temperature for 10 minutes or longer until the solution reaches the desired color intensity; Termination: Add 100 μL HCl stop solution to each well; Measurement: Within 20 minutes of adding the stop solution, measure the absorbance of the solution in each well at a wavelength of 450 nm using a microplate reader. APTT assay: Add 75 μL of diluted chimeric antibody (10 μg / ml) to each well of a 96-well plate. Add 75 μL of PBS to each well (2.5 μg / ml + 0.625 μg / ml blank control). Take thawed standard plasma and centrifuge at 1000 rpm for 1 min to remove the precipitate. Add 75 μL of standard plasma to each well of the 96-well plate. Mix the supernatant with the standard plasma thoroughly. Seal the plate and incubate at 37℃ for 10 min. Remove the seal and transfer the samples to 1.5 ml centrifuge tubes. Cut off the caps and place the tubes in the loading slots in the order A1, B1, C1… Preheat the APTT reagent and calcium chloride solution (NXS0190, SECCO) according to the instructions and place them in the corresponding reagent slots. Double-click the blank area under APTT to prepare the sample for testing and start the experiment. Data will be automatically recorded. Instrument: Fully automated coagulation analyzer (SF-8050, SECCO).

[0321] The experimental results are shown in Tables 1 and 2. Different clones bind to different domains of FXI, mainly binding to the FXI-A2 and FXI-CD regions. Meanwhile, the 11H4 and 13E12 clones can significantly prolong APTT, comparable to Abelacimab, and both have certain species cross-activity with rabbits and dogs.

[0322] Table 1. In vitro activity data of chimeric antibodies "+" indicates that it binds to the corresponding protein, and "-" indicates that it does not bind to the corresponding protein. "APTT" refers to the activation time of partial thromboplastin.

[0323] Table 2. Effects of chimeric antibodies on APTT in different species

[0324] Example 3: Humanization of FXI chimeric antibody

[0325] The chimeric antibody described above was humanized to reduce its immunogenicity. The CDR region of the murine monoclonal antibody was combined with the relatively conserved human variable framework region (FR), and the corresponding protein was expressed after gene recombination. The chimeric antibody of this invention was humanized according to the following steps: ① The CDR sequence was determined according to the IMGT extended CDR partitioning method; ② The closest homologous sequence was found in the human genotype sequence database for each V / J region of the heavy and light chains; ③ The genotypes that best matched the heavy and light chains and the minimum amount of reversion mutations were screened; ④ The CDR region of the chimeric antibody was constructed onto the human backbone region; ⑤ The amino acid positions in the backbone region that maintain the CDR function were determined using sequence and structural features; ⑥ Reversion mutations were performed at the identified important sequence positions. ⑦ Optimize the amino acids at risk sites; Obtain the light chain variable region and heavy chain variable region of the humanized antibody, of which 13E12 clones obtained 4 VH and 4 VL sequences, named 13E12H1, 13E12H2, 13E1H3, 13E12H4, 13E12L1, 13E12L2, 13E12L3, 13E12L4, respectively, which are combined to form humanized molecules 13E12Z01-13E12Z24, and the original light chain variable region and heavy chain variable region are named L0 and H0, respectively;

[0326] Table 3.1 Correspondence between the names of 13E12 humanized clones and heavy / light chains

[0327] The 11H4 clone also yielded four VH and four VL sequences, named 11H4H1, 11H4H2, 11H4H3, 11H4H4, 11H4L1, 11H4L2, 11H4L3, and 11H4L4, respectively. These sequences were combined to form humanized molecules 11H4Z01-11H4Z24. The original light chain variable region and heavy chain variable region were named L0 and H0, respectively.

[0328] Table 3.2 Correspondence between the names of 11H4 humanized clones and heavy / light chains

[0329] Similar to Example 2.2, plasmids of humanized molecules were transfected into 293F cells. Cell supernatant was collected on day 5 and purified by Protein A. The obtained antibodies were then dialyzed and replaced with 1×PBS buffer. Antibodies of acceptable purity were screened by ELISA (Figure 1), and humanized molecules 11H4Z12 and 13E12Z08 were selected.

[0330] The sequence information of the humanized light / heavy variable region is shown below:

[0331] The preferred humanized light / heavy variable region sequence information is shown below:

[0332] Example 4: Binding activity of FXI antibody to FXI and FXII proteins

[0333] From the above-mentioned humanized molecules, 13E12Z08 and 11H4Z12 humanized molecules were selected and their binding activity and selectivity with FXI and FXII proteins were investigated. For specific experimental procedures, please refer to Example 2. The experimental results are shown in Figure 2. Humanized 11H4Z12 and 13E1Z08 bind efficiently to human FXI protein, and at the same time have good cross-activity with monkey and rabbit FXI proteins, and do not bind to FXII protein, showing good selectivity.

[0334] Example 5: Detection of in vitro activated partial thromboplastin time (APTT) of anti-FXI antibody

[0335] The time required to activate partial thromboplastin was determined in the presence of humanized 11H4Z12 and 13E12Z08. Fresh human blood was collected from volunteers using sodium citrate tubes, centrifuged at 3000 rpm for 15 min, and the supernatant plasma was collected. This plasma was mixed with the FXI antibody of the present invention at a pre-set concentration (333.33 nM, 166.67 nM, followed by 4-fold serial dilutions). The anti-FXI antibody-plasma mixture and 25 μL of APTT reagent (NXS0190, SECCO) were incubated at 37°C for 3 min. Then, 30 μL of 25 mM calcium chloride solution was added to initiate coagulation. Finally, the coagulation time was determined using a fully automated coagulation analyzer (SF-8050, SECCO). As shown in Figure 3, the 11H4Z12 and 13E12Z08 molecules significantly prolonged the activated partial thromboplastin time (APTT), which was about three times that of the negative control group. Moreover, at a low concentration, they could achieve a 1.5-fold prolongation of APTT, which was superior to the control antibodies Abelacimab and Xisomab. They also had a significant inhibitory effect on FXI and could effectively inhibit the intrinsic coagulation pathway.

[0336] Example 6: In vitro FXIa enzyme activity inhibition assay of FXI antibody

[0337] To assess whether anti-FXI antibodies affect the function of their activated form—FXIa, this experiment used BIOPHEN. TM The FXIa kit was used to detect the effect of candidate molecule pairs on the catalytic activity of FXIa. The principle of the kit is as follows: In the presence of phospholipids (PLPs), calcium, and thrombin, FXIa in the test sample can activate FIX to form FIXa. FIXa can form an enzyme complex with thrombin-activated FVIII:C, activating FX to form FXa. FXa can hydrolyze the chromogenic substrate SXa-11, releasing p-nitroaniline (pNa). Finally, its absorbance at 405 nm was measured.

[0338] Add antibody solutions of different concentrations (prepared with Tris-BSA buffer, starting at 100 nM, serially diluted 3 times) and 10 mIU FXIa to a 96-well plate, mix well, and incubate at 37°C for 10 min. Then add FX and FCVIII:C solutions and FIX solutions from the kit, mix well, and incubate at 37°C for 2 min. Next, add the activation reagent (thrombin-calcium-phospholipid solution), mix well, and incubate at 37°C for 2 min. Add 50 μL of SXa-11 solution and incubate at 37°C for 5 min. Finally, add 2% citric acid to terminate the reaction. Measure the absorbance at 405 nm using a microplate reader. Analyze the data using GraphPad Prism software.

[0339] The experimental results are shown in Figure 4, where human IgG isotype was used as a negative control (NC). A graph was plotted with the logarithm of antibody concentration on the x-axis and the OD value (at 405 nm) on the y-axis, and the IC50 value was calculated using Graphpad Prism. Xisomab and 11H4Z12, which target and bind to the A2 region, showed no inhibitory activity against FXIa enzyme activity. 13E12Z08 effectively bound activated FXIa and exhibited significantly stronger inhibitory activity against FXIa enzyme activity than Abelacimab, with IC50 values ​​of 0.67 nM and 8.17 nM for 13E12Z08 and Abelacimab, respectively.

[0340] Example 7: In vivo antithrombotic activity of FXI antibody

[0341] To further investigate the antithrombotic ability of FXI antibodies in vivo, a thrombosis model was established on New Zealand white rabbits, and the blood flow velocity downstream of the arteriovenous junction was measured. At the same time, blood samples were collected at different time points to perform APTT / PT coagulation index, thrombus weight, and bleeding detection, etc., to evaluate the antithrombotic ability of FXI antibody drugs in vivo.

[0342] 7.1 Establishing a rabbit thrombosis model

[0343] Before thrombosis induction, eight male New Zealand white rabbits (Pengli Biotechnology) weighing 2.0-3.0 kg were anesthetized with isoflurane and their body temperature maintained. After disinfection, the neck was exposed, and the left common carotid artery and right jugular vein were isolated. An ultrasound probe was placed perpendicularly across the lateral aspect of the jugular vein near the heart to record blood flow changes. The left carotid artery and right jugular vein were connected using a PE tube containing a 10 cm surgical suture pre-filled with saline. Blood flow changes were recorded throughout the connection. After connection, blood flowed through the surgical suture in the arteriovenous connection tube, allowing thrombus to accumulate and form. Thrombosis was monitored for 30 minutes. FXI antibody drug was administered to the animals 15 minutes before arteriovenous connection via injection into the left marginal ear vein at a dose of 10 mg / kg. Animals were randomly divided into 4 groups and administered the drug, with 2 animals in each group: G1: Vehicle (i.e., the PBS control group); G2: 11H4Z12 10 mg / kg; G3: 13E12Z08 10 mg / kg; G4: Abelacimab 10 mg / kg.

[0344] 7.2 Detection Indicators

[0345] 7.2.1 Weight

[0346] The weight of the animals was measured and recorded before and after the experiment. The average weight of each group of animals after the experiment is shown in Figure 5-2C.

[0347] 7.2.2 Blood Flow Velocity Recording

[0348] After thrombosis induction began, Doppler flowmeter (Transonic, T402PP, USA) was used to detect blood flow velocity downstream of the arteriovenous junction. The detection time interval was 1 minute, and the detection lasted for 30 minutes. The blood flow velocity of each group of animals is shown in Figure 5-2D.

[0349] 7.2.3 APTT / PT (Prothrombin Time) Test

[0350] For all experimental groups (G1-G4), approximately 0.2 mL of whole blood was collected from the saphenous vein or femoral vein of the hind limb before drug administration (-15 min), before the start of the model (0 min), after the end of the model (30 min), and at the end of the bleeding measurement (75 min), into a 3.2% sodium citrate anticoagulant tube. The blood and anticoagulant (0.109 M trisodium citrate) were mixed at a volume ratio of 9:1 and centrifuged at 1600g for 8 min to obtain a plasma sample (100 μL). The APTT / PT of the plasma was measured using a coagulation analyzer.

[0351] For details of the APTT experiment, please refer to Example 5;

[0352] Prothrombin time (PT) testing primarily reflects the status of the extrinsic coagulation system. It determines whether the coagulation time is prolonged by calculating the ratio of the coagulation time measured in blood samples from animals treated with the antibody to that of control blood samples from animals not treated with the antibody. A ratio of 1:1 or less indicates no delay or acceleration of the coagulation time, while a ratio greater than 1:1 indicates prolonged coagulation time. The results are shown in Figure 5-1C.

[0353] The prothrombin time (PT) in the presence of different antibodies (diluted in PBS) was determined using a PT prothrombin time assay kit (NXS0189, SECCO). The obtained plasma samples were mixed with pre-warmed PT reagent, and the clotting time was measured using a Coatron M4 coagulation analyzer (TECO, MC-4000, Germany). The PT value was then obtained. The results are shown in Figure 5-1D.

[0354] 7.2.4 Thrombus weight

[0355] After the model was completed, the blood flow at both ends of the connecting tube was closed, the connecting tube was quickly removed, the surgical suture inside the tube was carefully removed, the blood was wiped off on absorbent filter paper, and the wet weight of the thrombus was weighed using a balance. The results are shown in Figure 5-1A.

[0356] 7.2.5 Bleeding Test

[0357] Thirty-five minutes after modeling, a toe amputation bleeding test was performed. The amount and duration of bleeding were measured using qualitative filter paper, with a maximum duration of 40 minutes. Bleeding times exceeding 40 minutes were counted as 40 minutes. The bleeding weight and duration are shown in Figures 5-2A and 5-2B.

[0358] 7.2.6 Thrombosis Inhibition Rate

[0359] The thrombus inhibition rate is calculated as follows:

[0360] The result of (thrombus weight in vehicle control group - thrombus weight in experimental group) / thrombus weight in vehicle control group is shown in Figure 5-1B.

[0361] As shown in Figure 5-1, the wet weight of thrombi in the humanized 11H4Z12 and 13E12Z08 groups was significantly lower than that in the control group, with a statistically significant difference. This indicates that while prolonging activated partial thromboplastin time, it did not affect plasma prothrombin time. Figure 5-2 shows that the humanized 11H4Z12 and 13E12Z08 molecules did not cause significant bleeding risk during antithrombotic activity, demonstrating a high safety index. Regarding bleeding duration and weight, the amount of the positive control Abelacimab was significantly greater than that in the groups treated with humanized 11H4Z12 and 13E12Z08, while the thrombus inhibition rates were similar across the three groups. Therefore, the antibodies of this invention have higher safety during antithrombotic activity.

[0362] Example 8: Construction of FXI bispecific antibody molecule

[0363] Preliminary experiments showed that the combined use of Xisomab (targeting the A2 region) and Abelacimab / Osocimab (targeting the CD region) to synergistically block FXI-CD and the A2 domain significantly prolonged the activation of partial thromboplastin time, with better results than using anti-FXI-CD / A2 molecules alone.

[0364] Therefore, humanized 13E12Z08 (targeting the CD region) and 11H4Z12 (targeting the A2 region) were arranged and combined to construct six bispecific antibodies BIS01-BIS06, the structures of which are shown in Figure 6 and the sequences are shown in the table below.

[0365] The peptide chains corresponding to the aforementioned bispecific antibodies were cloned into the Ptt5 vector to obtain antibody expression plasmids. 293F cells in good logarithmic growth phase were seeded into 250 mL cell culture flasks and cultured in 50 mL of culture medium. The vectors carrying the heavy and light chains of the antibody molecules were transfected into the 293F cells using chemical transfection. PEI (purchased from Polysciences) was used as the chemical transfection reagent, and the cultured 293F cells were transiently transfected according to the manufacturer's protocol. Cell supernatant was collected on day 5 post-transfection culture, centrifuged at 4000 rpm for 20 min, and the supernatant was purified using Protein A medium (purchased from BorgL). The antibody was then further purified by dialysis to PBS pH 7.2 buffer to obtain the corresponding bispecific antibodies.

[0366] Example 9: Binding activity of FXI bispecific antibody molecule to human FXI and FXII proteins

[0367] Different forms of bispecific antibody molecules exhibit varying functional activities. Therefore, the binding activity of BIS01-BIS06 with FXI and FXII proteins was further investigated, specifically referring to Example 4. The experimental results are shown in Figure 7. The binding activity of the bispecific antibody molecules with the full-length FXI / FXI-CD protein was significantly better than that of the monoclonal antibody molecules, with BIS02 showing the best binding activity. Meanwhile, BIS01-BIS06 did not bind to FXII, demonstrating high selectivity.

[0368] The binding activity of the monoclonal and bispecific antibodies of the present invention with family proteins FVII, FIX and FX was also investigated. Specifically, refer to Example 4. The experimental results are shown in Figure 8. The monoclonal and bispecific antibodies of the present invention do not bind to family proteins FVII, FIX and FX, and have high selectivity.

[0369] Example 10: Extracellular activity assay of FXI dual antibody

[0370] The anticoagulant ability and bleeding risk of the bispecific antibody molecules BIS01-BIS06 were investigated by the FXIa enzyme activity inhibition function test (refer to Example 6), the activated partial thromboplastin time (APTT) test (refer to Example 5), and the prothrombin time (PT) test (refer to Example 7).

[0371] The experimental results are shown in Figure 9. In the FXIa enzyme activity inhibition assay, the inhibitory capacity of BIS01, 02, and 06 molecules (IC50 = 1.5 nM) was comparable to that of monoclonal antibodies and significantly superior to Abelacimab (IC50 = 45 nM). The dual antibody molecules can simultaneously inhibit FXI and FXIa, effectively inhibiting the intrinsic coagulation pathway. In the APTT assay, the activation time of partial thromboplastin by BIS01, 02, and 06 molecules was approximately 135 s, which prolonged the clotting time by about 30 s compared to monoclonal antibodies. In the PT assay, BIS01, 02, and 06 molecules did not affect prothrombin time and did not increase the risk of bleeding, further demonstrating the effectiveness and safety of selecting FXI targets as antithrombotic therapy.

[0372] Example 11. Affinity experiment between dual-antibody molecule and FcRn

[0373] IgG binds to the Fc receptor (FcRn) in a pH-dependent manner, meaning it binds under acidic conditions (pH 6.0-6.5) and dissociates under neutral conditions (pH 7.0-7.5). This invention modifies the antibody Fc using a combination of M252Y / S254T / T256E mutations to enhance the interaction between the Fc terminus and FcRn.

[0374] The binding kinetics (KD) of BIS02, BIS06, and Abelacimab to human FcRn protein were detected using a Gator™ label-free biomolecular analyzer (Gator Prime, Gatorbio) with Bio-Layer Interferometry (BLI). SA-hFcRn protein (FCM-H82W4, Acro) was diluted to 2 μg / mL and immobilized onto the SA biosensor. Antibodies were then diluted to six concentration gradients (125 nM, 41.67 nM, 13.89 nM, 4.63 nM, 1.54 nM, and 0.51 nM) to bind to the protein. The results were fitted using a 1:1 model to calculate the equilibrium dissociation constant (KD) value of the samples.

[0375] The experimental results are detailed in Figure 10. BIS02 and BIS06 have higher affinity for FcRn and slower dissociation rates under acidic conditions. The calculated KD ratios of BIS02, BIS06, and Abelacimab to human FcRn at pH 7.4 and pH 6.0 are 223, 184, and 148.9, respectively, indicating that BIS02 and BIS06 are more likely to participate in FcRn-mediated antibody recycling and have a prolonged half-life in the blood.

[0376] This invention further investigated the binding affinity of BIS06 and Abelacimab to human FXI and FXI-A2. Simultaneously, since FXI must first be activated into FXIa before exerting its coagulation activity during intrinsic coagulation, the binding affinity of the bispecific antibody molecules to FXIa was also tested.

[0377] The binding affinity of the above-mentioned bispecific antibody molecules to FXI, FXI-A2, and FXIa proteins was determined by surface plasmon resonance (Biacore 8K; Cytiva).

[0378] Protein A (Cytiva; 29127556) was immobilized on the sensor chip for antibody capture (experimental channel Fc2), while the reference channel Fc1 did not require ligand capture. The antibody molecules were diluted to 5 μg / mL with the running reagent (10 mM N-(2-hydroxyethyl)piperazine-N-2-sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM ethylenediaminetetraacetic acid (EDTA), 0.005% Tween-20, pH adjusted to 7.4) and injected into Fc2 at a flow rate of 10 μL / min for approximately 200 RU. Human FXI (ACRO, FXI-H52H5), FXI-A2, and FXIa (HFXIa 1111a, Enzyme Research) proteins were serially diluted 2-fold with the running reagent. The diluted protein was sequentially injected into the experimental and reference channels at a flow rate of 30 μL / min, with binding and dissociation times recorded. After each concentration analysis, the chip was regenerated for 30 s with glycine hydrochloride at pH 1.5 at a flow rate of 20 μL / min to wash away ligands and undissociated analytes. Finally, the KD value of each molecule was calculated using Biacore 8K analysis software (Biocore Insight Evaluation Software). The reference channel (Fc1) was used for background subtraction.

[0379] The results are shown in Figure 11, which indicates that the affinity of BIS06 for binding FXI and FXIa is comparable to that of Abelacimab. Furthermore, BIS06 has an affinity of 2.62 nM for binding FXI-A2, while Abelacimab shows no significant binding.

[0380] Example 12. In vivo efficacy evaluation of dual-antibody molecular cynomolgus monkeys

[0381] 12.1 Detection of APTT in monkey plasma

[0382] The experimental steps in Example 5 were repeated in collected monkey plasma (Pengli Biotechnology) to determine the time required for the bispecific antibody molecules to activate partial thromboplastin in monkey plasma.

[0383] The results are shown in Figure 12: In monkey plasma, BIS02 and BIS06 significantly prolonged APTT at low concentrations compared to KN060.

[0384] 12.2 Detection of APTT and PT in cynomolgus monkeys

[0385] Following the validation in experiment 12.1, further evaluation was conducted on cynomolgus monkeys (Pengli Biotechnology, male, 5-8.5 kg body weight). Twelve cynomolgus monkeys were randomly divided into four groups of three animals each based on their body weight. The specific drug administration regimens are shown in Table 1.

[0386] Table 1: Grouping and Dosing Regimens

[0387] Sampling time points: 0h, 0.083h (i.e., 5min), 1h, 4h, 6h, day1, day3, day5, day7, day14, day21, day28, day35, day42, day49, and day56, a total of 15 time points. At each time point, 0.4mL of whole blood was collected, anticoagulated with 3.2% sodium citrate (anticoagulant to whole blood volume ratio 1:9), and centrifuged at 1600g at room temperature for 8mins to separate plasma samples. The APTT / PT of fresh plasma was measured using a blood coagulation analyzer, referring to Examples 5 and 7.

[0388] The experimental results are shown in Figure 13, which demonstrate that BIS02 and BIS06 molecules can persistently prolong APTT in cynomolgus monkeys. The maximum APTT ratio of BIS06 is approximately 3, comparable to that of KN060 and significantly superior to Abelacimab (≤7 days). The APTT ratio of BIS06 is superior to KN060A (≥14 days) and comparable to Abelacimab at ≥21 days. Further studies revealed that the antibody molecules had no significant effect on PT in monkeys, indicating that the candidate bispecific antibody molecules can persistently inhibit FXI-mediated intrinsic coagulation function in non-human primates without affecting the extrinsic coagulation pathway, thus reducing the risk of bleeding.

[0389] 12.3 Detection of free FXI in cynomolgus monkeys

[0390] Furthermore, the concentration of free FXI in cynomolgus monkeys was detected using an FXI kit (Abcam, ab108834). Pre-coated anti-Factor XI antibody was bound to FXI in whole blood samples collected at different time points. Biotin-labeled anti-FXI antibody from the kit was added to form an immune complex. Horseradish peroxidase (HRP)-labeled streptavidin was added to bind to the unbound biotinylated antibody. Finally, TMB chromogenic solution was added; HRP catalyzed the formation of a blue product, which turned yellow upon acidification. The color intensity was directly proportional to the FXI concentration. OD values ​​were measured at 450 nm using a microplate reader with 570 nm correction.

[0391] Figure 14 shows that BIS06 significantly reduced the level of free FXI in cynomolgus monkeys compared to Abelacimab, indicating that it has stronger antithrombotic activity.

[0392] 12.4 PK Detection in Crab-Eating Mammals

[0393] Similar to the APTT / PT assays performed at 15 time points, 0.3 mL of whole blood was collected at each time point and anticoagulated with 3.2% sodium citrate (anticoagulant to whole blood volume ratio 1:9). The samples were centrifuged at 1600g at room temperature for 8 min to obtain approximately 0.15 mL of plasma. The plasma was immediately frozen at -60 to -80℃ for PK analysis. In a 96-well microplate, 100 μL of 1 μg / mL FXI protein (HFXI 1111, Enzyme Research) was added to each well for coating. The plate was then washed and blocked with 2% BSA solution. After washing, plasma samples and antibody standards (BIS06 or Abelacimab) were added, and the plate was incubated at room temperature for 1 hour. Following this, the plate was washed again, and 100 μL of Anti-human Fc HRP (Goat anti-Human IgG Fc Secondary Antibody, HRP; Invitrogen / A18817) was added to each well, and the plate was incubated at room temperature for 0.5 hours. Finally, TMB substrate solution was added for color development, and the absorbance was measured at 450 nm using a microplate reader. The results are shown in Figure 15, which indicates that the BIS06 molecule has similar PK properties to Abelacimab, supporting its excellent antithrombotic activity.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to FXI, wherein the heavy chain variable region comprises the three heavy chain CDRs contained in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, and the light chain variable region comprises the three light chain CDRs contained in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 (IMGT numbering scheme).

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29-32, 37-40, and 45-48 or has at least 80% sequence identity to the sequence set forth above, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 33-36, 41-44, and 49-53 or has at least 80% sequence identity to the sequence set forth above.

3. An antibody or antigen-binding fragment thereof that specifically binds to FXI, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain CDRs, HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises three light chain CDRs, LCDR1, LCDR2, and LCDR3, wherein (i) the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87, the HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88, the HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, the LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90, the LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 91, and the LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 92, or (ii) the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 79, the HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 80, the HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 81, the LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 82, the LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 83, and the LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:

84.

4. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein 1) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12; (ii) the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 81, the HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 82, the H ​ ​ ​ 2) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34; 3) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8; or 4) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO:

43.

5. The anti-FXI antibody or antigen-binding fragment thereof of any one of claims 1-4, further comprising a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is or is from an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region, e.g., a human IgGl, IgG2, IgG3 or IgG4 constant region, e.g., the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 79 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the set forth sequence; and / or the light chain constant region is or is from a lambda or Kappa light chain constant region, preferably a Kappa light chain constant region, e.g., a human lambda or Kappa light chain constant region, e.g., the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 80 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the set forth sequence.

6. The anti-FXI antibody or antigen-binding fragment thereof of claim 5, wherein the heavy chain constant region comprises, relative to a wild-type heavy chain constant region (i) a mutation that reduces Fc region effector function, e.g., a mutation that reduces or abrogates binding of the Fc region to FcyR (FcyRI, FcyRII, FcyRIII) and Clq, e.g., L234A / L235A mutation and / or P329G mutation, and / or (ii) a mutation that increases binding of the Fc region to FcRn, e.g., M252Y / S254T / T256E mutation; Preferably, the heavy chain constant region comprises, relative to a wild-type heavy chain constant region, L234A / L235A / M252Y / S254T / T256E / P329G mutations; Preferably, the IgGl constant region with L234A / L235A / M252Y / S254T / T256E / P329G mutations comprises or consists of the amino acid sequence set forth in SEQ ID NO: 101 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

7. The anti-FXI antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, and a humanized antibody.

8. The anti-FXI antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of a Fab, a Fab', a Fv fragment, a F(ab')2, a scFv, a di-scFv, and a dAb.

9. The anti-FXI antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the anti-FXI antibody comprises a multispecific antibody, preferably a bispecific antibody.

10. A bispecific antibody comprising the FXI antibody of any one of claims 1-8, which contains a first antigen-binding domain and a second antigen-binding domain; the first and second antigen-binding domains bind to different domains on FXI; the first antigen-binding domain binds to the FXI apple domain (A1-A4) and the second antigen-binding domain binds to the FXI catalytic domain (CD).

11. The bispecific antibody of claim 10, wherein the first antigen-binding domain binds to FXI apple domain A1, A2, A3, or A4, preferably to apple domain A2.

12. The bispecific antibody of claim 10 or 11, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein the first heavy chain variable region (VH1) comprises the three heavy chain CDRs contained in SEQ ID NO: 5, 7, 15, 19, or 25; and the first light chain variable region (VL1) comprises the three light chain CDRs contained in SEQ ID NO: 6, 8, 16, 20, or 26, preferably, the first heavy chain variable region (VH1) comprises the three heavy chain CDRs contained in SEQ ID NO: 7; and the first light chain variable region (VL1) comprises the three light chain CDRs contained in SEQ ID NO:

8.

13. The bispecific antibody of claim 10 or 11, wherein the first antigen-binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein the first heavy chain variable region comprises three heavy chain CDRs, HCDR1, HCDR2, and HCDR3, and the first light chain variable region comprises three light chain CDRs, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 81, the HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 82, the HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 83, the LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 84, the LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 85, and the LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:

86. ​ ​ 13. The bispecific antibody of any one of claims 10-12, wherein the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein, the second heavy chain variable region (VH2) comprises the 3 heavy chain CDRs contained in SEQ ID NO: 1, 3, 9, 11, 17, or 27; and the second light chain variable region (VL2) comprises the 3 light chain CDRs contained in SEQ ID NO: 2, 4, 10, 12, 18, or 28, preferably, the second heavy chain variable region (VH2) comprises the 3 heavy chain CDRs contained in SEQ ID NO: 11; and the second light chain variable region (VL2) comprises the 3 light chain CDRs contained in SEQ ID NO: 12; preferably, the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein the second heavy chain variable region comprises 3 heavy chain CDRs, HCDR1, HCDR2, and HCDR3, and the second light chain variable region comprises 3 light chain CDRs, LCDR1, LCDR2, and LCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88, HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90, LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 91, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:

92.

14. The bispecific antibody of any one of claims 10-13, wherein the first antigen binding domain comprises a first heavy chain variable region (VH1) and a first light chain variable region (VL1), wherein, the first heavy chain variable region (VH1) comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5, 7, 15, 19, 25, 37-40, or an amino acid sequence that is at least 80%, 85%, 90, 95%, or 99% identical to any one of SEQ ID NOs: 5, 7, 15, 19, 25, 37-40, or an amino acid sequence that has one or more, preferably 1-10, more preferably 1-5, additions, deletions, and / or substitutions of amino acids compared to any one of SEQ ID NOs: 5, 7, 15, 19, 25, 37-40; the first light chain variable region (VL1) comprises an amino acid sequence as set forth in SEQ ID NOs: 6, 8, 16, 20, 26, 41-44, or an amino acid sequence that is at least 80%, 85%, 90, 95%, or 99% identical to SEQ ID NOs: 6, 8, 16, 20, 26, 41-44, or an amino acid sequence that has one or more, preferably 1-10, more preferably 1-5, additions, deletions, and / or substitutions of amino acids compared to SEQ ID NOs: 6, 8, 16, 20, 26, 41-44; Preferably, wherein the VH1 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 7, and the VL1 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 8; or the VH1 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 40, and the VL1 comprises or consists of an amino acid sequence as set forth in SEQ ID NO:

43.

15. The bispecific antibody of any one of claims 10-14, wherein the second antigen binding domain comprises a second heavy chain variable region (VH2) and a second light chain variable region (VL2), wherein, the second heavy chain variable region (VH2) comprises an amino acid sequence as set forth in SEQ ID NO: 1, 3, 9, 11, 17, 27, 29-32, or an amino acid sequence that is at least 80%, 85%, 90, 95% or 99% identical to SEQ ID NO: 1, 3, 9, 11, 17, 27, 29-32, or an amino acid sequence that has one or more, preferably 1-10, more preferably 1-5, additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 1, 3, 9, 11, 17, 27, 29-32; the second light chain variable region (VL2) comprises an amino acid sequence as set forth in SEQ ID NO: 2, 4, 10, 12, 18, 28, 33-36, or an amino acid sequence that is at least 80%, 85%, 90, 95% or 99% identical to SEQ ID NO: 2, 4, 10, 12, 18, 28, 33-36, or an amino acid sequence that has one or more, preferably 1-10, more preferably 1-5, additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 2, 4, 10, 12, 18, 28, 33-36; preferably, the VH2 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 11, and the VL2 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 12; or the VH2 comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 32, and the VL2 comprises or consists of an amino acid sequence as set forth in SEQ ID NO:

34.

16. The bispecific antibody of any one of claims 10-15, wherein the first antigen binding domain is a scFv and the second antigen binding domain is a Fab.

17. The bispecific antibody of any one of claims 10-16, wherein the bispecific antibody comprises an Fc region of an immunoglobulin, wherein, The Fc region is an IgG Fc region, e.g., a Fc region of human IgGl, IgG2, IgG3 or IgG4, preferably a Fc region of human IgGl; for example the human IgGl Fc comprises an amino acid sequence as set forth in SEQ ID NO: 96, or an amino acid sequence that is at least 80%, 85%, 90, 95% or 99% identical to said amino acid sequence; Optionally, the Fc region comprises (i) a mutation that reduces the effector function of the Fc region, e.g., a mutation that reduces or abrogates the binding of the Fc region to FcyR (FcyRI, FcyRII, FcyRIII) and Clq, e.g., a L234A / L235A mutation and / or a P329G mutation, and / or (ii) a mutation that increases the binding of the Fc region to FcRn, e.g., a M252Y / S254T / T256E mutation; Preferably, the Fc region comprises L234A / L235A / M252Y / S254T / T256E mutations relative to a wild-type Fc region; preferably the Fc region with L234A / L235A / M252Y / S254T / T256E mutations comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 103, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; Preferably, the Fc region comprises L234A / L235A / M252Y / S254T / T256E / P329G mutations relative to a wild-type Fc region; Preferably, the Fc region with L234A / L235A / M252Y / S254T / T256E / P329G mutations comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 99, or an amino acid sequence at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

18. The bispecific antibody of claim 17, wherein the bispecific antibody comprises two different Fc regions, wherein one Fc region comprises a Knob mutation and the other Fc region comprises a Hole mutation, for example, (a) the knob mutation is T366W and the hole mutations are T366S, L368A, and Y407V; (b) the knob mutations are S354C and T366W and the hole mutations are Y349C, T366S, L368A, and Y407V; numbering according to the EU index. Preferably, one Fc region comprises an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 100 and comprises mutations S354C and T366W, and optionally L234A / L235A / M252Y / S254T / T256E / P329G mutations, and the other Fc region comprises an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 65 and comprises mutations Y349C, T366S, L368A and Y407V, and optionally L234A / L235A / M252Y / S254T / T256E / P329G mutations; or one Fc region comprises an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 104 and comprises mutations S354C and T366W, and optionally L234A / L235A / M252Y / S254T / T256E mutations, and the other Fc region comprises an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence set forth in SEQ ID NO: 105 and comprises mutations Y349C, T366S, L368A and Y407V, and optionally L234A / L235A / M252Y / S254T / T256E mutations.

19. The bispecific antibody of any one of claims 10-18, comprising or consisting of a first chain, a second chain and a third chain, wherein the first chain comprises or consists of VH2-CH1 -first Fc; the second chain comprises or consists of VH1 -linker 1 -VL1 -linker 2-second Fc; the third chain comprises or consists of VL2-CL; wherein the VH1 and VL1 are as defined in claim 12 or 14, the VH2 and VL2 are as defined in claim 13 or 15, preferably the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 40, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 43, the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 32, and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 34; optionally the Fc regions are as defined in claim 17, CL is a light chain constant region; CH1 is a heavy chain constant region CH1.

20. The bispecific antibody of any one of claims 10-18, comprising or consisting of a first chain and a second chain, preferably two first chains and two second chains, wherein the first chain comprises or consists of VH2-CH1 -linker 3-VH1 -linker 1 -VL1 -linker 2-Fc; the second chain comprises or consists of VL2-CL; wherein said VH1 and VL1 are as defined in claim 12 or 14, said VH2 and VL2 are as defined in claim 13 or 15, preferably said VH1 comprises the amino acid sequence of SEQ ID NO: 40, said VL1 comprises the amino acid sequence of SEQ ID NO: 43, said VH2 comprises the amino acid sequence of SEQ ID NO: 32, and said VL2 comprises the amino acid sequence of SEQ ID NO: 34; said Fc region is as defined in claim 17, CL is a light chain constant region; CH1 is a heavy chain constant region CH1.

21. The bispecific antibody of claim 19 or 20, wherein the light chain constant region is or is from a lambda or Kappa light chain constant region, preferably a Kappa light chain constant region, e.g. a human lambda or Kappa light chain constant region, e.g. the light chain constant region comprises the amino acid sequence of SEQ ID NO: 80 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown; and / or the heavy chain constant region CH1 is or is from a CH1 of a human IgG heavy chain constant region, e.g. a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; e.g. the heavy chain constant region is a CH1 of a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; e.g. the CH1 comprises the amino acid sequence of SEQ ID NO: 95 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown.

22. The bispecific antibody of any one of claims 19-21, wherein the linker comprises an amino acid sequence selected from the group consisting of (G4)n, (G4S)n and GS(G4S)n, wherein n is an integer selected from 1 -5, preferably the linker comprises an amino acid sequence as set forth in GGGG or GGGGS; preferably the linker 1 comprises or consists of the amino acid sequence of SEQ ID NO: 97, and / or the linker 2 comprises or consists of the amino acid sequence of SEQ ID NO: 98, and / or the linker 3 comprises or consists of the amino acid sequence of SEQ ID NO: 102 (GGGGS).

23. The bispecific antibody of claim 19, wherein the first chain comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 67; and the second chain comprises the amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:

68. the second chain comprises the amino acid sequence set forth in SEQ ID NO: 68, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 68; and the third chain comprises the amino acid sequence set forth in SEQ ID NO: 57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57; Preferably, the first chain comprises the amino acid sequence set forth in SEQ ID NO: 67, the second chain comprises the amino acid sequence set forth in SEQ ID NO: 68, and the third chain comprises the amino acid sequence set forth in SEQ ID NO: 57; or the first chain comprises the amino acid sequence set forth in SEQ ID NO: 93, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 93; the second chain comprises the amino acid sequence set forth in SEQ ID NO: 94, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 94; and the third chain comprises the amino acid sequence set forth in SEQ ID NO: 57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57; Preferably, the first chain comprises the amino acid sequence set forth in SEQ ID NO: 93, the second chain comprises the amino acid sequence set forth in SEQ ID NO: 94, and the third chain comprises the amino acid sequence set forth in SEQ ID NO:

57.

24. The bispecific antibody of claim 20, wherein the first chain comprises the amino acid sequence set forth in SEQ ID NO: 56, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 56; the second chain comprises the amino acid sequence set forth in SEQ ID NO: 57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57; Preferably, the first chain comprises the amino acid sequence set forth in SEQ ID NO: 56, the second chain comprises the amino acid sequence set forth in SEQ ID NO:

57.

25. An isolated polynucleotide encoding the anti-FXI antibody or antigen binding fragment of any one of claims 1-9 or the bispecific antibody of any one of claims 10-24.

26. A vector comprising the polynucleotide of claim 25, wherein the vector is an expression vector, preferably the vector is a pTT5 vector.

27. A host cell comprising the polynucleotide of claim 25 or the vector of claim 26, preferably the host cell is a mammalian cell.

28. A method for producing the anti-FXI antibody or antigen binding fragment of any one of claims 1-9 or the bispecific antibody of any one of claims 10-24, the method comprising: culturing a host cell comprising a polynucleotide encoding a polypeptide chain of the antibody under conditions suitable for expression of the polypeptide chain; and assembling the polypeptide chains under conditions suitable for assembly of the polypeptide chains into the antibody to produce the antibody.

29. A pharmaceutical composition comprising the anti-FXI antibody or antigen binding fragment of any one of claims 1-9 or the bispecific antibody of any one of claims 10-24 and a pharmaceutically acceptable carrier and / or excipient.

30. A pharmaceutical combination comprising the anti-FXI antibody or antigen binding fragment of any one of claims 1-9 or the bispecific antibody of any one of claims 10-24 or the pharmaceutical composition of claim 29, and one or more other therapeutic agents, wherein the other therapeutic agent is for example one or more other anticoagulant or antithrombotic drug, preferably selected from vitamin K antagonists (e.g. warfarin), thrombin inhibitors (e.g. direct thrombin inhibitors such as Gandiprost, dabigatran; indirect thrombin inhibitors such as low molecular heparin; or specific thrombin inhibitors such as FXa inhibitors, e.g. edoxaban, rivaroxaban or apixaban; or antiplatelet drugs (e.g. COX inhibitors, ADP receptor antagonists, GPIIb / IIIa inhibitors, PDE inhibitors); fibrinolytic drugs (e.g. profastigil).

31. A method of anticoagulation or prevention or treatment of a disease or disorder associated with FXI activity or expression in a subject, comprising administering to the subject the anti-FXI antibody or antigen binding fragment of any one of claims 1-9 or the bispecific antibody of any one of claims 10-24 or the pharmaceutical composition of claim 29 or the pharmaceutical combination of claim 30.

32. The method of claim 31, wherein the disease or disorder associated with FXI activity or expression comprises, without limitation, any of the following: a thrombotic or thromboembolic disease, a thrombotic or thromboembolic complication, an arrhythmia, an ischemic stroke, disseminated intravascular coagulation; preferably, the thrombotic or thromboembolic disease or complication thereof is selected from the group consisting of: cardiac coronary artery disease, myocardial infarction with ST segment elevation, myocardial infarction without ST segment elevation, stable angina pectoris, unstable angina pectoris, reocclusion and restenosis after coronary intervention, leading to peripheral arterial occlusive disease, pulmonary embolism, venous thromboembolism, venous thrombosis, transient ischemic attack, thrombotic and thromboembolic stroke, pulmonary disease caused by chronic thromboembolism, pulmonary arterial hypertension caused by CTEPH, myocardial infarction, ischemic stroke, pulmonary thromboembolism, atrial fibrillation, medical device related thromboembolic disorders, severe systemic inflammatory response syndrome, thromboembolism formed in extracorporeal circulation such as cardiopulmonary bypass, hemodialysis and ECMO, arterial thrombosis, end-stage renal disease, antiphospholipid syndrome, metastatic cancer or infectious disease, preferably the disease is venous thromboembolism.

33. The method of claim 31 or 32, wherein the medicament is used in combination with at least one other therapeutic agent, such as one or more other anticoagulant or anti-coagulation or anti-thrombotic agent, preferably selected from the group consisting of vitamin K antagonists (e.g. warfarin), thrombin inhibitors (e.g. direct thrombin inhibitors such as Gandiprost, dabigatran; indirect thrombin inhibitors such as low molecular weight heparins; or specific thrombin inhibitors such as FXa inhibitors, e.g. edoxaban, rivaroxaban or apixaban; or anti-platelet agents (e.g. COX inhibitors, ADP receptor antagonists, GPIIb / IIIa inhibitors, PDE inhibitors); fibrinolytic agents (e.g. profiromase).

34. A kit comprising an anti-FXI antibody or antigen-binding fragment according to any one of claims 1-9 or a bispecific antibody according to any one of claims 10-24 or an anti-FXI antibody or antigen-binding fragment or bispecific antibody prepared according to the method of claim 28, a polynucleotide according to claim 25, a vector according to claim 26, a host cell according to claim 27, a pharmaceutical composition according to claim 29 or a pharmaceutical combination according to claim 30.

Images