Factor XI (FXI) binding protein

Abstract

This invention relates to the field of pharmaceutical biology and discloses a single-domain antibody against coagulation factor XI (FXI) and its derived protein. Specifically, this invention discloses a coagulation factor XI (FXI) binding protein derived from a single-domain antibody against coagulation factor XI (FXI) and its uses.

Description

[0001] This application is a divisional application of the invention patent application filed on July 2, 2021, with application number 202180047622.8 and invention title "Coagulation Factor XI (FXI) Binding Protein". Technical Field

[0002] This invention relates to the field of pharmaceutical biology and discloses a single-domain antibody against coagulation factor XI (FXI) and its derived protein. Specifically, this invention discloses a coagulation factor XI (FXI) binding protein derived from a single-domain antibody against coagulation factor XI (FXI) and its uses. Background of the Invention Coagulation factors are various protein components involved in the blood clotting process. Their physiological function is to be activated when blood vessels bleed, adhering to platelets and sealing leaks in the blood vessels. This process is called coagulation. They are partially produced by the liver and can be inhibited by coumarin. For standardized nomenclature, the World Health Organization uses Roman numerals to number them according to the order of their discovery, including coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, and XIII.

[0003] Coagulation factor XI (FXI) is a dimer composed of identical 80 kDa subunits, each of which consists of four apple domains (A1, A2, A3, and A4) and a catalytic domain, starting from the N-terminus. FXI is a proenzyme that cycles in complex with high molecular weight kininogen (HK). HK binds to the A2 domain of FXI and is a physiological cofactor for the activation of FXIIa from FXI to FXIa. The remaining apple domains in FXI also mediate important physiological functions. For example, the binding site of FXI is located in A3, while the binding site for FXIIa is in A4. Residues critical for FXI dimerization are also located in A4.

[0004] Studies have shown that FXI plays a crucial role in the pathological process of thrombosis, contributing relatively little to hemostasis, and is therefore a promising target for thrombosis control. In the Ionis Pharmaceuticals Inc. FXI antisense oligonucleotide (ASO) phase II trial (Buller et al., N Engl J Med 2015, 372:232-240), in patients undergoing total knee arthroplasty, FXI ASO resulted in a significant reduction in venous thromboembolism (VTE) with a trend toward less bleeding compared to enoxaparin. Human genetic and epidemiological studies (Duga et al., Semin Thromb Hemost 2013; Chen et al., Drug Discov Today 2014; Key, Hematology Am Soc Hematol Educ Program 2014, 2014:66-70) indicate that severe FXI deficiency (hemophilia C) reduces the risk of ischemic stroke and deep vein thrombosis; conversely, increased FXI levels are associated with a higher risk of VTE and ischemic stroke. Furthermore, multiple preclinical studies have shown that FXIa inhibition or loss of function mediates thrombotic protection without impairing hemostasis (Chen et al., Drug Discov Today 2014). Notably, in a baboon AV branch thrombosis model, monoclonal antibodies 14E11 and 1A6 produced significant thrombosis reduction (US Patent No. 8,388,959; Tucker et al., Blood 2009, 113:936-944; Cheng et al., Blood 2010, 116:3981-3989). Furthermore, 14E11 (in its cross-reactivity with mouse FXI) provided protection in a mouse model of acute ischemic stroke (Leung et al., Transl Stroke Res 2012, 3:381-389). Other mAb studies targeting FXI in preclinical models have also been reported, confirming FXI as an antithrombotic target with a minimal bleeding risk (van Montfoort et al., Thromb Haemost 2013, 110; Takahashi et al., Thromb Res 2010, 125:464-470; van Montfoort, Ph.D. Thesis, University of Amsterdam, Amsterdam, Netherlands, November 14, 2014). Inhibition of FXI is therefore a promising strategy for novel antithrombotic therapy, offering an improved benefit-risk ratio compared to current standard anticoagulants. Brief description of the attached diagram Figure 1The blocking activity of the FCI single-domain antibody Fc fusion protein against FCI is shown (ATPP assay).

[0005] Figure 2 The blocking activity of the humanized FCI single-domain antibody Fc fusion protein against FCI is shown (ATPP assay).

[0006] Figure 3 This study demonstrates the inhibitory effect of the huFE bispecific antibody Fc fusion protein on human FXI activity.

[0007] Figure 4 The inhibitory activity of the huFE bispecific antibody Fc fusion protein on APTT in human whole plasma is shown.

[0008] Figure 5 The inhibitory activity of the huFE bispecific antibody Fc fusion protein on APTT in whole monkey plasma is shown.

[0009] Figure 6 The inhibitory activity of the huFE bispecific antibody Fc fusion protein on APTT in rabbit whole plasma is shown.

[0010] Figure 7 The effects of the FCI single-domain antibody Fc fusion protein and the bispecific antibody on rabbit venous thrombosis are shown. Detailed Implementation

[0011] definition Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0012] Unless otherwise stated, the interchangeable terms “antibody” or “immunoglobulin” used herein, whether referring to heavy-chain antibodies or conventional four-chain antibodies, are used generally to include full-length antibodies, their individual chains, and all their portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). Furthermore, the term “sequence” as used herein (e.g., in the terms “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence,” etc.) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said sequence, unless a more specific interpretation is required herein.

[0013] As used herein, the term (of a polypeptide or protein) “domain” refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without loss of the function of the rest of the protein and / or the domain itself.

[0014] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., the chain of a conventional four-chain antibody or a heavy-chain antibody), or to a polypeptide that is essentially composed of such globular regions. Immunoglobulin domains are characterized by their ability to maintain the immunoglobulin folding characteristics of antibody molecules.

[0015] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain essentially composed of four "frame regions," referred to in the art and hereinafter as "frame region 1" or "FR1," "frame region 2" or "FR2," "frame region 3" or "FR3," and "frame region 4" or "FR4," respectively, wherein these frame regions are separated by three "complementarity-determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antibody specificity for antigens by possessing antigen-binding sites.

[0016] As used herein, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains. An example of an immunoglobulin single variable domain in the context of this invention is a "domain antibody," such as the immunoglobulin single variable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin single variable domain is the "VHH domain" (or simply "VHH") of the camelid family as defined below.

[0017] The “VHH domain,” also known as heavy chain single-domain antibody, VHH, VHH domain, VHH antibody fragment, and VHH antibody, is a variable domain of antigen-binding immunoglobulin called a “heavy chain antibody” (i.e., “antibody lacking a light chain”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH domain” is used to distinguish this variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) present in conventional 4-chain antibodies and the light chain variable domain (referred herein as the “VL domain”) present in conventional 4-chain antibodies. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain.

[0018] In the context of this invention, the terms "heavy chain single-domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", and "VHH antibody" are used interchangeably.

[0019] For example, Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999). Figure 2As shown, the amino acid residues used in the VHH domain of camelids can be numbered according to the general numbering method for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0020] Alternative methods for numbering amino acid residues in the VH domain are known in the art, and these alternative methods can also be similarly applied to the VHH domain. For example, the Chothia CDR refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM CDR represents a compromise between the Kabat hypervariable region and the Chothia structural loop, and is used in Oxford Molecular's AbM antibody modeling software. The "Contact" CDR is based on the analysis of the available crystal structure of the complex. The residue descriptions of the CDRs from each method are as follows:

[0021] Antibody CDRs can also be IMGT-CDRs, which is a CDR definition method based on IMGT antibody encoding. This encoding is obtained by synthesizing structural information from over 5000 sequences. In the VH CDR encoding of IMGT, CDR1: 27-38; CDR2: 56-65; CDR3: 105-117.

[0022] However, it should be noted that, as is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence.

[0023] For example, a CDR may include “extended CDRs”, such as: 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2) and 89-97 or 89-96 (LCDR3) in VL; 26-35 (HCDR1), 50-65 or 49-65 (HCDR2) and 93-102, 94-102 or 95-102 (HCDR3) in VH.

[0024] The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0025] The VHH domain and other structural characteristics and functional properties of peptides containing it can be summarized as follows: The VHH domain (which is naturally "designed" to functionally bind to antigens in the absence of light chain variable domains and without interaction with them) can be used as a single and relatively small functional antigen-binding structural unit, domain, or peptide. This distinguishes the VHH domain from the VH and VL domains of conventional 4-chain antibodies, which are generally not suitable on their own for practical applications as a single antigen-binding protein or a single variable domain of an immunoglobulin, but need to be combined in one or more forms to provide a functional antigen-binding unit (e.g., in the form of a conventional antibody fragment such as a Fab fragment; or in the form of an scFv consisting of VH domains covalently linked to a VL domain).

[0026] Due to these unique properties, using VHH domains—alone or as part of a larger polypeptide—offers many significant advantages over using conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., Fab- or F(ab')2- fragments): Only a single domain is required to bind antigens with high affinity and selectivity, thus eliminating the need for two separate domains or ensuring they are in appropriate spatial conformation and configuration (e.g., scFv typically requires specially designed linkers); VHH domains can be expressed from a single gene without post-translational folding or modification; VHH domains can be easily modified into multivalent and multispecific formats (formatted); VHH domains are highly soluble and do not aggregate; VHH domains... It is highly stable to heat, pH, proteases, and other denaturing agents or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thus saving costs, time, and the environment; the VHH domain is easy to prepare and relatively inexpensive, even at the scale required for production; the VHH domain is relatively small compared to conventional 4-chain antibodies and their antigen-binding fragments (approximately 15 kDa or 1 / 10 the size of conventional IgG), thus exhibiting higher tissue permeability and allowing for higher doses compared to conventional 4-chain antibodies and their antigen-binding fragments; the VHH domain can exhibit so-called cavity-binding properties (especially due to its elongated CDR3 loop compared to conventional VH domains), thereby reaching targets and epitopes that are inaccessible to conventional 4-chain antibodies and their antigen-binding fragments.

[0027] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185–195; Li et al., J Biol Chem., 287 (2012) 13713–13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0028] The VHH domain derived from the Camelidae family can be "humanized" (also referred to herein as "sequence optimization," which, in addition to humanization, may also encompass other modifications to the sequence by one or more mutations that provide improved VHH properties, such as removal of potential post-translational modification sites) by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a conventional human 4-chain antibody. The humanized VHH domain may contain one or more fully human framework regions. Humanization can be accomplished using protein surface amino acid resurfacing methods and / or CDR grafting to a universal framework, for example, as exemplified in the examples.

[0029] As used herein, the term “epitope” or the interchangeable term “antigenic determinant” refers to any antigenic determinant on an antigen to which the complementary site of an antibody binds. Antigenic determinants typically contain chemically active surface groups of a molecule, such as amino acid or sugar side chains, and typically possess specific three-dimensional structural features as well as specific charge features. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation, and can be a “linear” epitope or a “conformal” epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformal epitope, points of interaction are separated by protein amino acid residues.

[0030] Epitopes of a given antigen can be identified using many epitope mapping techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). For instance, linear epitopes can be determined by, for example, the simultaneous synthesis of a large number of peptides on a solid support, wherein these peptides correspond to portions of a protein molecule, and by reacting these peptides with an antibody while still attached to the support. These techniques are known in the art and described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes can be identified by determining the spatial configuration of amino acids, such as by, for example, X-ray crystallography and 2D nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (ibid.).

[0031] Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competitive and cross-competitive studies can be performed to obtain antibodies that compete or cross-competitively bind to the antigen. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Application WO03 / 48731. Therefore, antibodies and their antigen-binding fragments that compete with the antibody molecules of the present invention for binding to the same epitope on FXI can be obtained using conventional techniques known to those skilled in the art.

[0032] Generally, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (e.g., the immunoglobulin single variable domain of the present invention) can bind. Specificity can be determined based on the affinity and / or cohesion of the antigen-binding protein. Affinity, expressed as the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein, is a measure of the strength of binding between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the stronger the binding between the epitope and the antigen-binding protein (or, affinity can also be expressed as the association constant (KA), which is 1 / KD). As those skilled in the art will understand, affinity can be determined in a known manner depending on the specific antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding protein (e.g., an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing it) and the associated antigen. Affinity relates to both the affinity between the antigen and the antigen-binding site on the antigen-binding protein and the number of associated binding sites present on the antigen-binding protein.

[0033] As used herein, the term "coagulation factor XI (FXI) binding protein" means any protein capable of specifically binding to coagulation factor XI (FXI). FXI binding proteins may include antibodies against FXI as defined herein. FXI binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR transplantation molecules.

[0034] The "FXI-binding protein" of the present invention may comprise at least one immunoglobulin single variable domain, such as VHH, that binds to FXI. In some embodiments, the "FXI-binding molecule" of the present invention may comprise 2, 3, 4, or more immunoglobulin single variable domains, such as VHH, that bind to FXI. In addition to the immunoglobulin single variable domain that binds to FXI, the FXI-binding protein of the present invention may also comprise a linker and / or an effector-functional portion, such as a half-life-extending portion (e.g., an immunoglobulin single variable domain that binds to serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, the "FXI-binding protein" of the present invention also encompasses bispecific antibodies containing immunoglobulin single variable domains that bind to different antigens or different regions of the same antigen (e.g., different epitopes).

[0035] Typically, the FXI-binding protein of the present invention will be measured in a preferred 10 as in a Biacore, KinExA, or Fortibio assay. -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 moles per liter, or even more preferably 10 -9 Up to 10 -10 Or a lower dissociation constant (KD), and / or at least 10 7 M -1 Preferably at least 10 8 M -1 More preferably at least 10 9 M -1 More preferably at least 10 10 M -1 The association constant (KA) of the antigen (i.e., FXI) binds to the antigen it wants to bind. Any antigen greater than 10... -4 The KD value of M is generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays) as described herein.

[0036] Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as known and agreed upon in the art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a specified number of amino acid residues at a position in a reference sequence compared to the other sequence. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, meaning that an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and that has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are well known in the art. For example, the conserved amino acid substitutions are preferably the substitution of one amino acid in the following groups (i)-(v) by another amino acid residue in the same group: (i) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln, or Glu; Met is substituted by Leu, Tyr, or Ile; Phe is substituted by Met, Leu, or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; Val is substituted by Ile or Leu.

[0037] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of identical or conserved amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, the BLAST program in the NCBI database can be used to determine identity. For determining sequence identity, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0038] A polypeptide or nucleic acid molecule is considered "isolated" when it has been separated from at least one other component (e.g., another protein / peptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) that is normally associated with it in that source or medium (culture medium), compared to its natural biological source and / or the reaction medium or culture medium from which the molecule is obtained. Specifically, a polypeptide or nucleic acid molecule is considered "isolated" when it has been purified at least 2-fold, particularly at least 10-fold, more particularly at least 100-fold, and up to 1000-fold or more. "Isolated" polypeptide or nucleic acid molecules are preferably substantially homogeneous, as determined by suitable techniques (e.g., suitable chromatographic techniques, such as polyacrylamide gel electrophoresis).

[0039] "Effective amount" means that the amount of the FXI binding protein or pharmaceutical composition of the present invention can lead to a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of damage or disability caused by disease suffering.

[0040] As used in this article, "thrombosis" refers to the formation or presence of a clot (also called a "thrombus") within a blood vessel, obstructing blood flow through the circulatory system. Thrombosis is typically caused by abnormalities in blood composition, the quality of the vessel wall, and / or the nature of blood flow. Clot formation is usually caused by damage to the vessel wall (such as damage from trauma or infection) and a slowing or stagnation of blood flow through the site of damage. In some cases, coagulation abnormalities lead to thrombosis.

[0041] As used herein, "without impairing hemostasis" means that minimal or no detectable bleeding is observed in a subject after administration of the FXI-binding protein or pharmaceutical composition of the present invention. In the case of targeting FXI, inhibiting the conversion of FXI to FXIa or inhibiting the activation of FXIa by FXIa by FXIa inhibits coagulation and related thrombosis without bleeding.

[0042] As used in this article, the term "object" refers to mammals, especially primates, and particularly humans.

[0043] The FXI binding protein of the present invention In one aspect, the present invention provides an FXI-binding protein comprising at least one immunoglobulin single variable domain capable of specifically binding to FXI.

[0044] In some embodiments, the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of any of the VHHs shown in SEQ ID NO:1-23. The CDR may be Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR.

[0045] In some embodiments, the at least one immunoglobulin single variable domain comprises a set of CDR1, CDR2, and CDR3 selected from the following:

[0046] In some embodiments, at least one immunoglobulin single variable domain in the FXI-binding protein of the present invention is a VHH. In some embodiments, the VHH comprises any amino acid sequence of SEQ ID NO:1-23.

[0047] In some embodiments, at least one immunoglobulin single variable domain in the FXI binding protein of the present invention is a humanized VHH.

[0048] In some embodiments, at least one immunoglobulin single variable domain of the FXI-binding protein of the present invention is a humanized VHH, said humanized VHH comprising an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with any of the sequences in SEQ ID NO:1-23. In some embodiments, the amino acid sequence of said humanized VHH comprises one or more amino acid substitutions compared to any of SEQ ID NO:1-23, preferably conserved amino acid substitutions. For example, it comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions.

[0049] In some embodiments, at least one immunoglobulin single variable domain of the FXI binding protein of the present invention is a humanized VHH, wherein the humanized VHH comprises any amino acid sequence in SEQ ID NO:300-335.

[0050] In some embodiments, the at least one immunoglobulin single variable domain binds to an epitope within the Apple2 domain of FXI. An exemplary amino acid sequence of the Apple2 domain of FXI is shown in SEQ ID NO:338. For example, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in any of SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:14. In some embodiments, the immunoglobulin single variable domain comprises a set of CDR1, CDR2, and CDR3 selected from SEQ ID NO:60-62, SEQ ID NO:63-65, SEQ ID NO:66-68, SEQ ID NO:69-71, SEQ ID NO:132-134, SEQ ID NO:135-137, SEQ ID NO:138-140, SEQ ID NO:141-143, SEQ ID NO:180-182, SEQ ID NO:183-185, SEQ ID NO:186-188, and SEQ ID NO:189-191. In some specific embodiments, the immunoglobulin single variable domain comprises any of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:10, or SEQ ID NO:14. In some specific embodiments, the immunoglobulin single variable domain comprises any of the amino acid sequences shown in SEQ ID NO:306-323.

[0051] In some embodiments, the at least one immunoglobulin single variable domain does not bind to an epitope within the Apple2 domain of FXI. In some embodiments, the at least one immunoglobulin single variable domain does not bind to the Apple2 domain of FXI. In some embodiments, the at least one immunoglobulin single variable domain does not bind to isolated Apple2 domain peptides of FXI.

[0052] In some embodiments, the at least one immunoglobulin single variable domain binds to an epitope within the Apple3 domain of FXI. An exemplary amino acid sequence of the Apple3 domain of FXI is shown in SEQ ID NO:339. For example, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:17. In some embodiments, the immunoglobulin single variable domain comprises a set of CDR1, CDR2, and CDR3 selected from SEQ ID NO:216-218, SEQ ID NO:219-221, SEQ ID NO:222-224, and SEQ ID NO:225-227. In some specific embodiments, the immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:17. In some specific embodiments, the immunoglobulin single variable domain comprises any of the amino acid sequences shown in SEQ ID NO:324-329.

[0053] In some embodiments, the at least one immunoglobulin single variable domain binds to an epitope within the Apple4 domain of FXI. An exemplary amino acid sequence of the Apple4 domain of FXI is shown in SEQ ID NO:340. For example, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:1. In some embodiments, the immunoglobulin single variable domain comprises a set of CDR1, CDR2, and CDR3 selected from SEQ ID NO:24-26, SEQ ID NO:27-29, SEQ ID NO:30-32, and SEQ ID NO:33-35. In some specific embodiments, the immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:1. In some specific embodiments, the immunoglobulin single variable domain comprises any of the amino acid sequences shown in SEQ ID NO:300-305.

[0054] In some embodiments, the at least one immunoglobulin single variable domain binds to an epitope within the Apple1-2 region of FXI (the region between the Apple1 and Apple2 domains). An exemplary amino acid sequence of the Apple1-2 region of FXI is shown in SEQ ID NO:341.

[0055] In some embodiments, the at least one immunoglobulin single variable domain binds to an epitope within the Apple2-3 region of FXI (the region between the Apple2 and Apple3 domains). An exemplary amino acid sequence of the Apple2-3 region of FXI is shown in SEQ ID NO:342. For example, the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:20. In some embodiments, the immunoglobulin single variable domain comprises a set of CDR1, CDR2, and CDR3 selected from SEQ ID NO:252-254, SEQ ID NO:255-257, SEQ ID NO:258-260, and SEQ ID NO:261-263. In some specific embodiments, the immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:20. In some specific embodiments, the immunoglobulin single variable domain comprises any of the amino acid sequences shown in SEQ ID NO:330-335.

[0056] In some embodiments, the at least one immunoglobulin single variable domain binds to an epitope within the Apple3-4 region of FXI (the region between the Apple3 and Apple4 domains). An exemplary amino acid sequence of the Apple3-4 region of FXI is shown in SEQ ID NO:343.

[0057] In some embodiments, the FXI-binding protein comprises a single variable immunoglobulin domain that specifically binds to FXI.

[0058] In some embodiments, the FXI-binding protein comprises at least two, such as 2, 3, 4 or more, immunoglobulin single variable domains that specifically bind to FXI.

[0059] In some implementations, the at least two immunoglobulin single variable domains bind to the same region or epitope of FXI, or competitively bind to or partially competitively bind to the same region or epitope of FXI, for example, the at least two immunoglobulin single variable domains are identical.

[0060] In some implementations, the at least two immunoglobulin single variable domains bind to different regions or epitopes of FXI, or do not compete to bind to the same regions or epitopes of FXI.

[0061] Whether two antibodies or immunoglobulins bind or compete to bind to the same region or epitope can be determined by epitope binning using biomembrane interferometry (BLI), as exemplified in the embodiments of this application.

[0062] In some implementations, the at least two immunoglobulin single variable domains that specifically bind to FXI are directly interconnected.

[0063] In some embodiments, the at least two immunoglobulin single variable domains that specifically bind to FXI are interconnected by a linker. The linker may be a non-functional amino acid sequence of 1-20 or more amino acids in length, without secondary or higher-order structures. For example, the linker is a flexible linker, such as GGGGS, GS, GAP, (GGGGS) x 3, etc.

[0064] In some embodiments, the FXI-binding protein includes a first immunoglobulin single variable domain capable of specifically binding to FXI and a second immunoglobulin single variable domain capable of specifically binding to FXI, wherein the first immunoglobulin single variable domain and the second immunoglobulin single variable domain bind different epitopes on FXI.

[0065] In some embodiments, the first immunoglobulin single variable domain binds to an epitope within the Apple2 domain of FXI, and the second immunoglobulin single variable domain binds to an epitope within the Apple3 domain of FXI; or The first immunoglobulin single variable domain binds to the epitope within the Apple2 domain of FXI, and the second immunoglobulin single variable domain binds to the epitope within the Apple4 domain of FXI; or The first immunoglobulin single variable domain binds to an epitope within the Apple2 domain of FXI, and the second immunoglobulin single variable domain binds to an epitope within the Apple2-3 region of FXI; or The first immunoglobulin single variable domain binds to the epitope within the Apple3 domain of FXI, and the second immunoglobulin single variable domain binds to the epitope within the Apple4 domain of FXI; or The first immunoglobulin single variable domain binds to epitopes within the Apple3 domain of FXI, and the second immunoglobulin single variable domain binds to epitopes within the Apple2-3 region of FXI; or The first immunoglobulin single variable domain binds to the epitope within the Apple4 domain of FXI, and the second immunoglobulin single variable domain binds to the epitope within the Apple2-3 region of FXI.

[0066] In some embodiments, the FXI-binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein, The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:4; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:9; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:10; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:14; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:17; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:1, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:20; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:4, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:9; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:4, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NO:10; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:4, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:14; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:4, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:17; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:4, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:20; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:10; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:14; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:17; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:20; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:10, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:14; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:10, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:17; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:10, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:20; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:14, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:17; or The first immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:14, and the second immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of VHH shown in SEQ ID NO:20; or The first immunoglobulin single variable domain includes CDR1, CDR2 and CDR3 of VHH shown in SEQ ID NO:17, and the second immunoglobulin single variable domain includes CDR1, CDR2 and CDR3 of VHH shown in SEQ ID NO:20.

[0067] In some implementations, CDR1, CDR2, and CDR3 in the VHH represented by SEQ ID NO:1, 4, 9, 10, 14, 17, or 20 are shown in the table below:

[0068] In some embodiments, the FXI-binding protein comprises a first immunoglobulin single variable domain and a second immunoglobulin single variable domain, wherein, The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:1, 300-305, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:4, 306-311; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO:1 or 300-305, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:9; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:1, 300-305, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:10, 312-317; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:1, 300-305, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:14, 318-323; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:1, 300-305, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:17, 324-329; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:1, 300-305, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:20, 330-335; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO:4, 306-311, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:9; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:4, 306-311, and the second immunoglobulin single variable domain comprises the amino acid sequence in VHH shown in any one of SEQ ID NO:10, 312-317; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:4, 306-311, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:14, 318-323; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:4, 306-311, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:17, 324-329; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:4, 306-311, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:20, 330-335; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO:10 or 312-317; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO:14 or 318-323; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO:17 or 324-329; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in SEQ ID NO:9, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in one of SEQ ID NO:20 or 330-335; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:10, 312-317, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:14, 318-323; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:10, 312-317, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:17, 324-329; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:10, 312-317, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:20, 330-335; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:14, 318-323, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:17, 324-329; or The first immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:14, 318-323, and the second immunoglobulin single variable domain comprises the amino acid sequence shown in any one of SEQ ID NO:20, 330-335; or The first immunoglobulin single variable domain contains the amino acid sequence shown in any one of SEQ ID NO:17, 324-329, and the second immunoglobulin single variable domain contains the amino acid sequence shown in any one of SEQ ID NO:20, 330-335.

[0069] In some embodiments, the first immunoglobulin single variable domain is located at the N-terminus of the second immunoglobulin single variable domain. In other embodiments, the second immunoglobulin single variable domain is located at the N-terminus of the first immunoglobulin single variable domain.

[0070] In some embodiments, the FXI-binding protein of the present invention, in addition to at least one immunoglobulin single variable domain capable of specifically binding FXI, also includes an immunoglobulin Fc region. The inclusion of an immunoglobulin Fc region in the FXI-binding protein of the present invention allows the binding molecule to form a dimer. The Fc region usable in the present invention can be derived from different immunoglobulin subtypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

[0071] In some implementations, mutations can be introduced into the wild-type Fc sequence to alter Fc-mediated activities. These mutations include, but are not limited to: a) mutations altering Fc-mediated CDC activity; b) mutations altering Fc-mediated ADCC activity; or c) mutations altering FcRn-mediated in vivo half-life. Such mutations are described in the following literature: Leonard G Presta, Current Opinion in Immunology 2008, 20:460–470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4: 443-446; Paul R. Hinton et al., J Immunol, 2006, 176:346-356. For example, mutations in amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in the CH2 region can increase or decrease Fc-mediated ADCC or CDC activity, or enhance or weaken the affinity of FcRn. Furthermore, mutations in amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in the hinge region can increase protein stability.

[0072] In some implementations, mutations can be introduced into the Fc sequence, making the mutated Fc more likely to form homodimers or heterodimers. For example, the knob-hole model, which utilizes the spatial interaction of amino acid side chain groups at the Fc contact interface, as mentioned in Ridgway, Presta et al. 1996 and Carter 2001, makes it easier for different Fc mutations to form heterodimers. Another example is found in CN 102558355A or CN 103388013A, where the charge of the amino acids at the Fc contact interface is changed, thereby altering the ionic interaction forces between the Fc contact interfaces, making it easier for different Fc mutation pairs to form heterodimers (CN 102558355A), or for Fcs with the same mutation to form homodimers (CN103388013A).

[0073] The immunoglobulin Fc region is preferably a human immunoglobulin Fc region, such as the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some specific embodiments, the amino acid sequence of the immunoglobulin Fc region is shown in SEQ ID NO:336.

[0074] In some specific embodiments, in the FXI binding protein of the present invention, the immunoglobulin Fc region (e.g., the Fc region of human IgG1) is directly or indirectly linked to the C-terminus of the immunoglobulin single variable domain (e.g., VHH) via a linker (e.g., a peptide linker).

[0075] In some embodiments, the FXI-binding protein of the present invention comprises a single variable immunoglobulin domain that specifically binds to FXI, which is directly or via a linker to the Fc region of an immunoglobulin, the Fc region of which allows the FXI-binding protein to form a dimer molecule comprising two FXI-binding domains. Such an FXI-binding protein is also referred to as a divalent FXI-binding protein. In some embodiments, the dimer is a homodimer.

[0076] In some embodiments, the FXI-binding protein of the present invention comprises two specific FXI-binding immunoglobulin single variable domains interconnected directly or via a linker, and an immunoglobulin Fc region, wherein the immunoglobulin Fc region allows the FXI-binding protein to form a dimer molecule comprising four FXI-binding domains. Such an FXI-binding protein is also referred to as a tetravalent FXI-binding protein. In some embodiments, the dimer is a homodimer. In some embodiments, the two specific FXI-binding immunoglobulin single variable domains in the FXI-binding protein bind to different regions or different epitopes of FXI, respectively.

[0077] In some embodiments, the FXI-binding protein of the present invention can inhibit the activity of FXI. In some embodiments, the FXI-binding protein of the present invention can inhibit the coagulation function of FXI.

[0078] Nucleic acid, vector, host cell In another aspect, the present invention relates to a nucleic acid molecule encoding the FXI-binding protein of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA. According to one embodiment of the present invention, the nucleic acid of the present invention is a substantially isolated nucleic acid.

[0079] The nucleic acids of the present invention may also be in vector form, and may be present in and / or part of a vector, such as plasmids, sticky-terminal plasmids, or YACs. The vectors may be, in particular, expression vectors, providing a means for expressing the FXI-binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically contains at least one nucleic acid of the present invention, operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a particular host is common knowledge to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the FXI-binding protein of the present invention include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0080] The nucleic acids of the present invention can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides of the present invention given herein, and / or can be isolated from suitable natural sources.

[0081] In another aspect, the present invention relates to recombinant host cells that express or are capable of expressing one or more of the FXI-binding proteins of the present invention and / or contain the nucleic acids or vectors of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0082] Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0083] Suitable fungal cells include cells from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells from species of the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichia methanolica*), and *Hansenula*.

[0084] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0085] However, the present invention may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0086] This invention also provides a method for generating the FXI-binding protein of this invention, the method typically comprising the following steps: - Culture the host cells of the present invention under conditions that allow expression of the FXI-binding protein of the present invention; and -Recover the FXI-binding protein expressed by the host cells from the culture; and -Optional further purification and / or modification of the FXI-binding protein of the present invention.

[0087] The FXI binding protein of the present invention can be produced in cells as described above in an intracellular manner (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), followed by isolation from the host cell and optionally further purification; or it can be produced in an extracellular manner (e.g., in a culture medium for culturing host cells), followed by isolation from the culture medium and optionally further purification.

[0088] Methods and reagents for recombinantly generating polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, protein separation and purification techniques suitable for the methods of producing the FXI-binding protein of the present invention are well known to those skilled in the art.

[0089] However, the FXI binding protein of the present invention can also be obtained by other protein-generating methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0090] Pharmaceutical Composition On the other hand, the present invention provides a composition, such as a pharmaceutical composition, containing one or a combination of the FXI-binding proteins of the present invention formulated with a pharmaceutically acceptable carrier. Such compositions may contain one or a combination (e.g., two or more different) of the FXI-binding proteins of the present invention. For example, the pharmaceutical compositions of the present invention may contain a combination of antibody molecules that bind to different epitopes on a target antigen (FXI).

[0091] The term "pharmaceutically acceptable carrier" as used herein includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, may be encapsulated in a material to protect it from acids and other natural conditions that could inactivate it.

[0092] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as palmitic acid ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0093] These compositions may also contain preservatives, wetting agents, emulsifiers, and dispersants.

[0094] The presence of microorganisms can be prevented through sterilization procedures or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, and phenolic sorbic acid. In many cases, the composition preferably contains isotonic agents, such as sugars, polyols such as mannitol, sorbitol, or sodium oxide. Prolonged absorption of injectable drugs can be achieved by adding delayed absorption agents, such as monostearate and gelatin, to the composition.

[0095] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the provisional preparation of sterile injections or dispersions. The use of these media and reagents for pharmaceutically active substances is well known in the art. Conventional media or reagents, except for any ranges incompatible with the active compound, may be used in the pharmaceutical compositions of the present invention. Additional active compounds may also be incorporated into the compositions.

[0096] Therapeutic compositions must generally be sterile and stable under preparation and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, by using coatings, such as lecithin, appropriate flowability can be maintained in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0097] Sterile injection solutions can be prepared by mixing the active compound in a suitable solvent in the required amount, and adding one or a combination of the components listed above as needed, followed by aseptic microfiltration. Dispersants are typically prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other desired components listed above. For sterile powders used to prepare sterile injection solutions, preferred preparation methods include vacuum drying and freeze-drying (lyophilization), yielding a powder containing the active ingredient plus any additional desired components from a pre-sterile filtered solution.

[0098] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the target population and the specific route of administration. Generally, the amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation is the amount of the composition that produces the therapeutic effect. Typically, this amount, expressed as 100%, ranges from about 0.01% to about 99% of the active ingredient, for example, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0099] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be proportionally reduced or increased as needed for an emergency in the treatment situation. It is particularly advantageous to formulate the parenteral composition into easily administered and uniformly dosed unit forms. The term "dose unit form" as used herein refers to a physically discontinuous unit suitable as a unit dose for use on the subject being treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect when combined with the desired drug carrier. Specific descriptions of the dose unit forms of the invention are limited to and directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art for formulating such active compounds for treating individual sensitivities.

[0100] For the administration of antibody molecules, the dosage range is approximately 0.0001 to 100 mg / kg, more typically 0.01 to 30 mg / kg of recipient body weight. For example, the dosage may be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight, 10 mg / kg body weight, 20 mg / kg body weight, or 30 mg / kg body weight, or in the range of 1–30 mg / kg. Exemplary treatment regimens may require weekly, bi-weekly, tri-weekly, quadri-weekly, monthly, 3-monthly, 3–6-monthly dosing, or slightly shorter initial dosing intervals (e.g., weekly to tri-weekly) followed by longer intervals (e.g., monthly to 3–6-monthly).

[0101] Alternatively, antibody molecules can be administered as a sustained-release formulation, in which case less frequent dosing is required. Dosage and frequency vary based on the antibody molecule's half-life in the patient. Typically, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, relatively low doses are administered at less frequent intervals over a longer period. Some patients continue treatment for the rest of their lives. In therapeutic use, sometimes higher doses are required at shorter intervals until disease progression subsides or ceases, preferably until the patient shows partial or complete improvement in disease symptoms. Afterward, the patient can be given prophylactic medication.

[0102] The actual dose level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration, without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention or its esters, salts, or amides, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment, and similar factors known in the medical field.

[0103] The compositions of the present invention can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the FXI-binding proteins of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. The phrase “parenteral administration” as used herein refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrasheath, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0104] Alternatively, the FXI binding protein of the present invention can also be administered via non-parenteral routes, such as local, epidermal or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual or local administration.

[0105] Disease treatment and / or prevention In one aspect, the present invention also provides a method for treating and / or preventing thromboembolic symptoms or diseases in a subject, comprising administering to the subject a therapeutically effective amount of the FXI-binding protein of the present invention or a pharmaceutical composition of the present invention.

[0106] In some implementations, the subject has or is at risk of having: myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolic disorders, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (such as cardiopulmonary bypass, hemodialysis, and ECMO), arterial thrombosis, advanced renal disease, antiphospholipid syndrome, stroke, metastatic cancer, or infectious disease.

[0107] In some implementations, the object has pathological activation of FXI.

[0108] In one aspect, the present invention further provides the use of the FXI binding protein of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating and / or preventing thromboembolic conditions or diseases.

[0109] In some specific implementations, the thromboembolic condition or disease is myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, 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, stroke, metastatic cancer, or infectious disease.

[0110] In one aspect, the present invention provides a method for inhibiting FXI activation in a subject (e.g., by factor XIIa (FXIIa)), comprising: (a) selecting a subject to be treated, wherein the subject to be treated has thrombosis or is at risk of thrombosis; and (b) administering to the subject an effective amount of the FXI-binding protein of the present invention or the pharmaceutical composition of the present invention, thereby inhibiting FXI activation.

[0111] In some implementation schemes, the recipients of treatment are individuals who have or are at risk of having the following conditions: myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, 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, stroke, metastatic cancer, or infectious diseases.

[0112] In some implementations, the subjects requiring treatment are those with pathological activation of FXI.

[0113] In some embodiments, the effective amount of the FXI-binding protein of the present invention or the pharmaceutical composition of the present invention is an amount sufficient to inhibit the activation of FXI by at least 10%, 20%, 30%, 40%, or 50%.

[0114] On the other hand, the present invention provides a method for inhibiting coagulation and associated thrombosis in a subject without impairing hemostasis, comprising administering a therapeutically effective amount of the FXI-binding protein of the present invention or the pharmaceutical composition of the present invention to the subject, thereby inhibiting coagulation and associated thrombosis in the subject without impairing hemostasis.

[0115] In some implementations, the subject has or is at risk of having: myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolic disorders, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (such as cardiopulmonary bypass, hemodialysis, and ECMO), arterial thrombosis, advanced renal disease, antiphospholipid syndrome, stroke, metastatic cancer, or infectious disease.

[0116] In some implementations, the object is a pathologically activated object with FXI.

[0117] In another aspect, the present invention also provides the use of the FXI binding protein of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for inhibiting coagulation and related thrombosis without impairing hemostasis.

[0118] In some embodiments of various aspects of the present invention, the FXI binding protein of the present invention or the pharmaceutical composition of the present invention is administered to the subject via parenteral administration.

[0119] Detection In another aspect, the present invention also provides a method for detecting the presence and / or amount of FXI in a biological sample, comprising contacting the biological sample and a control sample with the FXI-binding protein of the present invention under conditions where a complex can be formed between the FXI-binding protein of the present invention and FXI. The formation of the complex is then detected, wherein the difference in complex formation between the biological sample and the control sample indicates the presence and / or amount of FXI in the sample.

[0120] In some embodiments, the FXI binding protein of the present invention is further conjugated with fluorescent dyes, chemicals, peptides, enzymes, isotopes, tags, etc., which can be used to detect or can be detected by other reagents.

[0121] Reagent test kit The scope of this invention also includes kits for use with the methods of this invention, the kits comprising the FXI binding protein of this invention, and instructions for use. Kits generally include a label indicating the intended use of the kit contents. Terminology labels include any written or documented material provided on or with the kit or otherwise with the kit.

[0122] Example The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the described embodiments.

[0123] Example 1: Screening of single-domain antibodies against FXI heavy chain 1.1 Construction of the Library Before immunization, 5 mL of arterial blood was collected from Bactrian camels in a vacuum blood collection tube, and the supernatant was collected as pre-immunization serum. For the initial immunization, a healthy 2-year-old Xinjiang Bactrian camel was selected, and 300 μg of recombinant human factor XI (hFXI, prepared in-house, sequence referenced from the Uniprot database, accession number P03951) was used as antigen and mixed with an equal volume of complete Freund's adjuvant to ensure complete protein emulsification. The mixture was then injected into the Bactrian camel's neck muscles at multiple sites. For subsequent immunizations, an equal volume of antigen was mixed with an equal volume of incomplete Freund's adjuvant each time. Immunizations were performed weekly, for a total of six immunizations. At the end of the final immunization, 5 mL of arterial blood was collected from the Bactrian camel in a vacuum blood collection tube, and the supernatant was collected as post-immunization serum.

[0124] Lymphocytes were isolated using density gradient centrifugation, and total RNA was extracted using an RNA extraction kit provided by QIAGEN. The extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRANDSUPERMIX kit according to the manufacturer's instructions. Nested PCR was then used to amplify the nucleic acid fragment encoding the variable region of the heavy chain antibody.

[0125] The target heavy chain single-domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pMECS using restriction endonucleases (NEB), PstI, and NotI. The product was then electroporated into *E. coli* electroporated competent cells TG1 to construct a phage display library of recombinant human factor XI and the library was validated. The library size was calculated to be 1.5 × 10⁻⁶ cells after serial dilution plating. 8 To determine the insertion rate of the library, 50 clones were randomly selected for sequencing. All 50 clones had the correct foreign fragment insertion, resulting in a 100% accuracy rate. Analysis and alignment of the DNA and amino acid sequences of the sequenced clones confirmed that all sequences were camel VHH sequences, estimating a diversity of over 95%.

[0126] 1.2 Screening of heavy chain single-domain antibodies against FXI For the first screening, 5 μg / well of hApple-chis protein (prepared in-house, sequence referenced from the Uniprot database, accession number P03951, selecting the first 387 amino acids, and adding a His-tag to the C-terminus for purification) was used to coat plates, and the plates were incubated overnight at 4°C. The next day, the plates were blocked with 2% skim milk at room temperature for 2 hours, followed by the addition of 100 μL of phage (approximately 10 μg / well). 8 -10 9 PFU (from a 1.1 hFXI-Chis single-domain antibody display library) was incubated at room temperature for 2 hours. Afterwards, the cells were washed 25 times with PBST (PBS containing 0.05% Tween 20) to remove unbound phages. Finally, the phages specifically bound to hApple-chis were dissociated using Glycine (100 mM, pH 2.0).

[0127] For the second screening, 3 μg / well of mApple-chis protein (prepared in-house, sequence referenced from the Uniprot database, accession number Q91Y47, selecting the first 389 amino acids, and adding a His-tag to the C-terminus for purification) was used to coat plates, and the plates were incubated overnight at 4°C. The next day, the plates were blocked with 2% skim milk powder at room temperature for 2 hours, followed by the addition of 100 μL of phage (approximately 10 μg / well). 8 -10 9PFU (from the 1.1 hFXI-Chis single-domain antibody display library) was incubated at room temperature for 2 hours. Afterwards, the cells were washed 25 times with PBST (PBS containing 0.05% Tween 20) to remove unbound phages. Finally, the phages specifically bound to mApple-chis were dissociated with Glycine (100 mM, pH 2.0) and used to infect logarithmic-phase E. coli TG1 to generate and purify phages for the next round of screening. In the second round, plates were coated with 10 μg / well of hApple-chis protein, with the remaining procedures the same as above.

[0128] Thus, positive clones were enriched, achieving the goal of using phage display technology to screen for FXI-specific antibodies in the antibody library.

[0129] 1.3 Screening for specific single positive clones using enzyme-linked immunosorbent assay (ELISA). The FXI-positive phages obtained after the above selection were used to infect blank *E. coli* and plated. Subsequently, 190 single colonies were randomly selected and named iFE 1 to iFE 190, and inoculated onto 2TY-AG plates. When the OD600 reached approximately 0.8, IPTG was added to a final concentration of approximately 1 mM, and the plates were incubated overnight at 25°C to induce expression. Single-domain antibody expression occurred in the periplasm of *E. coli*. The next day, bacteria were harvested, lysed, and the supernatant was used for ELISA detection. Plates were coated with hFXI, hApple, and mApple respectively and incubated overnight at 4°C. The obtained sample lysis supernatant (control group: blank *E. coli* lysis supernatant) was added, and the reaction was carried out at room temperature for 2 hours. After washing, secondary antibody Goat anti-HA tag HRP (purchased from Abcam) was added, and the reaction was carried out at room temperature for 2 hours. After washing, TMB chromogenic buffer was added, and the absorbance values ​​at 450 nm and 650 nm were read. The final absorbance value was obtained by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. When the OD value of the sample well is more than twice that of the control well, it is considered a positive clone. The positive clone is then sent to Genewiz for sequencing.

[0130] The protein sequences of each clone were analyzed using the sequence alignment software BioEdit. Clones with >90% homology to CDR1, CDR2, and CDR3 sequences were considered the same antibody strain. A total of 23 different antibodies were obtained. The results are shown in Table 1 below. It can be seen that iFE96, iFE97, iFE148, iFE163, iFE166, and iFE168 bind to hFXI, hApple, and mApple simultaneously; iFE29, iFE30, iFE5, iFE7, iFE13, iFE15, iFE35, iFE56, iFE11, iFE17, iFE22, iFE43, iFE49, iFE50, iFE70, iFE108, and iFE128 bind to hFXI and hApple, but not to mApple.

[0131] Table 1. Binding characteristics of 23 antibody strains

[0132] 1.4 Prokaryotic expression and purification of positive clones The seed culture of the positive clones obtained in section 1.3 (E. coli TG1 expression system, vector pMECS, containing HA and HIS tags) cultured overnight in 2TY-AG medium was transferred to 50 mL of 2TY-AG medium and cultured at 37°C until the OD600 reached approximately 0.8. Then, IPTG was added to a final concentration of approximately 1 mM, and expression was induced overnight at 25°C. The cells were harvested the next day, resuspended in Tris buffer, and sonicated to break down the bacteria. The supernatant obtained was purified using Ni column affinity chromatography with the His tag on the single-domain antibody to obtain the corresponding target protein.

[0133] 1.5 Affinity detection of prokaryotic expressed proteins to hApple and mApple Plates were coated with hApple and mApple proteins at a concentration of 0.5 μg / well and incubated overnight at 4°C. A serially diluted series of candidate single-domain antibodies with His and HA tags obtained in section 1.4 were added, and the reaction was carried out at room temperature for 2 hours. After washing, horseradish peroxidase-labeled chicken anti-HA tag secondary antibody (streptavidin-HRP, abcam) was added, and the reaction was carried out at room temperature for 2 hours. After washing, chromogenic buffer was added, and absorbance values ​​were read at 450 nm and 650 nm. The final absorbance value was obtained by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. Data processing and graphical analysis were performed using SotfMax Pro v5.4 software. Binding curves and EC50 values ​​of the candidate single-domain antibodies against FXI with hApple and mApple were obtained through four-parameter fitting to reflect the affinity of these candidate antibodies for hApple and mApple.

[0134] The results are shown in Table 2. It can be seen that all of these candidate single-domain antibodies bind to hApple. Among them, iFE43, iFE50, and iFE96 have weaker affinity for the hApple protein; and iFE96, iFE97, iFE148, iFE163, iFE166, and iFE168 also bind to mApple.

[0135] Table 2. Binding affinity of candidate antibodies for hApple and mApple

[0136] Example 2: Preparation of Fc fusion protein of FXI single-domain antibody using mammalian cells 2.1 Preparation of Fc fusion plasmid for FXI single-domain antibody Primers were designed for PCR amplification of the FXI single-domain antibody VHH fragment (amino acid sequence shown in SEQ ID NO: 1-23; to improve protein stability, individual amino acids in the FR2 region of some sequences were mutated), fused with a DNA fragment encoding human IgG1-Fc (amino acid sequence SEQ ID: 336), and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the FXI single-domain antibody-Fc fusion protein in mammals. Different VHH fragments were amplified using universal primers and fused with the human IgG1-Fc DNA fragment. The universal primers are as follows: Upstream primer cccACCGGTCAGGTGCAGCTGCAGGAGTC Downstream primer: cccGGATCCTGAGGAGACGGTGACCTGG.

[0137] 2.2 Preparation of Fc fusion protein of FXI single-domain antibody The vector constructed in section 2.1 was transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmid was diluted with Freestyle293 medium and PEI (Polyethylenimine) solution was added for transformation. Each plasmid / PEI mixture was added to HEK293 cell suspension and incubated at 37°C with 5% CO2. After 5-6 days of culture, the supernatant of the transient expression culture was collected, and the target FXI single-domain antibody-Fc fusion protein was purified by Protein A affinity chromatography. The purity of the obtained proteins was initially determined by SDS-PAGE and SEC-HPLC. The expression and purity analysis of each protein are shown in Table 3 below.

[0138] Table 3. One-step purification results of the obtained anti-FXI single-domain antibody-FC fusion protein after transient transfection.

[0139] As can be seen, the expression levels of FXI single-domain antibody-Fc fusion proteins iFE7m-Fc, iFE15m-Fc, iFE17EREG-Fc, iFE29m-Fc, and iFE30m-Fc were very low, while the expression levels of the others were all above 290 mg / L. After one-step purification using a Protein A affinity chromatography column, the target protein with stable concentration and high purity was obtained.

[0140] Example 3: Identification of the function of the FXI single-domain antibody-Fc fusion protein 3.1 Binding curves of FXI single-domain antibody-Fc fusion protein to mApple and hApple Plates were coated with mApple and hApple proteins, 0.5 μg / well, with a blank control included, and incubated overnight at 4°C. A serially diluted series of the FXI single-domain antibody-Fc fusion protein obtained in Example 2.2 were added, and the reaction was carried out at room temperature for 2 hours. After washing, Goat anti-human IgG-HRP (purchased from SIGMA) was added, and the reaction was carried out at room temperature for 2 hours. After washing, chromogenic buffer was added, and absorbance values ​​were read at 450 nm and 650 nm. The final absorbance value was obtained by subtracting the absorbance value at 650 nm from the absorbance value at 450 nm. Data processing and graphical analysis were performed using SotfMax Pro v5.4 software. Four-parameter fitting was used to obtain antibody binding curves and EC50 values ​​for mApple and hApple to reflect the antibody's affinity for mApple and hApple.

[0141] The results are shown in Table 4. All antibodies bound well to the hApple protein, and antibodies iFE96, iFE97, iFE148, iFE163, iFE166, and iFE168 also bound well to the mApple protein.

[0142] Table 4. Binding of FXI single-domain antibody-Fc fusion protein to mApple and hApple

[0143] 3.2 Detection of affinity for FXI single-domain antibody-Fc fusion protein (Biomembrane interference technique, BLI) The binding kinetics of the FXI single-domain antibody-Fc fusion protein obtained in the above embodiments against the recombinant proteins hApple and mApple were measured using a molecular interaction analyzer via biolayer interferometry (BLI). FXI single-domain antibody-Fc fusion proteins iFE5-Fc, iFE13-Fc, iFE35-Fc, iFE56-Fc, iFE97-Fc, and iFE5-Fc were diluted to a final concentration of 10 μg / mL and directly immobilized onto a ProteinA biosensor. For kinetic measurements, hApple was diluted with 0.02% PBST20 to five concentrations: 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM. mApple was diluted to five concentrations: 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM. The injection time was 150 s, the dissociation time was 900 s, and regeneration was performed with 10 mM glycine-HCl (pH 1.7) for 5 s. The binding rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Languir binding model (Octet K2 Data Analysis Software Version 9.0). The equilibrium dissociation constant (kD) was calculated as the ratio kdis / kon.

[0144] The results are shown in Tables 5 and 6. Table 5 shows that the binding affinity of the FXI single-domain antibody-Fc fusion protein to hApple is comparable, while Table 6 shows that the antibodies iFE97-Fc and iFE148-Fc have better affinity to mApple.

[0145] The positive control 14E11 was synthesized according to the sequence in patent WO2010080623, and then prepared by transient expression in 293 cells according to the above method.

[0146] Table 5. Affinity with hApple

[0147] Table 6. Affinity with mApple

[0148] 3.3 Detection of different epitopes binding to FCI single-domain antibody-Fc fusion protein and Apple protein (Biomembrane interferometry, BLI: epitope binning) Using the in-tandem method, happy-chis-biotin and mapple-chis-biotin were diluted to 10 μg / mL with 0.02% PBST20 and immobilized onto an SA biosensor for 100 s, resulting in a height of approximately 1 nm. The FXI single-domain antibody-Fc fusion protein was diluted to 200 nM with 0.02% PBST20 and divided into two groups. The antibody binding time for both groups was 300 s, and the regeneration buffer was 10 mM glycine-HCl (pH 1.7). The first antibody (saturating antibody) bound to the sensor until saturation, and then the second antibody (competitive antibody) competed with the first antibody at the same concentration. The percentage was calculated using the formula: Ab2 with Ab1 / Ab2 without Ab1.

[0149] The measurement results are shown in Tables 7, 8, and 9. Based on the above results, Table 7 shows that iFE13-Fc, iFE35-Fc, and 14E11 compete; the iFE13-Fc epitope overlaps with 14E11, and the iFE35-Fc epitope partially overlaps with 14E11. iFE56-Fc and iFE35-Fc compete; the iFE56-Fc epitope has partial steric hindrance to 14E11. iFE5-Fc and iFE30-Fc compete, sharing the same epitope, and do not overlap with 14E11. Tables 8 and 9 show that iFE97-Fc and iFE163-Fc compete, sharing the same epitope, and do not overlap with 14E11. iFE148-Fc recognizes both humans and mice; its epitope does not overlap with other single-domain antigens or 14E11.

[0150] Table 7 hApple

[0151] Table 8 happle

[0152] Table 9 mapple

[0153] 3.4 Detection of non-specific binding of FCI single-domain antibody-Fc fusion protein to empty cells CHOK1 and 293F empty cells were resuspended in 3% BSA-PBS and the cell count was adjusted to 6 × 10⁶ cells / mL. 6Cells / mL. Following the results of Example 3.1, the final concentration of the FXI single-domain antibody-Fc fusion protein was selected as 100 μg / mL. Negative and blank controls were also included. Cells were incubated on ice for 60 min. After washing, Biolegend secondary antibody FITC anti-human IgG FC was added, and the cells were incubated on ice for 30 min. After washing, the cells were resuspended in 300 μL of 1% PBS-BSA buffer and analyzed by flow cytometry.

[0154] The results are shown in Tables 10 and 11. The candidate antibodies did not bind nonspecifically to the blank cells.

[0155] Table 10

[0156] 3.5 Identification of the blocking activity of the FCI single-domain antibody Fc fusion protein against FCI (ATPP assay) Using an optical detection method, pre-warmed plasma and reagents are rapidly mixed and incubated. The absorbance is measured at a wavelength of 660 nm. As the incubation time increases, fibrinogen converts to fibrin, increasing the turbidity of the mixture and causing a change in the intensity of scattered light. The instrument detects the change in scattered light intensity due to the increase in sample turbidity, thereby determining the coagulation time. Commercially available coagulation factor XI is used as a standard. A standard curve coagulation method is employed, with coagulation time as the Y-axis and the activity percentage of reference plasma as the X-axis. The activity of the sample is calculated based on the standard curve.

[0157] The antibodies were diluted to 1 μg / mL with FXI-deficient plasma, with a buffer used as a negative control. 50 μL of each treated sample was incubated at 37°C for 2 h, then loaded into an automated coagulation analyzer. The samples were automatically mixed with Dade Actin Activated Cephaloplastin Reagent, Calcium Chloride Solution, and FACTOR IX DEFICIENT reagent, incubated, and the absorbance was measured at 660 nm. The results are shown in Table 11 below. iFE15, iFE29, iFE96, iFE166, and iFE168 showed no blocking activity.

[0158] Table 11

[0159] Based on the results in Table 11, antibodies iFE13, iFE35, iFE97, iFE5, iFE148, iFE56, and iFE30 were serially diluted with standard human plasma, and their activity curves were detected. A positive control, 14E11, was also included. The results are as follows: Figure 1It can be seen that the inhibitory effects of iFE5, iFE13, iFE35, iFE56, and iFE97 are significantly better than those of the positive control 14E11; the inhibitory effects of the remaining antibodies iFE148 and iFE30 are comparable to those of the positive control.

[0160] 3.6 Binding of FXI single-domain antibodies to different apple domains The FXI single-domain anti-Fc fusion protein obtained in the above examples, and the positive control 14E11, were selected. The binding kinetics of these proteins to different human Apple domain proteins were investigated using biolayer interferometry (BLI). The sequences of the different Apple domain proteins were derived from Uniprot (accession number P03951). Some Apple domains had a His-tag added to their C-terminus for purification (the name includes "Chis"), while others had a mouse Fc fragment added to their C-terminus for purification (the name includes "muFc"). The proteins were transiently transfected into 293 cells and then further purified using IMAC. The specific procedures were similar to those described above. The assay samples used were self-prepared hApple1-2muFc, hApple2-3muFc, hApple3-4muFc, hApple2-chis, hApple4-chis, hApple1-muFc, and hApple3-muFc.

[0161] The binding of candidate FCI single-domain antibody Fc fusion proteins to different Apple domains is shown in Tables 12 and 13 below.

[0162] Table 12

[0163] Table 13

[0164] Example 4: Humanization of FXI single-domain antibody The humanization method was achieved by resurfacing amino acids on the protein surface and grafting the VHH humanized universal framework (CDR).

[0165] The humanization steps are as follows: Homology modeling was performed on antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, and iFE148 using Modeller9 software. The reference homologous sequence was the NbBcII10 antibody (PDB number: 3DWT), and the relative solvent accessibility of amino acids was calculated based on the protein's three-dimensional structure. If any amino acid in antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, or iFE148 was exposed to a solvent, it was replaced with the amino acid at the same position in the reference human antibody 10HQ sequence, thus completing all replacements.

[0166] The specific steps of the VHH humanized universal framework transplantation method are as follows: First, obtain the universal humanized VHH framework hNbBcII10FGLA (PDB number: 3EAK), designed by Cécile Vincke et al. based on sequence homology. This framework design is based on the nanobody NbBcII10 antibody (PDB number: 3DWT). Referencing the human antibody 10HQ, protein surface amino acid humanization was performed, and certain amino acids VLP in VHH sequence framework 1, GL in VHH sequence framework 2, RSKRAAV in VHH sequence framework 3, and L in VHH sequence framework 4 were modified. We directly used hNbBcII10FGLA as the framework, replacing the CDRs with the CDR regions of antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, and iFE148 to complete the antibody humanization.

[0167] Antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, and iFE148 were humanized to obtain six humanized variants, huFE. The sequences of these humanized variants are suitable for SEQ ID NO:300-335, respectively.

[0168] Example 5: Preparation of Fc fusion protein of humanized single-domain antibody 5.1 Preparation of Fc fusion plasmids for humanized single-domain antibodies The humanized huFE sequence from Example 4 was synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and restriction enzyme sites were added to both ends.

[0169] The VHH fragment of the huFE single-domain antibody was digested with two enzymes, fused with a DNA fragment encoding human IgG1FC, and cloned into a conventional mammalian expression vector to obtain a recombinant plasmid for expressing the huFE single-domain antibody Fc fusion protein in mammals.

[0170] 5.2 Preparation of Fc fusion protein of humanized single-domain antibody The vectors constructed in section 4.1 were transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmids were diluted with Freestyle293 medium and PEI (Polyethylenimine) solution was added for transformation. Each plasmid / PEI mixture was added to HEK293 cell suspension and cultured at 37°C, 5% CO2, and 130 rpm. After four hours, EXCELL293 medium and 2 mM glutamine were added, and the cells were cultured at 130 rpm. After 24 hours, 3.8 mM VPA was added, and after 72 hours, 4 g / L glucose was added. After 5-6 days of culture, the transient expression culture supernatant was collected, and the target huFE single-domain antibody Fc fusion protein was purified by Protein A affinity chromatography. The purity of the proteins was preliminarily assessed by SDSPAGE and SECHPLC. The expression levels of each protein exceeded 350 mg / L, and the SEC purity after one-step purification was greater than 95%.

[0171] Example 6: Identification of the function of huFE single-domain antibody-Fc fusion protein 6.1 Affinity of huFE single-domain antibody Fc fusion protein The binding kinetics of the huFE single-domain antibody Fc fusion protein obtained in the above embodiments against the hApple-CHis protein were measured using a molecular interaction analyzer via biolayer interferometry (BLI). The huFE single-domain antibody Fc fusion protein obtained in Example 5.2 was diluted to a final concentration of 10 μg / mL and directly immobilized onto an AHC biosensor. Kinetic measurements were performed by diluting the hApple-CHis protein to five concentrations: 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM. The baseline time was 60 s, binding time was 120 s, and dissociation time was 900 s. The dilution buffer was kinetic buffer, the regeneration buffer was glycine-HCl (pH 1.7), and the neutralization buffer was the dilution buffer. The biosensor was Protein A. A simple one-to-one Languir binding model (Octet K2 data analysis software version 9.0) was used to calculate the binding rate (kon) and dissociation rate (kdis). The equilibrium dissociation constant (kD) is calculated as the ratio kdis / kon.

[0172] The measured affinity of the huFE single-domain antibody Fc fusion protein for the hApple-Chis protein is shown in Table 14.

[0173] Table 14

[0174] The binding kinetics of the huFE single-domain antibody Fc fusion protein obtained in the above embodiments against the hFXI-CHis protein were measured using a molecular interaction analyzer via biolayer interferometry (BLI). The specific procedure was the same as described above, and the hFXI-Chis protein was used as the detection analyte. 4E11 (as described above) and B1213790-F11a (prepared according to the sequence in WO2018134184) were used as controls.

[0175] The measured affinity of the huFE single-domain antibody Fc fusion protein for the hFXI-CHis protein is shown in Table 15.

[0176] Table 15

[0177] The results showed that the humanized single-domain antibody-Fc fusion protein bound well to both hApple-CHis and hFXI-CHis, with no significant differences between the various humanized versions. Furthermore, its binding affinity was comparable to that of the two control antibodies.

[0178] A subset of the huFE single-domain anti-Fc fusion proteins obtained in the above embodiments were selected, and their binding kinetics to New Zealand rabbit FXI protein were investigated using biolayer interferometry (BLI). The specific procedures were the same as described above, and the assay sample used was a self-prepared New Zealand rabbit FXI protein (the relevant protein sequence is referenced from the Uniprot database accession number Q95ME7). The measured affinity of the huFE single-domain antibody Fc fusion protein for the RabFXI-Apple-CHis protein is shown in Table 16 below.

[0179] Table 16

[0180] A subset of the huFE single-domain anti-Fc fusion proteins obtained in the above embodiments were selected, and their binding kinetics to cynomolgus monkey FXI protein were investigated using biolayer interferometry (BLI). The specific procedures were the same as described above, and the analyte used was a self-prepared cynomolgus monkey FXI protein (the relevant protein sequence is referenced from the Uniprot database accession number A0A2K5VVK2). The affinity of the measured huFE single-domain antibody Fc fusion protein for the cynomolgus monkey FXI-CHis protein is shown in Table 17.

[0181] Table 17

[0182] The above experimental results indicate that most candidate antibodies can simultaneously recognize coagulation factor F11 in humans, New Zealand rabbits, and cynomolgus monkeys. However, binding to New Zealand rabbits is relatively reduced. Furthermore, antibodies 35 and 56 showed virtually no activity in New Zealand rabbits, while antibody 5 showed virtually no activity in cynomolgus monkeys. There was no significant difference in activity among the various humanized versions of the protein.

[0183] A subset of the huFE single-domain anti-Fc fusion proteins obtained in the above embodiments were selected, and their binding kinetics to activated human factor 11 protein (hFXIa) were investigated using biolayer interferometry (BLI). The specific procedures were the same as described above, and commercially available hFXIa was used as the detection analyte. 14E11 (as described above) and B1213790-F11a (as described above) were used as controls.

[0184] The measured affinity of the huFE single-domain antibody Fc fusion protein for the hFXIa protein is shown in Table 18.

[0185] Table 18

[0186] The results above show that the humanized FCI single-domain antibody Fc fusion protein can effectively bind to activated FCI factors. Its binding affinity is lower than that of B1213790-F11a, which directly binds to the activation site of FCI factors.

[0187] Furthermore, combining previous results on the binding of inactive FXI factors, it was found that 14E11, which also binds to the Apple domain (Apple2), exhibits increased binding activity to activated FXIa. In contrast, the candidate antibody of this invention maintains comparable binding activity to activated FXIa compared to inactive FXI, or its binding activity to activated FXIa is reduced. This result, from another perspective, demonstrates that even if some candidate antibodies of this invention have some overlap with 14E11 in binding epitopes, their binding modes are significantly different.

[0188] 6.2 Inhibitory effect of huFE single-domain antibody Fc fusion protein on human FXI factor activity The activity of human FXI in standard human plasma (purchased from WHO) was 92%. Combined with FXI-deficient plasma, the inhibitory effects of adding different single-domain antibody Fc fusion proteins and a control (14E11) on FXI factor activity in plasma were tested. The activity results of FXI factor mixed with different antibodies are shown in Table 19. Figure 2These humanized FXI antibodies all showed good inhibitory activity against the FXI factor. 14E11 protein was used as a positive control.

[0189] Table 19

[0190] Example 7: Preparation of Fc fusion protein of huFE bispecific antibody using mammalian cells 7.1 Preparation of Fc fusion plasmid for huFE bispecific antibody The gene of the huFE single-domain antibody Fc fusion protein obtained in Example 4 was molecularly cloned to obtain a recombinant plasmid for expressing the huFE bispecific antibody Fc fusion protein in mammals, which was used to prepare the following bispecific antibody proteins: huFE97n13-Ld-Fc, huFE13n97-Ld-Fc, huFE97n56-Ld-Fc, huFE56n97-Ld-Fc, huFE97di-Ld-Fc, huFE56n13-Ld-Fc, huFE13n56-Ld-Fc. c, huFE97n148-Ld-Fc, huFE148n97-Ld-Fc, huFE5n97-Ld-Fc, huFE97n5-Ld-Fc, huFE148n5-Ld-Fc, huFE5n148-Ld-F c, huFE148n56-Ld-Fc, huFE56n148-Ld-Fc, huFE56n5-Ld-Fc, huFE5n56-Ld-Fc, huFE5n13-Ld-Fc, huFE13n5-Ld-Fc.

[0191] Meanwhile, the tetravalent monospecific antibody HuFE97di-Ld-Fc was also used as a control antibody via the above method.

[0192] 7.2 Preparation of Fc fusion protein of huFE bispecific antibody The vector constructed in section 7.1 was transfected into HEK293 cells for transient antibody expression. The recombinant expression plasmid was diluted with Freestyle293 medium and PEI (Polyethylenimine) solution was added for transformation. Each plasmid / PEI mixture was added to HEK293 cell suspension and cultured at 37°C in 5% CO2. After 5-6 days of culture, the supernatant was collected, and the target huFE bispecific antibody Fc fusion protein was purified by Protein A affinity chromatography. The purity of the protein was preliminarily assessed by SDSPAGE and SECHPLC.

[0193] The expression levels of all proteins ranged from 250 mg / L to 400 mg / L, and the purity of SEC after one-step purification was above 95%. These results preliminarily demonstrate that these bispecific antibodies exhibit excellent solubility and stability, making them suitable as drug candidate molecules.

[0194] Example 8: High-Temperature Accelerated Development of huFE Bispecific Antibody-Fc Fusion Protein 12 mg of each of the huFE bispecific antibody Fc fusion protein obtained in the above examples was concentrated to 10 mg / ml by ultrafiltration and then placed in PBS solution. The solution was then stored at 40°C, and samples were periodically taken to detect changes in protein content, SEC purity, etc., to assess its stability. The results are shown in Tables 20 and 21 below.

[0195] Table 20. Protein content of 7 proteins after acceleration (OD280nm).

[0196] Table 21 SE-HPLC Detection

[0197] Conclusion: After 20 days at high temperature, the purity decreased slightly, but remained within an acceptable range.

[0198] Example 9: Identification of the function of the huFE bispecific antibody-Fc fusion protein 9.1 Affinity of huFE bispecific antibody Fc fusion protein The binding kinetics of the huFE bispecific antibody Fc fusion protein obtained in the above examples against hApple-Chis and hFXI-Chis proteins were measured using a molecular interaction analyzer via biolayer interferometry (BLI). The huFE bispecific antibody Fc fusion protein obtained in Example 7.2 was diluted to a final concentration of 10 μg / mL and directly immobilized onto an AHC biosensor. Kinetic measurements were performed by diluting hApple-Chis or hFXI-Chis proteins at five different concentrations, with a baseline of 60 s, binding at 120 s, and dissociation at 900 s. The dilution buffer was kinetic buffer, the regeneration buffer was glycine-HCl (pH 1.7), and the neutralization buffer was the dilution buffer. The biosensor was Protein A. A simple one-to-one Languir binding model (Octet K2 data analysis software version 9.0) was used to calculate the binding rate (kon) and dissociation rate (kdis). The equilibrium dissociation constant (kD) was calculated as the ratio kdis / kon.

[0199] The affinity of the measured huFE bispecific antibody Fc fusion protein for the hFXI-Chis protein is shown in Table 22 below.

[0200] Table 22

[0201] The measured affinity of the huFE bispecific antibody Fc fusion protein for the hApple-Chis protein is shown in Table 23 below.

[0202] Table 23

[0203] The above results indicate that all candidate FXI bispecific antibodies exhibited good affinity for human FXI factor protein and human Apple domain, which was higher than that of the maternal monospecific antibody.

[0204] 9.2 Specificity of the huFE bispecific antibody Fc fusion protein The FXI bispecific antibodies obtained in the above examples were selected, and their nonspecific binding to other coagulation-related proteins was investigated using biolayer interferometry (BLI). The antibody proteins were immobilized onto an AHC chip, and the proteins examined were commercially available FVII, FIX, FV, FXII, pro-thrombin, α-kallikrein, FVIIa, FIXa, FVa, FXIIa, and Thrombin. Experimental results showed that none of the bispecific antibodies bound to FVII, FIX, FV, FXII, pro-thrombin, α-kallikrein, FVIIa, FIXa, FVa, FXIIa, or Thrombin.

[0205] 9.3 Inhibitory effect of huFE bispecific antibody Fc fusion protein 9.3.1 Inhibitory effect on human FXI activity The activity of FXI in standard human (WHO) was 92%, and the activity of FXI in standard human plasma (purchased from Sigma) was 87.5%. Using FXI-deficient plasma, the effects of adding different single-domain antibody Fc fusion proteins and controls (14E11, MAA868-F11) on the inhibition of FXI factor activity in plasma were detected. The activity results of FXI factor mixed with different antibodies are shown in [the table below]. Figure 3 These FXI antibodies all showed good inhibitory activity against FXI factors and were superior to the control positive antibodies. Among them, 14E11 protein (prepared as above) and MAA868-F11 (prepared according to patent application US15 / 739414) were used as positive controls.

[0206] 9.3.2 Inhibitory activity against APTT in human whole plasma FXI antibodies were diluted to different concentrations with standard human plasma (purchased from Sigma), incubated at 37°C for 3 minutes, and then the APTT time of whole blood was measured. 14E11 and B1213790-F11a were used as positive controls.

[0207] Figure 4 The results show the change in APTT time with antibody concentration. These results demonstrate that all candidate FXI antibody proteins can effectively prolong the whole blood APTT clotting time; and the bispecific antibody exhibits superior anticoagulation effects.

[0208] 9.3.3 Inhibitory activity against APTT in whole monkey plasma The FXI antibody was diluted to different concentrations with monkey plasma (purchased from Sigma), incubated at 37°C for 3 minutes, and then the APTT time in whole blood was measured. 14E11 and B1213790-F11a were used as positive controls.

[0209] See results Figure 5 .

[0210] 9.3.4 Inhibitory activity against APTT in rabbit whole plasma The Fc fusion protein of the Shili FXI antibody and the standard were diluted to different concentrations using rabbit plasma (purchased from Sigma), and the APTT time in whole blood was measured. 14E11 and B1213790-F11a were used as positive controls.

[0211] See results Figure 6 .

[0212] The above experimental results indicate that most FXI candidate antibodies, regardless of whether they are bispecific, exhibit good APTT inhibitory activity in human or monkey plasma. Some antibodies showed no activity in rabbits.

[0213] The activity of candidate antibodies in human plasma is generally superior to that of control positive antibodies. Furthermore, the activity of bispecific antibodies is often superior to that of monospecific antibodies.

[0214] 9.4 Effects of FXI single-domain antibody Fc fusion protein and bispecific antibody on rabbit venous thrombosis Rabbits were fasted overnight. Blood was collected via the marginal ear vein. Anesthesia was administered via the marginal ear vein. The jugular vein was ligated at both the distal and proximal ends, with the interval between the two ligatures maintained at approximately 3.0 cm. Fifteen minutes before modeling, 1 mg / kg of the test sample (the huFE single-domain antibody Fc fusion protein obtained in Example 5.2) or a PBS negative control was injected via the marginal ear vein (see Table 24). Arterial clamps were applied to both the proximal and distal ends of the jugular vein. Blood was aspirated from the facial vein using a syringe. 0.3 ml of a 5 mg / ml agonist was then injected into the blocked segment of the vessel. After incubation for 5 minutes, the agonist was withdrawn with a syringe, and the vessel was flushed twice with saline. The arterial clamps were then released to restore blood flow, maintaining the vessel diameter at 0.8 mm to induce thrombus formation. Twenty-five minutes after blood flow was restored, the distal and proximal ends of the blocked vein were clamped with arterial clamps, and the previously ligated surgical sutures for the proximal and distal ends of the vein were tightened. The blocked vein was then cut open, the thrombus was removed, and its wet weight was immediately weighed and recorded. The thrombus was then dried in a 60-degree oven for 20 hours, and its dry weight was weighed and recorded (see Table 25).

[0215] The observation indicator was thrombus weight. Experimental data were expressed as mean ± standard deviation (X±SD). GraphPad Prism 5-way ANOVA was used for significance testing (see [link to relevant documentation]). Figure 7 ).

[0216] Due to the cross-species interaction between different antibodies and rabbits, only some antibodies showed good thrombosis inhibition effects. Subsequent in vivo activity tests will be conducted on monkeys, which have better species cross-species interaction.

[0217] Table 24

[0218] Table 25

[0219] Sequence information Amino acid sequences and corresponding CDR sequences of 23 heavy chain single-domain antibodies iFE5 QVQLQESGGGSVQAGGSLRLSCAAS GYTYSFNSMG WFRQAPGKEREGVAV IYLGSTHYADSVKG RFTISQDNAKNMVYLQMNSLKPEDTAMYYCAA DLLEYDGTWFGSTYNY WGQGTQVTVSS(SEQ ID NO:1)

[0220] >iFE7m QVQLQESGGGLVQPGGSLRLSCAAS GLTFS LYDMS WVRQAPGKGLEGVS GINSGDGSTYYADSVKG RFTISRDNAKNTLYLQMNSLKTEDTAVYYCAT GRTRETYFREDD WGQGTQVTVSS (SEQ ID NO:2)

[0221] >iFE11m QVQLQESGGGLVQPGGSLRLSCAAS GLRFSVYDMS WVRQAPGKGLEGVS GIDSGGGSTYYADSVKG RFTISRDDAKNTLYLQMNSLKTEDTAVYYCAT GLGGSWYREPD WGQGTQVTVSS (SEQ ID NO:3)

[0222] >iFE13 QVQLQESGGGSVQAGGSLRLSCAGD GYTYSMGAMA WFRQAPGKEREGVA LIATSSGFISYTDSVKG RFTISQDDAKIYLQMNSLKPEDTAMYYCAA GTQFSWSPGSYNY WGQGTQVTVSS (SEQ ID NO:4)

[0223] >iFE15m QVQLQESGGGLVQPGGSLRLSCAAS GFTFSNYDMS WVRQAPGKGLEGVS GINSGGGHPHYADSVKG RFTISRDNAKNTLYLQMNSLKTEDTAVYFCAT DEGERYGEDFGY WGQGTQVTVSS (SEQ ID NO:5)

[0224] >iFE17EREG QVQLQESGGGLVQPGGSLRVSCAAS GFTFSLYDMS WVRQAPGKEREGVS GINSDDGSTSYADSVKG RFTISRDNAKNTLYLQMNSLKTEDTAVYYCAT GVGARWFREDD WGQGTQVTVSS (SEQ ID NO:6)

[0225] >iFE22m QVQLQESGGGLVQPGGSLRLSCAAS GLRFEIYDMS WVRQAPGKGLEGVS AINSGGGSTYYADSVKG RFTISRDDVKNTLYLQMNSLKTEDTAVYYCAT GDGGSWYRDGD WGQGTQVTVSS (SEQ ID NO:7)

[0226] >iFE29m QVQLQESGGGLVQPGGSLRLSCVAS GLTFSLYDMS WVRQAPGKGLEGVS GISGESGGSTYYADSVKG RFTISRDNAKNTLYLQMNSLKTEDTAVYYCAT GDRGTWYREDD WGQGTQVTVSS (SEQ ID NO:8)

[0227] >iFE30m QVQLQESGGGLVQPGGSLRLSCAAS GLRFSIYDMS WVRQAPGKGLEGVS GINSGGGSTYYADSVKG RFTISRDNAKNTLYLQMNSLKTEDTAVYYCAT GLGGSWFREDD WGQGTQVTVSS (SEQ ID NO:9)

[0228] >iFE35 QVQLQESGGGSVQAGGSLGLSCTAS GFTFDDSDMG WYRQAPGNECELVS TISSDGRGFYADSVKG RFTISQDRAKNTVYLHMNSLKPEDTAVYYCAA GWYGSFCSG LGQGTQVTVSS (SEQ ID NO:10)

[0229] >iFE43 QVQLQESGGGSVQAGGSLRLSCAAS GLRFSIYDWS WVRQAPGKGLEWVS AINSGGGVTSYADSVKG RVTISRDNAKNTLYLQMHSLKTEDTAVYYCAT GDGGSWYRDGD WGQGTQVTVSS (SEQ ID NO:11)

[0230] >iFE49 QVQLQESGGDSVQAGGSLRLSCAAS GLISTGLISTRHCMS WFRQAPGKEREEVA VIGSDGSTRYGDFA KG RFTISKDTAKNTLYLQMNSLEPEDTAMYYCAA GPLVSTASRRRTRCPEGPRENIY WGQGTQVTVSS (SEQ ID NO:12)

[0231] >iFE50 QVQLQESGGGLVQPGGSLRLSCAAS GFTFSLYDMS WVRQAPGQGLEWVS AITNPGGSTYYADSVKD RFTISRDNAKNTLYLQMNSLKTEDTAVYCAI GRRATFQREKD WGQGTQVTVSS (SEQ ID NO:13)

[0232] >iFE56 QVQLQESGGGSVQAGGSLRLSCAAS GHTYSSNYCMA WFRQAPGKEREGVA AIYSDGSTSYADSVKG RFTISKDNAKNTLYLQIDSLKPEDTSLYYCAA TAYEGSWTGKQPLCLLYEYTY WGQGTQVTVSS (SEQ ID NO:14)

[0233] >iFE70m QVQLQESGGGLVQPGGSLRLSCAAS GFTFSLYDMS WVRQAPGKGLDGVS GIGGDGGSTYYADSVKG RFAISRDDAKNTLYLQMNSLKIEDTAVYCAL GRPPYTDYDS RGQGTQVTVSS (SEQ ID NO:15)

[0234] >iFE96 QVQLQESGGGSVQAGGSLRLSCAAS GFTASSNFMG WFRQAPGKEREGVA AIYTGGASTFYADSVKGRFTISQDNAKNTVYLEMDTLKPEDTAMYYCAA APRWIAPLRADRYEY WGQGTQVTVSS (SEQ ID NO:16)

[0235] >iFE97 QVQLQESGGGSVQAGGSLRLSCTAS EFTFDDSDMA WYRQAPGNECELVS TITSDGGTYYADSVKG RFTISQDNAKNTMYLQMNNLKPEDTAVYYCAA DQWGSAEGDCTSSYPGGY WGQGTQVTVSS (SEQ ID NO:17)

[0236] >iFE107m QVQLQESGGGLVQPGGSLRLSCAAS GFTFSLYDMS WVRQSPGKGLEGVS AIDSGDGRTYYADSVKG RFTISRDDAKNTLYLQMNSLKTGDTAVYYCAN GGRGSWFRESD WGQGTQVTVSS (SEQ ID NO:18)

[0237] >iFE128m QVQLQESGGRLVQPGGSLRLSCVAS GFTFEYYDMS WVRQAPGKGLEGVS AIDARSITDYADAVKG RFTISRDNAKNTLYLQLNNLKTEDTAMYYCAN GGRIGWFREDD WGQGTQVTVSS (SEQ ID NO:19)

[0238] >iFE148 QVQLQESGGGSVQAGGSLRLSCVAS GYTFNNNFVG WFRQAPGKEREGVA AIYSLGGSTYYADSVKG RFTISQDNAKNTVYLQMDSLKPEDTATYYCAG AITWVPPLSHRRYTY WGQGTQVTVSS (SEQ ID NO:20)

[0239] >iFE163 QVQLQESGGDSVQAGGSLRLSCIAS GFTFDDSDMG WYRQAPGHQCEFVS HITNDGTTYYADSVKG RFTISRDNAKNTVYLQMNSLKPEDAAVYYCAA DAWGGGRSTCVNPIGY WGQGTQVTVSS (SEQ ID NO:21)

[0240] >iFE166 QVQLQESGGGSVQAGGSLRLSCVAS GFTGCRNIMS WYRQAPGKDREFVS AIDSDGVDYADSVKG RFSISQHNDKKTMYLQMTSLKLEDTAMYYCRR VDFGTTSQCRGNY WGQGTQVTVSS (SEQ ID NO:22)

[0241] >iFE168 QVQLQESGGGSVQAGGSLRLSCAAS GYTFSNNFVG WFRQAPGKKREGVA TIYTLGSSTYYADSVKG RFTISQDNAKNTLYLEMNSLKPEDTAMYYCAA ARGWVSPLDPARFVY WGQGTQVTVSS (SEQ ID NO:23)

[0242] >huFE5huv1 QVQLVESGGG S VQ A GGSLRLSCAASGYTYSFNSMGWFRQAPGK ER EGV A VIYLGSTHYADSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCAADLLEYDGTWFGSTYNYWGQGTLVTVSS (SEQ ID NO:300) >huFE5huv2 QVQLVESGGG L VQ P GGSLRLSCAASGYTYSFNSMGWFRQAPGK GL EGV S VIYLGSTHYADSVKGRFTIS R DNSKNTL YLQMNSL RA EDTAVYYCAADLLEYDGTWFGSTYNYWGQGTLVTVSS (SEQ ID NO:301) >huFE5huv3 QVQLVESGGG L VQ P GGSLRLSCAASGYTYSFNSMGWFRQAPGK ON EGV A VIYLGSTHYADSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCAADLLEYDGTWFGSTYNYWGQGTLVTVSS (SEQ ID NO:302) >huFE5huv4 QVQLVESGGG S VQ A GGSLRLSCAASGYTYSFNSMGWFRQAPGK GL EGV S VIYLGSTHYADSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCAADLLEYDGTWFGSTYNYWGQGTLVTVSS (SEQ ID NO:303) >huFE5huv5 QVQLVESGGG S VQ A GGSLRLSCAASGYTYSFNSMGWFRQAPGK GL EGV S VIYLGSTHYADSVKGRFTIS Q DNSKNT L YLQMNSL KP EDTAVYYCAADLLEYDGTWFGSTYNYWGQGTLVTVSS (SEQ ID NO:304) >huFE5huv6 QVQLVESGGG L VQ P GGSLRLSCAASGYTYSFNSMGWFRQAPGK GL EGV SVIYLGSTHYADSVKGRFTIS Q DNSKNT L YLQMNSL KP EDTAVYYCAADLLEYDGTWFGSTYNYWGQGTLVTVSS (SEQ ID NO:305) >huFE13huv1 QVQLVESGGG S VQ A GGSLRLSCAASGYTYSMGAMAWFRQAPGK ON EGV A LIATSSGFISYTDSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCA A GTQFSWSPGSYNYWGQGTLVTVSS (SEQ ID NO:306) >huFE13huv2 QVQLVESGGG L VQ P GGSLRLSCAASGYTYSMGAMAWFRQAPGK GL EGV S LIATSSGFISYTDSVKGRFTIS R DNSKNT L YLQMNSL RA EDTAVYYCA A GTQFSWSPGSYNYWGQGTLVTVSS (SEQ ID NO:307) >huFE13huv3 QVQLVESGGG L VQ P GGSLRLSCAASGYTYSMGAMAWFRQAPGK ON EGV A LIATSSGFISYTDSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCA A GTQFSWSPGSYNYWGQGTLVTVSS (SEQ ID NO:308) >huFE13huv4 QVQLVESGGG S VQA GGSLRLSCAASGYTYSMGAMAWFRQAPGK GL EGV S LIATSSGFISYTDSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCA A GTQFSWSPGSYNYWGQGTLVTVSS (SEQ ID NO:309) >huFE13huv5 QVQLVESGGG S VQ A GGSLRLSCAASGYTYSMGAMAWFRQAPGK GL EGV S LIATSSGFISYTDSVKGRFTIS Q DNSKNT L YLQMNSL KP EDTAVYYCA A GTQFSWSPGSYNYWGQGTLVTVSS (SEQ ID NO:310) >huFE13huv6 QVQLVESGGG L VQ P GGSLRLSCAASGYTYSMGAMAWFRQAPGK GL EGV S LIATSSGFISYTDSVKGRFTIS Q DNSKNT L YLQMNSL KP EDTAVYYCA A GTQFSWSPGSYNYWGQGTLVTVSS (SEQ ID NO:311) >huFE35huv1 QVQLVESGGG S VQ A GGSLRLSC T ASGFTFDDSDMGWYRQAPGN EC ELV S TISSDGRGFYADSVKGRFTIS Q DNSKNT V YLQMNSL RAEDTAVYYCAAGWYGSFCSGLGQGTLVTVSS (SEQ ID NO:312) >huFE35huv2 QVQLVESGGG L VQ P GGSLRLSC A ASGFTFDDSDMGWYRQAPGN EC ELV S TISSDGRGFYADSVKGRFTIS R DNSKNT L YLQMNSL RA EDTAVYYCAAGWYGSFCSGLGQGTLVTVSS (SEQ ID NO:313) >huFE35huv3 QVQLVESGGG L VQ P GGSLRLSC T ASGFTFDDSDMGWYRQAPGN EC ELV S TISSDGRGFYADSVKGRFTIS R DNSKNT V YLQMNSL RA EDTAVYYCAAGWYGSFCSGLGQGTLVTVSS (SEQ ID NO:314) >huFE35huv4 QVQLVESGGG S VQ A GGSLRLSC T ASGFTFDDSDMGWYRQAPGN EC ELV S TISSDGRGFYADSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCAAGWYGSFCSGLGQGTLVTVSS (SEQ ID NO:315) >huFE35huv5 QVQLVESGGG S VQ A GGSLRLSC T ASGFTFDDSDMGWYRQAPGN EC ELV STISSDGRGFYADSVKGRFTIS Q DNSKNT L YLQMNSL KP EDTAVYYCAAGWYGSFCSGLGQGTLVTVSS (SEQ ID NO:316) >huFE35huv6 QVQLVESGGG L VQ P GGSLRLSC T ASGFTFDDSDMGWYRQAPGN EC ELV S TISSDGRGFYADSVKGRFTIS R DNSKNT L YLQMNSL KP EDTAVYYCAAGWYGSFCSGLGQGTLVTVSS (SEQ ID NO:317) >huFE56huv1 QVQLVESGGG S VQ A GGSLRLSCAASGHTYSSNYCMAWFRQAPGK ON EGV A AIYSDGSTSYADSVKGRFTIS K DNSKNTLYLQ MN SL RA EDTAVYYCAATAYEGSWTGKQPLCLLYEYTYWGQGTLVTVSS (SEQ ID NO:318) >huFE56huv2 QVQLVESGGG L VQ P GGSLRLSCAASGHTYSSNYCMAWFRQAPGK ON EGV A AIYSDGSTSYADSVKGRFTIS R DNSKNTLYLQ MN SL RA EDTAVYYCAATAYEGSWTGKQPLCLLYEYTYWGQGTLVTVSS (SEQ ID NO:319) >huFE56huv3 QVQLVESGGG L VQ PGGSLRLSCAASGHTYSSNYCMAWFRQAPGK ON EGV A AIYSDGSTSYADSVKGRFTIS K DNSKNTLYLQ MN SL RA EDTAVYYCAATAYEGSWTGKQPLCLLYEYTYWGQGTLVTVSS (SEQ ID NO:320) >huFE56huv4 QVQLVESGGG S VQ A GGSLRLSCAASGHTYSSNYCMAWFRQAPGK GL EGV S AIYSDGSTSYADSVKGRFTIS K DNSKNTLYLQ MN SL RA EDTAVYYCAATAYEGSWTGKQPLCLLYEYTYWGQGTLVTVSS (SEQ ID NO:321) >huFE56huv5 QVQLVESGGG S VQ A GGSLRLSCAASGHTYSSNYCMAWFRQAPGK GL EGV S AIYSDGSTSYADSVKGRFTIS K DNSKNTLYLQ MN SL KP EDTAVYYCAATAYEGSWTGKQPLCLLYEYTYWGQGTLVTVSS (SEQ ID NO:322) >huFE56huv6 QVQLVESGGG L VQ P GGSLRLSCAASGHTYSSNYCMAWFRQAPGK GL EGV S AIYSDGSTSYADSVKGRFTIS K DNSKNTLYLQ MN SL KP EDTAVYYCAATAYEGSWTGKQPLCLLYEYTYWGQGTLVTVSS (SEQ ID NO:323) >huFE97huv1 QVQLVESGGG S VQ A GGSLRLSC T ASEFTFDDSDMAWYRQAPGN EC ELV S TITSDGGTYYADSVKGRFTIS Q DNSKNT M YLQMNSL RA EDTAVYYC A ADQWGSAEGDCTSSYPGGYWGQGTLVTVSS (SEQ ID NO:324) >huFE97huv2 QVQLVESGGG L VQ P GGSLRLSC A ASEFTFDDSDMAWYRQAPGN EC ELV S TITSDGGTYYADSVKGRFTIS R DNSKNT L YLQMNSL RA EDTAVYYC A ADQWGSAEGDCTSSYPGGYWGQGTLVTVSS (SEQ ID NO:325) >huFE97huv3 QVQLVESGGG L VQ P GGSLRLSC T ASEFTFDDSDMAWYRQAPGN EC ELV S TITSDGGTYYADSVKGRFTIS Q DNSKNT M YLQMNSL RA EDTAVYYC A ADQWGSAEGDCTSSYPGGYWGQGTLVTVSS (SEQ ID NO:326) >huFE97huv4 QVQLVESGGG S VQ A GGSLRLSC T ASEFTFDDSDMAWYRQAPGN EC ELV STITSDGGTYYADSVKGRFTIS R DNSKNT M YLQMNSL RA EDTAVYYC A ADQWGSAEGDCTSSYPGGYWGQGTLVTVSS (SEQ ID NO:327) >huFE97huv5 QVQLVESGGG S VQ A GGSLRLSC T ASEFTFDDSDMAWYRQAPGN EC ELV S TITSDGGTYYADSVKGRFTIS R DNSKNT L YLQMNSL KP EDTAVYYC A ADQWGSAEGDCTSSYPGGYWGQGTLVTVSS (SEQ ID NO:328) >huFE97huv6 QVQLVESGGG L VQ P GGSLRLSC T ASEFTFDDSDMAWYRQAPGN EC ELV S TITSDGGTYYADSVKGRFTIS R DNSKNT L YLQMNSL KP EDTAVYYC A ADQWGSAEGDCTSSYPGGYWGQGTLVTVSS (SEQ ID NO:329) >huFE148huv1 QVQLVESGGG S VQ A GGSLRLSC A ASGYTFNNNFVGWFRQAPGK ON EGV A AIYSLGGSTYYADSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCA G AITWVPPLSHRRYTYWGQGTLVTVSS (SEQ ID NO:330) >huFE148huv2 QVQLVESGGG L VQ P GGSLRLSC A ASGYTFNNNFVGWFRQAPGK GL EGV S AIYSLGGSTYYADSVKGRFTIS R DNSKNT L YLQMNSL RA EDTAVYYCA A AITWVPPLSHRRYTYWGQGTLVTVSS (SEQ ID NO:331) >huFE148huv3 QVQLVESGGG L VQ P GGSLRLSC A ASGYTFNNNFVGWFRQAPGK ON EGV A AIYSLGGSTYYADSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCA G AITWVPPLSHRRYTYWGQGTLVTVSS (SEQ ID NO:332) >huFE148huv4 QVQLVESGGG S VQ A GGSLRLSC A ASGYTFNNNFVGWFRQAPGK GL EGV S AIYSLGGSTYYADSVKGRFTIS Q DNSKNT V YLQMNSL RA EDTAVYYCA G AITWVPPLSHRRYTYWGQGTLVTVSS (SEQ ID NO:333) >huFE148huv5 QVQLVESGGG S VQ A GGSLRLSC A ASGYTFNNNFVGWFRQAPGK GL EGV SAIYSLGGSTYYADSVKGRFTIS Q DNSKNT L YLQMNSL KP EDTAVYYCA G AITWVPPLSHRRYTYWGQGTLVTVSS (SEQ ID NO:334) >huFE148huv4 QVQLVESGGG S VQ A GGSLRLSC A ASGYTFNNNFVGWFRQAPGK GL EGV S AIYSLGGSTYYADSVKGRFTIS R DNSKNT L YLQMNSL KP EDTAVYYCA G AITWVPPLSHRRYTYWGQGTLVTVSS (SEQ ID NO:335) >IgG1-FC EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:336) >Apple1 ECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISA(SEQ ID NO:337) >Apple2 HQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLA(SEQ ID NO:338) >Apple3 LSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVF(SEQ ID NO:339) >Apple4 IPVFCHSSFYHDTDFLGEELDIVAAKSHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDNESTTK(SEQ ID NO:340) >Apple1-2 ECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLA(SEQ ID NO:341) >Apple2-3 HQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVF(SEQ ID NO:342) >Apple 3-4 HQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVF(SEQ ID NO:343)。

Claims

1. Use of a coagulation factor XI (FXI) binding protein in the preparation of a medicament for treating and / or preventing thromboembolism, inhibiting FXI activation, or inhibiting coagulation and related thrombus formation in subjects of need without impairing hemostasis, wherein the FXI binding protein comprises at least one immunoglobulin single variable domain capable of specifically binding to FXI, wherein the at least one immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 as CDR1, CDR2, and CDR3 of VHH shown in any of SEQ ID NO: 14, 4, and 10. The CDR mentioned therein is a Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR.

2. The use according to claim 1, wherein CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO:14 are selected from any one of the following groups: when the CDR is a Kabat CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:180-182, respectively; when the CDR is an AbM CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:183-185, respectively; when the CDR is a Chothia CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:186-188, respectively; when the CDR is an IMGTCDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:189-191, respectively.

3. The use according to claim 2, wherein the amino acid sequence of the at least one immunoglobulin single variable domain consists of the amino acid sequence shown in one of SEQ ID NO: 14, 318-323.

4. The use according to claim 1, wherein CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO:4 are selected from any one of the following groups: when the CDR is a Kabat CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:60-62, respectively; when the CDR is an AbM CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:63-65, respectively; when the CDR is a Chothia CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:66-68, respectively; when the CDR is an IMGT CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:69-71, respectively.

5. The use according to claim 4, wherein the amino acid sequence of the at least one immunoglobulin single variable domain consists of the amino acid sequence shown in any one of SEQ ID NO:4, 306-311.

6. The use according to claim 1, wherein CDR1, CDR2, and CDR3 in the VHH shown in SEQ ID NO:10 are selected from any one of the following groups: when the CDR is a Kabat CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:132-134, respectively; when the CDR is an AbM CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:135-137, respectively; when the CDR is a Chothia CDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:138-140, respectively; when the CDR is an IMGTCDR, the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:141-143, respectively.

7. The use according to claim 6, wherein the amino acid sequence of the at least one immunoglobulin single variable domain consists of the amino acid sequence shown in one of SEQ ID NO: 10, 312-317.

8. The use according to any one of claims 1-7, wherein the FXI-binding protein binds to the Apple2 domain of FXI.

9. The use according to any one of claims 1-7, wherein the FXI binding protein further comprises an immunoglobulin Fc region.

10. The use according to claim 9, wherein the immunoglobulin Fc region is the human immunoglobulin Fc region.

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