Solidification factor XI (FXI) binding tank

Abstract

The present invention relates to the field of biopharmaceuticals and discloses a single domain antibody against coagulation factor XI (FXI) and its derivative protein. Specifically, the present invention discloses a coagulation factor XI (FXI) binding protein derived from a single domain antibody against coagulation factor XI (FXI) and uses thereof.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals and discloses a single-domain antibody against coagulation factor XI (FXI) and its derivative proteins. 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 technology]

[0002] Coagulation factors are various protein components involved in the blood coagulation process. Their physiological role is to be activated when blood vessels bleed, binding with platelets to seal the leak. This process is called coagulation. Some of these factors are produced by the liver. They are inhibited by coumarin. The World Health Organization has numbered them with Roman numerals in the order of their discovery, such as coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, etc., to standardize the naming convention.

[0003] Coagulation factor XI (FXI) is a dimer composed of identical 80 kDa subunits, each subunit consisting of four Apple domains (A1, A2, A3, and A4) from the N-terminus and a catalytic domain. FXI is a zymogen that circulates in complex with high molecular weight kininogen (HK). HK binds to the A2 domain of FXI and is a physiological cofactor of FXIIa, activating FXI to FXIa. The other Apple domains of FXI mediate important physiological functions. For example, the FIX-binding ectocyte is located at A3, while the FXIIa-binding site is at A4. Residues important for FXI dimerization are also located at A4.

[0004] FXI plays a crucial role in the pathological process of thrombus formation, but studies have shown that it contributes little to blood loss, making it a promising target for thrombosis. In a Phase II trial of FXI antisense oligonucleotides (ASOs) by Ionis Pharmaceuticals Inc. (Buller et al., N Engl J Med 2015, 372:232-240), FXI ASOs tended to cause less bleeding than enoxaparin in patients undergoing total knee arthroplasty, resulting in a significant reduction in venous thromboembolism (VTE). 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) have shown that severe FXI deficiency (hemophilia C) reduces the risk of ischemic stroke and deep vein thrombosis, while conversely, increased FXI levels are associated with a higher risk of VTE and ischemic stroke. Furthermore, multiple preclinical studies have revealed that FXIa inhibition or loss of function mediates thrombosis prevention without impairing hemostasis (Chen et al., Drug Discov Today 2014). Notably, in a baboon AV shunt thrombus formation model, the monoclonal antibodies 14E11 and 1A6 resulted in significant thrombus reduction (U.S. Patent No. 8,388,959; Tucker et al., Blood 2009, 113:936-944; Cheng et al., Blood 2010, 116:3981-3989). Furthermore, 14E11 provided protection in a mouse acute ischemic stroke experimental model (through cross-reactivity with mouse FXI) (Leung et al., Transl Stroke Res 2012, 3:381-389).Other mAb studies using preclinical models targeting FXI have also been reported, confirming that FXI, an antithrombotic target, carries a very low risk of bleeding (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, 14 Nov, 2014). Therefore, FXI inhibition is a promising novel antithrombotic treatment with an improved benefit-risk ratio compared to conventional standard anticoagulants. [Brief explanation of the drawing]

[0005] [Figure 1] This shows the blocking activity (APTT detection) of the FXI single-domain antibody-Fc fusion protein against FXI. [Figure 2] This shows the blocking activity (APTT detection) of a humanized FXI single-domain antibody-Fc fusion protein against FXI. [Figure 3] This study demonstrates the inhibitory effect of a huFE bispecific antibody-Fc fusion protein on the activity of human FXI. [Figure 4] This study demonstrates the inhibitory activity of a huFE bispecific antibody-Fc fusion protein against APTT in human whole plasma. [Figure 5] This study demonstrates the inhibitory activity of a huFE bispecific antibody-Fc fusion protein against APTT in whole monkey plasma. [Figure 6] This study demonstrates the inhibitory activity of a huFE bispecific antibody-Fc fusion protein against APTT in rabbit whole plasma. [Figure 7] This study demonstrates the effects of FXI single-domain antibody-Fc fusion protein and bispecific antibodies on rabbit venous thrombosis. [Modes for carrying out the invention]

[0006] definition Unless otherwise specified or defined, all terms used have their ordinary meanings in the art as known to those skilled in the art. For example, see the manuals Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd Ed.), Vol 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York, (1990); Roitt et al., “Immunology” (2nd Ed.), Gower Medical Publishing, London, New York (1989), and the general prior art cited herein. Furthermore, unless otherwise specified, any methods, steps, techniques, and procedures not detailed herein can and have already been performed in known ways, and such ways are known to those skilled in the art. See also, for example, the manuals, the general prior art mentioned above, and other references cited therein.

[0007] Unless otherwise specified, the interchangeable terms “antibody” or “immunoglobulin” are used herein as general terms encompassing full-length antibodies, their single chains, and all parts, domains, or fragments thereof (including, but not limited to, antigen-binding domains or fragments, with VHH domains or VH / VL domains being examples, respectively), whether referring to heavy-chain antibodies or typical four-chain antibodies. Furthermore, the terms “sequence” as used herein (e.g., in terms such as “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence”) should generally be understood to include not only the relevant amino acid sequence but also the nucleic acid sequence or nucleotide sequence encoding the above sequence, unless further restrictive interpretation is required herein.

[0008] As used herein, the term “domain” (of a polypeptide or protein) refers to a folded protein structure that can maintain its tertiary structure independently of other parts of the protein. Generally, a domain is responsible for a single functional property of the protein and may be added, removed, or transferred to another protein without impairing the function of other parts of the protein and / or the domain itself.

[0009] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a typical four-chain antibody or a chain of a heavy-chain antibody), or a polypeptide substantially composed of such globular regions. The immunoglobulin domain is characterized by maintaining the immunoglobulin folding properties of the antibody molecule.

[0010] As used herein, the term "variable domain of immunoglobulin" refers substantially to an immunoglobulin domain composed of four "framework regions" referred to in this field and below as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively, the framework regions being separated by three "complementarity-determining regions" or "CDRs" referred to in this field and below 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 the variable domain of immunoglobulin may be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Because the variable domain of immunoglobulin has an antigen-binding site, it confers specificity to the antibody against the antigen.

[0011] As used herein, the term "immunoglobulin monovariable domain" refers to a variable domain of an immunoglobulin that can specifically bind to an antigen epitope without pairing with other immunoglobulin variable domains. An example of an immunoglobulin monovariable domain dealt with in this invention is a "domain antibody," such as the immunoglobulin monovariable domains VH and VL (VH domain and VL domain). Another example of an immunoglobulin monovariable domain is the "VHH domain" (or abbreviated as "VHH") of the camelid family, as defined below.

[0012] The "VHH domain," also known as a heavy chain single-domain antibody, VHH, VHH domain, VHH antibody fragment, or VHH antibody, is the variable domain of antigen-binding immunoglobulin in "heavy chain antibodies," which are antibodies 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 the above variable domain from the heavy chain variable domain (referred to herein as the "VH domain") and the light chain variable domain (referred to herein as the "VL domain") present in typical four-chain antibodies. The VHH domain specifically binds to an epitope without requiring other antigen-binding domains (this is the opposite of the VH or VL domains in typical four-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a stable, efficient, and compact antigen-recognition unit formed by a single immunoglobulin domain.

[0013] In the context of the present invention, the terms "heavy chain single-domain antibody," "VHH domain," "VHH," "VHH domain," "VHH antibody fragment," and "VHH antibody" are interchangeable.

[0014] For example, as shown in Figure 2 of Riechmann & Muyldermans, J. Immunol. Methods 231, 25 - 38 (1999), it can be numbered based on the general numbering method of the VH domain proposed by Kabat et al. for the amino acid residues used in the VHH domain of camelids (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0015] Alternative methods for numbering the amino acid residues of the VH domain are known in the art, and the above alternative methods are also applicable 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 an intermediate state 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 available complex crystal structures. The following are the descriptions of the CDR residues by each method.

[0016] [Table 1]

[0017] The CDR of an antibody may be an IMGT - CDR, which is a CDR definition method based on the IMGT antibody code. This code is obtained by analyzing the structural information of more than 5000 sequences. In the VH CDR code of IMGT, CDR1: 27 - 38, CDR2: 56 - 65, CDR3: 105 - 117.

[0018] It should also be noted that, as is known in this field 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 based on the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number). This generally means that the numbers based on Kabat may or may not correspond to the actual numbers of amino acid residues in the actual sequence.

[0019] For example, a CDR may include "extended CDRs," such as VL 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 (LCDR3), and VH 26-35 (HCDR1), 50-65 or 49-65 (HCDR2), and 93-102, 94-102 or 95-102 (HCDR3).

[0020] The total number of amino acid residues in the VHH domain is generally in the range of 110 to 120, and is often between 112 and 115. Note that both short and long sequences are suitable for the purposes described herein.

[0021] The VHH domain and other structural and functional properties of polypeptides containing it can be summarized as follows:

[0022] VHH domains (naturally "designed" to functionally bind to antigens without the presence of a light chain variable domain and without interaction with a light chain variable domain) can be utilized as a single, relatively small, functional antigen-binding structural unit, domain, or polypeptide. VHH domains are distinguished from conventional four-chain antibodies by their VH and VL domains, which themselves are generally not suitable for practical use as single antigen-binding proteins or immunoglobulin single variable domains, but need to be combined in specific or alternative 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 a VH domain covalently bonded to a VL domain).

[0023] These unique properties make the use of VHH domains, either alone or as part of relatively large polypeptides, a significant advantage over using conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., Fab- or F(ab')2- fragments). A single domain can provide high-affinity, high-selectivity binding antigens, eliminating the need for two separate domains or ensuring their proper spatial conformation and arrangement (e.g., scFv generally requires a specially designed linker). VHH domains can be expressed from a single gene without requiring post-translational folding or modification. Multivalent and multispecific forms can be obtained from VHH domains with simple operations (formats). VHH domains exhibit excellent solubility and a lack of aggregation tendency. VHH domains are highly stable to heat, pH, proteases, and other denaturing agents or conditions, eliminating the need for refrigeration during manufacturing, storage, or transport, resulting in cost and time savings and environmental protection. VHH domains are not only easy to manufacture but also inexpensive, offering similar advantages at production scale. Because the VHH domain is smaller than that of a typical quadruple antibody and its antigen-binding fragment (approximately 15 kDa or 1 / 10 the size of a typical IgG), it exhibits higher tissue permeability and allows for higher dose administration. The VHH domain has so-called cavity binding properties (particularly the CDR3 loop is extended compared to a typical VH domain), allowing it to reach targets and epitopes that cannot be reached by a typical quadruple antibody and its antigen-binding fragment.

[0024] Methods for obtaining VHH that bind to specific antigens or epitopes have already been disclosed in the following publications: 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 Jun, 2009, and WO94 / 04678.

[0025] VHH domains derived from camelids can be “humanized” by substituting one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues located at the corresponding positions in the VH domain of a normal human four-chain antibody (hereinafter also referred to as “sequence optimization,” which, in addition to humanization, may include other modifications made to the sequence by one or more mutations that provide improved characteristics of the VHH, such as the removal of potential post-translational modification sites). The humanized VHH domain may contain one or more fully human framework region sequences. Humanization can be performed, for example, by resurfacing amino acids on the protein surface and / or CDR grafting to a humanized universal framework, as shown in the examples.

[0026] As used herein, the term “epitope” or the interchangeable term “antigenic determinant” refers to any antigenic determinant of an antigen to which an antibody paratope binds. Antigenic determinants generally consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural features and specific charge properties. For example, an epitope generally consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids as a unique spatial conformation, and may be a “linear” epitope or a “conformational” epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). In a linear epitope, all interaction sites between the protein and the interacting molecule (e.g., antibody) are linearly located along the primary amino acid sequence of the protein. In conformational epitopes, interaction sites are located across amino acid residues of proteins that are separated from each other.

[0027] The epitopes of given antigens can be identified by many well-known epitope mapping techniques in this field. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996). For example, linear epitopes can be determined by simultaneously synthesizing a large number of peptides on a solid support, corresponding to different parts of a protein molecule, and reacting them with an antibody while still attached to the support. These techniques are known in this field and are described, for example, U.S. Patent No. 4,708,871; Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al., (1986) Molec. Immunol. 23:709-715. Similarly, conformational epitopes can be identified by determining the conformation of amino acids, for example, by X-ray crystallography or two-dimensional nuclear magnetic resonance. For example, see Epitope Mapping Protocols (ibid.).

[0028] Antibodies can be screened for competitiveness in binding to the same epitope by conventional techniques known to those skilled in the art. For example, by performing competitive and cross-competition tests, antibodies that compete with each other or cross-compete to bind to the antigen can be obtained. A high-throughput method for obtaining antibodies that bind to the same epitope through such cross-competition is described in international patent application WO03 / 48731. Accordingly, antibodies and their antigen-binding fragments that compete with the antibody molecule of the present invention to bind to the same epitope in FXI can be obtained by conventional techniques known to those skilled in the art.

[0029] Generally, the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein (e.g., the immunoglobulin monovariate domain according to the present invention) can bind. The specificity can be determined based on the affinity and / or avidity of the antigen-binding protein. Affinity, indicated by the dissociation equilibrium constant (KD) of the antigen and the antigen-binding protein, is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein. A smaller KD value indicates a stronger binding strength between the epitope and the antigen-binding protein (or affinity may be expressed as an association constant (KA) where 1 / KD). As is known to those skilled in the art, affinity may be measured by known methods depending on the antigen of interest. Avidity is a measure of the binding strength between an antigen-binding protein (e.g., immunoglobulin, antibody, immunoglobulin monovariate domain or polypeptide containing the same) and the associated antigen. Avidity is related to the affinity to the antigen-binding site on the antigen-binding protein and the number of associated binding sites present on the antigen-binding protein.

[0030] As used herein, the term “coagulation factor XI (FXI) binding protein” means a protein that can specifically bind to coagulation factor XI (FXI). An FXI binding protein may include an antibody against FXI as defined herein. An FXI binding protein may also include an immunoglobulin superfamily antibody (IgSF) or a CDR implantation molecule.

[0031] The "FXI-binding protein" according to the present invention is at least one immunoglobulin single variable domain that binds to FXI, and may include, for example, VHH. In some embodiments, the "FXI-binding molecule" according to the present invention is two, three, four or more immunoglobulin single variable domains that bind to FXI, and may include, for example, VHH. The FXI-binding protein according to the present invention may, in addition to the immunoglobulin single variable domain that binds to FXI, include a linker and / or a portion having a factor function (e.g., a half-life extension 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" according to the present invention also includes a bispecific antibody containing immunoglobulin single variable domains that bind to different antigens or different regions (e.g., different epitopes) of the same antigen.

[0032] Generally, the FXI-binding protein according to the present invention has a dissociation constant (KD) measured in a Biacore or KinExA or Fortibio assay, preferably 10 -7 ~10 -10 mol / L (M), more preferably 10 -8 ~10 -10 mol / L, even more preferably 10 -9 ~10 -10 or less, and / or an association constant (KA) of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , even more preferably at least 10 10 M -1 to bind to the target antigen (i.e., FXI). 10 -4Any KD value greater than M is generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be measured by any known suitable method, such as the surface plasmon resonance (SPR) assay, Scatchard assay, and / or competitive binding assays described herein (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay).

[0033] Amino acid residues can be represented by standard three-letter or one-letter amino acid codes that are well known and consistent in this art. When comparing two amino acid sequences, the term "amino acid difference" refers to the insertion, deletion, or substitution of a predetermined number of amino acid residues at specific positions in a reference sequence compared to another sequence. In the case of substitutions, the substitution is preferably a conservative amino acid substitution, where the amino acid residue is replaced by another amino acid residue that has a chemically similar structure and has little or no effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in this art, and for example, a conservative amino acid substitution is preferably one in the following groups (i) to (v) replaced by another amino acid residue from the same group. (i) Small 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) Large aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (v) Aromatic residues: Phe, Tyr and Trp. Particularly preferred conservation amino acid substitutions are: substitution of Ala with Gly or Ser, substitution of Arg with Lys, substitution of Asn with Gln or His, substitution of Asp with Glu, substitution of Cys with Ser, substitution of Gln with Asn, substitution of Glu with Asp, substitution of Gly with Ala or Pro, substitution of His with Asn or Gln, substitution of Ile with Leu or Val, substitution of Leu with Ile or Val, substitution of Lys with Arg, Gln or Glu, substitution of Met with Leu, Tyr or Ile, substitution of Phe with Met, Leu or Tyr, substitution of Ser with Thr, substitution of Thr with Ser, substitution of Trp with Tyr, substitution of Tyr with Trp or Phe, and substitution of Val with Ile or Leu.

[0034] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are 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 generally measured using sequence analysis software. For instance, identity can be determined using the BLAST program in the NCBI database. For information on 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; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0035] A polypeptide or nucleic acid molecule is considered "separated" when it is separated from at least one other component generally associated with the source or medium (e.g., another protein / polypeptide, another nucleic acid, another biological component or macromolecule, or at least one contaminant, impurity or trace component) in relation to the reaction medium or culture medium used to obtain the polypeptide or nucleic acid molecule of its natural origin. In particular, a polypeptide or nucleic acid molecule is considered "separated" when it has been purified at least twice, especially at least ten times, and further at least 100 times and 1000 times or more. The "separated" polypeptide or nucleic acid molecule, as determined by appropriate techniques (e.g., appropriate chromatography techniques, e.g., polyacrylamide gel electrophoresis), is preferably substantially homogeneous.

[0036] "Effective amount" means the amount of FXI-binding protein or pharmaceutical composition according to the present invention that leads to a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of pain-related injury or disability due to the disease.

[0037] As used herein, “thrombus formation” refers to the obstruction of blood flow in the circulatory system by a blood clot (also called a “thrombus”) formed or present within a blood vessel. Thrombobus formation is generally caused by abnormalities in blood components, the condition of the blood vessel wall, and / or blood flow characteristics. Clot formation is generally caused by damage to the blood vessel wall (e.g., damage to the blood vessel wall due to trauma or infection) or by a delay or stagnation of blood flow at the site of the damage. In some cases, coagulation abnormalities cause thrombus formation.

[0038] As used herein, “uninterrupted hemostasis” means that after administration of the FXI-binding protein or pharmaceutical composition according to the present invention to the subject, very little bleeding is observed or no detectable bleeding is observed. When targeting FXI, inhibiting the conversion of FXI to FXIa or inhibiting the activation of FXIIIa to FIX inhibits coagulation and associated thrombus formation without bleeding.

[0039] As used herein, the term "subject" means mammals, in particular primates, and especially humans.

[0040] FXI-binding protein according to the present invention In one embodiment of the present invention, an FXI-binding protein is provided that includes at least one immunoglobulin monovariable domain capable of specifically binding to FXI.

[0041] In some embodiments, the above-mentioned at least one immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in any of SEQ ID NOs: 1 to 23. The above-mentioned CDR may be Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR.

[0042] In some embodiments, the above-mentioned at least one immunoglobulin monovariable domain comprises a set of CDR1, CDR2, and CDR3 selected from the following:

[0043] [Table 2]

[0044] In some embodiments, at least one immunoglobulin monovariate domain of the FXI-binding protein according to the present invention is VHH. In some embodiments, the VHH comprises any of the amino acid sequences of SEQ ID NOs: 1 to 23.

[0045] In some embodiments, at least one immunoglobulin monovariate domain of the FXI-binding protein according to the present invention is humanized VHH.

[0046] In some embodiments, at least one immunoglobulin monovariate domain of the FXI-binding protein according to the present invention is a humanized VHH, the 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 of SEQ ID NOs: 1 to 23. In some embodiments, the amino acid sequence of the humanized VHH comprises one or more amino acid substitutions, preferably conserved amino acid substitutions, compared to any of the sequences of SEQ ID NOs: 1 to 23. For example, it comprises one, two, three, four, five, six, seven, eight, nine, or ten conserved amino acid substitutions.

[0047] In some embodiments, at least one immunoglobulin monovariate domain of the FXI-binding protein according to the present invention is a humanized VHH, the humanized VHH comprising any of the amino acid sequences of SEQ ID NOs. 300 to 335.

[0048] In some embodiments, the above-mentioned immunoglobulin monovariable domain binds to an epitope in 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 above-mentioned immunoglobulin monovariable domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 14. In some embodiments, the above-mentioned immunoglobulin monovariable domain includes a set of CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 60-62, SEQ ID NOs: 63-65, SEQ ID NOs: 66-68, SEQ ID NOs: 69-71, SEQ ID NOs: 132-134, SEQ ID NOs: 135-137, SEQ ID NOs: 138-140, SEQ ID NOs: 141-143, SEQ ID NOs: 180-182, SEQ ID NOs: 183-185, SEQ ID NOs: 186-188, and SEQ ID NOs: 189-191. In some specific embodiments, the immunoglobulin monovariable domain comprises the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 14. In some specific embodiments, the immunoglobulin monovariable domain comprises the amino acid sequence shown in SEQ ID NOs: 306-323.

[0049] In some embodiments, the at least one immunoglobulin monovariable domain does not bind to an epitope in the Apple2 domain of FXI. In some embodiments, the at least one immunoglobulin monovariable domain does not bind to the Apple2 domain of FXI. In some embodiments, the at least one immunoglobulin monovariable domain does not bind to the isolated Apple2 domain polypeptide of FXI.

[0050] In some embodiments, the above-mentioned single variable immunoglobulin domain binds to an epitope in 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 above-mentioned single variable immunoglobulin domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17. In some embodiments, the above-mentioned single variable immunoglobulin domain includes a set of CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 216-218, SEQ ID NOs: 219-221, SEQ ID NOs: 222-224, and SEQ ID NOs: 225-227. In some specific embodiments, the above-mentioned single variable immunoglobulin domain includes the amino acid sequence shown in SEQ ID NO: 17. In some specific embodiments, the above-mentioned single variable immunoglobulin domain includes the amino acid sequence shown in any of SEQ ID NOs: 324-329.

[0051] In some embodiments, the above-mentioned immunoglobulin monovariate domain binds to an epitope in 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 above-mentioned immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1. In some embodiments, the above-mentioned immunoglobulin monovariate domain includes a set of CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 24-26, SEQ ID NOs: 27-29, SEQ ID NOs: 30-32, and SEQ ID NOs: 33-35. In some specific embodiments, the above-mentioned immunoglobulin monovariate domain includes the amino acid sequence shown in SEQ ID NO: 1. In some specific embodiments, the above-mentioned immunoglobulin monovariate domain includes the amino acid sequence shown in any of SEQ ID NOs: 300-305.

[0052] In some embodiments, the above-mentioned at least one immunoglobulin monovariate domain binds to an epitope in the Apple1-2 region of FXI (the region between the Apple1 domain and the Apple2 domain). An exemplary amino acid sequence of the Apple1-2 region of FXI is shown in SEQ ID NO: 341.

[0053] In some embodiments, the above-mentioned single immunoglobulin variable domain binds to an epitope in 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 above-mentioned single immunoglobulin variable domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20. In some embodiments, the above-mentioned single immunoglobulin variable domain includes a set of CDR1, CDR2, and CDR3 selected from SEQ ID NOs: 252-254, SEQ ID NOs: 255-257, SEQ ID NOs: 258-260, and SEQ ID NOs: 261-263. In some specific embodiments, the above-mentioned single immunoglobulin variable domain includes the amino acid sequence shown in SEQ ID NO: 20. In some specific embodiments, the above-mentioned single immunoglobulin variable domain includes the amino acid sequence shown in any of SEQ ID NOs: 330-335.

[0054] In some embodiments, the above-mentioned at least one immunoglobulin monovariate domain binds to an epitope in 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.

[0055] In some embodiments, the FXI-binding protein comprises a single immunoglobulin variable domain that specifically binds to FXI.

[0056] In some embodiments, the FXI-binding protein comprises at least two, for example, two, three, four or more immunoglobulin monovariable domains that specifically bind to FXI.

[0057] In some embodiments, the at least two immunoglobulin monovariable domains bind to the same region or epitope of FXI, or bind competitively or partially competitively to the same region or epitope of FXI, for example, the at least two immunoglobulin monovariable domains are the same.

[0058] In some embodiments, the at least two immunoglobulin monovariable domains bind to different regions or epitopes of FXI, or bind to the same region or epitope of FXI without competition.

[0059] Whether two antibody or immunoglobulin monovariate domains bind to the same region or epitope, or compete to bind to it, can be determined by epitope binning using biofilm interferometry (BLI), as shown, for example, in the examples of this application.

[0060] In some embodiments, the at least two immunoglobulin monovariable domains that specifically bind to FXI are directly interconnected.

[0061] In some embodiments, the at least two immunoglobulin monovariable domains that specifically bind to FXI are interconnected via linkers. The linkers may be non-functional amino acid sequences with a length of 1 to 20 or more amino acids and no secondary or higher structures. For example, the linkers are flexible linkers, such as GGGGS, GS, GAP, (GGGGS)×3, etc.

[0062] In some embodiments, the FXI-binding protein comprises a first immunoglobulin monovariable domain that can specifically bind to FXI and a second immunoglobulin monovariable domain that can specifically bind to FXI, wherein the first immunoglobulin monovariable domain and the second immunoglobulin monovariable domain bind to different epitopes of FXI.

[0063] In some embodiments, the first immunoglobulin monovariable domain binds to an epitope in the Apple2 domain of FXI, and the second immunoglobulin monovariable domain binds to an epitope in the Apple3 domain of FXI, or The first immunoglobulin monovariate domain binds to an epitope in the Apple2 domain of FXI, and the second immunoglobulin monovariate domain binds to an epitope in the Apple4 domain of FXI, or The first immunoglobulin monovariate domain binds to an epitope in the Apple2 domain of FXI, and the second immunoglobulin monovariate domain binds to an epitope in the Apple2-3 region of FXI, or The first immunoglobulin monovariate domain binds to an epitope in the Apple3 domain of FXI, and the second immunoglobulin monovariate domain binds to an epitope in the Apple4 domain of FXI, or The first immunoglobulin monovariate domain binds to an epitope in the Apple3 domain of FXI, and the second immunoglobulin monovariate domain binds to an epitope in the Apple2-3 region of FXI, or The first immunoglobulin monovariate domain binds to an epitope in the Apple4 domain of FXI, and the second immunoglobulin monovariate domain binds to an epitope in the Apple2-3 region of FXI.

[0064] In some embodiments, the FXI-binding protein comprises a first immunoglobulin monovariable domain and a second immunoglobulin monovariable domain, of which, The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, and the second immunoglobulin monovariate domain includes CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20.

[0065] In some embodiments, the CDR1, CDR2, and CDR3 of the VHH shown in SEQ ID NOs: 1, 4, 9, 10, 14, 17, or 20 are as shown in the table below.

[0066] [Table 3]

[0067] In some embodiments, the FXI-binding protein comprises a first immunoglobulin monovariable domain and a second immunoglobulin monovariable domain, of which, The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 4, 306-311, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 10, 312-317, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 14, 318-323, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 4, 306-311, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 4, 306-311, and the second immunoglobulin monovariate domain contains the amino acid sequence in VHH shown in SEQ ID NO: 10, 312-317, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 4, 306-311, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 14, 318-323, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 4, 306-311, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs. 4, 306-311, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs. 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 10, 312-317, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any of SEQ ID NOs: 14, 318-323, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any of SEQ ID NOs: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any of SEQ ID NOs: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 10, 312-317, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 14, 318-323, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 10, 312-317, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs: 10, 312-317, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs: 14, 318-323, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any of SEQ ID NOs: 14, 318-323, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any of SEQ ID NOs: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs: 17, 324-329, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NOs: 20, 330-335.

[0068] In some embodiments, the first immunoglobulin monovariable domain is located at the N-terminus of the second immunoglobulin monovariable domain. In other embodiments, the second immunoglobulin monovariable domain is located at the N-terminus of the first immunoglobulin monovariable domain.

[0069] In some embodiments, the FXI-binding protein according to the present invention further comprises an immunoglobulin Fc region in addition to at least one immunoglobulin monovariable domain that can specifically bind to FXI. By including an immunoglobulin Fc region in the FXI-binding protein according to the present invention, the binding molecule can form a dimer. The Fc region available according to the present invention may be derived from different subtypes of immunoglobulins, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM.

[0070] In some embodiments, the wild-type Fc sequence may be modified to alter the associated activity mediated by Fc. These modifications include, but are not limited to, a) mutations that alter Fc-mediated CDC activity, b) mutations that alter Fc-mediated ADCC activity, or c) mutations that alter FcRn-mediated in vivo half-life. Such mutations are described in the following references: 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, by mutating one, two, three, four, five, six, seven, eight, nine, or ten amino acids in the CH2 region, it is possible to improve or eliminate Fc-mediated ADCC or CDC activity, or to enhance or eliminate FcRn affinity. Furthermore, by mutating one, two, three, four, five, six, seven, eight, nine, or ten amino acids in the hinge region, it is possible to improve protein stability.

[0071] In some embodiments, introducing mutations into the Fc sequence makes mutant Fc more likely to form homodimers or heterodimers. As described in Ridgway, Presta et al. 1996 and Carter 2001, the knob-hole model, which utilizes the steric interactions of amino acid side chain groups at the Fc contact interface, facilitates heterodimer formation between different Fc mutations. Alternatively, as described in CN 102558355A or CN 103388013A, altering the charge of amino acids at the Fc contact interface, and further altering the ionic interaction force between the Fc contact interfaces, facilitates heterodimer formation between different Fc mutation pairs (CN 102558355A) or homodimer formation between Fc with the same mutation (CN 103388013A).

[0072] The Fc region of the above-mentioned immunoglobulin is preferably the Fc region of a human immunoglobulin, for example, the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some specific embodiments, the amino acid sequence of the Fc region of the above-mentioned immunoglobulin is as shown in SEQ ID NO: 336.

[0073] In some specific embodiments, the FXI-binding protein according to the present invention connects the Fc region of the immunoglobulin (e.g., the Fc region of human IgG1) directly or indirectly via a linker (e.g., a peptide linker) to the C-terminus of the immunoglobulin single variable domain (e.g., VHH).

[0074] In some embodiments, the FXI-binding protein according to the present invention comprises one immunoglobulin monovariate domain that specifically binds to FXI, either directly or via a linker to the Fc region of an immunoglobulin, the Fc region of the immunoglobulin enabling the FXI-binding protein to form a dimer molecule containing two FXI-binding domains. Such an FXI-binding protein is also called a divalent FXI-binding protein. In some embodiments, the dimer is a homodimer.

[0075] In some embodiments, the FXI-binding protein according to the present invention comprises two immunoglobulin monovariable domains that specifically bind to FXI and an immunoglobulin Fc region, interconnected directly or via a linker, wherein the immunoglobulin Fc region allows the FXI-binding protein to form a dimer molecule containing four FXI-binding domains. Such an FXI-binding protein is also called a tetravalent FXI-binding protein. In some embodiments, the dimer is a homodimer. In some embodiments, the two immunoglobulin monovariable domains of the FXI-binding protein that specifically bind to FXI each bind to a different region or different epitope of FXI.

[0076] In some embodiments, the FXI-binding protein according to the present invention can inhibit the activity of FXI. In some embodiments, the FXI-binding protein according to the present invention can inhibit the coagulation function of FXI.

[0077] nucleic acids, vectors, host cells Another aspect of the present invention relates to a nucleic acid molecule encoding an FXI-binding protein according to the present invention. The nucleic acid according to the present invention may be RNA, DNA, or cDNA. According to one embodiment of the present invention, the nucleic acid according to the present invention is a substantially isolated nucleic acid.

[0078] The nucleic acid according to the present invention may be in the form of a vector, or it may be located inside and / or part of a vector, such vector being, for example, a plasmid, cosmid, or YAC. In particular, the vector may be an expression vector, i.e., a vector capable of achieving the expression of an 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 generally includes at least one nucleic acid according to the present invention, operably attached to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). The selection of the above elements and their sequences for expression in a particular host is common sense to those skilled in the art. Specific examples of regulatory elements and other elements useful or essential for the expression of an FXI-binding protein according to the present invention include, for example, promoters, enhancers, terminators, integrated host factors, selection markers, reader sequences, and reporter genes.

[0079] The nucleic acids according to the present invention can be manufactured or obtained by known methods (e.g., automated DNA synthesis and / or recombinant DNA technology) based on the amino acid sequence information of the polypeptide according to the present invention as described herein, and / or isolated from suitable natural sources.

[0080] Another aspect of the present invention relates to recombinant host cells that express or are capable of expressing one or more FXI-binding proteins according to the present invention and / or contain nucleic acids or vectors according to the present invention. The host cells according to the present invention are preferably bacterial cells, fungal cells or mammalian cells.

[0081] Suitable bacterial cells include Gram-negative bacteria (e.g., Escherichia coli strains, Proteus strains, Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, Lactococcus strains).

[0082] Suitable fungal cells include cells of the species Trichoderma, Neurospora, and Aspergillus, or cells of the species Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris, Pichia methanolica), and Hansenula.

[0083] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, and COS cells.

[0084] Furthermore, in this invention, amphibian cells, insect cells, plant cells, and any other cells in the field used to express heterologous proteins may also be used.

[0085] The present invention further provides a method for producing FXI-binding protein according to the present invention, the above method generally, The steps include culturing host cells according to the present invention under conditions that enable the expression of the FXI-binding protein according to the present invention, A step of recovering the FXI-binding protein expressed by the host cells from the culture, Optionally, the process further includes the step of purifying and / or modifying the FXI-binding protein according to the present invention.

[0086] The FXI-binding protein according to the present invention may be produced in the above-mentioned cells by an intracellular method (for example, in the cytoplasm, periplasm, or within an inclusion body), then separated from the host cell and optionally further purified, or it may be produced by an extracellular method (for example, in the culture medium of the host cell), then separated from the medium and optionally further purified.

[0087] Methods and reagents for recombinant polypeptide production, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are already known in the art. Similarly, techniques for separating and purifying proteins suitable for the method for producing FXI-binding proteins according to the present invention are well known to those skilled in the art.

[0088] Furthermore, the FXI-binding protein according to the present invention can be obtained by other protein production methods known in the art, such as chemical synthesis including solid-phase or liquid-phase synthesis.

[0089] Pharmaceutical composition In another embodiment of the present invention, a composition is provided which comprises, for example, one or a combination of the FXI-binding proteins of the present invention formulated with a pharmaceutically acceptable carrier. Such a composition may comprise one or a combination (e.g., two or more different) of the FXI-binding proteins of the present invention. For example, the pharmaceutical composition of the present invention may comprise a combination of antibody molecules that bind to different epitopes on a target antigen (FXI).

[0090] As used herein, “pharmaceutically acceptable carriers” include any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, absorption retarders, etc. Preferably, the carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or epidermal administration (e.g., via injection or infusion). Depending on the route of administration, the active compound, i.e., antibody molecule, can be protected from the action of acids or other natural conditions that would inactivate the compound by encapsulating it in a specific material.

[0091] The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium pyrosulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbic acid palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0092] The above composition may further contain preservatives, humectants, emulsifiers, dispersants, and the like.

[0093] The presence of microorganisms can be prevented by a sterilization process or by including various antimicrobial and antifungal agents such as parahydroxybenzoic acid esters, chlorobutanol, and phenolsorbic acid. In many cases, the composition preferably contains, for example, sugar, polyol (e.g., mannitol, sorbitol), or sodium oxide as an isotonic agent. By adding, for example, monostearate salts or gelatin to the composition as absorption retarders, the absorption of injectable drugs can be extended.

[0094] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, sterile injection solutions, or powders for the immediate preparation of dispersions. The use of media and reagents for pharmaceutically active substances is well known in the art. Any conventional media or reagent may be used in the pharmaceutical composition according to the present invention, provided that it is compatible with the active compound. The active compound may be supplemented to the composition.

[0095] Therapeutic compositions are generally sterile and must be stable under their manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other regular configurations suitable for high drug concentrations. The carrier may be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, in the case of dispersions, a coating (e.g., lecithin) can be used to maintain a predetermined particle size, and a surfactant can be used to maintain appropriate fluidity.

[0096] A sterile injection solution can be prepared by mixing the required amount of the active compound with a suitable solvent, adding one or a combination thereof of the components listed above as needed, and then performing sterile microfiltration. Generally, a dispersant is prepared by mixing the active compound with a sterile carrier containing a basic dispersion medium and other necessary components listed above. For the preparation of sterile powder for sterile injection solutions, vacuum drying and freeze-drying are preferred, where the active component is obtained from a solution that has been previously sterile filtered, and powders of any necessary components are added.

[0097] The amount of active ingredient used to produce a unit dose form in combination with a carrier material varies depending on the target of treatment and the prescribed administration method. Generally, the amount of active ingredient used to produce a unit dose form in combination with a carrier material is the amount of composition that yields a therapeutic effect. Generally, on a 100% basis, this range is approximately 0.01% to 99% of the active ingredient, for example, approximately 0.1% to 70%, or approximately 1% to 30%, when combined with a pharmaceutically acceptable carrier.

[0098] The dose plan can be adjusted to provide the best desired response (e.g., therapeutic response). For example, it may be administered as a single bolus, in several divided doses over time, or proportionally reduced or increased depending on the urgency of treatment. Particularly useful is preparing parenteral compositions in a unit dose form that is easy to administer and has a uniform dose. A unit dose form, as used herein, refers to a physically non-contiguous unit suitable for use in a therapeutic target as a unit dose, containing a predetermined amount of the active compound per unit, and calculated to produce a predetermined therapeutic effect when the predetermined amount of the active compound is combined with the necessary pharmaceutical carrier. The specific description of the unit dose form according to the present invention is limited by and directly depends on (a) the specific properties of the active compound and the desired specific therapeutic effect, and (b) the limitations inherent in the art regarding the preparation of susceptible active compounds for such therapeutic individuals.

[0099] Regarding the administration of antibody molecules, the dose range is approximately 0.0001 to 100 mg / kg, with a more common range being 0.01 to 30 mg / kg of the patient's body weight. For example, the dose 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 within the range of 1 to 30 mg / kg. Exemplary treatment plans include administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months, or with shorter initial dosing intervals (e.g., from once a week to once every three weeks) followed by longer dosing intervals later on (e.g., from once a month to once every three to six months).

[0100] Alternatively, antibody molecules may be administered as a sustained-release formulation, in which case infrequent administration is required. The dosage and frequency vary depending on the half-life of the antibody molecule in the patient. Generally, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency vary depending on whether the treatment is for prevention or treatment. For preventive use, low doses are administered at infrequent intervals over a long period. Some patients receive continuous treatment until death. For therapeutic use, high doses at short intervals may be required and continued until the progression of the disease is reduced or stopped, preferably until the patient's symptoms of the disease are partially or completely resolved. Thereafter, the patient may be administered according to a preventive plan.

[0101] The actual dose level of the active ingredient in the pharmaceutical composition according to the present invention may be modified to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and administration method, and that is not toxic to the patient. The selection of the dose level is determined from a variety of pharmacokinetic factors, including the activity of the particular composition or its ester, salt, or amide according to the present invention, the route of administration, the time of administration, the excretion rate of the particular compound, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition, the age, sex, weight, condition, general health status, medical history, and other factors well known in the medical field of the patient being treated.

[0102] The compositions according to the present invention may be administered by one or more routes of administration using one or more methods well known in the art. Those skilled in the art will understand that the route and / or method of administration will vary depending on the desired result. Preferred routes of administration for the FXI-binding protein according to the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, intraspinal, or other parenteral routes, such as injection or infusion. As used herein, “parenteral administration” refers to routes of administration other than enteral and topical administration, and generally includes, but is not limited to, injections and infusions of intravenous, intramuscular, intra-arterial, intra-shearing, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0103] Alternatively, the FXI-binding protein according to the present invention may be administered via parenteral routes, such as topical, epidermal, or mucosal routes, for example, intranasal, oral, vaginal, rectal, sublingual, or topical administration.

[0104] Treatment and / or prevention of disease In one embodiment of the present invention, a method for treating and / or preventing thromboembolic conditions or diseases in a subject is provided, comprising administering a therapeutically effective amount of the FXI-binding protein or the pharmaceutical composition according to the present invention to the subject.

[0105] In some embodiments, the subjects have or are at risk of having myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolism, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis, ECMO), arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

[0106] In some embodiments, the subject has pathological activation of FXI.

[0107] In one aspect of the present invention, the invention further provides uses for the FXI-binding protein or pharmaceutical composition according to the present invention in the manufacture of drugs for treating and / or preventing thromboembolic conditions or diseases.

[0108] In some specific embodiments, the thromboembolic condition or disease described above is myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-associated thromboembolism, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis, ECMO), arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

[0109] In one aspect of the present invention, a method for inhibiting the activation of FXI (for example, by factor XIIa (FXIIa)) in a subject is provided, comprising: (a) selecting a subject in need of treatment, wherein the subject has a thrombus or is at risk of thrombus formation; and (b) inhibiting the activation of FXI by administering an effective amount of the FXI-binding protein or pharmaceutical composition according to the present invention to the subject.

[0110] In some embodiments, subjects requiring treatment are those who have or are at risk of having myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolism, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis, ECMO), arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

[0111] In some embodiments, the subjects requiring treatment are those who have pathological activation of FXI.

[0112] In some embodiments, the effective amount of the FXI-binding protein or the pharmaceutical composition according to the present invention is sufficient to inhibit FXI activation by at least 10%, 20%, 30%, 40%, or 50%.

[0113] In another embodiment of the present invention, a method is provided for inhibiting coagulation and associated thrombus formation in a target subject with unimpeded hemostasis, comprising administering a therapeutically effective amount of the FXI-binding protein or the pharmaceutical composition according to the present invention to the target subject, thereby inhibiting coagulation and associated thrombus formation in the target subject with unimpeded hemostasis.

[0114] In some embodiments, the subjects have or are at risk of having myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolism, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation (e.g., cardiopulmonary bypass, hemodialysis, ECMO), arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

[0115] In some embodiments, the subject is one that has pathological activation of FXI.

[0116] In another aspect of the present invention, the invention further provides applications of the FXI-binding protein or pharmaceutical composition according to the present invention in the manufacture of drugs for inhibiting coagulation and associated thrombus formation in conjunction with unimpeded hemostasis.

[0117] In some embodiments of each aspect of the present invention, the FXI-binding protein or the pharmaceutical composition according to the present invention is administered parenterally.

[0118] detection In another embodiment of the present invention, a method for detecting the presence and / or amount of FXI in a biological sample is provided, comprising: contacting the biological sample and a control sample with the FXI-binding protein according to the present invention under conditions that enable the FXI-binding protein according to the present invention to form a complex with FXI; then detecting the formation of the complex, and the difference in complex formation between the biological sample and the control sample indicating the presence and / or amount of FXI in the sample.

[0119] In some embodiments, the FXI-binding protein according to the present invention is further conjugated with a fluorescent dye, chemical substance, polypeptide, enzyme, isotope, tag, etc., which can be detected or detected by other reagents.

[0120] kit Kits applied to the methods according to the present invention are also within the scope of the present invention, and such kits include the FXI-binding protein according to the present invention and instructions for use. Kits generally include labels that describe the intended use of the contents of the kit. The term “label” includes any written or recorded material on or provided with the kit or otherwise provided with the kit. [Examples]

[0121] The present invention will be further described below with reference to examples, however, the present invention is not limited to the examples described.

[0122] Example 1: Screening for FXI heavy chain single-domain antibody 1.1 Building the Library Prior to immunization, 5 mL of Bactrian camel arterial blood was collected using a vacuum blood collection tube, and the supernatant was collected as preimmune serum. For the first immunization, healthy 2-year-old Xinjiang Bactrian camels were selected, and 300 μg of recombinant human factor XI (hFXI, self-prepared, sequence referenced from Uniprot database accession number P03951) was used as the antigen and homogeneously mixed with an equal volume of complete Freund's adjuvant. After the protein was completely emulsified, the mixture was intramuscularly injected into multiple sites in the neck of the Bactrian camels. For the later immunizations, an equal volume of antigen was homogeneously mixed with an equal volume of incomplete Freund's adjuvant each time, and immunization was performed once a week, for a total of six immunizations of Bactrian camels in the later stages. At the end of the final immunization, 5 mL of Bactrian camel arterial blood was collected using a vacuum blood collection tube, and the supernatant was collected as postimmune serum.

[0123] Lymphocytes were isolated using density gradient centrifugation, and total RNA was extracted using a QIAGEN RNA extraction kit. All extracted RNA was reverse transcribed into cDNA using the Super-Script III FIRST STRANDSUPERMIX kit according to the instructions, and nucleic acid fragments encoding the variable region of the heavy chain antibody were amplified by nested PCR.

[0124] Nucleic acid fragments of the target heavy chain single-domain antibody were recovered and cloned into the phage display vector pMECS using restriction enzymes (purchased from NEB) PstI and NotI. The product was then electrotransformed into E. coli electrocompetent cells TG1 to construct a phage display library of immunosingle-domain antibodies against recombinant human factor XI, and the library was validated. After serial dilution plating, the library volume size was 1.5 × 10⁶. 8 This was calculated. To detect the insertion rate of the library, 50 clones were randomly selected and sequenced. All 50 clones had the correct foreign fragment inserted, resulting in an accuracy of 100%. By analyzing and comparing the DNA and amino acid sequences of the sequenced clones, it was confirmed that all sequences were camel VHH sequences, and the diversity can be estimated to be over 95%.

[0125] 1.2 Panning of heavy chain single-domain antibodies against FXI For the first screening, plates were coated with 5 μg / well of the protein hApple-chis (a self-prepared protein, the sequence was selected from the Uniprot database accession number P03951, with the amino acid sequence at position 387 selected and a His-tag added to the C-terminus for purification) and left to stand overnight at 4 °C. The following day, the plates were blocked with 2% skim milk at room temperature for 2 hours, and then 100 μL of phage was added (approximately 10 8 ~10 9 The cells were incubated with pfu (derived from a 1.1 hFXI-Chis single-domain antibody display library) at room temperature for 2 hours. Subsequently, the cells were washed 25 times with PBST (PBS containing 0.05% polysorbate 20) to remove unbound phages. Finally, phages specifically bound to hApple-chis were dissociated with glycine (100 mM, pH 2.0).

[0126] In the second screening, the plates were coated with 3 μg / well of the protein mApple-chis (a self-prepared protein, the sequence of which was selected from the first 389 amino acids using accession number Q91Y47 in the Uniprot database, with a His-tag added to the C-terminus for purification) and left to stand overnight at 4°C. The following day, the plates were blocked with 2% skim milk powder at room temperature for 2 hours, and then 100 μL of phage was added (approximately 10 8 ~10 9 The cells were incubated with pfu (from a 1.1 hFXI-Chis single-domain antibody display library) at room temperature for 2 hours. Subsequently, the cells were washed 25 times with PBST (PBS containing 0.05% polysorbate 20) to remove unbound phages. Finally, phages specifically bound to mApple-chis were dissociated with glycine (100 mM, pH 2.0), and these were used to infect E. coli TG1 in the logarithmic growth phase to produce and purify phages in preparation for the next screening. For the second time, the plates were coated with 10 μg / well of the protein hApple-chis, and the other procedures were the same as above.

[0127] In this way, positive clones were enriched, and the objective of screening FXI-specific antibodies from the antibody library using phage display technology was achieved.

[0128] 1.3 Screening for specific single-positive clones by enzyme immunosorbent assay (ELISA) The FXI-conjugated phages obtained by the panning method described above were used to infect blank E. coli, which were then plated. Subsequently, 190 single colonies were randomly selected and named iFE1 to iFE190, respectively. Each colony was inoculated onto 2TY-AG and cultured until the OD600 reached approximately 0.8. IPTG was then added to achieve a final concentration of approximately 1 mM. Expression was induced overnight at 25 °C, and single-domain antibodies were expressed in the E. coli periplasm. The cells were collected the following day, lysed, and the supernatant was used for ELISA detection. The plates were coated with hFXI, hApple, and mApple respectively and kept overnight at 4 °C. Sample lysis supernatant (blank E. coli lysis supernatant for the control group) was added, and the mixture was incubated at room temperature for 2 hours. After washing, the secondary antibody Goat anti-HA tag HRP (purchased from abcam) was added, and the mixture was incubated at room temperature for 2 hours. After washing, TMB colorimetric solution was added, and absorbance values ​​were read at wavelengths of 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. A sample well was identified as a positive clone well if its OD value was more than twice that of the control well. Sequencing of the positive clones was performed by Genewiz.

[0129] The protein sequences of each clone were analyzed using the sequence comparison software BioEdit. Clones with more than 90% CDR1, CDR2, and CDR3 sequence homology were considered to be the same antibody strain. A total of 23 different antibody strains were ultimately obtained. The binding detection results are shown in Table 1. It was found that iFE96, iFE97, iFE148, iFE163, iFE166, and iFE168 bound simultaneously to hFXI, hApple, and mApple, while iFE29, iFE30, iFE5, iFE7, iFE13, iFE15, iFE35, iFE56, iFE11, iFE17, iFE22, iFE43, iFE49, iFE50, iFE70, iFE108, and iFE128 bound to hFXI and hApple but not to mApple.

[0130] [Table 4]

[0131] 1.4 Prokaryotic expression and purification of positive clones A seed solution of a single colony of a positive clone screened in 1.3 (E. coli TG1 expression system containing HA and His tags, with the vector pMECS) was cultured overnight in 2TY-AG medium and transferred to 50 mL of 2TY-AG medium. When the OD600 reached approximately 0.8, IPTG was added to a final concentration of approximately 1 mM, and expression was induced overnight at 25 °C. The bacterial cells were collected the following day, resuspended in Tris buffer, and disrupted by sonication. The supernatant was obtained and purified using affinity chromatography with a Ni column, utilizing the His tag on the single-domain antibody, to obtain the corresponding target protein.

[0132] 1.5 Detection of the affinity of prokaryotic expression proteins for hApple and mApple Plates were coated with 0.5 μg / well of hApple and mApple protein, respectively, and held overnight at 4 °C. Serial dilutions of candidate single-domain antibodies with His and HA tags obtained in 1.4 were added and reacted at room temperature for 2 hours. After washing, chicken anti-HA tagged secondary antibody (streptavidin-HRP, abcam) labeled with horseradish peroxidase was added and reacted at room temperature for 2 hours. After washing, chromogenic solution was added, and absorbance values ​​were read at wavelengths of 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 plotting analysis were performed using SotfMax Pro v5.4 software, and binding curves and EC between candidate single-domain antibodies and hApple and mApple against FXI were obtained by 4-parameter fitting. 50 By obtaining these values, the affinity of these candidate antibodies for hApple and mApple can be reflected.

[0133] The results in Table 2 show that all of these candidate single-domain antibodies bound to hApple, iFE43, iFE50, and iFE96 had weak affinity for the hApple protein, and iFE96, iFE97, iFE148, iFE163, iFE166, and iFE168 bound to mApple simultaneously.

[0134] [Table 5]

[0135] Example 2: Production of FXI single-domain antibody-Fc fusion protein using mammalian cells 2.1 Production of FXI single-domain antibody-Fc fusion plasmids We designed primers and performed PCR amplification of VHH fragments of the FXI single-domain antibody (amino acid sequences referencing SEQ ID NOs: 1-23, with individual amino acids in the FR2 region of some sequences mutated to improve protein stability). These fragments were then fused with a DNA fragment encoding human IgG1-Fc (amino acid sequence: SEQ ID NO: 336), and cloned into a standard mammalian expression vector to obtain recombinant plasmids 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

[0136] 2.2 Production of FXI single-domain antibody-Fc fusion protein The vector constructed in 2.1 was transfected into HEK293 cells to induce transient antibody expression. Recombinant expression plasmids were diluted in Freestyle293 medium, and the PEI (Polyethylenimine) solution required for transformation was added. The plasmid / PEI mixtures for each group were added to the HEK293 cell suspension, and the cells were cultured in suspension at 37 °C × 5% CO2. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target FXI single-domain antibody-Fc fusion protein. The purity of the proteins was detected preliminaryly by SDS-PAGE and SEC-HPLC. The expression status and purity analysis of each protein are shown in Table 3.

[0137] [Table 6]

[0138] It was found that the expression levels of the 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. Furthermore, after one-step purification by Protein A affinity chromatography, the target protein was obtained with stable concentration and high purity.

[0139] Example 3: Functional identification of FXI single-domain antibody-Fc fusion protein 3.1 Binding curves of FXI single-domain antibody-Fc fusion protein to mMapple and hApple Plates were coated with 0.5 μg / well of the proteins mApple and hApple, respectively, and a blank control was provided. The plates were kept at 4 °C overnight. A serial dilution series of the FXI single-domain antibody-Fc fusion protein obtained in Example 2.2 was added and reacted at room temperature for 2 hours. After washing, Goat anti-human IgG-HRP (purchased from Sigma) was added and reacted at room temperature for 2 hours. After washing, a chromogenic solution was added, and absorbance values ​​were read at wavelengths of 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 graphic analysis were performed using the software SoftMax Pro v5.4, and the binding curves and EC of the antibodies to mApple and hApple were obtained by 4-parameter fitting. 50 By obtaining these values, the affinity of the antibody for mApple and hApple can be reflected.

[0140] As can be seen from the results 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.

[0141] [Table 7]

[0142] 3.2 Affinity detection of FXI single-domain antibody-Fc fusion protein (Biofilm Interferometry BLI) The binding dynamics of the FXI single-domain antibody-Fc fusion proteins obtained in the above examples to recombinant proteins hApple and mApple were measured using biolayer interferometry (BLI) and an intermolecular interaction analyzer. 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 on a Protein A biosensor for pharmacokinetic analysis. hApple was diluted to five concentrations of 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM with 0.02% PBST20, and mApple was diluted to five concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM. The samples were injected over 150 seconds, with a dissociation time of 900 seconds, and regeneration was performed with 10 mM glycine-HCl (pH 1.7) for 5 seconds. The binding rate constant (kon) and dissociation rate constant (kdis) were calculated using a simple one-to-one Languir coupling model (Octet K2 data analysis software version 9.0). The dissociation equilibrium constant (KD) was calculated as the ratio kdis / kon.

[0143] The results are shown in Tables 5 and 6. Table 5 shows that the FXI single-domain antibody-Fc fusion protein has equivalent binding affinity to hApple, while Table 6 shows that the antibodies iFE97-Fc and iFE148-Fc have superior affinity to mApple.

[0144] The positive control 14E11 was prepared by transient expression in 293 cells following gene synthesis using the sequence described in patent WO2010080623.

[0145] [Table 8]

[0146] [Table 9]

[0147] 3.3 Detection of different epitopes of FXI single-domain antibody-Fc fusion protein binding to Apple protein (Biofilm Interferometry BLI: epitope binning) Using the in-tandem method, hApple-chis-biotin and mApple-chis-biotin were each diluted to 10 μg / mL in 0.02% PBST20 and immobilized on an SA biosensor for 100 seconds, with a height of approximately 1 nm. The FXI single-domain antibody-Fc fusion protein was diluted to 200 nM in 0.02% PBST20 and divided into two groups. The antibody binding time for both groups was 300 seconds. The regeneration solution was 10 mM glycine-HCl (pH 1.7). After the first antibody (saturated antibody) bound to the sensor and became saturated, the second antibody (competitive antibody) competed with the first antibody at the same concentration, and the percentage was calculated. The percentage calculation formula was Ab2 with Ab1 / Ab2 without Ab1.

[0148] The measurement results are shown in Tables 7, 8, and 9. To summarize the results above, Table 7 shows that iFE13-Fc, iFE35-Fc and 14E11 are competing, iFE13-Fc has an epitope that overlaps with 14E11, iFE35-Fc has a partial epitope that overlaps with 14E11, iFE56-Fc and iFE35-Fc are competing, iFE56-Fc has partial steric hindrance in its epitope with 14E11, iFE5-Fc and iFE30-Fc are competing, their epitopes are the same and do not overlap with 14E11, and Tables 8 and 9 show that iFE97-Fc and iFE163-Fc are competing, their epitopes are the same and do not overlap with 14E11, iFE148-Fc recognizes both humans and mice, and its epitope does not overlap with other single-domain antigens or 14E11.

[0149] [Table 10]

[0150] [Table 11]

[0151] [Table 12]

[0152] 3.4 Detection of nonspecific binding of FXI single-domain antibody-Fc fusion protein to null cells CHOK1 null cells and 293F null cells were resuspended in 3% BSA-PBS, and the number of cells was increased to 6 × 10⁶. 6 After adjusting the concentration to cells / mL, the final concentration of the FXI single-domain antibody-Fc fusion protein was set to 100 μg / mL based on the results of Example 3.1. Negative and blank controls were provided, and the cells were bathed in ice for 60 minutes. After washing, the Biolegend secondary antibody FITC anti-human IgG FC was added, and the cells were bathed in ice for 30 minutes. After washing, the cells were resuspended in 300 μL of 1% PBS-BSA Buffer and detected by flow cytometry.

[0153] The results in Tables 10 and 11 indicate that none of the candidate antibodies bound nonspecifically to null cells.

[0154] [Table 13]

[0155] 3.5 Identification of the blocking activity of FXI single-domain antibody-Fc fusion protein against FXI (APTT detection) Using an optical detection method, pre-warmed plasma and reagents were rapidly mixed and incubated. The absorbance value at a wavelength of 660 nm was detected. As the incubation time increased, fibrinogen was converted to fibrin, increasing the turbidity of the mixture and changing the intensity of scattered light. The coagulation time was measured by detecting the intensity of scattered light, which changed with the increase in sample turbidity, using an instrument. A calibration curve was created using a coagulation method with a commercially available coagulation factor XI as the standard substance, with coagulation time on the Y axis and the activity percentage of reference plasma on the X axis. The activity of the sample was calculated based on the calibration curve.

[0156] Antibodies were diluted to 1 μg / mL in FXI-deficient plasma, and 50 μL of each treated sample was incubated at 37 °C for 2 hours with Buffer as a negative control. The samples were then loaded into an automated coagulation system and automatically mixed with Dade Actin Activated Cephaloplastin Reagent, Calcium Chloride Solution, and FACTOR IX DEFICIENT reagent. After incubation, the absorbance at 660 nm was detected. As can be seen from the results in Table 11, iFE15, iFE29, iFE96, iFE166, and iFE168 do not exhibit blocking activity.

[0157] [Table 14]

[0158] As can be seen from the results in Table 11, the antibodies iFE13, iFE35, iFE97, iFE5, iFE148, iFE56, and iFE30 were serially diluted in standard human plasma, and their activity curves were detected. A positive control 14E11 was established, and from the results in Figure 1, it was found that the inhibitory effects of iFE5, iFE13, iFE35, iFE56, and iFE97 were clearly superior to those of the positive control 14E11, while the inhibitory effects of the other antibodies, iFE148 and iFE30, were equivalent to those of the positive control.

[0159] 3.6 Binding of FXI single-domain antibodies to different Apple domains The FXI single-domain antibody-Fc fusion proteins obtained in the above examples, along with the positive control 14E11, were selected, and their binding dynamics to different human Apple domain proteins were investigated by bio-layer interferometry (BLI). The sequences of the different Apple domain proteins were derived from Uniprot (accession number P03951), and some Apple domains had a His-tag added to their C-terminus for purification (those with Chis in their name), while others had a mouse Fc fragment added to their C-terminus for purification (those with muFc in their name). The proteins were subjected to transient transfection with 293 cells, followed by IMAC purification. The detailed procedure was the same as above, and self-prepared hApple1-2muFc, hApple2-3muFc, hApple3-4muFc, hApple2-chis, hApple4-chis, hApple1-muFc, and hApple3-muFc were used as the detection products.

[0160] For information on the binding status of candidate FXI single-domain antibodies-Fc fusion proteins to different Apple domains, please refer to Tables 12 and 13.

[0161] [Table 15]

[0162] [Table 16]

[0163] Example 4: Humanization of FXI single-domain antibody For humanization, we employed resurfacing of amino acids on the protein surface and CDR grafting to a humanized universal framework.

[0164] The humanization steps were as follows: Homological modeling was performed on antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, and iFE148 using Modeller9 modeling 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 specific amino acids in antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, or iFE148 were exposed to the solvent, they were substituted with the amino acids at the same position in the reference human antibody 10HQ sequence, until all amino acids were substituted.

[0165] The specific steps for transplanting VHH into the humanized universal framework were as follows: First, the universal humanized VHH framework hNbBcII10FGLA (PDB number 3EAK), designed by Cecile Vincke et al. based on sequence homology, was obtained. The design of this framework was completed by humanizing the amino acids on the protein surface based on the nanobody NbBcII10 antibody (PDB number 3DWT) and referencing the human antibody 10HQ. This was done by manipulating some amino acids VLP of VHH sequence framework 1, some amino acids GL of VHH sequence framework 2, some amino acids RSKRAAV of VHH sequence framework 3, and some amino acids L of VHH sequence framework 4. We directly used hNbBcII10FGLA as a framework and completed the humanization of the antibodies by substituting the CDR region of antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, and iFE148.

[0166] The antibody strains iFE5, iFE13, iFE35, iFE56, iFE97, and iFE148 were humanized, yielding six humanized variants (huFE) for each antibody strain. The sequences of these humanized variants correspond to SEQ ID NOs. 300-335, respectively.

[0167] Example 5: Production of a humanized single-domain antibody-Fc fusion protein 5.1 Production of Humanized Single-Domain Antibody-Fc Fusion Plasmids The gene synthesis of the huFE humanized sequence in Example 4 was performed by Suzhou Hongxun Biotechnology Co., Ltd., and enzymatic cleavage sites were added to both ends.

[0168] By bienzyme digestion of the VHH fragment of a huFE single-domain antibody, fusion with a DNA fragment encoding human IgG1FC, and cloning into a standard mammalian expression vector, a recombinant plasmid for expressing a huFE single-domain antibody-Fc fusion protein in mammals was obtained.

[0169] 5.2 Production of Humanized Single-Domain Antibody-Fc Fusion Proteins The vector constructed in 4.1 was transfected into HEK293 cells to induce transient antibody expression. Recombinant expression plasmids were diluted in Freestyle293 medium, and the PEI (Polyethylenimine) solution required for transformation was added. The plasmid / PEI mixtures for each group were added to the HEK293 cell suspension, and the cells were incubated at 37 °C × 5% CO2 at 130 rpm. After 4 hours, EXCELL293 medium and 2 mM glutamine were added, and the cells were incubated 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 incubation, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target huFE single-domain antibody-Fc fusion protein. The purity of the proteins was preliminaryly examined by SDSPAGE and SECHPLC. The expression level of each protein exceeded 350 mg / L, and the SEC purity after one-step purification exceeded 95% for all proteins.

[0170] Example 6: Functional identification 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 examples to the hApple-CHis protein were measured using biolayer interferometry (BLI) with an intermolecular interaction analyzer. 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 on an AHC biosensor to measure pharmacokinetics. The hApple-CHis proteins were diluted to five concentrations: 200 nM, 100 nM, 50 nM, 25 nM, and 12.5 nM, respectively, and the assay was performed with baseline at 60 seconds, binding at 120 seconds, and dissociation at 900 seconds. The diluent was Kinetic buffer, the regeneration solution was glycine-HCl (pH 1.7), and the neutralization solution was the diluent. The biosensor was ProteinA. The binding rate constant (kon) and dissociation rate constant (kdis) were calculated using a simple one-to-one Languir coupling model (Octet K2 data analysis software version 9.0). The dissociation equilibrium constant (KD) was calculated as the ratio kdis / kon.

[0171] For the results of measuring the affinity of the huFE single-domain antibody-Fc fusion protein to the hApple-Chis protein, please refer to Table 14.

[0172] [Table 17]

[0173] The binding dynamics of the huFE single-domain antibody-Fc fusion protein obtained in the above examples to the hFXI-CHis protein were measured using bio-layer interferometry (BLI) and an intermolecular interaction analyzer. The specific procedure was the same as described above, and the hFXI-Chis protein was used as the detection product. 4E11 (see above) and B1213790-F11a (self-prepared based on the sequence described in WO2018134184) were used as controls.

[0174] For the results of measuring the affinity of the huFE single-domain antibody-Fc fusion protein to the hFXI-CHis protein, please refer to Table 15.

[0175] [Table 18]

[0176] The results show that the humanized single-domain antibody-Fc fusion protein bound well to both hApple-CHis and hFXI-CHis, indicating no significant differences among the various humanized versions. Furthermore, its binding ability was comparable to that of the two control antibodies.

[0177] A portion of the huFE single-domain antibody-Fc fusion proteins obtained in the above examples were selected, and their binding kinetics to New Zealand rabbit FXI protein were investigated by bio-layer interferometry (BLI). The specific procedure was the same as described above, and the detected material was self-prepared New Zealand rabbit FXI protein (relevant protein sequence is shown in Uniprot database accession number Q95ME7).

[0178] For the results of measuring the affinity of the huFE single-domain antibody-Fc fusion protein to the RabFXI-Apple-CHis protein, please refer to Table 16.

[0179] [Table 19]

[0180] A portion of the huFE single-domain antibody-Fc fusion proteins obtained in the above examples were selected, and their binding kinetics to cynomolgus monkey FXI protein were investigated by bio-layer interferometry (BLI). The specific procedure was the same as described above, and the detected product was a self-prepared cynomolgus monkey FXI protein (see Uniprot database accession number A0A2K5VVK2 for the relevant protein sequence).

[0181] For the results of measuring the affinity of the huFE single-domain antibody-Fc fusion protein to the cynomolgus monkey FXI-CHis protein, please refer to Table 17.

[0182] [Table 20]

[0183] The experimental results described above indicate that most of the candidate antibodies can simultaneously recognize coagulation factor XI in humans, New Zealand rabbits, and cynomolgus monkeys. However, binding to New Zealand rabbits was reduced. Furthermore, antibodies 35 and 56 showed almost no activity in New Zealand rabbits, and antibody 5 showed almost no activity in cynomolgus monkeys. There were no significant differences in activity among the various humanized versions of the protein.

[0184] A portion of the huFE single-domain antibody-Fc fusion proteins obtained in the above examples were selected, and their binding kinetics to activated human factor XI protein (hFXIa) were investigated by biofilm interferometry (BLI). The specific procedure was the same as described above, and commercially available hFXIa was used as the detection material. 14E11 (see above) and B1213790-F11a (see above) were used as controls.

[0185] For the results of measuring the affinity of the huFE single-domain antibody-Fc fusion protein to the hFXIa protein, please refer to Table 18.

[0186] [Table 21]

[0187] The results above indicate that the humanized FXI single-domain antibody-Fc fusion protein can effectively bind to activated FXI factor. However, its binding ability was lower than that of B1213790-F11a, which directly binds to the activation site of FXI factor.

[0188] Furthermore, when combined with the above results regarding binding to inactivated FXI factor, it was found that 14E11, which binds to the Apple domain (Apple2), exhibits improved binding ability to activated FXIa. The candidate antibody according to the present invention has equivalent binding activity to activated FXIa and inactivated FXI, or reduced binding to activated FXIa. This result also indicates that although the candidate antibody according to the present invention shares several binding epitopes with 14E11, there is a clear difference in their binding modes.

[0189] 6.2 Inhibitory effect of huFE single-domain antibody-Fc fusion protein on the activity of human factor FXI Standard human plasma (purchased from the WHO) showed 92% human FXI activity. The inhibitory effects on FXI factor activity in plasma were detected after combining it with FXI-deficient plasma and adding different single-domain antibody-Fc fusion proteins and a reference substance (14E11). See Table 19 and Figure 2 for activity results after mixing with different antibodies against FXI factor. All of these humanized FXI antibodies showed good inhibitory activity against FXI factor. 14E11 protein was used as a positive control.

[0190] [Table 22]

[0191] Example 7: Production of huFE bispecific antibody-Fc fusion protein using mammalian cells 7.1 Production of huFE bispecific antibody-Fc fusion plasmid Molecular cloning of the gene for the huFE single-domain antibody-Fc fusion protein obtained in Example 4 yielded a recombinant plasmid for expressing the huFE bispecific antibody-Fc fusion protein in mammals, and this plasmid was used to express the bispecific antibody proteins huFE97n13-Ld-Fc, huFE13n97-Ld-Fc, huFE97n56-Ld-Fc, huFE56n97-Ld-Fc, huFE97di-Ld-Fc, huFE56n13-Ld-Fc, and huFE1 3n56-Ld-Fc, huFE97n148-Ld-Fc, huFE148n97-Ld-Fc, huFE5n97-Ld-Fc, huFE97n5-Ld-Fc, huFE148n5-Ld-Fc, huFE5n148-Ld-Fc, huFE148n56-Ld-Fc, huFE56n148-Ld-Fc, huFE56n5-Ld-Fc, huFE5n56-Ld-Fc, huFE5n13-Ld-Fc, and huFE13n5-Ld-Fc can be manufactured.

[0192] Furthermore, a tetravalent monospecific antibody of HuFE97di-Ld-Fc was obtained using the method described above and used as a control antibody.

[0193] 7.2 Production of huFE bispecific antibody-Fc fusion protein The vector constructed in 7.1 was transfected into HEK293 cells to induce transient antibody expression. The recombinant expression plasmid was diluted in Freestyle293 medium, and the PEI (Polyethylenimine) solution required for transformation was added. The plasmid / PEI mixture for each group was added to the HEK293 cell suspension, and the cells were cultured in suspension at 37 °C × 5% CO2. After 5-6 days of culture, the transient expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the target huFE bispecific antibody-Fc fusion protein. The purity of the protein was preliminaryly examined by SDSPAGE and SECHPLC.

[0194] The expression levels of all proteins ranged from 250 mg / L to 400 mg / L, and the SEC purity after one-step purification was over 95% for all. These results preliminarily demonstrate that these bispecific antibodies exhibit excellent solubility and stability, making them suitable as drug candidate molecules.

[0195] Example 8: High-temperature accelerated assay of huFE bispecific antibody-Fc fusion protein Using 12 mg each of the huFE bispecific antibody-Fc fusion protein obtained in the above examples, the solution was concentrated to 10 mg / mL by ultrafiltration and placed in PBS solution. The solution was then allowed to stand at 40 °C, and stability was examined by periodically taking samples and detecting changes in protein content, SEC purity, etc. See Tables 20 and 21 for the results.

[0196] [Table 23]

[0197] [Table 24]

[0198] Conclusion: After 20 days at high temperatures, the purity had decreased, but it was within an acceptable range.

[0199] Example 9: Functional identification of huFE bispecific antibody-Fc fusion protein 9.1 Affinity of huFE bispecific antibody-Fc fusion protein The binding dynamics of the huFE bispecific antibody-Fc fusion protein obtained in the above examples to the hApple-Chis and hFXI-Chis proteins were measured using biolayer interferometry (BLI) with an intermolecular interaction analyzer. 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 on an AHC biosensor to measure pharmacokinetics. The hApple-Chis or hFXI-Chis proteins were each diluted to 5 concentrations and measured at baseline 60 seconds, binding 120 seconds, and dissociation 900 seconds. The diluent was Kinetic buffer, the regeneration solution was glycine-HCl (pH 1.7), and the neutralization solution was the diluent. The biosensor was Protein A. The binding rate constant (kon) and dissociation rate constant (kdis) were calculated using a simple 1:1 Languir binding model (Octet K2 data analysis software version 9.0). The dissociation equilibrium constant (KD) was calculated as the ratio kdis / kon.

[0200] For the results of measuring the affinity of the huFE bispecific antibody-Fc fusion protein for the hFXI-Chis protein, please refer to Table 22.

[0201] [Table 25]

[0202] For the results of measuring the affinity of the huFE bispecific antibody-Fc fusion protein to the hApple-Chis protein, please refer to Table 23.

[0203] [Table 26]

[0204] The results above indicate that all candidate FXI bispecific antibodies have better affinity for human FXI factor protein and human Apple domain than the parental monospecific antibody.

[0205] 9.2 Specificity of huFE bispecific antibody-Fc fusion protein The 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 on AHC chips, and the target proteins were commercially available FVII, FIX, FV, FXII, pro-thrombin, α-kallikrein, FVIIa, FIXa, FVa, FXIIa, and Thrombin. The experimental results showed that none of the bispecific antibodies bound to any of the following proteins: FVII, FIX, FV, FXII, pro-thrombin, α-kallikrein, FVIIa, FIXa, FVa, FXIIa, or Thrombin.

[0206] 9.3 Inhibitory effect of huFE bispecific antibody-Fc fusion protein 9.3.1 Inhibitory effect on human FXI activity The activity of standard human FXI (provided by WHO) was 92%, and the activity of standard human plasma (purchased from Sigma) was 87.5%. The inhibitory effect on FXI factor activity in plasma was detected after adding different single-domain antibody-Fc fusion proteins and reference substances (14E11, MAA868-F11) in combination with FXI-deficient plasma. See Figure 3 for activity results after mixing with different antibodies against FXI factor. All of these FXI antibodies showed good inhibitory activity against FXI factor and were superior to the control-positive antibody. Of these, the 14E11 protein (manufacturing method the same as above) and MAA868-F11 (manufactured based on patent application US15 / 739414) were used as positive controls.

[0207] 9.3.2 Inhibitory activity of human whole plasma on APTT FXI antibodies were diluted to different concentrations in standard human plasma (purchased from Sigma), incubated at 37°C for 3 minutes, and then the APTT time in whole blood was detected. 14E11 and B1213790-F11a were used as positive controls.

[0208] Figure 4 shows the change in APTT time according to antibody concentration. These results indicate that all candidate FXI antibody proteins can effectively prolong the APTT coagulation time in whole blood, and that bispecific antibodies have a superior coagulation inhibitory effect.

[0209] 9.3.3 Inhibitory activity of whole monkey plasma against APTT FXI antibodies were diluted to different concentrations in monkey plasma (purchased from Sigma), incubated at 37°C for 3 minutes, and then the APTT time in whole blood was detected. 14E11 and B1213790-F11a were used as positive controls.

[0210] See Figure 5 for the results.

[0211] 9.3.4 Inhibitory activity of rabbit whole plasma against APTT The APTT time in whole blood was detected by diluting the FXI antibody-Fc fusion protein and standard substances of the example to different concentrations in rabbit plasma (purchased from Sigma). 14E11 and B1213790-F11a were used as positive controls.

[0212] See Figure 6 for the results.

[0213] The experimental results described above indicate that most FXI candidate antibodies exhibit good APTT inhibitory activity in human or monkey plasma, regardless of whether they are bispecific. Some antibodies show no activity in rabbits.

[0214] The activity of candidate antibodies in human plasma is generally superior to that of control-positive antibodies. Bispecific antibodies generally exhibit superior activity compared to monospecific antibodies.

[0215] 9.4 Effects of FXI single-domain antibody-Fc fusion protein and bispecific antibody on rabbit venous thrombosis The rabbits were fasted overnight, blood was collected from the marginal vein of the ear, anesthetic was injected from the marginal vein of the ear for anesthesia, the distal and proximal ends of the jugular vein were ligated, and the distance between the two ligatures was maintained at about 3.0 cm. 15 minutes before modeling, the test substance (huFE single-domain antibody-Fc fusion protein of Example 5.2) or PBS negative reference substance (see Table 24) was injected into the marginal vein of the ear at 1 mg / kg. The proximal and distal ends of the jugular vein were clamped with arterial clips respectively, the blood in the blood vessel was completely extracted from the facial vein using a syringe, and then 0.3 mL of 5 mg / mL agonist was injected into the blood vessel of the closed part. After incubating with the agonist for 5 minutes, the agonist was extracted with a syringe, rinsed twice with physiological saline, the arterial clip was opened, the blood flow was restored, and the diameter of the blood vessel was maintained at 0.8 mm to induce thrombosis. After the blood flow recovered for 25 minutes, the distal and proximal ends of the vein of the closed part were clamped with arterial clips, the surgical threads at the proximal and distal ends of the vein ligated previously were tightened, the closed vein was cut, the thrombus was taken out, and the wet weight of the thrombus was measured and recorded immediately. After the thrombus was dried in an oven at 60 °C for 20 hours, the dry weight of the thrombus was measured and the data was recorded (see Table 25).

[0216] The observation index was the thrombus weight, the experimental data was shown as X±SD, and a significance test was performed by GraphPad Prism 5 1 way ANOVA (see Figure 7).

[0217] Due to the influence of the species cross-reactivity of different antibodies with rabbits, only some antibodies showed a good thrombus inhibitory effect. In the future, monkeys with better species cross-reactivity were planned to be selected for corresponding in vivo activity detection.

[0218]

Table 27

[0219]

Table 28

[0220] Array information:

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34

Table 35

Table 36

Table 37

Table 38

Table 39

Table 40

Claims

1. A coagulation factor XI (FXI) binding protein comprising a first immunoglobulin monovariable domain that can specifically bind to FXI, and a second immunoglobulin monovariable domain that can specifically bind to FXI, Of these, the first immunoglobulin monovariable domain and the second immunoglobulin monovariable domain bind to different epitopes in FXI. The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 1, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 4, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 10, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 14, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, or The first immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 17, and the second immunoglobulin monovariate domain comprises CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NO: 20, and is an FXI-binding protein.

2. The FXI-binding protein according to claim 1, wherein the CDR is Kabat CDR, AbM CDR, Chothia CDR, or IMGT CDR.

3. The FXI-binding protein according to claim 1 or 2, wherein CDR1, CDR2, and CDR3 of VHH as shown in SEQ ID NOs: 1, 4, 9, 10, 14, 17, or 20 are as shown in the table below. Table 1

4. The FXI-binding protein comprises a first immunoglobulin monovariable domain and a second immunoglobulin monovariable domain, of which, The first immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 1, 300-305, and the second immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 4, 306-311, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 10, 312-317, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 14, 318-323, or The first immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 1, 300-305, and the second immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 1, 300-305, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 4,306-311, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, or The first immunoglobulin monovariate domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 4, 306-311, and the second immunoglobulin monovariate domain comprises the amino acid sequence in VHH shown in any one of SEQ ID NOs: 10, 312-317, or The first immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 4, 306-311, and the second immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 14, 318-323, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 4, 306-311, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 17, 324-329, or The first immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 4, 306-311, and the second immunoglobulin monovariable domain contains the amino acid sequence shown in any one of SEQ ID NOs: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 10, 312-317, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 14, 318-323, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in SEQ ID NO: 9, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 10, 312-317, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 14, 318-323, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 10, 312-317, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 10, 312-317, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 20, 330-335, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 14, 318-323, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 17, 324-329, or The first immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 14, 318-323, and the second immunoglobulin monovariate domain contains the amino acid sequence shown in any one of SEQ ID NOs: 20, 330-335, or The FXI-binding protein according to any one of claims 1 to 3, wherein the first immunoglobulin monovariate domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 17, 324-329, and the second immunoglobulin monovariate domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 20, 330-335.

5. The FXI-binding protein according to any one of claims 1 to 4, wherein the first immunoglobulin monovariable domain is located at the N-terminus of the second immunoglobulin monovariable domain, or the second immunoglobulin monovariable domain is located at the N-terminus of the first immunoglobulin monovariable domain.

6. The FXI-binding protein according to any one of claims 1 to 5, further comprising the Fc region of an immunoglobulin.

7. The FXI-binding protein according to claim 6, wherein the Fc region of the immunoglobulin is the Fc region of human immunoglobulin.

8. The FXI-binding protein according to claim 6, wherein the Fc region of the immunoglobulin is the Fc region of human IgG1, IgG2, IgG3, or IgG4.

9. The amino acid sequence of the Fc region of the immunoglobulin is the FXI-binding protein according to claim 6, as shown in SEQ ID NO:

336.

10. A nucleic acid molecule encoding an FXI-binding protein according to any one of claims 1 to 9.

11. An expression vector comprising a nucleic acid molecule according to claim 10, which is operably connected to an expression control element.

12. Recombinant cells comprising the nucleic acid molecule described in claim 10, or transformed with the expression vector described in claim 11, and capable of expressing the FXI-binding protein.

13. A method for producing an FXI-binding protein according to any one of claims 1 to 9, a) The step of culturing the recombinant cells according to claim 12 under conditions in which the FXI binding protein can be expressed, b) A step of recovering the FXI-binding protein expressed by the recombinant cells from the culture obtained from step a), c) optionally further comprising the step of purifying and / or modifying the FXI-binding protein obtained from step b).

14. A pharmaceutical composition comprising an FXI-binding protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, for the treatment and / or prevention of thromboembolic conditions or diseases in the subject.

16. The pharmaceutical composition according to claim 15, wherein 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 thromboembolism, severe systemic inflammatory response syndrome, arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

17. Use of the FXI-binding protein according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 14 in the manufacture of a drug for treating and / or preventing a thromboembolic condition or disease.

18. The use according to claim 17, wherein the thromboembolic condition or disease is myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolism, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation, arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

19. The pharmaceutical composition according to claim 14, for inhibiting the activation of FXI in a target.

20. The pharmaceutical composition according to claim 19, wherein the subject requiring the aforementioned treatment is a subject having or at risk of having myocardial infarction, ischemic stroke, pulmonary thromboembolism, venous thromboembolism (VTE), atrial fibrillation, disseminated intravascular coagulation, medical device-related thromboembolism, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation, arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

21. The pharmaceutical composition according to claim 14, which is for inhibiting coagulation and related thrombus formation in the target area in conjunction with proper hemostasis.

22. The pharmaceutical composition according to claim 21, wherein 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 thromboembolism, severe systemic inflammatory response syndrome, thromboembolism formed during extracorporeal circulation, arterial thrombosis, end-stage renal disease, antiphospholipid antibody syndrome, stroke, metastatic cancer, or infectious disease.

23. Use of the FXI binding protein according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 14 in the manufacture of a drug for inhibiting coagulation and related thrombus formation in conjunction with unimpeded hemostasis.

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