Antithrombotic antibody

Abstract

Provided are a novel antithrombotic antibody targeting a blood coagulation factor FIXa-FVIIIa binding site and an application thereof.SOLUTION: The present invention provides a novel antithrombotic antibody, which targets FIXa and has unique properties, specifically targets the binding site of blood coagulation factors FIXa and FVIIIa, reduces the formation of FVIIIa-FIXa complex, blocks the conversion of FX to FXa, and exerts an antithrombotic effect. The antibody of the present invention has an appropriate antithrombotic performance and a large effective therapeutic concentration window, but does not increase the bleeding risk, which can achieve the need for moderate antithrombosis and effectively avoid the bleeding problem caused by excessive action in clinical applications. The present invention also discloses methods of screening for drugs that target the FIXa-FVIIIa binding site.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to the fields of immunology and pharmacology, and more particularly to a novel antithrombotic antibody that targets the binding site of blood coagulation factor FIXa-FVIIIa. [Background technology]

[0002] Thromboembolic disease is a common clinical condition characterized by the formation or embolism of arterial, venous, and microvascular thrombi. These diseases involve the obstruction of blood flow after thrombus formation or the detachment of the thrombus, resulting in downstream blood flow disruption, leading to ischemia and necrosis of tissues and organs. Approximately 17.9 million people worldwide die from cardiovascular disease, accounting for 31% of global deaths. Thrombus formation is a key factor in the development of various serious cardiovascular and cerebrovascular diseases, making thrombotic disease the most significant threat to human health and life in modern society. The primary treatment for thromboembolic disease is antithrombotic therapy, including anticoagulation, antiplatelet, and thrombolytic therapy. Anticoagulation therapy primarily targets different blood coagulation factors in the blood coagulation cascade, thereby blocking the blood coagulation process.

[0003] Blood coagulation factors are various protein components involved in the blood clotting process. Their physiological function is to become activated during vascular bleeding, bind to platelets, and fill leaks in blood vessels. This process is called blood coagulation. The entire blood coagulation process can be roughly divided into two stages: activation of prothrombin and formation of a fibrin gel. For uniform naming, the World Health Organization (WHO) has numbered blood coagulation factors (F) I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, etc., in the order of their discovery. The addition of "a" to the number of some blood coagulation factors indicates their activated form; for example, the activated form of blood coagulation factor IX (FIX) is FIXa, and the activated form of blood coagulation factor VIII (FVIII) is FVIIIa.

[0004] Anticoagulants currently in clinical use include heparin and its derivatives, vitamin K antagonists (e.g., warfarin), and the small molecule inhibitors rivaroxaban and dabigatran. All of these drugs act on the common coagulation pathway (coagulation factors IIa and Xa) of the blood coagulation cascade, inevitably affecting physiological hemostatic function and therefore increasing the risk of serious bleeding (especially cerebral hemorrhage). Although the intrinsic coagulation pathway is closely related to pathological thrombus formation, it is not essential for hemostatic function. Therefore, selective inhibitors of intrinsic coagulation factors have become a hot topic in research into new anticoagulant drugs.

[0005] FIXa is a key blood coagulation factor in the intrinsic blood coagulation pathway and the only soluble form of blood coagulation protein. FIXa can efficiently diffuse from tissue factor-bearing cells to platelets, where it is a key link between the initiation and amplification stages of the blood coagulation chain. FIXa can also be directly activated by FXIa on aggregated platelets. FIXa activates FX by forming a FIXa-FVIIIa complex. However, the binding site and binding action between FVIIIa and FIXa have not yet been elucidated in the art. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a novel antithrombotic antibody that targets the binding site of blood coagulation factor FIXa-FVIIIa and its application. [Means for solving the problem]

[0007] In a first aspect, the present invention provides an antithrombotic monoclonal antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain CDR1 of said monoclonal antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 3, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 4, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 5; the amino acid sequence of the light chain CDR1 is set forth in SEQ ID NO: 6, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 7, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 8.

[0008] In one preferred example, the monoclonal antibody includes: (a) an antibody having a heavy chain variable region amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region amino acid sequence set forth in SEQ ID NO: 2; or (b) an antibody having a heavy chain variable region amino acid sequence that is 80% or more (e.g., 85%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence set forth in SEQ ID NO: 1 and a light chain variable region amino acid sequence that is 80% or more (e.g., 85%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence set forth in SEQ ID NO: 2, and having the antibody function of (a).

[0009] In another preferred embodiment, the monoclonal antibody comprises a murine antibody, a chimeric antibody, or a humanized antibody; or the monoclonal antibody or antigen-binding fragment thereof comprises a single-chain antibody (scFV), a domain antibody, a Fab fragment, a Fab' fragment, an Fd fragment, or an F(ab')2 fragment.

[0010] In another preferred embodiment, the antithrombotic monoclonal antibody or its antigen-binding fragment specifically targets the binding site of blood coagulation factors FIXa and FVIIIa, reduces the formation of FVIIIa-FIXa complexes, blocks the conversion of FX to FXa, and exerts an antithrombotic effect.

[0011] In another preferred example, the monoclonal antibody or antigen-binding fragment thereof extends the activated partial thromboplastin time (APTT) by 2 to 4 times (e.g., 2.5, 3, or 3.5 times; preferably, based on the normal activated partial thromboplastin time in the natural body, the normal time being, for example, 20-40 seconds, preferably 25-36 seconds); or the monoclonal antibody or antigen-binding fragment thereof extends the activated partial thromboplastin time (APTT) by 75 seconds or more, preferably 80 seconds or more (e.g., 80 to 120 seconds, more specifically 82, 85, 88, 90, 95, 100, or 110 seconds).

[0012] In another preferred embodiment, the monoclonal antibody or its antigen-binding fragment acts on the binding site between FIXa and FVIIIa or an adjacent site on FIXa, thereby reducing the formation of a FVIIIa-FIXa complex; preferably, the binding site between FIXa and FVIIIa or an adjacent site on FIXa includes Asn93, Lys132, Arg165, and Thr175, more preferably further includes Ala95, Lys98, Asp164, Lys173, and Tyr177, and more preferably includes Lys126, Asn129, Asn178, Lys230, Arg233, and Asn236.

[0013] In another preferred embodiment, the numbering of the amino acid residues at each site follows the numbering of chymotrypsin.

[0014] In another preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof does not affect the catalytic activity of FIXa.

[0015] In another preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof does not bind to the catalytically active site of FIXa, and the catalytically active site of FIXa is, for example, the His57-Asp102-Ser195 site.

[0016] In another preferred embodiment, the monoclonal antibody or antigen-binding fragment thereof does not affect PT time.

[0017] Another aspect of the present invention provides an isolated polynucleotide or a construct containing the polynucleotide, wherein the polynucleotide encodes any one of the antithrombotic monoclonal antibodies or antigen-binding fragments thereof; preferably, the construct is an expression vector.

[0018] In another aspect of the present invention, there is provided an antibody expression system, said expression system comprising the construct described above or having an exogenous polynucleotide integrated into its genome; preferably, said expression system is a cellular expression system.

[0019] In another aspect of the present invention, there is provided a method for preparing any one of the above antithrombotic monoclonal antibodies or antigen-binding fragments thereof, comprising expressing the antibody using the above antibody expression system under conditions suitable for expression of the antibody; preferably, also including purifying and isolating the antibody.

[0020] Another aspect of the present invention provides a fusion protein comprising any one of the above antithrombotic monoclonal antibodies or antigen-binding fragments thereof and a fusion partner operably linked thereto; preferably, the fusion partner includes, but is not limited to, a protein or active domain having the effect of extending half-life in the body, or a protein or active domain having a function of enhancing the effect on or binding to an effector (to exert one or more functions); more preferably, the protein or active domain having the effect of extending half-life in the body includes, but is not limited to, an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, serum albumin (e.g., human HSA), or a fragment thereof.

[0021] In one preferred example, the immunoglobulins are one or more combinations selected from IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is one or more combinations selected from IgG1, IgG2, IgG3, and IgG4 subtypes.

[0022] In another preferred embodiment, a linker peptide is present between the antithrombotic monoclonal antibody or its antigen-binding fragment and the fusion partner operably linked thereto; the linker peptide is preferably selected from flexible polypeptide chains consisting of alanine and / or serine and / or glycine, and the length of the linker peptide is preferably 3 to 30 amino acids.

[0023] In another aspect of the present invention, there is provided an immunoconjugate comprising any one of the above anti-thrombus monoclonal antibodies or antigen-binding fragments thereof, or the above fusion protein, and a functional molecule attached thereto (including, but not limited to, covalently binding, coupling, attachment, or adsorption); preferably, the functional molecule includes, but is not limited to, a molecule that targets a blood cell (e.g., platelet) surface marker, a hydrophilic polymer (e.g., polyethylene glycol, polyethylene glycol-liposome complex, etc.), or a detectable marker (e.g., including, but not limited to, a fluorescent marker, a colorimetric marker).

[0024] In another aspect of the present invention, there is provided a pharmaceutical composition comprising the antithrombotic monoclonal antibody or its antigen-binding fragment, the fusion protein, or the immunoconjugate; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0025] In another aspect of the present invention, there is provided the use of any one of the antithrombotic monoclonal antibodies or antigen-binding fragments thereof, the fusion protein, or the immunoconjugate, or a pharmaceutical composition containing them, in the preparation of a formulation or kit for alleviating or treating thromboembolic diseases.

[0026] In one preferred example, the thromboembolic disease includes, but is not limited to, venous, arterial or capillary thrombosis, thrombus formation in the heart, thrombus formation during and / or after contact of blood with artificial surfaces, interstitial lung disease (e.g., fibroproliferative and / or idiopathic pulmonary fibrosis), inflammation, neuroinflammatory disease, complement activation, fibrinolysis, angiogenesis, clot formation due to FVIIIa-FIXa complex formation, clot formation due to FX activation, clot formation due to FIIa amplification, and retinal vascular permeability-related diseases (such as embolism); preferably, diseases associated with arterial or capillary thrombosis include, but are not limited to, myocardial infarction, stroke, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome or Paget-Schroetter disease.

[0027] In another aspect of the present invention, there is provided a kit comprising the antithrombotic monoclonal antibody or antigen-binding fragment thereof, the fusion protein, or the immunoconjugate, or a pharmaceutical composition containing any of them.

[0028] In another aspect of the present invention, there is provided a method for screening a substance (including a potential substance) having antithrombotic function, the method comprising: (1) A candidate substance is added to a system containing FIXa and FVIIIa, where the two interact with each other (e.g., form an FVIIIa-FIXa complex); (2) Detecting the interaction between FIXa and FVIIIa in the system; if the candidate substance binds to FIXa in competition with FVIIIa and reduces the formation of the FVIIIa-FIXa complex, it indicates that the candidate substance is a substance (including a potential substance) with antithrombotic function; preferably, predicting the binding site of the candidate substance and the FIXa complex by a method for measuring the docking of FIXa and FVIIIa proteins, more preferably, observing the situation in which the candidate substance acts on the binding site of FIXa and FVIIIa on FIXa or an adjacent site, thereby detecting the candidate substance. The function (competitive binding ability) of the co-substance is determined, and the binding site or adjacent sites include the following sites: Asn93, Lys132, Arg165, Thr175, preferably the following sites: Ala95, Lys98, Asp164, Lys173, Tyr177, more preferably the following sites: Lys126, Asn129, Asn178, Lys230, Arg233, Asn236; preferably, the amino acid residues of the above sites form a cluster and occupy a common surface located between the FIXa protein c170-helix and c131-helix.

[0029] In one preferred example, when the function of a candidate substance is determined by observing the situation in which the candidate substance acts on the binding site of FIXa and FVIIIa or an adjacent site on FIXa, if the candidate substance shows strong (significant) binding to the above site, it is a substance (including a potential substance) with antithrombotic function.

[0030] In another preferred embodiment, the "reduction" (also referred to as weakening, attenuation, etc.) is a statistically significant or significant reduction, for example, a reduction of the FVIIIa-FIXa complex by 5%, 10%, 15%, 20%, 30%, 50%, 60%, 80%, 90%, 95% or more.

[0031] In another preferred embodiment, a control group is set up to clearly identify differences in the interaction between FIXa and FVIIIa in the test group compared to the control group.

[0032] In another preferred example, the candidate substance includes, but is not limited to, a regulatory molecule designed against FIXa or its upstream or downstream proteins or genes, such as an antibody, an interference molecule (e.g., an interfering RNA), a small molecule compound, a gene modification or gene editing construct, etc.

[0033] Based on the disclosure herein, other aspects of the present invention will be apparent to those skilled in the art. [Brief explanation of the drawings]

[0034] [Figure 1A] FIG. 1A shows the affinity of the FIXa-4 antibody to FIXa. [Figure 1B] FIG. 1B shows the activated partial thromboplastin time of the FIXa-4 antibody. [Figure 1C] FIG. 1C shows the prothrombin time of the FIXa-4 antibody. [Figure 2] FIG. 2 shows the effect of FIXa-4 antibody on FIXa enzyme activity. [Figure 3] Figure 3 shows the binding interface between FIXa-4 antibody and FIXa. (A) The catalytic trimer His57-Asp102-Ser195 (green, indicated by an arrow) of FIXa (blue, left-hand structure) is not inhibited by the binding of FIXa-4 antibody (red, right-hand structure). (B) The contact area between FIXa-4 antibody (red, right-hand structure) and FIXa (blue, left-hand structure) covers part of the predicted binding site between FIXa and FVIIIa (yellow, indicated by an arrow). (C) A reverse view of Figure B. The left side of the figure is the FIXa-4 antibody structure, and the right side is the FIXa structure; the arrow indicates part of the predicted binding site between FIXa and FVIIIa. [Figure 4A] FIG. 4A shows the inhibitory effect of FIXa-4 antibody on FXa production. [Figure 4B] FIG. 4B shows that FVIIIa corrects the inhibitory effect of FIXa-4 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0035] After extensive research, the present inventors have disclosed a high-affinity anti-FIXa antibody (FIXa-4) with unique properties that targets FIXa. It specifically targets the binding site between the blood coagulation factor FIXa and FVIIIa, rather than directly binding to the substrate catalytic site of FIXa, thereby reducing the formation of the FVIIIa-FIXa complex and blocking the conversion of FX to FXa, thereby exerting its antithrombotic effect. The antibody of the present invention has adequate antithrombotic activity and a wide therapeutic window without increasing the risk of bleeding. Furthermore, the antibody of the present invention targets the enzyme active site that does not directly bind to FIXa, so that once excessive anticoagulation effect occurs, it can be rescued by supplementing exogenous FVIII, thereby achieving the clinical needs for moderate antithrombotic effect and effectively avoiding bleeding problems caused by excessive effect.

[0036] term As used herein, blood coagulation factor IX (sequence: GenBank Accession No.: 2158) is also referred to as blood coagulation factor 9, blood coagulation factor nine, factor IX, FIX, F9, etc.; FIXa is the activated form of FIX. For example, FXIa is a Ca 2+ Upon the participation of FIX, it cleaves FIX, rendering it active FIX (FIXa). In some embodiments, it also includes variant forms, such as variant proteins obtained by one or more (1-20, more particularly 2, 3, 4, 5, or 10) amino acid substitutions, deletions, or insertions, while retaining the activity of FIX or FIXa.

[0037] As used herein, "antibody" or "immunoglobulin" is used herein as a general term to include full-length antibodies, single-chain antibodies, and all portions, domains, or fragments thereof (including, but not limited to, antigen-binding domains or fragments). Additionally, the term "sequence" as used herein (e.g., terms such as "immunoglobulin sequence," "antibody sequence," "single variable domain sequence," "VHH sequence," or "protein sequence") should generally be understood to include the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said sequence, unless a more restrictive interpretation is required herein.

[0038] As used herein, "monoclonal antibody" refers to a preparation of antibody molecules consisting of a single molecule. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0039] As used herein, "fusion partner" (FP) refers to another polypeptide fused to a target polypeptide, where the fusion partner can affect the functional properties of the fusion protein through several different mechanisms, such as extending the half-life of the target polypeptide in vivo. The fusion partner may perform one or more functions, including, but not limited to, a protein or active domain that acts to extend half-life in vivo, or a protein or active domain that acts to enhance or bind to an effector.

[0040] As used herein, the term "conjugate" refers to a product formed by covalently or non-covalently binding a functional molecule (including a polypeptide, a small molecule compound, a hydrophilic polymer, or a marker) to a monoclonal antibody described herein, wherein the hydrophilic polymer and the polypeptide can be attached at any suitable position, for example, at the N-terminus, C-terminus, or central portion of the polypeptide. Examples of the hydrophilic polymer include polysaccharides, polyalkylene glycols such as polyethylene glycol (PEG), polypropylene glycol (PPG), polyoxyethylene (PEO), copolymers of ethylene glycol and propylene glycol, and polyvinyl alcohol.

[0041] As used herein, "antithrombotic" can also be interpreted as "increasing vascular permeability" or "anticoagulation."

[0042] "Sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between sequences. "Sequence similarity" refers to the percentage of identical or conservative amino acid substitutions. Methods for assessing the level of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is usually measured using sequence analysis software. For example, identity can be determined using the BLAST program in the NCBI database.

[0043] An "effective amount" of a drug means the amount necessary to cause a physiological change in a cell or tissue to which it is administered.

[0044] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, means an amount that effectively achieves a desired therapeutic or prophylactic result, at dosages and for periods of time necessary. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0045] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is human.

[0046] The term "drug composition" refers to a formulation whose form effects the biological activity of the active ingredient contained therein and does not contain other ingredients that are unacceptably toxic to the subject receiving the composition.

[0047] "Pharmaceutically acceptable carrier" means an ingredient in a drug composition, other than an active ingredient, that is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0048] The term "treatment / prevention" refers to altering natural processes to treat disease in an individual, and may be a clinical intervention performed for prophylaxis or during the clinical pathological process. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing the direct or indirect pathological consequences of disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and relieving or improving prognosis.

[0049] The term "humanized antibody" refers to a molecule having an antigen-binding site substantially derived from a non-human immunoglobulin, where the remaining immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise complete variable domains fused to constant domains, or may comprise only the complementarity-determining regions (CDRs) grafted into appropriate framework regions within the variable domains. The antigen-binding site may be wild-type or may be modified by one or more amino acid substitutions to more closely resemble human immunoglobulins. Some forms of humanized antibodies retain all CDR sequences. Other forms have one or more CDRs altered relative to the original antibody.

[0050] The term "detectable marker" refers to a marker that can be bound to an antibody to determine the presence, absence, and amount of a specific target in a sample to be measured. The "detectable marker" may be, but is not limited to, an enzyme, a fluorescent marker, a nuclide, a quantum dot, colloidal gold, etc. More specifically, the detectable marker may be selected from the group consisting of horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, and glucose amylase.

[0051] antibody In the present invention, monoclonal antibodies targeting the blood coagulation factor FIXa are screened to investigate their antithrombotic function and mechanism of action. After extensive research and screening, an anti-FIXa antibody is provided that specifically targets the binding site between the blood coagulation factors FIXa and FVIIIa, reduces the formation of the FVIIIa-FIXa complex, blocks the conversion of FX to FXa, and exerts an antithrombotic effect. The present invention also includes antigen-binding fragments of the anti-FIXa antibody.

[0052] The inventors prepared highly purified monoclonal antibodies using hybridoma technology and monoclonal antibody cell expression and purification techniques. They evaluated the antithrombotic effects of the monoclonal antibodies using activated partial thromboplastin time (APTT) and prothrombin time (PT), measured their effects on FIXa enzyme activity using a chromogenic substrate assay, predicted the FIXa-antibody interaction binding site using protein-protein docking, and verified this binding site through competition experiments (indirectly using a chromogenic substrate assay). As a result, a high-affinity anti-FIXa monoclonal antibody, FIXa-4, was obtained. FIXa-4 significantly prolonged the APTT in a concentration-dependent manner. Mechanistic studies revealed that FIXa-4 occupies the binding region between FIXa and FVIIIa, rather than directly binding to the substrate catalytic site of FIXa. Therefore, the present invention provides a unique monoclonal antibody, FIXa-4, which binds to FIXa in competition with FVIIIa, inhibits the formation of the FVIIIa-FIXa complex, blocks the conversion of FX to FXa, and exerts antithrombotic effects.

[0053] In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain CDR1 of the anti-FIXa antibody or the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO: 3, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 5; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 8.

[0054] The anti-FIXa antibodies provided by the present invention may comprise framework regions FR, such as those listed in Table 1. However, the framework regions are not limited to the sequences listed in Table 1, and antibodies in which partial or entire sequences of the framework regions have been modified, such as chimeric or humanized antibodies formed by modification, are also included in the present invention.

[0055] The antigen-binding properties of an antibody are usually determined by the complementarity-determining regions (CDRs), which are regularly arranged with the FR regions, which are not directly involved in the binding reaction. These CDRs form a ring-like structure and are close to each other in a spatial structure through the β-sheet formed in the FRs between them, constituting the antigen-binding site of the antibody. The CDR regions are sequences of proteins of immunological interest, and the CDR regions of the antibodies of the present invention are completely novel.

[0056] The antibodies of the present invention may be intact immunoglobulin molecules or antigen-binding fragments, including, but not limited to, Fab fragments, Fd fragments, Fv fragments, F(ab)2 fragments, complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), domain antibodies, bivalent single-chain antibodies, single-chain phage antibodies, bispecific two-chain antibodies, triple-chain antibodies, quadruplex antibodies, and the like.

[0057] In a preferred embodiment of the present invention, the anti-FIXa antibody has a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 2. The present invention also includes antibodies having a heavy chain variable region amino acid sequence that is 85% or more identical to the sequence shown in SEQ ID NO: 1 and a light chain variable region amino acid sequence that is 85% or more identical to the sequence shown in SEQ ID NO: 2, which have the same functions as the antibodies described in the Examples of the present invention; preferably, the amino acids in the CDR regions of the heavy chain variable region / light chain variable region of the antibody are conserved.

[0058] The present invention includes functional variants of the above antibodies. These variants can specifically bind to FIXa in competition with the parent antibody, and their ability to recognize FIXa and their location of action are similar to those of the specific antibodies provided in embodiments of the present invention (which target the blood coagulation factor FIXa-FVIIIa binding site). The above functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions, and deletions. These modifications can be introduced by standard techniques known in the art (e.g., targeted mutagenesis and random PCR-mediated mutagenesis), and may include natural and unnatural nucleotides and amino acids. Preferably, the sequence modifications occur in regions other than the CDR regions of the above antibodies.

[0059] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof described in the present invention prolongs the activated partial thromboplastin time (APTT) by approximately 3.5-fold, has adequate antithrombotic properties, has a wide effective therapeutic concentration window, but does not increase the risk of bleeding.

[0060] Constructs and antibody expression systems The present invention also provides a construct comprising the isolated polynucleotide described herein. Methods for constructing such constructs are known to those skilled in the art. For example, the construct can be constructed by in vitro recombinant DNA technology, DNA synthesis technology, in vitro recombination technology, etc. More specifically, the construct can be constructed by inserting the isolated polynucleotide into a polycloning site of an expression vector. The expression vector of the present invention generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. The vector may contain one or more regulatory sequences operably linked to the polynucleotide sequence, and the regulatory sequence may include a suitable promoter sequence. The promoter sequence is usually operably linked to a sequence encoding the amino acid sequence to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, including mutated, truncated, and heterologous promoters, and may be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence may be a suitable transcription terminator sequence, which is a sequence recognized by a host cell to terminate transcription. The terminator sequence is linked to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that functions in the selected host cell can be used in the present invention.

[0061] Suitable vectors typically contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. These promoters include, but are not limited to, the lac or trp promoters of E. coli, the phage lambda PL promoter, eukaryotic promoters such as the CMV immediate-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the Pichia yeast alcohol oxidase promoter, and promoters containing other known controllable genes expressed in prokaryotic or eukaryotic cells or their viruses. Marker genes can be used to provide a phenotypic trait for selection of transformed host cells, and include, but are not limited to, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector may also contain an enhancer sequence, which, when inserted into the vector, enhances transcription. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs long, that act on a promoter to enhance transcription of a gene.

[0062] Another aspect of the present invention provides an antibody expression system, which comprises the above-described construct or has the above-described exogenous polynucleotide integrated into its genome. Any cell suitable for expressing an expression vector can be a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, including, but not limited to, one or more of the following: Escherichia coli, Streptococcus sp., Salmonella typhimurium bacterial cells; fungal cells, such as yeast, filamentous fungi, and plant cells; Drosophila S2 or Sf9 insect cells; and animal cells, such as CHO, COS, HEK293, or Bowes melanoma cells. Methods for constructing the expression system are known to those skilled in the art and include, but are not limited to, one or more combinations of microinjection, particle delivery, electroporation, viral-mediated transformation, electron bombardment, calcium phosphate precipitation, and the like.

[0063] Fusion Proteins / Immunoconjugates The present invention includes fusion proteins comprising a first domain of an antibody according to the present invention and a second domain for extending in vivo half-life and / or for binding to an effector or effector cell.

[0064] In the second domain, the fragment for extending the in vivo half-life may comprise serum albumin or a fragment thereof, a domain that binds to serum albumin (for example, an anti-serum albumin antibody), or the like.

[0065] In the second domain, the fragment having binding activity to an effector or effector cell may include an immunoglobulin Fc region, preferably selected from a human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations to alter Fc-mediated effector function, which may include one or more combinations of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin may be one or more combinations selected from IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG may be one or more combinations selected from IgG1, IgG2, IgG3, or IgG4 subtypes, etc. The immunoglobulin Fc region contained in the antibody fusion protein allows the fusion protein to form a dimer, thereby extending the in vivo half-life of the fusion protein and enhancing the relevant Fc-mediated activity. In one specific embodiment of the present invention, the immunoglobulin Fc region may be a human IgG1 Fc region, more specifically, a wild-type IgG1 Fc sequence, and mutations to alter Fc-mediated effector functions, such as a) mutations that alter Fc-mediated CDC activity; b) mutations that alter Fc-mediated ADCC activity; or c) mutations that alter Fc-mediated ADCP activity, can be introduced into the sequence. Such mutations are described in the following documents: 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.

[0066] In the anti-FIXa antibody fusion protein provided by the present invention, a linker peptide may be inserted between the first and second domains. The linker peptide may be a flexible polypeptide chain consisting of alanine (A) and / or serine (S) and / or glycine (G). The linker peptide may be 3 to 30 amino acids long, preferably 3-9, 9-12, 12-16, or 16-20 amino acids long. In another specific embodiment of the present invention, the linker peptide may be 8 or 15 amino acids long.

[0067] The present invention also provides an isolated polynucleotide, which encodes an antibody of the present invention or encodes the fusion protein; the polynucleotide may be RNA, DNA, cDNA, etc. Methods for providing the isolated polynucleotide will be known to those skilled in the art, and may be obtained by preparation, for example, automated DNA synthesis and / or recombinant DNA techniques, or may be isolated from a suitable natural source.

[0068] The present invention also provides immunoconjugates comprising an antibody according to the present invention or a fusion protein according to the present invention, which typically include a functional molecule attached (including, but not limited to, covalently bonded, coupled, attached, or adsorbed) to the antibody or fusion protein, including, but not limited to, a hydrophilic polymer, a detectable marker, a radioisotope, a biologically active protein, a molecule that targets a blood cell (e.g., platelet) surface marker, or the like, or a combination thereof.

[0069] The hydrophilic polymers include, but are not limited to, polyethylene glycol, polyethylene glycol-liposome complexes, polysaccharides, polyalkylene glycols, polypropylene glycol (PPG), polyoxyethylene (PEO), copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol, etc., or combinations thereof.

[0070] Methods for producing the immunoconjugates should be known to those skilled in the art. For example, the antibodies and / or fusion proteins can be linked to functional molecules directly or via a spacer of appropriate length, and the linking method may be chemical crosslinking or genetic engineering fusion expression, thereby obtaining the immunoconjugates.

[0071] The immunoconjugate may comprise an antibody or fusion protein of the invention and a detectable marker. The detectable marker may include, but is not limited to, a fluorescent marker, a colorimetric marker, or a protein label; for example, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron-emitting metal, and a non-radioactive paramagnetic metal ion. The immunoconjugate may comprise one or more markers. The marker with which the antibody is labeled for detection and / or analysis and / or diagnosis will depend on the particular detection / analysis / diagnostic technique and / or method used (e.g., immunohistochemical staining (tissue) samples, flow cytometry, etc.). Markers suitable for detection / analysis / diagnostic techniques and / or methods known in the art will be familiar to those of skill in the art.

[0072] The antibodies or fusion proteins of the present invention can be coupled with a marker group (a labeled polypeptide) and then used, for example, for diagnostic purposes. Suitable marker groups include or include radioisotopes (e.g., those described above) or groups containing radioisotopes or radionuclides, fluorescent groups (e.g., fluorescent proteins such as GFP and RFP, dyes, rhodamine, fluorescein and their derivatives, e.g., FITC, cyanine dyes), enzyme groups (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase), chemiluminescent groups, biotin groups, metal particles (e.g., gold particles), magnetic particles (e.g., those with a core containing magnetite (Fe3O4) and / or maghemite (Fe2O3)), and certain polypeptide groups.

[0073] The immunoconjugate may comprise an antibody or fusion protein of the present invention and a molecule that targets a surface marker on blood cells, such as platelets, which can recognize blood cells and facilitate the delivery of the antibody of the present invention to the blood cells.

[0074] Drug Compositions and Kits The present invention also provides a pharmaceutical composition comprising an anti-FIXa antibody of the present invention, or a fusion protein of an anti-FIXa antibody of the present invention, or an immunoconjugate of the present invention.

[0075] The pharmaceutical compositions may also contain various pharmaceutically acceptable carriers known in the art. Pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; chlorhexidine diammonium; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; hydrocarbyl p-hydroxybenzoates such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as serum proteins, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, and dextrin; chelating agents such as EDTA; tonicity adjusters such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose, or sorbitol; surfactants such as polysorbates; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEE®, PLURONICS®, or polyethylene glycol (PEG). Drug formulations for internal administration are typically sterile, and methods for achieving sterility in drug formulations should be well known to those skilled in the art and can be achieved, for example, by methods such as sterile filtration membrane filtration. Those skilled in the art can also select appropriate pharmaceutically acceptable carriers depending on the dosage form required for the drug composition, and prepare it into different dosage forms. For example, the drug composition of the present invention includes, but is not limited to, various dosage forms such as tablets, injections, and freeze-dried preparations.

[0076] The content of the monoclonal antibody, fusion protein, or immunoconjugate in the pharmaceutical composition is typically an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined based on the therapeutic target and the specific administration method. For example, the content of the monoclonal antibody, fusion protein, or immunoconjugate in the pharmaceutical composition may range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99% of the total mass of the pharmaceutical composition.

[0077] The monoclonal antibodies, fusion proteins, and immunoconjugates of the present invention may be administered as a single active ingredient or in combination therapy, i.e., with other drugs. For example, the combination therapy may comprise the monoclonal antibody, fusion protein, or immunoconjugate in combination with at least one other antithrombotic drug. For further example, the combination therapy may comprise the monoclonal antibody, fusion protein, or immunoconjugate in combination with an antibody targeting another blood cell-specific antigen.

[0078] The present invention also provides a test kit containing the antibody, fusion protein, or immunoconjugate described in the present invention. The kit may optionally include a container, a control (negative or positive control), a buffer, an auxiliary agent, etc., which can be selected by those skilled in the art according to the specific situation. The kit may also include an instruction manual to facilitate operation by those skilled in the art.

[0079] Purpose The present invention also provides the use of the antibody, fusion protein, immunoconjugate or pharmaceutical composition of the present invention in the preparation of a formulation, kit for alleviating or treating a thromboembolic disorder.

[0080] In the present invention, the "thromboembolic disease" includes venous, arterial or capillary thrombus formation, thrombus formation in the heart, thrombus formation during and / or after contact of blood with artificial surfaces, interstitial lung diseases (e.g. fibroproliferative and / or idiopathic pulmonary fibrosis), inflammation, neuritis diseases, complement activation, fibrinolysis, angiogenesis, clot formation due to FVIIIa-FIXa complex formation, clot formation due to FX activation, clot formation due to FIIa amplification, and retinal vascular permeability-related diseases (e.g., embolism).

[0081] However, diseases associated with arterial or capillary thrombosis include myocardial infarction, stroke, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome, or Paget-Schroetter disease.

[0082] A "therapeutically effective amount" of the fusion protein, immunoconjugate, or pharmaceutical composition provided herein preferably reduces the severity of disease symptoms, increases the frequency and duration of disease-free periods, or prevents damage or disability due to disease affliction. For example, in the treatment of thrombotic diseases, a "therapeutically effective amount" preferably reduces thrombus formation (or increases vascular permeability) by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, or more preferably at least about 80% relative to a subject not treated or a disease-free period in the same subject. The ability to inhibit thrombus formation can be evaluated in an in vitro reaction system, a cell model, or an animal model system. Those skilled in the art can select an appropriate therapeutically effective amount depending on the actual situation, such as the size of the subject, the severity of the subject's symptoms, and the specific composition or administration route selected. Treatment prescriptions (e.g., dosage determinations) can be determined by a physician and generally take into consideration factors including, but not limited to, the disease being treated, the patient's individual condition, the delivery site, the method of administration, and other factors. A prophylactically effective amount refers to an amount effective to achieve the desired prophylactic effect at the necessary dosage and for the necessary time. Because a prophylactic dose is usually, but not necessarily, administered to a subject before the onset of disease or at an early stage of disease, a "prophylactically effective amount" will usually be lower than a "therapeutically effective amount."

[0083] The present invention further provides a method for detecting FIXa antigen using the antibody, including, but not limited to, qualitative detection, quantitative detection, and localization detection, specifically, the detection method includes, but is not limited to, immunofluorescence assay, immunohistochemistry, radioimmunoassay, etc.

[0084] A method for detecting the presence of FIXa antigen in a sample may include contacting the sample with an antibody of the present invention and observing whether an antibody complex is formed, where the formation of an antibody complex indicates the presence of FIXa antigen in the sample. The sample may be a cell and / or tissue sample; the sample may be fixed or lysed in a medium; and the level of FIXa antigen in the fixed or lysed sample is detected. In some embodiments, the target of detection may be a cell-containing sample present in a cell preservation solution. In another embodiment, the antibody is conjugated to a fluorescent dye, chemical, polypeptide, enzyme, isotope, label, etc. that can be used for detection or detected by other reagents.

[0085] In the present invention, the FIXa-4 antibody, unlike previously reported FIXa inhibitors, does not directly act on the catalytically active site of FIXa but occupies a large portion of the binding region between FIXa and FVIIIa, blocking the formation of the FIXa-FVIIIa complex and further affecting the catalysis of the conversion of FX to FXa, resulting in anticoagulant effects. The inventors also observed that FIXa-4 antibody can substantially block FXa production in vitro, while supplementation with FVIIIa can correct this inhibitory effect. Thus, the antithrombotic effect of FIX-4 antibody is partially reversed with increasing FVIIIa concentrations. Compared with existing antithrombotic drugs, the reversibility of the antithrombotic effect must be considered when developing new, safe, and effective antithrombotic drugs. This property can be utilized in clinical administration to prevent or treat uncontrollable bleeding side effects caused by excessive anticoagulation. Therefore, the inventor's discovery is particularly significant, and by taking into consideration the FIXa-4 antibody and the FIXa mode of action, it is possible to screen or design a series of drugs that similarly enable such competitive binding, which could potentially become drugs that combine blood coagulation regulation activity with safety.

[0086] Based on the new discovery of the present inventors, a method for screening substances with antithrombotic function is provided, which comprises the following steps: (1) adding a candidate substance to a system containing FIXa and FVIIIa, where the two interact with each other (for example, forming an FVIIIa-FIXa complex); (2) detecting the interaction between FIXa and FVIIIa in the system of (1); if the candidate substance binds to FIXa in competition with FVIIIa and reduces the formation of the FVIIIa-FIXa complex, this indicates that the candidate substance has antithrombotic function; preferably, the candidate substance binds to the FI on FIXa. The function of the candidate substance is determined by observing its effect on the binding site between Xa and FVIIIa or an adjacent site, which includes the following sites: Asn93, Lys132, Arg165, Thr175, preferably the following sites: Ala95, Lys98, Asp164, Lys173, Tyr177, more preferably the following sites: Lys126, Asn129, Asn178, Lys230, Arg233, Asn236. The amino acid residues at these sites, or portions thereof, form a cluster and occupy a common surface located between the c170-helix and the c131-helix of the FIXa protein.

[0087] Furthermore, when the function of a candidate substance is determined by observing the state in which the candidate substance acts on the FIXa-FVIIIa binding site or an adjacent site on FIXa, if the candidate substance shows enhanced binding to the above site, it is a substance with antithrombotic function.

[0088] Methods for screening substances that act on proteins or genes or specific regions thereof as targets are well known to those skilled in the art, and any of these methods can be used in the present invention. The candidate substances may be selected from peptides, polymeric peptides, pseudopeptides, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences. Depending on the type of substance to be screened, those skilled in the art will understand how to select an appropriate screening method. In some more specific embodiments, the candidate substances include, but are not limited to, regulatory molecules designed against FIXa or its upstream or downstream proteins or genes, such as antibodies, interference molecules (e.g., interfering RNA), small molecule compounds, gene modification or gene editing constructs, etc.

[0089] Through extensive screening, a series of potential substances that specifically act on the above-mentioned sites of interest and have regulatory effects can be obtained.

[0090] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely illustrative of the present invention and do not limit the scope of the present invention. Experimental methods in the following examples that do not specify specific conditions are usually carried out according to the general conditions described in J. Sambrook et al., Guide to Molecular Cloning, Third Edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0091] When an embodiment indicates a range of values, it should be understood that any value between the two endpoints of each range can be selected unless otherwise specified. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, the present invention can be realized using any methods, equipment, and materials in the prior art that are similar or equivalent to the methods, equipment, and materials described in the examples of the present invention, based on the understanding of the prior art by those skilled in the art and the description of the present invention.

[0092] 1. Materials and Methods 1.1 Production of monoclonal antibodies targeting FIXa 1.1.1 Immunized mice and cell fusion Four 6-8 week-old SPF BALB / c male mice (purchased from the Department of Animal Science, School of Medicine, Shanghai Jiao Tong University) were immunized. For the first immunization, purchased full-length 46 KD FIXa antigen (Enzyme Research Laboratories) and complete Freund's adjuvant (Sigma) were mixed to a final FIXa concentration of 100 μg / 100 μl, and the mixture was injected into both footpads at 50 μg / 50 μl. For the second immunization, complete Freund's adjuvant was replaced with incomplete Freund's adjuvant (Sigma). For the third immunization, FIXa antigen was diluted with PBS to 100 μg / 100 μl and injected intraperitoneally. For the fourth immunization, a booster immunization was performed by tail vein injection. A total of four immunizations were performed, with two-week intervals between immunizations.

[0093] Three days after the immunization, spleen cells from the immunized mice were fused with myeloma cells (laboratory cell line SP2 / 0) under the effect of PEG to obtain hybridoma cells.

[0094] 1.1.2 Screening and cloning of antibody-positive cell lines After 7 days of hybridoma cell culture, antibody positivity was tested using ELISA. ELISA plates (Corning) were coated with 0.1 μg / 100 μl of antigen FIXa and incubated at 37°C for 1.5 h. Blocking was performed with 2% BSA at 37°C for 1 h. Primary antibody (cultured cell supernatant) was then added and incubated at 37°C for 1.5 h. Mouse secondary antibody was then added and incubated at 37°C for 30 min. Finally, chromogenic substrate TMB (Thermo) was added and incubated at room temperature for 5 min in the dark. The reaction was then stopped by adding 2M H2SO4. Antibody-positive cell lines were screened based on absorbance values ​​measured at 450 nm using a spectrophotometer (Thermo Fisher Scientific). Clonal culture was then performed in semi-solid medium (STEMCELL). After 7 days, single clones were selected into 96-well plates and rescreened using the ELISA method described above. Finally, antibody-positive single clones were obtained.

[0095] 1.1.3 Antibody sequencing A sufficient amount of cell sediment was collected, RNA was extracted, and then reverse transcribed into cDNA. Then, appropriate primers were selected to perform PCR, and finally, the PCR products were sent to a company for sequencing.

[0096] 1.1.4 Preparation of ascites fluid and antibody purification Four 6- to 8-week-old male nude mice (purchased from the Department of Animal Science, Shanghai Jiao Tong University School of Medicine) were selected. Pristane (Sigma) was injected at 0.5 ml per mouse 7 days prior to the injection, and then 0.5-1 × 10 sera were intraperitoneally injected into the mouse. 6 After 7-10 days, abdominal swelling of the nude mice was observed, and the left lower abdominal cavity was pierced with a needle. The ascites was collected in an EP tube and centrifuged at 5000 rpm for 10 minutes. The supernatant was collected and sodium azide was added to a final concentration of 0.02%.

[0097] Following the instructions for the Protein G Agarose Purification Resin (YEASEN), the collected ascites was balanced, loaded, washed, and eluted. Finally, the eluate was concentrated by ultrafiltration using a 30 kD ultrafiltration tube to obtain highly purified monoclonal antibodies.

[0098] 1.2 Antibody subtype and affinity detection According to the instructions of the Sigma subtype detection kit, the following steps were performed sequentially: antigen coating, blocking, incubation with the primary antibody (0.1 μg / 100 μl), incubation with the isotype-specific reagent at room temperature for 30 minutes, incubation with the secondary antibody (R-antiGoat-HRP), color development, and reaction with the stop solution. Finally, the absorbance at 450 nm was measured. Antibody affinity detection was the same as the above ELISA method.

[0099] 1.3 Blood coagulation measurement The blood coagulation activity of the antibody was evaluated by activated partial thromboplastin time (APTT) and prothrombin time (PT).

[0100] 1.3.1 Activated partial thromboplastin time (APTT) First, the instrument and related reagents were preheated to 37°C. Then, the antibody was diluted with OVB buffer (SIEMENS), the APTT program was selected, and the reaction cup was placed in the instrument. 50 μl of APTT reagent, 50 μl of normal pooled plasma, and magnetic beads were added, and the start key was pressed to oscillate the magnetic beads. Next, 25 μl of antibody was added, and the timing key was pressed to initiate timing. After 180 s, 25 μl of 50 mM CaCl2 (SIEMENS) was added, and the measurement key on the handle was simultaneously pressed. The oscillation of the magnetic beads was observed until the plasma clot occurred, and the number of seconds was recorded on the instrument. Finally, a curve was plotted based on the logarithm of the final antibody concentration in the plasma versus the blood clotting time in seconds.

[0101] 1.3.2 Prothrombin time (PT) First, the instrument and reagents were preheated to 37°C. Then, the antibody was diluted with normal pooled plasma, the PT program was selected, and the reaction cup was placed in the reaction vessel. Magnetic beads were added, and the start key was pressed to oscillate the magnetic beads. 50 μl of antibody-containing normal pooled plasma was added, and the timing key was pressed to initiate timing. After 60 seconds, 100 μl of PT reagent was added, and the measurement key on the handle was simultaneously pressed. The oscillation of the magnetic beads was observed until the plasma clotted, and the number of seconds was recorded on the instrument. A curve was plotted based on the logarithm of the final antibody concentration in the plasma versus the number of seconds.

[0102] 1.4 Detection of the effect of antibodies on enzyme activity by the chromogenic substrate method for FIXa First, the spectrophotometer was set to 37°C, and the kinetic cycle settings were set to 10 min total time, 5 s interval, and 121 cycles. Following the instructions for the FIXa chromogenic substrate SPECTROZYME (IMMBIOMED), 100 μl Tris Buffer (50 mM TRIS, 100 mM NaCl, 5 mM CaCl2, pH 7.4, 33% ethylene glycol), 10 μl 2 μM FIXa protein, 2.5 μl antibody (final concentrations: 1000, 10, 1, 0.1, 0.01 μg / ml), and control TBS were added to a 96-well plate in that order; finally, 12.5 μl 10 mM FIXa chromogenic substrate was added to initiate the reaction. The change in absorbance at 405 nm (ΔOD / min) was measured.

[0103] 1.5Prediction of the interaction binding site between FIXa and FIXa-4 monoclonal antibody 1.5.1 Building the FIXa model A three-dimensional model of FIXa was constructed based on the crystal structure of the FIXa catalytic domain in an inhibitor complex (PDB entry: 3 LC 3).

[0104] 1.5.2 Construction of FIXa-4 monoclonal antibody model The three-dimensional structures of the antibody variable regions were predicted by Abody Builder in SabPred (Venkateswarlu D. Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: a computational protein-protein docking and molecular dynamics refinement study. Biochem Biophys Res Commun. 2014 Sep 26;452(3):408-14.).

[0105] 1.5.3 Antibody-FIXa docking The antibody and FIXa were docked using ClusPro, and the antibody pattern was used to mask the non-CDR regions of the antibody during the docking process (Dunbar J et al., SAbPred: a structure-based antibody prediction server. Nucleic Acids Res. 2016 Jul 8;44(W1):W474-8).

[0106] 1.5.4 Description of antibody-FIXa interactions A graph describing the interaction between FIXa and the antibody was generated using the open-source PyMOL (version 2.5.0).

[0107] 1.6FIXa-4 antibody binds to FIXa in competition with FVIIIa 1.6.1 FVIII activation To 125 μl of buffer (20 mM Hepes, 300 mM NaCl, 2.5 mM CaCl), 100 μl of 10 U / ml thrombin and 25 μl of 4 mg / ml FVIII were added and incubated at 37°C for 10 minutes to obtain FVIIIa (final concentration: approximately 0.4 mg / ml). Finally, 1 U of Hirudin was added to prevent degradation of FVIIIa.

[0108] 1.6.2 Antibodies inhibit the FIXa-FVIIIa complex from activating FX First, 50ng / 2μl of phosphatidylcholine (PC), phosphatidylserine (PS) mixture, 5μl of 20nM FIXa, and 10μl of 3μM FX were mixed, followed by 8μl of premixed 80μg / ml FVIIIa and 5μl of antibody, and the mixture was incubated at 37℃ for 15 minutes. The reaction was terminated by adding 20μl of 20mM EDTA, and finally, the mixture was diluted with TBS-Ca containing 0.1% PEG 8000. 2+ 150 μl of buffer was added to dilute the reaction product.

[0109] The microplate reader was then set to 37°C, and the kinetic cycle settings were 5 min total time, 10 s interval, and 31 cycles. 40 μl of the reaction mixture was added to a 96-well plate, followed by 40 μl of chromogenic substrate S2765 (1 mM), to initiate the reaction. The change in absorbance at 405 nm (ΔOD / min) was measured.

[0110] 1.6.3 FVIIIa counteracts the antibody-mediated inhibitory effect on FX activation by the FIXa-FVIIIa complex An appropriate antibody working concentration was selected in reaction 1.6.2, and in the same manner, 5 μl of antibody in the above reaction system was replaced with 2.5 μl of FVIIIa and 2.5 μl of antibody (the final concentration remained unchanged), and the change in absorbance value at 405 nm, ΔOD / min, was measured.

[0111] 1.6.4 Establishing a standard curve of FXa production and substrate degradation rate First, 25 μl of FX (200 nM), 25 μl of Russell Viper Venom (RVV) (42 nM), and 50 μl of TBS-Ca 2+ After mixing with a buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl2, 0.1% BSA) and incubating at 37°C for 30 minutes, FX was activated to FXa (final concentration 10 nM).

[0112] The spectrophotometer was then set to 37°C, and the kinetic cycle settings were set to a total time of 5 min, 10 s interval, and 31 cycles. The reaction was initiated by adding 40 μl of a gradient of FXa and 40 μl of the chromogenic substrate S2765 (1 mM) to a 96-well plate. The change in absorbance at 405 nm (ΔOD / min) was measured and used as the substrate degradation rate. A standard curve was plotted based on the final FXa concentration and the corresponding reaction rate. [Example]

[0113] Antibody acquisition Through extensive research and screening using mouse immunization, hybridoma fusion, antibody sequencing, cell expression and purification techniques, the inventors have isolated a monoclonal antibody with high affinity to human FIXa.

[0114] The half maximal effective concentration (EC50) of the antibody was measured, and the result was EC50=94.67 ng / ml, as shown in FIG. 1A.

[0115] The present inventors named the monoclonal antibody FIXa-4 antibody.

[0116] The amino acid sequence of the heavy chain variable region of the FIXa-4 antibody is shown below (SEQ ID NO: 1): QVTLKESGPGILKPSQTLSLTCSFS GFSLNTPGMG VGWIRQPSGKGLEWLAH IWWDDDK YYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYC ARSDDVSYALDY WGQGTSVTVSS The amino acid sequence of the light chain variable region of the FIXa-4 antibody is shown below (SEQ ID NO: 2): DIQMTQSPASLSASVGETVTITCRAS ENIDSY LAWYQQKQGKSPQLLVY NAK TLADGVPSRFSGSGSGTQFSLKIDSLQPEDFGSHYC QHHDGTTWT FGGGTKLEIK However, the sequences of the CDR regions and their adjacent framework regions are shown in Table 1.

[0117] [Table 1] [Example]

[0118] Evaluate the anticoagulant effect of monoclonal antibodies using APTT and PT Next, we examined the effects of the monoclonal antibody on activated partial thromboplastin time (APTT) and prothrombin time (PT) to evaluate its role in the intrinsic and extrinsic blood coagulation pathways. We added different concentrations of the monoclonal antibody (final concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0.78 μg / ml) to the APTT or PT reaction system.

[0119] The experimental results showed that FIXa-4 antibody significantly prolonged the APTT compared with the control group. Furthermore, with increasing FIXa-4 antibody concentration, the APTT-prolonging effect also increased, reaching a maximum of 88.8 seconds, 3.5 times longer than the control group's 25.5 seconds (IC50 = 7.705 μg / ml, as shown in Figure 1B). This prolongation effect is significantly higher than the 2.5-fold APTT prolongation required by current antithrombotic drugs. Furthermore, with increasing antibody concentration, uncontrolled prolongation of the APTT did not occur, and the APTT prolongation rate was limited to a 3.5-fold range. These results indicate that FIXa-4 has a wide therapeutic window and is unlikely to increase the risk of bleeding, which is highly in line with clinical practice.

[0120] At the same time, measurements on prothrombin time (PT) show that FIXa-4 antibody does not substantially affect PT, as shown in FIG. 1C.

[0121] The above experimental data revealed that FIXa-4 antibody specifically inhibits the intrinsic blood coagulation pathway, but does not act on blood coagulation factors in the extrinsic blood coagulation pathway and the common blood coagulation pathway, such as FX and FII. [Example]

[0122] FIXa-4 antibody does not directly bind to the catalytic active site of FIXa In the intrinsic blood coagulation pathway, FIXa acts as an enzyme, catalyzing the formation of FXa from FX, mediating the blood coagulation cascade. Therefore, to investigate the anticoagulation mechanism of FIXa-4 antibody, we first examined its effect on FIXa catalytic activity. The enzyme-catalyzed decomposition rate of the substrate is used to reflect the catalytic activity of the enzyme. We mixed different concentrations of FIXa-4 antibody (final concentrations of 1000, 10, 1, 0.1, and 0.01 μg / ml) with FIXa protein, incubated for 3 minutes, and then added the mixture to a FIXa chromogenic substrate reaction system. OD 405 nm was monitored in real time. ΔOD 405 / min reflected the catalytic activity of the enzyme.

[0123] The experimental results, as shown in FIG. 2, show that FIXa-4 antibody has no obvious effect on the enzymatic catalytic activity of FIXa.

[0124] These experimental results revealed that the FIXa-4 antibody does not exert its anticoagulant activity by directly binding to the catalytically active site of FIXa. [Example]

[0125] Prediction of the site of action of FIXa-4 antibody and FIXa To further explore the mechanism of the anticoagulant effect of the FIXa-4 antibody, we used protein docking to predict the binding site between the FIXa-4 antibody and FIXa. The protein database (PDB) does not contain a complete experimentally determined structure of the full-length FIXa protein. Considering the primary focus of this study on the effect of antibodies on FIXa catalytic activity, we constructed a 3D model of the FIXa catalytic domain based on the inhibitor complex structure of the FIXa catalytic domain (PDB entry: 3LC3). At the same time, we used an antibody structure prediction tool to construct a 3D model of the FIXa-4 antibody binding region and predicted the optimal conformational ensemble for binding between the two by protein-protein docking. This conformational ensemble contained a total of 979 docking conformations, organized into 30 clusters. Observation of representative conformations in each cluster revealed that the majority of the binding conformations between FIXa and the FIXa-4 antibody share a single binding surface, with the frequently contacting residues listed in Table 2. In all conformations, binding between the FIXa-4 antibody and FIXa did not inhibit substrate entry or exit from the FIXa catalytic domain (the catalytic trimer formed by His57-Asp102-Ser195) (Figure 3A). Although conformational changes of FIXa and the antibody itself were not considered during the docking process, given the large distance between the binding site and the catalytic domain, the relatively stable three-dimensional structure of the catalytic domain, and the low likelihood of significant deformation due to binding, we can initially confirm that binding of the FIXa-4 antibody does not directly affect FIXa catalysis.

[0126] These contact residues overlap considerably with the previously predicted FIXa-FVIIIa binding interface [DeLano WL (2002) The PyMOL molecular graphics system] (Fig. 3B and 3C). For example, residues such as Asn-c93, Lys-c132, Arg-c165, and Thr-c175 of FIXa in Table 1 also bind to FVIIIa. Furthermore, residues Ala-c95, Lys-c98, Asp-c164, Lys-c173, and Tyr-c177 are close in sequence to the predicted FIXa-FVIIIa binding residues, each of which is less than two residues long. In the 3D structure, these residues occupy a common surface between the c170-helix and c131-helix of the FIXa protein, which is the key binding region between the 558-helix of FVIIIa and FIXa, so the binding of the FIXa-4 antibody may compete with the binding of FVIIIa and FIXa. That is, according to the prediction of the FIXa-4 antibody and the FIXa catalytic domain binding site, the FIXa-4 antibody does not reduce the activity of FIXa by directly binding to the catalytic active site, but rather it binds to FIXa in competition with FVIIIa and inhibits the formation of the FVIIIa-FIXa complex, thereby suppressing the activation of FIXa by FVIIIa in the intrinsic blood coagulation pathway and ultimately reducing the catalytic activity of FIXa.

[0127] [Table 2] All residues in the table are numbered according to chymotrypsin. Considering residues within 3 Å of FIXa to the antibody, the frequency is

[0128]

number

[0129] FIXa-4 binds to FIXa in competition with FVIIIa As previously mentioned, FIXa-4 binds to FIXa in competition with FVIIIa, and the effect of FIXa-4 antibodies weakens with increasing FVIIIa concentration. First, we constructed an in vitro model to simulate FXa production from the FIXa-FVIIIa complex in vivo. PS / PC, FIXa, FVIIIa, and FX were added to this system, and FXa was generated by the reaction. The amount of FXa produced was then measured using an FXa chromogenic substrate assay.

[0130] We added different concentrations of FIXa-4 antibody (final concentrations: 1560, 780, 390, 156, 78, 39, 15.6, and 7.8 pM) to this system and found that FIXa-4 antibody inhibited FXa production in a dose-dependent manner (Fig. 4A). At a FIXa-4 antibody concentration of 400 pM, FXa production was almost completely inhibited. In this system, we increased the amount of FVIIIa to different concentrations (final concentrations: 1.1, 1.6, 2.5, 3.7, 5.6, 8.3, and 12.5 nM). With increasing FVIIIa concentrations, the inhibitory effect of the antibody was gradually corrected, reaching a correction rate of nearly 50% (Fig. 4B).

[0131] These experimental results demonstrated that FIXa-4 binds to FIXa in competition with FVIIIa and exerts its anticoagulant effect.

[0132] From the above, the monoclonal antibody FIXa-4 obtained by the present inventors, which targets FIXa, clearly has anticoagulant properties. Its mechanism of action is to bind to a site on FIXa in competition with FVIIIa, thereby inhibiting the formation of the FVIIla-FIXa complex and exerting its anticoagulant properties.

[0133] These findings provide a new target for antithrombotic therapy and indirect evidence for the binding site of FVIIIa and FIXa interaction.

[0134] Consider Currently used anticoagulants have various limitations, including unpredictable pharmacokinetics, lack of reversibility, and in some cases immunogenicity. The development of safe and effective antithrombotic drugs remains a hot topic in modern medicine. There are two important considerations when selecting a target blood coagulation factor. First, interrupt the blood coagulation cascade before significant amplification occurs. Second, target the rate-limiting step to provide effective anticoagulation activity within the broadest therapeutic range. FIXa is a key blood coagulation factor in the intrinsic blood coagulation pathway and the only soluble form of blood coagulation protein. FIXa can diffuse from tissue factor-bearing cells (TBCs) to platelets and is a key link between the initiation and amplification stages of the blood coagulation chain.

[0135] Monoclonal antibodies have strong antigen-binding specificity and a long half-life. With the development of monoclonal antibody technology, antibody drugs have played an important role in the prevention, diagnosis, and treatment of diseases. In this invention, a new anti-FIXa monoclonal antibody was obtained through hybridoma technology, antibody sequencing, cell expression, and purification techniques. The antithrombotic properties of FIXa-4 were demonstrated through APTT and PT function assays. The APTT-prolonging effect of FIXa-4 is dose-dependent. However, increasing the antibody concentration significantly did not result in uncontrolled APTT prolongation, and the APTT prolongation rate was limited to approximately 3.5-fold. These results demonstrate that FIXa-4 has a wide effective therapeutic concentration window and is unlikely to increase the risk of bleeding, meeting actual clinical needs.

[0136] The inventors have investigated the mechanism of action of this antithrombotic activity and found that, unlike previously reported FIXa inhibitors, the FIXa-4 antibody does not directly act on the catalytically active site of FIXa, but rather occupies a large portion of the binding region between FIXa and FVIIIa, blocking the formation of the FIXa-FVIIIa complex and further affecting the catalysis of the conversion of FX to FXa, resulting in anticoagulant effects. Interestingly, FIXa-4 antibody can substantially block FXa production in vitro, while supplementation with FVIIIa can reverse this inhibitory effect. Thus, the antithrombotic effect of FIX-4 antibody is partially reversed with increasing FVIIIa concentrations. Compared with existing antithrombotic drugs, the reversibility of the antithrombotic effect is currently a key consideration for developing new, safe, and effective antithrombotic drugs. This property can be utilized in clinical administration to prevent or treat uncontrollable bleeding side effects caused by excessive anticoagulation.

[0137] Based on the above, the inventor has prepared a new type of antithrombotic antibody that targets the FIXa-FVIIIa binding site, which not only provides a new concept for thrombosis treatment but also provides new evidence for the mode of action of the currently unclear FVIIIa-FIXa binding site.

[0138] All references mentioned in this application are incorporated herein by reference as if individually incorporated by reference. It should be understood that, based on the above disclosure of the present invention, one skilled in the art may make various changes or modifications to the present invention, and equivalents thereof are also included within the scope defined in the claims appended hereto.

Claims

1. The amino acid sequence of the heavy chain CDR1 of the antithrombotic monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO: 3, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 5; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 6, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 8; The antithrombotic monoclonal antibody or its antigen-binding fragment, characterized in that the antithrombotic monoclonal antibody retains FIXa-binding activity, and the antithrombotic monoclonal antibody prolongs activated partial thromboplastin time but does not affect prothrombin time.

2. The monoclonal antibody is (a) an antibody whose heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 1 and whose light chain variable region amino acid sequence is set forth in SEQ ID NO: 2; or (b) an antibody having a heavy chain variable region amino acid sequence that is 80% or more identical to the sequence shown in SEQ ID NO: 1 and a light chain variable region amino acid sequence that is 80% or more identical to the sequence shown in SEQ ID NO: 2, and having the antibody function of (a); The antithrombotic monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising:

3. The monoclonal antibody may be a murine antibody, a chimeric antibody, or a humanized antibody; or the monoclonal antibody or antigen-binding fragment thereof may be a single chain antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a F(ab') fragment, a ２ The antithrombotic monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that it contains a fragment thereof.

4. The antithrombotic monoclonal antibody or antigen-binding fragment thereof targets FIXa at the binding site between FIXa and FVIIIa or a site adjacent thereto, and reduces the formation of a FVIIIa-FIXa complex; preferably, in FIXa, the binding site between FIXa and FVIIIa or a site adjacent thereto includes Asn93, Lys132, Arg165, and Thr175, more preferably also includes Ala95, Lys98, Asp164, Lys173, and Tyr177, more preferably also includes Lys1 26, Asn129, Asn178, Lys230, Arg233, and Asn236; more preferably, the anti-thrombotic monoclonal antibody or antigen-binding fragment thereof does not affect the catalytic activity of FIXa; more preferably, the anti-thrombotic monoclonal antibody or antigen-binding fragment thereof does not bind to the catalytic site of FIXa comprising the His57-Asp102-Ser195 site.

5. An isolated polynucleotide, wherein the polynucleotide encodes the antithrombotic monoclonal antibody or its antigen-binding fragment described in claim 1, wherein the antithrombotic monoclonal antibody retains FIXa-binding activity.

6. A construct comprising the polynucleotide of claim 5, wherein the polynucleotide encodes the antithrombotic monoclonal antibody or its antigen-binding fragment of claim 1; the antithrombotic monoclonal antibody retains FIXa-binding activity; preferably, the construct is an expression vector.

7. An antibody expression system comprising the construct of claim 6; preferably a cell expression system.

8. A method for preparing an antithrombotic monoclonal antibody or its antigen-binding fragment, comprising expressing the antibody using the antibody expression system described in claim 7 under conditions suitable for expression; preferably also purifying and isolating the antibody; wherein the antithrombotic monoclonal antibody retains its FIXa-binding activity.

9. A fusion protein comprising the antithrombotic monoclonal antibody or its antigen-binding fragment according to claim 1 and a fusion partner operably linked thereto, wherein the fusion partner preferably comprises a protein or active domain having an effect of extending half-life in vivo, or a protein or active domain having a function of enhancing the effect on an effector or a binding activity; more preferably, the protein or active domain having an effect of extending half-life in vivo comprises an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, serum albumin or a fragment thereof, and preferably, the immunoglobulin is selected from the group consisting of IgG, IgA1, IgA2, IgD, and IgE. and IgM or a combination thereof, wherein the IgG is selected from one of IgG1, IgG2, IgG3, or IgG4, or a combination thereof, and preferably there is a linker peptide between the anti-thrombus monoclonal antibody or its antigen-binding fragment and the fusion partner operably linked thereto, the linker peptide being preferably selected from a flexible polypeptide chain consisting of alanine and / or serine and / or glycine, and the length of the linker peptide is preferably 3 to 30 amino acids; and the anti-thrombus monoclonal antibody retains FIXa-binding activity.

10. 10. An immunoconjugate comprising the antithrombotic monoclonal antibody or its antigen-binding fragment according to claim 1, or a fusion protein comprising said antithrombotic monoclonal antibody or its antigen-binding fragment; and a functional molecule connected thereto; wherein said antithrombotic monoclonal antibody retains FIXa-binding activity.

11. A pharmaceutical composition comprising the antithrombotic monoclonal antibody or its antigen-binding fragment described in claim 1, a fusion protein comprising said antithrombotic monoclonal antibody or its antigen-binding fragment, or an immunoconjugate comprising said antithrombotic monoclonal antibody or its antigen-binding fragment; preferably, said pharmaceutical composition further comprises a pharmaceutically acceptable carrier; and said antithrombotic monoclonal antibody retains its activity of binding to FIXa.

12. Use of the antithrombotic monoclonal antibody or its antigen-binding fragment according to claim 1, a fusion protein comprising said antithrombotic monoclonal antibody or its antigen-binding fragment, or an immunoconjugate comprising said antithrombotic monoclonal antibody or its antigen-binding fragment, or a pharmaceutical composition comprising any of them, in the preparation of a formulation or kit for alleviating or treating a thromboembolic disease; wherein said antithrombotic monoclonal antibody retains FIXa-binding activity; Preferably, the thromboembolic diseases include venous, arterial or capillary thrombosis, thrombus formation in the heart, thrombus formation during and / or after contact of blood with artificial surfaces, interstitial lung diseases, inflammation, neuroinflammatory diseases, complement activation, fibrinolysis, angiogenesis, clot formation due to FVIIIa-FIXa complex formation, clot formation due to FX activation, clot formation due to FIIa amplification, retinal vascular permeability related diseases; preferably, diseases associated with arterial or capillary thrombosis include myocardial infarction, stroke, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome or Paget-Schroetter disease.

13. A kit comprising the antithrombotic monoclonal antibody or its antigen-binding fragment according to claim 1, a fusion protein comprising said antithrombotic monoclonal antibody or its antigen-binding fragment, or an immunoconjugate comprising said antithrombotic monoclonal antibody or its antigen-binding fragment, or a pharmaceutical composition comprising any of them; wherein said antithrombotic monoclonal antibody retains FIXa-binding activity.

14. A method for screening a substance having antithrombotic function, comprising: (1) adding a candidate substance to a system containing FIXa and FVIIIa, wherein the FIXa and FVIIIa interact with each other; (2) A step of detecting the interaction between FIXa and FVIIIa; if the candidate substance binds to FIXa in competition with FVIIIa and reduces the formation of the FVIIIa-FIXa complex, the candidate substance is indicated to have antithrombotic function; preferably, the binding site of the candidate substance and the FIXa complex can be predicted by a protein docking method for measuring the binding of FIXa and FVIIIa proteins, and more preferably, the situation in which the candidate substance affects the binding of FIXa and FVIIIa at the FIXa binding site or a site adjacent thereto is observed. and more preferably, the binding site or adjacent sites comprise: Asn93, Lys132, Arg165, Thr175, more preferably also: Ala95, Lys98, Asp164, Lys173, Tyr177, more preferably also: Lys126, Asn129, Asn178, Lys230, Arg233, Asn236; preferably, the amino acid residues of said sites can form a cluster and occupy a common surface located between the c170-helix and the c131-helix of the FIXa protein; A method comprising:

15. The method of claim 14, wherein when the function of the candidate substance is determined by observing the situation in which the candidate substance affects the binding of FIXa and FVIIIa at the FIXa binding site or a site adjacent to it, if the candidate substance shows a strong binding activity, it is a substance with antithrombotic function, and preferably a control group is also included so as to clearly distinguish the difference between the interaction between FIXa and FVIIIa in the test group and the interaction between FIXa and FVIIIa in the control group.

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