Anti-TFPI monoclonal antibody

Abstract

Disclosed are monoclonal antibodies or antigen-binding fragments thereof that target tissue factor pathway inhibitor (TFPI), and their pharmaceutical uses.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof against tissue factor pathway inhibitor (TFPI) and its use as a medicine. [Background technology]

[0002] Blood coagulation is the process by which blood forms a stable clot to stop bleeding. This process involves many zymogens and cofactors (or "clotting factors") in the blood. These zymogens and cofactors interact in various ways to be converted sequentially or simultaneously to their activated forms. Ultimately, this process leads to the activation of prothrombin to thrombin by activated factor X (FXa) in the presence of factor Va, calcium ions, and platelets. Activated thrombin then induces platelet aggregation and converts fibrinogen to fibrin, which is then cross-linked by activated factor XIII (FXIIIa) to form a clot.

[0003] There are two unique ways to activate factor X: the contact activation pathway (formerly called the intrinsic pathway) and the tissue factor pathway (formerly called the extrinsic pathway). It is now known that the tissue factor pathway is the primary pathway that initiates blood clotting.

[0004] Factor X is activated by the complex formation of tissue factor (TF) and activated factor VII (FVIIa). The complex of FVIIa and its essential cofactor TF is a potent initiator of the coagulation cascade.

[0005] The tissue factor pathway of coagulation is negatively regulated by tissue factor pathway inhibitor (TFPI). TFPI is a natural, FXa-dependent feedback inhibitor of the FVIIa / TF complex and belongs to the multivalent Kunitz-type serine protease inhibitor family. Physiologically, TFPI binds to activated factor X (FXa) to form a heterodimeric complex, which then interacts with and inhibits the activity of the FVIIa / TF complex, shutting down the tissue factor pathway of coagulation. In principle, inhibition of TFPI activity can restore the activity of FXa and FVIIa / TF, prolonging the duration of action of the tissue factor pathway and amplifying FXa production. Hemophilia A and hemophilia B lack FXa.

[0006] There remains a need in the art for pharmaceutical agents that can inhibit TFPI activity, such as TFPI-specific antibodies, to treat coagulation-related disorders. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows the results of alignment of the amino acid sequences of the humanized light chain and mouse light chain (A), and the humanized heavy chain variant and mouse heavy chain (B). [Figure 2] FIG. 2 shows the affinity of the humanized h7G6 antibody to hTFPI. [Figure 3] FIG. 3 shows inhibition of FXa-TFPI interaction by humanized h7G6 antibody. BEST MODE FOR CARRYING OUT THE INVENTION

[0008] I definition In the present invention, all scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art, unless otherwise defined. Furthermore, all terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and experimental procedures used in this specification are terms commonly used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the terms are provided below.

[0009] As used herein, the term "tissue factor pathway inhibitor" or "TFPI" refers to variants, isoforms, and homologs of human TFPI that are naturally expressed by cells. A representative human TFPI comprises the amino acid sequence set forth in SEQ ID NO:28.

[0010] As used herein, "antibody" refers to immunoglobulins and immunoglobulin fragments, whether natural, partially synthetically, or wholly synthetically (e.g., recombinantly) produced, including fragments comprising at least a portion of the variable region of an immunoglobulin molecule and having the binding specificity of a full-length immunoglobulin. Thus, antibodies include proteins having a binding domain that is homologous or substantially homologous to the antigen-binding domain (antibody combining site) of an immunoglobulin. Antibodies also include antibody fragments, such as anti-tumor cell antibody fragments. Thus, as used herein, the term antibody includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intrabodies, and antibody fragments, including, but not limited to, Fab, Fab', F(ab')2, and Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fab (scFab) fragments, diabodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of these antibodies. As used herein, antibody includes immunoglobulin types (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses (e.g., IgG2a and IgG2b).

[0011] As used herein, an "antibody fragment" or an "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is shorter than the full length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antigen-binding sites), and thus retains at least a portion of the specific binding ability and binding specificity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding site that binds to the antigen to which the antibody derived from the antibody fragment binds. Antibody fragments include antibody derivatives obtained by treating a full-length antibody with an enzyme, and synthetically obtained derivatives, such as recombinantly obtained derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). Fragments may comprise multiple chains interconnected by, for example, disulfide bonds and / or peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids. Antigen-binding fragments include those that immunospecifically bind to an antigen (i.e., at least (about) 10 7 ~10 8 M -1 The term "antibody fragment" includes any antibody fragment that is inserted into an antibody framework (e.g., by replacing the corresponding region) to obtain an antibody exhibiting a Ka of 0.05 or less.

[0012] As used herein, the term "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This characteristic is the opposite of a polyclonal population of antibodies, which contains antibodies with a wide variety of sequences. Monoclonal antibodies can be produced by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917 and Nelson et al., J. Clin. Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared from immortalized B cells, such as by fusing them with myeloma cells to obtain a hybridoma cell line or by infecting B cells with a virus such as EBV. Recombinant technology can also be used to prepare antibodies in vitro from a cloned population of host cells by transforming the host cells with a plasmid containing an artificial sequence of nucleotides encoding the antibody.

[0013] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (usually a myeloma or lymphoma cell) obtained by fusing an antibody-producing lymphocyte with a cancer cell that does not produce an antibody. As known to those skilled in the art, a hybridoma can grow and provide a continuous supply of a specific monoclonal antibody. Methods for preparing hybridomas are known in the art (see, e.g., Harlow & Lane, 1988). The term "hybridoma" or "hybridoma cell" also includes subclones and progeny cells of a hybridoma.

[0014] As used herein, a "traditional antibody" refers to an antibody comprising two heavy chains (which may be denoted H and H'), two light chains (which may be denoted L and L'), and two antigen-binding sites, where each heavy chain may be a full-length immunoglobulin heavy chain or a functional region thereof having antigen-binding ability (e.g., a heavy chain may include a V H chain, V H -C H 1 chain and V H -C H 1-C H 2-CH 3 chains), each light chain can be a full-length light chain or a functional region thereof (e.g., a light chain can include, but is not limited to, a V L Chain and V L -C L Each heavy chain (H and H') is paired with one light chain (L and L', respectively).

[0015] As used herein, a full-length antibody is defined as an antibody that comprises two full-length heavy chains (e.g., V H -C H 1-C H 2-C H 3 or V H -C H 1-C H 2-C H 3-C H 4), two full-length light chains (V L -C L ) and hinge region, for example, in natural antibodies produced by antibody-secreting B cells or synthetic antibodies containing the same domains.

[0016] As used herein, dsFv refers to V H -V L Refers to an Fv that has an artificial intermolecular disulfide bond that stabilizes the pair.

[0017] As used herein, a Fab fragment is an antibody fragment obtained by papain digestion of a full-length immunoglobulin or a fragment of the same structure synthesized, for example, recombinantly. L and C L one light chain containing the variable domain of the heavy chain (V H ) and one constant domain (C H 1) and another chain containing

[0018] As used herein, an F(ab')2 fragment is an antibody fragment obtained by digesting an immunoglobulin with pepsin at pH 4.0-4.5, or a fragment of the same structure synthesized, for example, by recombinant means. An F(ab')2 fragment essentially contains two Fab fragments, with each heavy chain containing several additional amino acids, including a cysteine, that form a disulfide bond between the two fragments.

[0019] As used herein, a Fab' fragment is a fragment containing half (heavy and light chains) of an F(ab')2 fragment.

[0020] As used herein, an scFv fragment refers to a variable light chain (VLC) covalently linked by a polypeptide linker in any order. L ) and variable heavy chain (V H The linker length is such that the two variable domains can be bridged without substantial interference. A typical linker contains multiple Glu or Lys residues (Gly-Ser), which are advantageous for solubility. n It is a residue.

[0021] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0022] "Humanized antibody" refers to non-human (e.g., murine) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity-determining regions (CDRs) of the recipient antibody are replaced by residues from CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0023] Furthermore, humanization can involve mutation of amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL, thereby improving one or more binding characteristics (e.g., affinity) of the antibody. For example, mutations can be introduced by PCR mutagenesis, and their effect on antibody binding or other functional characteristics can be assessed by in vitro or in vivo assays described herein. Conservative mutations are typically introduced. Such mutations can be amino acid substitutions, additions, or deletions. Furthermore, mutations in the CDRs are generally limited to one or two or less. Therefore, the humanized antibodies of the present invention also include antibodies with one or two amino acid mutations in the CDRs.

[0024] As used herein, the term "epitope" refers to an antigenic determinant on an antigen to which a complementary portion of an antibody binds. Generally, an epitope contains chemically active surface structures of molecules such as amino acids or sugar side chains, and usually has a specific three-dimensional structure and a specific charge.

[0025] As used herein, a variable domain or variable region is a specific Ig domain of an antibody heavy or light chain, which differs in amino acid sequence in different antibodies. Each light and heavy chain contains a variable region V L and V H The variable domain provides antigen specificity and is involved in antigen recognition. Each variable region consists of CDRs, which are part of the antigen-binding site domain, and framework regions (FRs).

[0026] As used herein, the terms "antigen-binding domain" and "antigen-binding site" are used synonymously to refer to the domain in an antibody that recognizes and physically interacts with an antigen. A conventional, naturally occurring, full-length antibody molecule has two antigen-binding sites, each containing a heavy chain variable region and a light chain variable region. A conventional antigen-binding site contains the loops connecting the antiparallel β strands within the variable domain. An antigen-binding site may also contain other portions of the variable region. Each conventional antigen-binding site contains three hypervariable regions in the heavy chain and three hypervariable regions in the light chain. The hypervariable regions are also known as complementarity-determining regions (CDRs).

[0027] As used herein, the terms "hypervariable region," "HV," "complementarity-determining region," "CDR," and "antibody CDR" are used interchangeably to refer to the portions of each variable domain that together form the antigen-binding site of an antibody. Each variable domain contains three CDRs: CDR1, CDR2, and CDR3. For example, a light chain variable domain contains three CDRs: VL CDR1, VL CDR2, and VL CDR3; a heavy chain variable domain contains three CDRs: VH CDR1, VH CDR2, and VH CDR3. The three CDRs of a variable region are discontinuous in the linear amino acid sequence but are adjacent to each other in the folded polypeptide. The CDRs are located within the loops connecting the parallel beta-folded chains in the variable domain.

[0028] As explained herein and known to those skilled in the art, CDRs can be defined by Kabat or Chothia numbering (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917). Other methods for numbering the amino acid residues of CDRs are also known in the art. For example, AbM CDRs are a compromise between Kabat hypervariable regions and Chothia structural loops, and use AbM from Oxford Molecular, an antibody modeling software. "Contact" CDRs are based on analysis of available complex crystal structures. The residues of the CDRs according to each method are as follows:

[0029] [Table A]

[0030] On the other hand, as is well known in the art, it should be noted that the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat numbering. (i.e., one or more positions according to Kabat numbering may not be present in the actual sequence, or the actual sequence may contain more amino acid residues than indicated by Kabat numbering.) This generally means that the Kabat numbering may or may not correspond to the numbering of amino acid residues in the actual sequence. For example, CDRs may include extended CDRs such as 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 (LCDR3) for VL; and 26-35 (HCDR1), 50-65 or 49-65 (HCDR2), and 93-102, 94-102, or 95-102 (HCDR3) for VH.

[0031] In this specification, framework regions (FR) are domains of antibody variable regions within the β-fold; in terms of amino acid sequence, FR regions are relatively less variable than hypervariable regions.

[0032] As used herein, a "constant region" refers to a domain of an antibody heavy or light chain that contains an amino acid sequence that is relatively less variable than the amino acid sequence of the variable domain. In a conventional full-length antibody molecule, each light chain contains a single light chain constant region (C L ), and each heavy chain has C H 1. C H 2. C H 3 and C H one or more heavy chain constant regions (C H The full-length IgA, IgD, and IgG isoforms have C H 1. C H 2. C H 3 and hinge region, and IgE and IgM are C H 1. C H 2. C H 3 and C H 4. C H 1 and C LThe constant domains extend the Fab portion of the antibody molecule, facilitate interaction with antigens, and allow rotation of the Fab portion of the antibody. The constant regions of the antibody contribute to effector functions, including, but not limited to, eliminating antigens, pathogens, and toxins that specifically bind to the antibody by interacting with various cells, biomolecules, and tissues.

[0033] As used herein, "specific binding" or "immunospecific binding" of an antibody or antigen-binding fragment thereof are used interchangeably and refer to the ability of the antibody or antigen-binding fragment to form one or more non-covalent bonds with the antigen through non-covalent interactions between the antibody and the antibody-binding site of the antigen. The antigen may be an isolated antigen or may be present on tumor cells. Generally, an antibody that immunospecifically (or specifically) binds to an antigen binds to the antigen at a concentration of approximately 1 x 10 7 M -1 or 1 x 10 8 M -1 The affinity constant Ka (or 1 × 10 -7 M or 1 x 10 -8 The dissociation constant K d Affinity constants can be determined by standard kinetic methods of antibody reactions, such as immunoassays and surface plasmon resonance (SPR) (Rich and Myzka (2000) Curr. Opin. Biotechnology 11: 54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989)). Methods for detecting and monitoring binding kinetics in real time are known, and such instruments are commercially available (BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; see Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0034] As used herein, the term "compete" with respect to antibodies means that a first antibody or antigen-binding fragment thereof binds to an epitope in a manner similar to that of a second antibody or antigen-binding fragment thereof, and that binding of the first antibody to the corresponding epitope is detectably reduced in the presence of the second antibody compared to its absence. Alternatively, binding of the second antibody to its epitope in the presence of the first antibody can be, but is not necessarily, detectably reduced. That is, a first antibody may inhibit binding of a second antibody to its epitope without inhibiting binding of the second antibody to its epitope. However, if each antibody detectably inhibits binding of another antibody to its corresponding epitope or ligand, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes, regardless of whether the degree of binding is the same, greater, or less. All competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which this competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or fragment thereof), the present invention covers the competing antibodies and / or cross-competing antibodies and, based on the teachings of the present invention, will be understood by those skilled in the art to be usable in the methods disclosed herein.

[0035] As used herein, a "polypeptide" refers to two or more covalently linked amino acids. The terms "polypeptide" and "protein" are used interchangeably herein.

[0036] An "isolated protein," "isolated polypeptide," or "isolated antibody" means that the protein, polypeptide, or antibody is (1) not associated with components with which it naturally coexists, (2) free from other proteins of the same species, (3) expressed in the cells of another species, or (4) not found in nature. Thus, a chemically synthesized polypeptide or a polypeptide produced by cells other than those in nature that produce the polypeptide is "separated" from natural components. A protein can also be rendered substantially free of natural components, i.e., separated using protein purification techniques well known in the art.

[0037] Suitable conservative amino acid substitutions of peptides or proteins are known to those of skill in the art and can generally be made without altering the biological activity of the resulting molecule. In general, those skilled in the art will appreciate that single amino acid substitutions in non-essential regions of a polypeptide will not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0038] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing at least two nucleotides or nucleotide derivatives linked together, typically by phosphodiester bonds, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0039] As used herein, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules with which it occurs in nature. An "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or culture medium when recombinantly produced, or substantially free of chemical precursors or other chemical components in chemical synthesis. Exemplary isolated nucleic acid molecules herein include isolated nucleic acid molecules encoding the antibodies or antigen-binding fragments described herein.

[0040] Sequence "identity" has an art-established meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated by published techniques. Sequence identity can be determined over the entire length of a polynucleotide or polypeptide, or over a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). There are many methods for determining identity between two polynucleotides or polypeptides, and the term "identity" is widely known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0041] As used herein, "operably linked" with respect to a nucleic acid sequence, region, element, or domain means that the regions of nucleic acid are functionally related to each other. For example, a promoter may be operably linked to a nucleic acid that encodes a polypeptide, such that the promoter controls or mediates transcription of the nucleic acid.

[0042] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The amount of polypeptide expression can be determined by methods known in the art, including, for example, methods for determining the amount of polypeptide produced by a host cell. Such methods include, but are not limited to, quantification of polypeptide in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0043] As used herein, a "host cell" is a cell that receives, maintains, replicates, and amplifies a vector. A host cell can also be used to express a polypeptide encoded by a vector. When the host cell divides, the nucleic acid contained in the vector is replicated and amplified. A host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, Hela cells, and HEK cells such as HEK293 cells.

[0044] "Codon optimization" refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell by replacing at least one codon (e.g., approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon more frequently or most frequently used in the host cell's genes while maintaining the original amino acid sequence. Different species have different preferences for specific codons for amino acids. Codon preference (i.e., differences in codon usage between organisms) is often related to the efficiency of messenger RNA (mRNA) translation and is thought to depend on the characteristics of the codon being translated and the availability of transfer RNA (tRNA) molecules. The advantages of the tRNAs selected by a cell generally reflect the codons most frequently used in peptide synthesis. Therefore, genes can be custom-designed for optimal gene expression in a specific organism based on codon optimization. Codon usage tables are readily available, for example, from the Codon Usage Database available at www.kazusa.or.jp / codon / , and can be adapted to various methods. See Nakamura Y. et al., codon usage tabulated from the international DNA sequence databases: status for the year 2000. nucl. acids Res., 28:292 (2000).

[0045] As used herein, a "vector" is a replicable nucleic acid capable of expressing one or more heterologous proteins when the vector is transformed into an appropriate host cell. Vectors generally include vectors into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced by restriction digestion and ligation. Vectors also include those containing a nucleic acid encoding a polypeptide. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express / display the polypeptide encoded by the nucleic acid. Vectors are typically empty or can be engineered to integrate genes or portions thereof into a chromosome for genome integration. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles are well known to those skilled in the art.

[0046] As used herein, vector also includes "viral vectors." A viral vector is a virus designed to be operatively linked to a foreign gene to transfer the foreign gene into a cell (as a vehicle or shuttle).

[0047] As used herein, the term "expression vector" includes vectors capable of expressing DNA that is operably linked to regulatory sequences (e.g., promoter regions) that may affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct (e.g., a plasmid, bacteriophage, recombinant virus, or other vector that results in expression of the cloned DNA when introduced into an appropriate host cell). Suitable expression vectors are well known to those of skill in the art and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells, either free or integrated into the genome of the host cell.

[0048] As used herein, "treatment" of an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment. Treatment therefore includes prevention, amelioration, and / or cure. Prevention means preventing potential disease and / or preventing the worsening of symptoms or the onset of disease. Use of the antibodies or antigen-binding fragments thereof herein and the compositions herein as pharmaceuticals is also included in treatment.

[0049] As used herein, "efficacy" refers to the effect that results from treating an individual, typically altering (usually improving or alleviating) or eliminating a disease or condition.

[0050] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a substance, compound, material, or composition containing such a compound that is sufficient to produce at least a therapeutic effect after administration to a subject, i.e., an amount necessary to prevent, cure, ameliorate, block, or partially block the symptoms of a disease or disorder.

[0051] As used herein, the term "patient" refers to a mammal, such as a human.

[0052] II Anti-TFPI monoclonal antibody In one aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof against TFPI, the monoclonal antibody comprising a light chain variable region and a heavy chain variable region, The light chain variable region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence in which one or two amino acid residues are substituted, deleted or added relative to the amino acid sequence shown in SEQ ID NO: 12; VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence in which one or two amino acid residues are substituted, deleted or added relative to the amino acid sequence set forth in SEQ ID NO: 13; and VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence in which one or two amino acid residues have been substituted, deleted, or added relative to the amino acid sequence set forth in SEQ ID NO: 14. Including, The heavy chain variable region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which one or two amino acid residues are substituted, deleted or added relative to the amino acid sequence shown in SEQ ID NO: 7; VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence in which one or two amino acid residues are substituted, deleted or added relative to the amino acid sequence set forth in SEQ ID NO: 8; and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence in which one or two amino acid residues are substituted, deleted or added relative to the amino acid sequence set forth in SEQ ID NO: 9; Includes.

[0053] In one aspect, the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, The light chain variable region VL CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12; VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and VL CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14; Including, The heavy chain variable region VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7; VH CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8; VH CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; Includes.

[0054] In some embodiments, the monoclonal antibody is a humanized antibody.

[0055] In one embodiment, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 11. In one embodiment, the light chain variable region comprises an amino acid sequence that has about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0056] In certain embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:6, or an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:6. In certain embodiments, the heavy chain variable region comprises an amino acid sequence that has about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:6.

[0057] In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 (humanized heavy chain variable region #1). In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 (humanized heavy chain variable region #2).

[0058] In one embodiment, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17 (humanized light chain variable region #1). In one embodiment, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 (humanized light chain variable region #2). In one embodiment, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19 (humanized light chain variable region #3).

[0059] In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 17. In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18. In one embodiment, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0060] In one embodiment, the heavy chain of the monoclonal antibody further comprises a human IgG4 constant region or a variant thereof, for example, the variant of the human IgG4 constant region comprises the amino acid sequence set forth in SEQ ID NO:20.

[0061] In one embodiment, the heavy chain of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22.

[0062] In one embodiment, the light chain of the monoclonal antibody further comprises a human Igκ constant region or a variant thereof, for example, the human Igκ constant region comprises the amino acid sequence set forth in SEQ ID NO:23.

[0063] In one embodiment, the light chain of the monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0064] In one embodiment, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 24. In one embodiment, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In one embodiment, the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0065] In one aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof against TFPI, which competes for binding to TFPI with an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6.

[0066] In one aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof against TFPI, which competes with an antibody comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 6 for binding to the same epitope on TFPI.

[0067] In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention specifically bind to TFPI. In certain embodiments, the monoclonal antibodies or antigen-binding fragments thereof of the present invention can inhibit the interaction of FXa and TFPI.

[0068] III. Methods for producing nucleic acids, vectors, and antibodies In another aspect, the present invention relates to an isolated nucleic acid molecule encoding the above-described antibody or antigen-binding fragment thereof of the present invention. For example, the nucleic acid molecule may encode the light chain and / or the heavy chain of the above-described antibody or antigen-binding fragment thereof of the present invention.

[0069] In one embodiment, the base sequence of the nucleic acid molecule is codon optimized for the host cell used for expression.

[0070] In one embodiment, the nucleic acid molecule comprises the base sequence set forth in SEQ ID NO:5 and / or SEQ ID NO:10.

[0071] In one aspect, the nucleic acid molecules of the invention are operably linked to regulatory sequences for expression.

[0072] The present invention also relates to an expression vector comprising the above-described nucleic acid molecule of the present invention.

[0073] The present invention also relates to a host cell transformed with the above-described nucleic acid molecule or expression vector of the invention.

[0074] In another aspect, the invention relates to a method for producing an antibody or antigen-binding fragment thereof of the invention, the method comprising: (i) culturing a host cell of the invention under conditions suitable for expression of the nucleic acid molecule or expression vector; and (ii) isolating and purifying the antibody or antigen-binding fragment thereof expressed by the host cell; Includes.

[0075] The present invention also includes isolated antibodies or antigen-binding fragments thereof obtained by the methods of the present invention that are capable of specifically binding to TFPI and / or inhibiting the interaction of FXa and TFPI.

[0076] IV. Medicinal Use The monoclonal antibodies or antigen-binding fragments thereof of the present invention can be used to treat coagulation-related disorders, such as inherited or acquired coagulation factor disorders. For example, the monoclonal antibodies or antigen-binding fragments thereof of the present invention can be used to inhibit the interaction between TFPI and FXa or prevent the inhibition of TF / FVIIa activity by TFPI. Furthermore, the monoclonal antibodies or antigen-binding fragments of the present invention can be used to restore TF / FVIIa-driven FXa production and circumvent the lack of FVIII- or FIX-dependent FXa amplification.

[0077] The monoclonal antibodies or antigen-binding fragments thereof of the invention can be used to treat coagulation-related disorders such as thrombocytopenia, platelet disorders, and bleeding disorders (eg, hemophilias such as hemophilia A, hemophilia B, and hemophilia C).

[0078] Accordingly, the present invention relates to methods for treating coagulation-related disorders, such as thrombocytopenia, platelet disorders, and bleeding disorders (e.g., hemophilias such as hemophilia A, hemophilia B, and hemophilia C), comprising administering to a patient in need thereof a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0079] The monoclonal antibodies or antigen-binding fragments thereof of the invention can also be used to treat uncontrolled bleeding, such as trauma or hemorrhagic stroke.

[0080] Accordingly, the present invention further relates to a method for reducing bleeding time, comprising administering to a patient in need thereof a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0081] The monoclonal antibodies or antigen-binding fragments thereof of the invention can be used in monotherapy or in combination with other therapies to treat coagulation-related disorders. For example, the monoclonal antibodies or antigen-binding fragments thereof of the invention can be administered simultaneously with a coagulation factor such as factor VII, factor VIII, or factor IX to treat hemophilia.

[0082] Accordingly, the present invention relates to a method for treating a coagulation-related disorder, such as an inherited or acquired coagulation factor disorder, comprising administering a monoclonal antibody or antigen-binding fragment thereof of the invention in combination with a coagulation factor. In one embodiment, the coagulation factor is factor VII, factor VIII, or factor IX. In one embodiment, the inherited or acquired coagulation factor disorder is, for example, hemophilia.

[0083] The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0084] As used herein, a "pharmaceutically acceptable carrier" is a substance that may be added to an active pharmaceutical ingredient to aid in the formulation or stabilization of a formulation without causing significant toxicity to the patient, and includes, but is not limited to, a disintegrant, adhesive, filler, buffer, isotonicity agent, stabilizer, antioxidant, surfactant, or lubricant.

[0085] In some embodiments, the pharmaceutical composition also includes a clotting factor such as Factor VII, Factor VIII, or Factor IX.

[0086] The monoclonal antibody or antigen-binding fragment thereof, or the pharmaceutical composition of the present invention can be administered to a patient in need thereof by injection or continuous infusion. For example, the antigen-binding fragment of the monoclonal antibody of the present invention administered by injection may be 0.0025 to 100 mg, 0.025 to 0.25 mg, 0.010 to 0.10 mg, or 0.10 to 0.50 mg per kg of body weight. When administered by continuous infusion, the antigen-binding fragment of the monoclonal antibody of the present invention may be administered at a rate of 0.001 to 100 mg / kg body weight / min, 0.0125 to 1.25 mg / kg body weight / min, 0.010 to 0.75 mg / kg body weight / min, 0.010 to 1.0 mg / kg body weight / min, or 0.10 to 0.50 mg / kg body weight / min over 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours. For full-length monoclonal antibodies of the present invention, the dosage may be approximately 1 to 10 mg / kg body weight, 2 to 8 mg / kg body weight, or 5 to 6 mg / kg body weight. Such full-length antibodies are typically infused over 30 minutes to 3 hours. The frequency of administration depends on the severity of the condition. The frequency of administration may range from three times a week to once every two or three weeks.

[0087] Furthermore, the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention can be subcutaneously injected into a patient. For example, the monoclonal antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention can be subcutaneously injected into a patient at a dose of 10 to 100 mg weekly, every two weeks, or monthly. [Example]

[0088] The present invention will be further described by the following examples, but the scope of the present invention is not limited to these examples.

[0089] Example 1: Production of anti-TFPI monoclonal antibody 1.1 Immunization and Fusion Mice were immunized with segmented TFPI containing only the first two Kunitz domains (sequence number 27), and multiple mouse spleen cells that showed a strong antibody-specific response to the hTFPI antigen (sequence number 28) were collected and fused to prepare hybridoma cells.

[0090] 1.2 Preliminary screening of positive hybridoma cells by ELISA binding assay The same concentration of hTFPI was added to the TFPI hybridoma cell supernatant-coated ELISA plate and incubated until the anti-TFPI antibody in the cell supernatant was fully bound to hTFPI. Peroxidase-labeled anti-6xHis tag antibody was then added. After complete incubation, TMB substrate was added, which hydrolyzed the peroxide with peroxidase to generate oxygen free radicals, oxidizing TMB to yield a blue product. The reaction was stopped with sulfuric acid, resulting in a yellow color. The OD reading at 450 nm was then read. The affinity of the anti-TFPI antibody was assessed by the OD reading; a higher OD reading indicated a stronger affinity. Finally, 35 cells with the strongest affinity for hTFPI were selected. The specific results are shown in Table 1.

[0091] [Table 1]

[0092] 1.3 Preliminary screening of positive hybridoma cells by competitive ELISA assay Plates were coated with 2 μg / mL FXa protease (Neb, batch no. 0941404), blocked with BSA, and incubated thoroughly with the same diluted supernatant of TFPI hybridoma cells and 50 ng / mL hTFPI1 (batch no. TE20140825, in-house production). Subsequently, a 1:1500 dilution of mouse THE™ His Tag Antibody [HRP] mAb (GenScript, batch no. 14C000744) was added, and the color was developed with TMB. The color development was stopped by adding 1 M H2SO4 stop solution, and the readings were read on a microplate reader. OD values were measured at 450 nm and 650 nm (reference wavelength). Based on the detection data, the inhibition of FXa-hTFPI binding by anti-TFPI antibodies was analyzed, and cell clones with superior inhibitory activity were screened. The lower the OD value, the better the inhibition by anti-TFPI antibodies. Table 2 shows the results of the competition assay of 35 hybridoma cell supernatants, with 7G6 demonstrating superior inhibition.

[0093] [Table 2]

[0094] 1.4 Determination of hybridoma cell subclones by ELISA binding assay The TFPI-7G6 cell line was subcloned. 31 subcloned cells showed strong responses to hTFPI in ELISA binding assays. The assay method was the same as in 1.1.

[0095] [Table 3]

[0096] Then, the 31 7G6 subclone cell supernatants were subjected to inhibition experiments at various dilutions. The experimental method was the same as in 1.2.

[0097] [Table 4]

[0098] Considering the inhibitory effect and cell conditions, 7G6-2G8, 7G6-5A10, 7G6-1C12, and 7G6-5F11 were selected as the final cell lines.

[0099] Example 2 Cloning and sequencing of the murine TFPI-7G6 antibody The heavy and light chain sequences of the mouse anti-TFPI antibody were cloned from four hybridoma cells: 7G6-2G8, 7G6-5A10, 7G6-1C12, and 7G6-5F11. Total DNA was extracted from each of the four hybridoma cells using the RANiso Plus kit (Takara) and used as a cDNA template. First-strand cDNA was synthesized from total RNA using PrimeScript RTase (Takara). HC and LC variable region fragments were amplified by PCR, and A was added to the ends.

[0100] Amplification of LC variable region primer pair:

[0101] [ka]

[0102] Amplification of HC variable region primer pair:

[0103] [ka]

[0104] The PCR products were separated by gel electrophoresis, and the target gene fragments for the heavy and light chain variable regions were recovered using the AxyPrep DNA Gel Extraction Kit (AXYGEN). They were then ligated into a T-vector and transformed into chemically competent Mach1-T1. Colony PCR was performed on colonies selected using M13F / M13R. Positive clones were sequenced using primer M13F(-47) to determine the sequences of the heavy and light chain variable regions. The specific sequences are as follows:

[0105] [ka]

[0106] Example 3 Design and construction of humanized TFPI-7G6 antibody 3.1 Humanization of TFPI-7G6 antibody Humanization was performed by humanizing amino acids on the protein surface (resurfacing) and CDR grafting onto a universal framework to humanize VH and VL.

[0107] The humanization method was as follows: The VH and VL of the antibody strain 7G6 were subjected to homology modeling using the software Modeller 9, with the PDB serial numbers for the VL homologous sequence: 1nldL and VLK2; and the PDB serial numbers for the VH homologous sequence: 1xgyl and VH1B, respectively. The CDR regions were then grafted onto the framework of the humanized homologous sequence according to the Kabat numbering system. Meanwhile, the relative solvent accessibility of amino acids was calculated based on the three-dimensional structure of the protein. Amino acids not exposed to the solvent were appropriately substituted with amino acids at the same positions in the original antibody.

[0108] TFPI-7G6 was humanized to obtain two humanized heavy chain sequences, h7G6VH-v1 (SEQ ID NO: 15) and hu7G6VH-v2 (SEQ ID NO: 16), and three humanized light chain sequences, h7G6VL-v1 (SEQ ID NO: 17), h7G6VL-v2 (SEQ ID NO: 18), and 7G6VL-v3 (SEQ ID NO: 19). Alignment of these humanized variants with the murine antibody is shown in Figure 1.

[0109] 3.2 Preparation of expression vector for humanized h7G6 Following the humanization design of the above antibody, the DNA sequences of humanized h7G6-2-VH1, h7G6-2-VH2, h7G6-2-VL1, h7G6-2-VL2, and h7G6-2-VL3 were synthesized (Genewiz). The constructs contained the LC or HC signal peptide and Kozak sequence (5'-GCCACC-3') immediately upstream of the start codon.

[0110] Based on the amino acid sequence of the human IgG4 constant region (P01861) in the protein database uniprot, Ser at position 108 was substituted with Pro to eliminate the formation of a monomeric antibody fragment (i.e., a "half antibody" consisting of one LC and one HC), yielding the amino acid sequence of the human IgG4-Fc region (SEQ ID NO: 20). A nucleic acid fragment encoding human IgG4-Fc was obtained by codon optimization and gene synthesis, followed by digestion and ligation to obtain an amino acid fragment encoding the heavy chain variable region of the h7G6 antibody obtained in the above Example. This fragment was then cloned into a conventional mammalian expression vector to obtain heavy chain 1 (SEQ ID NO: 21) and heavy chain 2 (SEQ ID NO: 22) of the h7G6 antibody. The final sequence of the construct was verified by DNA sequencing.

[0111] The amino acid sequence of the human Igκ constant region (SEQ ID NO: 23) was obtained according to the amino acid sequence of the human Igκ constant region (P01834) in the protein database uniprot. A nucleic acid fragment encoding the human Igκ constant region was obtained by codon optimization and gene synthesis, followed by digestion and ligation to obtain the amino acid fragment encoding the light chain variable region of the h7G6 antibody obtained in the above example. This fragment was then cloned into a conventional mammalian expression vector to obtain light chain 1 (SEQ ID NO: 24), light chain 2 (SEQ ID NO: 25), and light chain 3 (SEQ ID NO: 26) of the h7G6 antibody. The final sequence of the construct was verified by DNA sequencing.

[0112] 3.3 Selection of plasmid combinations for protein expression A mixture of 0.2 μg of HC vector DNA and 0.3 μg of LC vector DNA was used per mL of medium. The combination of antibody heavy chain 1 and antibody light chain 1 was Hu7G61, the combination of antibody heavy chain 2 and antibody light chain 2 was Hu7G62-v1, and the combination of antibody heavy chain 2 and antibody light chain 3 was Hu7G62-v2. HEK293 cells were transfected with the mixed DNA for antibody expression. Comparisons were made with the original mouse antibody. The expression levels and purities of the three humanized sequences and the original mouse sequence are shown in the table below.

[0113] [Table 5]

[0114] All three humanized antibodies were shown to be expressed with a purity of over 90%. Among them, the expression levels and purity of the last two humanized sequences were significantly superior to those of the original mouse antibodies, and the last humanized antibody, Hu7G62-v2, had the highest expression level.

[0115] 3.4 Production of h7G6 antibody protein For antibody expression, HEK293 cells were transfected with a mixture of antibody heavy chain 2 and antibody light chain 3 plasmids. The recombinant expression plasmids were diluted in Freestyle 293 medium, and the PEI (polyethyleneimine) solution required for transformation was added. Each plasmid / PEI mixture was then added to the HEK293 cell suspension and cultured at 37°C, 10% CO2, and 90 rpm; during this time, 50 μg / L IGF-1 was added. After 4 hours, EX293 medium, 2 mM glutamine, and 50 μg / L IGF-1 were added and cultured at 135 rpm. After 24 hours, 3.8 mM VPA was added. After 5–6 days of culture, the supernatant of the transient expression culture was collected and purified by protein A affinity chromatography to obtain the target hu7G6 protein.

[0116] Example 4 Verification of the function of the hu7G6 antibody protein 4.1 Affinity of humanized h7G6 antibody to hTFPI 1) ELISA method The plate was coated with 0.5 μg of hTFPI161 protein per well overnight at 4°C. After washing, a gradient dilution series of the h7G6 antibody protein obtained in the above Example was added and incubated for 2 hours at 25°C ± 2°C. After washing, 100 μL / well of a 1:2000 dilution of mouse anti-human IgG4 pFc' antibody [HP 6023] (HRP) was added and incubated for 2 hours at 25°C ± 2°C. After washing, a developer was added, and the absorbance at 450 / 650 nm was measured. For data processing and mapping analysis, the software SotfMax Pro v5.4 was used to obtain the binding curve of the h7G6 antibody to hTFPI161 and the EC50 value reflecting the affinity of the antibody for hTFPI161 using a four-parameter fitting.

[0117] The results are shown in the table below and in Figure 2. Here, the vertical axis represents OD450, and the horizontal axis represents the concentration (ng / mL) of the h7G6 antibody protein; the Hu7G62-v2 antibody protein showed excellent affinity for hTFPI161.

[0118] [Table 6]

[0119] 2) Detection by biolayer interferometry In this example, we used Bio-Layer Interferometry (BLI) technology to detect the affinity of h7G6 for human TFPI. We used a Fortibio K2 instrument. First, KN057 was diluted to 10 μg / mL and immobilized on a Protein A biosensor (model 18-5010). Then, hTFPI161-Chis was diluted to 30 nM, 15 nM, 7.5 nM, 3.75 nM, and 1.875 nM, respectively, and combined with h7G6 to detect binding signals of varying intensity. The equilibrium constant (KD) of the sample was calculated by fitting the results using a 1:1 model. The analytical results are as follows: the KD values of three batches of h7G6 stock solution for human TFPI (180727DS, 180808DS, and 180820DS) were 1.32E-09M, 1.32E-09M, and 1.47E-09M, respectively, with an average KD of (1.37±0.09)E-09M and an RSD% of 6.32%.

[0120] 4.2 Inhibition of the interaction between FXa and TFPI by the humanized hu7G6 antibody After coating with 0.2 μg of FXa protease per well overnight at 4°C and blocking with BSA, 100 μL of a gradient dilution series of the hu7G6 antibody protein obtained in the above example (containing 50 ng / mL hTFPI161) was added to each well and incubated at room temperature for 1 hour. After washing, a 1:1500 dilution of mouse THE™ His Tag Antibody [HRP] mAb was added and incubated at room temperature for 1 hour. After washing, a developer solution was added, and the absorbance at 450 / 650 nm was measured.

[0121] For data processing and mapping analysis, the software SotfMax Pro v5.4 was used to obtain the binding curve and EC50 value of the h7G6 antibody to FXa-TFPI by four-parameter fitting. The results are shown in Figure 3 below. Here, the vertical axis is OD450, and the horizontal axis is the concentration (ng / mL) of the h7G6 antibody protein; Hu7G62-v2 antibody protein was effective in inhibiting the interaction between FXa and TFPI.

[0122] [Table 7]

[0123] Example 5 In vivo studies Twenty-eight New Zealand rabbits were randomly divided into four groups based on body weight: 1 normal control group (n = 4); 2 model control group (n = 8); 3 positive control group (n = 8); and 4 test group (n = 8), with half of each sex. After anesthesia, all animals except the normal control group were injected with 600 μg / kg of BO2C11 antibody (a human coagulation factor VIII neutralizing antibody; its sequence was retrieved from the following literature: Structure of the factor VIII C2 domain-immunoglobulin G4k Fab complex: Identification of an inhibitory antibody epitope on the surface of factor VIII) into the marginal ear vein to establish a New Zealand rabbit model of hemophilia A. Ten minutes after modeling, 2 mg / kg of the test sample (h7G6 antibody protein) or control sample (TFPI2021, a Novartis control antibody) was injected into the marginal ear vein, and the normal and model control groups received the corresponding volume of PBS. 25 minutes after administration, the left forelimb of the animal was preheated with 45 mL of a solution containing physiological saline at 37°C. After 10 minutes, the top of the third nail of the left forelimb of the animal was cut with surgical scissors. During bleeding, a stopwatch was started to measure the bleeding time. The time was stopped when no more bleeding was observed from the wound, which was considered the end point of the coagulation, and the bleeding time was recorded.

[0124] Table 8 shows that the bleeding time significantly increased from the normal 6.3±2.3 minutes (control group) to 28.1±14.4 minutes (model group); the test sample and the positive control sample were able to shorten the bleeding time of New Zealand rabbits with hemophilia A after administration and exert similar functions, demonstrating that the Hu7G62-v2 antibody is effective in treating rabbits with hemophilia A.

[0125] [Table 8]

[0126] Example 6. Estimation of dose-effect relationships After anesthesia, New Zealand rabbits were injected with 1 mg / kg of anti-FVIII antibody (BO2C11) into the marginal ear vein to establish a New Zealand rabbit model of hemophilia A. Ten minutes after modeling, different concentrations (2 or 20 mg / kg) of Hu7G62-v2 antibody were injected into the marginal ear vein. Thirty-five minutes later, the upper part of the third nail of the left forelimb of the animals was clipped. The bleeding time was measured during bleeding and stopped when no bleeding from the wound was observed, which is considered the end point of this clotting. The upper limit of the bleeding time for animals was 60 minutes, and bleeding times longer than 60 minutes were recorded as 60 minutes. For grouping and dosing information, see Table 9.

[0127] [Table 9]

[0128] The results are shown in Table 10. The bleeding time of rabbits injected with anti-FVIII antibody increased significantly (P<0.01) from 6.0±1.9 minutes in the control group to 54.0±13.4 minutes. After a single intravenous administration of various concentrations of h7G6 antibody, the animals' bleeding time was significantly (P<0.05) shortened, demonstrating a certain dose-dependence. While the detection of hemoglobin may indicate the amount of bleeding in the animals under observation, the results show that the amount of hemoglobin significantly increased after modeling but decreased after administration of h7G6 antibody, indicating that the h7G6 antibody enabled shortening of bleeding time and reduced the risk of bleeding.

[0129] [Table 10]

[0130] Example 7 Evaluation of in vivo pharmacokinetics of anti-TFPI antibodies in cynomolgus monkeys The purpose of this example was to measure plasma drug concentrations of anti-TFPI antibodies in cynomolgus monkeys after a single intravenous administration and a single subcutaneous administration of multiple doses, and to investigate the in vivo pharmacokinetics in cynomolgus monkeys. The differences in exposure of anti-TFPI antibodies in cynomolgus monkeys after intravenous and subcutaneous administration were compared, and absolute bioavailability was calculated.

[0131] Cynomolgus monkeys were divided into four groups, three males and three females. The following doses and administration methods were used. The administration was performed once. Blood samples were taken before administration and at 0.5 hours, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours (3 days), 96 hours (4 days), 120 hours (5 days), 144 hours (6 days), 168 hours (7 days), 216 hours (9 days), and 264 hours (11 days) after subcutaneous injection, and plasma drug concentrations were measured.

[0132] [Table 11]

[0133] Pharmacokinetic parameters were calculated using Phoenix software (version 8.1) based on the obtained plasma drug concentration data.

[0134] The results are shown in the table below.

[0135] [Table 12]

[0136] Compared with the pharmacokinetic data results of Bayer TFPI antibody BAY1093884 in cynomolgus monkeys published by Jian-Ming Gu et al. in 2017 (Ref to Gu J, Zhao X, Schwarz T, et al. Mechanistic Modeling of the Pharmacodynamic and Pharmacokinetic Relationship of Tissue Factor Pathway Inhibitor-Neutralizing Antibody (BAY 1093884) in Cynomolgus Monkeys [J]. Aaps Journal, 2017, 19(4): 1186-1195), the in vivo half-life and mean drug retention time of the anti-TFPI antibody of the present invention administered subcutaneously at a dose of 3 mg / kg or more were found to be significantly shorter than those of BAY1093884 administered subcutaneously at 5 mg / kg (T 1 / 2 = 25 hr, MRT = 40 hr), and its relative drug exposure was also significantly higher than that of BAY1093884 (AUC norm The plasma clearance rate of the anti-TFPI antibody of the present invention administered intravenously at a dose of 10 mg / kg was significantly lower than that of BAY1093884 administered intravenously at doses of 5 mg / kg and 20 mg / kg (5 mg / mL group: 1.2 mL / hr / kg; 20 mg / kg group: 0.6 mL / hr / kg). These results indicate that the anti-TFPI antibody of the present invention has a longer circulation time and slower clearance in vivo compared with Bayer's BAY1093884; the lower the dose, the better the drug exposure.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof against TFPI, the monoclonal antibody comprises a light chain variable region and a heavy chain variable region; The light chain variable region VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12; a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13; and VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14 Including, The heavy chain variable region VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7; a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8; and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; An isolated monoclonal antibody or antigen-binding fragment thereof comprising:

2. 2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein the isolated monoclonal antibody is a humanized antibody.

3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence that has at least 85%, at least 90%, at least 95% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:

11.

4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence that has at least 85%, at least 90%, at least 95% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:

6.

5. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO:

16.

6. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the light chain variable region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17 to 19.

7. 3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

17.

8. 3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

18.

9. 3. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 2, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

19.

10. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the heavy chain of the monoclonal antibody further comprises a human IgG4 constant region or a variant thereof, for example, the variant of the human IgG4 constant region comprises the amino acid sequence set forth in SEQ ID NO:

20.

11. 11. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 10, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:

22.

12. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the light chain of the monoclonal antibody further comprises a human Igκ constant region or a variant thereof, for example, the human Igκ constant region comprises the amino acid sequence set forth in SEQ ID NO:

23.

13. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 12, wherein the light chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 24 to 26.

14. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 10 to 13, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

24.

15. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 10 to 13, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

25.

16. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 10 to 13, wherein the monoclonal antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 22 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

26.

17. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, and a pharmaceutically acceptable carrier.

18. 18. The pharmaceutical composition of claim 17, further comprising a coagulation factor such as Factor VII, Factor VIII or Factor IX.

19. 17. Use of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 10 to 16 in the manufacture of a medicament for treating a coagulation-related disorder.

20. 20. The use according to claim 19, wherein the coagulation-related disease is an inherited or acquired coagulation disorder.

21. 21. The use according to claim 19 or 20, wherein the coagulation-related disorder is hemophilia, such as hemophilia A, hemophilia B and hemophilia C.

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