Monoclonal antibodies against TFPI and uses thereof

Abstract

Antibodies or antigen-binding fragments against human TFPI, nucleic acid molecules encoding the antibodies, expression vectors and host cells for expressing the antibodies, and methods for producing the antibodies are provided, as well as pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof and their use in the manufacture of medicaments for the prevention and / or treatment of coagulation disorders in patients with genetic or acquired coagulation factor deficiencies.

Description

[Technical Field]

[0001] The present invention is in the field of therapeutic monoclonal antibodies, and more particularly relates to antibodies or antigen-binding fragments thereof against tissue factor pathway inhibitor (TFPI), and further to the use of said antibodies in the preparation of medicaments for the prevention or treatment of inherited or acquired coagulation factor deficiency diseases or conditions. [Background technology]

[0002] Hemophilia A and hemophilia B are inherited bleeding disorders characterized by impaired production of activated thrombin, prolonged clotting times, and a tendency to lifelong bleeding even after minor trauma. Severe patients may experience spontaneous bleeding even without apparent injury. Hemophilia A accounts for over 80% of all hemophilia cases and is characterized by clotting factor VIII (FVIII) deficiency or insufficiency, with a male incidence of 1:5,000. Hemophilia B is characterized by clotting factor IX (FIX) deficiency or insufficiency and affects approximately 1 in 30,000 newborns. Recurrent, spontaneous, and trauma-related bleeding are characteristic of severe hemophilia, with typical bleeding sites being joints and muscles. With minimal treatment, severe patients may experience 30 to 40 bleeding episodes per year, which can lead to irreversible joint damage and secondary disability. The most commonly used treatment is factor replacement therapy, which provides hemophilia patients with FVIII or FIX replacement. Current treatments require frequent infusions of FVIII or FIX two to four times per week to reduce spontaneous bleeding. While this treatment effectively prevents most spontaneous bleeding, it does not prevent trauma-induced bleeding. Furthermore, treatment efficacy can be affected by infusion frequency, dose, or missed or delayed administration, which is influenced by the factor's elimination half-life, the cost-effectiveness of the treatment plan, and patient acceptability. Novel treatment methods need to be explored to overcome the limitations of factor replacement therapy, improve patient compliance, and reduce patient burden.

[0003] Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein in the body that controls the initiation step of coagulation. It specifically inhibits the tissue factor-initiated coagulation pathway by binding to the TF / FVIIa complex, preventing the activation of factor X and suppressing coagulation. Neutralization of TFPI activity with anti-TFPI antibodies restores or enhances the coagulation process. TFPI is a glycoprotein consisting of 276 amino acid residues, containing three Kunitz domains (K1, K2, and K3), two linker regions, an N-terminus rich in acidic amino acids, and a C-terminus rich in basic amino acids. While domains K1 and K2 are essential for TFPI's anticoagulant activity, K3 is not essential for this role but enhances TFPI's ability to inhibit coagulation. Recent studies have shown that K3 and the C-terminus can bind heparin, membrane surface glycoproteins, and polysaccharides, and this region is also associated with TFPI's anti-inflammatory effects. TFPI inhibits the coagulation pathway in two steps. First, TFPI binds to activated factor X via K2 and competitively inhibits its activity. 2+ This is a reversible process that does not depend on factor Xa. Next, TFPI in the factor Xa / TFPI complex binds to the active site of the FVIIa / TF complex via K1, inhibiting the FVIIa / TF complex. Anti-TFPI antibodies act on the K2 domain of TFPI, competitively blocking the binding site of factor Xa to TFPI, increasing the level of factor Xa and also inhibiting the interaction between factor Xa and Ca. 2+ By blocking the inhibitory effect of TFPI on TF-FVIIa in the presence of , it increases factor Xa levels and thrombin generation, enhancing coagulation through a dual mechanism.

[0004] Anti-TFPI antibodies can be used in hemophilia patients with or without FVIII and FIX inhibitors, and the antibodies themselves have a longer half-life, allowing for less frequent dosing. Currently, hemophilia treatment antibodies targeting different Kunitz domains are being studied in clinical trials. For example, the humanized monoclonal antibody concizumab, developed by Novo Nordisk, is in Phase III clinical trials; the monoclonal antibody marstacimab, developed by Pfizer, is also in Phase III clinical trials; and BAY-1093884, developed by Bayer, is in Phase II clinical trials. Currently, there are no approved antibody drugs targeting TFPI on the market. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides an antibody capable of specifically binding to human TFPI, a nucleic acid encoding the antibody, a vector containing the nucleic acid, a host cell containing the vector, a method for producing the antibody, and a pharmaceutical composition for use in preventing or treating coagulation disorders in patients with inherited or acquired coagulation factor deficiencies, wherein the pharmaceutical composition contains the antibody as an active ingredient and is capable of inhibiting TFPI, thereby activating the coagulation pathway. The anti-human TFPI antibodies disclosed herein can be used to treat or prevent hemophilia.

[0006] In a first aspect of the present invention, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising: An antibody or antigen-binding fragment thereof capable of specifically binding to TFPI, wherein the heavy chain variable region (VH) contained in the antibody or antigen-binding fragment comprises: (i) an HCDR1 having a sequence as set forth in SEQ ID NO: 1 or 7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; (ii) an HCDR2 having a sequence as set forth in SEQ ID NO: 2, 8, 12 or 13, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; (iii) an HCDR3 having a sequence as set forth in SEQ ID NO: 3 or 9, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; and / or the light chain variable region (VL) contained in the antibody or antigen-binding fragment thereof comprises at least one, two, or three complementarity-determining regions (CDRs) selected from the group consisting of: (iv) an LCDR1 having a sequence as set forth in SEQ ID NO: 4 or 10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; (v) an LCDR2 having a sequence set forth in SEQ ID NO:5 or Gly-Thr-Ser (SEQ ID NO:11), or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (vi) an LCDR3 having a sequence as set forth in SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to the above sequence; At least one, two or three complementarity determining regions (CDRs) selected from the group An antibody or antigen-binding fragment thereof capable of specifically binding to TFPI is provided.

[0007] In some preferred embodiments, the substitution according to any one of (i) to (vi) is a conservative substitution.

[0008] In some preferred embodiments, HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region and / or LCDR1, LCDR2, and LCDR3 contained in the light chain variable region are defined according to the Kabat or IMGT numbering system. Table 2 in Example 3 exemplarily shows CDR amino acid sequences of mouse-derived antibodies defined according to the Kabat or IMGT numbering system.

[0009] In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises: (1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5 or 6, respectively, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; (2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the sequences set forth in SEQ ID NO: 7, 8, 9, 10, Gly-Thr-Ser (SEQ ID NO: 11) or SEQ ID NO: 6, respectively, or sequences having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; (3) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having a sequence set forth in SEQ ID NO: 1, 12, 3, 4, 5 or 6, respectively, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; (4) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 having the sequences shown in SEQ ID NO: 7, 13, 9, 10, Gly-Thr-Ser (SEQ ID NO: 11) or SEQ ID NO: 6, respectively, or sequences having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to any of the above sequences; The present invention comprises three VH variable region CDRs and three VL variable region CDRs selected from the group:

[0010] In some embodiments, the antibody or antigen-binding fragment thereof is murine or chimeric, and its heavy chain variable region comprises the heavy chain FR region of a murine IgG1, IgG2, IgG3, or mutant thereof, and its light chain variable region comprises the light chain FR region of a murine κ or λ chain, or mutant thereof. Table 3 in Example 3 shows the amino acid sequence numbers of the variable regions of preferred murine antibodies.

[0011] In some preferred embodiments, the murine or chimeric antibody or antigen-binding fragment thereof comprises: a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 14, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)), and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)); The following VH and VL sequences are included:

[0012] In some embodiments, the antibody or antigen-binding fragment thereof is humanized. Example 3 shows the basic outline of a humanization strategy, Table 2 shows exemplary CDR amino acid sequences of a new humanized antibody as defined by the Kabat or IMGT numbering system, and Table 3 shows amino acid sequence numbers of the variable regions of preferred humanized antibodies.

[0013] In some preferred embodiments, the murine or chimeric antibody or antigen-binding fragment thereof comprises: a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)), and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)); The following VH and VL sequences are included:

[0014] In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region derived from a human immunoglobulin.

[0015] More preferably, the antibody consists of the human kappa light chain constant region amino acid sequence (amino acid sequence shown in SEQ ID NO: 18).

[0016] More preferably, the antibody comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; even more preferably, the antibody comprises a heavy chain constant region selected from human IgG1, IgG2, and IgG4; the heavy chain constant region has a sequence that has one or more amino acid substitutions, deletions, or additions compared to a native sequence or the native sequence from which it is derived. For example, in one embodiment, the humanized antibody molecule comprises a heavy chain constant region of wild-type human IgG1 (having the amino acid sequence set forth in SEQ ID NO: 19). In another embodiment, the humanized antibody molecule comprises a heavy chain constant region of human IgG1 with M252Y, S254T, T256E, and M428L mutations according to EU numbering (having the amino acid sequence set forth in SEQ ID NO: 20). In another embodiment, the humanized antibody molecule comprises a heavy chain constant region of wild-type human IgG2 (having the amino acid sequence set forth in SEQ ID NO: 21). In another embodiment, the humanized antibody molecule comprises a human IgG4 with a mutation at position 228 (such as S to P) according to EU numbering (having the amino acid sequence as shown in SEQ ID NO: 22).

[0017] In an exemplary embodiment, the humanized antibody molecule has a heavy chain amino acid sequence as shown in SEQ ID NO:23 and a light chain amino acid sequence as shown in SEQ ID NO:24.

[0018] In any of the foregoing embodiments, the antibody or antigen-binding fragment thereof of the present invention can bind to TFPI with a KD of 10 nM or lower, preferably, can bind to TFPI with a KD of 1 nM or lower, preferably, can bind to TFPI with a KD of 100 pM or lower, and preferably, can bind to TFPI with a KD of 10 pM or lower.

[0019] In a second aspect of the invention, there is provided a DNA molecule encoding the antibody or antigen-binding fragment thereof.

[0020] In a third aspect of the present invention, there is provided a vector comprising the DNA molecule described above.

[0021] In a fourth aspect of the present invention, there is provided a host cell comprising said vector; said host cell consists of a prokaryotic cell, a yeast cell or a mammalian cell, such as a CHO cell, an NSO cell or other mammalian cell, preferably a CHO cell.

[0022] In a fifth aspect of the invention, there is further provided a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and a medicamentable excipient, vector or diluent.

[0023] In a sixth aspect of the present invention, The method comprises the steps of: (a) obtaining a gene encoding an antibody or antigen-binding fragment thereof and constructing an expression vector for the antibody or antigen-binding fragment thereof; (b) transfecting the expression vector into a host cell by genetic engineering techniques; (c) culturing the host cell under conditions that permit the production of the antibody or antigen-binding fragment thereof; and (d) isolating and purifying the produced antibody or antigen-binding fragment thereof. Further provided is a method for preparing an antibody or antigen-binding fragment thereof according to the invention.

[0024] Wherein, the expression vector described in step (a) is one or more selected from the group consisting of a plasmid, a bacterium and a virus, and preferably, the expression vector is pcDNA3.1.

[0025] In step (b), the vector constructed by genetic engineering techniques is transfected into a host cell, which may be a prokaryotic cell, yeast, or mammalian cell, such as a CHO cell, an NS0 cell, or another mammalian cell, preferably a CHO cell.

[0026] In step (d), the antibody or antigen-binding fragment thereof is isolated and purified by conventional immunoglobulin purification methods, including protein A affinity chromatography and ion exchange.

[0027] In a seventh aspect of the present invention, there is provided a use of an antibody or antigen-binding fragment in the manufacture of a pharmaceutical product for the prevention or treatment of inherited or acquired coagulation factor deficiencies or diseases or events resulting therefrom. For example, the antibodies or antigen-binding fragments provided herein can be used to inhibit the interaction between TFPI and FXa or to prevent TFPI-dependent inhibition of TF / FVIIa activity. Furthermore, the human monoclonal antibodies can be used to circumvent the lack of FVIII- or FIX-dependent FXa enhancement and restore TF / FVIIa-driven FXa production. Furthermore, diseases include hemophilia A and hemophilia B, spontaneous bleeding events, traumatic bleeding events, or bleeding events during prophylactic treatment, preoperative management, or surgical treatment.

[0028] In another aspect of the invention, there is provided a method for preventing or treating inherited or acquired coagulation factor deficiencies or diseases or conditions resulting therefrom, comprising administering an effective amount of an antibody or antigen-binding fragment, or a pharmaceutical composition.

[0029] Abbreviations and Definitions The following abbreviations are used in this paper: CDR - Complementarity-Determining Region, the variable region of an immunoglobulin that binds to antigen, as defined by the Kabat, IMGT, Chothia, or AbM numbering systems (see terms "highly variable region," "CDR region," or "complementarity-determining region"). EC 50 -Concentration producing 50% efficacy or binding ELISA - Enzyme-linked immunosorbent assay FR - antibody framework region: immunoglobulin variable region other than the CDR region HRP - Horseradish Peroxidase I C 50 - concentration producing 50% inhibition IgG - immunoglobulin G Kabat - An immunoglobulin amino acid sequence alignment and numbering system proposed by Elvin A. Kabat. PCR - Polymerase Chain Reaction V region - IgG chain segments with sequence variability between different antibodies, spanning Kabat residue 109 in the light chain and residue 113 in the heavy chain. KD - equilibrium dissociation constant ka - binding rate constant kd - dissociation rate constant

[0030] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures used in cell culture, biochemistry, nucleic acid chemistry, immunology laboratory, etc. are all common procedures widely used in the corresponding fields. In addition, to better understand the present invention, the following definitions and interpretations of related terms are provided.

[0031] The term "tissue factor pathway inhibitor" or "TFPI" refers to any variant, isoform, and various cognate forms of human TFPI that are naturally expressed by cells.

[0032] The term "EU Numbering System" (or "Scheme") refers to the first human IgG1 immunoglobulin, named Eu, isolated and purified by Gerald M. Edelman and colleagues in the late 1960s (1968-1969). Its amino acid sequence was measured and numbered (Edelman GM et al., 1969, Proc Natl Acad USA, 63:78-85). The heavy chain constant regions of other immunoglobulins are numbered by matching their amino acid sequences with Eu, and the corresponding amino acid positions are numbered with Eu numbers. The EU numbering system primarily applies to the heavy chain constant regions of immunoglobulins, including CH1, CH2, CH3, and the hinge region.

[0033] The term "Kabat Numbering System" (or "Scheme") was coined in 1979 when Kabat et al. proposed the first standardized numbering scheme for human immunoglobulin variable regions (Kabat EA, Wu TT, Bilofsky H. Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain and β2-Microglobulins. 1979. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health). In the book "Immunologically Related Protein Sequences" (Kabat EA, Wu TT, Perry HM, Gottesman KS, Foeller C. 1991. Sequences of Proteins of Immunological Interest, 5th edition. Bethesda, MD: US Department of Health and Human Services, National Institutes for Health), Kabat et al. collated and numbered the amino acid sequences of antibody light and heavy chains. They found that these analyzed sequences represented variable lengths, and that default and inserted amino acids or amino acid fragments appeared only at specific positions. Interestingly, insertion points were often located within the CDRs, but could also appear at certain positions in the framework regions. In the Kabat numbering scheme, light chain variable regions are numbered up to position 109, and heavy chain variable regions are numbered up to position 113, with inserted amino acids in the light and heavy chains identified and annotated by alphabetical characters (e.g., 27a, 27b, etc.). All Lambda light chains do not contain residue 10, but Lambda and Kappa light chains are encoded by two different genes and located on different chromosomes.Lambda and Kappa light chains can be distinguished by differences in the amino acid sequences of their constant regions. Unlike the EU numbering system, which only covers the heavy chain constant region, the numbering scope of the Kabat numbering system covers the full-length immunoglobulin sequence and includes the light and heavy chain variable and constant regions of the immunoglobulin.

[0034] The term "antibody" generally refers to a protein-binding molecule with immunoglobulin-like functions. Typical examples of antibodies are immunoglobulins and their derivatives or functional fragments, which may display the required binding specificity. Techniques for preparing antibodies are well known in the art. "Antibodies" includes different types of naturally occurring immunoglobulins (e.g., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, IgA1, IgA2, etc.). "Antibodies" also includes non-naturally occurring immunoglobulins, such as single-chain antibodies, chimeric antibodies (e.g., human-derived murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof (e.g., Fab', F(ab')2, Fab, Fv, and rIgG). Further reference may be made to, for example, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co, Rockford, IL); Kuby J, Immunology, 3rd Ed., WH Freeman & Co, New York, 1997. An antibody may bind to one antigen, referred to as "monospecific," or to two different antigens, referred to as "bispecific," or to more than one different antigen, referred to as "multispecific." An antibody may be monovalent, bivalent, or multivalent, i.e., it can bind to one, two, or more antigen molecules at a time. An antibody "monovalently" binds to a particular protein when one antibody molecule binds to only one molecule of the protein, but the antibody may also bind to different proteins. When an antibody binds only to molecules of each of two different proteins, the antibody "monovalently" binds to each protein, and the antibody "bispecifically" and "monovalently" binds to each of two different proteins. An antibody may be "monomeric," i.e., comprise a single polypeptide chain. An antibody may also comprise multiple polypeptide chains ("multimeric"), or may comprise two ("dimeric"), three ("trimeric"), or four ("tetrameric") polypeptide chains.If the antibody is a multimer, the antibody may be a homomultimer, i.e., the antibody comprises more than one molecule of only one type of polypeptide chain, including a homodimer, homotrimer, or homotetramer. Preferably, the multimeric antibody may be a heteromultimer, i.e., the antibody comprises more than one different type of polypeptide chain, including a heterodimer, heterotrimer, or heterotetramer.

[0035] The term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population of antibodies, in which each individual antibody within the population is identical except for possible minor mutations, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. Monoclonal antibodies are generated by methods known to those skilled in the art, e.g., by fusing myeloma cells with immune spleen cells to prepare heterozygous antibody-producing cells. They are synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. Monoclonal antibodies may also be obtained by recombinant, phage display, synthetic, or other conventional techniques.

[0036] The term "complete antibody" refers to an antibody composed of two antibody heavy chains and two antibody light chains. A "complete antibody heavy chain" is composed of, from the N-terminus to the C-terminus, an antibody heavy chain variable domain (VH), antibody constant heavy chain domain 1 (CH1), antibody hinge region (HR), antibody heavy chain constant domain 2 (CH2), and antibody heavy chain constant domain 3 (CH3), and is abbreviated as VH-CH1-HR-CH2-CH3. In the case of an IgE subclass antibody, it preferably further contains antibody heavy chain constant domain 4 (CH4). Preferably, a "complete antibody heavy chain" is a polypeptide composed of, from the N-terminus to the C-terminus, VH, CH1, HR, CH2, and CH3. A "complete antibody light chain" is a polypeptide composed of, from the N-terminus to the C-terminus, an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), and is abbreviated as VL-CL. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). Intact antibody chains are connected via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and interpolypeptide disulfide bonds between the hinge regions of the intact antibody heavy chains. Typical examples of intact antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE.

[0037] The term "antibody fragment" or "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs that retain the ability to specifically bind to an antigen, and typically contain at least a portion of the antigen-binding or variable region of the parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, they are expressed in molar units (K D), an antibody fragment retains at least 10% of the binding activity of the parent. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the binding affinity of the parent antibody for the target. Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, complementarity-determining region (CDR) fragments, disulfide bond-stable proteins (dsFv), etc., linear antibodies, single-chain antibodies (e.g., scFv monoclonal antibodies), monoclonal antibodies (Unibody, technology derived from Genmab), bivalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (e.g., VH domain antibodies), domain antibodies (Domantis, technology derived from Domantis), nanobodies (nanobodies, technology derived from Ablynx), multispecific antibodies formed from antibody fragments (e.g., three-chain antibodies, four-chain antibodies, etc.), engineered antibodies such as chimeric antibodies (e.g., human-derived mouse antibodies), heteroconjugate antibodies, etc. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0038] The term "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody. It is a recombinant protein consisting of a heavy chain variable region (VH) and a light chain variable region (VL) connected by a linker, which bridges these two domains to form an antigen-binding site. The linker sequence is generally composed of a flexible peptide, such as, but not limited to, G2(GGGGS)3. The size of an scFv is generally one-sixth that of a complete antibody. A single-chain antibody is preferably a single amino acid sequence encoded by a single nucleotide chain. For a review of scFvs, see Pluckthun A, 1994. Antibodies from Escherichia coli, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenberg M and Moore GP (eds.), Springer-Verlag, New York, pp. 269-315. Further reference may be made to International Patent Application Publication No. WO 88 / 01649 and US Pat. Nos. 4,946,778 and 5,260,203.

[0039] The term "VL domain" refers to the amino-terminal variable region domain of an immunoglobulin light chain.

[0040] The term "VH domain" refers to the amino-terminal variable region domain of an immunoglobulin heavy chain.

[0041] The term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region may be divided into three distinct domains: the upper, middle, and lower hinge domains (Roux KH et al., 1998, J Immunol, 161:4083-4090).

[0042] The term "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions and is the minimum fragment containing a complete antigen recognition and binding site.

[0043] The term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper hinge region, middle hinge region, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide used herein may comprise a polypeptide chain having a CH1 domain, a polypeptide having a CH1 domain, at least a portion of a hinge domain, and a CH2 domain, a polypeptide chain having a CH1 domain and a CH3 domain, a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain having a CH1 domain, at least a portion of a hinge structure, a CH2 domain, and a CH3 domain. In another example, a polypeptide of the present application comprises a polypeptide chain having a CH3 domain. An antibody used herein may also lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As noted above, those skilled in the art will appreciate that heavy chain constant regions may be altered so that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0044] The term "light chain constant region" comprises an amino acid sequence derived from the light chain of an antibody. Preferably, said light chain constant region comprises at least one of a constant kappa domain and a constant lambda domain.

[0045] The term "Fc region" or "Fc fragment" refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the hinge region, CH2 domain, and CH3 domain, which mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Fc regions include native-sequence Fc regions and variant Fc regions.

[0046] Typically, the human IgG heavy chain Fc region extends from the amino acid residue at position Cys226 or Pro230 to the carboxyl terminus, although the boundaries may vary. The C-terminal lysine of the Fc region (residue 447, according to the EU numbering system) may or may not be present. Fc may refer to this region independently or in the context of a protein polypeptide containing Fc; for example, an "Fc region-containing binding protein" is also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). Native-sequence Fc regions in the antibodies of the present invention include IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4 from mammals (e.g., humans). In some embodiments, there are approximately 10 single amino acid substitutions, insertions, and / or deletions per 100 amino acids in the amino acid sequences of the two Fc polypeptide chains relative to the amino acid sequence of the mammalian Fc polypeptide. In some embodiments, the amino acid differences in the Fc region may be Fc modifications that extend half-life, increase FcRn binding, enhance Fcγ receptor (FcγR) binding, and / or enhance ADCC, ADCP, and / or CDC.

[0047] As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen-presenting cells, macrophages, or granulocytes) and soluble macromolecules (including antibodies, cytokines, and complement) produced by immune cells or the liver, which result in selective damage, destruction, or removal from the human body of invading pathogens, cells or tissues infected by pathogens, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation. In the present invention, the term "antigen-specific T cell response" refers to an immune response produced by T cells, which is produced when an antigen specific to the T cell stimulates the T cell. Non-limiting examples of responses produced by T cells upon antigen-specific stimulation include T cell proliferation and cytokine (e.g., IL-2) production.

[0048] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), allowing these cytotoxic effector cells to specifically bind to antigen-bound target cells and then kill the target cells by secreting cytotoxins. Methods for detecting ADCC activity of an antibody are known in the art and can be assessed, for example, by measuring the binding activity between the antibody to be measured and an Fc receptor (e.g., CD16a).

[0049] As used herein, the term "complement-dependent cytotoxicity (CDC)" refers to a form of cytotoxicity that activates the complement cascade by binding the complement component C1q to an antibody Fc. Methods for detecting CDC activity of an antibody are known in the art, and can be assessed, for example, by measuring the binding activity between the antibody to be measured and an Fc receptor (e.g., C1q).

[0050] In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the CH2 and CH3 constant domains of each of the antibody's two heavy chains, while the Fc region of IgM and IgE comprises three heavy chain constant domains (CH2-4 domains) in each polypeptide chain.

[0051] The term "humanized antibody" refers to a genetically engineered antibody of non-human origin, whose amino acid sequence has been modified to improve homology with that of a human antibody. Most or all of the amino acids outside the CDR regions of the non-human antibody, e.g., a mouse antibody, are replaced with corresponding amino acids from a human immunoglobulin, while most or all of the amino acids within one or more CDR regions remain unchanged. Amino acid additions, deletions, insertions, substitutions, or modifications are permissible as long as they do not eliminate the antibody's ability to bind to a specific antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody. The origin of the CDRs is not particularly limited and may be from any animal. For example, CDR regions from a mouse antibody, rat antibody, rabbit antibody, or non-human primate (e.g., cynomolgus monkey) antibody can be used. Human antibody germline sequences can be obtained by searching the IMGT antibody germline database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). Generally, human germline antibody sequences that are highly homologous to the modified non-human antibody are selected as framework regions for humanized antibodies.

[0052] The term "hypervariable region" or "CDR region" or "complementarity determining region" refers to the amino acid residues of an antibody that are responsible for antigen binding and are non-contiguous amino acid sequences. The CDR region sequence may be the amino acid residues within the variable region as defined by the Kabat, Chothia, or IMGT method (Lefranc et al., 2003, Dev Comparat Immunol, 27:55-77) or identified by any method for determining CDR region sequences well known in the art. For example, hypervariable regions are amino acid residues (Kabat numbering system) derived from the "complementarity determining regions" or "CDRs" defined by sequence matching (e.g., residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in the light chain variable domain and residues 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in the heavy chain variable domain; see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health). Health, Bethesda, Md.) and / or residues from structurally defined "hypervariable links" (HVLs) (Chothia numbering system) (e.g., residues 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3) in the light chain variable domain and residues 26-32 (CDR-H1), 53-55 (CDR-H2), and 96-101 (CDR-H3) in the heavy chain variable domain; see Chothia C and Lesk AM, 1987, J Mol Biol, 196:901-917; Chothia C et al, 1989, Nature, 342:878-883). "Framework" or "FR" residues are variable domain residues other than the hypervariable region residues as defined herein. In some embodiments, the CDRs contained in an antibody or antigen-binding fragment thereof of the present invention are preferably defined according to the Kabat, IMGT, or Chothia numbering system.Those skilled in the art can unambiguously assign each numbering system to any variable domain sequence without relying on any experimental data other than the sequence itself. For example, the Kabat residue numbering system for a given antibody can be determined by comparing the homologous regions of the antibody sequence with each "standard" numbered sequence. It is well within the ordinary skill of one of ordinary skill in the art to determine the number of any variable domain sequence in the sequence listing based on the sequence numbering scheme herein.

[0053] As used herein, the term "isolated" with respect to nucleic acids (e.g., DNA or RNA) refers to molecules separated from other DNAs or RNAs, respectively, that occur as naturally occurring large molecules. The term "isolated" also refers to nucleic acids or polypeptides that are substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when prepared by chemical synthesis. "Isolated nucleic acids" also refers to nucleic acid fragments that are not naturally occurring and would not be found in the natural state. The term "isolated" is also used herein to refer to cells or polypeptides that have been separated from other cellular proteins or tissues. Isolated polypeptides are meant to include purified and recombinant polypeptides.

[0054] The term "cross-reactivity" refers to the ability of an antibody of the present invention to bind to an antigen from a different species. Cross-reactivity can be measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or by detecting binding to cells that physiologically express the antigen, or by interacting with the function of cells that physiologically express the antigen in other ways. Examples of assays known in the art to measure binding affinity include surface plasmon resonance (e.g., Biacore) or similar technologies (e.g., Kinexa or octet).

[0055] The terms "immune binding" and "immune binding properties" refer to the non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immune binding interaction is determined by the equilibrium dissociation constant (K D ), where K D A lower value indicates a higher affinity. The immunobinding properties of a selected polypeptide can be measured by methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen / antibody complex. The "association rate constant" (K a or K on ) and "dissociation rate constant" (K d or K off Both k and k can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, 1993, Nature, 361:186-187). d / k a The ratio of the equilibrium dissociation constant K D (See Davies DR et al., 1990, Annual Rev Biochem 59:439-473). K can be calculated by any effective method. D , k a and k d The value can be measured.

[0056] The term "host cell" refers to a cell in which a vector can be propagated and its DNA expressed, and may be a prokaryotic or eukaryotic cell. The term also includes any progeny of the subject host cell. It is understood that such progeny are included and that not all progeny are identical to the parent cell because mutations may occur during replication. Host cells include prokaryotic, yeast, or mammalian cells, such as CHO cells, NS0 cells, or other mammalian cells.

[0057] The term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. If a position in each of the two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., if a position in each of two DNA molecules is occupied by adenine, or if a position in each of two polypeptides is occupied by lysine), the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared, then multiplied by 100. For example, if six of ten positions in two sequences match, the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three of six positions match). Typically, two sequences are compared to generate maximum identity. Such matching can be easily performed using a computer program, such as the Align program (DNAstar, Inc.), and can be achieved using the method of Needleman and Wunsch (Needleman SB and Wunsch CD, 1970, J Mol Biol, 48:443-453).

[0058] The terms "mutant," "variant," and "mutation" refer to a substitution, deletion, or insertion of one or more nucleotides or amino acids, respectively, compared to a native nucleic acid or polypeptide (i.e., a reference sequence that can be used to define the wild type).

[0059] The term "conservative modification" means that the amino acid modification does not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as point mutation and PCR-mediated mutagenesis. A conservative amino acid substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been well described in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the invention can be substituted with other amino acid residues from the same side chain family.

[0060] The antibodies of the present invention or nucleic acids or polynucleotides encoding the antibodies of the present application can be used to prepare pharmaceutical or sterile compositions, for example, by mixing the antibodies with pharmaceutically acceptable vectors, excipients, or stabilizers. Pharmaceutical compositions may contain one or a combination (e.g., two or more different) of the antibodies of the present invention. For example, pharmaceutical compositions of the present invention may contain a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on the target antigen. Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing them with pharmaceutically acceptable vectors, excipients, or stabilizers in the form of lyophilized powder, slurry, aqueous solution, or suspension. The term "pharmaceutically acceptable" means that the molecular entity, molecular fragment, or composition does not elicit adverse, allergic, or other adverse reactions when appropriately administered to animals or humans. Specific examples of some substances that can be used as pharmaceutically acceptable vectors or components thereof include sugars (e.g., lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyhydric alcohols (e.g., propylene glycol), alginic acid, etc. The antibodies of the invention or nucleic acids or polynucleotides encoding the antibodies of the present application may be used alone or in combination with one or more other therapeutic agents, such as, for example, vaccines.

[0061] The term "pharmaceutically acceptable vector and / or excipient and / or stabilizer" refers to a vector and / or excipient and / or stabilizer that is pharmacologically and / or physiologically compatible with the subject and active ingredient, and whose dosage and concentration used are non-toxic to cells or mammals exposed thereto. These may include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH adjusters may include, but are not limited to, phosphate buffers. Surfactants may include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers may include, but are not limited to, sodium chloride. Preservatives may include, but are not limited to, various antibacterial and antifungal agents, such as parahydroxybenzoates, chlorobutanal, phenol, sorbic acid, etc. Agents for maintaining osmotic pressure may include, but are not limited to, sugars, NaCl, and analogs thereof. Agents that delay absorption may include, but are not limited to, monostearate salts and gelatin. Diluents may include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols and polyalcohols (e.g., glycerin), etc. Preservatives may include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parahydroxybenzoates, chlorobutanal, phenol, sorbic acid, etc. Stabilizers have meanings commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the formulation, and may include, but are not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, glucan, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or their degradation products (e.g., lactalbumin hydrolysate), etc.

[0062] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired effect, or at least partially achieve it. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, inhibit, or delay the onset of a disease. A disease-treating effective amount refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. Determining such effective amounts is entirely within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously.

[0063] The following examples further illustrate embodiments of the present invention, but it should be understood by those skilled in the art that the following figures and examples are merely illustrative of the present invention and are not intended to further limit the present invention. [Brief explanation of the drawings]

[0064] [Figure 1] Measurement of binding of anti-human TFPI antibodies to human TFPI protein. [Figure 2] Assay of FXa activity recovery by anti-human TFPI antibody. [Figure 3] TFPI neutralization assay using anti-human TFPI antibody. [Figure 4] Measurement of binding of anti-human TFPI antibodies to HUVEC cells. [Figure 5] Assay of TFPI neutralization on HUVEC cells by anti-human TFPI antibody. DETAILED DESCRIPTION OF THE INVENTION

[0065] The invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the invention.

[0066] Unless otherwise specified, the molecular biological experimental methods and immunoassays used in the present invention are essentially performed with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M.A.usubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995, and restriction enzymes are used in accordance with the conditions recommended by the product manufacturers. Those skilled in the art will understand that the examples describe the present invention in an illustrative manner and are not intended to limit the scope of the claims of the present invention.

[0067] Example 1: Preparation of mouse-derived monoclonal antibody against human TFPI Human TFPI antigen (protein sequence: NCBI accession number NP_006278.1) was thoroughly emulsified in complete Freund's adjuvant at 50 μg / mouse and immunized into male Balb / C mice using a multipoint immunization method with an immunization cycle of once every three weeks. Ten days after the third immunization, blood was collected from the tail vein and the anti-human TFPI antibody titer in the plasma was tested by ELISA to monitor the immune response of the mice. Subsequently, mice with the highest anti-human TFPI antibody titer were boosted once during the first three days of fusion. Three days later, the mice were sacrificed and their spleens were removed and fused with the mouse myeloma Sp2 / 0 cell line. 2×10 8 2 × 10 Sp2 / 0 cells and 2 × 10 8 The spleen cells were mixed with 5 × 10 spleen cells and fused in a solution of 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO). The spleen cells were fused at a concentration of 5 × 10 spleen cells in Iscove's medium (containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μg thymidine). 5The solution was adjusted to 1 mL / mL, and 0.3 mL was placed in a 96-well culture plate well and placed in a 37°C, 5% CO2 incubator. After 10 days of culture, clones with high affinity binding to TFPI and antibodies in the supernatant were detected using a high-throughput ELISA. The fused cells in the monoclonal antibody wells were then subcloned and further selected to obtain hybridoma cell line #3-71.

[0068] Clones that produced specific antibodies were cultured in RPMI 1640 medium supplemented with 10% FCS at a cell density of approximately 5 × 10 5 When the cell density reached 100 cells / mL, the medium was replaced with serum-free medium. After 2-4 days, the culture medium was centrifuged and the culture supernatant was collected. A protein G column was used to purify the antibody. The monoclonal antibody eluate was dialyzed against 150 mM NaCl. The dialyzed solution was filtered through a 0.22 μM filter to sterilize it, and the purified mouse-derived monoclonal antibody mAb3-71 was obtained and ready for testing.

[0069] Example 2. Affinity measurements and kinetic studies of TFPI mouse-derived antibodies The binding affinity constant of purified anti-human TFPI mouse monoclonal antibody (mAb3-71) to human TFPI was measured using biofilm interference (BLI) technology on a PALL ForteBio octet RED&QK system. The concentration gradient for multichannel parallel quantitative analysis was set at 3.125, 6.25, 12.5, 25, 50, and 100 nM, and 10 μg / mL His-tagged human TFPI was coupled to a Ni-NTA sensor. The affinity measurement results are shown in Table 1. The results indicated that the mouse monoclonal antibody had extremely high binding affinity for human TFPI, with a 10 -12 It has been shown that it is possible to reach the order of M.

[0070] [Table 1]

[0071] Example 3. Humanization of anti-human TFPI mouse-derived antibodies CDR grafting was used to humanize mouse antibodies. The basic principle of CDR grafting is to graft the CDR regions of a mouse antibody onto a human antibody template, while simultaneously introducing several key mouse FR region residues (backmutations) that stabilize the CDR conformation and are important for antigen-antibody binding. This aims to maintain the affinity of the mouse antibody while reducing its immunogenicity. In addition to the CDR grafting procedure, we also calculated the isoelectric point (PI), hydrophobic aggregation, post-translational modifications (PTMs, e.g., glycosylation, cleavage, isomerization sites, etc.), and immunogenicity of the humanized antibody after CDR grafting. We then mutated amino acids that could cause problems in these four areas to ensure that the humanized antibody would be clinically effective.

[0072] The specific steps for antibody humanization are as follows. The IMGT human antibody germline database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) was searched to obtain human antibody templates with high similarity to the mouse antibody. Discovery Studio was used to annotate the CDR regions of the mouse antibody and human antibody templates, and the CDR regions were defined according to the Kabat or IMGT scheme. The six CDR regions of the human antibody template were replaced with the six CDR regions of the mouse antibody. Individual CDR regions among the six grafted CDR regions may be defined according to either Kabat or IMGT. After CDR grafting, back mutations were performed on the mouse antibody to the FR region of the humanized template. The following four amino acid residues were included in the mouse antibody FR region, which stabilize the antibody CDR conformation and are important for antigen-antibody binding. 1) amino acids buried under the antibody surface within 6 Å of the CDR region, 2) amino acids exposed on the antibody surface within 6 Å of the CDR region, 3) amino acids at the interface between the antibody light and heavy chain domains, and 4) vernier zone residues that stabilize the antibody CDR region conformation (Foote J and Winter G, 1992, J Mol Biol, 224:487-499). These four important residues in the mouse antibody FR region were identified by establishing a three-dimensional structural model of the mouse antibody. For these four amino acids in the human-derived template that did not match the mouse antibody sequence, three-dimensional structural analysis was used to select amino acids important for maintaining the CDR conformation and antigen-antibody binding, and amino acids were then transplanted or substituted from the mouse antibody to the human-derived template. The isoelectric point, hydrophobic aggregation, post-translational modification, and immunogenicity of the humanized antibody generated after the four amino acid transplants were then further calculated, and the problematic amino acids were mutated to obtain the final humanized antibody sequence.

[0073] Using the above method, a humanized antibody was constructed based on the CDRs of the mouse-derived antibody mAb3-71 and named AB8D. The amino acid sequences of the CDR regions contained in the variable regions of the above mouse antibody and humanized antibody are shown in Table 2, and the amino acid sequences of the heavy and light chain variable regions are shown in Table 3.

[0074] [Table 2]

[0075] [Table 3]

[0076] To obtain a full-length antibody sequence consisting of two heavy chains and two light chains, antibody heavy chain constant region (preferably human IgG1, IgG2 or IgG4) and light chain constant region (preferably human kappa light chain, the amino acid sequence of which was as shown in SEQ ID NO: 18) sequences of the VH and VL sequences shown in Table 3 were stitched or assembled using conventional techniques. Preferably, the heavy chain constant region is a human wild-type heavy chain constant region or a variant thereof.

[0077] In one exemplary embodiment, the humanized antibody molecule consists of the heavy chain constant region of wild-type human IgG1 (amino acid sequence shown in SEQ ID NO: 19). In another embodiment, the humanized antibody molecule consists of the heavy chain constant region of human IgG1 mutated at M252Y, S254T, T256E, and M428L according to EU numbering (amino acid sequence shown in SEQ ID NO: 20). In another embodiment, the humanized antibody molecule consists of the heavy chain constant region of wild-type human IgG2 (amino acid sequence shown in SEQ ID NO: 21). Alternatively, a modified human IgG4 constant region sequence is employed; in one embodiment, the humanized antibody molecule consists of a human IgG4 mutated at position 228 according to EU numbering (e.g., from S to P) (amino acid sequence as shown in SEQ ID NO: 22).

[0078] In an exemplary embodiment, the humanized antibody molecule has a heavy chain amino acid sequence as shown in SEQ ID NO:23 and a light chain amino acid sequence as shown in SEQ ID NO:24.

[0079] Example 4: Construction, expression, and preparation of an expression vector for anti-human TFPI antibody Based on the heavy and light chain sequences obtained in the above examples, coding cDNAs were designed and inserted into the pcDNA3.1 eukaryotic expression vector to construct a humanized expression vector. The expression vector plasmid contains the cytomegalovirus early gene promoter-enhancer required for high-level expression in mammalian cells. The vector plasmid also contains a selectable marker gene that confers ampicillin resistance in bacteria and G418 resistance in mammalian cells. The vector plasmid also contains a dihydrofolate reductase (DHFR) gene, allowing co-amplification of the antibody gene and the DHFR gene in appropriate host cells using methotrexate (MTX).

[0080] The recombinant expression vector plasmid constructed above was transfected into a mammalian host cell line to express the humanized antibody. To stabilize high-level expression, the preferred host cell line is DHFR-deficient Chinese hamster ovary (CHO) cells (see U.S. Patent No. 4,818,679). The preferred transfection method is electroporation, although other methods, including calcium phosphate coprecipitation, lipofection, and protoplast fusion, may also be used. For electroporation, a GenePulser (Bio-Rad Laboratories) was used, set to a 300 V electric field and a capacitance of 1050 μFd, and 2 × 10 cells were transfected into a cuvette. 7Cells were added and suspended in 0.8 mL of PBS, containing 20 μg of expression vector plasmid. Two days after transfection, 0.2 mg / mL G418 and 200 nM MTX (Sigma) were added. To achieve high-level expression, the transfected antibody gene was co-amplified with the DHFR gene, which was suppressed by MTX. Subcloned transfectants were subjected to limiting dilution, and the secretion rate of each cell line was measured using ELISA to select cell lines that expressed high levels of antibody. The conditioned medium of the antibody was collected and used to measure its in vitro and in vivo biological activity.

[0081] Example 5: Purification and characterization of anti-human TFPI antibodies The purification and characterization methods for humanized antibody AB8D are described in this Example. Cell culture supernatant was clarified by high-speed, low-temperature centrifugation or depth filtration, followed by 0.22 μm sterile filtration. The antibody was then purified by three chromatography steps: Protein A affinity, anion exchange, and cation exchange. The first step of capture was Protein A affinity chromatography, equilibrated with PBS buffer. Purification was performed by linear elution with elution buffer (50 mM NaAc-HAc, pH 3.7). The pH of the Protein A elution product was adjusted to 3.6-3.8 with 1 M glacial acetic acid. The virus was inactivated by incubation at room temperature for 60 minutes, followed by neutralization with 2 M Tris and pH 5.5. For intermediate purification, the anion exchange sorbent Q Bestarose FF was selected to remove residual DNA, endotoxins, and impurity proteins. The equilibration solution was 50 mM NaAc-HAc, pH 5.5. The protein flowed down from the anionic medium was directly bound to the cation exchange medium MonomixHC45-SP. The equilibration solution for cation exchange chromatography was 50 mM NaAc-HAc (pH 5.5), and linear elution was performed using an eluent (50 mM NaAc-HAc, 0.2 M NaCl, pH 5.5). The protein solution eluted from the cationic medium was filtered through a nanofiltration membrane, concentrated by ultrafiltration, and decontaminated and filtered to obtain a stock solution.

[0082] SEC-HPLC revealed that the purity of the main peak of the purified antibody was 99% or higher. The theoretical molecular weight of the AB8D antibody was approximately 145 KD, and SDS-PAGE electrophoresis revealed that the light chain was approximately 25 KD and the heavy chain was approximately 50 KD, which were essentially the theoretical values under reducing conditions.

[0083] Example 6: Evaluation of in vitro biological function of anti-human TFPI antibodies 6.1 Determination of the affinity of anti-human TFPI antibodies for human TFPI protein. Human TFPI was encapsulated at 0.1 μg / mL and incubated overnight at 4°C in 100 μL per well of an enzyme-labeled plate. The encapsulation solution was discarded, and the plate was sealed with 1% BSA in PBS (PBSB). 200 μL per well was added and incubated at 37°C for 1 hour. A series of diluted concentrations of AB8D was then added at 100 μL per well and incubated at 37°C for 1 hour. The plate was washed with 0.05% PBST, and HRP-conjugated goat anti-human IgG (H+L) (Jackson Laboratory) was added as a detection antibody and incubated at 37°C for 1 hour. After washing three times with 0.05% PBST, 100 μL of TMB was added per well and the plate was allowed to develop at room temperature for 5 minutes. The reaction was terminated by adding 0.2 M H2SO4 (50 μL per well). The absorbance values were read at two wavelengths, 450 and 620 nm, using an enzyme marker. The reaction was analyzed using GraphPad Prism 6 software, with OD450 nm-OD620 nm on the Y axis and antibody concentration on the X axis, and EC 50 As shown in Figure 1, AB8D exhibits affinity for human TFPI protein and exhibits an EC 50 was 1.65 ng / mL.

[0084] 6.2. FXa activity measurement test Human TFPI was diluted to a concentration of 10 μg / mL in Buffer 1 (containing 20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, and 0.5 mg / mL BSA, pH 8.0) and added at 50 μL per well to a 96-well flat-bottom plate. FXa (purchased from Hematologic Technologies) was diluted to a concentration of 1 μg / mL in Buffer 1, 50 μL added per well, and incubated at 37°C for 30 minutes. Substrate S-2765 (purchased from Chromogenix) was diluted to 2 mM in Buffer 1 and 50 μL added per well. The absorbance at 405 nm was measured using an enzyme marker after the reaction. Graphs were then created using GraphPad Prism 6 software, with OD at 405 nm on the Y-axis and antibody concentration on the X-axis. As shown in Figure 2, AB8D inhibited TFPI-inhibited FXa activity with an EC of 6.9 μg / mL. 50 and recovered.

[0085] 6.3. TFPI neutralization test: measurement of TF / FVIIa / FXa inhibitory effect TF (tissue factor, purchased from Beijing Bosi Technology Co., Ltd.) or FVIIa (WHO) was diluted in Buffer 1 to a concentration of 1.4 ng / mL for TF and 45 IU / mL for FVIIa. 25 μL of each solution was added to each well of a 96-well plate and incubated at 37°C for 10 minutes. AB8D was then diluted at various concentrations and added at 25 μL per well. Human TFPI was diluted in Buffer 1 to a concentration of 10 μg / mL and added at 25 μL per well of the 96-well plate and incubated at 37°C for 30 minutes. FX (purchased from Hematologic Technologies) was diluted in Buffer 1 to a concentration of 44.2 μg / mL and added at 25 μL per well of the 96-well plate and incubated at 37°C for 10 minutes. The reaction was terminated by adding 10 μL of 75 mM EDTA per well. Substrate S-2765 was diluted to 2 mM with Buffer 1 and added at 50 μL per well. After the reaction, the absorbance at 405 nm was measured using an enzyme counter. OD 405 nm was plotted on the Y-axis and antibody concentration on the X-axis. Analysis was performed using GraphPad Prism 6 software, and EC was calculated using a four-parameter fitting curve. 50 As shown in Figure 3, at the molecular level, AB8D neutralized the inhibitory effect of TFPI and restored the TFPI-mediated FXa inhibitory activity of FVIIa / TF in a dose-dependent manner, resulting in an EC 50 was 3.17 μg / mL.

[0086] 6.4. FACS method to detect binding to TFPI-expressing HUVECs Cultured HUVEC cells (human umbilical vein endothelial cell line, Shanghai Enzyme Research Biotechnology Co., Ltd.) were centrifuged and collected. The cells were resuspended in 1% PBSB to a cell density of 1 × 10 6The cells were adjusted to 100 μL per well and added to a 96-well U-bottom plate and blocked at 4°C for 30 minutes. The cells were washed once with 1% PBSB. AB8D was then diluted at 100 μL per well and incubated at 4°C for 1 hour. The supernatant was removed by centrifugation, washed twice with 1% PBSB, and diluted AF647 sheep anti-human IgG (H+L) antibody (Jackson Immuno Research Inc., 1:400 dilution) was added at 100 μL per well and incubated at 4°C for 1 hour in the dark. The supernatant was removed by centrifugation, the plate was washed twice with 1% PBSB, and the cells were resuspended in 1% PBSB. 150 μL was added per well. Signal intensity was detected by flow cytometry. The mean fluorescence intensity (Y axis) and antibody concentration (X axis) were plotted on the Y axis. EC values were calculated using a four-parameter fitting curve in GraphPad Prism 6 software. 50 As shown in Figure 4, AB8D bound to HUVECs via TFPI and the EC 50 was 28.6 ng / mL.

[0087] 6.5. Neutralization of TFPI in HUVECs by anti-human TFPI antibodies When the HUVEC cells reached a confluence rate of 80% or more and a good appearance, the medium was discarded and the dishes were washed with sterile phosphate buffer. 1 mL of cell digestion solution was added to each dish and incubated at room temperature for 1 minute. Cell digestion was completed by adding 2 mL of ECM medium containing 10% fetal bovine serum to each dish and resuspending the cells. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded, and an appropriate amount of medium was added to resuspend the cells and gently mixed. Cell counting: An appropriate amount of cells for counting was collected and the cell density was adjusted to 5 x 10 5After adjusting the concentration to cells / mL, 100 μL of cells were spread per well in a 96-well plate and cultured overnight in a CO2 incubator. The next day, the cell culture medium was discarded, the plates were washed twice with DPBS, and 25 μL of prepared TNFα (400 ng / mL) and IL-1β (400 ng / mL) were added to each well. AB8D diluted in Buffer 2 (25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, pH 7.4) was added at a series of concentrations to each well. FVIIa was diluted to a concentration of 45 IU / mL in Buffer 3 (25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, 5 mM CaCl2, 1 mg / mL BSA, pH 7.4) and added at a concentration of 25 μL to each well. FX was diluted with Buffer 3 to a concentration of 400 nM, 25 μL per well, and incubated at 37°C for 40 minutes. 40 μL of the supernatant was transferred to a new 96-well plate, and 10 μL of 75 mM EDTA was added per well to terminate the reaction. Substrate S-2765 was diluted to 1.2 mM with Buffer 3, 50 μL per well. After the reaction, the absorbance at 405 nm was read using the enzyme marker. Next, a graph was plotted using GraphPad Prism 6 software, with OD at 405 nm on the Y axis and antibody concentration on the X axis. EC values were calculated using a four-parameter fitting curve. 50 As shown in Figure 5, at the cellular level, AB8D neutralized TFPI in HUVEC cells, blocked TFPI-mediated FVIIa / TF inhibition, and suppressed FXa activity with an EC value of 117.3 ng / mL. 50 and recovered.

[0088] Example 7: Evaluation of in vivo efficacy of anti-human TFPI antibody Twenty-four male New Zealand rabbits (weight 2.0–2.5 kg) purchased from Shanghai Jie Si Jie Laboratory Animal Co., Ltd. were randomly divided into eight groups based on body weight: G1 (vehicle control group), G2 (AB8D-10 mg / kg group), G3 (AB8D-5 mg / kg group), G4 (AB8D-2.5 mg / kg group), G5 (vehicle control group), G6 (AB8D-20 mg / kg group), G7 (AB8D-10 mg / kg group), and G8 (AB8D-5 mg / kg group).

[0089] Each group received subcutaneous injections of either vehicle control or different concentrations of the AB8D test substance. On days 4 and 7 after administration, groups G1-G4 and G5-G8 received a single injection of 15 mg / kg of anticoagulant factor VIII neutralizing antibody (anti-FVIII neutralizing antibody) via the marginal ear vein to establish the hemophilia model. Forty-five minutes after administration of the anti-FVIII neutralizing antibody, the rabbits were anesthetized with 2-5% isoflurane. The anesthetized rabbits were placed in a prone position, with one of their front paws pre-immersed in a beaker containing 37°C saline. Cutting of the third nail was performed to induce cuticle bleeding. After cutting, the paw was naturally placed in the beaker containing 37°C saline, with the front paw submerged below the surface of the saline. The bleeding time was recorded, and the simultaneous bleeding volume was measured gravimetrically. Bleeding times exceeding 60 minutes were recorded as 60 minutes.

[0090] As shown in Tables 4 and 5, after a single administration of AB8D on days 4 and 7, AB8D at doses of 5 mg / kg or more had an inhibitory effect on both the amount of keratinous bleeding and the bleeding time in hemophilia model rabbits.

[0091] [Table 4]

[0092] [Table 5]

[0093] While preferred embodiments of the present invention have been illustrated and described, it should be understood that those skilled in the art, following the teachings herein, can make various modifications which do not depart from the scope of the invention.

[0094] All documents mentioned in this application are incorporated herein by reference as if each document were incorporated by reference individually. It should be understood that, after reading the above teachings of the present invention, those skilled in the art may make various modifications or changes to the present invention, and the equivalent forms thereof will also fall within the scope defined by the claims appended hereto.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to TFPI, The heavy chain variable region (VH) contained in the antibody or antigen-binding fragment thereof comprises: (i) an HCDR1 having a sequence set forth in SEQ ID NO: 1 or 7, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (ii) an HCDR2 having a sequence set forth in SEQ ID NO: 2, 8, 12, or 13, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (iii) an HCDR3 having a sequence set forth in SEQ ID NO: 3 or 9, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; and at least one, two or three complementarity determining regions (CDRs) selected from the group and / or The light chain variable region (VL) contained in the antibody or antigen-binding fragment thereof comprises: (iv) an LCDR1 having a sequence set forth in SEQ ID NO: 4 or 10, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (v) an LCDR2 having a sequence set forth in SEQ ID NO: 5 or Gly-Thr-Ser, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (vi) an LCDR3 having a sequence set forth in SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three substitutions, deletions or additions) compared to the above sequence; and at least one, two or three complementarity determining regions (CDRs) selected from the group wherein the heavy chain variable region containing the CDRs and / or the light chain variable region containing the CDRs are defined according to the Kabat or IMGT numbering system; Preferably, the substitutions according to any one of (i) to (vi) are conservative substitutions. An antibody or antigen-binding fragment thereof capable of specifically binding to TFPI.

2. The antibody or antigen-binding fragment thereof (1) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each having a sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, or 6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (2) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each having a sequence set forth in SEQ ID NO: 7, 8, 9, 10, Gly-Thr-Ser, or SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (3) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each having a sequence set forth in SEQ ID NO: 1, 12, 3, 4, 5, or 6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; (4) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each having a sequence set forth in SEQ ID NO: 7, 13, 9, 10, Gly-Thr-Ser, or SEQ ID NO: 6, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three substitutions, deletions, or additions) compared to any of the above sequences; comprising three VH variable region CDRs and three VL variable region CDRs selected from the group The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof is mouse-derived or chimeric, and its heavy chain variable region comprises a heavy chain FR region of mouse-derived IgG1, IgG2, IgG3, or a mutant thereof, and its light chain variable region comprises a light chain FR region of mouse-derived κ or λ chain, or a mutant thereof. The antibody or antigen-binding fragment thereof according to claim 2.

4. The antibody or antigen-binding fragment thereof a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 14, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)); and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 15, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)). The following VH and VL sequences are included: The antibody or antigen-binding fragment thereof according to claim 3.

5. the antibody or antigen-binding fragment thereof is humanized; The antibody or antigen-binding fragment thereof according to claim 2.

6. The antibody or antigen-binding fragment thereof a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 16, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)); and a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 17, or a sequence substantially identical thereto (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity, or having one or more amino acid substitutions (e.g., conservative substitutions)). The following VH and VL sequences are included: The antibody or antigen-binding fragment thereof according to claim 5.

7. The antibody comprises a heavy chain constant region and a light chain constant region derived from a human immunoglobulin, and preferably the heavy chain constant region is selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, and IgG4, and the heavy chain constant region has a native sequence or a sequence having one or more amino acid substitutions, deletions, or additions compared to the native sequence from which it is derived, and the light chain constant region is preferably the constant region of the human κ appa chain shown in SEQ ID NO:

18. The antibody described in claim 6.

8. The heavy chain constant region contained in the antibody is (i) a wild-type human IgG1 heavy chain constant region as set forth in SEQ ID NO: 19; (ii) a heavy chain constant region of human IgG1 comprising the M252Y, S254T, T256E, and M428L mutations shown in SEQ ID NO: 20; (iii) a wild-type human IgG2 heavy chain constant region as set forth in SEQ ID NO: 21; and (iv) a heavy chain constant region of human IgG4 containing the S228P mutation shown in SEQ ID NO: 22; selected from the group The antibody described in claim 7.

9. The antibody of claim 7, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 23 and the light chain comprises the amino acid sequence of SEQ ID NO:

24.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment binds to TFPI with a KD of 10 nM or lower.

11. Encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. DNA molecule.

12. comprising the DNA molecule of claim 11. vector.

13. including prokaryotic, yeast, or mammalian cells, preferably CHO cells; A host cell comprising the vector of claim 12.

14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, a pharmaceutical excipient, a vector, or a diluent. Pharmaceutical compositions.

15. a step (a) of obtaining a gene encoding an antibody or antigen-binding fragment thereof and constructing an expression vector for the antibody or antigen-binding fragment thereof; a step (b) of transfecting the expression vector into a host cell by genetic engineering techniques; a step (c) of culturing the host cell under conditions that permit the production of the antibody or antigen-binding fragment thereof; and a step (d) of isolating and purifying the produced antibody or antigen-binding fragment thereof; Wherein, the expression vector in step (a) is one or more selected from the group consisting of a plasmid, a bacterium, and a virus, and preferably, the expression vector is a pcDNA3.1 vector; In step (b), the vector constructed by genetic engineering techniques is transfected into a host cell, and the host cell includes a prokaryotic cell, a yeast cell, or a mammalian cell, and preferably a CHO cell. wherein step (d) isolating and purifying the antibody or antigen-binding fragment thereof by conventional immunoglobulin purification methods, including protein A affinity chromatography and ion exchange; A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a medicament for the prevention or treatment of a disease or event associated with inherited or acquired coagulation factor deficiency.

17. Use in the preparation of a medicament for the prevention or treatment of a disease or event associated with an inherited or acquired deficiency of a coagulation factor, wherein the disease is hemophilia; use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 9.

Images