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HK1156635A - Monoclonal antibodies against tissue factor pathway inhibitor (tfpi) - Google Patents

Monoclonal antibodies against tissue factor pathway inhibitor (tfpi) Download PDF

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Publication number
HK1156635A
HK1156635A HK11110795.3A HK11110795A HK1156635A HK 1156635 A HK1156635 A HK 1156635A HK 11110795 A HK11110795 A HK 11110795A HK 1156635 A HK1156635 A HK 1156635A
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Hong Kong
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seq
chain variable
variable region
polypeptide
light chain
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HK11110795.3A
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Chinese (zh)
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HK1156635B (en
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Zhuozhi Wang
John E. Murphy
Junliang Pan
Haiyan Jiang
Bing Liu
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拜耳医药保健有限公司
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Publication of HK1156635B publication Critical patent/HK1156635B/en

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Description

Monoclonal antibodies against Tissue Factor Pathway Inhibitor (TFPI)
Sequence Listing submission
The sequence listing associated with this application is submitted in electronic form via an EFS network and is hereby incorporated by reference in its entirety into the specification. The name of the text file containing the Sequence Listing is MSB7329PCT _ Sequence _ Listing _ ST 25.
Technical Field
Isolated monoclonal antibodies that bind to human Tissue Factor Pathway Inhibitor (TFPI) and fragments thereof and related inventions are provided.
Background
Blood coagulation (blood coagulation) is the process by which blood forms a stable clot to stop bleeding. This process involves many proenzymes and pro-cofactors (or "clotting factors") circulating in the blood. Those proenzymes and pro-cofactors interact via several pathways that convert them into the activated form sequentially or simultaneously. Finally, the process results in the activation of prothrombin to thrombin by activated factor x (fxa) in the presence of factor Va, ionic calcium, and platelets. The activated thrombin in turn induces platelet aggregation and converts fibrinogen to fibrin, which is then cross-linked by activated factor xiii (fxiiia) to form a clot.
The process leading to factor X activation can be carried out by two distinct pathways: the contact activation pathway (formerly known as the intrinsic pathway) and the tissue factor pathway (formerly known as the extrinsic pathway). It was previously thought that the coagulation cascade consisted of two pathways of equal importance connected to a common pathway. It is now known that the main pathway for initiating blood coagulation is the tissue factor pathway.
Factor X can be activated by Tissue Factor (TF) in combination with activated factor vii (fviia). The complex of factor VIIa and its essential cofactor TF is a powerful initiator of the coagulation (clotting) cascade.
The tissue factor pathway of coagulation is negatively controlled by tissue factor pathway inhibitor ("TFPI"). TFPI is a natural, FXa-dependent feedback inhibitor of the FVIIa/TF complex. It is a member of the multivalent Kunitz-type serine protease inhibitor. Physiologically, TFPI binds activated factor x (fxa) to form a heterodimeric complex, which then interacts with the FVIIa/TF complex to inhibit its activity, thus closing the tissue factor pathway of coagulation. In principle, blocking TFPI activity can restore FXa and FVIIa/TF activity, thus prolonging the duration of action of the tissue factor pathway, and amplifying FXa production, which is a common defect of hemophilia a and B.
Indeed, some preliminary experimental evidence has indicated that blocking TFPI activity by antibodies against TFPI normalizes prolonged clotting time or shortens bleeding time. For example, Nordfang et al show that the extended dilution prothrombin time of hemophilia plasma (dilute prothrombin time) is normalized after treatment of the plasma with an antibody against TFPI (Thromb. Haemost, 1991, 66 (4): 464-. Similarly, Erhardtsen et al showed a significant reduction in bleeding time in a hemophilia A rabbit model by anti-TFPI antibodies (Blood clotting and Fibrinolysis, 1995, 6: 388-394). These studies suggest that inhibition of TFPI by anti-TFPI antibodies may be useful for treating hemophilia a or B. Only polyclonal anti-TFPI antibodies were used in these studies.
Monoclonal antibodies against recombinant human tfpi (rhtfpi) were prepared and identified using hybridoma technology. See Yang et al, chi.med.j., 1998, 111 (8): 718-721. The effect of monoclonal antibodies on the diluted Prothrombin Time (PT) and the Activated Partial Thromboplastin Time (APTT) was tested. Experiments have shown that anti-TFPI monoclonal antibodies shorten the diluted thromboplastin clotting time of factor IX deficient plasma. The tissue factor pathway has been suggested to play an important role not only in physiological coagulation but also in hemophilia bleeding (Yang et al, Hunan Yi Ke Da Xue Xue Xue Bao, 1997, 22 (4): 297-300).
U.S. patent No. 7,015,194 to Kjalke et al discloses a composition comprising FVIIa and a TFPI inhibitor (including polyclonal or monoclonal antibodies or fragments thereof) for use in the treatment or prevention of bleeding episodes or coagulation treatments. Also disclosed is the use of such compositions to reduce clotting time in normal mammalian plasma. It is further suggested that factor VIII or variants thereof may be included in the disclosed compositions of FVIIa and TFPI inhibitors. No combinations of FVIII or factor IX with TFPI monoclonal antibodies were suggested.
In addition to treating hemophilia, it has also been suggested that TFPI inhibitors, including polyclonal or polyclonal antibodies, may be used for cancer treatment (see Hung, U.S. patent No. 5,902,582).
Thus, antibodies specific for TFPI are needed to treat hematologic diseases and cancer.
Generally, genetic engineering has been used to create murine, chimeric, humanized or fully human antibodies to generate therapeutic antibodies for human diseases. Murine monoclonal antibodies have been shown to have limited utility as therapeutic agents due to short serum half-life, inability to trigger human effector functions, and production of human anti-mouse antibodies. Brekke and Sandlie, "Therapeutic additives for Human Diseases at the Dawn of the twenties-first center," Nature 2, 53, 52-62 (month 1 2003). Chimeric antibodies have been shown to elicit human anti-chimeric antibody responses. Humanized antibodies further minimize the mouse component of the antibody. However, fully human antibodies completely avoid the immunogenicity associated with murine elements. Thus, there is a need to develop fully human antibodies to avoid the immunogenicity associated with other forms of genetically engineered monoclonal antibodies. In particular, if antibodies with murine components or murine origin are used, long-term (chronoic) prophylactic treatment, such as would be required for hemophilia treatment with anti-TFPI monoclonal antibodies, has a high risk of developing an immune response to the treatment due to the frequent dosing and long duration of therapy required. For example, antibody therapy for hemophilia a may require dosing weekly for the lifetime of the patient. This can be a continuous challenge to the immune system. Thus, there is a need for fully human antibodies for antibody therapy for hemophilia and related genetic and acquired deficiencies or defects in coagulation.
Therapeutic antibodies have been generated via hybridoma technology as described by Koehler and Milstein in "Continuous Cultures of Fused Cells secreted antibodies of refined Specificity," Nature 256, 495-497 (1975). Fully human antibodies can also be recombinantly produced in prokaryotes and eukaryotes. The therapeutic antibody is preferably produced recombinantly in the host cell rather than by a hybridoma. Recombinant production has the advantage of greater product consistency, possibly higher production levels, and controlled production, which minimizes or eliminates the presence of animal-derived proteins. For these reasons, it is desirable to have a recombinantly produced monoclonal anti-TFPI antibody.
Summary of The Invention
Monoclonal antibodies directed against human Tissue Factor Pathway Inhibitor (TFPI) are provided. Further provided are isolated nucleic acid molecules encoding the same. Also provided are pharmaceutical compositions comprising anti-TFPI monoclonal antibodies and methods of treating genetic and acquired deficiencies or deficiencies in coagulation, such as hemophilia a and B. Methods for reducing bleeding time by administering an anti-TFPI monoclonal antibody to a patient in need thereof are also provided. Methods for generating monoclonal antibodies that bind to human TFPI according to the invention are also provided.
Brief Description of Drawings
FIG. 1: representative examples of Fab selected by panning and screening for binding activity to human TFPI ("h-TFPI") and mouse TFPI ("m-TFPI"). Control Fab against estradiol-BSA ("EsB") and 12 Fab's (1-4 and 6-13) selected by panning TFPI were tested. The Y-axis represents the fluorescence units of the ELISA results.
FIG. 2: dose-dependent in vitro functional activity of 4 representative anti-TFPI antibodies (4B 7: TP-4B7, 2a 8: TP-2a8, 2G 6: TP-2G6, 2G 7: TP-2G7) obtained by panning and screening human antibody libraries, as shown by their shortened dPT. The experiment included incorporation of 0.5ug/mL mTFPI in TFPI depleted plasma.
FIG. 3: in vitro functional activity of an anti-TFPI Fab, i.e., Fab-2A8 (from TP-2A8), as tested in the ROTEM assay.
FIG. 4: binding activity of clone TP-2G6 ("2G 6") to human TFPI and mouse TFPI after conversion to IgG. And (delta): IgG-2G6 bound to mouse TFPI; □: IgG-2G6 binds to human TFPI; a tangle-solidup: control IgG bound to mouse TFPI; ■: control IgG bound human IgG.
FIG. 5: anti-TFPI antibodies TP-2A8 ("2 A8"), TP-3G1 ("3G 1"), and TP-3C2 ("3C 2") reduce whole blood clotting time in hemophilia a mice as tested in the ROTEM assay. Each dot represents an individual hemophilia a mouse.
FIG. 6: anti-TFPI monoclonal antibodies TP-2A10(SEQ ID NO: 18), TP-2B1(SEQ ID NO: 22), TP-2A2(SEQ ID NO: 2), TP-2G2(SEQ ID NO: 66), TP-2A5.1(SEQ ID NO: 6), TP-3A3(SEQ ID NO: 98), TP-2A8(SEQ ID NO: 14), TP-2B8(SEQ ID NO: 34), TP-2G7(SEQ ID NO: 82), TP-4H8(SEQ ID NO: 170), TP-2G4(SEQ ID NO: 70), TP-3F2(SEQ ID NO: 134), TP-2A6(SEQ ID NO: 10), TP-3A2(SEQ ID NO: 94), TP-2C1(SEQ ID NO: 42), TP-3E1(SEQ ID NO: 126), TP-3F1(SEQ ID NO: 130), TP-3D3(SEQ ID NO: 122), TP-4A7(SEQ ID NO: 150), TP-4G8(SEQ ID NO: 166), TP-2B3(SEQ ID NO: 26), TP-2F9(SEQ ID NO: 62), TP-2G5(SEQ ID NO: 74), TP-2G6(SEQ ID NO: 78), TP-2H10(SEQ ID NO: 90), TP-2B9(SEQ ID NO: 38), TP-2C7(SEQ ID NO: 46), TP-3G3(SEQ ID NO: 142), TP-3C2(SEQ ID NO: 114), TP-3B4(SEQ ID NO: 110), TP-2E5(SEQ ID NO: 58), TP-3C3(SEQ ID NO: 118), TP-3G1(SEQ ID NO: 138), Alignment of amino acid sequences between the variable light chains of TP-2D7(SEQ ID NO: 50), TP-4B7(SEQ ID NO: 158), TP-2E3(SEQ ID NO: 54), TP-2G9(SEQ ID NO: 86), TP-3C1(SEQ ID NO: 86), TP-3A4(SEQ ID NO: 102), TP-2B4(SEQ ID NO: 30), TP-3H2(SEQ ID NO: 146), TP-4A9(SEQ ID NO: 154), TP-4E8(SEQ ID NO: 162), and TP-3B3(SEQ ID NO: 106).
FIG. 7: anti-TFPI monoclonal antibodies TP-2A10(SEQ ID NO: 20), TP-3B3(SEQ ID NO: 108), TP-2G4(SEQ ID NO: 72), TP-2A5.1(SEQ ID NO: 8), TP-4A9(SEQ ID NO: 156), TP-2A8(SEQ ID NO: 16), TP-2B3(SEQ ID NO: 28), TP-2B9(SEQ ID NO: 40), TP-2H10(SEQ ID NO: 92), TP-3B4(SEQ ID NO: 112), TP-2C7(SEQ ID NO: 48), TP-2E3(SEQ ID NO: 56), TP-3C3(SEQ ID NO: 120), TP-2G5(SEQ ID NO: 76), TP-4B7(SEQ ID NO: 160), TP-2G6(SEQ ID NO: 80), TP-3C2(SEQ ID NO: 116), TP-2D7(SEQ ID NO: 52), TP-3G1(SEQ ID NO: 140), TP-2E5(SEQ ID NO: 60), TP-2B8(SEQ ID NO: 36), TP-3F1(SEQ ID NO: 132), TP-3A3(SEQ ID NO: 100), TP-4E8(SEQ ID NO: 164), TP-4A7(SEQ ID NO: 152), TP-4H8(SEQ ID NO: 172), TP-2A6(SEQ ID NO: 12), TP-2C1(SEQ ID NO: 44), TP-3G3(SEQ ID NO: 144), TP-2B1(SEQ ID NO: 24), TP-2G7(SEQ ID NO: 84), TP-3H2(SEQ ID NO: 148), TP-2A2(SEQ ID NO: 4), TP-3E1(SEQ ID NO: 128), TP-2G2(SEQ ID NO: 68), TP-3D3(SEQ ID NO: 124), TP-2G9(SEQ ID NO: 88), TP-2B4(SEQ ID NO: 32), TP-3A2(SEQ ID NO: 96), TP-2F9(SEQ ID NO: 64), TP-3A4(SEQ ID NO: 104), TP-3C1(SEQ ID NO: 136), TP-3F2(SEQ ID NO: 136), and TP-4G8(SEQ ID NO: 168).
FIG. 8: a graph showing survival rates at 24 hours after tail vein transection of mice treated with (1) anti-TFPI antibody TP-2A8 ("2 A8"), (2)2A8 and recombinant factor VIII, (3) mouse IgG, and (4) recombinant factor VIII.
FIG. 9: graphs showing clotting time and clot formation time assays for mice treated with the anti-TFPI antibody TP-2A8 ("2 A8"), factor vila, and a combination of 2A8 and factor vila.
FIG. 10: a graph showing the clotting time of normal human blood treated with F VIII inhibitor and anti-TFPI antibody TP-2A8 ("2A 8") and anti-TFPI antibody TP-4B7 ("4B 7") compared to F VIII inhibitor alone.
Detailed Description
Definition of
As used herein, the term "tissue factor pathway inhibitor" or "TFPI" refers to any variant, isoform and species homolog of human TFPI that is naturally expressed by a cell. In a preferred embodiment of the invention, binding of an antibody of the invention to TFPI reduces blood clotting time.
As used herein, "antibody" refers to whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. The term includes full-length immunoglobulin molecules (e.g., IgG antibodies) that occur naturally or are formed by the process of normal recombination of immunoglobulin gene fragments, or immunologically active portions of immunoglobulin molecules such as antibody fragments that retain specific binding activity. Regardless of structure, an antibody fragment binds to the same antigen recognized by a full-length antibody. For example, anti-TFPI monoclonal antibody fragments bind to epitopes of TFPI. The antigen binding function of an antibody may be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, i.e., a fragment consisting of VL、VH、CLAnd CH1Monovalent fragments of domain; (ii) f (ab')2A fragment, i.e. a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) fd fragment consisting of VHAnd CH1Domain composition; (iv) fv fragment consisting of V of a single arm of an antibodyLAnd VH(V) dAb fragment (Ward et al, (1989) Nature 341: 544-546) consisting of VHDomain composition; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although two domains of the Fv fragment, i.e., VLAnd VHAre encoded by different genes, but they can be joined using recombinant methods by synthetic linkers that enable them to be produced as a single protein chain, where VLAnd VHThe regions pair to form monovalent molecules (known as single chain fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the fragments are screened for efficacy in the same manner as intact antibodies.
As used herein, the terms "inhibit binding" and "block binding" (e.g., refer to inhibiting/blocking binding of TFPI ligand to TFPI) are used interchangeably and encompass both partial and complete inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in binding affinity of TFPI to a physiological substrate upon contact with an anti-TFPI antibody as compared to TFPI not contacted with an anti-TFPI antibody, e.g., blocking the interaction of TFPI with factor Xa or blocking the interaction of a TFPI-factor Xa complex with tissue factor, factor VIIa, or a complex of tissue factor/factor VIIa by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).
As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds TFPI is substantially free of antibodies that bind antigens other than TFPI). However, an isolated antibody that binds to an epitope, isoform or variant of human TFPI may have cross-reactivity with other relevant antigens, such as from other species (e.g., TFPI species homologs). Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.
As used herein, "specific binding" refers to the binding of an antibody to a predetermined antigen. Typically, the antibody is present in an amount of at least about 105M-1And binds with a different affinity than it binds to the predetermined antigen or closely related antigenUnrelated antigens (e.g., BSA, casein) bind the predetermined antigen with a high (e.g., at least two times greater) affinity. The phrases "antibody recognizing an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody specifically binding to an antigen".
As used herein, the term "high affinity" with respect to an IgG antibody refers to at least about 107M-1In some embodiments at least about 108M-1In some embodiments at least about 109M-1、1010M-1、1011M-1Or larger, e.g. up to 1013M-1Or greater binding affinity. However, for other antibody isotypes, "high affinity" binding may vary. For example, "high affinity" binding of IgM isotype means at least about 1.0X 107M-1Binding affinity of (4). As used herein, "isotype" refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.
"complementarity determining regions" or "CDRs" refer to one of the three hypervariable regions within the heavy chain variable region or the light chain variable region of an antibody molecule which form an N-terminal antigen-binding surface which is complementary to the three-dimensional structure of the antigen to which it binds. Starting from the N-terminus of the heavy or light chain, these complementarity determining regions are denoted "CDR 1", "CDR 2" and "CDR 3", respectively. The CDRs are involved in antigen-antibody binding, and the CDRs 3 contain unique regions for antigen-antibody binding specificity. Thus, the antigen binding site may comprise 6 CDRs comprising CDR regions from each of the heavy and light chain V regions.
As used herein, "conservative substitutions" refer to polypeptide modifications that involve the substitution of an amino acid with one or more amino acids having similar biochemical properties without resulting in a loss of biological or biochemical function of the polypeptide. "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). It is contemplated that the antibodies of the invention may have conservative amino acid substitutions, and still retain activity.
The term "substantial homology" (substantial homology) with respect to nucleic acids and polypeptides indicates that two nucleic acids or two polypeptides or designated sequences thereof are identical in at least about 80% of the nucleotides or amino acids, usually at least about 85%, preferably about 90%, 91%, 92%, 93%, 94%, or 95%, more preferably at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the nucleotides or amino acids, with appropriate nucleotide or amino acid insertions or deletions when optimally aligned and compared. Alternatively, the segment will have substantial homology to the nucleic acid when hybridized to the complement of the strand under selective hybridization conditions. The invention includes nucleic acid sequences and polypeptide sequences having substantial homology to the specific nucleic acid sequences and amino acid sequences recited herein.
The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% homology-the number of identical positions/total number of positions x 100), which takes into account the number of gaps and the length of each gap, which need to be introduced to make an optimal alignment of the two sequences. Mathematical algorithms (such as, without limitation, VectorNTI) may be usedTMAlignX of (Invitrogen Corp., Carlsbad, Calif.)TMModule) to achieve sequence comparison and percent identity determination between two sequences. For AlignXTMDefault parameters for multiple alignments are: gap opening penalty: 10; gap extension penalty: 0.05; gap separation penalty range: 8; % identity of alignment delay: 40. (further)See http:// www.invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-C organizations/Vector-NTI-Community/Sequence-analysis-and-data-management-for-PCs/align X-Module-for-Vector-NTI-Advance. us. html).
Another method (also referred to as global sequence alignment) for determining the best overall match between a query sequence (a sequence of the invention) and a subject sequence can be determined using the CLUSTALW Computer program (Thompson et al, Nucleic Acids Research, 1994, 2 (22): 4673-) -4680, which is based on the algorithm of Higgins et al (Computer Applications in the Biosciences (CABIOSs), 1992, 8 (2): 189-) -191. In sequence alignment, both the query and subject sequences are DNA sequences. The results of the global sequence alignment are in percent identity. Preferred parameters used to calculate percent identity via pairwise alignment in CLUSTALW alignments of DNA sequences are: the matrix IUB, k-tuple 1, number of top diagonals 5, gap penalty 3, gap open penalty 10, gap extension penalty 0.1. For multiple alignments, the following CLUSTALW parameters are preferred: gap opening penalty of 10, gap extension parameter of 0.05; gap separation penalty range 8; the% identity of alignment delay was 40.
The nucleic acid may be present in the whole cell, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "rendered substantially pure" when purified away from other cellular components with which they are normally associated in their natural environment. For isolating nucleic acids, standard techniques can be used such as the following: alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques known in the art.
Monoclonal antibodies
44 TFPI-binding antibodies were identified by panning and screening human antibody libraries against human TFPI. The heavy chain variable region and the light chain variable region of each monoclonal antibody were sequenced and their CDR regions were identified. The sequence identifier numbers ("SEQ ID NOs") corresponding to these regions of each monoclonal antibody are summarized in table 1.
Table 1. A summary of sequence identifier numbers ("SEQ ID NOs") for the heavy chain variable region ("VH") and the light chain variable region ("VL") of each TFPI-binding monoclonal antibody. Sequence identifier numbers for the CDR regions ("CDR 1", "CDR 2", and "CDR 3") of each of the heavy and light chains are also provided. N.A.: a nucleic acid sequence; A.A.: an amino acid sequence.
In one embodiment, an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor is provided, wherein the antibody comprises CDR3, wherein CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-430 amino acid sequence. These CDRs 3 were identified from the heavy chain of the antibodies identified during panning and screening. In yet another embodiment, the antibody further comprises (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 302-344, (b) a CDR2 comprising the amino acid sequence selected from SEQ ID NOs: 345, 387, or (c) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 302-344 and a CDR1 comprising the amino acid sequence selected from SEQ ID NOs: 345 and 387, and a CDR 2.
In another embodiment, antibodies are provided that share a CDR3 from one of the light chains of the antibodies identified during panning and screening. Thus, the present invention relates to an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises CDR3, wherein CDR3 comprises an amino acid sequence selected from the group consisting of seq id NOs: 259-301. In other embodiments, the antibody further comprises (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 173-215, (b) a CDR2 comprising the amino acid sequence selected from SEQ ID NOs: 216-258, or (c) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 173-215 and a CDR1 comprising the amino acid sequence selected from SEQ ID NOs: 216, 258, and a CDR 2.
In another embodiment, the antibody comprises CDR3 from the heavy chain and CDR3 from the light chain of the antibody identified by screening and panning. Thus, there is provided an antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 388-430 and a CDR3 comprising an amino acid sequence selected from SEQ ID NO: 259 and 301. In yet another embodiment, the antibody further comprises (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 302-344, (b) a CDR2 comprising the amino acid sequence selected from SEQ ID NOs: 345 and 387, (c) a CDR1 comprising the amino acid sequence selected from SEQ ID NOs: 173-215, and/or (d) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 216-258.
In other specific embodiments, the antibody comprises heavy and light chain variable regions comprising:
(a) comprising a polypeptide comprising SEQ ID NO: 173. 216 and 259 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 302. 345 and 388;
(b) comprising a polypeptide comprising SEQ ID NO: 174. 217 and 260 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 303. 346 and 389;
(c) comprising a polypeptide comprising SEQ ID NO: 175. 218 and 261 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 304. 347 and 390;
(d) comprising a polypeptide comprising SEQ ID NO: 176. 219 and 262 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 305. 348 and 391;
(e) comprising a polypeptide comprising SEQ ID NO: 177. 220 and 263 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 306. 349 and 392;
(f) comprising a polypeptide comprising SEQ ID NO: 178. 221 and 264 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 307. 350 and 393;
(g) comprising a polypeptide comprising SEQ ID NO: 179. 222 and 265 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 308. 351 and 394;
(h) comprising a polypeptide comprising SEQ ID NO: 180. 223 and 266 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 309. 352 and 395;
(i) comprising a polypeptide comprising SEQ ID NO: 181. 224 and 267 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 310. 353 and 396;
(j) comprising a polypeptide comprising SEQ ID NO: 182. 225 and 268 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 311. 354 and 397;
(k) comprising a polypeptide comprising SEQ ID NO: 183. 226 and 269 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 312. 355 and 398;
(l) Comprising a polypeptide comprising SEQ ID NO: 184. 227 and 270 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 313. 356 and 399;
(m) comprises a polypeptide comprising SEQ ID NO: 185. 228 and 271 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 314. 357 and 400;
(n) comprises a polypeptide comprising SEQ ID NO: 186. 229 and 272 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 315. 358 and 401;
(o) comprises a polypeptide comprising SEQ ID NO: 187. 230 and 273 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 316. 359, and 402;
(p) comprises a polypeptide comprising SEQ ID NO: 188. 231 and 274 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 317. 360 and 403;
(q) comprises a polypeptide comprising SEQ ID NO: 189. 232 and 275 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 318. 361 and 404;
(r) comprises a polypeptide comprising SEQ ID NO: 190. 233 and 276 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 319. 362 and 405;
(s) comprises a polypeptide comprising SEQ ID NO: 191. 234 and 277 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 320. 363 and 406;
(t) comprises a polypeptide comprising SEQ ID NO: 192. 235 and 278 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 321. 364 and 407, respectively;
(u) comprises a polypeptide comprising SEQ ID NO: 193. 236 and 279 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 322. 365 and 408;
(v) comprising a polypeptide comprising SEQ ID NO: 194. 237 and 280 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 323. 366 and 409;
(w) comprises a polypeptide comprising SEQ ID NO: 195. 238 and 281 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 324. 367 and 410;
(x) Comprising a polypeptide comprising SEQ ID NO: 196. 239 and 282 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 325. 368 and 411;
(y) comprises a polypeptide comprising SEQ ID NO: 197. 240 and 283 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 326. 369 and 412;
(z) comprises a polypeptide comprising SEQ ID NO: 198. 241 and 284 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 327. 370 and 413;
(aa) comprises a polypeptide comprising SEQ ID NO: 199. 242 and 285 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 328. 371 and 414;
(bb) comprises a polypeptide comprising SEQ ID NO: 200. 243 and 286 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 329. 372 and 415;
(cc) comprises a polypeptide comprising SEQ ID NO: 201. 244 and 287 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 330. 373 and the amino acid sequence of seq id no;
(dd) comprises a nucleotide sequence comprising SEQ ID NO: 202. 245 and 288 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 331. 374 and 417;
(ee) comprises a polypeptide comprising SEQ ID NO: 203. 246 and 289 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 332. 375 and 418;
(ff) comprises a polypeptide comprising SEQ ID NO: 204. 247 and 290 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 333. 376 and 419;
(gg) comprises a polypeptide comprising SEQ ID NO: 205. 248 and 291 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 334. 377 and 420;
(hh) comprises a polypeptide comprising SEQ ID NO: 206. 249, and 292 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 335. the heavy chain variable region of the amino acid sequences of 378 and 421;
(ii) comprising a polypeptide comprising SEQ ID NO: 207. 250 and 293 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 336. 379 and 422;
(jj) comprises a polypeptide comprising SEQ ID NO: 208. 251 and 294 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 337. 380 and 423;
(kk) comprises a polypeptide comprising SEQ ID NO: 209. 252 and 295 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 338. 381 and 424;
(ll) comprises a polypeptide comprising SEQ ID NO: 210. 253 and 296 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 339. 382, and 425;
(mm) comprises a polypeptide comprising SEQ ID NO: 211. 254 and 297 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 340. 383 and 426;
(nn) comprises a polypeptide comprising SEQ ID NO: 212. 255 and 298 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 341. 384 and 427;
(oo) comprises a polypeptide comprising SEQ ID NO: 213. 256 and 299 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 342. 385 and 428;
(pp) comprises a polypeptide comprising SEQ ID NO: 214. 257 and 300 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 343. 386 and 429;
(qq) comprises a polypeptide comprising SEQ ID NO: 215. 258 and 301 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 344. a heavy chain variable region of the amino acid sequence of 387 and 430; or
(rr) comprises a polypeptide comprising SEQ ID NO: 194. 237 and 280 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 335. 378 and 421.
In another embodiment, the invention relates to an antibody comprising:
(a) having the polypeptide sequence SEQ ID NO: 2 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 4, a heavy chain variable region;
(b) having the polypeptide sequence SEQ ID NO: 6 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8, a heavy chain variable region;
(c) having the polypeptide sequence SEQ ID NO: 10 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12, a heavy chain variable region;
(d) having the polypeptide sequence SEQ ID NO: 14 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16;
(e) having the polypeptide sequence SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20, a heavy chain variable region;
(f) having the polypeptide sequence SEQ ID NO: 22 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 24, a heavy chain variable region;
(g) having the polypeptide sequence SEQ ID NO: 26 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 28;
(h) having the polypeptide sequence SEQ ID NO: 30 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 32, a heavy chain variable region;
(i) having the polypeptide sequence SEQ ID NO: 34 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 36, a heavy chain variable region;
(j) having the polypeptide sequence SEQ ID NO: 38 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40, a heavy chain variable region;
(k) having the polypeptide sequence SEQ ID NO: 42 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44, a heavy chain variable region;
(l) Having the polypeptide sequence SEQ ID NO: 46 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 48, a heavy chain variable region;
(m) a polypeptide having the polypeptide sequence of SEQ ID NO: 50 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 52, a heavy chain variable region;
(n) a polypeptide having the polypeptide sequence of SEQ ID NO: 54 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 56;
(o) a polypeptide having the polypeptide sequence of SEQ ID NO: 58 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 60;
(p) a polypeptide having the polypeptide sequence of SEQ ID NO: 62 and a light chain variable region having the polypeptide sequence of SEQ id no: 64;
(q) a polypeptide having the polypeptide sequence of SEQ ID NO: 66 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 68;
(r) a polypeptide having the polypeptide sequence of SEQ ID NO: 70 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72, a heavy chain variable region;
(s) a polypeptide having the polypeptide sequence of SEQ ID NO: 74 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 76;
(t) a polypeptide having the polypeptide sequence of SEQ ID NO: 78 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 80;
(u) a polypeptide having the polypeptide sequence of SEQ ID NO: 82 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 84, the heavy chain variable region;
(v) having the polypeptide sequence SEQ ID NO: 86 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 88;
(w) a polypeptide having the polypeptide sequence of SEQ ID NO: 90 and a light chain variable region having the polypeptide sequence SEQ ID NO: 92, the heavy chain variable region;
(x) Having the polypeptide sequence SEQ ID NO: 94 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 96, the heavy chain variable region;
(y) a polypeptide having the polypeptide sequence of SEQ ID NO: 98 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100, a heavy chain variable region;
(z) has the polypeptide sequence of SEQ ID NO: 102 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 104;
(aa) a polypeptide having the polypeptide sequence of SEQ ID NO: 106 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 108;
(bb) has the polypeptide sequence of SEQ ID NO: 110 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 112;
(cc) a polypeptide having the polypeptide sequence of SEQ ID NO: 114 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 116, the heavy chain variable region;
(dd) a polypeptide having the polypeptide sequence of SEQ ID NO: 118 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 120, a heavy chain variable region;
(ee) has the polypeptide sequence of SEQ ID NO: 122 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 124, the heavy chain variable region;
(ff) a polypeptide having the polypeptide sequence of SEQ ID NO: 126 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128, a heavy chain variable region;
(gg) has the polypeptide sequence of SEQ ID NO: 130 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 132;
(hh) has the polypeptide sequence of SEQ ID NO: 134 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136, the heavy chain variable region;
(ii) having the polypeptide sequence SEQ ID NO: 138 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 140;
(jj) has the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144, the heavy chain variable region;
(kk) having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148;
(ll) has the polypeptide sequence of SEQ ID NO: 150 and a light chain variable region having the polypeptide sequence SEQ ID NO: 152;
(mm) has the polypeptide sequence of SEQ ID NO: 154 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156;
(nn) has the polypeptide sequence of SEQ ID NO: 158 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 160, a heavy chain variable region;
(oo) has the polypeptide sequence of SEQ ID NO: 162 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 164, heavy chain variable region;
(pp) has the polypeptide sequence of SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 168, a heavy chain variable region;
(qq) has the polypeptide sequence of SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 172, the heavy chain variable region; or
(rr) has the polypeptide sequence of SEQ ID NO: 86 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136, or a heavy chain variable region of the light chain.
Also provided is an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises a human heavy chain variable region comprising an amino acid sequence at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 4. SEQ ID NO: 8. SEQ ID NO: 12. SEQ ID NO: 16. SEQ ID NO: 20. SEQ ID NO: 24. SEQ ID NO: 28. SEQ ID NO: 32. SEQ ID NO: 36. SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48. SEQ ID NO: 52. SEQ ID NO: 56. SEQ ID NO: 60. SEQ ID NO: 64. SEQ ID NO: 68. SEQ ID NO: 72. SEQ ID NO: 76. SEQ ID NO: 80. SEQ ID NO: 84. SEQ ID NO: 88. SEQ ID NO: 92. SEQ ID NO: 96. SEQ ID NO: 100. SEQ ID NO: 104. SEQ ID NO: 108. SEQ ID NO: 112. SEQ ID NO: 116. SEQ ID NO: 120. SEQ ID NO: 124. SEQ ID NO: 128. SEQ ID NO: 132. SEQ ID NO: 136. SEQ ID NO: 140. SEQ ID NO: 144. SEQ ID NO: 148. SEQ ID NO: 152. SEQ ID NO: 156. SEQ ID NO: 160. SEQ ID NO: 164. SEQ ID NO: 168. and SEQ ID NO: 172, or a fragment thereof.
Also provided is an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises a human light chain variable region comprising an amino acid sequence at least 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 14. SEQ ID NO: 18. SEQ ID NO: 22. SEQ ID NO: 26. SEQ ID NO: 30. SEQ ID NO: 34. SEQ ID NO: 38. SEQ ID NO: 42. SEQ ID NO: 46. SEQ ID NO: 50. SEQ ID NO: 54. SEQ ID NO: 58. SEQ ID NO: 62. SEQ ID NO: 66. SEQ ID NO: 70. SEQ ID NO: 74. SEQ ID NO: 78. SEQ ID NO: 82. SEQ ID NO: 86. SEQ ID NO: 90. SEQ ID NO: 94. SEQ ID NO: 98. SEQ ID NO: 102. SEQ ID NO: 106. SEQ ID NO: 110. SEQ ID NO: 114. SEQ ID NO: 118. SEQ ID NO: 122. SEQ ID NO: 126. SEQ ID NO: 130. SEQ ID NO: 134. SEQ ID NO: 138. SEQ ID NO: 142. SEQ ID NO: 146. SEQ ID NO: 150. SEQ ID NO: 154. SEQ ID NO: 158. SEQ ID NO: 162. SEQ ID NO: 166. and SEQ ID NO: 170, or a pharmaceutically acceptable salt thereof.
In addition to antibody descriptions relying on the use of sequence identifiers discussed above, some embodiments may also be described by reference to Fab clones isolated in the experiments described herein. In some embodiments, the recombinant antibody comprises the heavy and/or light chain CDR3 of the following clones: TP-2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A10, TP-2B1, TP-2B3, TP-2B4, TP-2B8, TP-2B9, TP-2C1, TP-2C7, TP-2D7, TP-2E3, TP-2E5, TP-2F9, TP-2G2, TP-2G4, TP-2G5, TP-2G6, TP-2G7, TP-2G9, TP-2H10, TP-3A2, TP-3A3, TP-3A4, TP-3B3, TP-3B4, TP-3C1, TP-3D 8672, TP-3A 1, TP-3F1, TP-3G1, TP-F3G, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H 8. In some embodiments, the antibodies may further comprise the CDR2 of these antibodies, and still further comprise the CDR1 of these antibodies. In other embodiments, the antibody may further comprise any combination of CDRs.
Thus, in another embodiment, there is provided an anti-TFPI antibody comprising: (1) a human heavy chain framework region, a human heavy chain CDR1 region, and a human heavy chain CDR1 region, wherein the human heavy chain CDR1 region is TP-2A1, TP-2A5.1, TP-2A1, TP-2B1, TP-2B1, TP-2C1, TP-2D 1, TP-2E 1, TP-2F 1, TP-2G 1, TP-2H1, TP-3A 1, TP-3B 3C 3B 1, TP-3C1, TP-3C1, A heavy chain CDR3 of TP-3D3, TP-3E1, TP-3F1, TP-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H 8; and (2) a human light chain framework region, a human light chain CDR region, and a human light chain CDR region, wherein the human light chain CDR region is TP-2A, TP-2A5.1, TP-2A, TP-2B, TP-2C, TP-2D, TP-2E, TP-2F, TP-2G, TP-2H, TP-3A, TP-3B, TP-3C, TP-2H, TP-3A, TP-3B, TP-3C, TP-2C, or TP-2C, TP-3D3, TP-3E1, TP-3F1, TP-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or the light chain CDR3 of TP-4H8, wherein the antibody binds TFPI. The antibody may further comprise TP-2A, TP-2A5.1, TP-2A, TP-2B, TP-2C, TP-2D, TP-2E, TP-2F, TP-2G, TP-2H, TP-3A, TP-3B, TP-3C, TP-3D, TP-3E, TP-3F, TP-3G, TP-2E, TP-2F, TP-2D, TP-2E, TP-3A, TP, TP-3G3, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or the heavy chain CDR2 and/or light chain CDR2 of TP-4H 8. The antibody may further comprise TP-2A, TP-2A5.1, TP-2A, TP-2B, TP-2C, TP-2D, TP-2E, TP-2F, TP-2G, TP-2H, TP-3A, TP-3B, TP-3C, TP-3D, TP-3E, TP-3F, TP-3G, TP-2E, TP-2F, TP-2D, TP-2E, TP-3A, TP, TP-3G3, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or the heavy chain CDR1 and/or light chain CDR1 of TP-4H 8.
The CDRs 1, 2, and/or 3 regions of the engineered antibodies described above may comprise precise amino acid sequences, TP-2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A10, TP-2B1, TP-2B3, TP-2B4, TP-2B8, TP-2B9, TP-2C1, TP-2C7, TP-2D7, TP-2E3, TP-2F 3, TP-2G 3, 36-2G 3, TP-2H 3, TP-3A3, TP-3B 3C3, TP-3C3, TP-2A 3, and the like, TP-3C3, TP-3D3, TP-3E1, TP-3F1, TP-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H 8.
However, one of ordinary skill will appreciate that TP-2A2, TP-2A5.1, TP-2A2, TP-2B1, TP-2B 2, TP-2C 2, TP-2D 2, TP-2E 2, TP-2F 2, TP-2G2, TP-2H 2, TP-3A2, TP-3B 2, TP-3C 72, TP-3C 3D 2, TP-3F2, TP-3C2, TP-3F2, TP-3C2, TP-3F2, TP-3F 36, Some deviation from the exact CDR sequences of TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H8 may be possible, so long as the ability of the antibody to effectively bind TFPI is still retained. Thus, in another embodiment, an engineered antibody may be composed of one or more CDRs corresponding, for example, to TP-2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A10, TP-2B1, TP-2B3, TP-2B4, TP-2B8, TP-2B9, TP-2C1, TP-2D 1, TP-2E 1, TP-2F 1, TP-2G 1, TP-2H1, TP-3A 1, TP-3B 1, TP-3C1, TP-2C1, TP-2F 1, TP-F1, or combinations thereof, One or more CDRs of TP-3D3, TP-3E1, TP-3F1, TP-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H8 are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical.
The antibody may be of any of a variety of classes of antibodies, such as, but not limited to, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies.
In one embodiment, an isolated fully human monoclonal antibody to a human tissue factor pathway inhibitor is provided.
In another embodiment, isolated fully human monoclonal antibodies to Kunitz domain 2 of human tissue factor pathway inhibitor are provided.
Nucleic acids
Isolated nucleic acid molecules encoding any of the monoclonal antibodies described above are also provided.
Methods of making antibodies to TFPI
Monoclonal antibodies can be recombinantly produced by expressing in a host cell a nucleotide sequence encoding the variable region of a monoclonal antibody according to embodiments of the invention. With the aid of expression vectors, the nucleic acids containing the nucleotide sequences can be transfected and expressed in host cells suitable for production. Thus, there is also provided a method for generating a monoclonal antibody that binds to human TFPI, comprising:
(a) transfecting a nucleic acid molecule encoding a monoclonal antibody of the invention into a host cell,
(b) culturing said host cell so as to express said monoclonal antibody in the host cell, and optionally
(c) Isolating and purifying the monoclonal antibody produced, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the monoclonal antibody of the invention.
In one example, to express an antibody or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into an expression vector to operably link the genes to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to refer to the linkage of an antibody gene into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of the antibody gene fragment and complementary restriction sites on the vector, or blunt-ended ligation if no restriction sites are present). Can be used forTo create full-length antibody genes of any antibody isotype using the light and heavy chain variable regions of the antibodies described herein by inserting them into expression vectors that already encode the heavy and light chain constant regions of the desired isotype such that VHC in sections and carriersHThe segments being operatively connected and having VLC in sections and carriersLThe segments are operatively connected. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
In addition to the antibody chain encoding gene, the recombinant expression vectors of the invention carry regulatory sequences that control expression of the antibody chain gene in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel; gene Expression technology methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). It will be appreciated by those skilled in the art that the design of the expression vector (including the choice of regulatory sequences) may depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, and the like. Examples of regulatory sequences for expression by mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from Cytomegalovirus (CMV), simian virus 40(SV40), adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the β -globin promoter.
In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017 all by Axel et al). For example, typically, a selectable marker gene confers resistance to a drug such as G418, hygromycin or methotrexate to a host cell into which the vector has been introduced. Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells by methotrexate selection/amplification) and the neo gene (for G418 selection).
For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" of various forms is intended to cover a wide variety of commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably mammalian host cells, is most preferred because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a correctly folded and immunologically active antibody.
Examples of mammalian host cells for expression of recombinant antibodies include Chinese hamster ovary (CHO cells), including DHFR-CHO cells used with DHFR selection markers (e.g., as described in R.J.Kaufman and P.A.Sharp (1982) mol.biol.159: 601-621, described in Urlaub and Chasin, (1980) Proc.Natl.Acad.Sci.USA 77: 4216-4220), NSO myeloma cells, COS cells, HKB11 cells, and SP2 cells. When a recombinant expression vector encoding a gene for an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is cultured. The antibody can be recovered from the culture medium using standard protein purification methods such as ultrafiltration, size exclusion chromatography, ion exchange chromatography, and centrifugation.
Use of partial antibody sequences to express whole antibodies
Antibodies interact with the target antigen primarily via amino acid residues located in the 6 heavy and light chain CDRs. For this reason, the amino acid sequences within the CDRs are more diverse between antibodies than sequences outside the CDRs. See, e.g., fig. 6 and 7, where CDR regions in the light and heavy variable chains, respectively, of a monoclonal antibody according to the invention are identified. Since the CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that comprise CDR sequences from a particular naturally occurring antibody grafted to framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al, 1998, Nature 332: 323-327; Jones, P. et al, 1986, Nature 321: 522-. Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from the mature antibody gene sequences in that they will not include the fully assembled variable genes that are formed by v (d) J junctions during B cell maturation. It is not necessary to obtain the entire DNA sequence of a particular antibody to recreate a complete recombinant antibody with binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequences spanning the CDR regions are generally sufficient for this purpose. Partial sequences are used to determine which germline variable and linking gene segments contribute to the recombinant antibody variable gene. Germline sequences are then used to fill in the missing portions of the variable regions. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. For this reason, it is necessary to use the corresponding germline leader sequence for the expression construct. To add the missing sequence, the cloned cDNA sequence can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping oligonucleotides and combined by PCR amplification to create a fully synthesized variable region clone. This approach has certain advantages, such as the elimination or inclusion of specific restriction sites, or the optimization of specific codons.
The nucleotide sequences of the heavy and light chain transcripts were used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with the same amino acid coding capacity as the native sequence. The synthetic heavy and kappa chain sequences may differ from the natural sequence in three ways: interrupting the repetitive nucleotide base string to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites were incorporated according to the rules of Kozak (Kozak, 1991, J.biol.chem.266: 19867-19870); and engineering a HindIII site upstream of the translation initiation site.
The optimized coding and corresponding non-coding strand sequences are broken down into 30-50 nucleotide portions approximately at the midpoint of the corresponding non-coding oligonucleotides for both the heavy and light chain variable regions. Thus, for each strand, oligonucleotides can be assembled into overlapping double-stranded sets spanning a segment of 150-400 nucleotides. The pool was then used as a template to generate a PCR amplification product of 150-400 nucleotides. Typically, a single set of variable region oligonucleotides will be broken down into two pools, which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include overlapping fragments of the heavy or light chain constant regions in PCR amplification to generate fragments that can be easily cloned into expression vector constructs.
The reconstructed heavy and light chain variable regions are then combined with cloned promoter, translation initiation, constant region, 3' untranslated polyadenylation, and transcription termination sequences to form expression vector constructs. The heavy and light chain expression constructs may be combined into a single vector, co-transfected, serially transfected, or separately transfected into a host cell, which is then fused to form a host cell expressing both chains.
Thus, in another aspect, structural features of human anti-TFPI antibodies (e.g., TP2A8, TP2G6, TP2G7, TP4B7, etc.) are used to create structure-associated human anti-TFPI antibodies that retain the function of binding TFPI. More specifically, one or more CDRs of the well-identified heavy and light chain regions of the monoclonal antibodies of the invention can be recombined with known human framework regions and CDRs to create additional recombinantly engineered human anti-TFPI antibodies of the invention.
Thus, in another embodiment, there is provided a method for making an anti-TFPI antibody, comprising: an antibody comprising (1) a human heavy chain framework region and human heavy chain CDRs, wherein the human heavy chain CDRs 3 comprise a sequence selected from the group consisting of SEQ ID NOs: 388-430 and/or (2) a human light chain framework region and human light chain CDRs, wherein the light chain CDRs 3 comprise amino acid sequences selected from the group consisting of SEQ ID NOs: 259-301 amino acid sequence; wherein the antibody retains the ability to bind TFPI. In other embodiments, the methods are performed using other CDRs of the invention.
Pharmaceutical composition
Also provided are pharmaceutical compositions comprising a therapeutically effective amount of an anti-TFPI monoclonal antibody and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" is a substance that can be added to an active ingredient to help formulate or stabilize a formulation without causing significant adverse toxicological effects to the patient. Examples of such carriers are well known to those skilled in the art and include water, sugars such as maltose or sucrose, albumin, salts such as sodium chloride, and the like. Other carriers are described, for example, in Remington's Pharmaceutical Sciences of e.w. martin. Such compositions will contain a therapeutically effective amount of at least one anti-TFPI monoclonal antibody.
Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Preferably, the composition is formulated for parenteral injection. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases, it will contain isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and the other desired ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Pharmaceutical use
Monoclonal antibodies can be used for therapeutic purposes to prepare genetic and acquired deficiencies or defects in coagulation. For example, monoclonal antibodies in the embodiments described above may be used to block TFPI interaction with FXa, or to prevent TFPI-dependent inhibition of TF/FVIIa activity. In addition, monoclonal antibodies can also be used to restore TF/FVIIa driven FXa production to circumvent deficiencies in FVIII or FIX dependent FXa amplification.
Monoclonal antibodies have therapeutic use in the treatment of hemostatic disorders such as thrombocytopenia (thrombocytopenia), platelet disorders, and bleeding disorders (e.g., hemophilia a and hemophilia B). Such conditions may be treated by administering to all patients in need thereof a therapeutically effective amount of an anti-TFPI monoclonal antibody. Monoclonal antibodies also have therapeutic use in treating uncontrolled bleeding in indications such as trauma and hemorrhagic stroke (hemorrhagic stroke). Thus, a method for reducing bleeding time is also provided comprising administering to a patient in need thereof a therapeutically effective amount of an anti-TFPI monoclonal antibody of the invention.
The antibodies can be used as monotherapy or in combination with other therapies to address hemostatic disorders. For example, co-administration of one or more antibodies of the invention with a clotting factor, such as factor VIIa, factor VIII, or factor IX, is believed to be useful for treating hemophilia. In one embodiment, a method is provided for treating genetic and acquired deficiencies or defects in coagulation comprising administering (a) a first amount of a monoclonal antibody that binds to a human tissue factor pathway inhibitor and (b) a second amount of factor VIII or factor IX, wherein the first and second amounts together are effective to treat the deficiency or defect. In another embodiment, a method is provided for treating genetic and acquired deficiencies or defects in coagulation comprising administering (a) a first amount of a monoclonal antibody that binds to a human tissue factor pathway inhibitor and (b) a second amount of factor VIII or factor IX, wherein the first and second amounts together are effective to treat the deficiency or defect, and further wherein factor VII is not co-administered. The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of a combination of a monoclonal antibody of the invention and factor VIII or factor IX, wherein the composition is free of factor VII. "factor VII" includes factor VII and factor VIIa. These combination therapies have the potential to reduce the necessary infusion frequency of coagulation factors. Co-administration or combination therapy means administration of two therapeutic agents each formulated separately or co-formulated in one composition and administered at approximately the same time or at different times when formulated separately, but within the same treatment period.
The pharmaceutical composition may be administered parenterally to subjects with hemophilia a or B at a dose and frequency that may vary with the severity of the bleeding event, or in the case of prophylactic therapy, the severity of the patient's coagulopathy.
The composition may be administered to a patient in need thereof as a bolus (bolus) or by continuous infusion. For example, a bolus administration of an inventive antibody present as a Fab fragment may be in an amount of 0.0025 to 100mg/kg body weight, 0.025 to 0.25mg/kg, 0.010 to 0.10mg/kg, or 0.10-0.50 mg/kg. For continuous infusion, the inventive antibody present as a Fab fragment may be administered at 0.001 to 100mg/kg body weight/minute, 0.0125 to 1.25 mg/kg/minute, 0.010 to 0.75 mg/kg/minute, 0.010 to 1.0 mg/kg/minute, or 0.10-0.50 mg/kg/minute for a period of 1-24 hours, 1-12 hours, 2-12 hours, 6-12 hours, 2-8 hours, or 1-2 hours. For administration of the inventive antibody as a full length antibody (with intact constant regions), the dosage amount may be about 1-10mg/kg body weight, 2-8mg/kg, or 5-6 mg/kg. Such full length antibodies will typically be administered by infusion over a period of 30 minutes to 3 hours. The frequency of administration will depend on the severity of the condition. The frequency may range from three times a week to once every two or three weeks.
In addition, the composition may be administered to the patient via subcutaneous injection. For example, a dose of 10 to 100mg of anti-TFPI antibody may be administered to a patient weekly, biweekly, or monthly via subcutaneous injection.
As used herein, "therapeutically effective amount" refers to the amount of anti-TFPI monoclonal antibody or combination of said antibody with factor VIII or factor IX that is effective to increase clotting time in vivo or in other cases to elicit a measurable in vivo benefit to a patient in need thereof. The precise amount will depend on many factors including, but not limited to, the ingredients and physical characteristics of the therapeutic composition, the intended patient population, individual patient considerations, and the like, and can be readily determined by one skilled in the art.
Examples
Versatile materials and methods
Example 1: panning and screening human antibody libraries against human TFPI
Panning human antibody libraries against TFPI
anti-TFPI antibodies were selected by panning a phage display combinatorial human antibody library HuCal Gold (Rothe et al, J.mol.biol., 2008, 376: 1182-1200) against human TFPI (American Diagnostica). Briefly, 200 μ l TFPI (5 μ g/ml) was coated overnight at 4 ℃ on 96-well Maxisorp plates, and the plates were then blocked with PBS buffer containing 5% milk. After washing the plates with pbs (pbst) containing 0.01% Tween-20, aliquots of the combinatorial human antibody library were added to TFPI-coated wells and incubated for 2 hours. Unbound phage were washed away with PBST and antigen-bound phage were diluted with dithiothreitol, infected, and amplified in escherichia coli (e.coli) strain TG 1. The phage were rescued by helper phage for the next round of panning. A total of three rounds of panning were performed and clones from the first two rounds were screened for human TFPI in an ELISA assay.
Screening of antibody clones by antigen binding in ELISA
To select antibody clones that bound human TFPI, Fab genes from the second and third rounds of panning of phage clones were subcloned into bacterial expression vectors and expressed in e.coli strain TG 1. Bacterial lysates were added to the wells of human TFPI-coated Maxisorp plates. After washing, goat anti-human Fab coupled with HRP was used as a detection antibody, and the plate was developed by adding amplex red (invitrogen) with hydrogen peroxide. Signals at least 5 times higher than background were considered positive. Cross-reactivity of anti-human TFPI antibodies to mouse TFPI was determined by a similar mouse TFPI binding ELISA. Plates were coated with mouse TFPI (R & D System), BSA and lysozyme. The latter two antigens were used as negative controls. A representative data set is shown in fig. 1.
Sequences of anti-TFPI human antibodies
After panning and screening the HuCal Gold human antibody library against TFPI, DNA sequencing was performed on positive antibody clones to generate 44 unique antibody sequences (table 2). Of these antibody sequences, 29 were lambda light chains and 15 were kappa light chains. Our analysis of the variable regions of the heavy chain revealed 28 VH3 species, 14 VH6 species, 1 VH1 species and 1 VH5 species.
Table 2: peptide sequences of the variable regions of 44 anti-TFPI antibodies
Cross-reactivity with mouse TFPI
The 44 human TFPI-binding clones described above were also tested for binding to mouse TFPI in an ELISA. 19 antibodies were found to be cross-reactive with mouse TFPI. To facilitate studies using the mouse hemophilia model, we further characterized these 19 antibodies and 5 antibodies specific for human TFPI. A representative data set is shown in fig. 1. None of these antibodies bound BSA or lysozyme in ELISA.
Example 2: expression and purification of anti-TFPI antibodies
anti-TFPI antibodies (as Fab fragments) were expressed and purified from bacterial strain TG 1. Briefly, a single clone of bacterial strain TG1 containing the antibody expression plasmid was picked up and cultured overnight in 8ml of 2 × YT medium in the presence of 34 μ g/ml chloramphenicol and 1% glucose. A volume of 7ml of culture was transferred to 250ml of fresh 2 XYT medium containing 34. mu.g/ml chloramphenicol and 1% glucose. After 3 hours of incubation, 0.5mM IPTG was added to induce Fab expression. The culture was continued overnight at 25 ℃. The culture was centrifuged to pellet the bacterial cells. The pellet was then resuspended in Bug Buster lysis buffer (Novagen). After centrifugation, the supernatant of the bacterial lysis was filtered. The Fab fragments were affinity purified by Ni-NTA column (Qiagen) according to the manufacturer's instructions.
Example 3: EC of anti-TFPI antibody50And determination of binding affinity
Determination of EC of anti-TFPI antibodies on human or mouse TFPI Using purified Fab antibodies50. Evaluation of EC in ELISA similarly as described above50. Results were analyzed using SoftMax. The binding affinity of anti-TFPI antibodies was determined in a Biacore assay. Briefly, antigen (either human or mouse TFPI) was immobilized on CM5 chips using an amine coupling kit (GE HealthCare) according to the manufacturer's instructions. The amount of immobilized TFPI was adjusted relative to the mass of antigen to give about 300 RUs. The antibody Fab was analyzed in the mobile phase and purified antibodies were used in Biacore assays at least five different concentrations (0.1, 0.4, 1.6, 6.4 and 25 nM). Kinetics and binding affinities were calculated using Biacore T100 evaluation software.
As shown in Table 3, 24 anti-TFPI Fab showed multiple EC for human TFPI (0.09 to 792nM) and mouse TFPI (0.06 to 1035nM)50Thus the affinity determined by Biacore is different (1.25 to 1140nM) for human TFPI. In Biacore studies of Fab on mouse TFPI, the change in affinity was smaller (3.08 to 51.8 nM).
Table 3: the binding activity of 24 antibodies against human or mouse TFPI was determined by ELISA and Biacore (hTFPI: human TFPI; mTFPI: mouse TFPI; Neg: signal is less than twice background; ND, not completed).
Example 4: conversion of anti-TFPI Fab to IgG
All of the identified anti-TFPI antibodies are fully human fabs that can be feasibly converted to human IgG as a therapeutic agent. However, in this example, selected fabs were converted to chimeric antibodies containing mouse IgG constant regions, so they were more suitable for testing in a mouse model. The variable regions of the selected antibodies were grafted into a mammalian expression vector containing mouse constant regions. The fully assembled IgG molecules were then transfected and expressed in HKB11 cells (Mei et al, mol. Biotechnol., 2006, 34: 165-178). The culture supernatant was collected and concentrated. Affinity purification of anti-TFPI IgG molecules was performed by Hitrap protein G column (GE Healthcare) following the manufacturer's instructions.
Example 5: selection of anti-TFPI neutralizing antibodies
anti-TFPI neutralizing antibodies were selected based on their inhibition of TFPI activity under three experimental conditions. Using ACTICHROMETFPI activity assay (American diagnostic inc., Stamford, CT), a three-stage chromogenic assay used to measure the ability of TFPI to inhibit the catalytic activity of the TF/FVIIa complex to activate factor X to factor Xa, measures TFPI activity. The neutralizing activity of anti-TFPI antibodies is proportional to the amount of recovered FXa production. In a first context, purified anti-TFPI antibodies are combined with a specified concentration of human or mouse recombinant TFPI (R)&D System) were incubated together. After incubation, the sample is mixed with TF/FVIIa and FX, followed by the use of SPECTROZYMEFXa (a highly specific fXa chromogenic substrate) to measure the residual activity of the TF/FVIIa complex. This substrate is cleaved only by the FXa produced in the assay, releasing the p-nitroanilide (pNA) chromophore, which is measured at 405 nm. The TFPI activity present in the sample is interpolated from a standard curve constructed using known levels of TFPI activity. The assay was performed in endpoint mode. In two other settings, anti-TFPI antibodies were spiked into normal human plasma or hemophilia a plasma and recovered FXa production was measured.
Example 6: anti-TFPI antibodies reduce clotting time in a dilute prothrombin time (dPT) assay
Performing the dPT assay essentially as described by Welsch et al, Thrombosis Res., 1991, 64 (2): 213 and 222. Briefly, human normal plasma (FACT, George King biological), human TFPI-depleted plasma (American diagnostic) or hemophilia a plasma (George King biological) was prepared by mixing plasma with 0.1 volume of control buffer or anti-TFPI antibody. After incubation at 25 ℃ for 30 minutes, plasma samples (100. mu.l) were combined with 200. mu.l of suitably diluted (1: 500 dilution) Simplastin (biomerieux) as a source of thromboplastin and the clotting time was measured using a profilometer STA4 (Stago). Thromboplastin was diluted with PBS or 0.05M Tris-based buffer (pH 7.5) containing 0.1M sodium chloride, 0.1% bovine serum albumin and 20. mu.M calcium chloride.
Example 7: anti-TFPI antibodies alone or in combination with recombinant factor VIII or factor IX reduce blood clotting time in ROTEM assays
The ROTEM system (Pentapharm GmbH) includes a four-channel instrument, computer, plasma standard, activator, and disposable cup and needle. The thromboelastogram (thromboelastogram) parameters of the ROTEM hemostatic system include: coagulation Time (CT), which reflects the reaction time to start blood coagulation (the time required to obtain an amplitude of 2mm after starting data collection); coagulation Formation Time (CFT) and alpha angle to reflect coagulation propagation, and maximum amplitude and maximum modulus of elasticity (elastic modulus) to reflect clot firmness. In the ROTEM assay, 300 μ l of fresh citrated whole blood or plasma was evaluated. All components were reconstituted and mixed according to the manufacturer's instructions and data collection continued for the time period required for each system. Briefly, samples were mixed by taking/dispensing 300 μ Ι of blood or plasma into a ROTEM cup with an automated pipette, to which 20 μ Ι CaCl was added2(200mmol) followed by immediate mixing of the samples and initiation of sample collection. Data was collected for 2 hours using a computer controlled (software version 2.96) ROTEM system.
Exemplary results of the ROTEM assay in detecting the effect of anti-TFPI antibodies in reducing blood clotting time are shown in fig. 3 and 5. FIG. 3 shows that TP-2A8-Fab reduces clotting time in human hemophilia A plasma or mouse hemophilia A whole blood alone or in combination with recombinant FVIII when the ROTEM system is started with NATEM. FIG. 5 shows that IgG-format anti-TFPI antibodies (TP-2A8, TP-3G1, and TP-3C2) reduce clotting time compared to negative control mouse IgG antibodies. Based on these results and the knowledge in the art, the skilled artisan would expect that these anti-TFPI antibodies in combination with recombinant FIX also shorten clotting times compared to these antibodies alone.
Example 8: in vitro functional Activity of anti-TFPI antibodies
To investigate TFPI antibodies in blocking TFPI function, antibodies obtained by panning and screening were tested for functional activity using both chromogenic assay actichromame and diluted prothrombin time (dPT). In both assays, monoclonal rat anti-TFPI antibody (R & D System) was used as a positive control, and human polyclonal Fab was used as a negative control. In the chromogenic assay, 8 antibodies inhibited TFPI activity by more than 50% compared to rat monoclonal antibody (table 4). In the dPT assay, all 8 anti-TFPI fabs showed a high inhibitory effect, which shortened clotting time at 80 seconds, while 4 of the 8 fabs shortened dPT at 70 seconds. The dose dependence of 4 representative clones on shortened dPT is shown in figure 2. However, other human anti-TFPI fabs with low or no TFPI inhibitory activity also shorten clotting times in dPT. For example, TP-3B4 and TP-2C7, although exhibiting less than 25% inhibitory activity, still reduced dPT to less than 70 seconds. Simple linear regression analysis of inhibitory activity and dPT suggested a significant correlation (p 0.0095) but large variance (R square 0.258).
Table 4: the in vitro functional activity of an anti-TFPI antibody as determined by its inhibitory activity in the human TFPI assay and the dPT assay.
One of the anti-TFPI fabs, namely Fab-2A8, was also tested in the ROTEM assay, using either human hemophilia a plasma or mouse hemophilia a whole blood with low levels of factor VIII. As shown in figure 3, comparing polyclonal rabbit anti-TFPI antibodies, Fab-2A8 showed similar activity in human hemophilia a plasma, reducing Clotting Time (CT) from 2200 to about 1700 seconds. When using mouse hemophilia a whole blood, the control antibody rabbit anti-TFPI reduced CT from 2700 to 1000 seconds, whereas Fab-2A8 reduced CT from 2650 to 1700 seconds. These results indicate that Fab-2A8 can significantly shorten clotting time in both human plasma and mouse blood (p ═ 0.03).
Example 9: function of anti-TFPI antibody after conversion into chimeric IgG
In vitro assays for factor Xa generation and dilution of prothrombin time indicate that at least six of the 24 anti-TFPI Fab's, namely TP-2A8, TP-2B3, TP-2G6, TP-3C2, TP-3G1 and TP-4B7, block TFPI function. To facilitate in vivo studies using hemophilia a mice, we converted these six anti-TFPI human fabs to chimeric iggs using the murine IgG1 isotype. The IgG expression vector was transfected into HKB11 cells, and the expressed antibodies were collected in the culture supernatant and purified on a protein G column. 2G6-IgG showed EC in human TFPI when representative clone 2G6-Fab was converted to IgG50Binding was increased 2-fold (from 0.48nM to 0.22nM) and 10-fold (from 5.18nM to 0.51nM) against mouse TFPI. The results of IgG-2G6 binding to human and mouse TFPI are shown in FIG. 4.
Example 10: effect on survival in a model of Tail vein transection in hemophilia A (HemA) mice
A mouse tail vein transection model has been established for pharmacological evaluation. This model mimics a large panel of bleeding phenotypes observed between normal individuals and severe hemophiliacs. For these studies, male hemophilia a mice (8 weeks of age and 20 to 26 grams) were used. Mice were dosed prior to injury by tail vein infusion with anti-TFPI monoclonal antibody (40 μ g/mouse), alone or in combination with coagulation factors such as FVIII (0.1 IU/mouse). At 24 hours post dose administration, the left vein of the tail at 2.7mm (in diameter) from the apex was transected. Survival was observed within 24 hours after transection. Survival was shown to be dose dependent when administered with recombinant FVIII (data not shown). The data shown in fig. 8 were from two separate studies (n 15 and n 10, respectively). This result shows that TP-2A8-IgG significantly prolonged the survival of hemophilia a mice compared to control mouse IgG; and exhibits better survival in combination with recombinant FVIII than either agent alone.
Example 11: combination of anti-TFPI antibodies with recombinant factor VIIa further reduces clotting time and clot formation time
The combined effect of anti-TFPI antibodies and recombinant FVIIa (Novo Nordisk) was evaluated in a ROTEM system using EXTEM (1: 1000 dilution) and mouse hemophilia A whole blood. The indicated amounts of anti-TFPI antibody TP-2A8-IgG ("2 A8") and recombinant FVIIa ("FVIIa") were added to 300 μ l of citrated mouse hemophilia a whole blood and an EXTEM system was used to initiate blood clotting. FIG. 9 shows the reduction of clotting time and clot formation time, respectively, by addition of TP-2A8-IgG or recombinant FVIIa to mouse hemophilia A whole blood. The combination of TP-2A8-IgG and recombinant FVIIa ("2A 8+ FVIIa") further reduced clotting time and clot formation time, indicating that the combination of anti-TFPI antibodies with recombinant FVIIa is useful in treating hemophilia patients with or without inhibitors.
Example 12: anti-TFPI antibodies reduce clotting time in FVIII inhibitor-induced hemophilia blood
Selected anti-TFPI antibodies 2A8 and 4B7 were also tested in a ROTEM assay, which was performed using neutralizing FVIII antibodies to induce hemophilia in whole blood drawn from non-hemophiliacs. Figure 10 shows that normal human blood has a clotting time of about 1000 seconds. Coagulation time was extended to about 5200 seconds in the presence of FVIII neutralizing antibodies (PAH, 100 micrograms/mL). The extended clotting time was significantly shortened by the addition of anti-TFPI antibodies 2A8 or 4B7, indicating that anti-TFPI antibodies are useful in treating hemophilia patients with inhibitors.
Example 13: inhibitory anti-TFPI antibodies bind to the Kunitz domain of human TFPI
Western blot and ELISA were used to determine which domain/domains of TFPI were bound by inhibitory antibodies. These studies were performed using recombinant full-length human TFPI or TFPI domains. The ELISA was similar to example 3. In Western blots, human TFPI or domains were run on 4-12% Bis-Tris SDS PAGE running buffer MES (Invitrogen, Carlsbad, Calif.) and then transferred to cellulose membranes. After 10 minutes incubation with inhibitor antibody, the membrane was washed three times using the snap system (Millipore, Billerica, MA). Mu.l of horse anti-mouse antibody coupled with HRP (Pierce, Rockford, IL) diluted 1 to 10,000 was incubated with the membrane for 10 min. After a similar washing step, SuperSignal substrate (Pierce, Rockford, IL) was used to visualize the membrane. While control anti-Kunitz domain 1 antibodies bind to full-length TFPI, truncated TFPI, and the domain, inhibitory anti-TFPI antibodies only bind to TFPI containing Kunitz domain 2. This indicates that binding to Kunitz domain 2 is essential for the inhibitory function of the antibody.
Table 5: antibody-binding domains as determined by Western blotting and ELISA
Anti-K1 mlgG TP-2A8 TP-2B3 TP-2G6 TP-3C2 TP-3G1 TP-4B7
Full length + - + + + + + +
K1+K2+K3 + - + + + + + +
K1+K2 + - + + + + + +
K1 + - - - - - - -
While the invention has been described with reference to specific embodiments and examples, it will be understood that various modifications and changes may be made, and equivalents may be substituted, without departing from the true spirit and scope of the invention. The specification and examples are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Further, all articles, books, patent applications, and patents mentioned herein are hereby incorporated by reference in their entirety.

Claims (35)

1. An isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises CDR3, the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-430 amino acid sequence.
2. The isolated antibody of claim 1, wherein the antibody further comprises (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 302-344, (b) a CDR2 comprising the amino acid sequence selected from SEQ ID NOs: 345, 387, or (c) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 302-344 and a CDR1 comprising the amino acid sequence selected from SEQ ID NOs: 345 and 387, and a CDR 2.
3. An isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises CDR3, the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 259-301.
4. The isolated antibody of claim 3, wherein the antibody further comprises (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 173-215, (b) a CDR2 comprising the amino acid sequence selected from SEQ ID NOs: 216-258, or (c) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 173-215 and a CDR1 comprising the amino acid sequence selected from SEQ ID NOs: 216, 258, and a CDR 2.
5. The antibody of claim 1, wherein the antibody further comprises CDR3, the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 259-301.
6. The antibody of claim 5, wherein the antibody further comprises (a) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 302-344, (b) a CDR2 comprising the amino acid sequence selected from SEQ ID NOs: 345 and 387, (c) a CDR1 comprising the amino acid sequence selected from SEQ ID NOs: 173, and 215, and (d) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-258.
7. The antibody of claim 1, wherein the antibody comprises heavy and light chain variable regions comprising:
(a) comprising a polypeptide comprising SEQ ID NO: 173. 216 and 259 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 302. 345 and 388;
(b) comprising a polypeptide comprising SEQ ID NO: 174. 217 and 260 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 303. 346 and 389;
(c) comprising a polypeptide comprising SEQ ID NO: 175. 218 and 261 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 304. 347 and 390;
(d) comprising a polypeptide comprising SEQ ID NO: 176. 219 and 262 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 305. 348 and 391;
(e) comprising a polypeptide comprising SEQ ID NO: 177. 220 and 263 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 306. 349 and 392;
(f) comprising a polypeptide comprising SEQ ID NO: 178. 221 and 264 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 307. 350 and 393;
(g) comprising a polypeptide comprising SEQ ID NO: 179. 222 and 265 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 308. 351 and 394;
(h) comprising a polypeptide comprising SEQ ID NO: 180. 223 and 266 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 309. 352 and 395;
(i) comprising a polypeptide comprising SEQ ID NO: 181. 224 and 267 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 310. 353 and 396;
(j) comprising a polypeptide comprising SEQ ID NO: 182. 225 and 268 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 311. 354 and 397;
(k) comprising a polypeptide comprising SEQ ID NO: 183. 226 and 269 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 312. 355 and 398;
(l) Comprising a polypeptide comprising SEQ ID NO: 184. 227 and 270 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 313. 356 and 399;
(m) comprises a polypeptide comprising SEQ ID NO: 185. 228 and 271 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 314. 357 and 400;
(n) comprises a polypeptide comprising SEQ ID NO: 186. 229 and 272 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 315. 358 and 401;
(o) comprises a polypeptide comprising SEQ ID NO: 187. 230 and 273 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 316. 359, and 402;
(p) comprises a polypeptide comprising SEQ ID NO: 188. 231 and 274 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 317. 360 and 403;
(q) comprises a polypeptide comprising SEQ ID NO: 189. 232 and 275 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 318. 361 and 404;
(r) comprises a polypeptide comprising SEQ ID NO: 190. 233 and 276 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 319. 362 and 405;
(s) comprises a polypeptide comprising SEQ ID NO: 191. 234 and 277 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 320. 363 and 406;
(t) comprises a polypeptide comprising SEQ ID NO: 192. 235 and 278 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 321. 364 and 407, respectively;
(u) comprises a polypeptide comprising SEQ ID NO: 193. 236 and 279 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 322. 365 and 408;
(v) comprising a polypeptide comprising SEQ ID NO: 194. 237 and 280 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 323. 366 and 409;
(w) comprises a polypeptide comprising SEQ ID NO: 195. 238 and 281 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 324. 367 and 410;
(x) Comprising a polypeptide comprising SEQ ID NO: 196. 239 and 282 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 325. 368 and 411;
(y) comprises a polypeptide comprising SEQ ID NO: 197. 240 and 283 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 326. 369 and 412;
(z) comprises a polypeptide comprising SEQ ID NO: 198. 241 and 284 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 327. 370 and 413;
(aa) comprises a polypeptide comprising SEQ ID NO: 199. 242 and 285 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 328. 371 and 414;
(bb) comprises a polypeptide comprising SEQ ID NO: 200. 243 and 286 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 329. 372 and 415;
(cc) comprises a polypeptide comprising SEQ ID NO: 201. 244 and 287 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 330. 373 and the amino acid sequence of seq id no;
(dd) comprises a nucleotide sequence comprising SEQ ID NO: 202. 245 and 288 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 331. 374 and 417;
(ee) comprises a polypeptide comprising SEQ ID NO: 203. 246 and 289 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 332. 375 and 418;
(ff) comprises a polypeptide comprising SEQ ID NO: 204. 247 and 290 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 333. 376 and 419;
(gg) comprises a polypeptide comprising SEQ ID NO: 205. 248 and 291 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 334. 377 and 420;
(hh) comprises a polypeptide comprising SEQ ID NO: 206. 249, and 292 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 335. the heavy chain variable region of the amino acid sequences of 378 and 421;
(ii) comprising a polypeptide comprising SEQ ID NO: 207. 250 and 293 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 336. 379 and 422;
(jj) comprises a polypeptide comprising SEQ ID NO: 208. 251 and 294 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 337. 380 and 423;
(kk) comprises a polypeptide comprising SEQ ID NO: 209. 252 and 295 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 338. 381 and 424;
(ll) comprises a polypeptide comprising SEQ ID NO: 210. 253 and 296 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 339. 382, and 425;
(mm) comprises a polypeptide comprising SEQ ID NO: 211. 254 and 297 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 340. 383 and 426;
(nn) comprises a polypeptide comprising SEQ ID NO: 212. 255 and 298 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 341. 384 and 427;
(oo) comprises a polypeptide comprising SEQ ID NO: 213. 256 and 299 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 342. 385 and 428;
(pp) comprises a polypeptide comprising SEQ ID NO: 214. 257 and 300 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 343. 386 and 429;
(qq) comprises a polypeptide comprising SEQ ID NO: 215. 258 and 301 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 344. a heavy chain variable region of the amino acid sequence of 387 and 430;
or
(rr) comprises a polypeptide comprising SEQ ID NO: 194. 237 and 280 and a light chain variable region comprising an amino acid sequence comprising SEQ ID NO: 335. 378 and 421.
8. The monoclonal antibody of claim 1, comprising:
(a) having the polypeptide sequence SEQ ID NO: 2 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 4, a heavy chain variable region;
(b) having the polypeptide sequence SEQ ID NO: 6 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 8, a heavy chain variable region;
(c) having the polypeptide sequence SEQ ID NO: 10 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 12, a heavy chain variable region;
(d) having the polypeptide sequence SEQ ID NO: 14 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16;
(e) having the polypeptide sequence SEQ ID NO: 18 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 20, a heavy chain variable region;
(f) having the polypeptide sequence SEQ ID NO: 22 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 24, a heavy chain variable region;
(g) having the polypeptide sequence SEQ ID NO: 26 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 28;
(h) having the polypeptide sequence SEQ ID NO: 30 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 32, a heavy chain variable region;
(i) having the polypeptide sequence SEQ ID NO: 34 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 36, a heavy chain variable region;
(j) having the polypeptide sequence SEQ ID NO: 38 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 40, a heavy chain variable region;
(k) having the polypeptide sequence SEQ ID NO: 42 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44, a heavy chain variable region;
(l) Having the polypeptide sequence SEQ ID NO: 46 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 48, a heavy chain variable region;
(m) a polypeptide having the polypeptide sequence of SEQ ID NO: 50 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 52, a heavy chain variable region;
(n) a polypeptide having the polypeptide sequence of SEQ ID NO: 54 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 56;
(o) a polypeptide having the polypeptide sequence of SEQ ID NO: 58 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 60;
(p) a polypeptide having the polypeptide sequence of SEQ ID NO: 62 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 64;
(q) a polypeptide having the polypeptide sequence of SEQ ID NO: 66 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 68;
(r) a polypeptide having the polypeptide sequence of SEQ ID NO: 70 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72, a heavy chain variable region;
(s) a polypeptide having the polypeptide sequence of SEQ ID NO: 74 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 76;
(t) a polypeptide having the polypeptide sequence of SEQ ID NO: 78 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 80;
(u) a polypeptide having the polypeptide sequence of SEQ ID NO: 82 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 84, the heavy chain variable region;
(v) having the polypeptide sequence SEQ ID NO: 86 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 88;
(w) a polypeptide having the polypeptide sequence of SEQ ID NO: 90 and a light chain variable region having the polypeptide sequence SEQ ID NO: 92, the heavy chain variable region;
(x) Having the polypeptide sequence SEQ ID NO: 94 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 96, the heavy chain variable region;
(y) a polypeptide having the polypeptide sequence of SEQ ID NO: 98 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100, a heavy chain variable region;
(z) has the polypeptide sequence of SEQ ID NO: 102 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 104;
(aa) a polypeptide having the polypeptide sequence of SEQ ID NO: 106 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 108;
(bb) has the polypeptide sequence of SEQ ID NO: 110 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 112;
(cc) a polypeptide having the polypeptide sequence of SEQ ID NO: 114 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 116, the heavy chain variable region;
(dd) a polypeptide having the polypeptide sequence of SEQ ID NO: 118 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 120, a heavy chain variable region;
(ee) has the polypeptide sequence of SEQ ID NO: 122 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 124, the heavy chain variable region;
(ff) a polypeptide having the polypeptide sequence of SEQ ID NO: 126 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128, a heavy chain variable region;
(gg) has the polypeptide sequence of SEQ ID NO: 130 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 132;
(hh) has the polypeptide sequence of SEQ ID NO: 134 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136, the heavy chain variable region;
(ii) having the polypeptide sequence SEQ ID NO: 138 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 140;
(jj) has the polypeptide sequence of SEQ ID NO: 142 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 144, the heavy chain variable region;
(kk) having the polypeptide sequence of SEQ ID NO: 146 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 148;
(ll) has the polypeptide sequence of SEQ ID NO: 150 and a light chain variable region having the polypeptide sequence SEQ ID NO: 152;
(mm) has the polypeptide sequence of SEQ ID NO: 154 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156;
(nn) has the polypeptide sequence of SEQ ID NO: 158 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 160, a heavy chain variable region;
(oo) has the polypeptide sequence of SEQ ID NO: 162 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 164, heavy chain variable region;
(pp) has the polypeptide sequence of SEQ ID NO: 166 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 168, a heavy chain variable region;
(qq) has the polypeptide sequence of SEQ ID NO: 170 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 172, the heavy chain variable region; or
(rr) has the polypeptide sequence of SEQ ID NO: 86 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 136, or a heavy chain variable region of the light chain.
9. An isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises a human heavy chain variable region comprising an amino acid sequence having at least 96% identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 4. SEQ ID NO: 8. SEQ ID NO: 12. SEQ ID NO: 16. SEQ ID NO: 20. SEQ ID NO: 24. SEQ ID NO: 28. SEQ ID NO: 32. SEQ ID NO: 36. SEQ ID NO: 40. SEQ ID NO: 44. SEQ ID NO: 48. SEQ ID NO: 52. SEQ ID NO: 56. SEQ ID NO: 60. SEQ ID NO: 64. SEQ ID NO: 68. SEQ ID NO: 72. SEQ ID NO: 76. SEQ ID NO: 80. SEQ ID NO: 84. SEQ ID NO: 88. SEQ ID NO: 92. SEQ ID NO: 96. SEQ ID NO: 100. SEQ ID NO: 104. SEQ ID NO: 108. SEQ ID NO: 112. SEQ ID NO: 116. SEQ ID NO: 120. SEQ ID NO: 124. SEQ ID NO: 128. SEQ ID NO: 132. SEQ ID NO: 136. SEQ ID NO: 140. SEQ ID NO: 144. SEQ ID NO: 148. SEQ ID NO: 152. SEQ ID NO: 156. SEQ ID NO: 160. SEQ ID NO: 164. SEQ ID NO: 168. and SEQ ID NO: 172, or a fragment thereof.
10. An isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises a human light chain variable region comprising an amino acid sequence having at least 97% identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 2. SEQ ID NO: 6. SEQ ID NO: 10. SEQ ID NO: 14. SEQ ID NO: 18. SEQ ID NO: 22. SEQ ID NO: 26. SEQ ID NO: 30. SEQ ID NO: 34. SEQ ID NO: 38. SEQ ID NO: 42. SEQ ID NO: 46. SEQ ID NO: 50. SEQ ID NO: 54. SEQ ID NO: 58. SEQ ID NO: 62. SEQ ID NO: 66. SEQ ID NO: 70. SEQ ID NO: 74. SEQ ID NO: 78. SEQ ID NO: 82. SEQ ID NO: 86. SEQ ID NO: 90. SEQ ID NO: 94. SEQ ID NO: 98. SEQ ID NO: 102. SEQ ID NO: 106. SEQ ID NO: 110. SEQ ID NO: 114. SEQ ID NO: 118. SEQ ID NO: 122. SEQ ID NO: 126. SEQ ID NO: 130. SEQ ID NO: 134. SEQ ID NO: 138. SEQ ID NO: 142. SEQ ID NO: 146. SEQ ID NO: 150. SEQ ID NO: 154. SEQ ID NO: 158. SEQ ID NO: 162. SEQ ID NO: 166. and SEQ ID NO: 170, or a pharmaceutically acceptable salt thereof.
11. The antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the antibody is selected from the group consisting of: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies.
12. The antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein blood clotting time in the presence of the antibody is shortened as measured by dilution of prothrombin time.
13. The antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 which is an antibody fragment or a single chain antibody.
14. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 and a pharmaceutically acceptable carrier.
15. A method for treating genetic and acquired deficiencies or defects in coagulation comprising administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 14.
16. The method of claim 15, wherein the method treats hemophilia a or B.
17. A method for reducing bleeding time comprising administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 14.
18. A method for treating genetic and acquired deficiencies in coagulation comprising administering (a) a first amount of a monoclonal antibody that binds to a human tissue factor pathway inhibitor and (b) a second amount of factor VIII or factor IX, wherein said first and second amounts together are effective to treat said deficiency or deficiency, and further wherein factor VII is not co-administered.
19. A pharmaceutical composition comprising a therapeutically effective amount of a combination of (a) the monoclonal antibody of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 and (b) factor VIII or factor IX; wherein the composition is free of factor VII.
20. A method for treating genetic and acquired deficiencies or defects in coagulation comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 19.
21. A method for reducing bleeding time comprising administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 19.
22. An isolated fully human monoclonal antibody to a human tissue factor pathway inhibitor.
23. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of claim 22 and a pharmaceutically acceptable carrier.
24. A method for treating genetic and acquired deficiencies or defects in coagulation comprising administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 23.
25. The method of claim 24, further comprising administering factor VIII or factor IX.
26. An isolated nucleic acid molecule encoding an antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises CDR3, the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-430 amino acid sequence.
27. An isolated nucleic acid molecule encoding an antibody that binds to a human tissue factor pathway inhibitor, wherein the antibody comprises CDR3, the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 259-301.
28. A method for generating a fully human monoclonal antibody that binds to a human tissue factor pathway inhibitor comprising:
(i) transfecting a nucleotide sequence encoding said fully human monoclonal antibody into a host cell, and
(ii) culturing the host cell to express the monoclonal antibody.
29. The method of claim 28, wherein the monoclonal antibody comprises CDR3, the CDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-430 amino acid sequence.
30. A method for generating a monoclonal antibody that binds to a human tissue factor pathway inhibitor, comprising:
(ii) transfecting a nucleotide sequence encoding said monoclonal antibody into a host cell, and
(ii) culturing the host cell to express the monoclonal antibody,
wherein the monoclonal antibody comprises CDR3, the CDR3 comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 259-301.
31. The method of claim 29, wherein said monoclonal antibody comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 388-430 and heavy chain CDR3 comprising an amino acid sequence selected from SEQ ID NO: 259-301 amino acid sequence of light chain CDR 3.
32. An isolated fully human monoclonal antibody directed to Kunitz domain 2 of human tissue factor pathway inhibitor.
33. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody of claim 32 and a pharmaceutically acceptable carrier.
34. A method for treating genetic and acquired deficiencies or defects in coagulation comprising administering to a patient a therapeutically effective amount of the pharmaceutical composition of claim 33.
35. The method of claim 34, further comprising administering factor VII, factor VIII, or factor IX.
HK11110795.3A 2008-08-04 2009-08-04 Monoclonal antibodies against tissue factor pathway inhibitor (tfpi) HK1156635B (en)

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