JP2017222678A - Monoclonal antibodies against tissue factor pathway inhibitors (tfpi) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide monoclonal antibodies against human tissue factor pathway inhibitors (TFPI).SOLUTION: Disclosed herein is a therapeutic composition comprising an isolated human monoclonal IgG antibody that specifically binds to a binding site including the Kunitz domains 1 and 2 of TFPI, where the antibody heavy chain is constituted from an amino acid sequence having at least 80% homology to SEQ ID NO: 16, the antibody light chain is constituted from an amino acid sequence having at least 80% homology to SEQ ID NO: 14, and the heavy chain has a CDR-1 region constituted from an amino acid sequence having at least 80% homology to SEQ ID NO:305, a CDR-2 region constituted from an amino acid sequence having at least 80% homology to SEQ ID NO:348 and a CDR-3 region constituted from an amino acid sequence having at least 80% homology to SEQ ID NO:391, and the human monoclonal IgG inhibits 50% or more of TFPI activity.SELECTED DRAWING: None

Description

Submission of Sequence Listing The sequence listing associated with the present application has been filed in electronic form by EFS-Web, and is incorporated herein by reference in its entirety. The name of the text file containing the sequence listing is MSB7
329PCT_Sequence_Listing_ST25.

Field of Embodiments Isolated monoclonal antibodies and fragments thereof that bind to human tissue factor pathway inhibitor (TFPI) and related inventions are provided.

Background Art Blood clotting is the process by which blood forms a stable thrombus that stops bleeding. The process involves a number of proenzymes circulating in the blood and procofactors (proco).
factors) (or “coagulation factors”). Their proenzymes and procofactors interact through several pathways, during which they are converted to activated forms sequentially or simultaneously. Ultimately, the process results in the activation of prothrombin to thrombin by activated factor X (FXa) in the presence of factor Va, ionized calcium and platelets. 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 thrombus.

The process that results in the activation of factor X can be performed by two different pathways: the contact activation pathway (previously called the intrinsic pathway) and the tissue factor pathway (formerly the extrinsic pathway and Was called). Previously, the coagulation cascade was thought to consist of two equally important pathways added to a common pathway. It is now known that the main pathway for the initiation of blood clotting is the tissue factor pathway.

Factor X can be activated by tissue factor (TF) in combination with activated factor VII (FVIIa). The complex of FVIIa and its important cofactor, TF, is a potent initiator of the blood coagulation cascade.

The tissue factor pathway of blood clotting is negatively controlled by a tissue factor pathway inhibitor (“TFPI”). TFPI is a natural FXa-dependent feedback inhibitor of the FVIIa / TF complex. TFPI is a member of a multivalent Kunitz-type serine protease inhibitor. Physiologically, TFPI binds to activated factor X (FXa) to form a heterodimeric complex and then interacts with the FVIIa / TF complex to inhibit its activity, resulting in blood Inhibits the tissue factor pathway of clotting. In principle, disruption of TFPI activity restores FXa and FVIIa / TF activity, resulting in prolonged duration of action of the tissue factor pathway and the common loss of FXa in hemophilia A and B Production can be amplified.

Indeed, evidence from several preliminary experiments indicates that inhibition of TFPI activity by antibodies to TFPI normalizes delayed blood clotting time or shortens bleeding time. For example, Nordfang et al. Showed that the dilute prothrombin time of hemophilia plasma was normalized after treatment of the plasma with an antibody against TFPI (Thromb. Haemo).
st. 1991, 66 (4): 464-467). Similarly, Erhardtsen
et al. Showed that the bleeding time in hemophilia A rabbit model was significantly shortened by anti-TFPI antibody (Blood Coagulation and Fibrin)
olisis, 1995, 6: 388-394). These studies indicate that inhibition of TFPI by anti-TFPI antibodies may be useful in the treatment of hemophilia A or B.
Only polyclonal anti-TFPI antibodies were used in these studies.

Using hybridoma technology, monoclonal antibodies against recombinant human TFPI (rhTFPI) were generated and identified. Yang et al. , Chin. Med
. J. et al. , 1998, 111 (8): 718-721. The effects of monoclonal antibodies on diluted prothrombin time (PT) and activated partial thromboplastin time (APTT) were tested. Experiments have shown that anti-TFPI monoclonal antibodies reduce the blood clotting time of diluted thromboplastin in plasma lacking factor IX. It has been shown that the tissue factor pathway plays an important role not only in physiological blood clotting but also in haemophilia bleeding (Yang et al., Hunan Yi Ke).
Da Xue Xue Bao, 1997, 22 (4): 297-300).

Kjalke et al. U.S. Pat. No. 7,015,194 to FVIIa,
And a composition for treating or preventing bleeding symptoms or coagulation treatments, comprising a TFPI inhibitor comprising a polyclonal or monoclonal antibody, or fragment thereof.
The use of such compositions has also been disclosed to reduce clotting time in normal mammalian plasma. Further, Factor VIII or a variant thereof is FVIIa and TFPI
It has been shown that inhibitors can be included in the disclosed compositions. The combination of FVIII or Factor IX and the TFPI monoclonal antibody is not shown.

In addition to the treatment of hemophilia, it has also been shown that TFPI inhibitors, including polyclonal or monoclonal antibodies, can be used for cancer treatment (see US Pat. No. 5,902,582 to Hung). ).

Thus, antibodies specific for TFPI are needed to treat blood diseases and cancer.

In general, therapeutic antibodies for human disease have been produced by genetic modification to produce murine, chimeric, humanized or fully human antibodies. Mouse monoclonal antibodies have been shown to have limited use as therapeutic agents due to short serum half-life, inability to induce human effector function, and production of human anti-mouse antibodies. Brekke and
Sandlie, “Therapeutic Antibodies for Hum
an Diseases at the Dawn of the Twenty-fi
rst Century, "Nature 2, 53, 52-62 (Jan. 2
003). Chimeric antibodies have been shown to produce 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 mouse components. Thus, there is a need to develop fully human antibodies that circumvent the immunogenicity associated with other forms of genetically modified monoclonal antibodies. In particular, chronic prophylactic treatment, such as required for hemophilia treatment with anti-TFPI monoclonal antibodies, is possible because the mouse component or mouse origin is required for frequent administration and long duration of therapy. When required, there is a high risk of developing an immune response to the treatment. For example, antibody therapy for hemophilia A may require weekly administration for the lifetime of the patient. This can be an ongoing challenge to the immune system. Thus, a need exists for fully human antibodies for antibody treatment of hemophilia A and related congenital and acquired blood coagulation deficiencies or defects.

Therapeutic antibodies are described in “Continou” by Koehler and Milstein.
s Cultures of Fused Cells Secreting Anti
body of Predefined Specificity, “Nature
256, 495-497 (1975). Fully human antibodies can also be produced recombinantly in prokaryotes and eukaryotes. Recombinant production of antibodies in host cells is preferred for therapeutic antibodies over hybridoma production. Recombinant production has the advantage of controlled production that minimizes or eliminates the presence of higher product consistency, higher production levels, and animal-derived proteins. For these reasons, it is desirable to have a recombinantly produced anti-TFPI antibody.

Summary Monoclonal antibodies against human tissue factor pathway inhibitor (TFPI) are provided. In addition, an isolated nucleic acid molecule encoding the antibody is provided. Also provided are pharmaceutical compositions comprising anti-TFPI monoclonal antibodies and methods of treating congenital and acquired blood coagulation deficiencies or defects such as hemophilia A and B. Also provided is a method for reducing bleeding time by administering an anti-TFPI monoclonal antibody to a patient in need thereof. The invention also provides a method of producing a monoclonal antibody that binds to human TFPI.

Representative binding activity of Fabs selected from panning and screening against human TFPI (“h-TFPI”) and mouse TFPI (“m-TFPI”). Twelve Fabs (1-4 and 6-13) selected from control Fab and TFPI panning against estradiol-BSA (“EsB”) were tested. The Y axis shows the fluorescence unit of the ELISA result. Four representative anti-TFPI antibodies (4B7: TP-4B7, 2A8: TP-2A8, 2G6: TP-2G6, 2G7: obtained from panning and screening of human antibody libraries, as shown by truncated dPT: Dose-dependent in vitro functional activity of TP-2G7). The experiment included 0.5 ug / mL mTFPI spiked into TFPI-deficient plasma. In vitro functional activity of the anti-TFPI Fab, Fab-2A8 (from TP-2A8), tested in the ROTEM assay. Binding activity of human TFPI and mouse TFPI of clone TP-2G6 ("2G6") after conversion to IgG. Δ: IgG-2G6 binding to mouse TFPI; □: IgG-2G6 to human TFPI; ▲: control IgG binding to mouse TFPI; ■: control IgG binding to human IgG. Anti-TFPI antibodies TP-2A8 (“2A8”), TP-3G1 (“3G1”), and TP-3C2 (“3C2”) were tested for total blood clotting time in hemophilia A mice as tested in the ROTEM assay. Was shortened. Each point represents one individual hemophilia A mouse. 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: 6), 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), 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-4 Amino acid sequence alignment between the variable light chain of 8 (SEQ ID NO: 162), and TP-3B3 (SEQ ID NO: 106). 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 (sequence) 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 amino acid sequence alignment between the variable heavy chain (SEQ ID NO: 136), and TP-4G8 (SEQ ID NO: 168). (1) Anti-TFPI antibody TP-2A8 ("2A8"), (2) 2A8 and recombinant factor VIII, (3) mouse IgG, and (4) mice treated with recombinant factor VIII after tail vein amputation Schematic showing survival rate over 24 hours. Schematic showing blood clotting time and thrombus formation time assays for mice treated with anti-TFPI antibody TP-2A8 (“2A8”), Factor VIIa, and combinations of 2A8 and Factor VIIa. Compared to the FVIII inhibitor alone, blood clotting time for normal human blood treated with FVIII inhibitor and anti-TFPI antibody TP-2A8 ("2A8") and anti-TFPI antibody TP-4B7 ("4B7") Schematic shown.

DETAILED DESCRIPTION The term “tissue factor pathway inhibitor” or “TFPI” as used herein refers to
It means all variants, isoforms and species homologs of human TFPI that are naturally expressed in cells. 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 a whole antibody and any antigen-binding fragment thereof (ie,
“Antigen-binding portion”) or single chain. The term refers to a full-length immunoglobulin molecule, either naturally occurring or formed by a normal immunoglobulin gene fragment recombination process (
IgG antibody), or an immunologically active portion of an immunoglobulin molecule that retains a specific binding activity, eg, an antibody fragment. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody. For example, an anti-TFPI monoclonal antibody fragment binds to an epitope of TFPI. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include: (i) Fab fragments that are monovalent fragments consisting of V _L , V _H , C _L and C _H1 domains. (Ii) a F (ab ′) ₂ fragment that is a divalent fragment comprising two Fab fragments joined by a disulfide bridge in the hinge region; (iii) an F consisting of V _H and C _H1 domains;
d fragment; (iv) an Fv fragment consisting of the _VL and V _H domains of a single arm of the antibody; (v)
DAb fragment consisting of V _H domain (Ward et al., (1989) Nature
341: 544-546); and (vi) an isolated complementarity determining region (CDR). In addition, the two domains of the Fv fragment, _VL and _VH , are encoded by separate genes, but using recombinant methods, they are joined by a synthetic linker that joins them as a single protein chain. Where V _L and V _H pair to form a monovalent molecule (known as single chain Fv (scFv); for example, Bird et a
l. (1988) Science 242: 423-426; and Huston et
al (1988) Proc. Natl. Acad. Sci. USA 85: 5
879-5883). 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 use in the same manner as intact antibodies.

As used herein, the terms “inhibit binding” and “prevent binding” (see, eg, inhibition / intervention of binding of a TFPI ligand to TFPI) are synonymous, partial and Includes both complete inhibition or interference. Inhibition and interference are also anti-TFP
Any measurable decrease in binding affinity between TFPI and a physiological substrate when contacted with an anti-TFPI antibody compared to TFPI not contacted with an I antibody, eg, at least about 10%, 20% 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95
Up to%, 96%, 97%, 98%, 99%, or 100% of TFPI and factor Xa interaction, or TFPI-factor Xa complex and tissue factor, factor VIIa or tissue factor / factor It is intended to include interfering with the complex of factor VIIa.

The term “monoclonal antibody” or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Thus, the term “human monoclonal antibody” refers to an antibody that exhibits single binding specificity with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention are introduced by amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). Mutations).

As used herein, an “isolated antibody” is intended to mean an antibody that is substantially free of other antibodies with different antigen specificities (eg, an isolated antibody that binds to TFPI is
There is virtually no antibody binding to antigens other than TFPI). However, human TFP
An isolated antibody that binds to an epitope, isoform, or variant of I has cross-reactivity with other related antigens (eg, TFPI species homologs) from other species, for example. further,
An isolated antibody can be substantially free of other cellular material and / or chemicals.

As used herein, “specific binding” refers to an antibody that binds to a predetermined antigen. In general, an antibody binds with an affinity of at least about 10 ⁵ M ⁻¹ and has a higher affinity than that of binding to a given antigen or an unrelated antigen other than a closely related antigen (eg, BSA, casein). For example, it binds to a predetermined antigen with at least twice the affinity. The terms “an antibody recognizing an antigen” and “an antibody specific for an antigen” are synonymous with the term “an antibody which specifically binds to an antigen” herein.

As used herein, the term “high affinity” for an IgG antibody is at least about 1
0 ⁷ M ⁻¹ , in certain embodiments, at least about 10 ⁸ M ⁻¹ , in certain embodiments, at least about 10 ⁹ M ⁻¹ , 10 ¹⁰ M ⁻¹ , 10 ¹¹ M ⁻¹ or more, such as 10
It means a binding affinity up to ¹³ M ⁻¹ or higher. However, “high affinity”
Binding can vary for other antibody isotypes. For example, “high affinity” binding for an IgM isotype means a binding affinity of at least about 1.0 × 10 ⁷ M ⁻¹ .
As used herein, “isotype” refers to the antibody class (eg, IgM or IgG1) that is encoded by heavy chain constant region genes.

“Complementarity determining regions” or “CDRs” are the three variable regions of the heavy or light chain of an antibody molecule that form an N-terminal antigen-binding surface that is complementary to the three-dimensional structure of the antigen to which it binds. Means one of the hypervariable regions. In order from the N-terminus of the heavy or light chain, these complementarity determining regions are designated as “CDR1”, “CDR2” and “CDR3”, respectively. CDRs are involved in antigen-antibody binding, and CDR3 contains specific regions specific for antigen-antibody binding.
Thus, the antigen binding site may comprise 6 CDRs including the CDR regions from each of the heavy and light chain V regions.

As used herein, a “conservative substitution” includes the substitution of one or more amino acids with amino acids having similar biochemical properties, and the biological or biochemical function of the polypeptide. It means a modification of a polypeptide that does not result in a defect. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg, glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine,
Proline, phenylalanine, methionine, tryptophan), amino acids with β-chain side chains (eg, threonine, valine, isoleucine), and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). It is envisioned that the antibodies of the invention may have conservative amino acid substitutions and still retain activity.

For nucleic acids and polypeptides, the term “substantial homology” optimally aligns two nucleic acids or two polypeptides, or their designated sequences, with appropriate nucleotides or amino acid insertions or deletions. Compared to at least about 80% of 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 9
It shows that it is the same at 9.5%. Alternatively, substantial homology for nucleic acids is
Present when a fragment hybridizes to the complement of a strand under selective hybridization conditions. The invention includes nucleic acid and polypeptide sequences that have substantial homology to the specific nucleic acid and amino acid sequences described herein.

The percent identity between two sequences is a function of the number of gaps and the number of identical positions shared by the sequence considering the length of each gap (ie,% identity = number of identical positions /
Total number of positions x 100), it needs to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished by mathematical algorithms such as the VectorNTI ™ AlignX ™ module (Invit
rogen Corp. , Carlsbad, CA). Ali
For gnX ™, the default parameters for multiple alignment are as follows: gap opening penalty: 10; gap extension penalty: 0.05; gap separation penalty range (gap separation penalty). penalty range)
: 8;% identity for alignment delay: 40
. (For more details, see http://www.invitrogen.com/site.
/ Us / en / home / LINNEA-Online-Guides / LINNEA-
Communities / Vector-NTI-Community / Sequenc
e-analysis-and-data-management-software-
for-PCs / AlignX-Module-for-Vector-NTI-Adv
ance. reg. us. found in html).

Another method (also called global sequence alignment) for determining the best overall match between a query sequence (sequence of the invention) and a subject sequence is the CLUSTALW computer program (Thompson et al., Nucleic Acids Res.
arch, 1994, 2 (22): 4673-4680),
It is described in Higgins et al. Algorithm (Computer Apply
ations in the Biosciences (CABIOS), 1992
, 8 (2): 189-191). In sequence alignment, both the query and subject sequences are DNA sequences. The result of the global sequence alignment is% identity. Pairwise alignment (pairwise alignment)
In order to calculate% identity by s), the preferred parameters used in the CLUSTALW alignment of DNA sequences are as follows: matrix = IUB, k
-Tuple (k-tuple) = 1, number of Top Diagonals (Number o
f Top Diagnostics) = 5, gap penalty = 3, gap start penalty = 10, gap extension penalty = 0.1. For multiple alignment, the following CL
USTALW parameters are preferred: gap opening penalty = 10, gap extension parameter = 0.05; gap separation penalty range = 8;% identity for alignment delay = 40.

The nucleic acid can be present in whole cells, cell lysates, or partially purified or in substantially pure form. A nucleic acid is “isolated” or “substantially pure” when it is purified away from other cellular components normally associated with it in its natural environment. Standard techniques such as the following can be used to isolate nucleic acids: alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques known in the art.

Monoclonal antibodies 44 from panning and screening of human antibody libraries against human TFPI
TFPI-binding antibodies have been identified. The heavy chain and light chain variable regions of each monoclonal antibody were sequenced and their CDR regions were identified. The sequence identification numbers (“SEQ ID NOs”) corresponding to these regions of each monoclonal antibody are summarized in Table 1.

Table 1. Summary of the sequence identification number (“SEQ ID NO”) of the heavy chain variable region (“VH”) and light chain variable region (“VL”) of each TFPI binding monoclonal antibody. Sequence identification numbers for each heavy and light chain CDR regions (“CDR1”, “CDR2”, and “CDR3”) are also provided. N. A. : Nucleic acid sequence; A. : Amino acid sequence.

In one embodiment, an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, comprising a CD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-430
An antibody comprising R3 is provided. These CDR3s are identified from the antibody heavy chain identified during panning and screening. In a further 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 345-387,
Or (c) a CD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 302-344
CDR comprising an amino acid sequence selected from the group consisting of R1 and SEQ ID NOs: 345-387
2 is included.

In other embodiments, antibodies are provided that share CDR3s derived from one of the light chains of the antibodies identified during panning and screening. Accordingly, the present invention provides an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor comprising SEQ ID NOs: 259-301.
Directed to an antibody comprising a CDR3 comprising an amino acid sequence selected from the group consisting of In a further aspect, 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-258, Or (c) both a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 173-215 and a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-258.

In other embodiments, the antibody comprises a CDR3 derived from the heavy chain of the antibody identified during panning and screening and a CDR3 derived from the light chain. Accordingly, the present invention provides an antibody that binds to a human tissue factor pathway inhibitor, comprising a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 388-430 and an amino acid selected from the group consisting of SEQ ID NOs: 259-301 An antibody comprising a CDR3 comprising sequence is provided. In a further aspect, 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 an amino acid sequence selected from the group consisting of SEQ ID NOs: 345-387, (C) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 173-215, and / or (d) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 216-258.

In another specific embodiment, the antibody is:
(A) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 173, 216 and 259 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 302, 345 and 388;
(B) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 174, 217 and 260 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 303, 346 and 389;
(C) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 175, 218 and 261 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 304, 347 and 390;
(D) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 176, 219 and 262 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 305, 348 and 391;
(E) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 177, 220 and 263 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 306, 349 and 392;
(F) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 178, 221 and 264 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 307, 350 and 393;
(G) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 179, 222 and 265 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 308, 351 and 394;
(H) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 180, 223, and 266 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 309, 352, and 395;
(I) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 181, 224 and 267 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 310, 353 and 396;
(J) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 182, 225 and 268 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 311, 354 and 397;
(K) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 183, 226 and 269 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 312, 355 and 398;
(L) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 184, 227 and 270 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 313, 356 and 399;
(M) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 185, 228 and 271 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 314, 357 and 400;
(N) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 186, 229 and 272 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 315, 358 and 401;
(O) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 187, 230 and 273 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 316, 359 and 402;
(P) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 188, 231 and 274 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 317, 360 and 403;
(Q) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 189, 232 and 275 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 318, 361 and 404;
(R) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 190, 233 and 276 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 319, 362 and 405;
(S) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 191, 234 and 277 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 320, 363 and 406;
(T) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 192, 235 and 278 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 321, 364 and 407;
(U) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 193, 236 and 279 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 322, 365 and 408;
(V) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 194, 237 and 280 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 323, 366 and 409;
(W) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 195, 238 and 281 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 324, 367 and 410;
(X) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 196, 239 and 282 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 325, 368 and 411;
(Y) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 197, 240 and 283 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 326, 369 and 412;
(Z) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 198, 241, and 284 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 327, 370, and 413;
(Aa) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 199, 242 and 285 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 328, 371 and 414;
(Bb) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 200, 243 and 286 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 329, 372 and 415;
(Cc) a light chain variable region comprising amino acid sequences comprising SEQ ID NOS: 201, 244 and 287 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 330, 373 and 416;
(Dd) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 202, 245 and 288 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 331, 374 and 417;
(Ee) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 203, 246 and 289 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 332, 375 and 418;
(Ff) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 204, 247 and 290 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 333, 376 and 419;
(Gg) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 205, 248 and 291 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 334, 377 and 420;
(Hh) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 206, 249 and 292 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 335, 378 and 421;
(Ii) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 207, 250 and 293 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 336, 379 and 422;
(Jj) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 208, 251 and 294 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 337, 380 and 423;
(Kk) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 209, 252 and 295 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 338, 381 and 424;
(Ll) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 210, 253 and 296 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 339, 382 and 425;
(Mm) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 211, 254 and 297 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 340, 383 and 426;
(Nn) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 212, 255 and 298 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 341, 384 and 427;
(Oo) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 213, 256 and 299 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 342, 385 and 428;
(Pp) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 214, 257 and 300 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 343, 386 and 429;
(Qq) a light chain variable region comprising amino acid sequences comprising SEQ ID NOs: 215, 258 and 301 and a heavy chain variable region comprising amino acid sequences comprising SEQ ID NOs: 344, 387 and 430; or (rr) SEQ ID NOs: 194, 237 and 280 A light chain variable region comprising an amino acid sequence comprising and a heavy chain and light chain variable region comprising a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 335, 378 and 421.

In another embodiment, the present invention provides:
(A) a light chain variable region having the polypeptide sequence of SEQ ID NO: 2 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 4;
(B) a light chain variable region having the polypeptide sequence of SEQ ID NO: 6 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 8;
(C) a light chain variable region having the polypeptide sequence of SEQ ID NO: 10 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 12;
(D) a light chain variable region having the polypeptide sequence of SEQ ID NO: 14 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 16;
(E) a light chain variable region having the polypeptide sequence of SEQ ID NO: 18 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 20;
(F) a light chain variable region having the polypeptide sequence of SEQ ID NO: 22 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 24;
(G) a light chain variable region having the polypeptide sequence of SEQ ID NO: 26 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 28;
(H) a light chain variable region having the polypeptide sequence of SEQ ID NO: 30 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 32;
(I) a light chain variable region having the polypeptide sequence of SEQ ID NO: 34 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 36;
(J) a light chain variable region having the polypeptide sequence of SEQ ID NO: 38 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 40;
(K) a light chain variable region having the polypeptide sequence of SEQ ID NO: 42 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 44;
(L) a light chain variable region having the polypeptide sequence of SEQ ID NO: 46 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 48;
(M) a light chain variable region having the polypeptide sequence of SEQ ID NO: 50 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 52;
(N) a light chain variable region having the polypeptide sequence of SEQ ID NO: 54 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 56;
(O) a light chain variable region having the polypeptide sequence of SEQ ID NO: 58 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 60;
(P) a light chain variable region having the polypeptide sequence of SEQ ID NO: 62 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 64;
(Q) a light chain variable region having the polypeptide sequence of SEQ ID NO: 66 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 68;
(R) a light chain variable region having the polypeptide sequence of SEQ ID NO: 70 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 72;
(S) a light chain variable region having the polypeptide sequence of SEQ ID NO: 74 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 76;
(T) a light chain variable region having the polypeptide sequence of SEQ ID NO: 78 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 80;
(U) a light chain variable region having the polypeptide sequence of SEQ ID NO: 82 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 84;
(V) a light chain variable region having the polypeptide sequence of SEQ ID NO: 86 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 88;
(W) a light chain variable region having the polypeptide sequence of SEQ ID NO: 90 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 92;
(X) a light chain variable region having the polypeptide sequence of SEQ ID NO: 94 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 96;
(Y) a light chain variable region having the polypeptide sequence of SEQ ID NO: 98 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 100;
(Z) a light chain variable region having the polypeptide sequence of SEQ ID NO: 102 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 104;
(Aa) the light chain variable region having the polypeptide sequence of SEQ ID NO: 106 and SEQ ID NO: 108
A heavy chain variable region having the polypeptide sequence of
(Bb) the light chain variable region having the polypeptide sequence of SEQ ID NO: 110 and SEQ ID NO: 112
A heavy chain variable region having the polypeptide sequence of
(Cc) the light chain variable region having the polypeptide sequence of SEQ ID NO: 114 and SEQ ID NO: 116
A heavy chain variable region having the polypeptide sequence of
(Dd) a light chain variable region having the polypeptide sequence of SEQ ID NO: 118 and SEQ ID NO: 120
A heavy chain variable region having the polypeptide sequence of
(Ee) the light chain variable region having the polypeptide sequence of SEQ ID NO: 122 and SEQ ID NO: 124
A heavy chain variable region having the polypeptide sequence of
(Ff) the light chain variable region having the polypeptide sequence of SEQ ID NO: 126 and SEQ ID NO: 128
A heavy chain variable region having the polypeptide sequence of
(Gg) a light chain variable region having the polypeptide sequence of SEQ ID NO: 130 and SEQ ID NO: 132
A heavy chain variable region having the polypeptide sequence of
(Hh) the light chain variable region having the polypeptide sequence of SEQ ID NO: 134 and SEQ ID NO: 136
A heavy chain variable region having the polypeptide sequence of
(Ii) the light chain variable region having the polypeptide sequence of SEQ ID NO: 138 and SEQ ID NO: 140
A heavy chain variable region having the polypeptide sequence of
(Jj) the light chain variable region having the polypeptide sequence of SEQ ID NO: 142 and SEQ ID NO: 144
A heavy chain variable region having the polypeptide sequence of
(Kk) a light chain variable region having the polypeptide sequence of SEQ ID NO: 146 and SEQ ID NO: 148
A heavy chain variable region having the polypeptide sequence of
(Ll) a light chain variable region having the polypeptide sequence of SEQ ID NO: 150 and SEQ ID NO: 152
A heavy chain variable region having the polypeptide sequence of
(Mm) the light chain variable region having the polypeptide sequence of SEQ ID NO: 154 and SEQ ID NO: 156
A heavy chain variable region having the polypeptide sequence of
(Nn) a light chain variable region having the polypeptide sequence of SEQ ID NO: 158 and SEQ ID NO: 160
A heavy chain variable region having the polypeptide sequence of
(Oo) a light chain variable region having the polypeptide sequence of SEQ ID NO: 162 and SEQ ID NO: 164
A heavy chain variable region having the polypeptide sequence of
(Pp) a light chain variable region having the polypeptide sequence of SEQ ID NO: 166 and SEQ ID NO: 168
A heavy chain variable region having the polypeptide sequence of
(Qq) the light chain variable region having the polypeptide sequence of SEQ ID NO: 170 and SEQ ID NO: 172
Or (rr) a light chain variable region having the polypeptide sequence of SEQ ID NO: 86 and a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 136.

An isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, comprising 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: 9
6, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 11
6, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 13
6, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 15
6, an amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, and SEQ ID NO: 172 and at least 89%, 90%, 9
1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 9
Also provided is an antibody comprising a human heavy chain variable region comprising an amino acid sequence having 9.5% identity.

An isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor comprising 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: 9
4, 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
An amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, and SEQ ID NO: 170 and at least 93%, 94%, 95
Also provided is an antibody comprising a human light chain variable region comprising an amino acid sequence having%, 96%, 97%, 98%, 99%, or 99.5% identity.

In addition to relying on antibody description using the sequence identifier above, certain embodiments can also be described by Fab clones isolated in the experiments described herein. In certain embodiments, the recombinant antibody comprises the heavy and / or light chain CDR3 of the following clone: T
P-2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A10, TP-
2B1, TP-2B3, TP-2B4, TP-2B8, TP-2B9, TP-2C1, T
P-2C7, TP-2D7, TP-2E3, TP-2E5, TP-2F9, TP-2G2
, TP-2G4, TP-2G5, TP-2G6, TP-2G7, TP-2G9, TP-2
H10, TP-3A2, TP-3A3, TP-3A4, TP-3B3, TP-3B4, T
P-3C1, TP-3C2, TP-3C3, TP-3D3, TP-3E1, TP-3F1
, TP-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4
A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H8. In certain embodiments, the antibodies can further comprise CDR2 of these antibodies, and further CDRs of these antibodies.
1 may be included. In other embodiments, the antibody may further comprise any combination of CDRs.

Accordingly, in other embodiments, (1) a human heavy chain framework region, a human heavy chain CDR
1 region, a human heavy chain CDR2 region, and a human heavy chain CDR3 region, wherein the human heavy chain CD
R3 region consists of TP-2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2
A10, TP-2B1, TP-2B3, TP-2B4, TP-2B8, TP-2B9, T
P-2C1, TP-2C7, TP-2D7, TP-2E3, TP-2E5, TP-2F9
, TP-2G2, TP-2G4, TP-2G5, TP-2G6, TP-2G7, TP-2
G9, TP-2H10, TP-3A2, TP-3A3, TP-3A4, TP-3B3, T
P-3B4, TP-3C1, TP-3C2, TP-3C3, TP-3D3, TP-3E1
, TP-3F1, TP-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4
A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H8
And (2) human light chain framework region, human light chain CDR1
Region, human light chain CDR2 region, and human light chain CDR3 region, wherein the human light chain CDR
The three regions are TP-2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A
10, 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-2G
9, TP-2H10, TP-3A2, TP-3A3, TP-3A4, TP-3B3, TP
-3B4, TP-3C1, TP-3C2, TP-3C3, TP-3D3, TP-3E1,
TP-3F1, TP-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A
7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or TP-4H8 light chain CDR3), wherein an antibody that binds to TFPI is provided. The antibody further comprises TP-2A2, TP-2A5.1, TP-2A6, TP-2A8,
TP-2A10, TP-2B1, TP-2B3, TP-2B4, TP-2B8, TP-2
B9, 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-3
B3, TP-3B4, TP-3C1, TP-3C2, 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
It may comprise a heavy chain CDR2 and / or a light chain CDR2 of -4H8. The antibody further comprises TP-
2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A10, TP-2B
1, TP-2B3, TP-2B4, TP-2B8, TP-2B9, TP-2C1, TP-
2C7, TP-2D7, TP-2E3, TP-2E5, TP-2F9, TP-2G2, T
P-2G4, TP-2G5, TP-2G6, TP-2G7, TP-2G9, TP-2H1
0, TP-3A2, TP-3A3, TP-3A4, TP-3B3, TP-3B4, TP-
3C1, TP-3C2, TP-3C3, TP-3D3, TP-3E1, TP-3F1, T
P-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9
, TP-4B7, TP-4E8, TP-4G8, or TP-4H8 heavy chain CDR1 and / or light chain CDR1.

The CDR1, 2, and / or 3 regions of the modified antibody are represented by TP-2 described herein.
A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A10, TP-2B1
, TP-2B3, TP-2B4, TP-2B8, TP-2B9, TP-2C1, TP-2
C7, 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-3
C1, TP-3C2, TP-3C3, TP-3D3, TP-3E1, TP-3F1, TP
-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9,
It may comprise the exact amino acid sequence of CDR1, 2, and / or 3 regions of TP-4B7, TP-4E8, TP-4G8, or TP-4H8.

However, one of ordinary skill in the art retains the ability of antibodies to efficiently bind to TFPI, TP-
2A2, TP-2A5.1, TP-2A6, TP-2A8, TP-2A10, TP-2B
1, TP-2B3, TP-2B4, TP-2B8, TP-2B9, TP-2C1, TP-
2C7, TP-2D7, TP-2E3, TP-2E5, TP-2F9, TP-2G2, T
P-2G4, TP-2G5, TP-2G6, TP-2G7, TP-2G9, TP-2H1
0, TP-3A2, TP-3A3, TP-3A4, TP-3B3, TP-3B4, TP-
3C1, TP-3C2, TP-3C3, TP-3D3, TP-3E1, TP-3F1, T
P-3F2, TP-3G1, TP-3G3, TP-3H2, TP-4A7, TP-4A9
It is well understood that some deviation from the exact CDR sequence of TP-4B7, TP-4E8, TP-4G8, or TP-4H8 may be possible. Thus, in other embodiments, the modified antibody is, for example, TP-2A2, TP-2A5.1, TP-2A6, T
P-2A8, TP-2A10, TP-2B1, TP-2B3, TP-2B4, TP-2B
8, TP-2B9, TP-2C1, TP-2C7, TP-2D7, TP-2E3, TP-
2E5, TP-2F9, TP-2G2, TP-2G4, TP-2G5, TP-2G6, T
P-2G7, TP-2G9, TP-2H10, TP-3A2, TP-3A3, TP-3A
4, TP-3B3, TP-3B4, TP-3C1, TP-3C2, TP-3C3, TP-
3D3, TP-3E1, TP-3F1, TP-3F2, TP-3G1, TP-3G3, T
P-3H2, TP-4A7, TP-4A9, TP-4B7, TP-4E8, TP-4G8
, Or one or more CDRs of TP-4H8 and at least 90%, 91%, 92
%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% may consist of one or more CDRs that are identical.

Antibodies can be of various classes of antibodies, eg, IgG1, IgG2, IgG3, IgG4
, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies (but not limited to).

In one embodiment, an isolated fully human monoclonal antibody against a human tissue factor pathway inhibitor is provided.

In another aspect, an isolated fully human monoclonal antibody against Kunitz domain 2 of a human tissue factor pathway inhibitor is provided.

Nucleic acids Isolated nucleic acid molecules encoding any of the above monoclonal antibodies are also provided.

Methods for Producing Antibodies to TFPI Monoclonal antibodies can be produced recombinantly by expressing in a host cell a nucleotide sequence encoding the variable region of a monoclonal antibody according to embodiments of the invention. Nucleic acid containing nucleotide sequences can be introduced into a suitable host cell for production and expressed with the aid of an expression vector. Accordingly, a method for producing a monoclonal antibody that binds to human TFPI, comprising the following steps:
(A) introducing a nucleic acid molecule encoding the monoclonal antibody of the present invention into a host cell;
(B) culturing the host cell to express the monoclonal antibody in the host cell; (c) isolating and purifying the produced monoclonal antibody, wherein the nucleic acid molecule is of the present invention. Also provided is a method comprising a nucleotide sequence encoding a monoclonal antibody.

In one example, in order 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, so that A gene is operably linked to transcriptional and translational control sequences. As used herein, the term “operably linked” refers to antibodies linked to a vector such that transcriptional and translational control sequences within the vector perform their intended function of controlling the transcription and translation of the antibody gene. Is meant to mean The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more generally, both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (eg, ligation of complementary restriction enzyme sites on the antibody gene fragment and vector, or blunt end ligation in the absence of restriction enzyme sites). Using the light and heavy chain variable regions of the antibodies described herein, any antibody isotype can be inserted by inserting them into expression vectors that already encode the heavy and light chain constant regions of the desired isotype. Full-length antibody genes can be generated, so that the V _H fragment can be expressed as C _H in the vector.
Fragment and operatively linked, V _L fragment, operably linked to a C _L fragment in the compactors. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from a non-immunoglobulin protein).

In addition to the gene encoding the antibody chain, the recombinant expression vector of the present invention may have a control sequence that controls the 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 (eg, polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such control sequences are described, for example, by Goeddel; Gene Expression Technol.
ogy. Methods in Enzymology 185, Academic
Press, San Diego, Calif. (1990).
It will be appreciated by those skilled in the art that the design of an expression vector, including the selection of control sequences, may depend on factors such as the choice of host cell to be transformed, the level of protein expression desired, etc.
Examples of control sequences for mammalian host cell expression include viral elements directed to high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (eg, adenovirus). Major late promoter (AdMLP)) and promoters and / or enhancers derived from polyoma. Alternatively, non-viral regulatory sequences such as ubiquitin promoter or β-globin promoter can be used.

In addition to the antibody chain genes and control sequences, the recombinant expression vector may have additional sequences, such as sequences that control replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (eg, US Pat. No. 4,399,2 by Axel et al.).
16, No. 4,634,665 and 5,179,017). For example, in general, a selectable marker gene is a drug in a host cell into which a vector has been introduced
For example, conferring resistance to G418, hygromycin or methotrexate.
Examples of selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification).

For the expression of light and heavy chains, expression vectors encoding heavy and light chains are introduced into host cells by standard techniques. Various forms of the term “introduction” refer to exogenous DN
It is intended to encompass a wide range of techniques commonly used for introduction of A into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran introduction, and the like. While it is theoretically possible to express an antibody of the invention in prokaryotic or eukaryotic host cells, expression of the antibody in eukaryotic cells, and most preferably mammalian host cells, is such eukaryotic cells, and Most preferred because certain mammalian cells are likely to fold properly and build and secrete immunologically active antibodies when compared to prokaryotic cells.

Examples of mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) (Urlab and Chasin, (1980) Proc. Na.
tl. Acad. Sci. Dhf described in USA 77: 4216-4220.
r-CHO cells, including DHFR selectable markers such as R. J. et al. Kaufm
an and P.A. A. Sharp (1982) Mol. Biol. 159: 6
01-621), NSO myeloma cells, COS cells, H
Includes KB11 cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody allows expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is growing. Produced by culturing host cells for a sufficient period of time. Antibodies 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 Intact Antibodies Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. See, for example, FIGS. 6 and 7, where the CDR regions in each of the light and heavy chain variable regions of the monoclonal antibody according to the invention have been identified. CDR
Since sequences are involved in many antibody-antigen interactions, construct expression vectors containing CDR sequences derived from specific naturally occurring antibodies grafted on framework sequences derived from different antibodies with different properties By doing so, it is possible to express recombinant antibodies that mimic the properties of certain naturally occurring antibodies (eg, Riechmann, L. e.
t al. , 1998, Nature 332: 323-327; Jones, P.
. et al. , 1986, Nature 321: 522-525; and Qu
een, C.I. et al. , 1989, Proc. Natl. Acad.
Sci. U. S. A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. Because these germline sequences do not contain fully assembled variable genes (formed by V (D) J joining during B cell maturation), Is different. It is not necessary to obtain the entire DNA sequence of a particular antibody in order to reconstruct an intact recombinant antibody with similar binding properties as the original antibody (see WO 99/45662). Partial heavy and light chain sequences spanning the CDR regions are generally sufficient for this purpose. The partial sequence is used to determine which germline variable and linked gene fragments are involved in the recombinant antibody variable gene. The germline sequence is then used to fill in variable region deletions. The heavy and light chain leader sequences are cleaved during protein maturation and are not involved in the final antibody properties.
It is therefore necessary to use the corresponding germline leader sequence for the expression construct. To add a missing sequence, the cloned cDNA sequence can be combined with a synthetic oligonucleotide 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 an entire synthetic variable region clone. This process has certain advantages such as the elimination or inclusion of specific restriction enzyme sites, or optimization of specific codons.

The nucleotide sequences of the heavy and light chain transcripts are used to design a set of overlapping synthetic oligonucleotides, creating a synthetic V sequence that has the ability to encode the same amino acid as the natural sequence. Synthetic heavy and kappa chain sequences can differ from the native sequence in three ways: a string of repetitive nucleotide bases is used for oligonucleotide synthesis and PC
Blocked to promote R amplification; optimal translation start site is Kozak rule (Kozak
k, 1991, J.A. Biol. Chem. 266: 19867-19870)
And the HindIII site is modified upstream of the translation start site.

For heavy and light chain variable regions, the optimal coding strand sequence and the corresponding non-coding strand sequence are cleaved into 30-50 nucleotide sections approximately in the middle of the corresponding non-coding oligonucleotide. Thus, for each strand, oligonucleotides can be constructed in overlapping duplex sets spanning fragments of 150-400 nucleotides. The pool is then used as a template to produce a 150-400 nucleotide PCR amplification product. In general, a single variable region oligonucleotide set is divided into two pools, which are amplified separately to produce two overlapping PCR products. These overlapping products are then combined by PCR amplification to form a complete variable region. It may also be desirable to include overlapping fragments of heavy or light chain constant regions in PCR amplification to create fragments that can be easily cloned into expression vector constructs.

The reconstructed heavy and light chain variable regions are then combined with a cloned promoter sequence, translation initiation sequence, constant region sequence, 3 ′ untranslated region sequence, polyadenylation sequence, and transcription termination sequence to construct an expression vector The body is formed. The heavy and light chain expression constructs are combined in a single vector and co-introduced, serially introduced, or separately introduced into the host cell, and then induced to form a host cell that expresses both chains.

Thus, in other aspects, human anti-TFPI antibodies, such as TP2A8, TP2G
6, structural features such as TP2G7, TP4B7 retain the ability to bind to TFPI,
Used to make structurally related human anti-TFPI antibodies. In particular, one or more CDRs of the specifically identified heavy and light chain regions of the monoclonal antibodies of the invention
Can be recombinantly linked to known human framework regions and CDRs, and further recombinantly modified human anti-TFPI antibodies of the invention can be generated.

Accordingly, in another aspect, a method for producing an anti-TFPI antibody comprising:
) Human heavy chain framework region and human heavy chain CDR, where human heavy chain CDR3 comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 388-430 and / or (2) human light chain framework Region and human light chain CDRs (where light chain CDR3 is
A method is provided comprising producing an antibody comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 259-301, wherein the antibody retains the ability to bind to TFPI. In other embodiments, the method is practiced with other CDRs of the invention.

Pharmaceutical compositions 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 aid in formulation or to stabilize the formulation and does not produce a significant side effect toxic effect on the patient. Examples of such carriers are 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 in, for example, E.I. W. Remington's Ph by Martin
described in armoral sciences. Such a composition is
A therapeutically effective amount of at least one anti-TFPI monoclonal antibody may be included.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and materials for pharmaceutically active substances is known in the art. The composition is preferably formulated for parenteral administration. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. In some cases,
It may 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 dose in a suitable solvent containing a combination of one or more of the ingredients listed above, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the required dispersant medium and the required other ingredients from those enumerated above. In the case of sterile powders for the production of sterile injectable solutions, some of the production methods are vacuum drying and freeze-drying techniques, which are further desired with the active ingredients from the sterile filtration solution described above Produce powder of ingredients.

Pharmaceutical Use Monoclonal antibodies can be used for therapeutic purposes to treat congenital and acquired blood coagulation deficiencies or defects. For example, the monoclonal antibody in the above embodiment is TF.
It can be used to block the interaction of PI and FXa or to block TFPI-dependent inhibition of TF / FVIIa activity. In addition, monoclonal antibodies can also be used with FXa TF /
It can be used to restore FVIIa-induced production and bypass deficiencies in FVIII or FIX dependent amplification of FXa.

Monoclonal antibodies have therapeutic use in the treatment of hemostatic disorders, such as thrombocytopenia, platelet disorders and bleeding disorders (eg, hemophilia A and hemophilia B). Such disorders can be treated by administering a therapeutically effective amount of an anti-TFPI monoclonal antibody to a patient in need thereof. Monoclonal antibodies also have therapeutic use in the treatment of uncontrollable bleeding in indications such as trauma and stroke. Accordingly, there is also provided a method for reducing bleeding time 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, one or more antibodies of the invention and Factor VIIa, Factor VIII
Co-administration of a blood clotting factor such as factor or factor IX is considered useful for treating hemophilia. In one embodiment, congenital and acquired 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. A method of treating a blood coagulation deficiency or defect is provided, wherein the first amount and the second amount are both effective to treat the deficiency or defect. In other embodiments, congenital and acquired 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. Provided is a method of treating a blood coagulation deficiency or defect, wherein the first amount and the second amount are both effective to treat the deficiency or deficiency, and further no factor VII is co-administered . The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody of the invention and a combination of Factor VIII or Factor IX but no Factor VII. “Factor VII” includes Factor VII and Factor VIIa. These combination therapies can reduce the required infusion frequency of blood clotting factors. Co-administration or combination therapy refers to the administration of two therapeutic agents, each formulated separately, or co-formulated together in one composition, and when formulated separately, It is administered at the same time point or at different time points, but with the same treatment period.

The pharmaceutical composition may vary with the severity of the bleeding episode or, in the case of prophylactic treatment, at a dose and frequency that may vary with the severity of the patient's blood coagulation defect.
Alternatively, it can be administered parenterally to a subject suffering from B.

The composition can be administered to a patient in need thereof as a bolus dose or by continuous infusion. For example, bolus administration of an antibody of the invention present as a Fab fragment is 0.00
It may be in an amount of 25 to 100 mg / kg body weight, 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg or 0.10-0.50 mg / kg. For continuous infusion, the antibodies of the present invention present as Fab fragments can have 1-24 hours, 1-12 hours,
0.001 to 10 for 2-12 hours, 6-12 hours, 2-8 hours, or 1-2 hours
0 mg / kg body weight / min, 0.0125 to 1.25 mg / kg / min, 0.010 to 0.75 mg / kg / min, 0.010 to 1.0 mg / kg / min, or 0. 10-
It can be administered at 0.50 mg / kg / min. For administration of an antibody of the invention present as a full length antibody (having a full length constant region), the dosage is about 1-10 mg / kg body weight, 2-8
It can be mg / kg, or 5-6 mg / kg. Such full length antibodies are generally 3
It can be administered by infusion extending from 0 minutes to 3 hours. The frequency of administration can depend on the severity of the condition. The frequency can range from 3 times a week to once every 2 or 3 weeks.

In addition, the composition can be administered to the patient by subcutaneous injection. For example, 10 to 100 m
A dose of g anti-TFPI antibody can be administered to a patient by subcutaneous injection once a week, once every two weeks, or once a month.

As used herein, a “therapeutically effective amount” is an anti-reactivity necessary to effectively increase in vivo blood clotting time or produce a measurable benefit in vivo for a patient in need thereof. It means the amount of a TFPI monoclonal antibody or a combination of said antibody and factor VIII or factor IX. The exact amount will depend on many factors, including but not limited to, the components and physical characteristics of the therapeutic composition, the intended patient population, individual patient considerations, etc., and is readily determined by one skilled in the art. obtain.

EXAMPLES General Materials and Methods Example 1. Panning and screening of human antibody library against human TFPI Panning of human antibody library against TFPI Phage display combinatorial human antibody library against human TFPI (American Diagnostica) HuCal Gold (Rothe et
al. , J. et al. Mol. Biol. , 2008, 376: 1182-1200.
) Was panned to select anti-TFPI antibodies. In summary, 96 well Ma
Xisorp plates were coated with 200 μl TFPI (5 μg / ml) overnight at 4 ° C., then the plates were blocked with PBS buffer containing 5% milk. 0.
After washing the plate with PBS containing 01% Tween-20 (PBST), aliquots of combinatorial human antibody libraries were added to TFPI-coated wells and incubated for 2 hours. Unbound phages were washed away with PBST, antigen-bound phages were eluted with dithiothreitol. E. coli strain TG1 was infected and amplified. Phages were rescued with helper phage for the next panning. All three pannings were performed and the clones from the last two were screened against human TFPI in an ELISA assay.

Screening of antibody clones by antigen binding in ELISA In order to select antibody clones that bind to human TFPI, the Fab genes of the phage clones from the second and third panning were subcloned into a bacterial expression vector.
It was expressed in E. coli strain TG1. Bacterial lysate is human TFPI coated Maxiso
Added to wells of rp plate. After washing, the plate was expressed by adding AmplexRed (Invitrogen) containing hydrogen peroxide using HRP-conjugated goat anti-human Fab as the detection antibody. A signal at least 5 times higher than the background was considered positive. Cross-reactivity of anti-human TFPI antibody to mouse TFPI was determined by a similar mouse TFPI binding ELISA. Plate TFPI (R & D S
system), BSA and lysozyme. The last two antigens (BSA and lysozyme) were used as negative controls. A representative set of data is shown in FIG.

Anti-TFPI Human Antibody Sequences After panning and screening of the HuCal Gold human antibody library against TFPI, DNA sequencing was performed on positive antibody clones, yielding 44 specific antibody sequences (Table 2). Of these antibody sequences, 29 were lambda light chains and 15 were kappa light chains. Our analysis of the heavy chain variable region shows that 28 VH3, 14 VH6, 1 VH
1 and 1 VH5 are shown.

Table 2. Peptide sequences of the variable regions of 44 anti-TFPI antibodies

Cross reaction to mouse TFPI The 44 human TFPI binding clones were also tested for binding to mouse TFPI by ELISA. For 19 antibodies, a cross-reactivity to mouse TFPI was found. In order to facilitate testing with a mouse hemophilia model, we further characterized these 19 antibodies and 5 antibodies specific for human TFPI. A representative set of data is shown in FIG. None of these antibodies bound to BSA or lysozyme in the ELISA.

Example 2 Expression and purification of anti-TFPI antibodies Anti-TFPI antibodies (as Fab fragments) were expressed and purified in bacterial strain TG1. In summary, a single colony of bacterial strain TG1 containing the antibody expression plasmid was picked and
8 ml of 2xYT in the presence of g / ml chloramphenicol and 1% glucose
Grow overnight in medium. A 7 ml volume of the culture was transferred to 250 ml of fresh 2xYT medium containing 34 μg / ml chloramphenicol and 0.1% glucose. After 3 hours of incubation, 0.5 mM IPTG was added to induce Fab expression. Incubation was continued overnight at 25 ° C. The culture was centrifuged to precipitate bacterial cells. The precipitate was then resuspended in Bug Buster lysis buffer (Novagen). After centrifugation, the bacterial lysate supernatant was filtered. Ni-NT according to manufacturer's instructions
The Fab fragment was affinity purified through A column (Qiagen).

Example 3 FIG. Determination of EC ₅₀ and binding affinity of anti-TFPI antibodies Using purified Fab antibodies, anti-TFPI antibodies can be tested for human or mouse TFPI E
C ₅₀ was determined. As above, EC ₅₀ was evaluated by ELISA. SoftMa
The results were analyzed using x. The binding affinity of the anti-TFPI antibody was determined in a Biacore assay. In summary, antigen (ie human or mouse TFPI) was immobilized on a CM5-chip using an amine coupling kit (GE HealthCare) according to the manufacturer's instructions. The amount of immobilized TFPI was adjusted to the amount of antigen providing about 300 RU. Antibody Fab was analyzed in the mobile phase and at least 5 different concentrations of purified antibody (0.1, 0.4, 1.6, 6.4 and 25 nM) were used in the Biacore assay. Kinetics and binding affinities were calculated using Biacore T100 Evaluation software.

As shown in Table 3, the 24 anti-TFPI Fabs show various EC ₅₀ for human TFPI (0.09 to 792 nM) and mouse TFPI (0.06 to 1035 nM) and are thus determined by Biacore. The affinity for human TFP
For I (1.25 to 1140 nM). In the Biacore test of Fab against mouse TFPI, the variation in affinity was smaller (3.
08 to 51.8 nM).

Table 3. Human or mouse TFPI determined by ELISA and Biacore
Activity of 24 antibodies against hTFPI (hTFPI: human TFPI; mTFPI: mouse TF)
PI; Neg: signal was less than twice background; ND: not performed)

Example 4 Conversion of anti-TFPI Fab to IgG All identified anti-TFPI antibodies are fully human Fabs, which can be suitably converted to human IgG as a therapeutic agent. However, in this example, the selected Fab was converted to a chimeric antibody containing a more appropriate mouse IgG constant region for testing in a mouse model. The variable region of the selected antibody was transplanted into a mammalian expression vector containing a mouse constant region. Fully assembled IgG molecules were then introduced into HKB11 cells and expressed (Me
i et al. , Mol. Biotechnol. , 2006, 34: 165
-178). The culture supernatant was collected and concentrated. Hitra according to manufacturer's instructions
p Anti-TFPI via Protein G column (GE Healthcare)
IgG molecules were affinity purified.

Example 5 FIG. Selection of anti-TFPI neutralizing antibodies Anti-TFPI neutralizing antibodies were selected based on inhibition of TFPI activity under three experimental conditions. The activity of TFPI was measured using the ACTICHROME® TFPI activity assay (Ame
rican Diagnostica Inc. , Stamford, CT) (ie, a three-step chromogenic assay to measure the ability of TFPI to inhibit the catalytic activity of the TF / FVIIa complex to activate factor X to factor Xa). Anti-TFP
The neutralizing activity of the I antibody is proportional to the recovered FXa production. In an initial setting, purified anti-TFPI antibody was purified at the indicated concentrations to human or mouse recombinant TFPI (R & D Sys
tem). After incubation, the sample was TF / FVII
After mixing with a and FX, the residual activity of the TF / FVIIa complex was then measured using SPECTROZYME® FXa, a highly specific fXa chromogenic substrate. This substrate was cleaved only by FXa produced in the assay, releasing the p-nitroaniline (pNA) chromophore, which was measured at 405 nm. The TFPI activity present in the sample was interpolated from a standard curve constructed using known TFPI activity levels.
The assay was performed in an endpoint format. In the other two 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 blood clotting time in a diluted prothrombin time (dPT) assay. Essentially, Welsch et al. , Thrombosis Res. , 199
1, 64 (2): 213-222, the dPT assay was performed. In summary, human normal plasma (FACT, George King Biomedical)
Human TFPI deficient plasma (American Diagnostica) or hemophilia A
Plasma (George King Biomedical) was prepared by mixing plasma with 0.1 equivalent of control buffer or anti-TFPI antibody. After 30 minutes incubation at 25 ° C., plasma samples (100 μl) were diluted with 200 μl of appropriately diluted (1: 500 dilution) Simlastin (B
fibrometer STA4 (Stago) in combination with iometieux)
Was used to determine blood clotting time. PBS or 0.1 M sodium chloride, 0.1%
0.05 M Tris with bovine serum albumin and 20 μM calcium chloride
Thromboplastin was diluted with a buffer based on pH 7.5 (pH 7.5).

Example 7 Anti-TFPI antibody alone or a combination of anti-TFPI antibody and recombinant factor VIII or factor IX reduces blood clotting time in the ROTEM assay ROTEM system (Pentapharm GmbH) is a 4-channel device, computer, plasma standard, activator Disposable cups and pins (cups)
and pins). The thromboelastography parameters of the ROTEM hemostasis system included the following: blood clotting time (CT) reflecting the reaction time to initiate blood clotting (required to obtain 2 mm amplitude after starting data collection) Time taken to clot); thrombus formation time (CFT) and α angle reflecting blood coagulation amplification, and maximum amplitude and maximum elastic modulus reflecting thrombus hardness. In the ROTEM assay, 300 μl of fresh citrated whole blood or plasma was evaluated. Data was collected for the time period required for each system, with all components reconfigured and mixed according to the manufacturer's instructions. In summary, while adding 20 μl of CaCl ₂ (200 mmol),
Mix samples by collecting / dispensing μl of blood or plasma in a ROTEM cup using an automatic pipette,
Immediately thereafter, the sample was mixed and data collection was started. Data was collected for 2 hours using a computer controlled (software 2.96 version) ROTEM system.

Exemplary results of a ROTEM assay to detect the effect of anti-TFPI antibodies in reducing blood clotting time are shown in FIGS. FIG. 3 shows that TP-2A8-Fab alone or in combination with recombinant FVIII reduces blood clotting time in human hemophilia A plasma or mouse hemophilia A whole blood (ROTEM system Started with NATEM). FIG. 5 shows that anti-TFPI antibodies (TP-2A8, TP-3G1 and TP-3C2) in IgG mode shortened blood clotting time compared to negative control mouse IgG antibodies. Based on these results and an understanding in the art, one of ordinary skill in the art can predict that the combination of these anti-TFPI antibodies and recombinant FIX will also reduce blood clotting time compared to these antibodies alone. .

Example 8 FIG. In vitro functional activity of anti-TFPI antibodies To investigate TFPI antibodies for interference with TFPI, chromogenic assay ACTICH
ROME and diluted prothrombin time (dPT) were used to test the functional activity of the antibodies obtained from panning and screening. 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,
Eight antibodies inhibited TFPI activity by 50% or more compared to the rat monoclonal antibody (Table 4). In the dPT assay, all of these 8 anti-TFPI Fabs show high inhibitory activity (reducing blood clotting time to less than 80 seconds), 4 out of 8 Fabs
A dPT of less than 70 seconds was exhibited. The dose dependence of four representative clones in dPT shortening is shown in FIG. However, other human anti-TFPI Fabs that show low or no TFPI inhibitory activity also reduced blood clotting time in dPT. For example, TP-3B4 and TP-2C7 only showed an inhibitory activity of less than 25%, but could shorten dPT to less than 70 seconds. A simple linear regression analysis of inhibitory activity and dPT shows a significant correlation (p = 0.0095), but has a large variance (determining coefficient (R
square) = 0.258).

Table 4. Anti-T determined by inhibitory activity in human TFPI and dPT assays
In vitro functional activity of FPI antibodies

Fab-2A8, one of the anti-TFPI Fabs, was also tested in the ROTEM assay, where human hemophilia A plasma or mouse hemophilia A whole blood with low levels of factor VIII was used. As shown in FIG. 3, Fab-2A8 shows similar activity in human hemophilia A plasma when compared to polyclonal rabbit anti-TFPI antibody,
Blood clotting time (CT) was decreased from 0 seconds to about 1700 seconds. When mouse hemophilia A whole blood was used, the control antibody, rabbit anti-TFPI, shortened CT from 2700 seconds to 1000 seconds, and Fab-2A8 shortened CT from 2650 seconds to 1700 seconds. These results indicate that Fab-2A8 can significantly reduce blood clotting time in human plasma and mouse blood (p = 0.03).

Example 9 Function of anti-TFPI antibody after conversion to chimeric IgG An in vitro assay of factor Xa production and diluted prothrombin time was performed using 24 anti-T
FPI Fab, TP-2A8, TP-2B3, TP-2G6, TP-3C2, TP-3
Shows that at least 6 of G1 and TP-4B7 can interfere with TFPI function. To facilitate in vivo testing with hemophilia A mice, we converted these 6 anti-TFPI human Fab chimeric IgGs using the mouse IgG1 isotype. Ig
The G expression vector is introduced into HKB11 cells, and the expressed antibody is recovered in the culture supernatant.
It was purified with a rotein G column. When a representative clone, 2G6-Fab, is converted to IgG, 2G6-IgG increases the EC ₅₀ binding to human TFPI by a 2-fold increase (0.4
8 nM to 0.22 nM) and a 10-fold increase in EC ₅₀ binding to mouse TFPI (5.18 nM to 0.51 nM). The results for IgG-2G6 binding to human and mouse TFPI are shown in FIG.

Example 10 Effect on survival in hemophilia A (HemA) mouse tail vein amputation model A mouse tail vein amputation model was established for pharmacological evaluation. This model simulates the widespread bleeding phenotype observed between normal individuals and severe hemophilia. For these studies, hemophilia A male mice (8 weeks old, 20-26 g) were used. Anti-TFPI monoclonal antibody (40 μg / mouse) alone by tail vein administration before injury,
Alternatively, mice were administered with blood clotting factors such as FVIII (0.1 IU / mouse). 24 hours after administration, the left tail vein was cut at a diameter of 2.7 mm from the tip (in diameter). Survival was observed for 24 hours after cutting. When provided with recombinant FVIII, survival was shown to be dose dependent (data not shown). The data shown in FIG. 8 was from separate tests (n = 15 and n = 10, respectively). Results show that TP-2A8-IgG significantly prolonged the survival of hemophilia A mice compared to control mouse IgG and improved when combined with recombinant FVIII compared to the drug alone It was shown to show the survival rate.

Example 11 The combination of anti-TFPI antibody and recombinant factor Vila further reduced the blood clotting time and thrombus formation time. Using A whole blood, ROTE
Evaluated by M system. The indicated amount of anti-TFPI antibody, TP-2A8-IgG (“2
A8 "), and recombinant FVIIa (" FVIIa ") were added to 300 μl of citrated mouse hemophilia A whole blood to initiate blood clotting with the EXTEM system. FIG. 9 shows T
It is shown that the addition of P-2A8-IgG or recombinant FVIIa to mouse hemophilia A whole blood shortens blood clotting time and thrombus formation time, respectively. The combination of TP-2A8-IgG and recombinant FVIIa (“2A8 + FVIIa”) further reduces blood clotting time and thrombus formation time, which means that the combination of anti-TFPI antibody and recombinant FVIIa has or has an inhibitor. It is useful in the treatment of patients with hemophilia who do not.

Example 12 Anti-TFPI antibody selected in ROTEM assay using neutralizing FVIII antibody that induces hemophilia in whole blood collected from non-hemophilia patients with reduced blood clotting time in FVIII inhibitor-induced human hemophilia blood Anti-TFPI antibodies, 2A8 and 4B7, were also tested. FIG. 10 shows that normal human blood has a blood clotting time of about 1000 seconds.
In the presence of FVIII neutralizing antibody (PAH, 100 μg / mL), blood clotting time was extended to about 5200 seconds. Prolonged blood clotting time is anti-TFPI antibody 2
Significantly shortened by the addition of A8 or 4B7, indicating that anti-TFPI antibodies are useful in the treatment of hemophilia patients with inhibitors.

Example 13 Inhibitory anti-TFPI antibodies bind to Kunitz domain 2 of human TFPI Western blots and ELISAs were used to determine which domain of TFPI the inhibitory antibody binds to. Recombinant full-length human TFPI or TFPI domain was used for these studies. ELISA was performed in the same manner as in Example 3. In Western blots, human TFPI or domain was expressed as 4-12% Bis-Tris SD.
S PAGE running buffer MES (Invitrogen, Carlsbad
, CA) and then transferred to a cellulose membrane. After 10 minutes incubation with inhibitory antibodies, the SNAPid system (Millipore, Billerica)
, MA) and the membrane was washed three times. HRP-conjugated donkey anti-mouse antibody (Pierce, Rockford, IL) at a dilution of 1 to 10,000 was incubated with the membrane for 10 minutes. After a similar washing step, the SuperSignal substrate (Pierce, Rock)
Ford, IL) was used to express the membrane. Control anti-Kunitz domain 1 antibody binds to full-length TFPI, truncated TFPI and domain but inhibits anti-TFP
The I antibody binds only to TFPI containing Kunitz domain 2. This is Kunit
FIG. 5 shows that binding to z domain 2 is required for the inhibitory effect of the antibody.

Table 5. Domain to which antibody determined by Western blot and ELISA binds

Although the invention has been described with reference to specific embodiments and examples, it will be understood that various modifications and changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Should. The specification and examples are, therefore, to be regarded as illustrative rather than in a restrictive sense. Further, all articles, books, patent applications and patents mentioned in this specification are hereby incorporated by reference.

Claims (27)

Kunitz domain 1 and Kuni of human tissue factor pathway inhibitor (TFPI)
An isolated human monoclonal Ig that specifically binds to a binding site comprising tz domain 2
A therapeutic composition comprising a G antibody, wherein the antibody heavy chain is composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 16 and the antibody light chain has at least 80% homology with SEQ ID NO: 14 Composed of amino acid sequence, wherein the heavy chain is at least 80% with SEQ ID NO: 305
CDR-1 region composed of amino acid sequences having the homology of SEQ ID NO: 348, CDR-2 region composed of amino acid sequences having at least 80% homology with SEQ ID NO: 348, and at least 80% homology with SEQ ID NO: 391 The human monoclonal IgG exhibits a 50% or more inhibition of TFPI activity.
Composition.   The composition of claim 1 further comprising Factor VIII or Factor IX.   The composition of claim 2, wherein Factor VII is absent. 2. The composition of claim 1, wherein the antibody has a binding affinity for human TFPI of 1.25 to 1140 nM by Biacore assay. The composition of claim 1, wherein the antibody is formulated in a pharmaceutically acceptable carrier for subcutaneous injection. 2. The heavy chain CDR-2 region has at least 80% homology with SEQ ID NO: 348.
A composition according to 1. The heavy chain CDR-3 region has at least 80% homology with SEQ ID NO: 391.
A composition according to 1.   2. The composition of claim 1, wherein the antibody is a single chain fragment. CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 305, CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 348, and SEQ ID NO: 3
The composition according to claim 1, which has a CDR-3 region composed of the amino acid sequence shown in 91. CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 305, CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 348, and SEQ ID NO: 3
A CDR-1 region having the CDR-3 region composed of the amino acid sequence shown in 91, and the antibody light chain comprising the amino acid sequence shown in SEQ ID NO: 176, comprising the amino acid sequence shown in SEQ ID NO: 219 The composition of Claim 1 which has a CDR-3 region composed of the amino acid sequence set forth in SEQ ID NO: 262 and the CDR-2 region set forth above. The composition according to claim 1, wherein the antibody heavy chain is composed of an amino acid sequence represented by SEQ ID NO: 16. The composition according to claim 10, wherein the antibody light chain is composed of the amino acid sequence shown in SEQ ID NO: 14. 2. The composition of claim 1, wherein the antibody has a binding affinity for human TFPI of 1.25 nM by Biacore assay. 2. The composition of claim 1, wherein the antibody is formulated in a dose of 10 to 100 mg in a pharmaceutically acceptable carrier for injection. Kunitz domain 1 and Kuni of human tissue factor pathway inhibitor (TFPI)
An isolated human monoclonal Ig that specifically binds to a binding site comprising tz domain 2
A therapeutic composition comprising a G antibody, wherein the antibody heavy chain is composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 160, and the antibody light chain is at least 80% with SEQ ID NO: 158
The heavy chain is at least 8 from SEQ ID NO: 341.
CDR-1 region composed of amino acid sequence having 0% homology, CDR-2 region composed of amino acid sequence having at least 80% homology with SEQ ID NO: 384, and at least 80% with SEQ ID NO: 427 CDR- composed of amino acid sequences having homology
A composition having 3 regions and wherein the human monoclonal IgG exhibits 50% or more inhibition of TFPI activity.   16. The composition of claim 15, further comprising Factor VIII or Factor IX.   17. A composition according to claim 16, wherein factor VII is absent. 16. The composition of claim 15, wherein the antibody has a binding affinity for human TFPI of 15.8 nM by Biacore assay. 16. The composition of claim 15, wherein the antibody is formulated in a pharmaceutically acceptable carrier for injection at a dose of 10 to 100 mg. 2. The heavy chain CDR-1 region has at least 85% homology with SEQ ID NO: 341.
5. The composition according to 5. The heavy chain CDR-2 region has at least 85% homology with SEQ ID NO: 384.
5. The composition according to 5. 2. The heavy chain CDR-3 region has at least 85% homology with SEQ ID NO: 427.
5. The composition according to 5.   16. The composition of claim 15, wherein the antibody is a single chain fragment. CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 341, CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 384, and SEQ ID NO: 4
The composition according to claim 15, which has a CDR-3 region composed of the amino acid sequence shown in 27. The composition according to claim 15, wherein the antibody heavy chain is composed of an amino acid sequence represented by SEQ ID NO: 160. CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 341, CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 384, and SEQ ID NO: 4
A CDR-1 region comprising the amino acid sequence shown in SEQ ID NO: 212, having a CDR-3 region comprising the amino acid sequence shown in SEQ ID NO: 212 The composition according to claim 15, which has a CDR-2 region composed of the amino acid sequence set forth in SEQ ID NO: 298 and the CDR-2 region set forth in SEQ ID NO: 298. 16. The composition of claim 15, wherein the antibody heavy chain is composed of the amino acid sequence set forth in SEQ ID NO: 160 and the antibody light chain is composed of the amino acid sequence set forth in SEQ ID NO: 158.

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