JP6848016B2 - Monoclonal antibody against tissue factor pathway inhibitor (TFPI) - Google Patents

Description

Submission of Sequence Listing The sequence listing related to this application has been filed in electronic form via EFS-Web and is hereby incorporated by reference in its entirety. The name of the text file containing the sequence listing is MSB7.
329PCT_Sequence_Listing_ST25.

Areas of Embodiment Provided are an isolated monoclonal antibody and fragments thereof that bind to a human tissue factor pathway inhibitor (TFPI) and related inventions.

Background Technology Blood coagulation is the process by which blood forms a stable blood clot that stops bleeding. The process involves many proenzymes and procofactors circulating in the blood.
Factors) (or "coagulation factors"). Their proenzymes and procofactors interact through several pathways, during which they are converted to activated forms continuously or simultaneously. Finally, the process results in activation of prothrombin to thrombin by factor X (FXa) activated in the presence of factor Va, ionized calcium and platelets. Activated thrombin, in turn, induces platelet aggregation, converting fibrinogen to fibrin, which is then cross-linked by activated factor XIII (FXIIIa) to form a thrombus.

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

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

The tissue factor pathway of blood coagulation is negatively regulated 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 the multivalent Kunitz serine protease inhibitor. Physiologically, TFPI binds to activated factor X (FXa) to form a heterodimer complex, which then interacts with the FVIIa / TF complex to inhibit its activity, resulting in blood. Inhibits the tissue factor pathway of coagulation. In principle, disruption of TFPI activity restores FXa and FVIIa / TF activity, thus prolonging the duration of action of tissue factor pathways and is commonly deficient in hemophilia A and B. Production can be amplified.

Indeed, some preliminary experimental evidence indicates that inhibition of TFPI activity by antibodies to TFPI normalizes delayed blood clotting times or shortens bleeding times. For example, Nordfang et al. Showed that the extended prothrombin time of hemophiliac plasma is standardized after treating the plasma with an antibody against TFPI (Thromb. Haemo).
st. , 1991, 66 (4): 464-467). Similarly, Erhardtsen
et al. Showed that anti-TFPI antibody significantly reduced bleeding time in the hemophilia A rabbit model (Blood Coagulation and Fibrin).
olysis, 1995, 6: 388-394). These studies show 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.

Monoclonal antibodies against recombinant human TFPI (rhTFPI) were generated and identified using hybridoma technology. Yang et al. , Chin. Med
.. J. , 1998, 111 (8): 718-721. The effect of the monoclonal antibody on diluted prothrombin time (PT) and activated partial thromboplastin time (APTT) was tested. Experiments have shown that anti-TFPI monoclonal antibodies reduce diluted thromboplastin blood coagulation time in factor IX-deficient plasma. Tissue factor pathways have been shown to play an important role not only in physiological blood coagulation but also in hemophilia 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, by FVIIa,
Also disclosed are compositions for the treatment or prevention of bleeding symptoms or coagulation treatments, including TFPI inhibitors containing polyclonal or monoclonal antibodies, or fragments thereof.
The use of such compositions has also been disclosed to reduce coagulation time in normal mammalian plasma. In addition, Factor VIII or variants thereof are FVIIa and TFPI.
It has been shown that it can be included in the disclosed compositions of inhibitors. No combination of FVIII or Factor IX with a TFPI monoclonal antibody has been 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 the treatment of cancer (see US Pat. No. 5,902,582 by Hung). ).

Therefore, TFPI-specific antibodies are needed to treat blood disorders and cancers.

Generally, therapeutic antibodies for human diseases have been made by genetic modification to produce mouse, chimeric, humanized or fully human antibodies. Mouse monoclonal antibodies have been shown to have limited use as therapeutic agents due to their short serum half-life, non-inducible human effector function, and the production of human anti-mouse antibodies. Breake and
Sandlie, “Therapeutic Antibodies for Hum
an Diseases at the Down of the Twenty-fi
rst Century, "Nature 2, 53, 52-62 (Jan. 2)
See 003). Chimeric antibodies have been shown to produce a human anti-chimeric antibody response. Humanized antibodies also minimize the mouse component of the antibody. However, fully human antibodies completely avoid the immunogenicity associated with mouse components. Therefore, 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 measures such as those required for the treatment of hemophilia with anti-TFPI monoclonal antibodies are such that the mouse component or mouse origin is due to the required frequent doses and long-lasting treatment If required, there is a high risk of developing an immune response to the treatment. For example, antibody treatment for hemophilia A may require weekly administration throughout the patient's life. This can be a continuous challenge to the immune system. Therefore, there is a need for fully human antibodies for the treatment of hemophilia A and related congenital and acquired blood coagulation deficiency or deficiency antibodies.

Therapeutic antibody was described by Koehler and Milstein as "Continuou".
s Cultures of Forced Cells Secreting Anti
body of Predefined Specificity, "Nature
It can be made using the hybridoma technique described in 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 advantages of higher product consistency, higher production levels, and controlled production that minimizes or eliminates the presence of animal-derived proteins. For these reasons, it is desirable to have a recombinantly produced anti-TFPI antibody.

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

Binding activity of representative examples 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 Fabs and TFPI panning for 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 indicated by the shortened dPT. Dose-dependent in vitro functional activity of TP-2G7). Experiments included 0.5 ug / mL mTFPI spiked into TFPI-deficient plasma. In vitro functional activity of anti-TFPI Fab, Fab-2A8 (derived 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”) have been tested in the ROTEM assay for total blood coagulation time in hemophilia A mice. Was shortened. Each point indicates one individual hemophilia A mouse. Anti-TFPI monoclonal antibody 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: 140) SEQ ID NO: 70), TP-3F2 (SEQ ID NO: 134), TP-2A6 (SEQ ID NO: 10), TP-3A2 (SEQ ID NO: 94), TP-2C1 (SEQ ID NO: 42), TP-3E1 (SEQ ID NO: 126), TP-3F1 (SEQ ID NO: 130), TP-3D3 (SEQ ID NO: 122), TP-4A7 (SEQ ID NO: 150), TP-4G8 (SEQ ID NO: 166), TP-2B3 (SEQ ID NO: 26), TP-2F9 (SEQ ID NO: 26) 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: 118) 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-4E8 (SEQ ID NO: 162), and TP-3B3 (SEQ ID NO: 162). 106) Amino acid sequence alignment between variable light chains. Anti-TFPI monoclonal antibody 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: 112). SEQ ID NO: 48), TP-2E3 (SEQ ID NO: 56), TP-3C3 (SEQ ID NO: 120), TP-2G5 (SEQ ID NO: 76), TP-4B7 (SEQ ID NO: 160), TP-2G6 (SEQ ID NO: 80), TP-3C2 (SEQ ID NO: 116), TP-2D7 (SEQ ID NO: 52), TP-3G1 (SEQ ID NO: 140), TP-2E5 (SEQ ID NO: 60), TP-2B8 (SEQ ID NO: 36), TP-3F1 (SEQ ID NO: 36) 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: 24) 4), TP-3E1 (SEQ ID NO: 128), TP-2G2 (SEQ ID NO: 68), TP-3D3 (SEQ ID NO: 124), TP-2G9 (SEQ ID NO: 88), TP-2B4 (SEQ ID NO: 32), TP- 3A2 (SEQ ID NO: 96), TP-2F9 (SEQ ID NO: 64), TP-3A4 (SEQ ID NO: 104), TP-3C1 (SEQ ID NO: 136), TP-3F2 (SEQ ID NO: 136), and TP-4G8 (SEQ ID NO: 136). 168) Amino acid sequence alignment between variable heavy chains. After tail vein amputation for mice treated with (1) anti-TFPI antibody TP-2A8 (“2A8”), (2) 2A8 and recombinant factor VIII, (3) mouse IgG, and (4) recombinant factor VIII. A scheme showing the survival rate over 24 hours. Schematic showing a blood coagulation time and thrombus formation time assay for mice treated with the anti-TFPI antibody TP-2A8 (“2A8”), Factor VIIa, and a combination of 2A8 and Factor VIIa. Blood coagulation time for normal human blood treated with FVIII inhibitor and anti-TFPI antibody TP-2A8 (“2A8”) and anti-TFPI antibody TP-4B7 (“4B7”) compared to FVIII inhibitor alone. Diagram shown.

Detailed Description The term "tissue factor pathway inhibitor" or "TFPI" as used herein is used.
Means all variants, isoforms and species homologues of human TFPI that are naturally expressed in cells. In a preferred embodiment of the invention, binding of the antibody of the invention to TFPI reduces blood coagulation time.

As used herein, "antibody" refers to all antibodies and any antigen-binding fragment thereof (ie, that is.
"Antigen binding portion") or single strand. The term refers to a full-length immunoglobulin molecule that occurs naturally or is formed by a conventional immunoglobulin gene fragment recombination process.
For example, an IgG antibody), or an immunologically active portion of an immunoglobulin molecule that retains a particular binding activity, such as an antibody fragment. Regardless of the structure, the antibody fragment binds to the same antigen recognized by the full-length antibody. For example, an anti-TFPI monoclonal antibody fragment binds to an epitope of a TFPI. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments included within the term "antigen binding portion" of an antibody include: (i) Fab fragment, a monovalent fragment consisting of _VL , _VE , _CL and _{CH1 domains. (Ii) F (ab') 2} fragments, which are divalent fragments containing two Fab fragments linked by disulfide cross-linking in the hinge region; (iii) F consisting of _{V H} and C _{H1 domains.}
d fragment; (iv) _{Fv fragment consisting of a single arm VL} and _VH domain of the antibody; (v)
_{DAb fragment consisting of VH} domain (Word et al., (1989) Nature
341: 544-546); and (vi) isolated complementarity determining regions (CDRs). In addition, the two domains of the Fv fragment, _VL and _VH , are encoded by separate genes, but they are bound together by a synthetic linker that binds them as a single protein chain using a recombinant method. Where _VL and _VH are paired to form a monovalent molecule (known as single chain Fv (scFv); eg, Bird et a.
l. (1988) Science 242: 423-426; and Huston et.
al (1988) Proc. Natl. Acad. Sci. USA 85: 5
See 879-5883). Such single chain antibodies are also intended to be included within the term "antigen binding portion" of the antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art and the fragments are screened for use in a manner similar to intact antibodies.

As used herein, the terms "inhibit binding" and "interfere with binding" (see, eg, inhibition / inhibition of binding of a TFPI ligand to TFPI) are synonymous, partial and partial. Includes both complete inhibition or obstruction. Inhibition and interference are also anti-TFP
Any measurable reduction in binding affinity between TFPI and 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
Interfering with the interaction of TFPI and factor Xa up to%, 96%, 97%, 98%, 99%, or 100%, or TFPI-factor Xa complex and tissue factor, factor VIIa or tissue factor / th It is intended to include interfering with the interaction of the Factor VIIa complex.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" means a preparation of an antibody molecule of a single molecule composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" means an antibody exhibiting single binding specificity with variable and constant regions derived from a human germline immunoglobulin sequence. Human antibodies of the invention are introduced by amino acid residues not encoded by a human germline immunoglobulin sequence (eg, by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo. Mutations that have been made) can be included.

As used herein, "isolated antibody" is intended to mean an antibody in which no other antibody with different antigen specificity is substantially present (eg, an isolated antibody that binds to TFPI).
There are virtually no antibodies that bind to antigens other than TFPI). However, human TFP
An isolated antibody that binds to an epitope, isoform or variant of I has, for example, cross-reactivity with other related antigens (eg, TFPI species homologues) from other species. further,
The isolated antibody can be substantially free of other cellular and / or chemicals.

As used herein, "specific binding" means an antibody that binds to a given antigen. Generally, the antibody binds with an affinity of at least about 10 ⁵ M ^-1, predetermined antigen or a closely related unrelated antigens other than the antigen (e.g., BSA, casein) higher affinity than the affinity of binding to For example, it binds to a predetermined antigen with at least 2-fold affinity. The terms "antibody that recognizes an antigen" and "antibody specific to an antigen" are synonymous with the terms "antibody that specifically binds to an antigen" herein.

The term "high affinity" for IgG antibodies as used herein is at least about 1.
0 ⁷ M ^-1 , in some embodiments at least about 10 ⁸ M ^-1 , in some embodiments at least about 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 , 10 ¹¹ M ^-1 or more, eg, 10
¹³ M- ¹ or higher binding affinity. However, "high affinity"
Binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to a binding affinity of at least about 1.0x10 ⁷ M ^-1.
As used herein, "isotype" means an antibody class encoded by a heavy chain constant region gene (eg, IgM or IgG1).

The "complementarity determining regions" or "CDRs" are three of the heavy or light chain variable regions 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. It means one of the hypervariable regions. These complementarity determining regions are shown as "CDR1,""CDR2," and "CDR3," respectively, starting from the N-terminus of the heavy or light chain. CDRs are involved in antigen-antibody binding, and CDR3 contains specific regions specific for antigen-antibody binding.
Thus, the antigen binding site may include 6 CDRs, including CDR regions from each of the heavy and light chain V regions.

As used herein, "conservative substitutions" include substitutions of one or more amino acids with amino acids having similar biochemical properties, which are the biological or biochemical functions of the polypeptide. It means modification of a polypeptide that does not cause a defect. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues with similar side chains is 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, glutamate), amino acids with uncharged polar chains (eg, glycine, etc.). Aspartin, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanin, valine, leucine, isoleucine,
It contains 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 their activity.

For nucleic acids and polypeptides, the term "substantial homology" is optimally aligned with two nucleic acids or two polypeptides, or their designated sequences, with appropriate nucleotide or amino acid insertions or deletions. At least about 80% of nucleotides or amino acids, usually at least about 85%, preferably about 90%, when compared.
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
Shows the same at 9.5%. Alternatively, the substantial homology for nucleic acids is
It is present when the fragment hybridizes to a strand complement under selective hybridization conditions. The present invention includes nucleic acid sequences and polypeptide sequences that are substantially homologous to the particular nucleic acid and amino acid sequences described herein.

% Identity between two sequences is a function of the number of gaps and the number of identical positions shared by the sequences considering the length of each gap (ie,% identity = number of identical positions). /
The total number of positions x 100), it needs to be introduced for the optimal alignment of the two sequences. The comparison of sequences and the determination of% identity between two sequences can be done with mathematical algorithms such as the VectorNTI ™ AlignX ™ module (Invit).
rogen Corp. , Carlsbad, CA). Ali
For gnX ™, the default parameters for multiple alignment are: gap opening penalty: 10; gap extension penalty: 0.05; gap separation. penalty language)
: 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 / Sequence
e-analysis-and-data-management-software-
for-PCs / AlignX-Module-for-Vector-NTI-Adv
answer. reg. us. Found in html).

Another method (also called global sequence alignment) for determining the best overall match between a query sequence (the sequence of the invention) and a subject sequence is the ClustalW computer program (Thompson et al., Nucleic Acids Res).
It can be determined using earth, 1994, 2 (22): 4673-4680).
It is described in Highgins et al. Algorithm (Computer Appli)
cations in the Biosciences (CABIOS), 1992
, 8 (2): 189-191). In sequence alignment, both the query and the target sequence are DNA sequences. The result of the global sequence alignment is% identity. Pairwise alignment
Preferred parameters used in the CLUSTALW alignment of DNA sequences to calculate% identity by s) are: Matrix = IUB, k.
-Tuple (k-tuple) = 1, Number of Top Diagnostics (Number o)
f Top Diagnostics) = 5, Gap Penalty = 3, Gap Start Penalty = 10, Gap Extension Penalty = 0.1. About multiple alignment, CL below
The USTALW parameter is preferred: gap start penalty = 10, gap extension parameter = 0.05; gap separation penalty range = 8;% identity for alignment delay = 40.

Nucleic acid can be present in whole cell, cell lysate, or partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when separated from other cell components normally associated in the 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 From Panning and Screening of Human Antibody Libraries Against Human TFPI 44
A number of TFPI-binding antibodies have been identified. The heavy 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. A summary of the sequence identification numbers (“SEQ ID NOs”) of the heavy and light chain variable regions (“VH”) and light chain variable regions (“VL”) of each TFPI-binding monoclonal antibody. Sequence identification numbers for the CDR regions of each heavy and light chain (“CDR1”, “CDR2”, and “CDR3”) are also provided. N. A. : Nucleic acid sequence; A. A. : Amino acid sequence.

In one embodiment, a CD comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 388-430, an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor.
Antibodies, including R3, are provided. These CDR3s are identified from the heavy chains of antibodies identified during panning and screening. In a further embodiment, this antibody further
(A) CDR1, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 302-344,
(B) CDR2, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 345-387.
Or (c) a CD containing an amino acid sequence selected from the group consisting of SEQ ID NO: 302-344.
CDR containing an amino acid sequence selected from the group consisting of R1 and SEQ ID NO: 345-387
Includes both of 2.

In another embodiment, an antibody that shares CDR3 from one of the light chains of the antibody identified during panning and screening is provided. Therefore, the present invention is an isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, SEQ ID NO: 259-301.
It is directed to an antibody comprising CDR3 containing an amino acid sequence selected from the group consisting of. In a further embodiment, the antibody further comprises (a) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 173-215, (b) CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 216-258, Or (c) contains both CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 173-215 and CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 216-258.

In other embodiments, the antibody comprises CDR3 derived from the heavy chain and CDR3 derived from the light chain of the antibody identified during panning and screening. Therefore, the present invention is an antibody that binds to a human tissue factor pathway inhibitor and contains an amino acid sequence selected from the group consisting of SEQ ID NO: 388-430 and an amino acid selected from the group consisting of SEQ ID NO: 259-301. Antibodies are provided that include CDR3 containing the sequence. In a further embodiment, the antibody further comprises (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 302-344, and (b) a CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 345-387. (C) Contains CDR1 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 173-215, and / or (d) CDR2 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 216-258.

In other particular embodiments, the antibody is described below:
(A) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 173, 216 and 259 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 302, 345 and 388;
(B) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 174, 217 and 260 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 303, 346 and 389;
(C) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 175, 218 and 261 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 304, 347 and 390;
(D) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 176, 219 and 262 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 305, 348 and 391;
(E) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 177, 220 and 263 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 306, 349 and 392;
(F) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 178, 221 and 264 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 307, 350 and 393;
(G) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 179, 222 and 265 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 308, 351 and 394;
(H) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 180, 223 and 266 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 309, 352 and 395;
(I) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 181, 224 and 267 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 310, 353 and 396;
(J) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 182, 225 and 268 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 311, 354 and 397;
(K) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 183, 226 and 269 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 312, 355 and 398;
(L) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 184, 227 and 270 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 313, 356 and 399;
(M) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 185, 228 and 271 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 314, 357 and 400;
(N) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 186, 229 and 272 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 315, 358 and 401;
(O) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 187, 230 and 273 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 316, 359 and 402;
(P) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 188, 231 and 274 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 317, 360 and 403;
(Q) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 189, 232 and 275 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 318, 361 and 404;
(R) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 190, 233 and 276 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 319, 362 and 405;
(S) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 191, 234 and 277; and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 320, 363 and 406;
(T) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 192, 235 and 278 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 321, 364 and 407;
(U) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 193, 236 and 279 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 322, 365 and 408;
(V) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 194, 237 and 280 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 323, 366 and 409;
(W) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 195, 238 and 281 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 324, 376 and 410;
(X) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 196, 239 and 282 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 325, 368 and 411;
(Y) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 197, 240 and 283 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 326, 369 and 412;
(Z) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 198, 241 and 284 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 327, 370 and 413;
(Aa) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 199, 242 and 285 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 328, 371 and 414;
(Bb) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 200, 243 and 286 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 329, 372 and 415;
(Cc) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 201, 244 and 287 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 330, 373 and 416;
(Dd) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 202, 245 and 288 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 331, 374 and 417;
(Ee) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 203, 246 and 289 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 332, 375 and 418;
(Ff) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 204, 247 and 290 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 333, 376 and 419;
(Gg) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 205, 248 and 291 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 334, 377 and 420;
(Hh) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 206, 249 and 292 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 335, 378 and 421;
(Ii) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 207, 250 and 293 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 336, 379 and 422;
(Jj) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 208, 251 and 294 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 337, 380 and 423;
(KK) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 209, 252 and 295 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 338, 381 and 424;
(Ll) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 210, 253 and 296 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 339, 382 and 425;
(Mm) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 211, 254 and 297 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 340, 383 and 426;
(Nn) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 212, 255 and 298 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 341, 384 and 427;
(Oo) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 213, 256 and 299 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 342, 385 and 428;
(Pp) A light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 214, 257 and 300 and a heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 343, 386 and 429;
(Qq) Light chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 215, 258 and 301; or heavy chain variable region comprising an amino acid sequence comprising SEQ ID NOs: 344, 387 and 430; or (rr) SEQ ID NOs: 194, 237 and 280. Includes 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 aspect, the invention is described below:
(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) Light chain variable region having the polypeptide sequence of SEQ ID NO: 106 and SEQ ID NO: 108
Heavy chain variable region with the polypeptide sequence of;
(Bb) Light chain variable region having the polypeptide sequence of SEQ ID NO: 110 and SEQ ID NO: 112
Heavy chain variable region with the polypeptide sequence of;
(Cc) Light chain variable region having the polypeptide sequence of SEQ ID NO: 114 and SEQ ID NO: 116
Heavy chain variable region with the polypeptide sequence of;
(Dd) Light chain variable region having the polypeptide sequence of SEQ ID NO: 118 and SEQ ID NO: 120
Heavy chain variable region with the polypeptide sequence of;
(Ee) Light chain variable region having the polypeptide sequence of SEQ ID NO: 122 and SEQ ID NO: 124
Heavy chain variable region with the polypeptide sequence of;
(Ff) Light chain variable region having the polypeptide sequence of SEQ ID NO: 126 and SEQ ID NO: 128
Heavy chain variable region with the polypeptide sequence of;
(Gg) Light chain variable region having the polypeptide sequence of SEQ ID NO: 130 and SEQ ID NO: 132
Heavy chain variable region with the polypeptide sequence of;
(Hh) Light chain variable region having the polypeptide sequence of SEQ ID NO: 134 and SEQ ID NO: 136
Heavy chain variable region with the polypeptide sequence of;
(Ii) Light chain variable region having the polypeptide sequence of SEQ ID NO: 138 and SEQ ID NO: 140
Heavy chain variable region with the polypeptide sequence of;
(JJ) Light chain variable region having the polypeptide sequence of SEQ ID NO: 142 and SEQ ID NO: 144
Heavy chain variable region with the polypeptide sequence of;
(KK) Light chain variable region having the polypeptide sequence of SEQ ID NO: 146 and SEQ ID NO: 148
Heavy chain variable region with the polypeptide sequence of;
(Ll) Light chain variable region having the polypeptide sequence of SEQ ID NO: 150 and SEQ ID NO: 152
Heavy chain variable region with the polypeptide sequence of;
(Mm) Light chain variable region having the polypeptide sequence of SEQ ID NO: 154 and SEQ ID NO: 156
Heavy chain variable region with the polypeptide sequence of;
(Nn) Light chain variable region having the polypeptide sequence of SEQ ID NO: 158 and SEQ ID NO: 160
Heavy chain variable region with the polypeptide sequence of;
(Oo) Light chain variable region having the polypeptide sequence of SEQ ID NO: 162 and SEQ ID NO: 164
Heavy chain variable region with the polypeptide sequence of;
(Pp) Light chain variable region having the polypeptide sequence of SEQ ID NO: 166 and SEQ ID NO: 168.
Heavy chain variable region with the polypeptide sequence of;
(Qq) Light chain variable region having the polypeptide sequence of SEQ ID NO: 170 and SEQ ID NO: 172
A heavy chain variable region having the polypeptide sequence of (rr); or an antibody comprising 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, 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. At least 89%, 90%, 9 with the amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, and SEQ ID NO: 172.
1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 9
Antibodies are also provided that contain a human heavy chain variable region containing an amino acid sequence having 9.5% identity.

An isolated monoclonal antibody that binds to a human tissue factor pathway inhibitor, 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
, At least 93%, 94%, 95 with the amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, and SEQ ID NO: 170.
Antibodies are also provided that include 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 identifiers described above, certain embodiments can also be described by Fab clones isolated in the experiments described herein. In some embodiments, the recombinant antibody comprises the heavy and / or light chain CDR3 of the clones below: 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 some embodiments, the antibodies may further comprise CDR2 of these antibodies and further CDR of these antibodies.
1 may be included. In other embodiments, the antibody may further comprise any combination of CDRs.

Therefore, in other embodiments, (1) human heavy chain framework regions, human heavy chain CDRs.
1 region, human heavy chain CDR2 region, and human heavy chain CDR3 region (here, the human heavy chain CD
The R3 region is 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
(Heavy chain CDR3); and (2) Human light chain framework region, human light chain CDR1
Region, human light chain CDR2 region, and human light chain CDR3 region (where 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
An anti-TFPI antibody comprising (7, TP-4A9, TP-4B7, TP-4E8, TP-4G8, or light chain CDR3 of TP-4H8) that binds to TFPI is provided. Antibodies are also 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
-4H8 heavy chain CDR2 and / or light chain CDR2 may be included. The antibody is also 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 described herein in TP-2.
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 contain the exact amino acid sequence of the CDR1, 2 and / or 3 regions of TP-4B7, TP-4E8, TP-4G8, or TP-4H8.

However, those skilled in the art retain the ability of the antibody to bind efficiently 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
, TP-4B7, TP-4E8, TP-4G8, or some deviation from the exact CDR sequences of TP-4H8 is well understood. 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 at least 90%, 91%, 92 with one or more CDRs of TP-4H8
%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% can consist of one or more CDRs that are identical.

Antibodies are of various classes, such as IgG1, IgG2, IgG3, IgG4.
, IgM, IgA1, IgA2, secretory IgA, IgD, and IgE antibodies (but not limited to these).

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

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

Nucleic Acids An isolated nucleic acid molecule encoding any of the above monoclonal antibodies is also provided.

Method for Producing an Antibody Against TFPI A monoclonal antibody can be recombinantly produced by expressing a nucleotide sequence encoding a variable region of a monoclonal antibody according to an embodiment of the present invention in a host cell. Nucleic acids containing nucleotide sequences can be introduced and expressed in suitable host cells for production with the help of expression vectors. Therefore, it is a method for producing a monoclonal antibody that binds to human TFPI, and is described in the following step:
(A) A step of introducing a nucleic acid molecule encoding the monoclonal antibody of the present invention into a host cell;
(B) The step of culturing the host cell to express the monoclonal antibody in the host cell; (c) the step of isolating and purifying the produced monoclonal antibody, wherein the nucleic acid molecule is the present invention. A method is also provided that comprises a nucleotide sequence encoding a monoclonal antibody.

In one example, in order to express an antibody or antibody fragment thereof, DNA encoding a partial or full length light chain and heavy chain obtained by standard molecular biology techniques is inserted into the expression vector, resulting in Genes operably bind to transcriptional and translational regulatory sequences. As used herein, the term "manipulatively bound" refers to the ligation of an antibody to a vector such that the transcription and translation control sequences within the vector exert their intended function of controlling the transcription and translation of the antibody gene. It is intended to mean that it is done. The expression vector and expression control sequence are selected 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 antibody gene fragments and complementary restriction enzyme sites on the vector, or blunt-ended ligation in the absence of restriction enzyme sites). Any antibody isotype by using the light and heavy chain variable regions of the antibodies described herein by inserting them into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype. possible to prepare a full-length antibody genes, resulting, V _H fragment, C _H in the vector
Fragment and operatively linked, V _L fragment, operably linked to a C _L fragment in the compactors. Alternatively, the recombinant expression vector may encode a signal peptide that stimulates the secretion of the antibody from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide binds 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 strand, the recombinant expression vector of the invention may have a regulatory sequence that controls the expression of the antibody strand 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 transcription or translation of antibody chain genes. Such control sequences are described, for example, in Goeddel; Gene Expression Technique.
ogy. Methods in Energy 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 selection of the host cell to be transformed, the desired level of protein expression, and the like.
Examples of regulatory sequences for mammalian host cell expression include viral elements that are oriented towards high levels of protein expression in mammalian cells, such as cytomegalovirus (CMV), Simianvirus 40 (SV40), adenovirus (eg, adenovirus). Includes a major late promoter (AdMLP)) and a promoter and / or enhancer derived from polyoma. Alternatively, a non-viral control sequence, such as the ubiquitin promoter or the β-globin promoter, can be used.

In addition to antibody chain genes and control sequences, recombinant expression vectors can have additional sequences, such as sequences that control the replication of the vector in host cells (eg, replication initiation sites) and selectable marker genes. The selectable marker gene facilitates the selection of the host cell into which the vector has been introduced (eg, US Pat. No. 4,399,2 by Axel et al.).
See Nos. 16, 634,665 and 5,179,017). For example, in general, selectable marker genes are used in host cells into which the vector has been introduced.
For example, it confer 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 light and heavy chain expression, expression vectors encoding heavy and light chains are introduced into the host cell by standard techniques. The various forms of the term "introduction" are extrinsic DN
It is intended to include a wide range of techniques commonly used for the introduction of A into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran introduction and the like. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably mammalian host cells, is such eukaryotic cells, and Most preferred because certain mammalian cells are more likely to fold appropriately and build and secrete immunologically active antibodies when compared to eukaryotic cells.

Examples of mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) (Urlauban and China, (1980) Proc. Na.
tl. Acad. Sci. USA 77: 4216-4220 dhf
DHFR selectable markers containing r-CHO cells, eg, R. J. Kaufm
and P. A. Sharp (1982) Mol. Biol. 159: 6
(Used with those described in 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 the expression of the antibody in the host cell or the secretion of the antibody into the culture medium in which the host cell is growing. It is produced by culturing the host cells for a sufficient time. The antibody can be recovered from the culture medium using standard protein purification methods such as ultrafiltration, size exclusion chromatography, ion exchange chromatography and centrifugation.

Use of partial antibody sequences to express intact antibodies Antibodies interact primarily with target antigens via amino acid residues located on the 6 heavy and light chain CDRs. For this reason, the amino acid sequences within the CDRs are more diverse among the individual antibodies than the sequences outside the 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 the sequence is involved in many antibody-antigen interactions, construct an expression vector containing a CDR sequence derived from a particular naturally occurring antibody transplanted into a framework sequence derived from a different antibody with different properties. By doing so, it is possible to express a recombinant antibody that mimics the properties of a particular naturally occurring antibody (eg, Richmann, 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. S. A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences. These germline sequences are associated with mature antibody gene sequences because they do not contain fully assembled variable genes (formed by V (D) J binding 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 that has similar binding properties to the original antibody (see WO 99/45962). Partial heavy and light chain sequences across the CDR regions are generally sufficient for this purpose. The partial sequence is used to determine which germline variable and linking gene fragments are involved in the recombinant antibody variable gene. Germline sequences are then used to fill the missing portion of the variable region. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the final antibody properties.
Therefore, it is necessary to use the corresponding germline leader sequence for the expression construct. To add the missing sequence, the cloned cDNA sequence can be combined with a synthetic oligonucleotide by ligation or PCR amplification. Alternatively, the fully variable region can be synthesized as a set of short overlapping oligonucleotides and combined by PCR amplification to produce an entire synthetic variable region clone. This step has certain advantages, such as 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 overlapping sets of synthetic oligonucleotides to create synthetic V sequences that have the ability to encode the same amino acids as the native sequence. Synthetic heavy and κ chain sequences can differ from natural sequences in three ways: repeated nucleotide base strings are oligonucleotide synthesis and PC.
Blocked to promote R amplification; the optimal translation initiation site is the Kozak rule (Koza)
k, 1991, J.M. Biol. Chem. 266: 19867-19870)
And the HindIII site is modified upstream of the translation initiation site.

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

The reconstructed heavy and light chain variable regions were 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 expressing both chains.

Therefore, 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.
It is used to make structurally relevant human anti-TFPI antibodies. Among other things, the CDRs of one or more of the particularly 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 made.

Therefore, in another embodiment, the method for producing an anti-TFPI antibody is as follows: (1)
) Human heavy chain framework region and human heavy chain CDR (where human heavy chain CDR3 includes an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 388-430) and / or (2) human light chain framework. Region and human light chain CDR (where light chain CDR3 is
A method is provided comprising producing an antibody comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 259-301, wherein the antibody retains the ability to bind to TFPI. In other embodiments, the method is performed using the other CDRs of the invention.

Pharmaceutical Compositions Pharmaceutical compositions comprising therapeutically effective amounts of anti-TFPI monoclonal antibodies and pharmaceutically acceptable carriers are also provided. A "pharmaceutically acceptable carrier" is a substance that can be added to the active ingredient to aid formulation or stabilize the formulation and does not produce significant side-effect toxic effects on the patient. Examples of such carriers are known to those of skill in the art and include water, sugars such as maltose or sucrose, albumin, salts such as sodium chloride and the like. Other carriers include, for example, E. coli. W. Remington's Ph by Martin
It is described in the architectural Sciences. Such compositions
A therapeutically effective amount of at least one anti-TFPI monoclonal antibody may be included.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersants and sterile powders for the immediate preparation of sterile injectable solutions or dispersants. The medium and use of such substances for pharmaceutically active substances is known in the art. The composition is preferably formulated for non-enteral administration. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.) and a suitable mixture thereof. In some cases
It may contain isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

Sterilized injection solutions can be prepared by incorporating the active compound in a suitable solvent containing a combination of one or more of the components listed above at the required dose, followed by filter sterilization. Dispersants are generally made by incorporating the active compound into a sterile vehicle containing the essential dispersant medium and other required components listed above. In the case of sterile powders for the production of sterile injectable solutions, some of the methods of manufacture are vacuum drying and lyophilization techniques, which are the active ingredient from the sterile filtered solution described above and any further desired. Produces powder of ingredients.

Pharmaceutical Use Monoclonal antibodies can be used for therapeutic purposes to treat congenital and acquired blood coagulation deficiencies or deficiencies. For example, the monoclonal antibody in the above embodiment is TF.
It can be used to interfere with the interaction of PI and FXa or to interfere with the TFPI-dependent inhibition of TF / FVIIa activity. In addition, monoclonal antibodies are also FXa TF /
It can be used to restore FVIIa-induced production and bypass the inadequacy of 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 uncontrolled bleeding in indications such as trauma and stroke. Therefore, methods for reducing bleeding time are also provided, including administering a therapeutically effective amount of an anti-TFPI monoclonal antibody of the invention to a patient in need thereof.

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, VIII.
Co-administration of blood coagulation factors such as factors or factor IX may be 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 a factor VIII or factor IX. Provided are methods of treating a blood coagulation deficiency or deficiency, wherein both the first and second amounts are effective in treating the deficiency or deficiency. 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 a factor VIII or factor IX. Provided is a method of treating a blood coagulation deficiency or deficiency, wherein both the first and second amounts are effective in treating the deficiency or deficiency, and factor VII is not co-administered. .. The invention also comprises a pharmaceutical composition comprising a therapeutically effective amount of a combination of a monoclonal antibody of the invention and a factor VIII or factor IX, but not factor VII. "Factor VII" includes Factor VII and Factor VIIa. These combination therapies can reduce the frequency of infusions of blood coagulation factors required. Co-administration or combination therapy refers to the administration of two therapeutic agents, each formulated separately or together in one composition, and when formulated separately, approximately Administered at the same or different time points, but for the same duration of treatment.

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

The composition can be administered to patients in need of it either as a bolus dose or by continuous infusion. For example, a bolus dose of an antibody of the invention present as a Fab fragment is 0.00.
It can be 25 to 100 mg / kg body weight, 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg or 0.10 to 0.50 mg / kg. For continuous infusion, the antibodies of the invention present as Fab fragments are 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 the antibody of the present invention, which exists as a full-length antibody (having a full-length constant region), the dose 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 extended from 0 minutes to 3 hours. The frequency of administration may 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 of the anti-TFPI antibody can be administered to the patient by subcutaneous injection once a week, once every two weeks, or once a month.

As used herein, a "therapeutically effective amount" is the anti-antibody needed to effectively increase blood coagulation time in vivo or to produce measurable benefits in vivo for patients in need of it. It means the amount of a TFPI monoclonal antibody or a combination of the antibody and a factor VIII or factor IX. The exact amount depends on and is easily determined by one of ordinary skill in the art, depending on many factors including, but not limited to, the components and physical properties of the therapeutic composition, the intended patient population, individual patient considerations, etc. obtain.

Examples General materials and methods Examples 1. Panning and Screening of Human Antibody Library for Human TFPI Panning of Human Antibody Library for TFPI Phage Display Combinatrial Human Antibody Library for Human TFPI (American Tissue Factor) HuCal Gold (Rothe et et)
al. , J. Mol. Biol. , 2008, 376: 1182-1200
) Was panned to select an anti-TFPI antibody. In summary, 96-well Ma
The xisorp plate was coated with 200 μl TFPI (5 μg / ml) overnight at 4 ° C., then the plate was blocked with PBS buffer containing 5% milk. 0.
After washing the plates with PBS (PBST) containing 01% Tween-20, an aliquot of the combinatorial human antibody library was added to the TFPI-coated wells and incubated for 2 hours. The unbound phage were rinsed with PBST and the antigen-bound phage were eluted with dithiothreitol. The coli strain TG1 was infected and amplified. Phage were rescued with helper phage for the next panning. All three pannings were performed and clones from the last two were screened against human TFPI in an ELISA assay.

Screening of antibody clones by antigen binding in ELISA To select antibody clones that bind to human TFPI, the Fab gene of the phage clones from the second and third pannings was subcloned into a bacterial expression vector and E. coli.
It was expressed in the coli strain TG1. Bacterial lysate coated with human TFPI Maxiso
Added to the wells of the rp plate. After washing, HRP-conjugated goat anti-human Fab was used as a detection antibody, and the plate was expressed by adding AmplexRed (Invitrogen) containing hydrogen peroxide. Signals that were at least 5 times higher than the background were considered positive. The cross-reactivity of anti-human TFPI antibodies to mouse TFPI was determined by a similar mouse TFPI binding ELISA. Mouse TFPI (R & D S) plate
It was coated with ystem), 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 to yield 44 specific antibody sequences (Table 2). Of these antibody sequences, 29 were λ light chains and 15 were κ light chains. Our analysis of the heavy chain variable region is 28 VH3, 14 VH6, 1 VH.
1 and 1 VH5 are shown.

Table 2. Variable region peptide sequences of 44 anti-TFPI antibodies

Cross-reactivity to mouse TFPI The 44 human TFPI-binding clones described above were also tested for binding to mouse TFPI by ELISA. Cross-reactivity to mouse TFPI was found for 19 antibodies. To facilitate testing with a mouse hemophilia model, we further characterized 19 of these 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 ELISA.

Example 2. Expression and purification of anti-TFPI antibody Anti-TFPI antibody (as a Fab fragment) was expressed and purified in bacterial strain TG1. In summary, a single colony of bacterial strain TG1 containing the antibody expression plasmid was removed and 34 μm.
8 ml 2xYT in the presence of g / ml chloramphenicol and 1% glucose
It was grown overnight in medium. 7 ml volumes of culture were transferred to 250 ml 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. Culturing 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 supernatant of bacterial lysates was filtered. Ni-NT according to the manufacturer's instructions
Fab fragments were affinity purified via the A column (Qiagen).

Example 3. With _{EC 50} and binding affinities determined purification of Fab antibody anti TFPI antibodies, E to human or mouse TFPI anti TFPI antibodies
C ₅₀ was determined. In the same manner as described above, was an _{EC 50} was evaluated by ELISA. SoftMa
The results were analyzed using x. The binding affinity of the anti-TFPI antibody was determined in the Biacore assay. In summary, the 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 an amount of antigen that provided 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. Dynamics and binding affinities were calculated using the Biacore T100 Evaluation software.

As shown in Table 3, 24 of anti-TFPI Fab illustrate various _{EC 50} for human TFPI (0.09 from 792 nM) and mouse TFPI (0.06 from 1035 nM), therefore, is determined by Biacore Affinity is human TFP
It varied for I (1.25 to 1140 nM). In Fab's Biacore test against mouse TFPI, the variation in affinity was smaller (3.
08 to 51.8 nM).

Table 3. Human or mouse TFPI as determined by ELISA and Biacore
Binding activity of 24 antibodies against (hTFPI: human TFPI; mTFPI: mouse TF)
PI; Neg: signal was less than twice the background; ND: no implementation)

Example 4. Conversion of Anti-TFPI Fab to IgG All identified anti-TFPI antibodies are fully human Fab, which can be appropriately converted to human IgG as a therapeutic agent. However, in this example, the selected Fab was converted to a chimeric antibody containing a more suitable 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. The perfectly assembled IgG molecule was then introduced into HKB11 cells and expressed (Me).
i et al. , Mol. Biotechnol. , 2006, 34: 165
-178). Culture supernatants were collected and concentrated. According to the manufacturer's instructions, Hitra
Anti-TFPI via p Protein G column (GE Healthcare)
The IgG molecule was purified by affinity.

Example 5. Selection of Anti-TFPI Neutralizing Antibodies Anti-TFPI Neutralizing Antibodies were selected based on inhibition of TFPI activity under three experimental conditions. TFPI activity, ACTICHROME® TFPI activity assay (Ame)
rican Diagnostica Inc. , Stamford, CT) (ie, a three-step color assay for measuring the ability of TFPI to inhibit the catalytic activity of the TF / FVIIa complex that activates factor X to factor Xa). Anti-TFP
The neutralizing activity of the I antibody is proportional to the recovered FXa production. In the initial setting, the purified anti-TFPI antibody is subjected to human or mouse recombinant TFPI (R & D Sys) at the indicated concentrations.
Incubated with tem). After incubation, sample TF / FVII
It was mixed with a and FX, and then the residual activity of the TF / FVIIa complex was measured using SPECTROZYME® FXa, a highly specific fXa chromogenic substrate. This substrate was cleaved only by the FXa produced in the assay to release 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 endpoint fashion. 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 coagulation time in diluted prothrombin time (dPT) assays, essentially Welsch et al. , Thrombosis Res. , 199
A dPT assay was performed as described in 1, 64 (2): 213-222. In summary, human normal plasma (FACT, George King Biomedical)
, Human TFPI-deficient plasma (American Disease) or hemophilia A
Plasma (George King Biomedical) was prepared by mixing plasma with 0.1 equivalents of control buffer or anti-TFPI antibody. After incubation at 25 ° C. for 30 minutes, plasma samples (100 μl) were appropriately diluted (1: 500 diluted) with 200 μl of Thromboplastin origin Simblastin (B).
In combination with iometieux), fibrometer STA4 (Stag)
Blood coagulation time was determined using. PBS or 0.1 M sodium chloride, 0.1%
0.05 M Tris containing bovine serum albumin and 20 μM calcium chloride
Thromboplastin was diluted with a buffer based on (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 coagulation time in ROTEM assays The ROTEM system (Pentapharm GmbH) is a 4-channel device, computer, plasma standard, activator. And disposable cups and pins (cups)
and pins) was included. The thromboelastography parameters of the ROTEM hemostasis system included: Blood coagulation time (CT), which reflects the reaction time to initiate blood coagulation (required to obtain an amplitude of 2 mm after the start of data collection). Time); Thrombus formation time (CFT) and α-angle, which reflect blood coagulation amplification, and maximum amplitude and maximum elastic coefficient, which reflect the hardness of the thrombus. In the ROTEM assay, 300 μl of fresh whole citrate or plasma was evaluated. According to the manufacturer's instructions, data was collected for the period required for each system, reconstructing and mixing all components. In summary, 300 with the addition of 20 μl CaCl _{2 (200 mmol).}
Samples are mixed by collecting / distributing μl of blood or plasma in a ROTEM cup using an automatic pipette.
Immediately after that, the samples were mixed and data collection was started. Data were collected for 2 hours using a computer-controlled (software 2.96 version) ROTEM system.

Illustrative results of the ROTEM assay for detecting the effect of anti-TFPI antibodies on shortening blood coagulation time are shown in FIGS. 3 and 5. FIG. 3 shows that when TP-2A8-Fab alone or in combination with recombinant FVIII, it reduces blood coagulation time in human hemophilia A plasma or mouse hemophilia A whole blood (ROTEM system). Started with NATEM). FIG. 5 shows that anti-TFPI antibodies in IgG mode (TP-2A8, TP-3G1 and TP-3C2) shortened blood coagulation time compared to negative control mouse IgG antibodies. Based on these results and 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 coagulation time compared to the case of these antibodies alone. ..

Example 8. In vitro functional activity of anti-TFPI antibody Coloring assay ACTICH to examine TFPI antibody for TFPI dysfunction
ROME and diluted prothrombin time (dPT) were used to test the functional activity of antibodies obtained from panning and screening. In both assays, a monoclonal rat anti-TFPI antibody (R & D System) was used as a positive control and a human polyclonal Fab was used as a negative control. In the color development assay
Eight antibodies inhibited TFPI activity by more than 50% compared to rat monoclonal antibodies (Table 4). In the dPT assay, all of these 8 anti-TFPI Fabs showed high inhibitory activity (shortening blood coagulation time to less than 80 seconds), and 4 out of 8 Fabs
It showed dPT less than 70 seconds. The dose dependence of four representative clones in shortening dPT is shown in FIG. However, other human anti-TFPI Fabs that show low TFPI inhibitory activity or no TFPI inhibitory activity also shortened blood coagulation time in dPT. For example, TP-3B4 and TP-2C7 showed less than 25% inhibitory activity, but could shorten dPT to less than 70 seconds. Simple linear regression analysis of inhibitory activity and dPT shows a significant correlation (p = 0.0095) but has a large variance (coefficient of determination (R)).
square) = 0.258).

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

One of the anti-TFPI Fabs, Fab-2A8, 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 showed similar activity in human hemophilia A plasma when compared to the polyclonal rabbit anti-TFPI antibody, 220.
Blood coagulation time (CT) was reduced from 0 seconds to about 1700 seconds. Using mouse hemophilia A whole blood, 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 coagulation time in human plasma and mouse blood (p = 0.03).

Example 9. Function of anti-TFPI antibody after conversion to chimeric IgG Factor Xa production and dilution prothrombin time in vitro assay showed 24 anti-T
FPI Fab, TP-2A8, TP-2B3, TP-2G6, TP-3C2, TP-3
It is shown 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 mouse IgG1 isotypes. Ig
The G expression vector was introduced into HKB11 cells, the expressed antibody was recovered in the culture supernatant, and P.
Purified on a rotain G column. When the 2G6-Fab is a representative clones converted to IgG, 2G6-IgG is 2-fold increase in _{EC 50 s} for binding to human TFPI (0.4
10 fold increase of _{EC 50} of binding from 8 nM to 0.22 nM) and mouse TFPI (showed 0.51 nM) from 5.18 nM. The results of IgG-2G6 binding to human and mouse TFPI are shown in FIG.

Example 10. Effect on survival rate 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. Hemophilia A male mice (8 weeks old, 20-26 g) were used for these tests. By tail vein administration, anti-TFPI monoclonal antibody (40 μg / mouse) alone before injury
Alternatively, they were administered to mice with blood coagulation factors such as FVIII (0.1 IU / mouse). Twenty-four hours after administration, the left tail vein was cut 2.7 mm from the tip (in diameter). Survival was observed for 24 hours after amputation. When provided with recombinant FVIII, survival was shown to be dose-dependent (data not shown). The data shown in FIG. 8 were from separate tests (n = 15 and n = 10, respectively). The results showed 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 drug alone. It was shown to show the survival rate.

Example 11. The combination of anti-TFPI antibody and recombinant Factor VIIa further reduced the blood coagulation time and thrombosis time, and the combined effect of anti-TFPI antibody and recombinant FVIIa (Novo Nordisk) was enhanced by EXTEM (1: 1000 dilution) and mouse thrombosis. Rote using A whole blood
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 citrate mouse hemophilia A whole blood and blood coagulation was initiated by 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 reduces blood coagulation time and thrombus formation time, respectively. The combination of TP-2A8-IgG and recombinant FVIIa ("2A8 + FVIIa") further reduces blood coagulation time and thrombosis time, which means that the combination of anti-TFPI antibody and recombinant FVIIa has or has an inhibitor. Show that it is useful in the treatment of patients with hemophilia.

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

Example 13. Inhibitive anti-TFPI antibodies used Western blots and ELISAs that bind to Kunitz domain 2 of human TFPI to determine which domain of TFPI the inhibitory antibody binds to. A 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 blot, human TFPI or domain, 4-12% Bis-Tris SD
SPAGE Running Buffer MES (Invitrogen, Carlsbad
, CA) and then transferred to a cellulose membrane. After 10 minutes incubation with inhibitory antibody, SNAPid system (Millipore, Billerica)
, MA) was used to wash the membrane three times. HRP-conjugated donkey anti-mouse antibodies (Pierce, Rockford, IL) at 1 to 10,000 dilutions were incubated with membranes for 10 minutes. After a similar washing step, the SuperSignal substrate (Pierce, Rock)
Membranes were expressed using ford, IL). The control anti-Kunitz domain 1 antibody binds to the full-length TFPI, the truncated TFPI and the domain, but is an inhibitory anti-TFP.
The I antibody binds only to TFPI containing Kunitz domain 2. This is Kunit
It is shown that binding to z-domain 2 is required for the inhibitory action of the antibody.

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

Although the present invention has been described with respect to specific embodiments and examples, it is understood that various modifications and variations are possible and that equivalents can be replaced without departing from the true spirit and scope of the invention. Should be. Therefore, the specification and examples should be taken as exemplary rather than in a limiting sense. In addition, all articles, books, patent applications and patents described herein are hereby incorporated by reference.
The present invention provides the following in one aspect.
[Item 1]
A therapeutic composition comprising an isolated human monoclonal IgG antibody that specifically binds to a binding site containing Kunitz domain 1 and Kunitz domain 2 of a human tissue factor pathway inhibitor (TFPI), wherein the antibody heavy chain is sequenced. The antibody light chain is composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 16, the antibody light chain is composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 14, and the heavy chain is composed of at least 80% with SEQ ID NO: 305. CDR-1 region composed of an amino acid sequence having the homology of, CDR-2 region composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 348, and at least 80% homology with SEQ ID NO: 391. A composition comprising a CDR-3 region composed of an amino acid sequence comprising, and wherein the human monoclonal IgG exhibits 50% or more inhibition of TFPI activity.
[Item 2]
The composition of item 1, further comprising factor VIII or factor IX.
[Item 3]
The composition according to item 2, wherein Factor VII is absent.
[Item 4]
The composition of item 1, wherein the antibody has a binding affinity for human TFPI of 1.25 to 1140 nM by Biacore assay.
[Item 5]
The composition of item 1, wherein the antibody is formulated in a pharmaceutically acceptable carrier for subcutaneous injection.
[Item 6]
The composition of item 1, wherein the heavy chain CDR-2 region has at least 80% homology with SEQ ID NO: 348.
[Item 7]
The composition of item 1, wherein the heavy chain CDR-3 region has at least 80% homology with SEQ ID NO: 391.
[Item 8]
The composition according to item 1, wherein the antibody is a single-strand fragment.
[Item 9]
The antibody heavy chain is composed of the CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 305, the CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 348, and the amino acid sequence shown in SEQ ID NO: 391. The composition according to item 1, which has a CDR-3 region.
[Item 10]
The antibody heavy chain is composed of the CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 305, the CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 348, and the amino acid sequence shown in SEQ ID NO: 391. The CDR-1 region has a CDR-3 region and the antibody light chain is composed of the amino acid sequence shown in SEQ ID NO: 176, the CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 219, and the sequence. The composition according to item 1, which has a CDR-3 region composed of the amino acid sequence shown by No. 262.
[Item 11]
The composition according to item 1, wherein the antibody heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 16.
[Item 12]
The composition according to item 10, wherein the antibody light chain is composed of the amino acid sequence shown in SEQ ID NO: 14.
[Item 13]
The composition of item 1, wherein the antibody has a binding affinity for 1.25 nM human TFPI by Biacore assay.
[Item 14]
The composition of item 1, wherein the antibody is formulated in a pharmaceutically acceptable carrier for injection at a dose of 10 to 100 mg.
[Item 15]
A therapeutic composition comprising an isolated human monoclonal IgG antibody that specifically binds to a binding site containing Kunitz domain 1 and Kunitz domain 2 of a human tissue factor pathway inhibitor (TFPI), wherein the antibody heavy chain is sequenced. The antibody light chain is composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 160, the antibody light chain is composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 158, and the heavy chain is composed of at least 80% with SEQ ID NO: 341. CDR-1 region composed of an amino acid sequence having the homology of, CDR-2 region composed of an amino acid sequence having at least 80% homology with SEQ ID NO: 384, and at least 80% homology with SEQ ID NO: 427. A composition comprising a CDR-3 region composed of an amino acid sequence comprising, and wherein the human monoclonal IgG exhibits 50% or more inhibition of TFPI activity.
[Item 16]
The composition of item 15, further comprising factor VIII or factor IX.
[Item 17]
The composition according to item 16, wherein Factor VII is absent.
[Item 18]
The composition of item 15, wherein the antibody has a binding affinity for 15.8 nM human TFPI by Biacore assay.
[Item 19]
15. The composition of item 15, wherein the antibody is formulated in a pharmaceutically acceptable carrier for injection at a dose of 10 to 100 mg.
[Item 20]
The composition of item 15, wherein the heavy chain CDR-1 region has at least 85% homology with SEQ ID NO: 341.
[Item 21]
The composition of item 15, wherein the heavy chain CDR-2 region has at least 85% homology with SEQ ID NO: 384.
[Item 22]
The composition of item 15, wherein the heavy chain CDR-3 region has at least 85% homology with SEQ ID NO: 427.
[Item 23]
The composition according to item 15, wherein the antibody is a single-strand fragment.
[Item 24]
The antibody heavy chain is composed of the CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 341, the CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 384, and the amino acid sequence shown in SEQ ID NO: 427. The composition according to item 15, which has a CDR-3 region.
[Item 25]
The composition according to item 15, wherein the antibody heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 160.
[Item 26]
The antibody heavy chain is composed of the CDR-1 region composed of the amino acid sequence shown in SEQ ID NO: 341, the CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 384, and the amino acid sequence shown in SEQ ID NO: 427. The CDR-1 region has a CDR-3 region and the antibody light chain is composed of the amino acid sequence shown in SEQ ID NO: 212, the CDR-2 region composed of the amino acid sequence shown in SEQ ID NO: 255, and the sequence. The composition according to item 15, which has a CDR-3 region composed of the amino acid sequence shown in No. 298.
[Item 27]
The composition according to item 15, wherein the antibody heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 160, and the antibody light chain is composed of the amino acid sequence shown in SEQ ID NO: 158.

Claims (18)

A therapeutic composition comprising an isolated human monoclonal IgG antibody that specifically binds to a binding site containing Kunitz domain 1 and Kunitz domain 2 of a human tissue factor pathway inhibitor (TFPI), wherein the antibody heavy chain is sequenced. The amino acid sequence is composed of an amino acid sequence having at least 89 % identity with No. 16, the antibody light chain is composed of an amino acid sequence having at least 93 % identity with SEQ ID NO: 14, and the heavy chain is the amino acid shown in SEQ ID NO: 305. CDR-1 region consists of sequences have CDR-3 region composed of the amino acid sequence shown in constructed CDR-2 region and SEQ ID NO: 391, the amino acid sequence shown in SEQ ID NO: 348, the light A CDR-1 region in which the chain is composed of the amino acid sequence shown in SEQ ID NO: 176, a CDR-2 region in which the chain is composed of the amino acid sequence shown in SEQ ID NO: 219, and a CDR composed of the amino acid sequence shown in SEQ ID NO: 262. A composition having a -3 region and the human monoclonal IgG exhibiting 50% or more inhibition of TFPI activity. The composition of claim 1, further comprising factor VIII or factor IX. The composition according to claim 2, wherein the factor VII is absent. The composition of claim 1, wherein the antibody has a binding affinity for human TFPI of 1.25 to 1140 nM by surface plasmon resonance analysis. The composition of claim 1, wherein the antibody is formulated in a pharmaceutically acceptable carrier for subcutaneous injection. The composition according to claim 1, wherein the antibody is a single-strand fragment. The composition according to claim 1, wherein the antibody heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 16. The composition according to claim 1 , wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 14. The composition according to claim 1, wherein the antibody has a binding affinity for 1.25 nM human TFPI by surface plasmon resonance analysis. The composition of claim 1, wherein the antibody is formulated in a pharmaceutically acceptable carrier for injection at a dose of 10 to 100 mg. A therapeutic composition comprising an isolated human monoclonal IgG antibody that specifically binds to a binding site containing Kunitz domain 1 and Kunitz domain 2 of a human tissue factor pathway inhibitor (TFPI), wherein the antibody heavy chain is sequenced. The amino acid sequence is composed of an amino acid sequence having at least 89 % identity with No. 160, the antibody light chain is composed of an amino acid sequence having at least 93 % identity with SEQ ID NO: 158, and the heavy chain is the amino acid shown in SEQ ID NO: 341. CDR-1 region consists of sequences have CDR-3 region composed of the amino acid sequence shown in constructed CDR-2 region and SEQ ID NO: 427, the amino acid sequence shown in SEQ ID NO: 384, the light The CDR-1 region in which the chain is composed of the amino acid sequence shown in SEQ ID NO: 212, the CDR-2 region in which the chain is composed of the amino acid sequence shown in SEQ ID NO: 255, and the CDR composed of the amino acid sequence shown in SEQ ID NO: 298. A composition having a -3 region and the human monoclonal IgG exhibiting 50% or more inhibition of TFPI activity. The composition of claim 11 , further comprising factor VIII or factor IX. The composition according to claim 12 , wherein the factor VII is absent. The composition according to claim 11 , wherein the antibody has a binding affinity for human TFPI of 15.8 nM by surface plasmon resonance analysis. 11. The composition of claim 11, wherein the antibody is formulated in a pharmaceutically acceptable carrier for injection at a dose of 10 to 100 mg. The composition according to claim 11 , wherein the antibody is a single-strand fragment. The composition according to claim 11 , wherein the antibody heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 160. The composition according to claim 11 , wherein the antibody heavy chain is composed of the amino acid sequence shown in SEQ ID NO: 160, and the antibody light chain is composed of the amino acid sequence shown in SEQ ID NO: 158.

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