ES2727706T3 - Antibodies that recognize the n-terminal part of the tissue factor pathway inhibitor capable of causing procoagulant activity - Google Patents

Abstract

An isolated antibody or fragment thereof that specifically binds to an epitope present in amino acid residues 1 to 79 of human TFPI (SEQ ID NO: 1), whose heavy chain comprises: - a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 6 (NYGVH); - a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 6 (VIWRGGSIDYNAAFMS); and - a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 6 (NSHGNYVGYAMDY); and whose light chain comprises: - a sequence of CDR1 corresponding to amino acids 24 to 34 of SEQ ID NO: 7 (KASQSVGPAVA); - a CDR2 sequence corresponding to amino acids 50 to 56 of SEQ ID NO: 7 (SASNRYT); and - a CDR3 sequence corresponding to amino acids 89 to 96 of SEQ ID NO: 7 (QQYTSYPT).

Description

DESCRIPTION

Antibodies that recognize the n-terminal part of the tissue factor pathway inhibitor capable of causing procoagulant activity

Field of the Invention

Procoagulant antibodies, and compositions thereof, which are capable of specifically binding to an epitope in the n-terminal part of the tissue factor pathway inhibitor (tfpi) are described herein. In addition, pharmaceutical and therapeutic uses of such antibodies are described.

Background of the invention

In the bleeding individual, coagulation is initiated by the activated Tissue Factor (TF) / Factor VII (FVIIa) complex when extravascular TF is exposed to FVIIa in the blood. The formation of TF / FVIIa complexes leads to the activation of Factor X (FX) to FXa which, together with activated Factor V (FVa), generates a limited amount of thrombin. The initial amount of thrombin activates platelets which, in turn, results in the surface exposure of platelet phospholipids that support the assembly and binding of the tenase complex, which consists of activated Factor VIII (FVIIIa) and Factor IX activated (FIXa). The tenase complex is a very efficient catalyst for the activation of FX and the FXa generated at this stage serves as the active protease in the FVa / FXa prothrombinase complex that is responsible for the final thrombin burst. Thrombin cleaves fibrinogen to generate fibrin monomers, which polymerize to form a fibrin network. The rapid and extensive burst of thrombin is a prerequisite for the formation of a solid and stable fibrin clot.

An inadequate spread of FXa and thrombin generation caused by FVIII or FIX deficiency is the underlying reason for hemorrhagic diathesis in patients with hemophilia A or B, respectively. In people with hemophilia, the generation of FXa is mainly driven by the TF / FVIIa complex: FVIII or FIX deficiency leads to the rudimentary generation of FXa through the tenase complex. The activation of FX to FXa mediated by TF / FVIIa is, however, temporary because the tissue factor pathway inhibitor (TFPI) inhibits the TF / FVIIa complex in a self-regulating loop. Inhibition of feedback leads to the formation of the TF / FVIIa / FXa / TFPI complex. Decreased inhibition mediated by TFPI prolongs activation of FX mediated by TF / FVIIa during the onset of coagulation and promotes hemostasis. Patients with hemophilia suffer from an altered activity of tenase due to, for example, deficiency of FVIII or FIX. Blocking the inhibition mediated by TFPI in these patients compensates for an inadequate generation of FXa and normalizes hemorrhagic diathesis.

TFPI is a competitive inhibitor of narrow, slow binding that regulates the activation of FX through inhibition of FXa and the TF / FVIIa complex. The TFPI contains three domains Kunitz type Protease Inhibitors arranged in tandem (KPI 1-3). The inhibition of the TFa-mediated FXa occurs in a biphasic reaction that initially leads to a relaxed TFPI-FXa complex, which is then slowly rearranged into a narrow-binding TFPI-FXa complex, where the k PI-2 binds and blocks the FXa active site. After the onset of coagulation, the FXa generation mediated by TF / FVIIa is negatively regulated, very accurately, by the TFPI. The TF / FVIIa complex is inhibited by TFPI in a process that, as a speed-limiting stage, seems to involve the inhibition of FXa mediated by the TFPI, either when FXa joins the TF / FVIIa complex or when it binds in the membrane cell near the TF / FVIIa complex (Baugh et al., J Biol Chem. 1998; 273 (8): 4378-86). KPI-1 contributes to the formation of the TFPI-FXa complex and binds directly and also blocks the active site of FVIIa linked to TF (Girard) et al., Nature 1989; 338: 518-520; Peraramelli et al., Thromb. Haemost 2012; 108: 266-276).

In vivo, TFPI is found in various cell compartments. A major fraction of TFPI is associated with the vascular endothelium and a smaller fraction circulates in the blood. Two variants of the TFPI splice, TFPI alpha (TFPI alpha) a) and TFPI beta (TFPI beta) P), have been described as being present in humans. Endothelial cells are the main sites for the production of TFPI and express both variants. The predominant form on the surface of endothelial cells is presumably TFPIp. TFPIa is secreted in plasma or found in intracellular deposits, which can be released before certain stimuli. Secreted TFPI circulates in the blood as a full-length protein (10%) or as modified proteins with different molecular masses (90%); the latter may be due, for example, to the truncation of the C-terminal region or to the association with lipoproteins. The secreted TFPI can also bind to the surface of the endothelial cells through interactions with, for example, glycoaminolicans. In addition, TFPIa is produced and stored in platelets.

The half-life of TFPI in humans is known to be approximately 60-120 minutes, and it is known that the total normal concentration of TFPI in human plasma is approximately 1.0-2.5 nM. On the contrary, it is known that the half-life of antibodies in humans is long: up to several weeks, depending on the subtype, origin and specific amino acid composition of immunoglobulins. The total concentration of TFPI (free TFPI plus TFPI / antibody complexes) in plasma may increase to concentrations as high as 20-40 nM (Augustsson et al., J Thromb Haemost. 2013, 11: s2, PA 4.14-2) when certain known TFPI antibodies, such as the AcM 0001 antibody described in Augustsson et al. higher up a complex with TFPI in circulation. In vivo the administration of TFPI antibodies may, therefore, cause TFPI / antibody complexes to accumulate at concentrations that are much higher than the plasma concentration prior to the dose of TFPI. In cases where the inhibitory activity of TFPI / antibody complexes is not completely neutralized, the accumulation can reverse the effect of a TFPI antibody in any other procoagulant manner, resulting in a net anticoagulant effect.

EP0539975 A1 describes a method for immunologically evaluating free TFPI in a human sample; that is, an ELISA. The method makes use of a first antibody immobilized in an insoluble carrier and a second labeled antibody, wherein one of the antibodies binds to a peptide within the K1 domain of the TFPI and the other binds to a peptide within the K3 domain of the TFPI .

It is conceived that the antibodies described in the present description, and the pharmaceutical compositions comprising them, address the limitations mentioned above.

Brief Description of the Invention

The scope of the present invention is defined by the claims and any information that does not fall within the claims is provided for information purposes only. In the present description an antibody that specifically binds to an epitope in the N-terminal part of the tissue factor pathway inhibitor is described.

In one aspect this antibody binds to a TFPI epitope comprising at least one of the following N-terminal amino acid residues Leu 16, Pro 17, Leu 19, Lys 20, Leu 21, Met 22, Phe 25, Cys 35, Ala 37, Met 39, Arg 41, Tyr 56, Gly 57, Gly 58, Cys 59, Glu 60, Gly 61, Asn 62, Gln 63, Arg 65, Phe 66, Glu 67, Glu 71 and Met 75 of the SEQ ID NO: 1.

In one aspect, the TFPI antibody epitope comprises the following N-terminal amino acid residues Arg 41, Arg 65 and Glu 67 of SEQ ID NO: 1.

In one aspect, the antibody neutralizes the inhibition of the TF / FVIIa complex mediated by TFPI, for example, which competes with TF / FVIIa to bind to TFPI-KPI-1. A TFPI binding antibody (1-79) that binds to, protects or modifies the conformation of the TF / FVIIa binding surface, or parts thereof, in the TFPI (s Eq ID NO: 1) and of this way functionally blocks the interaction of TFPI-TF / FVIIa, it can neutralize the inhibition of the TF / FVIIa complex mediated by TFPI.

In another aspect, the antibody neutralizes the stimulating effect of KPI-1 TFPI in reactions that lead to the formation of narrow complexes between TFPI KPI-2 and FXa.

In another aspect the TFPI antibody (1-79) binds and neutralizes all forms of TFPI.

In one aspect, the antibody neutralizes TFPI, even in circumstances where the total levels of TFPI in the circulation are high, and also normalizes thrombin generation under these conditions.

In the present description, in addition, a pharmaceutical formulation comprising at least one antibody capable of specifically binding to an epitope in TFPI (1-79) and at least one pharmaceutically acceptable excipient is described.

The antibody described in the present description, or a pharmaceutical formulation comprising it, can be used as a medicament - such as in the treatment of a subject with coagulopathy.

Brief description of the drawings

Figure 1 shows the effect of several TFPI antibodies (1-79) on thrombin generation (TGT assay), as measured by the use of a normal human plasma set (NHP) under conditions similar to hemophilia A which were obtained by the addition of 100 µg / mL of FVIII antibody.

Figure 2 shows the effect of several TFPI antibodies (1-79) on thrombin generation (TGT assay), as measured by the use of an NHP set under conditions of elevated TFPIa levels that were obtained by adding of full length recombinant TFPIa, 5 Nm, to NHP. Conditions similar to hemophilia A were obtained by adding 100 µg / mL of the FVIII antibody.

Brief description of the sequences

SEQ ID NO: 1 represents the amino acid sequence of human TFPIa.

SEQ ID NO: 2 represents the amino acid sequence of TFPI (1-79).

SEQ ID NO: 3 represents the amino acid sequence of TFPI (1-161).

SEQ ID NO: 4 represents the amino acid sequence of the heavy chain variable monoclonal antibody (AcM) 2F3.

SEQ ID NO: 5 represents the amino acid sequence of the variable light chain of the monoclonal antibody (AcM) 2F3.

SEQ ID NO: 6 represents the amino acid sequence of the heavy chain variable monoclonal antibody (AcM) 2F22.

SEQ ID NO: 7 represents the amino acid sequence of the variable light chain of monoclonal antibody (AcM) 2F22.

SEQ ID NO: 8 represents the amino acid sequence of the variable heavy chain of the monoclonal antibody (AcM) 2F45.

SEQ ID NO: 9 represents the amino acid sequence of the variable light chain of the monoclonal antibody (AcM) 2F45.

SEQ ID NO: 10 represents the amino acid sequence of the variable heavy chain of the monoclonal antibody (AcM) 1F91

SEQ ID NO: 11 represents the amino acid sequence of the variable light chain of the monoclonal antibody (AcM) 1F91.

SEQ ID NO: 12 represents the amino acid sequence of the variable heavy chain of the monoclonal antibody (AcM) 2F35.

SEQ ID NO: 13 represents the amino acid sequence of the variable light chain of the monoclonal antibody (AcM) 2F35.

SEQ ID NO: 14 represents the primer nucleic acid sequence used for cloning the antibody heavy chains.

SEQ ID NO: 15 represents the nucleic acid sequence of the primer used for cloning the antibody light chains.

SEQ ID NO: 16 represents the amino acid sequence of the truncated murine human chimeric heavy chain of Fab 0296.

SEQ ID NO: 17 represents the amino acid sequence of the human murine chimeric light chain of the monoclonal antibody AcM 0294 and Fab 0296.

SEQ ID NO: 18 represents the amino acid sequence of KPI-1 / N-terminal TFPI labeled.

SEQ ID NO: 19 represents the amino acid sequence of the human murine chimeric heavy chain of the monoclonal antibody AcM 0294.

SEQ ID NO: 20 represents the amino acid sequence of the murine human chimeric truncated heavy chain of Fab 0295.

Description

Mature human TFPIa is a 276 amino acid protein (SEQ ID NO: 1) consisting of an acid N-terminal region, three tandem Kunitz protease inhibitor domains (KPI-1, KPI-2 and KPI-3) interspersed by linker regions and a basic C terminal region. KPI-1, KPI-2 and KPI-3 are defined as residues 26-76, residues 97-147 and residues 189-239, respectively, of SEQ ID NO: 1. TFPIp is a 193 protein. Glycosylphosphatidylinositol (GPI) anchored amino acids of which the first 181 amino acids are identical to TFPIa (corresponding to residues 1-181 of SEQ ID NO: 1) but where the 12-terminal C-terminal amino acid sequence is not related to TFPIa and has a GPI junction added to residue 193.

In the present description an antibody is described that is capable of binding to a TFPI region, which represents the acid N-terminal region and the KPI-1 domain of TFPI, or any portion thereof.

The term "TFPI", as used herein, encompasses any form of naturally occurring TFPI that may be derived from any suitable organism. For example, the TFPI for use as described herein may be mammalian TFPI, such as human, mouse, rabbit, rat, primate, bovine, sheep, or pig TFPI. Preferably, the TFPI is human TFPI. TFPI can be a mature form of TFPI such as a TFPI protein that underwent post-translational processing within a suitable cell. Such mature TFPI protein may, for example, be glycosylated. TFPI can be a full length TFPI protein. The term TFPI also encompasses variants, isoforms and other homologues of such TFPI molecules. Variants of TFPI molecules can be characterized by having the same type of activity as naturally occurring TFPI, such as the ability to neutralize the catalytic activity of FXa, or the ability to inhibit a TF / FVIIa complex or TF ternary complex / FVIIa / FXa.

The term "antibody" in the present description refers to a protein, derived from a germline immunoglobulin sequence, which is capable of specifically binding to an antigen or a portion thereof: specifically TFPI (1-79). The term "antibody" includes full-length antibodies of any class (or isotype), that is, IgA, IgD, IgE, IgG, IgM and / or IgY. An antibody that specifically binds to an antigen, or a portion thereof, can bind exclusively to that antigen, or a portion thereof, or can bind to a limited number of homologous antigens, or portions thereof.

Full-length antibodies of natural origin generally comprise at least four polypeptide chains: two heavy chains (H) and two light chains (L) that are connected by disulfide bonds. In some cases, naturally occurring antibodies consist of less than four chains, as in the case of antibodies with only one heavy chain found in camelids (V ^h H fragments) and IgNARs found in Chondrichthyes. One class of immunoglobulins of particular pharmaceutical interest is the IgG class. In humans, the IgG class can be divided into four subclasses: IgG1, IgG2, IgG3 and IgG4, based on the sequence of the constant regions of its heavy chain. Light chains can be divided into two types: kappa and lambda chains, based on differences in their sequence composition. IgG molecules consist of two heavy chains, interconnected by two or more disulfide bonds, and two light chains, each attached to a heavy chain by a disulfide bond. An IgG heavy chain may comprise a heavy chain variable region (VH) and up to three heavy chain constant regions (CH): CH1, CH2 and CH3. A light chain may comprise a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR) or hypervariable regions (HVR), interspersed with regions that are more conserved, called framework regions (FR). The VH and VL regions are typically composed of three CDRs and four FRs, arranged from the amino terminal end to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable domains with the hypervariable regions of the heavy and light chains form a domain that is capable of interacting with an antigen, while the constant region of an antibody can mediate the binding of immunoglobulin to tissues or host factors, which include , but are not limited to various cells of the immune system (effector cells), Fc receptors and the first component (C1q) of the C1 complex of the classical complement system.

The antibodies described in the present description can be isolated. The term "isolated antibody" refers to an antibody that was separated and / or recovered from (one) other component (s) in the environment in which it was produced and / or that was purified from a mixture of components present in the environment in which it occurred.

The antibodies described in the present description may be monoclonal antibodies, in that they represent a set of unique heavy and light chain variable domain sequences expressed from a single B cell or by a clonal population of B cells. The antibodies described in the present description can be produced and purified by the use of various methods that are known to those skilled in the art. For example, antibodies can be produced from hybridoma cells. The antibodies can be produced by the expansion of B cells. The antibodies or fragments thereof can be expressed recombinantly in microbial or mammalian expression systems, or by in vitro translation . The antibodies or fragments thereof can be expressed recombinantly, in addition, as molecules bound to the cell surface, by means of, for example, a phage display, an exposure in bacteria, an exposure in yeasts, an exposure in mammalian cells , an exposure in ribosome or mRNA. Once produced, antibodies can be analyzed for their ability to bind TFPI, which includes subdomains such as TFPI (1-79) (SEQ ID NO: 2).

Various antigen binding fragments of the antibodies are further described in the present description, since it has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full length antibody. The term "antigen binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind or recognize an antigen, such as human TFPI (1-79) or other target molecule, as described in the present description. Examples of antigen binding fragments include Fab, Fab ', Fab ² , Fab' ² , FAbS, Fv (typically, the VL and Vh domains of a single arm of an antibody), single chain Fv (scFv; see for example , Bird et al., Science 1988; 242: 423-426; and Huston and other PNAS 1988; 85: 5879-5883), dsFv, Fd (typically, VH and CHI domains) and dAb fragments (typically a VH domain ); the VH, VL, VHh, and V-NAR domains; monovalent molecules comprising a single VH chain and a single VL chain; mini-bodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, for example, Ill et al. Protein Eng 1997; 10: 949-57); Camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed, for example, Holliger and Hudson, Nat Biotechnol 2005; 23: 1126-1136; WO2005040219, and US Pat. 20050238646 and 20020161201. These antibody fragments can be obtained by using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies.

The "Fab Fragments" of an antibody, which includes "Fab", "Fab '", and "Fab' ² ", are derived from said antibody by cleavage of the heavy chain in the hinge region at the N-terminal end or the C-terminal side of the cysteine residues of the hinge that connect the heavy chains of the antibody. A "Fab" fragment includes the variable and constant domains of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. The "Fab ' ² " fragments comprise a pair of "Fab" fragments that are generally covalently joined by their hinge cysteines. A Fab 'is formally derived from a Fab' ² fragment by cleavage of the disulfide bonds of the hinge that connect the heavy chains in the Fab ' ² . In addition, other chemical couplings of antibody fragments other than disulfide bonds are known in the art. A Fab fragment retains the ability of the parental antibody to bind to its antigen, potentially with a lower affinity. The Fab ' ² fragments are capable of divalent junctions, while Fab and Fab' fragments can be monovalently bound. Generally, Fab fragments lack the constant domains CH2 and CH3, that is the Fc part, where interaction with Fc receptors would occur. Therefore, Fab fragments generally lack effector functions. Fab fragments can be produced by methods known in the art, either by enzymatic cleavage of an antibody, for example, by using papain to obtain Fab or pepsin to obtain Fab ' ² , Fab fragments including Fab, Fab ', Fab' ² can also be produced recombinantly by using techniques well known to those skilled in the art.

An "Fv" fragment is an antibody fragment that contains a complete recognition and antigen binding site and generally comprises a dimer of a heavy variable chain domain and a light variable chain domain in an association that can be covalent in nature. ; for example, in a fragment of the single variable chain domain (scFv). It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions or a subset of these confer the antigen binding specificity for the antibody. However, even a single variable domain comprising only three hypervariable regions specific for an antigen can retain the ability to recognize and bind to the antigen, although usually at a lower affinity than the complete binding site (Cai & Garen, Proc. Natl Acad. Sci. United States, 93: 6280-6285, 1996). For example, naturally occurring antibodies to camelids that only have a heavy chain variable domain (VHH) can bind to the antigen (Desmyter et al., J. Biol. Chem., 277: 23645-23650, 2002; Bond et al., J. Mol. Biol. 2003; 332: 643-655).

The "single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the convenient structure for antigen binding. For a review of scFv, see Plückthun, 1994, in: The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore Ed. Springer-Verlag, New York, pp. 269-315.

The term "diabody" refers to small antibody fragments with two antigen binding sites, in which the fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same chain polypeptide (VH and VL). By using a linker that is too short to allow pairing between the two variable domains in the same chain, the variable domains are forced to mate with complementary domains of another chain, which creates two antigen binding sites. Diabodies are more fully described, for example, in EP 404,097; WO 93/11161; and Hollinger et al., 1993, Proc. Natl Acad. Sci. USA, 90: 6444-6448.

The term "linear antibodies" refers to antibodies as described in Zapata et al., 1995, Protein Eng., 8 (10): 1057-1062. Briefly, these antibodies contain a pair of tandem segments (VH-CH1-VH-CH1) which, together with the complementary light chain polypeptides, form a pair of antigen binding regions.

The term "monobody" as used herein, refers to an antigen-binding molecule with a heavy chain variable domain and no light chain variable domain. A monobody can bind to an antigen in the absence of light chains and typically has three hypervariable regions, for example the CDRs designated CDRH1, CDRH2, and CDRH3. An IgG heavy chain monobody has two heavy chain antigen binding molecules connected by a disulfide bond. The heavy chain variable domain comprises one or more hypervariable regions, preferably, a CDRH3 or HVL-H3 region.

The term "antigen" may refer, in the present description, to the entity used for the immunization of an immunocompetent vertebrate to produce the antibody, or fragment thereof, that recognizes it. Suitable antigens for immunization include full-length human TFPIa, human TFPI (1-79) and human TFPI (1-161). The term "antigen" is intended, in any other way, to include target molecules that are specifically recognized by the antibody, therefore that includes fragments or mimetics of the molecule used in the immunization process, or used for other isolation methods. generation and characterization of antibodies, for example phage exposure, ELISA or SPR.

The antibody described in the present description may compete with another molecule, such as a naturally occurring ligand or receptor or another antibody, to bind to TFPI and thus affect the functions associated with these interactions. The ability of an antibody to compete with a natural ligand / receptor can be assessed by various activity assays that measure the effect on apparent K ⁱ for TFPI-mediated inhibition. The values K ^d can then be deduced from the apparent values of K ⁱ .

Antibody fragments can be obtained, therefore by the use of conventional recombinant or protein engineering techniques and the fragments can be analyzed for binding to TFPI or sub-domains thereof, such as human TFPI (1-79) in the same manner. than the antibodies intact.

Antibody fragments can be made by truncation, for example, by removing one or more amino acids from the N and / or C terminal ends of a polypeptide. The fragments can also be generated by one or more internal deletions.

The antibodies can be human or humanized antibodies. The term "human antibody," as used herein, is intended to include antibodies that have variable regions in which at least a portion of a framework region and / or at least a portion of a CDR region are derived from sequences. of immunoglobulins of the human germ line. (For example, a human antibody may have variable regions in which both the framework and the CDR regions are derived from human germline immunoglobulin sequences). In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Such human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences ( eg, in vitro mutations , introduced by random or site-specific mutagenesis, or by in vivo somatic mutation ).

Such a human antibody can be a human monoclonal antibody. Such a human monoclonal antibody can be produced by a hybridoma that includes a B cell that is obtained from a transgenic non-human animal, for example, a transgenic mouse, which has a genome comprising the repertoires of the heavy chain gene segments and light human immunoglobulin, fused to an immortalized cell.

Human antibodies can be isolated from sequence libraries incorporated into human germ line sequence selections, further diversified with a variety of natural and synthetic sequences.

Human antibodies can be prepared by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with the Epstein-Barr virus.

The term "human antibody derivative" refers to any modified form of the human antibody, such as an antibody conjugate and another agent or antibody.

The term "humanized antibody", as used herein, refers to a human / non-human chimeric antibody that contains sequences (CDR regions or portions thereof) derived from a non-human immunoglobulin. A humanized antibody is, therefore, a human immunoglobulin (receptor antibody) in which at least the residues of a hypervariable region of the receptor are replaced by the residues of a hypervariable region of an antibody of a non-human species (donor antibody) such as a mouse, rat, rabbit or non-human primate, which has the specificity, affinity, sequence composition and functionality convenient. In some cases, the FR residues of the human immunoglobulin are replaced by the corresponding non-human residues. An example of such a modification is the introduction of one or more of the so-called reverse mutations, which are typically amino acid residues derived from the donor antibody. The humanization of an antibody can be accomplished by the use of recombinant techniques known to those skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. C. Lo). A suitable human receptor framework for both light and heavy chain variable domains can be identified by, for example, the structural sequence or homology. Alternatively, fixed receptor frames may be used, for example, based on knowledge of the structure, biophysical and biochemical properties. Receptor frames can be derived from the germ line or derived from a sequence of mature antibodies. The CDR regions of the donor antibody can be transferred by CDR grafting. The humanized antibody grafted with CDR can be optimized, subsequently, for example, by affinity, functionality and biophysical properties by identifying critical frame positions where the new introduction (reversal mutation) of the amino acid residue of the donor antibody has a beneficial impact on the properties of the humanized antibody. In addition to the reversal mutations derived from donor antibodies, the humanized antibody can be genetically modified by introducing germ line residues in the CDR or framework regions, elimination of immunogenic epitopes, site-directed mutagenesis, affinity maturation, and so on.

In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine the antibody performance. In general, a humanized antibody will comprise at least one - typically two - variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and in which all or substantially all of the FR residues are those of a sequence of human immunoglobulin. The humanized antibody may, optionally, further comprise at least a portion of a constant region of immunoglobulin (Fc), typically, that of a human immunoglobulin.

The term "humanized antibody derivative" refers to any modified form of the humanized antibody, such as an antibody conjugate and another agent or antibody.

The term "chimeric antibody", as used herein, refers to an antibody whose heavy and light chain genes have been constructed, typically, by genetic engineering, from region genes. constant and variable immunoglobulins that originate from different species. For example, the variable gene segments of a mouse monoclonal antibody can bind to human constant regions.

The crystallizable region of the fragment ("Fc Domain Fc region") of an antibody is the C-terminal region of an antibody, which comprises the constant domains CH2 and CH3. The Fc domain can interact with cell surface receptors called Fc receptors, as well as with some proteins of the complement system. The Fc region allows antibodies to interact with the immune system. In one aspect, antibodies can be genetically modified to include modifications within the Fc region, typically to alter one or more of its functional properties, such as serum half-life, complement fixation, Fc receptor binding, stability. of proteins and / or antigen-dependent cell cytotoxicity, or lack thereof, among others. In addition, the antibody can be chemically modified (for example, one or more chemical portions can bind to the antibody) or modified to alter its glycosylation, again to alter one or more functional properties of the antibody. An IgG1 antibody can carry a modified Fc domain comprising one or more, and perhaps all of the following mutations that will result in a lower affinity for certain Fc receptors (L234A, L235E and G237A) and a decrease in C1q-mediated complement fixation. (A330S and P331S), respectively (numbering of waste according to the EU index).

The isotype of the antibody described in the present description may be IgG, such as IgG1, such as IgG2, such as IgG4. If desired, the class of an antibody can be "changed" by known techniques. For example, an antibody that was originally produced as an IgM molecule can be changed class to an IgG antibody. Class change techniques can also be used to convert a subclass of IgG to another, for example: from IgG1 to IgG2 or IgG4; from IgG2 to IgG1 or IgG4; or from IgG4 to IgG1 or IgG2. In addition, the genetic modification of the antibodies can be made to generate chimeric molecules of constant region, by combining regions of different IgG subclasses.

In one aspect, the hinge region of CH1 is modified so that the number of cysteine residues in the hinge region is altered, for example, increases or decreases. This approach is further described, for example, in U.S. Patent No. 5,677,425 to Bodmer et al.

The constant region can be modified to stabilize the antibody, for example, to decrease the risk of a bivalent antibody separating into two monovalent VH-Vl fragments. For example, in a lgG4 constant region, residue S228 (numbering of residues according to the EU index) can be mutated to a proline residue (P) to stabilize the disulfide bridge formation between heavy chains in the hinge ( see, for example, Angal et al., Mol. Immunol. 1993; 30: 105-8).

Antibodies or fragments thereof can be defined in terms of their complementarity determining regions (CDR). The term "complementarity determining region" or "hypervariable region", when used in the present description, refers to regions of an antibody in which the amino acid residues involved in antigen binding are located. The hypervariability or CDR region can be identified as the regions with the greatest variability in amino acid alignments of antibody variable domains. The databases can be used for the identification of CDRs such as the Kabat database, the CDRs are defined as comprising amino acid residues 24-34 (L1), 50-56 (L2) and 89-97 (L3 ) of the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Alternatively, the CDRs can be defined as the residues of a "hypervariable loop" (residues 26-33 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1 ), 53 55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987; 196: 901-917). Typically, the numbering of amino acid residues in this region is done by the method described in Kabat and others, above. Phrases such as "Kabat position", "Kabat residue", and "according to Kabat" in the present description refer to this numbering system for heavy chain variable domains or light chain variable domains. By using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain few or additional amino acids that correspond to a shortening of, or insertion into, a frame (FR) or CDR of the variable domain. For example, a heavy chain variable domain may include amino acid insertions (residue 52a, 52b and 52c according to Kabat) after residue 52 of c Dr H2 and inserted residues ( e.g. residues 82a, 82b, and 82c, etc.) according to Kabat) after residue 82 of heavy chain FR. The Kabat numbering of the residues can be determined for a given antibody by alignment in regions of homology of the antibody sequence with a "standard" sequence numbered according to Kabat.

The original CDRs of an antibody can be genetically modified to improve, for example, its affinity, functionality and biophysical properties. The CDRs of the antibodies can be genetically modified by the introduction or addition of germline residues or residues that are not from the germline in any position. Modification of the amino acid composition in the C Dr may result in the elimination of predicted immunogenic epitopes, optimization of biophysical properties, such as viscosity, aggregation, elimination of hot spots of modification such as deamidation, formation of iso-aspartic acid or sites. of methionine oxidation. The removal of cysteine residues in the CDRs is also convenient, since this prevents the formation of an aberrant disulfide bond or reorganization. Optimization of the biochemical profile of an antibody can also be obtained, for example, by eliminating potential glycosylation sites. The optimization of the CDR regions can also result in an improved production profile. Modifications of the CDR may also result in the optimization of the antibody-antigen interface, by optimization or redesign of the paratope. Such optimization may include the introduction of amino acids where the chemical composition of the side chain allows novel interactions with the antigen. Optimization can also result in the elimination of steric obstructions in the interaction of epitopes and paratopes or in the optimization of the conformation, flexibility or stiffness of the CDR loop.

Such mutations can be introduced into the CDR regions by design, through the use, for example, of site-directed mutagenesis, or randomly by, for example, error-prone PCR or synthetically diversified DNA or PCR primers. The modification can also be introduced by rearranging CDR sequences from other antibodies that specifically bind to the same antigen.

The terms "framework region" or "FR" residues refer to amino acid residues VH or VL that are not within the CDRs, as defined in the present description.

The antibody described in the present description may contain a CDR region of one or more of the specific antibodies described herein, as a CDR region within SEQ ID NO: 4 to 13, as defined using Kabat or Chotia numbering or according to the sequential numbering of amino acids described in the present description.

The antibody may have a heavy chain comprising:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 4 (SYGVH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 4 (VIWRGGSTDFNAAFMS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; and / or • a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 4 (NSHGNYVGYAMDY), wherein one or two of these amino acid residues can be substituted by a different amino acid.

The antibody may have a light chain comprising:

• a CDR1 sequence corresponding to amino acids 24 to 34 (KASENVGAAVA) of SEQ ID NO: 5, wherein one, two or three of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 56 (SASNRYT) of SEQ ID NO: 5, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 89 to 96 (QQYTNYPT) of SEQ ID NO: 5, wherein one of these amino acid residues can be replaced by a different amino acid residue.

The antibody may have a heavy chain comprising:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 6 (NYGVH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 6 (VIWRGGSIDYNAAFMS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; and / or • a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 6 (NSHGNYVGYAMDY), wherein one or two of these amino acid residues can be substituted by a different amino acid.

The antibody may have a light chain comprising:

• a CDR1 sequence corresponding to amino acids 24 to 34 (KASQSVGPAVA) of SEQ ID NO: 7, wherein one, two or three of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 56 (SASNRYT) of SEQ ID NO: 7, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 89 to 96 (QQYTSYPT) of SEQ ID NO: 7, wherein one of these amino acid residues can be replaced by a different amino acid residue.

The antibody may have a heavy chain comprising:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 8 (GYGVH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 8 (VIWRGGSIDYNAAFMS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; and / or • a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 8 (NSHGNYVGYAMDY), wherein one or two of these amino acid residues can be substituted by a different amino acid.

The antibody may have a light chain comprising:

• a CDR1 sequence corresponding to amino acids 24 to 34 (KASQNVGTAVA) of SEQ ID NO: 9, wherein one, two or three of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 56 (SASNRYT) of SEQ ID NO: 9, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 89 to 96 (QQYTSYPT) of SEQ ID NO: 9, wherein one of these amino acid residues can be replaced by a different amino acid residue.

The antibody may have a heavy chain comprising:

• a CDR1 sequence corresponding to amino acids 31 to 36 of SEQ ID NO: 10 (SDYAWN), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 51 to 66 of SEQ ID NO: 10 (YISYSGSTSYNPSLKS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; and / or • a CDR3 sequence corresponding to amino acids 99 to 104 of SEQ ID NO: 10 (WAYDGP), wherein one of these amino acid residues can be substituted by a different amino acid.

The antibody may have a light chain comprising:

• a CDR1 sequence corresponding to amino acids 24 to 33 (RASSSVSHMH) of SEQ ID NO: 11, wherein one or two of these amino acid residues can be replaced by a different amino acid residue; and / or • a CDR2 sequence corresponding to amino acids 49 to 55 (ATSNLAS) of SEQ ID NO: 11, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 88 to 96 (QQWSSNPFT) of SEQ ID NO: 11, wherein one of these amino acid residues can be replaced by a different amino acid residue.

The antibody may have a heavy chain comprising:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 12 (DYYIH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 66 of SEQ ID NO: 12 (WIDPENGNTIFDPKFQG), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 99 to 105 of SEQ ID NO: 12 (RWYAMDY), wherein one of these amino acid residues can be substituted by a different amino acid.

The antibody may have a light chain comprising:

• a CDR1 sequence corresponding to amino acids 24 to 39 (KSSQSLLYTNGKTYLN) of SEQ ID NO: 13, wherein one, two or three of these amino acid residues may be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 55 to 61 (LVSKLDS) of SEQ ID NO: 13, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 94 to 102 (LQSTHFPWT) of SEQ ID NO: 13, wherein one of these amino acid residues can be replaced by a different amino acid residue.

The term "epitope," as used herein, is defined in the context of a molecular interaction between an "antigen-binding polypeptide," such as an antibody and its corresponding antigen. Generally, "epitope" refers to the area or region in an antigen to which an antibody specifically binds, that is, the area or region in physical contact with the antibody. Physical contact can be defined by the use of various criteria (for example, a distance limit of 2-6A, such as 3Á, such as 4A, such as 5Á; or solvent accessibility) for atoms in antibody molecules and the antigen. An epitope of a protein may comprise amino acid residues in the antigen that are directly involved in antibody binding (also called an immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as residues. of amino acids of the antigen that are effectively blocked by the antibody, ie amino acid residues within the "solvent excluded surface" and / or the "fingerprint" of the antibody.

The term "epitope" in the present description comprises both types of binding region in any particular region of TFPI that specifically binds to a TFPI antibody, or other specific TFPI agent, unless otherwise indicated. The TFPI may comprise a number of different epitopes, which may include, without limitation, (1) linear peptide epitopes (2) conformational epitopes consisting of one or more non-contiguous amino acids located near each other in the conformation of the mature TFP i; and (3) post-translational epitopes that consist, either in whole or in part, of molecular structures covalently linked to TFPI, such as carbohydrate groups.

The epitope for a given antibody / antigen pair can be described and characterized at different levels of detail by using a variety of experimental and computational methods of epitope mapping. Experimental methods include mutagenesis, X-ray crystallography, Nuclear Magnetic Resonance Spectroscopy (NMR), Hydrogen Deuterium Exchange Mass Spectrometry (HX-MS) and various competition binding methods; methods that are known in the art. Since each method depends on a unique principle, the description of an epitope is closely related to the method by which it has been determined. Therefore, depending on the epitope mapping method employed, the epitope for a given antibody / antigen pair can be described differently.

At its most detailed level, the epitope for the interaction between the antigen and the antibody can be described by the spatial coordinates that define the atomic contacts present in the antigen-antibody interaction, as well as information about their contributions related to thermodynamics of Union. At a less detailed level, the epitope can be characterized by the spatial coordinates that define the atomic contacts between the antigen and the antibody. At an even less detailed level the epitope can be characterized by amino acid residues comprising as defined by specific criteria such as the distance between, or solvent accessibility of, the atoms in the antibody: antigen complex. At a less detailed level, the epitope can be characterized through function, for example, by competition binding with other antibodies. In addition, the epitope can be defined more generically as comprising amino acid residues whose substitution by another amino acid will alter the characteristics of the interaction between the antibody and the antigen.

In the context of an X-ray-derived crystalline structure defined by spatial coordinates of a complex between an antibody, for example, a Fab fragment, and its antigen, the term epitope in the present description, unless otherwise specified. or is contradicted by the context, specifically defined, as being TFPI residues that have a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 A from a heavy atom in the antibody .

From the fact that epitope descriptions and definitions, depending on the epitope mapping method used, are obtained at different levels of detail, it follows that the comparison of epitopes for different antibodies in the same antigen can be performed similarly in Different levels of detail.

It is said that the epitopes described at the amino acid level, for example, determined from an X-ray structure, are identical if they contain the same set of amino acid residues. It is said that epitopes overlap if at least one amino acid is shared by epitopes. The epitopes are said to be separated (unique) if the epitopes do not share any amino acid residues.

The definition of the term "paratope" is derived from the previous definition of "epitope" by reversing the perspective. Therefore, the term "paratope" refers to the area or region in the antibody to which an antigen specifically binds, that is, with which it makes physical contact with the antigen.

Antibodies that bind to the same antigen can be characterized with respect to their ability to bind simultaneously to their common antigen and can undergo "competition binding" / "epitope mapping". In the present context, the term "binding" refers to a method for grouping antibodies that bind to the same antigen. The "binding" of antibodies can be based on competition binding of two antibodies to their common antigen in assays based on standard techniques such as surface plasmon resonance (SPR), Biocapa Interferometry, ELISA or flow cytometry.

An "epitope" of the antibody can be defined by the use of a single reference antibody or, alternatively, a group of reference antibodies. The resolution in the identification of the "epitope" for a given antibody will increase with the amount of reference antibodies used. When a single reference antibody is used, if a second antibody is not capable of binding an antigen at the same time as the reference antibody, the second antibody is said to belong to the same "epitope" as the reference antibody. In this case, the reference and the second antibody bind competitively to the same part of the antigen and are called "competitive antibodies." If a second antibody is capable of binding an antigen at the same time as the reference antibody, the second antibody is said to belong to a separate "epitope." In this case, the reference and the second antibody do not competitively bind to the same part of the antigen and are called "non-competitive antibodies." When a group of reference antibodies is used for the identification of the "epitope," said group of reference antibodies may comprise a group of known or novel antibodies that can be used to define the "epitopes" of individual antibodies by cross-competition analysis. where each Antibody within the group is evaluated by competence to determine antigen binding with each member of the group. Antibody A is said to belong to the same "epitope" as antibody B when they exhibit the same binding pattern in cross-competition analyzes. Antibody A is said to belong to a different "epitope" than antibody B, when they exhibit a different competition binding profile against one or more of the individual antibodies in the reference group. Competition binding profile is the compiled set of data where each antibody within the group is analyzed to determine the ability to bind to the antigen at the same time as another member of the group. For example, the antigen binding profile for antibody A relative to a reference group of antibodies 1, 2 and 3 is as follows: A + 1 = no A binding; A + 2 = union by A; A + 3 = binding by A. Antibody B has a different competition binding profile compared to antibody A and the two antibodies are said to belong to different "epitopes" if: B + 1 = binding by B; B + 2 = union by B; B + 3 = B binding. Antibody C has a similar binding profile compared to antibody A and the two antibodies are said to belong to the same "epitope" if: C + 1 = no C binding; C + 2 = union by C; C + 3 = C binding. As indicated in the identification of the "epitope" resolution for a given antibody will increase with the amount of reference antibodies used. Competitive binding assays do not provide information on binding affinities and the assay should be designed such that the antibodies evaluated are capable of individually binding to the antigen sufficiently to function as binding competitors.

By evaluating the binding of antibodies to variants of an antigen such as those carrying mutations, truncations, deletions, insertions as well as homologues of antigens (eg, species counterparts) can also be used for the "epitope mapping" "Of the antibody or to increase the resolution of the" epitope mapping "of the antibody.

The "epitope mapping" of the antibody does not provide direct information about the epitope. Competitive antibodies, that is, antibodies belonging to the same "epitope", can have identical epitopes, overlapping epitopes or even separate epitopes. The latter is the case if the reference antibody bound to its epitope in the antigen takes the space required for the second antibody to come into contact with its epitope in the antigen ("steric hindrance"). Non-competitive antibodies generally have separate epitopes.

The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, for example, an antibody, or fragment thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).

The binding affinity between two molecules, for example, an antibody, or fragment thereof, and an antigen, through a monovalent interaction can be quantified by determining the equilibrium dissociation constant (K ^d ). In turn, K ^d can be determined by measuring the kinetics of complex formation and dissociation, for example, by the SPR method. The velocity constants corresponding to the association and the dissociation of a monovalent complex are referred to as the constant of the association rate k ^a (ok ^on ) and constant of the dissociation rate k ^d (ok ^off ), respectively. K ^d is related to k ^a and k ^d through the equation K ^d = k ^d / k ^a .

After the above definition, binding affinities associated with different molecular interactions, such as comparing the binding affinity of different antibodies for a given antigen, can be compared by comparing the values of K ^d for individual antibody / complexes. antigen.

The antibody described in the present description may be able to compete with another molecule, such as a naturally occurring ligand or receptor or another antibody, to bind TFPI. Therefore, the antibody described in the present description may be able to bind to TFPI with a higher affinity than that of another molecule capable in addition to binding to TFP i. The ability of an antibody to compete with a natural ligand / receptor for binding to an antigen can be assessed by determining and comparing the value of K ^d for interactions of interest, such as a specific interaction between an antibody and an antigen, with that of the value of K ^d of an interaction that is not of interest. Typically, the K ^d for the antibody with respect to the target will be 2 times, preferably 5 times, more preferably 10 times less than the K ^d with respect to the other non-target molecule, such as unrelated material or accompanying material in the environment. More preferably, the K ^d will be 50 times less, such as 100 times less, or 200 times less; even more preferably 500 times less, such as 1000 times less or 10,000 times less.

The value of this dissociation constant can be determined directly by well known methods. Standard assays to assess the ability of ligand binding such as antibodies to targets are known in the art and include, for example, ELISA, Western blots, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody can also be evaluated by standard assays known in the art, such as SPR.

A competitive binding assay can be performed in which the binding of the antibody to the target is compared with the binding of the target by another ligand of that target, such as another antibody.

The antibody described in the present description may have a K ^d for its target of 1 x 10-7M or less, 1 x 10-8M or less, or 1 x 10-9M or less, or 1 x 10-10M or less, 1 x 10-11M or less, or 1 x 10-12M or less. The K ^d of the antibody may be less than 0.8 nM, such as less than 0.7 nM, such as less than 0.6 nM, such as less than 0.5 nM, such as less than 0.4 nM, such as less than 0.3 nM, such as less than 0.1 nM, such as less than 0.05 nM, such as less than 0.025 nM, such as less than 0.015 nM, such as between 0.015 nM and 0 nM.

In addition, compositions and formulations comprising the antibodies described in the present description are provided. For example, a pharmaceutical composition comprising one or more TFPI antibodies, formulated together with at least one pharmaceutically acceptable excipient is provided.

Accordingly, an objective is to provide a pharmaceutical formulation comprising such a TFPI antibody that is present in a concentration of 0.25 mg / mL to 250 mg / mL, and wherein said formulation has a pH of 2.0 to 10, 0. The formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer or a surfactant, as well as various combinations of these. The use of preservatives, isotonic agents, chelating agents, stabilizers and surfactants in the pharmaceutical compositions is well known to the experts. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th Edition, 1995.

In one aspect, the pharmaceutical formulation is an aqueous formulation. Such a formulation is typically a solution or a suspension but may also include colloids, dispersions, emulsions, and multiphase materials. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w of water. Likewise, the term "aqueous solution" is defined as a solution comprising at least 50% w / w of water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w of water.

In another aspect, the pharmaceutical composition is a lyophilized formulation, to which the doctor or the patient adds solvents and / or diluents before use.

In a further aspect, the pharmaceutical formulation comprises an aqueous solution of such an antibody, and a buffer, wherein the antibody is present in a concentration of 1 mg / mL or more, and wherein said formulation has a pH of about 2.0 to about 10.0.

The antibody described in the present description, or a formulation comprising it, can be used to treat a subject with coagulopathy.

The term "subject," as used herein, includes any human patient, or non-human vertebrate.

The term "coagulopathy", as used in the present description, refers to a greater bleeding tendency that can be caused by any qualitative or quantitative deficiency of any procoagulative component of the normal coagulation cascade, or by any positive regulation of fibrinolysis. Such coagulopathies can be congenital and / or acquired and / or iatrogenic and are identified by one skilled in the art.

Non-limiting examples of congenital hypocoagulopathies are hemophilia A, hemophilia B, Factor VII deficiency, Factor X deficiency, Factor XI deficiency, von Willebrand disease and thrombocytopenia such as Glanzmann thrombocytopenia and Bernard-Soulier syndrome. Said hemophilia A or B can be severe, moderate or mild. The clinical severity of hemophilia is determined by the concentration of FIX / FVIII functional units in the blood and is classified as mild, moderate, or severe. Severe hemophilia is defined by a level of the coagulation factor of <0.01 U / mL corresponding to <1% of the normal level, while moderate and mild patients have levels of 1-5% and> 5%, respectively. Hemophilia A with "inhibitors" (ie, alloantibodies against factor VIII) and hemophilia B with "inhibitors" (ie, alloantibodies against factor IX) are non-limiting examples of coagulopathies that are partially congenital and partially acquired .

A non-limiting example of acquired coagulopathy is serine protease deficiency caused by vitamin K deficiency; Such vitamin K deficiency can be caused by the administration of a vitamin K antagonist, such as warfarin. Acquired coagulopathy can also occur after extensive trauma. In this case, known in any other way as the “vicious cycle of bleeding”, it is characterized by hemodilution (thrombocytopenia due to dilution and dilution of coagulation factors), hypothermia, consumption of coagulation factors and metabolic disorders (acidosis). Fluid therapy and increased fibrinolysis can exacerbate this situation. This bleeding can be from any part of the body.

A non-limiting example of iatrogenic coagulopathy is an overdose of anticoagulant medication - such as heparin, aspirin, warfarin and other inhibitors of platelet aggregation - that may be prescribed to treat thromboembolic disease. A second non-limiting example of iatrogenic coagulopathy is that which is induced by excessive and / or inappropriate fluid therapy, such as that which can be induced by a blood transfusion.

In one embodiment, bleeding is associated with hemophilia A or B. In another modality, bleeding is associated with hemophilia A or B with acquired inhibitors. In another modality, bleeding is associated with thrombocytopenia. In another modality, bleeding is associated with von Willebrand disease. In another modality, bleeding is associated with severe tissue damage. In another modality, bleeding is associated with severe trauma. In another modality, bleeding is associated with surgery. In another embodiment, hemorrhage is associated with gastritis and / or hemorrhagic enteritis. In another embodiment, bleeding is profuse uterine bleeding, such as in placental abruption. In another embodiment, bleeding occurs in organs with a limited possibility of mechanical hemostasis, such as intracranial, intraauricularly or intraocularly. In another modality, bleeding is associated with anticoagulant therapy.

The term "treatment," as used herein, refers to the medical therapy of any human subject or other vertebrate that needs it. It is expected that said subject has undergone a physical examination by a doctor, or a veterinary medical professional, who has given a presumptive or definitive diagnosis that would indicate that the use of said specific treatment is beneficial to the health of said human or another vertebrate. The time and purpose of such treatment may vary from one individual to another, according to the status quo of the subject's health. Therefore, said treatment can be prophylactic, palliative, symptomatic and / or curative. In terms of the present invention, prophylactic, palliative, symptomatic and / or curative treatments may represent separate aspects of the invention.

The antibody described in the present description can be administered parenterally, such as intravenously, such as intramuscularly, such as subcutaneously. Alternatively, the antibody described in the present description can be administered by a non-parenteral route, such as through the mouth or topically. The antibody described in the present description can be administered prophylactically. The antibody described in the present description can be administered therapeutically (upon request).

This description also includes the aspects listed below, which will not be interpreted as a description of the claimed invention.

Aspects

1. An antibody, or fragment thereof, that is capable of specifically binding an epitope present in amino acid residues 1 to 79 of human TFPI (SEQ ID NO: 1).

2. The antibody or fragment thereof according to aspect 1, which is capable of binding to an epitope present in amino acid residues 26 to 76 (KPI-1) of human TFPI.

3. The antibody or fragment thereof according to any of aspects 1 to 2, having a Kd equal to or less than 1E-08 M, determined by the use of surface plasmon resonance.

4. The antibody or fragment thereof according to any of aspects 1 to 3, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 4 (SYGVH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 4 (VIWRGGSTDFNAAFMS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 4 (NSHGNYVGYAMDY), wherein one or two of these amino acid residues can be substituted by a different amino acid.

5. The antibody or fragment thereof according to any of aspects 1 to 3, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 6 (NYGVH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 6 (VIWRGGSIDYNAAFMS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 6 (NSHGNYVGYAMDY), wherein one or two of these amino acid residues can be substituted by a different amino acid.

6. The antibody or fragment thereof according to any of aspects 1 to 3, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 8 (GYGVH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 8 (VIWRGGSIDYNAAFMS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 8 (NSHGNYVGYAMDY), wherein one or two of these amino acid residues can be substituted by a different amino acid.

7. The antibody or fragment thereof according to any of aspects 4 to 6, whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 34 (KASENVGAAVA) of SEQ ID NO: 5, wherein one, two or three of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 56 (SASNRYT) of SEQ ID NO: 5, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 89 to 96 (QQYTNYPT) of SEQ ID NO: 5, wherein one of these amino acid residues can be replaced by a different amino acid residue.

8. The antibody or fragment thereof according to any of aspects 4 to 6, whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 34 (KASQSVGPAVA) of SEQ ID NO: 7, wherein one, two or three of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 56 (SASNRYT) of SEQ ID NO: 7, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 89 to 96 (QQYTSYPT) of SEQ ID NO: 7, wherein one of these amino acid residues can be replaced by a different amino acid residue.

9. The antibody or fragment thereof according to any of aspects 4 to 6, whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 34 (KASQNVGTAVA) of SEQ ID NO: 9, wherein one, two or three of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 56 (SASNRYT) of SEQ ID NO: 9, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 89 to 96 (QQYTSYPT) of SEQ ID NO: 9, wherein one of these amino acid residues can be replaced by a different amino acid residue.

10. The antibody or fragment thereof according to any of aspects 1 to 3, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 36 of SEQ ID NO: 10 (SDYAWN), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 51 to 66 of SEQ ID NO: 10 (YISYSGSTSYNPSLKS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 99 to 104 of SEQ ID NO: 10 (WAYDGP), wherein one of these amino acid residues can be substituted by a different amino acid.

11. The antibody or fragment thereof according to aspect 10, whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 33 (RASSSVSHMH) of SEQ ID NO: 11, wherein one or two of these amino acid residues can be replaced by a different amino acid residue; and / or • a CDR2 sequence corresponding to amino acids 49 to 55 (ATSNLAS) of SEQ ID NO: 11, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 88 to 96 (QQWSSNPFT) of SEQ ID NO: 11, wherein one of these amino acid residues can be replaced by a different amino acid residue.

12. The antibody or fragment thereof according to any of aspects 1 to 3, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 12 (DYYIH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 66 of SEQ ID NO: 12 (WIDPENGNTIFDPKFQG), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 99 to 105 of SEQ ID NO: 12 (RWYAMDY), wherein one of these amino acid residues can be substituted by a different amino acid.

13. The antibody or fragment thereof according to aspect 12, whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 39 (KSSQSLLYTNGKTYLN) of SEQ ID NO: 13, wherein one, two or three of these amino acid residues may be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 55 to 61 (LVSKLDS) of SEQ ID NO: 13, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 94 to 102 (LQSTHFPWT) of SEQ ID NO: 13, wherein one of these amino acid residues can be replaced by a different amino acid residue.

14. The antibody or fragment thereof according to any of aspects 1 to 3, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 4 (SYGVH), SEQ ID NO: 6 (NYGVH) or SEQ ID NO: 8 (GYGVH); Y

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 4 (VIWRGGSTDFNAAFMS), SEQ ID NO: 6 (VIWRGGSIDYNAAFMS) or SEQ ID NO: 8 (VIWRGGSIDYNAAFMS); Y • a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 (NSHGNYVGYAMDY); and whose light chain comprises:

a CDR1 sequence corresponding to amino acids 24 to 34 of SEQ ID NO: 5 (KASENVGAAVA), SEQ ID NO: 7 (KASQSVGPAVA) or SEQ ID NO: 9 (KASQNVGTAVA); Y

a CDR2 sequence corresponding to amino acids 50 to 56 of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 (SASNRYT); Y

a CDR3 sequence corresponding to amino acids 89 to 96 of SEQ ID NO: 5 (QQYTNYPT), SEQ D NO: 7 (QQYTSYPT) or SEQ ID NO: 9 (QQYTSYPT).

5. The antibody or fragment thereof according to aspect 10, whose heavy chain comprises:

a CDR1 sequence corresponding to amino acids 31 to 36 of SEQ ID NO: 10 (SDYAWN); and a CDR2 sequence corresponding to amino acids 51 to 66 of SEQ ID NO: 10 YISYSGSTSYNPSLKS); Y

a CDR3 sequence corresponding to amino acids 99 to 104 of SEQ ID NO: 10 (WAYDGP); and whose light chain comprises:

a CDR1 sequence corresponding to amino acids 24 to 33 (RASSSVSHMH) of SEQ ID NO: 11; and a CDR2 sequence corresponding to amino acids 49 to 55 (ATSNLAS) of SEQ ID NO: 11; and a CDR3 sequence corresponding to amino acids 88 to 96 (QQWSSNPFT) of SEQ ID NO: 11.

6. The antibody or fragment thereof according to aspect 12, whose heavy chain comprises:

a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 12 (DYYIH); and a CDR2 sequence corresponding to amino acids 50 to 66 of SEQ ID NO: 12 (WIDPENGNTIFDPKFQG); Y

a CDR3 sequence corresponding to amino acids 99 to 105 of SEQ ID NO: 12 (RWYAMDY); and whose light chain comprises:

a CDR1 sequence corresponding to amino acids 24 to 39 (KSSQSLLYTNGKTYLN) of SEQ ID NO: 3; Y

a CDR2 sequence corresponding to amino acids 55 to 61 (LVSKLDS) of SEQ ID NO: 13; and a CDR3 sequence corresponding to amino acids 94 to 102 (LQSTHFPWT) of SEQ ID NO: 13.

17. An antibody or fragment thereof that competes with the antibody according to any of the above modalities to bind TFPI (1-79), as determined by the use of Biocapa Interferometry.

18. An antibody or fragment thereof belonging to the same epitope as an antibody according to any of the above aspects, as determined by the use of Biocapa Interferometry.

19. The antibody or fragment thereof according to any of the above aspects, which is a monoclonal antibody.

20. The antibody or fragment thereof according to any of the above aspects, which is a humanized antibody.

21. The antibody fragment according to any of the above aspects, which is selected from the group consisting of a Fab fragment, a Fab ', a Fab ² , a Fab' ² and a scFv.

22. A pharmaceutical formulation comprising the antibody, or fragment thereof, according to any of aspects 1 to 21 and at least one pharmaceutically acceptable excipient.

23. The antibody or fragment thereof according to any of aspects 1 to 21, or the pharmaceutical formulation according to aspect 22, for use as a medicament.

24. The antibody or fragment thereof according to any of aspects 1 to 21, or the pharmaceutical formulation according to aspect 22, for use in the treatment of a subject with coagulopathy.

25. The antibody or fragment thereof for use according to aspect 24, wherein said subject has any congenital, acquired and / or iatrogenic coagulopathy, such as hemophilia A, with or without inhibitors, and hemophilia B, with or Without inhibitors

26. A method of treating a subject with coagulopathy, comprising administering to said subject the antibody or fragment thereof according to any of aspects 1 to 21.

The additional aspects are:

1. An isolated antibody or fragment thereof that specifically binds to an epitope present in amino acid residues 1 to 79 of human TFPI (SEQ ID NO: 1).

2. The antibody or fragment thereof according to aspect 1, which specifically binds to an epitope present in amino acid residues 26 to 76 (KPI-1) of human TFPI (SEQ ID NO: 1).

3. The antibody or fragment thereof according to aspect 1, which specifically binds to an epitope present in human TFPI wherein said epitope comprises at least one of the following amino acid residues L16, P17, L19, K20, L21 , M22, F25, C35, A37, M39, R41, Y56, G57, G58, C59, E60, G61, N62, Q63, R65, F66, E67, E71 and M75 of SEQ ID NO: 1.

4. The antibody or fragment thereof according to aspect 3, which specifically binds to an epitope present in human TFPI wherein said epitope comprises at least one of the following amino acid residues R41, R65 and E67 of SEQ ID NO: 1.

5. The antibody or fragment thereof according to aspect 1, which has a K ^d equal to or less than 1E-08 M, determined by the use of surface plasmon resonance.

6. An isolated antibody that specifically binds to human TFPI wherein the heavy chain of said antibody comprises amino acid residues

And in a position corresponding to position 2,

F in a position corresponding to position 27,

And in a position corresponding to position 32,

W in a position corresponding to position 52,

R in a position corresponding to position 53,

G in a position corresponding to position 54,

G in a position corresponding to position 55,

S in a position corresponding to position 56,

I in a position corresponding to position 57,

D in a position corresponding to position 58,

And in a position corresponding to position 59,

A in a position corresponding to position 61,

M in a position corresponding to position 64,

K in a position corresponding to position 97,

S in a position corresponding to position 99,

H in a position corresponding to position 100,

N in a position corresponding to position 102,

And in a position corresponding to position 103,

And in a position corresponding to position 104,

G in a position corresponding to position 105, and

And in a position corresponding to position 106

of SEQ ID NO: 16, and

wherein the light chain of said antibody comprises amino acid residues

P in a position corresponding to position 31,

A in a position corresponding to position 32,

And in a position corresponding to position 49,

S in a position corresponding to position 50,

N in a position corresponding to position 53,

And in a position corresponding to position 55,

T in a position corresponding to position 56,

And in a position corresponding to position 91,

T in a position corresponding to position 92,

S in a position corresponding to position 93, and

And in a position corresponding to position 94

of SEQ ID NO: 17.

7. The antibody according to aspect 1, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 4 (SYGVH), SEQ ID NO: 6 (NYGVH) or SEQ ID NO: 8 (Gy GVH), where one of these residues of amino acids can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 4 (VIWRGGSTDFNAAFMS), SEQ ID NO: 6 (VIWRGGSIDYNAAFMS) or SEQ ID NO: 8 (VIWRGGSIDYNAAFMS), where one, two or three of these amino acids can be substituted by a different amino acid residue; and / or • a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 4 (NSHGNYVGYAMDY), SEQ ID NO: 6 or SEQ ID NO: 8 (NSHGNYVGYAMDY), wherein one or two of these amino acid residues can be substituted for a different amino acid.

8. The antibody or fragment thereof according to aspect 1 or 7, whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 34 (KASENVGAAVA) of SEQ ID NO: 5, SEQ ID NO: 7 (KASQSVGPAVA) or SEQ ID NO: 9 (KASQNVGTAVA), where one, two or three of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 56 (SASNRYT) of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 (SASNRYT), where one of these amino acid residues can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 89 to 96 (QQYTNYPT) of SEQ ID NO: 5, SEQ ID NO: 7 (QQYTSYPT) or SEQ ID NO: 9 (QQYTSYPT), where one of these residues of Amino acids can be substituted for a different amino acid residue.

9. The antibody or fragment thereof according to aspect 1, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 4 (SYGVH), SEQ ID NO: 6 (NYGVH) or SEQ ID NO: 8 (GYGVH); Y

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 4 (VIWRGGSTDFNAAFMS), SEQ ID NO: 6 (VIWRGGSIDYNAAFMS) or SEQ ID NO: 8 (VIWRGGSIDYNAAFMS); and • a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 (NSHGNYVGYAMDY); and whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 34 of SEQ ID NO: 5 (KASENVGAAVA), SEQ ID NO: 7 (KASQSVGPAVA) or SEQ ID NO: 9 (KASQNVGTAVA); Y

• a CDR2 sequence corresponding to amino acids 50 to 56 of SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 (SASNRYT); Y

• a CDR3 sequence corresponding to amino acids 89 to 96 of SEQ ID NO: 5 (QQYTNYPT), SEQ ID NO: 7 (QQYTSYPT) or SEQ ID NO: 9 (QQYTSYPT).

10. The antibody or fragment thereof according to aspect 1, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 6 (NYGVH); Y

• a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 6 (VIWRGGSIDYNAAFMS); Y

• a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 6 (NSHGNYVGYAMDY); and whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 34 of SEQ ID NO: 7 (KASQSVGPAVA); and • a CDR2 sequence corresponding to amino acids 50 to 56 of SEQ ID NO: 7 (SASNRYT); and a CDR3 sequence corresponding to amino acids 89 to 96 of SEQ ID NO: 7 (QQYTSYPT).

11. The antibody or fragment thereof according to aspect 1, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 36 of SEQ ID NO: 10 (SDYAWN), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 51 to 66 of SEQ ID NO: 10 (YISYSGSTSYNPSLKS), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 99 to 104 of SEQ ID NO: 10 (WAYDGP), wherein one of these amino acid residues can be replaced by a different amino acid, and whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 33 (RASSSVSHMH) of SEQ ID NO: 11, wherein one or two of these amino acid residues can be replaced by a different amino acid residue; and / or • a CDR2 sequence corresponding to amino acids 49 to 55 (ATSNLAS) of SEQ ID NO: 11, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 88 to 96 (QQWSSNPFT) of SEQ ID NO: 11, wherein one of these amino acid residues can be replaced by a different amino acid residue.

12. The antibody or fragment thereof according to aspect 11, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 36 of SEQ ID NO: 10 (SDYAWN);

• a CDR2 sequence corresponding to amino acids 51 to 66 of SEQ ID NO: 10 (YISYSGSTSYNPSLKS); Y

• a CDR3 sequence corresponding to amino acids 99 to 104 of SEQ ID NO: 10 (WAYDGP); and whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 33 (RASSSVSHMH) of SEQ ID NO: 11; • a CDR2 sequence corresponding to amino acids 49 to 55 (ATSNLAS) of SEQ ID NO: 11; Y

• a CDR3 sequence corresponding to amino acids 88 to 96 (QQWSSNPFT) of SEQ ID NO: 11. 13. The antibody or fragment thereof according to aspect 1, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 12 (DYYIH), wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 50 to 66 of SEQ ID NO: 12 (WIDPENGNTIFDPKFQG), wherein one, two or three of these amino acids can be substituted by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 99 to 105 of SEQ ID NO: 12 (RWYAMDY), wherein one of these amino acid residues can be substituted by a different amino acid.

And whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 39 (KSSQSLLYTNGKTYLN) of SEQ ID NO: 13, wherein one, two or three of these amino acid residues may be substituted by a different amino acid residue; I

• a CDR2 sequence corresponding to amino acids 55 to 61 (LVSKLDS) of SEQ ID NO: 13, wherein one of these amino acid residues can be replaced by a different amino acid residue; I

• a CDR3 sequence corresponding to amino acids 94 to 102 (LQSTHFPWT) of SEQ ID NO: 13, wherein one of these amino acid residues can be replaced by a different amino acid residue.

14. The antibody or fragment thereof according to aspect 13, whose heavy chain comprises:

• a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 12 (DYYIH); Y

• a CDR2 sequence corresponding to amino acids 50 to 66 of SEQ ID NO: 12 (WIDPENGNTIFDPKFQG); Y

• a CDR3 sequence corresponding to amino acids 99 to 105 of SEQ ID NO: 12 (RWYAMDY); and whose light chain comprises:

• a CDR1 sequence corresponding to amino acids 24 to 39 (KSSQSLLYTNGKTYLN) of SEQ ID NO: 13; Y

• a CDR2 sequence corresponding to amino acids 55 to 61 (LVSKLDS) of SEQ ID NO: 13; and a CDR3 sequence corresponding to amino acids 94 to 102 (LQSTHFPWT) of SEQ ID NO: 13.

15. An isolated antibody or fragment thereof that competes with the antibody according to any of aspects 3 to 10 for binding to TFPI (1-79), as determined by the use of Biocapa Interferometry.

16. An isolated antibody or fragment thereof belonging to the same epitope as the antibody according to any of aspects 1 to 14, as determined by the use of Biocapa Interferometry.

17. A pharmaceutical formulation comprising at least one antibody or fragment thereof according to any of aspects 1 to 16 and at least one pharmaceutically acceptable excipient.

18. The antibody or fragment thereof according to any of aspects 1 to 16, or the pharmaceutical formulation according to aspect 17, for use as a medicament.

19. The antibody or fragment thereof according to any of aspects 1 to 16 for use in the treatment of a subject with coagulopathy.

20. The antibody or fragment thereof for use according to aspect 19, wherein said subject has congenital, acquired and / or iatrogenic coagulopathy, such as hemophilia A, with or without inhibitors, and hemophilia B, with or Without inhibitors

21. A method of treating a subject with coagulopathy, which comprises administering to said subject the antibody or fragment thereof according to any of aspects 1 to 16.

Examples

Example 1: Immunization, fusion and screening

RBF mice were immunized with human TFPI (SEQ ID NO: 1) or TFPI (1-161) (SEQ ID NO: 3). Mice were injected subcutaneously: 20 | ig of human TFPI was mixed with Freund's complete adjuvant for the first injection. For subsequent immunizations, incomplete Freund's adjuvant with the same antigen concentration was used. Ten days after the final immunization, the mice's eye blood was screened, using ELISA, for antibodies specific to human TFPI. Mice with positive serum titers received a 10 | ig booster dose of human TFPI or TFPI (1-161) by intravenous injection and were sacrificed after three days. The spleens were removed aseptically and dispersed in a suspension of individual cells. The fusion of spleen cells and myeloma cells was performed by means of the PEG method or by electrofusion. The resulting hybridoma cells were cloned by limiting dilution in microtiter plates. The supernatants of the individual hybridomas were initially screened by ELISA for the expression of antibodies capable of binding to full-length TFPIa or TFPI (1-161). To identify the hybridomas that produce TFPI-specific antibodies (1-79) the positive hybridomas will join the TFPI full length or TFPI (1-161) is contraseleccionaron for joining TFPI-KPI-2 fragment (represented by amino acid residues 97-147 of SEQ ID NO: 1). Hybridomas positive for binding to TFPI (1-161) and negative for binding to the TFPI-KPI-2 fragment were isolated and expanded for antibody production.

The antibodies were purified from supernatants by standard protein A affinity chromatography and used to determine the binding and affinity to human TFPIa and TFPI (1-79) and the neutralization activity of plasma TFPI (TGT assay). Hybridomas that produce antibodies of interest, that is, those specific to TFPI (1-79) were subcloned by limiting dilution and the original antibody profile was verified to the subcloned hybridoma material. Subcloned hybridoma cells were used for RNA isolation and subsequent antibody cloning and sequence identification.

Example 2: Cloning and sequencing of human anti-TFPI specific mouse AcM (1-79)

This example describes the cloning and sequencing of the murine heavy chain and light chain sequences of the TFPI antibodies AcM 2F3, AcM 2F22, AcM 2F45, AcM 1F56, AcM 1F91 and AcM 2F35. Total RNA was extracted from hybridoma cells by using the Qiagen RNeasy-Mini kit and used as a template for cDNA synthesis. The cDNA was synthesized in a 5'-RACE reaction by the use of the Clontech c Ma RT ™ RACE cD amplification kit. The subsequent directed amplification of the HC and LC sequences was performed by PCR using the Phusion Hot Start polymerase (Finnzymes) and the universal primer mix (UPM) included in the SMART ™ RACE kit as a direct primer. The reverse primer with the sequence shown in SEQ ID NO: 14 was used for the amplification of HC (VH domain) and the reverse primer with the sequence shown in SEQ ID NO: 15 was used for amplification of the LC. The PCR products were separated by gel electrophoresis, extracted by using the GFX PCR DNA & Gel Band Purification kit from GE Healthcare Bio-Sciences and cloned for sequencing by using a Zero Blunt TOPO PCR cloning kit and a chemically competent TOP10 E. coli (Life Technologies). Colony PCR was performed in selected colonies by using an AmpliTaq Gold Master mixture of Applied Biosystems and M13uni / M13rev primers. PCR cleaning of colonies was performed using the ExOsAP-IT (USB) enzyme mixture. Sequencing was performed at MWG Biotech, Martinsried Germany using M13uni (-21) / M13rev (-29) sequencing primers. The sequences were analyzed and annotated by using the Vector NTI Advance 11 program (Life Technologies). All kits and reagents were used according to the manufacturer's instructions.

A single single subclass of murine kappa LC and a single single subclass of murine HC, MiGg1 were identified for each of the hybridomas: AcM 2F3, AcM 2f22, AcM 2F45, AcM 1F91 and AcM 2F35. The LC and HC sequences of AcM 1F56 showed that this antibody is identical in sequence to AcM 1F91. The amino acid sequences for the variable heavy chain and variable light chain sequences (excluding the leader peptide sequences) are shown in SEQ ID NOs 4-13. SEQ ID NOs are listed in Table 1.

Generation of expression vectors for LC and HC of antibodies

CMV promoter-based expression vectors (pTT vectors) were generated for transient expression of human-murine chimeric versions of AcM 2F22. The pTT vectors were developed for transient protein expression by Yves Durocher (Durocher et al. Nucleic Acid Research, 2002). In addition to the CMV promoter, pTT-based vectors contain a pMB1 origin, an EBV origin and the Amp resistance gene.

An expression vector was generated for the expression of a light chain of chimeric AcM 2F22, which carries the murine light chain variable region 2F22 and a human kappa constant region (SEQ ID NO: 17). Expression vectors were generated for the expression of a heavy chain of chimeric AcM 2F22 carrying the murine heavy chain variable region 2F22 and a full-length human IgG4 constant region (S241P) for the expression of AcM 0294 (SEQ ID NO: 19) or the human IgG4 constant regions truncated for Fab expression 0296 (SEQ ID NO: 16) or Fab expression 0295 (SEQ ID NO: 20). SEQ IDs are NOT listed in Table 1.

The pTT-based LC expression vector was generated for the transient expression of a chimeric AcM 2F22 antibody and an antibody fragment. Initially, the region corresponding to the VL domains of the AcM 2F22 was amplified by PCR in a 2-stage reaction from an original TOPO sequencing clone, using specific primers for the N and C terminal end sequences. The original murine signal peptide was exchanged for the human CD33 signal peptide by 2-stage superimposed PCR. The primary sense primers carry the C-terminal part of the CD33 signal peptide sequence and the secondary sense primer contained an HIndiII restriction site for cloning purposes, a Kozak sequence (5'-GCCGCCACC-3 ') immediately in the direction 5 'of the ATG start codon and the N-terminal part of the CD33 signal peptide sequences. The antisense primer contains a BsiWI restriction site in the frame in the VL / CL transition sequence. The amplified fragment was cloned into a linearized pTT based vector containing the sequence for a human kappa CL domain and subsequently transformed into E. coli for selection. The sequence of the final construct was verified by DNA sequencing.

HC expression vectors based on pTT were generated for transient expression of an AcM 2F22 and chimeric Fab 2F22 fragments. The AcM and the chimeric fragments are called AcM 0294, Fab 0296 and Fab 0295, respectively. Initially, the region corresponding to the VH domain of the AcM 2F22 was amplified by PCR in a 2-stage reaction from an original TOPO sequencing clone, using specific primers for the N and C terminal end sequences. The original murine signal peptide was exchanged for the human CD33 signal peptide by 2-stage superimposed PCR. The primary sense primers carry the C-terminal part of the CD33 signal peptide sequence and the secondary sense primer contained an HIndiII restriction site for cloning purposes, a Kozak sequence (5'-GCCGCCACC-3 ') immediately in the direction 5 'of the ATG start codon and the N-terminal part of the CD33 signal peptide sequences. The antisense primer contains an NheI restriction site in frame at the VH / CH transition. For the generation of the full length HC expression vector (HC of AcM 0294), the PCR fragment of the generated VH domain was digested by restriction enzymes and cloned into a linearized vector based on pTT containing the sequence for a constant region of human IgG4 (S241P). Mutation of Serine 241 to Proline in the hinge region of IgG 4 is included to stabilize the IgG4 antibody by eliminating the formation of antibody halves. The mutated position on the hinge is referred to as S241P when it is numbered according to Kabat or alternatively S228P, when it is numbered according to the EU index.

For the generation of the truncated HC expression vector for Fab expression 0296, the PCR fragment of the generated VH domain was digested by restriction enzymes and cloned into a linearized vector based on pTT containing the sequence for a constant region. of truncated human IgG4. The HC based on IgG4 was truncated in the hinge region after lysine in the human IgG4 hinge as observed in the HC sequence of Fab 0296 (SEQ ID NO: 16). The cloning reaction was subsequently transformed into E. coli for selection. The sequence of the final construct was verified by DNA sequencing.

For the generation of the truncated HC expression vector for Fab expression 0295, the PCR fragment of the generated VH domain was digested by restriction enzymes and cloned into a linearized vector based on pTT containing the sequence for a second truncated human IgG4 constant region. The IgG4-based HC for Fab 0295 was truncated in the hinge region after the first cysteine of the human IgG4 hinge as observed in the Fab HC sequence 0295 (SEQ ID NO: 20). The cloning reaction was subsequently transformed into E. coli for selection. The sequence of the final construct was verified by DNA sequencing.

Expression and purification of AcM and Fab fragments

Anti-TFPI antibodies and Fab fragments were transiently expressed in suspension cultures of EXPI293F cells (Life Technologies), by co-transfection of the LC and HC expression vectors based on pTT. The following procedure describes the generic transfection protocol used for suspension-adapted EXPI293F cells.

EXPI293F Transfection

1) Separate dilutions of DNA and transfection reagent are initially prepared.

a) Use a total of 1 | jg of the DNA vector (0.5 ug LC vector and 0.5 ug HC vector) per mL of cell culture. Dilute the DNA in Opti-MEM medium (Gibco) 50 jL medium / jg DNA, mix and incubate at room temperature (23-25 ° C) for 5 min.

b) Use Expifectamin ™ 293 (Life Technologies) as a transfection reagent at a concentration of 2.7 pL per jg of DNA. Dilute the Expifectamin ™ 18.5X solution in Opti-MEM medium (Gibco), mix and incubate at room temperature (23-25 ° C) for 5 min.

2) Mix DNA and Expifectamin ™ 293 dilutions and allow to incubate at room temperature (23-25 ° C) for 10 min.

3) Add the DNA-Expifectamin ™ 293 mixture directly to the EXPI293F cell culture.

a) At the time of transfection the cell density of the EXPI293F culture should be 2.8-3.2 x 10 ⁶ cells / mL.

4) Transfer the transfected cell culture to an orbital shaking incubator at 36.5 ° C, 8% CO ² and 85-125 rpm.

5) 18 hours after transfection, add 5 uL of Expifectamin ™ 293 Transfection Enhancer1 / mL of culture and 50 uL of Expifectamin ™ 293 Transfection Enhancer2 / mL of culture and return the culture to an orbital shaking incubator at 36.5 ° C, 8% CO ² and 85-125 rpm.

6) 5 days after transfection, cell culture supernatants were collected by centrifugation, followed by filtration through a 0.22 pm PES filter unit (Corning).

General Purification Protocol

The AcM variants were purified by standard affinity chromatography using the GE Healthcare MabSelectSuRe resin according to the manufacturer's instructions. The purified antibodies were exchanged to PBS buffer pH 7.2.

Fab fragments were purified by standard affinity chromatography using the KapPaselect resin developed by GE Healthcare. The purified Fab fragments were exchanged to PBS buffer pH 7.2. Quality assessment and concentration determination were performed using SEC-HPLC.

Table 1. Summary of antibody fragments and antibodies identified by sequence

VH: heavy chain variable domain, VL: light chain variable domain, HC: heavy chain, LC: light chain.

Example 3: Analysis of binding interaction

Binding interaction data were obtained by means of Surface Plasmon Resonance on a Biacore T200 instrument. Polyclonal rabbit anti-mouse immunoglobulin (Mouse Antibody Capture Kit, GE Healthcare) was immobilized to an S-series CM4 sensor chip (GE Healthcare) in flow cells 1-4 using the protocol of Amine coupling available with the Biacore T-200 version 2.0 (GE Healthcare) control software and the reagents provided with the Amina Coupling Kit (GE Healthcare). The relevant mouse monoclonal antibody was captured in flow cell 2, 3, or 4 at a fixed concentration. Different concentrations of human TFPIa or TFPI (1-79) were evaluated by diluting the sample in run buffer (10 mM HEPES, pH 7.4, 300 mM NaCl, 5 mM CaCb, P200.05 Surfactant, BSA 1 mg / mL) Each sample was analyzed by using 300 seconds of contact time followed by 420-600 seconds of dissociation time at 30 pL / min flow rate. In addition, a control buffer was evaluated. The sensor surface was regenerated with 10 mM Glycine pH 1.7 either for 180 sec at 30 pL / min flow rate or with two cycles each 30 seconds at 30 pL / min flow rate.

The human anti-human IgG (Fc) mouse monoclonal antibody (Human Antibody Capture Kit, GE Healthcare) was immobilized to an S-series CM4 sensor chip (GE Healthcare) in flow cells 1-2 through use of the amine coupling protocol available with the Biacore T-200 control software (GE Healthcare) and the reagents provided with the Amina Coupling kit (GE Healthcare). The relevant antibody Bay 2A8, Bay 2A8-K95L or AcM 0294 (SEQ ID NO: 19 and 17) was captured in the flow cell 2 at a fixed concentration. Different concentrations of human TFPIa or TFPI (1-79) were evaluated as described above. The sensor surface was regenerated with 3M MgCb for either 180 sec at 30 pL / min flow rate or with two cycles, each 30 seconds at 30 pL / min flow rate.

The human Fab ligand (human Fab capture kit, GE Healthcare) was immobilized to an S-series CM4 sensor chip (GE Healthcare) in flow cells 1-4 to 9000-10,000 response units (RU) with the use of the amine coupling protocol available with the Biacore T-200 control software (GE Healthcare) and the reagents provided with the amine coupling kit (GE Healthcare). The relevant Fab fragments, Fab 0295 (SEQ ID NO: 20 and 17) or Fab 0296 (SEQ ID NO: 16 and 17) were injected at a fixed concentration (0.2 pg / mL) for 180 sec in the flow cell 2, 3 or 4 at 10 pl / min flow rate that should give a response of approximately 70-80 RU. Different concentrations of human TFPIa were evaluated by diluting the sample in run buffer (10 mM HEPES, pH 7.4, 300 mM NaCl, 5 mM CaCb, P200.05 Surfactant, 1 mg / mL BSA). Each sample was analyzed by using 300 seconds of contact time followed by 600 seconds of dissociation time at 50 pL / min flow rate. In addition, a control buffer was evaluated. The sensor surface was regenerated with 10 mM Glycine pH 2.1 with two cycles each of 30 seconds at 50 pL / min flow rate. The analysis temperature was 25 ° C and the temperature of the sample compartment 10 ° C.

The Biacore T200 Evaluation software (version 2.0) was used to analyze the data. The determination of binding constants (k ^a , k ^d , K ^d ) was obtained assuming a 1: 1 interaction of the TFPI and the monoclonal antibody of interest.

The data in Table 2 demonstrate that the antibodies that were evaluated (the AcM 1F56, 1F91, 2F3, 2F17, 2F22, 2F35, 2F37, 2F39, 2F45, 2F48) bind with high affinity to an epitope within human TFPI (1 -79), which encompasses the acid N-terminal peptide and the TFPI-KPI-1 domain.

The data in Table 3 demonstrate that AcM 2F22 expressed as a chimeric antibody AcM 0294: (SEQ ID NO: 17 and 19) and the chimeric Fab fragments, Fab 0296 (SEQ ID 17 and 16) and Fab 0295 (SEQ ID NO : 17 and 20) retain a high affinity binding to TFPI.

Table 2: Binding Constants

Binding constants (k ^a , k ^d , K ^d ) were obtained assuming a 1: 1 interaction of human TFPIa or TFPI (1-79) and the antibody of interest. Bay 2A8 is a human IgG4 (S241P) antibody variant of Fab 2A8 described in WO2010 / 017196. Bay 2A8-K95L is a variant of human IgG4 antibody (S241P) of Bay 2A8 that carries the K95L substitution (Kabat numbering) in HC of CDR3, identical to the substitution found in “Fab B” in relation to 2A8 as It is described in WO2012 / 135671.

Table 3: Binding Constants

Binding constants (k ^a , k ^d , K ^d ) were obtained assuming a 1: 1 interaction of human TFPIa or TFPI (1-79) and the antibody or Fab fragment of interest. nd: not determined.

Example 4: Antibody binding

The competition between TFPI antibodies 1F91, 2F3, 2F22, 2F35, 2F45, Bay 2A8-K95L, MBS532510 (MyBioSource.com), and 10R-T141A (Fitzgerald Industries International) for binding to full length TFPIa was measured by use of Biocapa Interferometry (Fortebio Octet RED384 instrument, PALL Life Sciences). The AcM 1F91, AcM 2F3, AcM 2F22, AcM 2F35, AcM 2f45, and Bay 2A8-K95L were randomly labeled with biotin in the lysine residues by using a ratio of 1: 1.2 mol of AcM: mol of biotin (Thermo Scientific cat # 21335) and in any other way following the manufacturer's specifications. The biotin-labeled antibody was captured on the Streptavidin Fortebio (PALL Life Sciences) sensors followed by the binding of human TFPIa followed by the binding of the AcM 1F91, AcM 2F3, AcM 2F22, AcM 2F35, AcM 2F45, Bay 2A8-K95L , MBS532510 or 10R-T141A. The results are shown in Table 4 (Black means that AcMs compete for TFPI binding. Gray means that AcMs do not compete for TFPI binding). These data show that antibodies are found in 4 different epitopes, indicating that they have different binding epitopes. Epitope1: AcM 1F91, MBS532510, and 10R-T141A. Epitope 2: AcM 2F3, 2F22 and 2F45. Epitope 3: AcM 2F35. Epitope 4: Bay2A8 K95L.

Table 4: Competition between indicated TFPI antibodies to bind to TFPI

Example 5: Effect of TFPI-specific antibodies on TFPI (1-79) in the generation of TF-induced thrombin in neutralized plasma for human FVIII

The effect of antibodies on thrombin generation was studied in normal human plasma (NHP, CricheCheck Normal Plasma). Coagulation onset was induced by recalcification and addition of 1 pM TF with 4 pM phospholipid vesicles (PPP-Low Reagent, Thrombinoscope). Hemophilia-like plasma A was obtained by adding 100 pg / mL of antibody generated in human anti-FVIII sheep (Haematologic Technologies Inc., PAHFVIII-S). The thrombin activity was continuously evaluated after the conversion of the Z-Gly-Gly-Arg-AMC.HCl (I-1140) fluorogenic substrate from Bachem (Bubendorf, Switzerland). Fluorescence was measured on a Fluorskan Ascent fluorometer microtiter plate (Thermo Labsystems, Helsinki, Finland) with excitation and emission wavelengths at 368 and 460 nm, respectively. A calibrator was used to allow the calculation of the amount of thrombin formed and the correction of the relative fluorescence units obtained for the effects of the internal filter and the consumption of the fluorogenic substrate. In addition, the contribution to substrate conversion was subtracted by the thrombin-a2-macroglobulin complex. These corrections were made automatically by means of the calibrated automated thrombogram (CAT) software (version 5.0.0.745) provided by Thrombinoscope BV (Maaestricht, The Netherlands). The first derivatives of the data that were obtained from the performance of the thrombin curve generation, allowed the calculation of i) delay time, ii) total area under the curve, the endogenous thrombin potential (ETP), iii) height thrombin maximum (maximum), iv) time to maximum (tt maximum) and v) maximum thrombin generation rate (Rate).

Figure 1 (curve (a)) shows the thrombin generation curve with NHP. The addition of the antibody generated in human anti-FVIII sheep, 100 pg / mL to the NHP to simulate a condition similar to hemophilia A strongly decreased thrombin generation (curve ( b)). The suppression of thrombin generation as a result of neutralization of FVIII was effectively reversed by the addition of 10 nM of the AcM 1F91 of TFPI-KPI-1 (curve ( c)), AcM 2F3 (curve ( d)), or AcM 2F22 (curve (e)). The parameters of the maximum thrombin height (maximum) and the time to the maximum (ttmax) derived from the curves in Figure 1 and for additional TFPI antibodies (1-79) are listed in Table 5. The data shown in Figure 1 and Table 5 suggest that 1) numerous TFPI antibodies (1-79) are inhibitors of TFPI activity, and 2) individual TFPI inhibitor antibodies (1-79) increased the maximum thrombin height and decrease the time for maximum height by a different measure.

Figure 2 (curve (a)) shows the thrombin generation curve with NHP. The NHP contains approximately 1.6 nM of TFPI, of which only approximately 0.2 nM are present as full-length human TFPIa. Figure 2 shows that the addition of 5 nM of the full length recombinant human TFPIa to the neutralized plasma for FVIII completely prevents the generation of measurable thrombin (curve ( b) b)). The addition of 10 nM of a high affinity antibody against KPI-1 (AcM 1F91) was completely unable to establish a significant thrombin generation in the presence of 5 nM TFPIa (curve (e)). Surprisingly, however, at these antibody concentrations the AcM 2F22 (curve ( d)) and to a lesser extent the AcM 2F3 (curve ( c)) could establish a solid thrombin generation that was comparable to the thrombin generation in human plasma normal.

The parameters of the maximum height of the thrombin (maximum) and the time to the maximum (ttmax) derived from the curves in Figure 2 and the additional TFPI mAbs (1-79) are listed in Table 5. The data suggest that some TFPI antibodies, but not all, effectively neutralize TFPI-mediated inhibition under conditions with elevated TFPI levels and restore thrombin generation to a level comparable to that of NHP. Similar results were found when selected antibodies (200 nM) were evaluated at an elevated TFPIa concentration of 20 nM (Table 6). TFPI 2F22 antibody was compared with different TFPI antibodies previously described (10R-T141A (Fitzgerald), MBS532510 (MyBioSource), ADG4903 (American Diagnostica GmbH), 2H8 (Mast et al., Arterioscler Thromb Vasc Biol. (2002) 22: 2099-2104)) for its ability to neutralize TFPI-mediated inhibition at elevated TFPI levels (20 nM). The thrombin generation parameters are listed in Table 7 and the data show that TFPI AcM 2F22 antibody efficiently neutralizes TFPI-mediated inhibition at elevated TFPI levels, while the KPI-1 TFPI antibodies described above do not. were able to neutralize the inhibition mediated by TFPI under these conditions. AcM 2F22 was expressed as a chimeric antibody AcM 0294 and chimeric antibody fragments Fab 0295 and Fab 0296. The data in Table 8 shows that the Fab fragment 0295 as a representative of AcM 0294 / AcM 2F22 (Fab 0295: SEQ ID NO : 17 and 20) retains a high neutralization capacity of TFPI-mediated inhibition in plasma neutralized for FVIII at normal or elevated levels of TFPI.

Table 5: Maximum thrombin height (maximum) and time to maximum height (maximum tt) derived from thrombin generation curves that were obtained in neutralized plasma for FVIII with or without 5 Nm of TFPIa

Table 6: Maximum thrombin height (maximum) and time to maximum height (maximum tt) derived from thrombin generation curves that were obtained in the neutralized plasma for FVIII with 20 nM TFPIa

Table 7: Maximum thrombin height (maximum) and time to maximum height (maximum tt) derived from thrombin generating curves that were obtained in the neutralized plasma for FVIII with 20nM TFPIa

Table 8 Maximum thrombin height (maximum) and time to maximum height (maximum tt) derived from thrombin generation curves that were obtained in the neutralized plasma for FVIII with and without 20 nM TFPIa

Example 6: Crystal structure of the Kunitz-like protease inhibitor domain of TFPI in complex with a Fab fragment of AcM 2F22

The 3D structure of the N-terminal part of human TFPI (1-79), which consists of an acid N-terminal region and a Kunitz 1 protease inhibitor domain (KPI-1) (SEQ ID NO: 2), in complex with a Fab fragment, Fab 0296 (SEQ ID NO: 16 and 17) of the AcM 2F22 was determined at high resolution by the use of X-ray crystallography. The results demonstrate that the antibody is capable of binding to the KPI-1 of the TFPI, and part of the previous N-terminal end. Residues resulting from the epitope of human TFPI include Leu 16, Pro 17, Leu 19, Lys 20, Leu 21, Met 22, Phe 25, Cys 35, Ala 37, Met 39, Arg 41, Tyr 56, Gly 57, Gly 58, Cys 59, Glu 60, Gly 61, Asn 62, Gln 63, Arg 65, Phe 66, Glu 67 , Glu 71 and Met 75 (SEQ ID NO: 2).

Materials and methods

For crystallographic purposes, CMV promoter-based expression vectors (pTT vectors) were generated for transient expression of the Fab fragment corresponding to the AcM 2F22 antibody fragment for crystallography as described in Example 2.

The Fab fragment of AcM 2F22 was expressed in a murine-human chimeric form of Fab 0296 (SEQ ID NO: 16 and 17) in EXPI293F cells and was purified by standard affinity chromatography using KappaSelect resin as described in the Example 2.

The human KPI-1 TFPI, which includes the N-terminal part of the human TFPI, and, additionally, a GSSGSSG tag attached at the N-terminal end (SEQ ID NO: 18) and Fab 0296 consisting of a light chain corresponding to the SEQ ID NO: 17 and a heavy chain fragment corresponding to SEQ ID NO: 17, both in a phosphate buffered saline buffer (PBS) (4 tablets in 2 liters of water, GIBCO Cat. No. 18912- 014, Invitrogen Corporation), were mixed with a slight molar excess (1.1: 1) of the TFPI species. Then, the complex was concentrated to approximately 10.0 mg / mL by using an Amicon Ultra-4 centrifuge filter with a molecular weight cut of 10,000 Da. The crystals were grown using the seated drop technique by using a 96-well TTP IQ plate from TTP Lab Tech no: 4150-05800 and 100 pl of precipitant solution per well. The precipitating solution contained PEG 335020% w / v, 200 mM potassium format and was mixed with the protein solution in a 3: 1 ratio. The initial total drop size was 200 nL and the crystals appeared after a few days. A cold freezing crystal was prepared by transferring 1 µl of a mixture of freezing solution containing 75% of the precipitating solution and 25% glycerol to the drop containing the crystal. The soak was left for about 2 minutes. The crystal was then taken, immediately frozen in liquid N ² and maintained at a temperature of 100 K by a stream of cryogenic N ² gas during data collection. Crystallographic data were collected, at a resolution of 1.65 A in the BL911-3 beam line in MAX-lab, Lund, Sweden. The determination, integration and scaling of data from the spacer group was carried out using the XDS software package [Kabsch, W., J.Appl.Crystallogr., (1993), Vol. 26, pages 795-800]. The spacer group was determined to be C2 and the cell parameters for the synchrotron data were determined as 89,010, 66,660, 106,110 A, respectively, and with a 111.18 ° angle. The R-sym at the resolution of 1.65 A was 8.4% and the whole 99.5%. The mean intensity / sigma (intensity) of single reflections was equal to 2.0 at approximately 1.8 A resolution.

The molecular replacement method (MR) was used to determine the structure by using the coordinates of a Fab molecule with 1NGZ access code [Yin, J. et al., Proc Natl Acad Sci US A.2003.Feb. 4, (100), Vol. 100 pages 856-861] of the Protein Data Bank (PDB) [Berman, HM et al., Nucleic Acids Res., (2000), Vol. 28, pages 235-242]. The Fab molecule was divided into two domains, the variable and constant domains, which each was used as a search model in the MR calculations. The Molrep software [Vagin, A. et al., J.Appl.Crystallogr., (1997), Vol. 30, pages 1022-1025] of the CCP4 package [Collaborative Computational Project, N., Acta crystallographica.Section D, Biological crystallography, (1994), Vol. 50, pages 760-763] was used to find the positions of the constant and variable Fab domains. The KPI-1 domain was not found in the MR stage, however, the electron density difference map indicated the approximate positions of the KPI-1 domain molecules at this stage. Improvements in electron density through the DM computer program of the CCP4 software package, followed by automated model and phase improvements through the use of the ARP-wARP software [Langer, G. et al., Nat Protoc, (2008) , Vol. 3, pages 1171-1179] [Murshudov, GN et al., Acta Crystallographica Section D Biological Crystallography, (2011), Vol. 67, pages 355-367] provided an almost complete structure of both the Fab 0296 molecule and the structure of the KPI-1 domain, and part of the N-terminal end that precedes the KPI-1 domain. For TFPI (SEQ ID NO: 2) residues from 15 to 77 were included in the X-ray model which, in addition to the KPI-1 domain also includes some residues of the N-terminal end of KPI-1 (residues 26-76 ). For the Fab 0296 fragment, light chain residues 1 to 212 and heavy chain residues 1 to 221 are observed. A procedure for inspecting computer graphics of electron density maps, model corrections and creation was recorded using the use of the Coot software [Emsley, P. et al., Acta Crystallogr.Sect.D-Biol.Crystallogr., (2004), Vol. 60, pages 2126-2132] followed by crystallographic improvements, through the use of computer programs Refmac5 [Murshudov, GN et al., Acta Crystallographica Section D Biological Crystallography, (2011), Vol. 67, pages 355-367] of the CCP4 software package. The procedure was subjected to a cycle until no further significant improvements could be made to the model. The final R and the free R for all data at a resolution of 1.65 A were 0.192 and 0.220, respectively.

Results

The calculation of the average areas excluded in peer interactions using the Areaimol software [Lee, B. et al., J Mol Biol, (1971), Vol. 55, pages 379-400] [Saff, EB et al., Math Intell , (1997), Vol. 19, pages 5-11] of the CCP4 program package [Collaborative Computational Project, N., Acta crystallographica. Section D, Biological crystallography, (1994), Vol. 50, pages 760-763] provided for the human TFPI fragment / anti-TFPI2F22 Fab the molecular complex of crystalline structure 1195 A2.

Direct contacts between the TFPI KPI-1, which includes the N-terminal part of the TFPI observed in the crystalline structure, (SEQ ID NO: 2) and Fab 0296 (SEQ ID Nos: 16 and 17), were identified by execution of the Contacts software of the CCP4 program package [Bailey, S., Acta Crystallogr.Sect.D-Biol.Crystallogr., (1994), Vol. 50, pages 760-763] by using a cutting distance of 4.0 A between Fab 0296 and TFPI fragment molecules. The results of the soluble TFPI fragment / complex crystal structure Fab 0296 are shown in Table 9.

It was found that the epitope resulting from TFPI KPI-1, which includes the N-terminal region of TFPI, for Fab 0296 comprises the following TFPI residues (by using the sequence numbering of SEQ ID NO: 2): Leu 16, Pro 17, Leu 19, Lys 20, Leu 21, Met 22, Phe 25, Cys 35, Ala 37, Met 39, Arg 41, Tyr 56, Gly 57, Gly 58, Cys 59, Glu 60, Gly 61, Asn 62, Gln 63, Arg 65, Phe 66, Glu 67, Glu 71 and Met 75. Evaluated from distances, charge-by-charge interactions, hydrogen bonds, polar and hydrophobic interactions and low solvent accessibility, the following residues They appear to be particularly important residues of the epitope: Arg 41, Arg 65 and Glu 67 (SEQ ID NO. 2).

Therefore, the TFPI epitope of the AcM 2F22 (represented by Fab 0296) comprises residues that precede the KPI-1 domain, which includes a short helix in the N-terminal, residues in the loop before the p-chains 1 of the KPI-1 domain and residues at the beginning of p-chain 1. In addition, it includes residues at the end of p-chain 2 and residues in the loop between p-chain 2 and the a-helix of end C- KPI-1 terminal and waste inside the helix of the C-terminal end of KPI-1.

Therefore, the results show that Fab 0296, and therefore the AcM 2F22 specifically bind TFPI KPI-1 and part of the previous N-terminal region.

The Fab 0296 paratope for TFPI KPI-1 includes Val 2, Phe 27, Tyr 32, Trp 52, Arg 53, Gly 54, Gly 55, Ser 56, Ile 57, Asp 58, Tyr 59, Ala 61, Met 64, Lys 97, Ser 99, His 100, Asn 102, Tyr 103, Val 104, Gly 105 and Tyr 106 of the heavy chain (H) (SEQ ID NO: 16, Table 9), and Pro 31 residues, Ala 32, Tyr 49, Ser 50, Asn 53, Tyr 55, Thr 56, Tyr 91, Thr 92, Ser 93 and Tyr 94 of the light chain (L) (SEQ ID NO: 17, Table 9).

Table 9: Data on the crystal structure complex of the TFPI / Fab fragment 0296

TFPI KPI-1, K chain, ( SEQ ID NO: 2) interactions with the heavy chain ( H chain) of Fab 0296 ( SEQ ID NO: 16) and the light chain ( L chain) of Fab 0296 ( SEQ ID NO : 17) for the crystallographic complex. A cut-off distance of 4.0 was used. The contacts were identified by the CONTACT computer software of the CCP4 package [Collaborative Computational Project, N., Acta crystallographica. Section D, Biological crystallography, ( 1994), Vol. 50, pages 760-763]. In the last column "***" indicates a strong possibility for a hydrogen bond in this contact (distance <3.3 A) as calculated by CONTACT, "*" indicates a weak possibility (distance> 3.3 A) . Blank indicates that the program considered that there is no possibility of a hydrogen bond. Hydrogen bonds are specific between a donor and an acceptor, are typically strong, and are easily identified.

Example 7: Effect of TFPI-specific antibodies for TFPI (1-79) on the affinity of FXa for TFPI and on the affinity of soluble FVIIa / TF for TFPI measured by Surface Plasmon Resonance

The ability of TFPI antibodies to inhibit the interaction between TFPI and FXa or TFPI and soluble FVIIa / TF (FVIIa / TFs) was evaluated by using a SPR assay with the Biacore T200 instrument. The affinity of FXa or FVIIa / TFs for TFPI bound to an antibody of TFP and KPI-1 was compared with the affinity of FXa or FVIIa / TFs for TFPI bound to the TFM AcM KPI-34F110 (WO2012 / 001087). As a control, the affinity of FXa or FVIIa / TFs for the TFPI linked to the TFPI AcM KPI-24F36 (WO2010 / 072691) was included in the assay.

Polyclonal rabbit anti-mouse immunoglobulin (Mouse Antibody Capture Kit, GE Healthcare) was immobilized to a series of CM4 S sensor chip (Ge Healthcare) in flow cells 1-4 as described in Example 3 For each experiment, the relevant N-terminal anti-TFPI antibodies (2F22, 2F3, 2F45, 2F48, 1F91, 2F35, 10R-T141A (Fitzgerald), MBS532510 (MyBioSource), ADG4903 (American Diagnostica Gmbh), 2H8 (Mast and others, Arterioscler Thromb Vasc Biol. (2002) 22: 2099-2104)), anti-TFPI KPI-2 antibody (4F36, WO2010 / 072691) or AcM anti-TFPI KPI-3 antibody (4F110 described in WO2012 / 001087) were injected for 180 sec into flow cells 2, 3 or 4 at 10 | iL / min flow rate, followed by a subsequent injection of 20 nM human TFPIa (SEQ ID NO: 1) for 180 sec at all flow cells (1-4) at 10 | iL / min flow rate. Different concentrations (25 nM, 12.5 nM, 6.25 nM and 0 nM) of FXa (Haematological Technologies) were evaluated by diluting the sample in run buffer (10 mM HEPES, pH 7.4, 150 mM NaCl , 10 mM CaCb, P200.05 surfactant, BSA 1 mg / mL). Each sample was analyzed by using 120 seconds of contact time followed by 180 seconds of dissociation time at 30 pL / min flow rate. The sensor surface was regenerated with 10 mM Glycine pH 1.7 with two cycles each of 30 seconds at 30 µl / min flow rate. Each FVIIa sample was prepared at a final concentration of 320 nM, 160 nM, 80 nM or 0 nM in run buffer containing 620 nM TFs. The soluble tissue factor 1-219 (TFs) was prepared according to the published procedures (Freskgard et al., 1996). The expression and purification of recombinant FVIIa was performed as described above (Thim et al., 1988; Persson and Nielsen, 1996). The FVIIa / TFs complex was incubated at room temperature for 10-15 min before starting the first sample injection. Each sample was analyzed by using 120 seconds of contact time followed by 180 seconds of dissociation time at 30 | jL / min flow rate. The sensor surface was regenerated with 10 mM Glycine pH 1.7 with two cycles each of 30 seconds at 30 µl / min flow rate. The Biacore T200 Evaluation software (version 2.0) was used to analyze the data. Determination of binding constants (k ^a , k ^d , K ^d ) was obtained assuming a 1: 1 interaction of TFPI and FXa or TFPI and FVIIa / TFs.

Some TFPI-specific anti-TFPI antibodies (1-79) (AcM 2F22, 2F3 and 2F45) decreased the affinity of FXa for TFPI more than 30 times compared to the affinity of FXa for TFPI linked to the anti-TFPI AcM KPI-34F110, while all other antibodies tested did not have or only had a minor effect (less than 10 times) on the affinity of FXa for TFPI (Table 10). The decrease in affinity is mainly due to a faster rate of dissociation (k ^d ).

All TFPI-specific anti-TFPI antibodies (1-79) blocked the binding of TFPI to FVIIa / TFs (Table 11).

Table 10: The affinity of FXa for human TFPIa bound to the indicated antibody was measured by SPR analysis. Determination of binding constants (ka, kd, KD) was obtained assuming a 1: 1 interaction of human TFPIa and Fxa

Table 11: The affinity of FVIIa / TFs for human TFPIa bound to the indicated antibody was measured by SPR analysis. Determination of binding constants (ka, kd, KD) was obtained assuming a 1: 1 interaction of human TFPIa and the FVIIa / TFs complex. n.b .: no detectable junction

Example 8: Effect of TFPI-specific anti-TFPI antibodies (1-79) on the inhibition of the amidolytic activity of the TFa-mediated FXa

The effect of the antibodies on TFPIa-mediated inhibition of the amidolytic activity of FXa (Enzyme Research Laboratories Ltd.) towards the chromogenic substrate S-2765 (Cromogenix) was analyzed in buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM CaCb, 0.1 mg / mL BSA. The anti-TFPI AcM (32 nM) was incubated with 8 nM TFPI (SEQ ID NO: 1) for 30 min at room temperature. S-2765 (0.5 mM) was added and incubated for 5 min. The reactions were initiated by the addition of FXa to a final concentration of 0.1 nM. Product formation was measured after 40 min incubation at 405 nM on a Spectramax 340 Microplate spectrophotometer. The activity in the absence of TFPIa was set at 100% and the activity in the presence of TFPIa, but in the absence of the antibody, it was set at 0%. The results are shown in Table 12. As expected, pre-incubation of TFPIa with KPI-2 AcM2021 (described in WO2010 / 072691) completely neutralized the activity of TFPI a towards FXa. The effect of TFPI-KPI-1 antibodies could be divided into two groups, a group (2F3, 2F22, 2F45) that partially neutralizes the inhibition of FXa mediated by TFPIa (> 20%) and another group, which did not affect the inhibition of FXa mediated by TFPIa appreciably (<20%).

Table 12: Effect of antibodies on inhibition of FXa mediated by TFPIa

Remaining activity of FXa measured after 40 min incubation with TFPIa. The activity in the absence of TFPIa was set at 100% and the activity in the presence of TFPIa, but in the absence of antibody, it was set at 0%.

Example 9: Reversal of TFPIa-mediated inhibition by TFPI-specific anti-TFPI antibodies (1-79) measured by the amidolytic activities of TFs / FVIIa- and Fxa

The effect of the antibodies on the inhibition of the activity of FVIIa / TFs mediated by TFPIa was studied in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM CaCb, 0.1% BSA. The reaction mixture contained 5 nM FVIIa, 10 nM soluble TF (TFs) and 0.5 mM chromogenic substrate S-2288 (HD-Ile-Pro-Arg-pNa, Chromogenix). The soluble tissue factor 1-219 (TFs) was prepared according to the published procedures (Freskgard et al., 1996). The expression and purification of recombinant FVIIa was performed as described above (Thim et al., 1988; Persson and Nielsen, 1996). The activity was measured at room temperature by the change in OD ^{405 nm} and the activity in the absence of TFPI was set at 100%. FVIIa / TFs activity was inhibited by the addition of 150 nM of TFPIa (SEQ ID NO: 1), and the inhibition was reversed by 200 nM of various antibodies against TFPI (1-79) as shown in Table 13. Some antibodies against TFPI (1-79) (1F91, 2F3, 2F22, 2F45) reversed the inhibition of FVIIa / TFs mediated by TFPIa under these conditions. A TFPI antibody (1-79) (2F35) and the KPI-2 antibody (2021 described in WO2010 / 072691) were without an appreciable effect.

The effect of KPI-1 antibodies on the reversal of inhibition of FXa activity mediated by TFPIa was then studied. The reaction mixture contained 1 nM FXa, and 0.5 mM of the S-2765 chromogenic substrate (ZD-Arg-Gly-Arg-pNa, Chromogenix) in 20 mM HEPES at pH 7.5, 150 mM NaCl, 5 mM CaCb , BSA 0.1%. The FXa was incubated with 4 nM TFPIa (SEQ ID NO: 1) for 15 min followed by the addition of S-27650.5 mM. Then, the activity was measured at room temperature by the change in OD ^{405 nm} . Reversal of the inhibition was induced after 15 min by the addition of 20 nM TFPI (1-79) antibodies (1F91, 2F3, 2F22, 2F35, 2F45) or KPI-2 (2021 described in WO2010 / 072691). The resulting progress of the curve was followed and the slope measured 100 min after the addition of the antibodies. The activity in the absence of the TFPI was set at 100%. The results are shown in the Table 13. Since KPI-2 binds to FXa, it was surprising that a number of anti-TFPI antibodies specific for TFPI (1-79) (2F3, 2F22, 2F45) more efficiently reversed inhibition of mediated FXa activity by the TFPIa that the AcM 2021 that binds to an epitope in the KPI-2 that interacts with the active FXa site. The results suggest that areas on TFPI (1-79), which are not the main contact area in KPI-2, are crucial for the union of TFPIa to FXa.

Table 13: Reversal of the inhibition of amidolytic activity of FVIIa / TFs and FXa mediated by TFPIa

Example 10: Effect of TFPI-specific antibodies (1-79) on the generation of Fxa

The effect of anti-TFPI antibodies on the inhibition of FXa generation mediated by TFPIa was analyzed in buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM CaCh, 1 mg / mL BSA, PEG80000, 1% (p / v). The antibody (64 nM) was incubated with 4 nM TFPIa for 10 min at room temperature, before adding a previously incubated mixture (5 min) of FVIIa (0.5 nM) and 30 pM of Dade Innovin (Dade Behring). The expression and purification of recombinant FVIIa was performed as described above (Thim et al., 1988; Persson and Nielsen, 1996). After the addition of 160 nM of FX (Enzyme Laboratories Ltd) the reactions were incubated in a total volume of 100 mL for 30 min. The reactions were quenched by the addition of 50 mL of stop buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 80 mM EDTA) and the amount of FXa generated by the addition of 50 mL of S-2765 chromogenic substrate (Chromogenix) 2 mM. The cleavage of S-2765 was monitored at 405 nm by using a Spectramax 340 Microplate spectrophotometer. The activity in the absence of TFPIa was set at 100% and the activity in the presence of TFPIa, but in the absence of antibody, it was set at 0%. The results suggest that anti-TFPI (1-79) 2F22 and 2F3 antibodies more efficiently neutralize the inhibition of FXa generation mediated by TFPIa (Table 14). As expected, a KPI-2 TFPI antibody (AcM 2021 described in WO2010 / 072691) completely neutralized the inhibition mediated by TFPI.

Table 14: FXa activity

FXa activity generated after 30 min incubation of FVIIa, Innovin, FX and TFPI with and without antibodies. The activity in the absence of TFPIa was set at 100% and the activity in the presence of TFPIa, but in the absence of antibody, it was set at 0%.

Example 11: Effect of TFPI antibodies specific for TFPI (1-79) on thromboelastography (TEG) in whole blood neutralized for FVIII

Claims (4)

1. An isolated antibody or fragment thereof that specifically binds to an epitope present in amino acid residues 1 to 79 of human TFPI (SEQ ID NO: 1), whose heavy chain comprises: • a CDR1 sequence corresponding to amino acids 31 to 35 of SEQ ID NO: 6 (NYGVH); • a CDR2 sequence corresponding to amino acids 50 to 65 of SEQ ID NO: 6 (VIWRGGSIDYNAAFMS); Y • a CDR3 sequence corresponding to amino acids 98 to 110 of SEQ ID NO: 6 (NSHGNYVGYAMDY); and whose light chain comprises: • a CDR1 sequence corresponding to amino acids 24 to 34 of SEQ ID NO: 7 (KASQSVGPAVA); • a CDR2 sequence corresponding to amino acids 50 to 56 of SEQ ID NO: 7 (SASNRYT); Y • a CDR3 sequence corresponding to amino acids 89 to 96 of SEQ ID NO: 7 (QQYTSYPT). 2. A pharmaceutical formulation comprising the isolated antibody, or fragment thereof, according to claim 1 and at least one pharmaceutically acceptable excipient. 3. The antibody, or fragment thereof, according to claim 1 or the pharmaceutical formulation according to claim 2 for use as a medicament. 4. The antibody, or fragment thereof, for use according to claim 3, wherein said subject has congenital, acquired and / or iatrogenic coagulopathy, such as hemophilia A, with or without inhibitors, and hemophilia B, with or without inhibitors.