Bispecific antibodies

Abstract

To provide improved compounds that can support prophylactic treatment with less frequent dosing and SC administration, for use in treating blood coagulation related diseases.SOLUTION: The invention relates to a bispecific antibody comprising a first antigen-binding site capable of binding Factor VII(a) and a second antigen-binding site capable of binding TLT-1, pharmaceutical formulations comprising such bispecific antibodies and uses thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to bispecific antibodies exhibiting improved pharmaceutical properties, compositions comprising such antibodies, and uses of such antibodies and compositions, including pharmaceutical and therapeutic uses.

[0002] Sequence Listing This application is submitted with a Sequence Listing in electronic format, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] The process of blood coagulation involves several proteins that act together after vascular injury to generate a clot that prevents severe loss of fluids and / or pathogenic invasion. The cascade of events that leads to the formation of a clot can be initiated through two pathways known as the intrinsic (contact) and extrinsic (tissue factor) pathways. Each pathway is composed of a series of zymogen activation steps in which a newly activated enzyme catalyzes the activation of the next zymogen in the series until prothrombin is converted to thrombin. Thrombin converts fibrinogen into a fibrin meshwork and activates platelets to form a platelet clot, which together form a stable clot. Initiation of the extrinsic coagulation pathway is mediated by the formation of a complex between membrane-bound tissue factor (TF), which is exposed as a result of injury to the vessel wall, and low levels of circulating factor VIIa (FVIIa). The FVIIa:TF complex initiates the coagulation cascade by activating small amounts of coagulation factor IX (FIX) and factor X (FX). During the early phase, low concentrations of FXa generate trace amounts of thrombin that can activate factor XI and cofactors VIII and V. During the proliferation phase, procoagulant complexes are assembled, which consist of the tenases FIXa, FVIIIa, and Ca, respectively. 2+ , phospholipids) and prothrombinase (FXa, FVa, Ca 2+ , phospholipid) complex significantly enhances the generation of FXa and thrombin.

[0004] In patients with hemophilia A and B (HA and HB, respectively), various steps of the coagulation cascade malfunction due to the absence or insufficient presence of functional FVIII and FIX, respectively. This leads to coagulation disorders and insufficient blood clotting, as well as potentially life-threatening bleeding or damage to internal organs such as joints.

[0005] Recombinant FVIIa (rFVIIa) is widely used as a bypass agent for on-demand (OD) treatment of bleeding in hemophilia (A and B) patients with inhibitors (HwI). rFVIIa has a short systemic half-life of 2-3 hours when administered intravenously (IV) and low bioavailability when administered subcutaneously (SC). The short systemic half-life of rFVIIa is thought to be due to the involvement of several mechanisms, including inhibition by the plasma inhibitors antithrombin III (AT) (Agersφ H et al. (2010) J. Thromb. Haemost. 9:333-8.) and alpha-2-macroglobulin (α2M) as well as renal clearance. The short systemic half-life and low SC bioavailability of rFVIIa make it difficult to utilize rFVIIa for prophylactic treatment. Furthermore, the low intrinsic activity of rFVIIa necessitates the administration of higher rFVIIa doses.

[0006] Therefore, there is a need for improved compounds that can support prophylactic treatment with less frequent dosing and with SC administration.

[0007] Roche has recently launched the bispecific antibody emicizumab, which is indicated for routine prophylaxis administered once weekly by SC administration in people with HA and HA with inhibitors (HAwI). Nevertheless, the development of safe and effective molecules with alternative mechanisms of action is an area of ​​significant interest to improve and complement the standard of care for patients with hemophilia. Summary of the Invention

[0008] The present invention relates to bispecific antibodies exhibiting improved pharmaceutical properties, in particular bispecific antibodies that may be used in the treatment of subjects with congenital and / or acquired coagulation disorders, such as for example people with hemophilia A or B, with or without inhibitors. Furthermore, the present invention is particularly related to bispecific antibodies capable of binding coagulation factor VII (FVII(a)) and TREM-like transcript 1 (TLT-1).

[0009] In one embodiment, a bispecific antibody of the invention comprises (i) a first antigen-binding site that binds FVII(a), and (ii) a second antigen-binding site that binds TLT-1.

[0010] In one embodiment of the invention, the bispecific antibody extends the active circulating half-life of endogenous FVIIa without loss of endogenous FVIIa activity and stimulates endogenous FVIIa activity by selectively localizing to activated platelets.

[0011] In one embodiment of the invention, the bispecific antibody comprises an Fc region, which mediates recycling of the bispecific antibody and extends its half-life in the circulation. In one embodiment of the invention, the first antigen-binding site competes for binding to FVII(a) in a competitive ELISA assay with any one of the anti-FVII(a) antibodies comprising a light chain variable domain (VL) and a heavy chain variable domain (VH) as set forth below. mAb0522 (VL: SEQ ID NO: 846 and VH: SEQ ID NO: 850), Fab0883 (VL: SEQ ID NO: 814 and VH: SEQ ID NO: 818), mAb0005 (VL: SEQ ID NO: 750 and VH: SEQ ID NO: 754), mAb0004 (VL: SEQ ID NO: 14 and VH: SEQ ID NO: 18), mAb0013 (VL: SEQ ID NO: 46 and VH: SEQ ID NO: 50), mAb0018 (VL: SEQ ID NO: 62 and VH: SEQ ID NO: 66), mAb0544 (VL: SEQ ID NO: 694 and VH: SEQ ID NO: 698), mAb0552 (VL: SEQ ID NO: 702 and VH: SEQ ID NO: 706), mAb0001 (VL: SEQ ID NO: 710 and VH: SEQ ID NO: 714), mAb0007 (VL: SEQ ID NO: 718 and VH: SEQ ID NO: 722), mAb0578 (VL: SEQ ID NO: 726 and VH: SEQ ID NO: 730), mAb0701 (VL: SEQ ID NO: 734 and VH: SEQ ID NO: 738), and mAb0587 (VL: SEQ ID NO: 742 and VH: SEQ ID NO: 746)

[0012] In a further embodiment of the invention, the first antigen-binding site of the bispecific antibody is capable of binding an epitope comprising amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO:1).

[0013] In one aspect of the invention, the bispecific antibody is formulated into a pharmaceutical formulation comprising the bispecific antibody of the invention and a pharma- ceutical acceptable carrier.

[0014] In one embodiment of the invention, the bispecific antibody is administered parenterally, such as intravenously, intramuscularly, or subcutaneously. In one embodiment of the invention, the bispecific antibody allows for prophylactic treatment of subjects with congenital and / or acquired coagulation disorders, such as hemophilia A, hemophilia B, hemophilia A with inhibitors, or hemophilia B with inhibitors. Thus, the bispecific antibody is designed to provide hemostatic coverage for bleeding. In one embodiment of the invention, the bispecific antibody is designed to be suitable for administration once a week, once a month, or less frequently.

[0015] Treatment with the bispecific antibodies of the invention may offer many advantages, such as longer duration between injections, more convenient administration, and potentially improved hemostatic protection between injections. Thus, the bispecific antibodies described herein may have a substantial impact on the quality of life of individuals with hemophilia A or B, with or without inhibitors.

[0016] Brief Description of the Sequence Listing Table 1 summarizes the antibodies and their corresponding SEQ ID NOs for the corresponding VL and VL domain sequences. The heavy chain constant domain type is as defined in Table 2a ("M" indicates the mouse IgG1 constant domain).

[0017] Table 2a summarizes the various formats used for recombinant expression of bivalent, monovalent (OA) and bispecific antibodies. SEQ ID NOs corresponding to the first heavy chain (HC-1) and the second heavy chain (HC-2, or truncated heavy chain (trHC) for OA antibodies) are listed. The light chain constant domain was human kappa corresponding to SEQ ID NO: 12 in all cases.

[0018] Table 2b is an overview of the mouse anti-FVII(a) antibodies of the invention. The correspondence between clone name, fully mouse (hybridoma-derived, except for recombinantly expressed mAb0765) antibodies and the corresponding mouse / human chimeric variants (mouse variable domains, human IgG4 S228P constant domain).

[0019] Table 2c is a summary of antibodies in the mouse and humanized 11F2 line.

[0020] Table 2d is a summary of the anti-TLT-1 mAb0012 series of antibodies. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 2] [Table 3] [Table 4] [Table 5]

[0021] In the present invention, all recombinantly produced antibodies were expressed in a human IgG4 background and all contained the standard hinge-stabilizing substitution S228P (EU numbering). In some variants, the C-terminal lysine of the heavy chain (K447, which is rapidly cleaved in vivo; see Cai et al. Biotechnol. Bioeng. 2011 vol.108, pp404-412) was omitted (referred to as delta-lys). According to Table 2a, additional substitutions were introduced into the heavy chain constant domain to ensure the desired chain pairing in bispecific and monovalent antibodies. (Duobody mutations F405L R409K (Labrijn et al. PNAS 2013,vol.110,pp.5145-5150) and knob-in-hole mutations (see Carter et al. J.Imm.Methods 2001,,vol.248,pp.7-15) T366W (knob) and T366S L368A Y407V (hole)), or extend half-life in vivo (YTE mutations M252Y S254T T256E (Dall'Acqua et al. J.Biol.Chem. 2006,vol.18,pp.23514-23524)). All recombinantly produced antibodies of the present invention have a human kappa light chain constant domain (SEQ ID NO:12). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention relates to the design and use of antibody compositions exhibiting improved pharmaceutical properties, in particular bispecific antibodies capable of binding the coagulation FVII(a) and TLT-1.

[0023] antibody As used herein, the term "antibody" refers to a protein derived from an immunoglobulin sequence capable of binding to an antigen or a portion thereof. The term antibody includes, but is not limited to, full-length antibodies of any class (or isotype), i.e., IgA, IgD, IgE, IgG, IgM, and / or IgY.

[0024] Of particular interest for therapeutic antibodies are the IgG subclasses, which in humans are divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequences of their heavy chain constant regions. Light chains can be divided into two types, kappa and lambda, based on differences in their sequence composition. IgG molecules are composed of two heavy chains linked by two or more disulfide bonds, and two light chains, each attached to a heavy chain by a disulfide bond. IgG heavy chains are composed of heavy chain variable domains (V H ) and up to three heavy chain constants (C H ) Domain: C H 1. C H 2, and C H 3. The light chain may comprise a light chain variable domain (V L ) and the light chain constant domain (C L ). V H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L A domain is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable domains, containing the hypervariable regions (CDRs), form a structure that can interact with an antigen, while the constant region of the antibody can mediate binding to the Fc receptor and C1q, the first component of the C1 complex of the classical complement system.

[0025] The term "antigen-binding site" or "binding portion" refers to the part of an antibody that enables antigen binding.

[0026] The term "antigen-binding fragment" of an antibody refers to a fragment of an antibody that retains the ability to bind to its cognate antigen, such as FVII(a), TLT-1, or another target molecule, as described herein. Examples of antigen-binding fragments include Fab, Fab', Fab2, Fab'2, Fv, single chain Fv (scFv), or single VFv. H Domain or V L These include (but are not limited to) domains.

[0027] As used herein, the term "one-armed antibody" refers to a particular type of monovalent antibody fragment composed of an antibody heavy chain, a truncated heavy chain lacking the Fab region, and a single light chain.

[0028] As used herein, the term "monospecific" antibody refers to an antibody capable of binding to one specific epitope (including but not limited to bivalent antibodies).

[0029] As used herein, the terms "bispecific antibody" and "biAb" refer to an antibody capable of binding to two different antigens, such as FVII(a) and TLT-1, or two different epitopes on the same antigen.

[0030] The bispecific antibody of the present invention is derived from an antibody or an antigen-binding fragment thereof. The bispecific antibody of the present invention may be a fusion or conjugate of an antibody and an antigen-binding fragment of an antibody, such as Fab, Fab', Fab2, Fab'2, or scFv. The bispecific antibody of the present invention may be a fusion or conjugate of an antibody fragment. A wide range of molecular formats of bispecific antibodies derived from antibodies and antibody fragments are known in the art, see for example (Spiess et al.: Molecular Immunology 67, (2015), pp. 95-106) and (Brinkmann and Kontermann: MABS, 9 (2017), pp 182-212).

[0031] Bispecific antibodies may be produced in various ways described in the art, see for example (Spiess et al.: Molecular Immunology 67, (2015), pp. 95-106) and (Brinkmann and Kontermann: MABS, 9 (2017), pp 182-212). For example, the desired heavy chain pairing can be achieved by engineering the dimerization interface of the Fc region to promote heterodimerization. One example of this is the so-called knob-in-hole mutation, where a sterically bulky side chain (knob) is introduced on one Fc that matches a sterically small side chain (hole) on the opposing Fc, thereby creating steric complementarity that promotes heterodimerization. Other methods for engineered heterodimerization Fc interfaces are electrostatic complementarity, fusion to non-IgG heterodimerization domains, or using the natural Fab arm exchange phenomenon of human IgG4 to perform heterodimerization in vitro. Examples of heterodimerized bispecific antibodies are described in detail in the literature, for example (Klein C, et al.; MAbs. 2012 4, pp653-663). Special attention should be paid to the light chain of the heterodimeric antibody. Correct pairing of LC and HC can be achieved by using a common light chain. Again, manipulation of the LC / HC interface can be used to facilitate heterodimerization or light chain crossover engineering, as in the case of CrossMab. In vitro reconstitution of antibodies under mild reducing conditions from two individual IgGs containing appropriate mutations can also be used to generate bispecific antibodies (e.g., Labrijn et al., PNAS, 110 (2013), pp5145-5150). A natural Fab arm exchange method has also been reported to ensure correct light chain pairing.

[0032] As used herein, the term "multispecific" antibody refers to an antibody that can bind to two or more different antigens or two or more different epitopes on the same antigen. Thus, multispecific antibodies include bispecific antibodies.

[0033] The antibodies herein may be combined with other antibodies and antibody fragments known in the art to create bispecific, trispecific, or multispecific antibody molecules.

[0034] In one aspect, the antibody of the present invention is a chimeric, human, or humanized antibody. Such antibodies can be generated, for example, by using a suitable antibody display or immunization platform, or any other suitable platform or method known in the art.

[0035] Furthermore, if the antibody contains a constant region, the constant region, or a portion thereof, also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0036] Human antibodies may be isolated from sequence libraries constructed based on a selection of human germline sequences, and further diversified with natural and synthetic sequence diversity. Human antibodies may be prepared by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus. Human antibodies may also be produced by recombinant methods known in the art.

[0037] The term "humanized antibody" as used herein refers to a human / non-human antibody that contains sequences (CDR regions or portions thereof) derived from a non-human immunoglobulin. Thus, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from at least the hypervariable region of the recipient are replaced with residues from the hypervariable region of an antibody from a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, sequence composition and functionality. In some cases, framework (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. An example of such a modification is the introduction of one or more so-called back mutations, which are typically amino acid residues derived from the donor antibody. Antibody humanization may be performed using recombinant techniques known to those skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. Lo). Suitable human recipient frameworks for both the light and heavy chain variable domains can be identified, for example, by sequence or structural homology. Alternatively, a fixed recipient framework may be used, for example, based on knowledge of structure, biophysical properties, and biochemical properties. The recipient framework may be derived from germline or from mature antibody sequences. The CDR regions from the donor antibody may be transferred by CDR grafting. The CDR-grafted humanized antibody may be further optimized, for example, for affinity, functionality, and biophysical properties, by identifying key framework positions where reintroduction of amino acid residues from the donor antibody (back mutation) beneficially affects the properties of the humanized antibody. In addition to back mutation from the donor antibody, the humanized antibody may be engineered by introducing germline residues into the CDR or framework regions, removing immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0038] Furthermore, 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 antibody performance. The humanized antibody may optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0039] The term "chimeric antibody" as used herein refers to an antibody that comprises antibody portions derived from two or more species. For example, the genes encoding such antibodies include genes encoding variable domains and genes encoding constant domains originating from two different species. For example, genes encoding the variable domains of a mouse monoclonal antibody may be combined with genes encoding the constant domains of an antibody of human origin.

[0040] Antibodies or fragments thereof may be defined in terms of their complementarity determining regions (CDRs). The term "complementarity determining region" as used herein refers to the regions of an antibody where the amino acid residues involved in antigen binding are typically located. CDRs may be identified as the regions with the highest variability between antibody variable domains. Databases such as the Kabat database may be used for CDR identification, which are defined as including, for example, amino acid residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain, and amino acid residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. As used herein, phrases such as "Kabat position", "Kabat residue" and "according to Kabat" refer to this numbering system for the heavy chain variable domain or the light chain variable domain. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to shortening of, or insertion into, the framework (FR) or CDR of the variable domain. For example, a heavy chain variable domain may include an amino acid insertion after residue 52 of CDR H2 (residues 52a, 52b, and 52c according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbering sequence at the homology region. Numbering is according to Kabat only where specifically stated, otherwise numbering is consecutive according to the designated SEQ ID NO:

[0041] The term "framework region" or "FR" residues, as defined herein, refer to those V residues that are not within the CDRs. H or V L Refers to amino acid residues.

[0042] An antibody of the invention may comprise the CDR regions from one or more of the specific antibodies disclosed herein.

[0043] The term "antigen" (Ag) refers to a molecular entity used to immunize an immunocompetent vertebrate to generate antibodies (Abs) that recognize the Ag. As used herein, Ag is referred to more broadly and is generally intended to include the target molecule recognized by an Ab.

[0044] The present invention encompasses variants of the antibodies of the invention or antigen-binding fragments thereof, which may contain 1, 2, 3, 4 or 5 amino acid substitutions and / or deletions and / or insertions in the specific sequences disclosed herein.

[0045] "Substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids.

[0046] As used herein, the term "epitope" is defined in terms of the molecular interaction between an "antigen-binding polypeptide" such as an antibody (Ab) and its corresponding antigen (Ag). In general, "epitope" refers to the area or region on an Ag to which an Ab binds, i.e., the area or region that is in physical contact with the Ab. In the present invention, an epitope is determined using an X-ray derived crystal structure to define the spatial coordinates of the complex between an Ab and its Ag, e.g., a Fab fragment. In the present specification, the term epitope is defined as an Ag (herein FVII(a) or TLT-1) residue that is characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 Å from a heavy atom in the Fab, unless otherwise specified or contradicted by context.

[0047] For example, epitopes described at the amino acid level as determined from X-ray structures are said to be identical if they contain the same set of amino acid residues. If at least one amino acid residue is shared by the epitopes, the epitopes are said to overlap. If no amino acid residue is shared by the epitopes, the epitopes are said to be separate (unique).

[0048] The definition of the term "paratope" is derived from the above definition of "epitope" by reversing perspective. Thus, the term "paratope" refers to the area or region on an antibody or fragment thereof to which an antigen binds, i.e., which is in physical contact with the antigen. The term paratope is defined herein as an Ab residue characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 Å from a heavy atom of FVII(a) or TLT-1, unless otherwise specified or contradicted by context.

[0049] An epitope on an antigen may contain one or more hotspot residues, i.e., residues that are particularly important for interaction with a cognate antibody, and interactions mediated by the side chains of said hotspot residues contribute significantly to the binding energy of the antibody / antigen interaction (Peng et al. PNAS 111, (2014), E2656-E2665). Hotspot residues can be identified by testing variants of an antigen in which a single epitope residue is replaced, for example, by alanine for binding to a cognate antibody. If substitution of the epitope residue with alanine strongly affects binding to the antibody, then said epitope residue is considered a hotspot residue and is therefore particularly important for binding of the antibody to the antigen.

[0050] Antibodies that bind to the same antigen can be characterized in terms of their ability to bind simultaneously to their common antigen and may be subject to "competitive binding" / "binning". In this context, the term "binning" refers to a method of grouping antibodies that bind to the same antigen. Antibody "binning" can be based on competitive binding of two antibodies to their common antigen in an assay based on standard techniques. Antibody "bins" are defined using a reference antibody. If the secondary antibody cannot bind to the antigen simultaneously with the reference antibody, the secondary antibody is said to belong to the same "bin" as the reference antibody. In this case, the reference antibody and the secondary antibody competitively bind to the same portion of the antigen and are called "competing antibodies". If the secondary antibody can bind to the antigen simultaneously with the reference antibody, the secondary antibody is said to belong to separate "bins". In this case, the reference antibody and the secondary antibody do not competitively bind to the same portion of the antigen and are called "non-competing antibodies".

[0051] Competitive assays for determining whether an antibody competes for binding with the anti-FVII(a) or anti-TLT-1 antibodies disclosed herein are known in the art. Exemplary competitive assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance analysis (e.g., using a BIAcore™ instrument), biolayer interferometry (ForteBio®), and flow cytometry.

[0052] Typically, a competitive assay involves the use of an antigen bound to a solid surface or expressed on a cell surface, a test FVII- or FVIIa-binding antibody, and a reference antibody. The reference antibody is labeled and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. Usually, the test antibody is present in excess (e.g., 1x, 5x, 10x, 20x, 100x, 1000x, 10000x or 100000x). Antibodies identified as competitive in a competitive assay (i.e., competing antibodies) include antibodies that bind to the same epitope as the reference antibody, or to overlapping epitopes, and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody to create steric hindrance.

[0053] In an exemplary competitive assay, reference anti-FVII or anti-FVIIa antibody is biotinylated using commercially available reagents. Biotinylated reference antibody is mixed with serial dilutions of test antibody or unlabeled reference antibody (self-competitive control), resulting in a mixture of various molar ratios (e.g., 1, 5, 10, 20, 100, 1000, 10000 or 100000 times) of test antibody (or unlabeled reference antibody) to labeled reference antibody. The antibody mixture is added to FVII or FVIIa polypeptide-coated ELISA plate. The plate is then washed, and horseradish peroxidase (HRP)-strepavidin is added to the plate as detection reagent. The amount of labeled reference antibody bound to the target antigen is detected after addition of chromogenic substrates known in the art (e.g., TMB (3,3',5,5'-tetramethylbenzidine) or ABTS (2,2"-azino-di-(3-ethylbenzthiazoline-6-sulfonate). Optical density readings (OD units) are taken using a spectrometer (e.g., a SpectraMax® M2 spectrometer (Molecular Devices)). The response (OD units) corresponding to zero percent inhibition is determined from wells that have no competing antibody. The response (OD units) corresponding to 100% inhibition, i.e., assay background, is determined from wells that have no labeled reference antibody or test antibody. The percentage inhibition of labeled reference antibody against FVII or FVIIa by test antibody (or unlabeled reference antibody) at each concentration is calculated as follows: % inhibition = (1 - (OD units - 100% inhibition) / (0% inhibition - 100% inhibition)) * 100.

[0054] Those skilled in the art will understand that similar assays can be performed to determine whether two or more anti-TLT-1 antibodies share binding regions, bins, and / or competitively bind antigens. Those skilled in the art will also understand that competitive assays can be performed using a variety of detection systems known in the art.

[0055] A test antibody competes with a reference antibody for binding to an antigen if an excess of one antibody (e.g., 1, 5, 10, 20, 100, 1000, 10000 or 100000 fold) inhibits binding of the other antibody, e.g., by at least 50%, 75%, 90%, 95%, or 99%, as measured in a competitive binding assay.

[0056] Unless otherwise stated, competition is determined using a competitive ELISA assay as described above and presented in Examples 7 and 32.

[0057] 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 a monovalent interaction.

[0058] The binding affinity between two molecules, e.g., an antibody or fragment thereof and an antigen, due to a monovalent interaction, is determined by the equilibrium dissociation constant (K D ) can be quantified by determining the K D can be determined by measuring the kinetics of complex formation and dissociation, for example, by surface plasmon resonance (SPR) as performed in Examples 6 and 16, or other methods known in the art. The rate constants corresponding to association and dissociation of monovalent complexes are the association rate constant k, respectively. a (or k on ), and the dissociation rate constant k d (or k off ) is called. D is the formula K D =k d / k a Through, k a and k d Related to:

[0059] According to the above definition, the binding affinities associated with different molecular interactions, such as the comparison of the binding affinities of different antibodies for a given antigen, are expressed as the K D The comparison may be by value comparison.

[0060] The K of the antibody of the invention against its target D may be less than 1 pM, such as less than 10 pM, for example less than 100 pM, such as less than 200 pM, for example less than 400 pM, such as less than 600 pM, for example less than 1 nM, such as less than 5 nM, for example less than 10 nM, such as less than 20 nM, for example less than 50 nM, such as less than 100 nM, for example less than 200 nM, such as less than 400 nM, for example less than 600 nM, such as less than 800 nM.

[0061] In one such embodiment the antibody has a K for FVIIa of less than 1 pM, such as less than 10 pM, for example less than 100 pM, such as less than 200 pM, for example less than 400 pM, such as less than 600 pM, for example less than 1 nM, such as less than 5 nM, for example less than 10 nM, such as less than 20 nM, for example less than 50 nM, such as less than 100 nM, for example less than 200 nM, such as less than 400 nM, for example less than 600 pM, such as less than 800 pM. D and an anti-FVII(a) arm having a K for TLT-1 of less than 1 pM, such as less than 10 pM, for example less than 100 pM, such as less than 200 pM, for example less than 400 pM, such as less than 600 pM, for example less than 1 nM, such as less than 5 nM, for example less than 10 nM, such as less than 20 nM, for example less than 50 nM, such as less than 100 nM, for example less than 200 nM, such as less than 400 nM, for example less than 600 nM, such as less than 800 nM. D and a second anti-TLT-1 arm having the following structure:

[0062] The term "identity" as known in the art refers to the relationship between the sequences of two or more polypeptides, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptides, as determined by the number of matches between two or more strings of amino acid residues. "Identity" measures the percentage of perfect matches between the smaller of two or more sequences, with gap alignment (if any) accommodated by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be easily calculated by known methods. In the present invention, similarity and identity were determined using Needleman (Needleman et al, J. Mol. Biol. 1970; 48: 443-453) from EMBOSS-6.6.0, using parameters of 10 and 0.5 for gap opening and gap extension, respectively (gapopen=10, gapextend=0.5).

[0063] The fragment crystallizable region of an antibody ("Fc region" / "Fc domain") is the C-terminal region of an antibody, which comprises the hinge and constant C domains. H 2 and C H Contains 3 domains.

[0064] The antibodies of the present invention may comprise an Fc region that may have a wild-type amino acid sequence or may comprise amino acid substitutions that modulate the effector function of the antibody, see, for example, (Wang et al.: Protein Cell. 9 (2018), pp. 63-73). A particular example of an Fc variant with modified effector function is a variant with reduced binding to Fcγ receptors. One specific example of such a variant is an IgG1 that contains substitutions of L234A, L235E, G237A, A330S and P331S (residue numbering according to the EU index) that reduce affinity for certain Fcγ receptors and C1q.

[0065] bispecific molecules The term "bispecific molecule" herein refers to a molecule that can bind to different targets, such as FVII(a) and TLT-1. The binding moieties of a bispecific molecule may be of antibody or non-antibody origin. One particular example of a bispecific molecule is a bispecific antibody.

[0066] In one aspect of the invention, the bispecific molecule comprises a first antigen-binding site capable of binding Factor VII(a) and a second antigen-binding site capable of binding TREM-like transcript 1 (TLT-1).

[0067] The bispecific molecules of the present invention may comprise non-antibody-derived binding moieties, also called alternative scaffolds. The bispecific molecules of the present invention may be fusions or conjugates of alternative scaffolds. The bispecific molecules of the present invention may be fusions or conjugates of antibodies and alternative scaffolds. The bispecific molecules of the present invention may also be fusions or conjugates of antibody fragments and alternative scaffolds.

[0068] A large number of different alternative scaffolds are known in the art, see, for example, (Simeon and Chen: Protein Cell 9 (2018), pp. 3-14), (Konning and Kolmar: Microbial Cell Factories (2018), pp. 17-32), and (Nygren and Skerra: Journal of Immunological Methods 290 (2004), pp. 3-28).

[0069] Specific examples of alternative scaffolds are Adnectins, Affilins, Anticalins, Avimers, Atrimers, FN3 scaffolds, Fynomers, Obodies, Kringle domains, Kunitz domains, Knottins, Affibodies, DARPins, bicyclic peptides, and Cys-knots.

[0070] Factor VII(a) The terms "Factor VII" and "FVII" herein refer to the zymogen of coagulation factor VII. The terms "Factor VIIa" and "FVIIa" herein refer to the activated coagulation factor VII, which is a serine protease. The terms "Factor VII(a)" and "FVII(a)" herein encompass the uncleaved zymogen, Factor VII (FVII), as well as the cleaved activated protease, Factor VIIa (FVIIa). The terms "Factor VII(a)" and "FVII(a)" herein include naturally occurring allelic variants of FVII(a) that may exist. One wild-type human Factor VII(a) sequence is presented in SEQ ID NO:1. Wild-type human coagulation factor VII(a) (SEQ ID NO: 1): ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASSPCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTKRSCRCHEGYSLLADGVSCTPTVEYPCGKIPILEKRNASKPQGRIVGGKVCPKGECPWQVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQSRRVAQVIIPSTYVPGTTNHDIALLRLHQPVVLTDHVVPLCLPERTFSERTLAFVRFSLVSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSRKVGDSPNITEYMFCAGYSDGSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQKLMRSEPRPGVLLRAPFP

[0071] Wild-type FVII(a) consists of 406 amino acid residues and is organized into four domains: an N-terminal gamma-carboxyglutamic acid-rich (Gla) domain in which 10 glutamic acid residues (highlighted in bold in the sequence above) may be gamma-carboxylated, followed by two epidermal growth factor (EGF)-like domains and a C-terminal serine protease domain. Both FVII and FVIIa are present in the circulation, but only in small amounts (approximately 1% of the total FVII(a) pool, Morrissey JH, Broze Jr GJ. Tissue factor and the initiation and regulation (TFPI) of coagulation. in Marder VJ, Aird WC, Bennett JS, Schulman S, White II GC, editors. Hemostasis and thrombosis: basic principles and clinical practice. 6th ed. Wolters Kluwer & Lippincott Williams & Wilkins: Philadelphia; 2013. p. 163-78). FVII can be activated to FVIIa by proteolytic cleavage between residues Arg152 and Ile153, resulting in a two-chain FVIIa molecule consisting of a light chain and a heavy chain. The two chains in FVIIa are linked by a disulfide bond. The light chain contains the Gla and EGF-like domains, and the heavy chain contains the protease domain. FVIIa requires binding to its cell surface cofactor tissue factor (TF) to reach its full biological activity.

[0072] The predicted full-length Macaca fascicularis FVII isoform X1 is composed of 406 amino acids with NCBI reference sequence ID XP_015295043.1. The term "cFVIIa-chimera" herein refers to a chimeric cynomolgus FVIIa construct. The amino acid sequence of the cFVIIa-chimera is such that the Gla and first EGF-like domain (amino acids 1-88 when aligned with the human FVIIa sequence) are composed of the human FVII sequence (Uniprot ID P08709), while the second EGF-like domain and the protease domain (amino acids 89-406 when aligned with the human FVIIa sequence) are composed of the cynomolgus FVII isoform X1 sequence (NCBI reference sequence ID XP_015295043.1).

[0073] The active half-life of recombinant FVIIa (and endogenous FVIIa) in humans is approximately 2-3 hours when administered intravenously. Factor VII(a) may be endogenous, plasma-derived, or recombinantly produced using well-known methods of production and purification. The degree and location of glycosylation, gamma-carboxylation, and other post-translational modifications may vary depending on the selected host cell and its growth conditions.

[0074] Factor VIIa may be found in various conformations. Factor VIIa circulates in blood in an inactive conformation or inactive form. This conformation does not have catalytic activity. FVIIa may also be found in an active conformation or form, and is herein referred to as fully active or fully activated FVIIa. The term FVIIa encompasses FVIIa in an inactive form or conformation, and FVIIa in an active form or conformation. For example, the active conformation of FVIIa may include a complex between FVIIa and tissue factor (FVIIa / TF), for example in the form of FVIIa / sTF(1-219), where sTF(1-219) is a truncated and soluble form of tissue factor, or the active conformation of FVIIa may include active site inhibited FVIIa (FVIIai). FVIIai is a catalytically inactive form of FVIIa that can be produced by treating FVIIa with dansyl-Glu-Gly-Arg chloromethylketone or Phe-Phe-Arg-chloromethylketone (FFR-chloromethylketone) (Wildgoose et al (1990) Biochemistry 29:3413-3420 and Shφrensen et al (1997) J Biol Chem 272:11863-11868). FVIIai retains its affinity for TF and is believed to adopt the same conformation that is induced in FVIIa by TF binding. Thus, FVIIai has an activated conformation, and the binding of a test compound to FVIIai suggests that the test compound also binds to the activated form of wild-type FVIIa.

[0075] The target molecule of the anti-FVII(a) antibody may be any FVII(a) molecule described herein.

[0076] TREM-like transcript 1 (TLT-1) Triggering receptors expressed on myeloid cells (TREMs) have a well-established role in the biology of various myeloid genes and play a key role in regulating innate and adaptive immunity. TREM-like transcript (TLT)-1 belongs to this protein family, but the TLT-1 gene is expressed only in a single lineage, namely megakaryocytes and thrombocytes (platelets), and is found only in the alpha granules of megakaryocytes and platelets. TLT-1 is a transmembrane protein that is exposed on the surface of activated platelets upon alpha granule release. So far, TLT-1 has not been found on the surface of resting platelets or on the surface of other cell types.

[0077] TLT-1 contains an extracellular globular head, a stalk region, a transmembrane domain, and an intracellular domain that contains an immunoreceptor tyrosine-based inhibitory motif (Washington et al. Blood, 2002; 100: 3822-3824). The extracellular globular head of human TLT-1 (hTLT-1) is a single immunoglobulin-like (Ig-like) domain. It is connected to the platelet membrane by a 37 amino acid linker region called the stalk (Gattis et al., Jour Biol Chem, 2006, 281, 19, 13396-13403).

[0078] The putative transmembrane segment of hTLT-1 is 20 amino acids long. TLT-1 also possesses cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that may function as intracellular signaling motifs.

[0079] A small portion of TLT-1 is shed upon platelet activation to form a soluble form (sTLT-1) (Gattis et al., Jour Biol Chem, 2006, 281, 19, 13396-13403). The cleavage site is located close to the platelet membrane. A shorter isoform of TLT-1 with a truncated intracellular domain is also present in platelets.

[0080] TLT-1 is involved in the control of coagulation and possibly inflammation at the site of injury. TLT-1 plays a role in platelet aggregation in response to suboptimal concentrations of some platelet agonists (Giomarelli et al,Thromb Haemost 2007;97:955-963.). The best described ligand for TLT-1 is fibrinogen (Washington et al.J Clin Invest 2009;119:1489-3824.). Recently, TLT-1 has been shown to also bind von Willebrand factor (Doerr A et al,abstract PB359 at International Society of Thrombosis and Haemostasis 2019). sTLT-1 has been suggested to be involved in the bleeding associated with sepsis by attenuating leukocyte activation and regulating platelet-neutrophil crosstalk (Derive,J Immunol 2012,188:5585-5592).

[0081] In the context of the present invention, TLT-1 may be from any vertebrate, such as any mammal, such as rodent (such as mouse, rat, or guinea pig), lagomorph (such as rabbit), artiodactyl (such as pig, cow, sheep, or camel), or primate (such as monkey or human). TLT-1 is preferably human TLT-1. TLT-1 may be translated from any naturally occurring genotype or allele that gives rise to a functional TLT-1 protein. One non-limiting example of human TLT-1 is the polypeptide sequence of SEQ ID NO:2.

[0082] The target molecule of the TLT-1 antibody may be any of the TLT-1 molecules described herein.

[0083] Anti-FVII(a) antibody The term "anti-FVII(a) antibody" herein refers to an antibody that has FVII(a) as its target. The anti-FVII(a) antibody can bind to the FVII(a) molecules described herein, including, but not limited to, endogenous FVII(a) present in human plasma, exogenous FVII(a) such as recombinant wild-type human FVII(a), and endogenous FVII(a) present in animal plasma, such as rabbit, mouse, rat, dog, or monkey.

[0084] The anti-FVII(a) antibody may be a monoclonal, monospecific antibody that has FVII(a) as its target. A monospecific anti-FVII(a) antibody typically contains two identical antigen binding sites that bind to FVII(a), and a non-limiting example is a monoclonal IgG4 anti-FVII(a) antibody.

[0085] Suitable anti-FVII(a) antibodies include, but are not limited to, any one of the anti-FVII(a) antibodies shown in Table 3. [Table 6]

[0086] An anti-FVII(a) antibody may potentially compete for binding to FVII(a) with any one of the antibodies shown in Table 3. Whether an anti-FVII(a) antibody competes for binding to FVII(a) with any one of the antibodies shown in Table 3 can be determined using well-known methods (i.e., competitive binding assays), such as surface plasmon resonance (SPR), ELISA, or flow cytometry. Example 7 describes how competitive binding to FVII(a) can be determined using competitive ELISA.

[0087] In one embodiment, the anti-FVII(a) antibody binds to FVII(a) with high affinity. The anti-FVII(a) antibody has a K for its target of less than 1 pM, such as less than 10 pM, for example less than 100 pM, such as less than 200 pM, for example less than 400 pM, for example less than 600 pM, such as less than 1 nM, for example less than 5 nM, for example less than 10 nM, such as less than 20 nM, for example less than 50 nM, for example less than 100 nM, such as less than 200 nM, for example less than 400 nM, for example less than 600 nM, such as less than 800 nM. D In vivo, high affinity anti-FVII(a) antibodies can reduce the clearance of FVII(a) by forming a complex with it, thereby increasing the half-life of FVII(a) and allowing it to accumulate in the circulation. In this way, the antibodies can result in an increase in the steady-state concentration of FVII(a).

[0088] In one embodiment, the anti-FVII(a) antibody does not interfere with the biological function of FVII(a). In one embodiment, the anti-FVII(a) antibody does not prevent endogenous FVII from being activated to FVIIa. For example, the anti-FVII(a) antibody does not interfere with the ability of FVII to convert to FVIIa (i.e., autoactivate) while bound to TF (as described in Example 12). It is also preferred that the anti-FVII(a) antibody does not prevent FVII(a) from forming the so-called initiation complex with tissue factor (TF) (as described in Example 10) and from activating factor X (FX) in a TF-dependent or independent manner. In one embodiment, the anti-FVII(a) antibody does not compete with FVII(a) substrates or cofactors. However, the anti-FVII(a) antibody may compete with inhibitors of FVIIa, such as antithrombin (AT) and alpha-2-macroglobulin (as described in Example 11).

[0089] Anti-TLT-1 antibody The term "anti-TLT-1 antibody" herein refers to an antibody that has TLT-1 as its target. The anti-TLT-1 antibody can bind to a TLT-1 molecule as described herein. The TLT-1 antibody can be a monoclonal, monospecific antibody.

[0090] Suitable anti-TLT-1 antibodies include, but are not limited to, the anti-TLT-1 antibodies shown in Table 4. [Table 7]

[0091] An anti-TLT-1 antibody may potentially compete with any one of the antibodies shown in Table 4 for binding to TLT-1. Whether an anti-TLT-1 antibody competes with any one of the antibodies shown in Table 4 for binding to TLT-1 can be determined using well-known methods (i.e., competitive binding assays) such as surface plasmon resonance (SPR), ELISA, or flow cytometry. Competitive binding to TLT-1 may be determined using competitive ELISA. Example 32 describes how competitive binding to TLT-1 can be determined using competitive ELISA.

[0092] The anti-TLT-1 antibody has a K for its target of less than 1 pM, such as less than 10 pM, for example less than 100 pM, such as less than 200 pM, for example less than 400 pM, such as less than 600 pM, for example less than 1 nM, such as less than 5 nM, for example less than 10 nM, such as less than 20 nM, for example less than 50 nM, such as less than 100 nM, for example less than 200 nM, such as less than 400 nM, for example less than 600 nM, such as less than 800 nM. D may have:

[0093] It is preferred that the anti-TLT-1 antibody does not interfere with the function of TLT-1, and in particular does not inhibit platelet aggregation.

[0094] In a preferred embodiment, the anti-TLT-1 antibody is capable of binding to TLT-1 without interfering with platelet aggregation.

[0095] In another preferred embodiment, the anti-TLT-1 antibody is capable of binding to TLT-1 without competing with fibrinogen for binding to TLT-1.

[0096] In another preferred embodiment, the TLT-1 antibody does not interfere with the shedding of TLT-1.

[0097] It is preferred that the anti-TLT-1 antibody does not bind to or shows little affinity for the triggering receptor expressed on any other myeloid cells (TREM) other than TLT-1, or for any other receptors on resting or activated platelets.

[0098] In one embodiment, the anti-TLT-1 antibody binds to the stalk of TLT-1.

[0099] Bispecific anti-FVII(a) / anti-TLT-1 antibody The bispecific antibodies of the invention comprise a first antigen-binding site capable of binding FVII(a) and a second antigen-binding site capable of binding TLT-1.

[0100] Anti-FVII(a) antigen binding site In one embodiment the bispecific antibody of the invention comprises a first antigen-binding site capable of binding FVII(a).

[0101] In some embodiments of the invention, the first antigen-binding site of the bispecific antibody competes for binding to FVII(a) with any one of the anti-FVII(a) antibodies identified in Table 3, has the same epitope as any one of the antibodies identified in Table 3, has the same CDR regions as any one of the antibodies identified in Table 3, and has the same VL and VH regions as any one of the antibodies identified in Table 3.

[0102] Anti-TLT-1 antigen binding site In one embodiment, the bispecific antibody of the invention comprises a second antigen-binding site capable of binding TLT-1.

[0103] In some embodiments of the invention, the second antigen-binding site of the bispecific antibody competes for binding to TLT-1 with any one of the anti-TLT-1 antibodies identified in Table 4, has the same epitope as any one of the antibodies identified in Table 4, has the same CDR regions as any one of the antibodies identified in Table 4, and has the same VL and VH regions as any one of the antibodies identified in Table 4.

[0104] Modified effector functions The bispecific antibodies of the present invention can comprise an Fc region that may have a wild-type amino acid sequence or may comprise amino acid substitutions that modulate the effector function of the antibody. See, for example, (Wang et al.: Protein Cell. 9 (2018), pp. 63-73). A specific example of an Fc variant with modified effector function is a variant with reduced binding to Fcγ receptors. One specific example of such a variant is an IgG1 with substitutions L234A, L235E, G237A, A330S and P331S (residue numbering according to the EU index), which has reduced affinity for certain Fcγ receptors and C1q.

[0105] A desired property of the bispecific antibody of the present invention is a long half-life in vivo. The bispecific antibody containing the Fc region may be recycled and rescued via the FcRn receptor, thereby resulting in the desired long half-life. For the bispecific antibody of the present invention lacking the Fc region, the half-life may be extended by other means. Various methods and principles for obtaining the extension of the half-life of polypeptides and antibodies are known in the art, see, for example, Kontermann: Expert Opinion on Biological Therapy, 16 (2016), pp. 903-915) and references therein.

[0106] In addition to Fc-based half-life extension of polypeptides and antibodies, fusion or conjugation to albumin or albumin variants has been demonstrated to be effective in extending half-life. Another approach is the attachment of polymers such as XTEN or PEG (insert reference). In addition, in vivo half-life extension can be obtained by attaching albumin binding moieties, see, for example, Tan et al.: Current Pharmaceutical Design 24 (2018), pp. 4932-4946; Kontermann: Expert Opinion on Biological Therapy, 16 (2016), pp. 903-915) and Kontermann: Current Opinion in Biotechnology 22 (2011), pp. 868-876).

[0107] Functional Features By binding to FVII(a), the bispecific antibody of the present invention can extend the active circulating half-life of FVIIa present in the circulation, and by binding to TLT-1, the bispecific antibody and the bound FVII(a) are directed to the surface of activated platelets. This in turn leads to increased accumulation of FVIIa on activated platelets, thus enhancing FVIIa procoagulant activity at the site of vascular injury. Thus, the bispecific antibody described herein can provide an endogenous pool of FVII(a) with improved pharmacokinetic (PK) and pharmacodynamic (PD) properties.

[0108] In humans, the active half-life of administered recombinant FVIIa is known to be approximately 2-3 hours. The short half-life of recombinant FVIIa is thought to be due to the involvement of several mechanisms, including inhibition by antithrombin III (AT), inhibition by alpha-2-macroglobulin (α2M), and renal clearance. Similar mechanisms are thought to apply to endogenous FVIIa, thus giving it a similarly short half-life.

[0109] In order to extend the active half-life of FVIIa, including the half-life of endogenous FVIIa, the bispecific antibodies described herein aim to block one or more of these clearance mechanisms by their anti-FVII arm or so-called "first antigen-binding site" without losing endogenous FVIIa activity. The Fc portion of the biAb:FVIIa complex mediates recycling of the complex in endosomes, via binding to FcRn, rescuing it from degradation. Furthermore, the high affinity anti-FVII arm of the bispecific antibody protects endogenous FVIIa from α2M inhibition and renal clearance due to the increased molecular size of the biAb:FVIIa complex compared to the size of free endogenous FVIIa. The anti-TLT-1 arm of the bispecific antibody selectively localizes the prolonged endogenous FVIIa to activated platelets. This localization of FVIIa to activated platelets enhances FVIIa activity without increasing its sensitivity to AT inhibition.

[0110] The bispecific antibodies of the invention are capable of increasing the mean residence time (MRT) of endogenous or exogenous FVII(a). Preferably, the bispecific antibodies are capable of enhancing the activity of FVII(a) in vivo.

[0111] Average residence time Mean residence time (MRT) is the average time that a molecule remains in the body and is available for therapeutic activity. MRT is calculated according to Equation 1 as a function of the volume of distribution at steady state (Vss) divided by the total body clearance (CL): MRT=Vss / CL Equation 1

[0112] The results are expressed in time. The MRT is calculated as the effective plasma half-life (t 1 / 2 ) correlates with MRT = ln(2)*t 1 / 2 formula 2

[0113] The ability of an antibody to increase the MRT of FVII(a) can be determined by well-known methods, such as those described in, for example, Pharmacokinetic and Pharmacodynamic Data Analysis: Concepts & Applications (Gabrielsson and Weiner). For example, by analysis of the plasma concentration or activity profile of FVII(a) after IV or SC administration to a laboratory animal, such as a mouse, rat, or monkey. The ability of an antibody to increase the functional MRT of FVII(a) can be determined by analysis of the plasma activity profile of FVII(a), as measured by an assay such as, but not limited to, the FVIIa activity assay described in Example 8.

[0114] The ability of antibodies to increase the MRT and functional MRT of FVII(a) may be determined, for example, as described in Examples 9 and 18.

[0115] In some embodiments, the bispecific antibodies of the invention are capable of increasing the MRT of FVII(a) by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, or at least 40-fold compared to administration of FVII(a) in the absence of the antibodies of the invention (FVII(a)) (FVII(a) polypeptide only).

[0116] In some embodiments, a bispecific antibody of the invention is capable of increasing the functional MRT of FVII(a) by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, or at least 40-fold compared to administration of FVII(a) in the absence of the bispecific antibody of the invention as measured in a FVIIa activity assay described in Example 8.

[0117] Accumulation of endogenous FVIIa The ability of a bispecific antibody of the invention to increase the level of circulating endogenous functionally active FVIIa can be determined by measuring the level of endogenous FVIIa before and after administration of the antibody to an experimental animal, e.g., a mouse, rat or monkey, as measured in an assay such as, but not limited to, the FVIIa activity assay described in Example 8.

[0118] The ability of antibodies to increase the level of circulating endogenous functionally active FVIIa can be determined, for example, as described in Examples 27 and 28.

[0119] In some embodiments, the bispecific antibodies of the invention are capable of increasing the level of circulating endogenous FVIIa by at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 80-fold, at least 160-fold, at least 320-fold, at least 640-fold compared to the level of circulating endogenous FVIIa in the absence of the administered bispecific antibody.

[0120] TLT-1 and TF-independent thrombin generation In one embodiment, the bispecific antibodies of the invention are capable of maintaining or increasing the TLT-1 and TF independent ability of Factor VIIa to generate thrombin.

[0121] The ability of an antibody to increase thrombin generation of a FVII(a) polypeptide can be determined by methods well known in the art, such as, for example, a thrombin generation assay as described in Example 5. In this assay, thrombin generation is measured in hemophilia A-induced human plasma in the presence of phospholipids, 25 nM FVIIa, and a concentration of antibody that approaches saturation of added FVIIa. Saturation is approached when >90% of FVIIa is bound by the antibody, according to the measured dissociation constants determined for the FVIIa-antibody interaction by SPR, for example, as exemplified in Example 6. Based on the ratio of peak thrombin formation in the presence and absence of the antibody, the antibodies are classified as stimulatory (>120%), inhibitory (<90%), or neutral (90-120%).

[0122] In some embodiments, the bispecific antibodies of the invention are capable of maintaining (neutral) / increasing (stimulating) the ability of a FVII(a) polypeptide to generate thrombin, as measured in a thrombin generation assay, compared to FVII(a) in the absence of the antibody.

[0123] In some embodiments, the bispecific antibodies of the invention are capable of increasing the ability of Factor VII(a) to generate thrombin by at least 20%, as compared to FVII(a) in the absence of the antibody, as measured in a thrombin generation assay.

[0124] Inhibition by antithrombin and / or alpha-2-macroglobulin In one embodiment, the bispecific antibodies of the invention are capable of reducing the susceptibility of FVIIa to inhibition by antithrombin (AT) and / or alpha-2-macroglobulin.

[0125] The ability of an antibody to reduce the inhibition of FVIIa by antithrombin (AT) and / or alpha-2-macroglobulin can be determined by methods well known in the art, such as those described in Examples 5 and 11.

[0126] In some embodiments, the bispecific antibodies of the invention are capable of reducing the inhibition of FVIIa by antithrombin (AT) and / or alpha-2-macroglobulin compared to the inhibition of FVIIa in the absence of the antibody.

[0127] TLT-1-dependent FXa generation (stimulatory activity assay) In one embodiment, the bispecific antibodies of the invention are capable of maintaining or increasing the ability of a FVIIa polypeptide to promote FX activation in the presence of a TLT-1-containing procoagulant membrane surface.

[0128] The ability of the bispecific antibodies of the invention to increase the ability of FVIIa polypeptides to promote FX activation can be determined by methods well known in the art, such as, for example, the TLT-1-dependent stimulatory activity assay described in Example 21. In this assay, FX activation is measured in the presence of a phospholipid membrane containing FX (150 nM), FVIIa (2.5 nM), TLT-1 (4 nM), and an anti-FVII(a) / anti-TLT-1 bispecific antibody. The so-called stimulatory activity of the bispecific antibody (expressed as a fold increase) is the amount of FXa generated in the presence of 100 nM of the bispecific antibody relative to the amount generated by FVIIa in the absence of the bispecific antibody.

[0129] In some embodiments, the stimulatory activity of bispecific antibodies of the invention is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 60-fold, at least 80-fold, at least 100-fold, or at least 150-fold.

[0130] TLT-1-dependent total thrombus formation In one embodiment, the bispecific antibodies of the invention are capable of promoting thrombus formation under hemophilia A conditions similar to or better than therapeutically effective concentrations of recombinant FVIIa.

[0131] The ability of the bispecific antibody to improve whole blood clot formation can be determined by methods well known in the art, such as, for example, a thromboelastography assay as described in Example 29. In this assay, clot formation is measured in human hemophilia-A induced whole blood to which the bispecific antibody is added together with FVII, FVIIa, and FVIIa:AT at concentrations that mimic the in vivo steady-state plasma levels of the corresponding endogenous factors upon repeated administration of the bispecific antibody as described in Examples 27, 28, and 29. Clotting is induced by the addition of the PAR1 agonist peptide SFLLRN and calcium. Clotting times under these conditions are compared to those achieved by the addition of 25 nM FVIIa in the absence of antibody.

[0132] In some embodiments, bispecific antibodies of the invention are capable of reducing the clotting time in human hemophilia A induced whole blood to a level similar to or below that achieved by the addition of 25 nM FVIIa. In some embodiments, bispecific antibodies of the invention are capable of reducing the clotting time in human hemophilia A induced whole blood to a level similar to or below that achieved by the addition of 2 nM FVIIa, 4 nM FVIIa, 6 nM FVIIa, 8 nM FVIIa, 10 nM FVIIa, 12 nM FVIIa, 16 nM FVIIa, or 20 nM FVIIa.

[0133] Pharmaceutical preparations In one aspect, the invention provides compositions and formulations comprising the bispecific antibodies described herein. For example, the invention provides pharmaceutical compositions comprising the bispecific antibodies formulated together with a pharma- ceutically acceptable carrier.

[0134] In one embodiment of the invention, the pharmaceutical formulation comprises a bispecific antibody present at a concentration of 80 mg / mL to 200 mg / mL, e.g., 100 to 180 mg / mL, and the formulation has a pH of 2.0 to 10.0. The formulation may further comprise one or more of a buffer system, a preservative, an isotonicity agent, a chelating agent, a stabilizer, or a surfactant, and various combinations thereof. The use of preservatives, isotonicity agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is well known to those skilled in the art. Remington: The Science and Practice of Pharmacy, 19 th edition, 1995 may be consulted.

[0135] In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multi-phase materials. The term "aqueous formulation" is defined as a formulation that contains at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution that contains at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension that contains at least 50% w / w water.

[0136] In another embodiment the pharmaceutical formulation is a lyophilized formulation, to which the physician or the patient adds solvents and / or diluents before use.

[0137] In a further aspect, the pharmaceutical formulation comprises an aqueous solution of a FVII(a) polypeptide, a bispecific antibody described herein, and a buffer, wherein the antibody is present at a concentration of 1 mg / mL or greater, and the formulation has a pH of about 2.0 to about 10.0.

[0138] The compositions of the invention may be administered parenterally, for example intravenously, for example intramuscularly, such as subcutaneously, preferably subcutaneously. The compositions of the invention may be administered prophylactically.

[0139] The pharmaceutical compositions of the present invention can be used to treat a subject having a coagulation disorder. As used herein, the term "subject" includes any human patient or non-human vertebrate having a coagulation disorder.

[0140] Medical Use As used herein, the term "treatment" refers to the medical therapy of any human or other animal subject in need thereof. The subject is expected to have undergone a physical examination by a physician who has given a provisional or definitive diagnosis indicating that the use of the particular treatment will be beneficial to the health of the human or other animal subject. The timing and purpose of said treatment may vary from individual to individual, depending on the current state of the subject's health. Prophylactic or preventive administration of the procoagulant compounds of the present invention is also contemplated, with prevention being defined as delaying or avoiding the onset or worsening of one or more symptoms of a disease or disorder. Thus, said treatment may be preventative, palliative, symptomatic ("on-demand") and / or curative.

[0141] In the context of the present invention, preventive, palliative and / or symptomatic treatment may represent separate aspects of the invention.

[0142] Coagulation abnormalities resulting in an increased tendency to bleed can be caused by any qualitative or quantitative deficiency of any procoagulant component of the normal coagulation cascade or any increased expression of fibrinolysis. Such coagulation abnormalities can be congenital and / or acquired and / or iatrogenic and are identified by the skilled artisan.

[0143] Non-limiting examples of congenital hypocoagulopathy include hemophilia A, hemophilia B, factor VII deficiency, factor X deficiency, factor XI deficiency, von Willebrand disease, and thrombocytopenias such as Glanzmann thrombasthenia and Bernard-Soulier syndrome.

[0144] A non-limiting example of an acquired coagulation disorder is a serine protease deficiency caused by vitamin K deficiency, which may be caused by administration of a vitamin K antagonist, such as warfarin. Acquired coagulation disorders may also occur after extensive trauma. In this case, otherwise known as a "hemorrhagic vicious circle," this is characterized by hemodilution (dilutional thrombocytopenia and dilution of clotting factors), hypothermia, consumption of clotting factors, and metabolic abnormalities (acidosis). Fluid therapy and increased fibrinolysis may exacerbate the situation. The bleeding may be from any part of the body.

[0145] Hemophilia A with "inhibitors" (i.e., alloantibodies to factor VIII) and hemophilia B with "inhibitors" (i.e., alloantibodies to factor IX) are non-limiting examples of coagulation disorders that are partly congenital and partly acquired.

[0146] Non-limiting examples of iatrogenic coagulation disorders are those induced by excessive and / or inappropriate fluid therapy, such as those that can be induced by blood transfusion.

[0147] In a preferred embodiment of the invention, the bleeding is associated with hemophilia A. In another preferred embodiment of the invention, the bleeding is associated with hemophilia B. In another preferred embodiment, the bleeding is associated with hemophilia A or B with acquired inhibitors. In another preferred embodiment, the bleeding is associated with FVII deficiency. In another preferred embodiment, the bleeding is associated with Glanzmann's thrombasthenia. In another embodiment, the bleeding is associated with von Willebrand's disease. In another embodiment, the bleeding is associated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is associated with surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis and / or enteritis. In another embodiment, the bleeding is profuse uterine bleeding, such as placental abruption. In another embodiment, the bleeding occurs in organs with limited possibilities for mechanical hemostasis, for example, intracranially, in the ear, or in the eye. In another embodiment, the bleeding is associated with anticoagulant therapy.

[0148] In a further embodiment, the bleeding may be associated with thrombocytopenia. In individuals with thrombocytopenia, the bispecific antibodies described herein can be co-administered with platelets.

[0149] Route of administration The bispecific antibodies described herein may be suitable for parenteral administration, preferably intravenous and / or subcutaneous administration. Subcutaneous administration is the preferred route of administration.

[0150] Dosing regimen The bioavailability and half-life of the bispecific antibodies described herein make them particularly attractive agents for prophylactic, subcutaneous treatment of subjects with acquired and / or congenital coagulation disorders. The bispecific antibodies described herein can be administered once a week, for example, once every two weeks, preferably once a month, to subjects with acquired and / or congenital coagulation disorders but who do not bleed. The bispecific antibodies described herein may be administered preoperatively to subjects with coagulation disorders who require invasive procedures such as surgery. The bispecific antibodies described herein may be administered to subjects with coagulation disorders who are undergoing invasive procedures such as surgery.

[0151] The bispecific antibodies described herein may also be co-administered with exogenous FVIIa, such as plasma-derived or recombinantly produced FVIIa, for on-demand or prophylactic treatment of subjects with coagulation disorders or experiencing bleeding episodes.

[0152] The dose administered to a subject with a coagulation disorder depends on the route of administration, whether it is administered prophylactically or on demand, and individual differences. Subcutaneous administration requires higher doses than intravenous administration.

[0153] In one embodiment of the present invention, the bispecific antibody is administered subcutaneously at a dose of 1.0 to 30.0 nmol / kg.

[0154] Unless otherwise indicated herein, terms provided in the singular also include the plural condition.

[0155] LIST OF EMBODIMENTS The present invention is further illustrated by the following non-limiting list of embodiments of the invention. 1. A bispecific antibody comprising: (i) a first antigen-binding site capable of binding Factor VII(a); and (ii) a second antigen-binding site capable of binding TREM-like transcript 1 (TLT-1). 2. The bispecific antibody of embodiment 1, wherein the first antigen-binding site competes for binding to FVII(a) with any one of the anti-FVII(a) antibodies in Table 3. [Table 8] 3. The bispecific antibody of embodiment 1, wherein the antibodies compete in a competitive ELISA assay as described in Example 7. 4. The bispecific antibody of embodiment 1, wherein the first antigen-binding site is capable of binding an epitope comprising amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO:1). 5. The bispecific antibody of embodiment 1, wherein the first antigen-binding site is capable of binding an epitope comprising one or more of amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO:1). 6. The bispecific antibody of embodiment 1, wherein the first antigen-binding site is capable of binding an epitope comprising the following amino acid residues R113, C114, H115, E116, G117, Y118, S119, L120, T130, V131, N184, T185, P251, V252, V253, Q388, M391, and R392 of FVII(a) (SEQ ID NO:1). 7. The bispecific antibody of embodiment 1, wherein the first antigen-binding site is capable of binding an epitope comprising one or more of the following amino acid residues R113, C114, H115, E116, G117, Y118, S119, L120, T130, V131, N184, T185, P251, V252, V253, Q388, M391, and R392 of FVII(a) (SEQ ID NO:1). 8. The bispecific antibody of embodiment 1, wherein the first antigen-binding site comprises: CDRL1 as represented by SEQ ID NO: 847, having 0, 1, 2 or 3 amino acid substitutions; CDRL2 represented by SEQ ID NO: 848, having 0, 1, 2 or 3 amino acid substitutions; CDRL3 represented by SEQ ID NO: 849, having 0, 1 or 2 amino acid substitutions; CDRH1 as represented by SEQ ID NO: 851, with 0 or 1 amino acid substitutions; CDRH2 represented by SEQ ID NO: 852, with 0 or 1 amino acid substitutions; CDRH3 represented by SEQ ID NO: 853, having 0, 1, 2 or 3 amino acid substitutions 9. The bispecific antibody of embodiment 1, wherein the first antigen-binding site comprises: CDRL1 represented by SEQ ID NO: 847, CDRL2 represented by SEQ ID NO: 848, CDRL3 represented by SEQ ID NO: 849, CDRH1 represented by SEQ ID NO: 851, CDRH2 represented by SEQ ID NO: 852, CDRH3 represented by SEQ ID NO: 853 10. The first antigen-binding site comprises a variable heavy chain domain sequence and a variable light chain domain sequence according to an antibody listed in Table 13 or 14, and has an affinity (K D 2. The bispecific antibody of embodiment 1, wherein the Cdc concentration is 1 nM or less. 11. The first antigen-binding site comprises a variable heavy chain domain sequence and a variable light chain domain sequence according to an antibody listed in Table 13 or 14, and has an affinity (K D 2. The bispecific antibody of embodiment 1, wherein the IgA concentration is 5 nM or less. 12. The first antigen-binding site comprises a variable heavy chain domain sequence and a variable light chain domain sequence according to an antibody listed in Table 13 or 14, and has an affinity (K D 2. The bispecific antibody of embodiment 1, wherein the CpG2 concentration is 10 nM or less. 13. The first antigen-binding site comprises a variable heavy chain domain sequence and a variable light chain domain sequence according to an antibody listed in Table 13 or 14, and has an affinity (K D 2. The bispecific antibody of embodiment 1, wherein the IgA concentration is 25 nM or less. 14. The antibody has an affinity (K) for FVII(a) of less than 1 pM, such as less than 10 pM, for example less than 100 pM, such as less than 200 pM, for example less than 400 pM, for example less than 600 pM, such as less than 1 nM, for example less than 5 nM, such as less than 10 nM, for example less than 20 nM, such as less than 50 nM, for example less than 100 nM, such as less than 200 nM, for example less than 400 nM, such as less than 600 nM, for example less than 800 nM. D 5. The bispecific antibody of any one of the preceding embodiments, 15. The bispecific antibody of embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850. 16. The bispecific antibody of embodiment 15, wherein the light chain variable domain and / or the heavy chain variable domain sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions. 17. The bispecific antibody of embodiment 1, wherein the second antigen-binding site competes for binding to TLT-1 with any one of the anti-TLT-1 antibodies of Table 4. [Table 9] 18. The bispecific antibody of embodiment 17, wherein the antibodies compete in a competitive ELISA assay as described in Example 32. 19. The bispecific antibody of embodiment 1, wherein the second antigen-binding site is capable of binding an epitope comprising the following amino acid residues of TLT-1 (SEQ ID NO: 13): K8, I9, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, A21. 20. The bispecific antibody of embodiment 1, wherein the second antigen-binding site comprises: CDRL1 as represented by SEQ ID NO: 855, having 0, 1, 2 or 3 amino acid substitutions; CDRL2 represented by SEQ ID NO: 856, having 0, 1 or 2 amino acid substitutions; CDRL3 represented by SEQ ID NO: 857, having 0, 1 or 2 amino acid substitutions; CDRH1 represented by SEQ ID NO: 859, with 0 or 1 amino acid substitutions; CDRH2 represented by SEQ ID NO: 860, with 0, 1, 2 or 3 amino acid substitutions; CDRH3 represented by SEQ ID NO: 861, having 0 or 1 amino acid substitutions 21. The bispecific antibody of embodiment 1, wherein the second antigen-binding site comprises: CDRL1 represented by SEQ ID NO: 855, CDRL2 represented by SEQ ID NO: 856, CDRL3 represented by SEQ ID NO: 857, CDRH1, represented by SEQ ID NO: 859, CDRH2 represented by SEQ ID NO: 860, CDRH3 represented by SEQ ID NO: 861 22. The bispecific antibody of embodiment 1, wherein the second antigen-binding site comprises: CDRL1 represented by SEQ ID NO: 871, CDRL2 represented by SEQ ID NO: 872, CDRL3 represented by SEQ ID NO: 873, CDRH1 represented by SEQ ID NO: 875, CDRH2 represented by SEQ ID NO: 876, and CDRH3 represented by SEQ ID NO: 877 23. The bispecific antibody of embodiment 1, wherein the second antigen-binding site comprises: CDRL1 represented by SEQ ID NO: 879, CDRL2 represented by SEQ ID NO: 880, CDRL3 represented by SEQ ID NO: 881, CDRH1, represented by SEQ ID NO: 883, the CDRH2 represented by SEQ ID NO: 884, and CDRH3 represented by SEQ ID NO: 885 24. The bispecific antibody of embodiment 1, wherein the second antigen-binding site comprises: CDRL1 represented by SEQ ID NO: 895, CDRL2 represented by SEQ ID NO: 896, CDRL3 represented by SEQ ID NO: 897, CDRH1 represented by SEQ ID NO: 899, CDRH2 represented by SEQ ID NO: 900, and CDRH3 represented by SEQ ID NO: 901 25. The bispecific antibody of embodiment 1, wherein the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858. 26. The bispecific antibody according to embodiment 25, wherein the light chain variable domain and / or the heavy chain variable domain sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions. 27. The antibody has an affinity (K) for TLT-1 of less than 1 pM, such as less than 10 pM, for example less than 100 pM, such as less than 200 pM, for example less than 400 pM, for example less than 600 pM, such as less than 1 nM, for example less than 5 nM, such as less than 10 nM, for example less than 20 nM, for example less than 50 nM, such as less than 100 nM, for example less than 200 nM, for example less than 400 nM, for example less than 600 nM, for example less than 800 nM. D 5. The bispecific antibody of any one of the preceding embodiments, 28. The bispecific antibody of embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, and the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858. 29. The bispecific antibody of any one of the preceding embodiments, wherein the antibody comprises an Fc region. 30. The bispecific antibody of embodiment 29, wherein the Fc region is an Fc variant of the Fc region of IgG1 comprising the following substitutions: L234A, L235E, G237A, A330S and P331S. 31. The bispecific antibody of any one of the preceding embodiments, wherein the antibody is a fusion or conjugate of an antigen-binding fragment of an antibody. 32. The bispecific antibody of embodiment 31, wherein one or more of the binding fragments is selected from the group of Fab, Fab', Fab2, Fab'2, and scFv. 33. The bispecific antibody of any one of the preceding embodiments, wherein the antibody is a multispecific antibody. 34. The bispecific antibody according to embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 5 and 4, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both defined by SEQ ID NO: 12. 35. The bispecific antibody according to embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 943 and 942, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both defined by SEQ ID NO: 12. 36. The bispecific antibody according to embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 7 and 6, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both defined by SEQ ID NO: 12. 37. The bispecific antibody according to embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 4 and 5, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both defined by SEQ ID NO: 12. 38. The bispecific antibody according to embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 942 and 943, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both defined by SEQ ID NO: 12. 39. The bispecific antibody according to embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 6 and 7, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both defined by SEQ ID NO: 12. 40. The bispecific antibody of embodiment 1, wherein the antibody competes for binding to FVII(a) and competes for binding to TLT-1 with one or more bispecific antibodies as described in Table 5 (constant domain abbreviations are defined according to Table 2a). [Table 10] 41. The bispecific antibody of embodiment 38, wherein the antibody competes for binding to FVII(a) and competes for binding to TLT-1 with one or more bispecific antibodies as described in Table 5 in a competitive ELISA. 42. The bispecific antibody of embodiment 38, wherein the antibodies compete in a competitive ELISA assay as described in Example 7 and Example 32. 43. The bispecific antibody according to embodiment 1, wherein the first antigen-binding site is capable of binding an epitope comprising amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO: 1), and the second antigen-binding site is capable of binding an epitope comprising the following amino acid residues K8, I9, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, A21 of TLT-1 (SEQ ID NO: 13). 44. The first antigen-binding site comprises: CDRL1 represented by amino acid residues (SEQ ID NO: 847) having 0, 1, 2 or 3 amino acid substitutions CDRL2 represented by amino acid residues (SEQ ID NO: 848) having 0, 1, 2 or 3 amino acid substitutions CDRL3 represented by amino acid residues (SEQ ID NO: 849) having 0, 1 or 2 amino acid substitutions said CDRH1 represented by the amino acid residue (SEQ ID NO: 851), CDRH2 represented by amino acid residues (SEQ ID NO: 852) with 0 or 1 amino acid substitutions, CDRH3 represented by amino acid residues (SEQ ID NO: 853) with 0, 1, 2 or 3 amino acid substitutions; and the second antigen-binding site comprises: CDRL1 represented by SEQ ID NO: 855, having 0, 1, 2 or 3 amino acid substitutions CDRL2 represented by SEQ ID NO: 856, having 0, 1 or 2 amino acid substitutions CDRL3 represented by SEQ ID NO: 857, having 0, 1 or 2 amino acid substitutions CDRH1 represented by SEQ ID NO: 859, having 0 or 1 amino acid substitutions CDRH2 represented by SEQ ID NO: 860, having 0, 1, 2 or 3 amino acid substitutions CDRH3 represented by SEQ ID NO: 861, having 0 or 1 amino acid substitutions 45. The bispecific antibody of any one of the preceding embodiments, wherein the antibody increases the functional MRT of FVII(a), as measured in a FVIIa activity assay described in Example 8. 46. ​​The bispecific antibody of embodiment 41, wherein the antibody increases the functional MRT of FVII(a) by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, or at least 40-fold compared to administration of FVII(a) in the absence of the bispecific antibody of the invention. 47. The bispecific antibody of any one of the preceding embodiments, wherein the antibody is capable of increasing the level of circulating endogenous functionally active FVIIa, as determined according to Examples 27 and 28, compared to the level of circulating endogenous FVIIa in the absence of the administered bispecific antibody. 48. The bispecific antibody according to embodiment 45, wherein the antibody increases the level of circulating endogenous functionally active FVIIa by at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 80-fold, at least 160-fold, at least 320-fold, at least 640-fold. 49. The bispecific antibody of any one of the preceding embodiments, wherein the antibody is capable of increasing the ability of a FVIIa polypeptide to promote FX activation as determined by the stimulatory activity assay described in Example 21. 50. The bispecific antibody of embodiment 43, wherein the stimulatory activity is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 60-fold, at least 80-fold, at least 100-fold, or at least 150-fold. 51. A pharmaceutical formulation comprising the bispecific antibody of any one of the preceding embodiments and a pharma- ceutical acceptable carrier. 52. The bispecific antibody according to embodiments 1 to 48, wherein the pharmaceutical formulation according to embodiment 49 is administered by subcutaneous administration. 53. A bispecific antibody according to embodiments 1 to 48, or a pharmaceutical formulation according to embodiment 49, for use as a medicament. 54. A bispecific antibody according to any one of embodiments 1 to 48 and 50 to 51, or a pharmaceutical preparation according to embodiment 49, for use in the treatment of a coagulation disorder, wherein the coagulation disorder is congenital and / or acquired. 55. The bispecific antibody of embodiment 52, wherein the coagulation disorder is selected from the group consisting of hemophilia A with or without inhibitors, or hemophilia B with or without inhibitors, FVII(a) deficiency and Glanzmann thrombasthenia. 56. The bispecific antibody according to embodiments 1-48 and 50-51 for the treatment of bleeding, wherein the bleeding is associated with congenital or acquired hemophilia A, congenital or acquired hemophilia B, hemophilia A with inhibitors, hemophilia B with inhibitors, or factor VII(a) deficiency. 57. The use according to any one of embodiments 1 to 48, wherein the bispecific antibody or pharmaceutical preparation according to embodiment 49 is administered parenterally, such as intravenously, intramuscularly or subcutaneously. EXAMPLES

[0156] Example 1: Generation of anti-FVII(a) mouse monoclonal antibodies using hybridoma technology Monoclonal antibodies were prepared by immunization of NMRCF1 mice (Charles River) with the FVIIa Q64C-sTF(1-219)G109C disulfide-linked complex as described in International Patent Application Publication No. WO07 / 115953.

[0157] Mice were given an initial subcutaneous injection of 20ug in FCA (Freund's complete adjuvant) followed by a booster intraperitoneal injection of 20ug of antigen in IFA (Freund's incomplete adjuvant). Spleens were aseptically removed and dispersed into a single cell suspension. Spleen cells were fused to X63Ag8.653 myeloma cells using electrofusion.

[0158] Cells were seeded in microtiter plates and cultured at 37°C, 5% CO2. Tissue culture medium (RPMI 1640 + 10% fetal bovine serum) containing HAT (Sigma) for selection was changed twice. After 10 days of growth, specific antibody-producing hybridoma clones were identified by ELISA screening using the following protocol: NUNC Maxisorb plates were coated with FVIIa Q64C-sTF(1-219)G109C disulfide-linked complex, or FVIIa (expressed and purified as described in Thim et al. (1988) Biochemistry 27:7785-7793 and Persson et al. (1996) FEBS Lett 385:241-243) at 1 μg / mL (HEPES buffer containing 5 mM CaCl2), 50 μl / well, and incubated overnight at 4°C. Plates were washed 5 times and blocked for 15 min in wash buffer (HEPES buffer, 5 mM CaCl2, 0.05% TWEEN 20). 50 μL of supernatant was transferred to each well and incubated for 1 h. Plates were washed 5 times and 50 μl of HRP-labeled goat anti-mouse was added (Fc gamma fragment specific, Jackson, working dilution 1 / 10000). Plates were incubated for 1 h, washed 5 times and developed with 50 μl of TMB (ready to use TMB ONE, Kem-En-Tec) for 10 min. The reaction was stopped by adding 50 μl of 4M H3PO4 and read at 450 and 620 nm on a FLUOStar Optima plate reader.

[0159] Hybridoma cells yielding positive results were subcloned at least twice by limiting dilution to ensure monoclonality. Antibody purification was performed using standard Protein A purification.

[0160] Hybridoma cells producing antibodies for use in PK studies were upscaled into T-flasks or shake flasks in RPMI1640+10%FBS medium. Conditioned medium was harvested by centrifugation and antibodies were purified by Protein A affinity chromatography and then desalted.

[0161] Example 2: Cloning and sequencing of mouse anti-FVII(a) antibodies This example describes the cloning and sequencing of murine heavy chain (HC) and light chain (LC) cDNA sequences encoding the variable domains of an anti-FVII(a) antibody.

[0162] Total RNA was extracted from hybridoma cells using the RNeasy-Mini Kit from Qiagen and used as a template for cDNA synthesis. cDNA was synthesized in a 5'-RACE reaction using the SMARTer RACE cDNA Amplification Kit from Clontech. Subsequent target amplification of the variable domains of the HC (designated VH) sequence and the variable domains of the LC (designated VL) sequence was performed by PCR using Phusion Hot Start polymerase (Finnzymes) and the Universal Primer Mix (UPM) included in the SMARTer™ RACE kit as forward primer. The sequence of the reverse primer used for VH amplification was 5'agctgggaaggtgtgcacac3'. The sequence of the reverse primer used for VL amplification was 5'gctctagactaacactcattcctgttgaagctcttg3'.

[0163] PCR products were separated by gel electrophoresis, extracted using the GFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciences, and cloned for sequencing using the Zero Blunt TOPO PCR Cloning Kit and chemically competent TOP10 E. coli (Invitrogen). Sequencing was performed at MWG Biotech, Martinsried Germany using M13uni(-21) / M13rev(-29) sequencing primers. All kits and reagents were used according to the manufacturers' instructions.

[0164] Example 3: Recombinant expression of bivalent antibodies, monovalent antibodies (OA antibodies), and antibody Fab fragments Bivalent antibodies, monovalent antibodies (referred to as one-armed, OA antibodies), and antibody Fab fragments were expressed using transient transfection of HEK293 suspension cells (293Expi, Invitrogen) essentially according to the manufacturer's instructions. 293Expi cells were subcultured every 3–4 days in Expi293F expression medium (Invitrogen, catalog no. A1435104) supplemented with 1% P / S (GIBCO catalog no. 15140-122). Expi293F cells were transfected at a cell density of 2.5–3 million cells / mL using Expifectamine. For each liter of Expi293F cells, transfections were performed by diluting a total of 1 mg of plasmid DNA into 50 mL of Optimem (GIBCO, Cat. No. 51985-026, Dilution A) and 2.7 mL of Expifectamine into 50 mL of Optimem (Dilution B). Bivalent antibodies were produced by cotransfecting VH-CH1-CH2-CH3 (HC) and VL-CL (LC) plasmids (1:1 ratio), and for Fab fragment plasmids, VH-CH1 and LC (1:1 ratio). For the production of OA antibodies, cells were transfected with three plasmids: the LC plasmid, the HC plasmid, and a third plasmid encoding a truncated HC (trHC). The HC of the OA antibody contained the hole mutations (T366S, L368A, Y407V) and the trHC was a knob (T366W) mutation, although the knob and hole can also be reversed. Knob / hole mutations are described in International Patent EP0979281B1 and are introduced to optimize the pairing of the desired heterodimers, i.e. HC to trHC, and suppress the undesired formation of homodimers, i.e. trHC to trHC and HC to HC. Dilutions A and B were mixed and incubated at room temperature for 10-20 min. After this, the transfection mixture was added to Expi293F cells and the cells were incubated at 37 °C in a humidified incubator with orbital rotation (85-125 rpm).One day after transfection, the transfected cells were supplemented with 5 ml of ExpiFectamine 293 Transfection Enhancer 1 and 50 ml of ExpiFectamine 293 Transfection Enhancer 2. Cell culture supernatants were harvested 4–5 days after transfection by centrifugation followed by filtration.

[0165] Bivalent and monovalent antibodies were purified by standard Protein A affinity chromatography known to those skilled in the art, and, if necessary, by additional purification steps such as gel filtration or ion exchange chromatography. Fab fragments were purified by affinity chromatography using an affinity resin that recognizes the kappa chain of the Fab.

[0166] Example 4: Preparation of bispecific and OA antibodies by in vitro assembly Bispecific antibodies prepared by in vitro assembly Bispecific antibodies were generated by in vitro assembly of two parent antibodies by a method similar to that described in Labrijn et al. PNAS 2013, vol. 110, pp. 5145-5150 and known as DuoBody® technology (Genmab). Instead of the IgG1 subtype used by Labrijn et al., IgG4 was used in the present invention. The exchange reaction was carried out in the presence of 75 mM 2-mercaptoethylamine (2-MEA) at 30°C for 4 hours. The resulting bispecific antibodies were purified by ion exchange chromatography to separate the residual parent antibodies from the bispecific antibodies. In this example, the heavy chain constant region of the first parent antibody (anti-FVII(a)) was IgG4 S228P F405L R409K and the heavy chain constant region of the second parent antibody (anti-TLT-1) was IgG4 S228P (both using EU numbering). The light chain constant domain was human kappa. The two parent antibodies were produced as described in Example 3. A bispecific anti-FVII(a) / anti-TLT-1 antibody may be constructed from a set of parent antibodies where the constant domain of the anti-FVII(a) antibody is IgG4 S228P and the constant domain of the anti-TLT-1 antibody is IgG4 S228P F405L R409K.

[0167] Monovalent antibodies prepared by in vitro assembly Monovalent antibodies were generated by in vitro assembly similar to that described above for bispecific antibodies, except that (1) instead of combining two antibodies to form a bispecific antibody, an antibody and a trHC dimer were combined to form a monovalent antibody. In this example, the trHC was IgG4 S228P F405L R409K and the HC was IgG4 S228P (both using EU numbering). Also, the light chain constant region was human kappa. Typically, the arm exchange reaction was performed with the trHC dimer in 20-50% molar excess to minimize the amount of bivalent antibody in the reaction mixture. The monovalent antibody was purified by size exclusion chromatography and optionally supplemented by additional purification steps, such as ion exchange chromatography, as desired.

[0168] Example 5: In vitro characterization of anti-FVIIa(a) in functional assays In order to promote the accumulation of endogenous FVIIa and allow it to exert its procoagulant activity, the anti-FVII(a) antibodies of the present invention should preferably not impair the activity of FVIIa and should also preferably not promote the inactivation of FVIIa by its main plasma inhibitor, antithrombin (Agersφ H, et al. (2011) J Thromb Haemost 9:333-338). To explore these aspects, the effect of anti-FVII(a) antibodies on the procoagulant activity of FVIIa and on the inactivation of FVIIa by antithrombin was determined in vitro using the assays described below.

[0169] Effect of anti-FVII(a) antibodies on thrombin generation in hemophilia A-induced human plasma The effect of bivalent or monovalent anti-FVII(a) antibodies on thrombin generation was measured in a kaolin-triggered thrombin generation assay (TGT) in a 96-well setup. Briefly, hemophilia A-induced plasma was prepared by adding anti-hFVIII antibody 4F30 (described in the international patent application with publication number WO2012 / 035050) to a final concentration of 37.5 μg / ml. Phospholipids at a final concentration of 10 μM (Rossix) were added to hemophilia A plasma and incubated at 37° C. for 15 minutes. Purified antibody (100 nM) and FVIIa (25 nM) were added to the mixture and incubated at room temperature for 10 minutes. Anti-FVII(a) antibodies were generated as described in Examples 1, 2, and 3. Triggering was performed by adding 10 μl of kaolin (Haemonetics) followed by 10 μl of FIIa FluCa-kit (Thrombinoscope BV) and fluorescence (excitation at 390 nm and emission at 460 nm) was measured for 2 h on an EnVision multilabel reader.

[0170] Thrombograms were calculated as the first derivative of the measured fluorescence. Thrombin peak height was calculated as the maximum value in the thrombogram. This was subsequently normalized and expressed as a percentage by dividing the observed peak height by the corresponding peak height observed for 25 nM FVIIa in the absence of antibody. On this basis, antibodies were classified as stimulatory (>120%), inhibitory (<90%), or neutral (90%-120%). Antibodies from all categories were identified, as shown in Table 6. Preferred antibodies were stimulatory or neutral in the TGT assay.

[0171] Effect of anti-FVII(a) antibodies on FVIIa inactivation by plasma-derived antithrombin Inhibition of FVIIa activity by plasma-derived antithrombin (AT) in the presence of bivalent or monovalent anti-FVII(a) antibodies was measured by incubating FVIIa (200 nM) with low molecular weight heparin (enoxaparin, 12 μM) and antibodies (200-1000 nM) for 10 min in 50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 0.1% PEG8000, 1 mg / ml BSA, pH 7.3. AT (5 μM) was then added and at 10, 20, 30, 40, 60, and 80 min samples were transferred to new microtiter plates where residual activity was measured at 405 nm for 5 min in a Spectramax instrument (Molecular Devices) in the presence of 1 mM S-2288 chromogenic substrate (Chromogenix), 200 nM soluble tissue factor (sTF, produced as described in Freskgard et al. (1996) Protein Sci 5:1531-1540), and polybrene (0.5 mg / ml). Samples in which buffer was substituted for AT provided uninhibited FVIIa activity.

[0172] Residual amidolytic activity was determined as a function of time relative to activity in the absence of inhibitor. Inhibition constants (kinh) were estimated by fitting the data to a one phase decay model. The kinh values ​​in the presence of FVII(a) antibodies (percent of the value determined in the absence of antibody) are referred to as kinh% and are reported for each antibody in Table 6.

[0173] Antibodies with an estimated kinh%<60% were classified as protecting FVIIa from AT inhibition. Antibodies with an estimated 60%≦kinh%≦150% were classified as neutral, while antibodies with an estimated kinh%>150% were classified as accelerating inhibition of FVIIa by AT. Antibodies from all three categories were identified, as reported in Table 6. Preferred antibodies were neutral or protected FVIIa from AT inhibition.

[0174] Of the antibodies tested, a subset of antibodies, including 11F2 (mAb005 and mAb0048, corresponding to the full mouse and mouse / human chimera, respectively), were found to have desirable in vitro properties as described above. [Table 11]

[0175] Example 6: SPR analysis of antibody binding to FVIIa and the effects of pH and calcium Binding of the antibodies from Example 5 to FVIIa was probed by surface plasmon resonance (Biacore T200). Anti-mouse IgG (from GE Healthcare) was immobilized on a CM4 sensor chip using a standard amine coupling chemistry kit (both supplied by GE Healthcare). Anti-FVII(a) antibodies (0.25 nM) purified according to Table 7 were injected for 1 min at a flow rate of 10 μl / min. Subsequently, 5, 15, 45 and 135 nM FVIIa were injected for 7 min at a flow rate of 30 μl / min to allow binding to the anti-FVII(a) antibodies, followed by dissociation from the anti-FVII(a) antibodies by a 9 min buffer injection. Running buffer was prepared by diluting 10xHBS-P buffer (provided by GE Healthcare) 10-fold and supplemented with 1 mg / ml BSA and 5 mM CaCl2 to obtain 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20 (P20), pH 7.4, 5 mM CaCl2, 1 mg / ml bovine serum albumin (BSA). Running buffer was also used for dilution of anti-FVII(a) antibody and FVII samples. Regeneration of the chip was achieved using a regeneration buffer consisting of 10 mM Gly-HCl, pH 1.7 (provided by GE Healthcare). Binding data were analyzed according to the 1:1 model using BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala, Sweden). The analysis yielded the binding constants reported in Table 7, with several antibodies demonstrating high affinity binding to FVIIa, including 11F2 (mAb005 and mAb0048, corresponding to the full mouse and mouse / human chimeras, respectively). [Table 12]

[0176] The effect of pH and CaCl2 on the binding of selected antibodies from Example 5 to FVIIa and cFVIIa-chimera was probed by surface plasmon resonance (Biacore T200) at 37°C. The cFVIIa-chimera sequences are presented in the section entitled Detailed Description of the Invention and are represented as outlined in Examples 16 and 26. Anti-mouse IgG (from GE Healthcare) was immobilized on a CM4 sensor chip using a standard amine coupling chemistry kit (both supplied by GE Healthcare). A pre-equilibrated mixture of 1.2 nM FVIIa and 0.5 nM 4F9 (NN-internal anti-FVII mouse Ab that binds to the FVIIa EGF1 domain) was injected for 1 min at a flow rate of 10 μl / min. Subsequently, 540, 180, 60, 20, 6.66, 2.22, 0.74, 0.25 nM of anti-FVII(a) antibody was injected for 7 min at a flow rate of 30 μl / min to allow binding to FVIIa, followed by a 9 min buffer injection to dissociate from FVIIa. Two running buffers were prepared. Buffer 1 was prepared by 10-fold dilution of 10×HBS-P+ (provided by GE Healthcare) and supplemented with 1 mg / ml BSA and 5 mM CaCl2 to give 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 7.4, 5 mM CaCl2, 1 mg / ml bovine serum albumin (BSA). Buffer 2 was prepared by diluting 10xHBS-P+ (provided by GE Healthcare) 10-fold and supplemented with 1 mg / mL BSA, 5 μM CaCl2, and pH adjusted to 6.0 (adjusted using 4 M HCl) to obtain 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 6.0, 5 μM CaCl2, 1 mg / ml bovine serum albumin (BSA). FVIIa, anti-FVII antibody 4F9, and anti-FVII(a) antibody were diluted separately in both running buffers. Regeneration of the chip was achieved using a regeneration buffer consisting of 10 mM Gly-HCl, pH 1.7 (provided by GE Healthcare).The binding data was analyzed according to a 1:1 model using BiaEvaluation 4.1 as supplied by the manufacturer (Biacore AB, Uppsala, Sweden). The analysis resulted in the binding constants reported in Table 7b, indicating retained high affinity binding between FVIIa and the anti-FVII(a) antibody. Table 7b shows the estimated binding constants and fold-differences for the interaction of FVIIa and anti-FVII(a) antibodies in two different buffers as determined by surface plasmon resonance (SPR) analysis according to Example 6. [Table 13]

[0177] Example 7: Identification of antibodies that compete with mAb0005 (11F2) for binding to FVII(a) in a competitive ELISA To determine whether anti-FVII(a) antibodies with the desired in vitro properties from Example 5 compete with mAb0005 (11F2) and antibodies derived therefrom for binding to FVIIa(a), a competition study was performed with the corresponding Fab fragment, Fab0076. FVIIa, HD-Phe-Phe-Arg chloromethylketone (FFR-cmk, Bachem, Switzerland) active site blocked FVIIa (FVIIai, see Example 9) was immobilized on NUNC maxisorp plates at a concentration of 125 ng / ml in dilution buffer (20 mM HEPES, 5 mM CaCl2 NaCl, pH 7.2) overnight at 4° C. Plates were washed and blocked with wash buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, 0.5 mL / L Tween 20, pH 7.2) for 15 min. 11F2-Fab0076 was biotinylated using a standard biotinylation kit (EZ-Link, Thermo) used according to the manufacturer's instructions. For competition studies, biotinylated Fab0076 at a final fixed concentration of 10 ng / ml was combined with a series of dilutions of anti-FVIIa(a) antibody. Final concentrations ranging from 100 mg / ml to 9.5 ng / ml were obtained in dilution buffer. The mixtures were added to the wells of the plate and incubated for 1 h. The plate was then washed and HRP-labeled streptavidin-HRPO (1:2000 in dilution buffer, Kirkegaard & Perry Labs) was added and incubated for 1 h. Finally, the plate was washed and developed with TMB ONE (KEMENTEC) for 10 min. The reaction was stopped by adding H3PO4 (4 M) and the plate was read on a FLUOStar Optima plate reader at 450 nm with subtraction of the background signal measured at 620 nm. All incubations were performed at room temperature unless otherwise specified, and plates were washed five times with wash buffer.

[0178] From the measured signal (in OD units), the competition at any given antibody concentration is calculated as follows: % Inhibition = (1-(OD units-100% inhibition) / (0% inhibition-100% inhibition))*100 where 0% inhibition was determined from the signal in wells that did not contain any competing anti-FVII(a) antibody and 100% inhibition was determined as the signal from wells that did not contain biotinylated Fab0076 (i.e., corresponding to assay background). An antibody was considered to compete with 11F2 (mAb0005) for binding to FVII(a) if at least 50% inhibition (% inhibition) was observed when antibody concentrations were tested up to 10000-fold excess of biotinylated Fab0076. The results are summarized in Table 8. [Table 14]

[0179] Example 8: FVIIa Activity Assay FVIIa activity was measured using reagents from the STAclot® VIIa-rTF kit (Diagnostica Stago) essentially as described in Morrissey JH et al Blood, 1993;81:734-44. The assay was performed on an ACLTOP500 automated coagulation instrument from Instrumentation Laboratory. The assay consisted of 40 μl diluted animal plasma samples, calibrators (recombinant FVIIa (rFVIIa) standards calibrated against the international WHO standard), or quality control (QC) samples consisting of 40 μl of FVII-deficient plasma and 40 μl of soluble tissue factor (sTF) mixed with phospholipids. To initiate the reaction, a volume of 40 μl of 25 mM CaCl2 was added and the clotting time was measured by the instrument. The clotting times of the samples and QC were compared to a rFVIIa calibration curve using plasma at the same concentration as the diluted samples to reduce plasma interference. The calibration curve range was 5-1000 mIU / mL and was fitted using a third order polynomial. QC and sample results were calculated by the software on the ACLTOP500 instrument. Results in IU / mL were converted to nM using the specific activity (IU / Pmol) of the administered compound.

[0180] Example 9: Pharmacokinetics of recombinant FVIIa in co-formulation with anti-FVII(a) antibody in rats Monoclonal anti-FVII(a) antibodies with desired in vitro properties from Example 5 were administered IV to male Sprague-Dawley rats in a co-formulation with 20 nmol / kg human FVIIa. The molar ratio of FVIIa to antibody was 1:1 or 1:5, as shown in Table 9. Animals had free access to food and water throughout the experiment. FVIIa plasma activity was measured using the FVIIa activity assay described in Example 8.

[0181] Pharmacokinetic analysis was performed by non-compartmental methods using WinNonlin. Mean residence time (MRT) was calculated from the data. The results are shown in Table 9.

[0182] Of the antibodies with the desired properties of Example 5, mAb0005 (11F2) and mAb0001 (11F26) conferred the longest mean residence times with human FVIIa of 7.9 and 7.5 hours, respectively, compared to a free mean residence time of 1.1 hours with free human FVIIa (see Table 9). mAb0005 and mAb0001 (mAb0759) are high affinity antibodies (Example 6) that show competition for binding to FVIIa (Example 7). [Table 15]

[0183] Example 10: Effect of 11F2 anti-FVII(a) antibody on the activation of FX by free FVII or TF-complexed FVIIa Preferably, the anti-FVII(a) antibodies of the invention should not interfere with the pharmacological action of FVIIa on membrane surfaces or the initiation of coagulation, i.e., activation of FX by the FVIIa-TF complex. To investigate these aspects, the effect of 11F2 on the TF-independent and TF-dependent proteolytic activity of FVIIa was determined in the absence or presence of lipidated TF, respectively. To avoid the binding activity effects resulting from the simultaneous binding of two FVIIa molecules to a normal bivalent antibody, a one-armed monovalent antibody format was used.

[0184] Effect of 11F2 antibody on FX activation by FVIIa in the presence of phospholipid membranes Activity measurements were performed in assay buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2 pH 7.3, 0.1% PEG8000 and 1 mg / ml BSA) containing 10 nM FVIIa, 0 or 200 nM antibody (see Table 10), and 25 μM 25:75 phosphatidylserine:phosphatidylcholine vesicles (Haematologic Technologies Inc.). Reactions were initiated by the addition of 0-300 nM human plasma-derived factor X (FX) and incubated for 20 min at room temperature in a total reaction volume of 100 μl in 96-well plates (n=2). After incubation, the reaction was quenched by the addition of 50 μl of quench buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 80 mM EDTA, pH 7.3), followed by the addition of 50 μl of 1 mM S-2765 chromogenic substrate (Chromogenix) in water. Conversion of the chromogenic substrate by the generated FXa was measured as the slope of the linear absorbance increase at 405 nm for 10 min using a Spectramax microplate spectrophotometer. By relating the measured slope to that generated under similar conditions with known amounts of plasma-derived human FXa, the initial rate of FXa generation on a molar basis could be estimated. Enzyme kinetic parameters were estimated by nonlinear curve fitting of the data to the Michaelis-Menten equation (v=kcat * [FX] * [FVIIa-TF] / (Km + [FX])) using GraphPad Prism.

[0185] As shown in Table 10, monovalent 11F2 did not affect the rate of FX activation by free FVIIa in the presence of phospholipid vesicles.

[0186] Effect of 11F2 antibody on FX activation by FVIIa in the presence of TF Activity measurements were performed in assay buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.3, 0.1% PEG8000 and 1 mg / ml BSA) containing 100 pM FVIIa, 0 or 200 nM antibody (see Table 10), and 2 pM E. coli-derived TF fragment 1-244 incorporated into 25:75 phosphatidylserine:phosphatidylcholine (PS:PC) vesicles as described in Smith and Morrissey (2005) J. Thromb. Haemost., 2:1155-1162. Reactions were initiated by the addition of 0-30 nM human plasma-derived factor X (FX) and incubated for 20 min at room temperature in a total reaction volume of 100 μl in 96-well plates (n=2). After incubation, reactions were quenched and FXa was quantified as described above.

[0187] As shown in Table 10, monovalent 11F2 (mAb0077(OA)) did not affect the rate of FX activation by the FVIIa / TF complex. [Table 16]

[0188] Example 11: Effect of 11F2 antibody on the inhibition of FVIIa by plasma inhibitors FVIIa exhibits a relatively short half-life in the circulation, in part due to its inactivation by the abundant plasma inhibitor antithrombin (AT). Similarly, animal studies have implicated another plasma inhibitor, alpha-2-macroglobulin, in the inactivation of FVIIa. Purified plasma-derived inhibitors were used to study the effect of monovalent 11F2 antibody on the inactivation of FVIIa by these inhibitors.

[0189] Effect of 11F2 antibody on the inhibition of FVIIa by antithrombin. Inhibition of FVIIa by human plasma-derived AT in the presence of monovalent 11F2 antibodies (mAb0048, mAb0077(OA)) was carried out under pseudo-first-order conditions. The assay was carried out at room temperature in a volume of 200 μl in assay buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 0.1% PEG8000, 1 mg / ml BSA, pH 7.3) containing 200 nM FVIIa, 12 μM low molecular weight heparin (enoxaparin, European Pharmacopoeia reference, code E0180000 batch 5.0, Id 00CK18), and either 0 or 1000 nM antibody. After a 10 min preincubation, the reaction was initiated by the addition of 5 μM AT (antithrombin III, Baxter, Lot VNB1M007; repurified on a heparin-sepharose column to remove serum albumin in the formulation). At selected time points, 10 μL aliquots were transferred to a total volume of 200 μl containing 0.5 mg / ml polybrene (hexadimethrine bromide, Sigma, Cat. No. H9268 Lot SLBC8683V), 200 nM sTF, and 1 mM S-2288 (Chromogenix) to quench the reaction and saturate FVIIa with sTF, allowing measurement of residual FVIIa activity by hydrolysis of a chromogenic substrate monitored at 405 nm for 5 min. Residual amidolytic activity was determined as the slope of the linear progress curve after blank subtraction, which was then fitted to a first-order exponential decay function using GraphPad Prism software to derive the pseudo-first-order rate constant (kapp) for the reaction. The apparent second-order rate constant (kinh) was estimated as kapp divided by the AT concentration.

[0190] This analysis revealed that the rate of inhibition of FVIIa in the presence of monovalent 11F2 (mAb0077(OA)) was 133±10 M-1s-1 compared to 124±7 M-1s-1 for inhibition of free FVIIa.

[0191] Effect of 11F2 antibody on inhibition of FVIIa by alpha-2-macroglobulin Inhibition of FVIIa by human plasma-derived alpha-2-macroglobulin in the presence of 0 or 1000 nM monovalent 11F2 antibody (mAb0077(OA)) was carried out under pseudo-first-order conditions. The assay was carried out at room temperature in a volume of 100 μl in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% PEG8000, 0.01% Tween 80, pH 7.3) containing 200 nM FVIIa and either 0 or 1000 nM antibody. The reaction was started by the addition of 0 or 1000 nM alpha-2-macroglobulin purified from human plasma according to Banbula et al. (2005) J. Biochem., 138:527-537. At selected time points, 10 μl aliquots were transferred to 190 μl of buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% PEG8000, 1 mg / ml BSA, pH 7.3) containing 200 nM sTF and 1 mM S-2288 (Chromogenix), allowing measurement of residual FVIIa activity by hydrolysis of a chromogenic substrate monitored at 405 nm for 5 min. Residual amidolytic activity was determined as the slope of the linear progress curve after blank subtraction, which was then fitted to a first-order exponential decay function using GraphPad Prism software to derive the pseudo-first-order rate constant (kapp) for the reaction. The apparent second-order rate constant (kinh) was estimated as kapp divided by the alpha-2-macroglobulin concentration.

[0192] In the absence of antibody, the apparent second-order rate constant for inhibition of FVIIa by alpha-2-macroglobulin was found to be 475 ± 21 M-1s-1. However, in the presence of monovalent 11F2 antibody (mAb0077(OA)), no apparent inhibition of FVIIa was observed until the last time point at 125 min. From these studies, it is concluded that 11F2 does not affect the inhibition of FVIIa by antithrombin, but protects FVIIa from inhibition by alpha-2-macroglobulin.

[0193] Example 12: Effect of 11F2 antibody on FVII autoactivation Upon vascular injury, endogenous FVII binds with high affinity to the cofactor tissue factor (TF) exposed on cells surrounding the vascular endothelium. During this process, FVII is converted to FVIIa by limited proteolysis. Activation is thought to occur as a result of TF-mediated FVIIa-FVII transactivation, also called autoactivation. To determine the effect of 11F2 antibody on FVII autoactivation, activation of FVII was measured in the presence of lipidated TF, FVII, limiting concentrations of FVIIa, and monovalent 11F2 antibody.

[0194] Activity measurements were performed in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, pH 7.3, containing 0.1% PEG8000 and 1 mg / ml BSA) containing 2 nM FVIIa, 145 nM FVII, 0 or 200 nM monovalent 11F2 antibody (mAb0077(OA)). Reactions were initiated by the addition of 2 nM lipidated E. coli-derived TF fragment 1-244 incorporated into 25:75 PS:PC vesicles as described by Smith and Morrissey (2005) J. Thromb Haemost., 2:1155-1162. Total reaction volume was 100 μl. FVIIa produced at selected time points (typically 5, 10, 15, 20, 30, 40, 50 and 60 min) was quantified according to the subsampling procedure described below.

[0195] Quantification of FVIIa by subsampling - At selected time points, 10 μl of sample was quenched by transferring into 140 μl of 50 mM HEPES, 100 mM NaCl, 5 mM EDTA containing 0.1% PEG8000, 1 mg / ml BSA and 215 nM soluble tissue factor (sTF). After collection of all samples, FVIIa chromogenic activity was measured by adding 50 μl of S-2288 (4 mM) in 60 mM CaCl2. Conversion of the chromogenic substrate by the generated FVIIa was measured as the slope of the linear absorbance increase at 405 nm over 10 min using a Spectramax plate reader. By relating the measured slope to the slope generated under similar conditions with a known amount of FVIIa, the molar concentration of FVIIa can be estimated.

[0196] Autoactivation of FVIIa was found to be TF-dependent and the measured FVIIa activities in Table 11 indicate that it is not impaired by the presence of monovalent 11F2 antibody. [Table 17]

[0197] Example 13: Crystal structure of 11F2-Fab0076 in complex with active site-blocked FVIIa and soluble tissue factor To determine the epitope recognized by the murine antibody 11F2 on FVII(a), the corresponding Fab fragment (Fab0076) was crystallized in complex with HD-Phe-Phe-Arg chloromethylketone (FFR-cmk; Bachem, Switzerland) active site blocked FVIIa (FVIIai) and soluble tissue factor fragment 1-219 (sTF) using the hanging-drop method according to Kirchhofer. D., et al., Proteins Structure Function and Genetics. (1995), 22, 419-425.

[0198] Crystallization Crystals of Fab0076 mixed with FVIIai / sTF complex in a 1:1 molar ratio were grown using the hanging drop vapor diffusion technique at 18°C. Crystals of the complex were obtained by mixing 1 μl of protein solution at 4.6 mg / ml in 10 mM HEPES, 50 mM NaCl, 5 mM CaCl2, pH 7.0 with 0.5 μl of 100 mM sodium citrate, pH 6.2 and 20% PEG 6000 as precipitant and incubating with 1 ml of the precipitant solution at a temperature of 18°C.

[0199] Diffraction data collection Crystals were cryoprotected in a solution consisting of 75 mM sodium citrate, pH 6.2, 15% PEG 6000, 4% glycerol, 4% ethylene glycol, 4.5% sucrose, and 1% glucose before flash cooling with liquid nitrogen. Diffraction data were collected at 100 K on the MAX-lab (Lund, Sweden) beamline I911-3 using a MAR Research marCCD225 detector. Automatic indexing, integration, and scaling of the data were performed using programs from the XDS package (diffraction data statistics are summarized in Table 12).

[0200] Structure determination and refinement The structure was determined by molecular replacement using Phaser as implemented in the program suite Phenix, with chains A and B of Protein Data Bank entry 1YY8 and Protein Data Bank entry 3ELA. The asymmetric unit contains two Fab:FVIIai / sTF complexes. The model was refined in COOT using Phenix refinement and manual rebuild steps. Refinement statistics are shown in Table 12. [Table 18]

[0201] The epitope (defined as residues in FVIIai having a non-hydrogen atom positioned within a distance of 4 Å or less from a non-hydrogen atom of Fab0076) was found to include the following residues according to SEQ ID NO:1: R113 C114 H115 E116 G117 Y118 S119 L120 T130 V131 N184 T185 I186 P251 V252 E265 M391 R392 E394 The paratope (defined as residues in Fab0076 having a non-hydrogen atom positioned within a distance of 4 Å or less from a non-hydrogen atom in FVIIai) comprises the following light chain residues according to SEQ ID NO:64: Q27 G28 S30 D31 Y32 K49 Y50 Q53 H92 S93 F94 and the following heavy chain residues according to SEQ ID NO:63: D32 Y54 N59 N101 Y102 Y103 G104 N105

[0202] Example 14: Humanized Mouse 11F2 We combined information from human germline-associated sequence identity, the crystal structure of the complex between Fab0076 (Example 12) and the active site inhibitor FVIIa (Example 9), and in vitro binding data to humanize the murine antibody 11F2 (mAb0005) with VH and VL domain sequences corresponding to SEQ ID NO:754 and SEQ ID NO:750, respectively, while preserving the high binding affinity to FVII(a), respectively. First, we used the blast algorithm to search the human germline database for human sequences of VH, VL, and VJ (for both heavy chain (HC) and light chain (LC)) with high sequence identity to the murine variable domain sequences of 11F2. The sequences with the highest sequence identity for VH of HC are IGHV4-30-4*01, IGHV4-28*01, IGHV4-28*06, and IGHV459*01, and for VJ segments of HC top germline: IGHJ5*01, IGHJ4*01. For LC, the top sequences for VL are IGKV6D-41*01 and IGKV3-11*01, and for VJ segments: IGKJ2*01 and IGKJ2*02 (Table 12). The differences between human germline and mouse VH and VL sequences were then mapped onto the crystal structure of the Fab0076 / FVIIa complex. Residues in the mouse variable domain that are more than 10 Å away from residues in the epitope were predicted to have no or little effect on binding affinity and were replaced with the corresponding human germline amino acid entities. Residues that make up the paratope were then considered more problematic to replace without affecting affinity, where the mouse amino acid entities were preserved. A subset of residues near the binding interface that could affect binding affinity was identified. Humanized variants were generated by mutating residues distal to the epitope to human amino acid entities from the germline alignment. Additionally, residues close to the paratope were mutated to human entities in the subset to bring the variants as close to the human germline as possible. The variants included in the set were those in which the mouse CDRs were grafted into the full human germline of HC and LC.From the initial analysis, 12 LCs and 13 HCs were generated, paired with 23 variants according to Table 13.

[0203] The binding affinity of the humanized antibodies to FVIIa (listed in Table 13) was measured using Bio-Layer Interferometry (Fortebio). All steps were performed at 30°C in running buffer (20 mM HEPES buffer (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.03% Tween 20, 1 mg / ml IgG-free BSA). Antibodies were captured on an anti-human chip (AHC, Fortebio) at a concentration of 10 μg / ml for 3 minutes. A 3-minute incubation was then performed to establish a baseline. This was followed by monitoring association for 3 minutes using four different concentrations of FVIIa (25 nm, 50 nM, 100 nM, and 200 nM), followed by dissociation for 3 minutes. The sensorgrams were analyzed using Fortebio data analysis software. Because Fortebio data was used for ranking, absolute affinity values ​​may deviate from those determined by SPR (Example 6, Table 7). [Table 19] [Table 20]

[0204] Variants from the first round of humanization that had affinity equal to or greater than the parental murine antibody were identified, and those mutations that were found to maintain or improve binding affinity were used to design second round variants. From this set of mutations, second round variants were generated by inserting many of these mutations onto the humanized HC and LC that already had the desired affinity. From the analysis, 19 VH sequences (corresponding to SEQ ID NOs: 314, 514, 522, 530, 538, 546, 554, 562, 570, 578, 586, 594, 602, 610, 618, 626, 634, 642 and 650) and 25 VL sequences (corresponding to SEQ ID NOs: 310, 318, 326, 334, 342, 350, 358, 366, 374, 382, ​​390, 398, 406, 414, 422, 430, 438, 446, 454, 462, 470, 478, 486, 494 and 502) were designed and all VL sequences were experimentally tested in combination with VL, resulting in a total of 475 combinations. As described above, the dissociation constant (KD) values ​​for binding to FVIIa were measured for the 475 resulting humanized antibodies using Bio-Layer Interferometry (Fortebio). The measured KD values ​​are listed in Table 14. [Table 21] [Table 22] [Table 23]

[0205] SPR analysis of humanized 11F2 variants The affinity of selected humanized 11F2 variants was determined by SPR analysis as detailed in Example 6. The measured dissociation constants are listed in Table 15 and demonstrate that the variants retain high affinity binding to human FVIIa. [Table 24]

[0206] Functional characterization of humanized 11F2 variants The effect of selected humanized 11F2 variants on the activity of FVIIa and its sensitivity to inhibition by antithrombin was determined as detailed in Example 5. The results are listed in Table 16 and show that the humanized variants retain desirable properties with respect to these parameters. [Table 25]

[0207] Pharmacokinetics of FVIIa coformulated with a humanized 11F2 variant in rats The humanized 11F2 variant family was co-formulated with 20 nmol / kg FVIIa and administered IV to male Sprague-Dawley rats as detailed in Example 9.

[0208] The results are presented in Table 17 and show that several of the humanized 11F2 variants confer the same long half-life to FVIIa as the parent antibody. [Table 26]

[0209] Example 15: Crystallization and epitope mapping of 11F2Fab0883 in complex with active site-blocked FVIIa and soluble tissue factor To determine the epitopes on FVII(a) recognized by humanized 11F2, i.e., mAb0705(OA) and mAb0842(OA), the corresponding Fab fragments Fab0883 were crystallized in complex with HD-Phe-Phe-Arg chloromethylketone (FFR-cmk; Bachem, Switzerland) active site-blocked FVIIa (FVIIai) and soluble tissue factor fragment 1-219 (sTF) using the hanging drop method according to Kirchhofer. D., et al., Proteins Structure Function and Genetics. (1995), 22, 419-425.

[0210] Crystallization Crystals of the SEC-purified complex of Fab complexed with FVIIai / sTF were grown using the sitting drop vapor diffusion technique at 18° C. 360 nl of protein solution of the protein complex (4.5 mg / ml) in 20 mM HEPES, 150 mM NaCl, 0.1 mM CaCl2, pH 7.4 was mixed with 360 nl of precipitating solution containing 0.15 M CsCl and 15% (w / v) polyethylene glycol 3350 and 360 nl of water and equilibrated against 80 μl of precipitating solution. Crystals grew within 6 weeks.

[0211] Diffraction data collection Crystals were cryoprotected in a solution consisting of 0.15 M CsCl, 15% (w / v) polyethylene glycol 3350, and 20% (v / v) glycerol before flash cooling with liquid nitrogen. Diffraction data were collected at 100 K on a Rigaku FRX rotating anode generator equipped with a Dectris Pilatus 1M detector. Data reduction was performed using programs from the XDS package (diffraction data statistics are summarized in Table 18).

[0212] Structure determination and refinement All crystallographic calculations were performed using the Phenix suite of crystallographic programs. The structure was determined by molecular replacement using the program Phaser with the coordinates of the complex structure obtained as described in Example 13 as a search model. The asymmetric unit contains four Fab:FVIIai / sTF complexes. Iterative cycles of manual reconstruction using COOT and Phenix refinement yielded the final model (Table 18). [Table 27]

[0213] Epitope mapping A residue is considered to be part of the epitope if all four independently determined FVIIa molecules in the crystal have a non-hydrogen atom of said residue located within a distance of 4 Å or less from a non-hydrogen atom in the Fab. Thus, the epitope was found to include the following residues according to SEQ ID NO:1: R113 C114 H115 E116 G117 Y118 S119 L120 T130 V131 N184 T185 P251 V252 V253 Q388 M391 R392

[0214] Paratope determination A residue is considered part of the paratope if all four independently determined Fab molecules in the crystal have a non-hydrogen atom of said residue located within a distance of 4 Å or less from a non-hydrogen atom in FVIIa. The paratope is comprised of the following light chain residues (according to SEQ ID NO: 814): Q27 G28 Y32 Y50 H92 S93 F94 and the following heavy chain residues (according to SEQ ID NO:818): D32 Y54 Y103 N105

[0215] Example 16: Hotspot analysis of 11F2 mAb0842(OA) using SPR Expression of alanine variants of FVIIa hFVII alanine variants were produced using QMCF technology (Icosagen), a stable episomal expression system. CHOEBNALT85 cells were cultured in Qmix1 medium (1L=1:1 CD-CHO and SFM II (NVO11514701) + 10ml Pen / Strep (Gibco, 15140-122) + 2ml Puromycin (Gibco, A11138-03)) in E125 flasks in a CO2 shaking incubator. On the day of transfection, 1x10e7 CHOEBNALT85 cells were transfected with 2μg of hFVII variant-encoding plasmid and 50μg of salmon sperm DNA using electroporation (Bio-Rad Gene Pulser Xcell Electroporation System, 300V, 900μF, 4mm cuvette). One day after transfection, G418 selection was started by transferring the cells into Qmix2 medium (1L = 1:1 CD-CHO and SFM II (NVO11514701) + 10ml Pen / Strep (Gibco, 15140-122) + 1ml K-vitamin (K.vit 13A 01311) + 14ml G418 (Gibco, 10131-027)). After 10-14 days of G418 selected cells reached >95% viability (Vi-Cell XR cell counter). Cells were split from 0.4x10e6 cells / ml into 2x250ml Qmix2 in 2xE1000 flasks. After 3-4 days cells reached a density of approximately 4-5x10e6 cells / mL. Expression was initiated by adding 20% ​​CHO CD Efficient Feed B (Gibco A10240) + 6 mM GlutaMax (Gibco, 35050). Four days after initiation, an additional 10% CHO CD Efficient Feed B + 6 mM GlutaMAX was added. Six days after initiation, the cultures were harvested and spun down (200 g, 5 min). The supernatant was collected and 15 mM HEPES (Gibco, 15630) and 5 mM CaCl2 (Sigma, 21115) were added. The supernatant was sterile filtered using a 0.22 μm bottle top filter (Corning, CLS430049).

[0216] Expression and purification of cFVIIa-chimera (22017-051) The cFVIIa-chimera was produced using a similar expression system as outlined above. The zymogen cFVII-chimera was purified from the culture medium using an affinity column prepared by coupling an in-house anti-FVII(a) antibody (F1A2) to Sepharose beads as described in Example 26. The anti-FVII(a) antibody F1A2 binds to the Gla domain of FVII(a) in a Ca++-dependent manner. The zymogen cFVII-chimera was activated using human FIXa and re-purified using F1A2 affinity purification to obtain the final cFVIIa-chimera.

[0217] Hotspot Analysis Hotspot analysis using monovalent humanized antibody mAb0842(OA) was performed by binding studies with a panel of 19 FVII(a) variants at 25°C using surface plasmon resonance (Biacore T200). 25 μg / ml of anti-FVII(a) antibody (NN internal Ab 4F6 (Nielsen AL et al, PNAS 114(47)12454-12459, 2017)) targeting the FVII(a)gla domain was immobilized on a CM4 sensor chip using a standard amine coupling chemistry kit (both supplied by GE Healthcare). According to Table 19, FVII(a) variants in cell culture supernatant (as described above) were diluted in running buffer and injected for 1 min at a flow rate of 10 μl / min to achieve capture levels between 5 and 55 RU. Each FVII(a) variant was captured by the immobilized anti-FVII(a)gla Ab. Subsequently, mAb0842(OA) at 540 nM (3-fold dilution) was injected at a flow rate of 30 μl / min for 7 min to bind to the captured FVII(a) variants, followed by a 9 min buffer injection to allow dissociation of the one-armed anti-FVII(a) antibody. Running buffer was prepared by diluting 10xHBS-P buffer (provided by GE Healthcare) and supplemented with 1 mg / ml BSA and 5 mM CaCl2 to give 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 7.4, 5 mM CaCl2, 1 mg / ml bovine serum albumin (BSA). Running buffer was also used for diluting the anti-FVII(a) antibody and FVII(a) samples. Chip regeneration was achieved using 10 mM HEPES, 150 mM NaCl, 20 mM EDTA, 0.05 v / v polysorbate 20, pH 7.4. Binding data were analyzed with a 1:1 kinetic model and steady-state analysis using BiaEvaluation 4.1, supplied by the manufacturer (Biacore AB, Uppsala, Sweden). Whenever possible, ka, kd, and KD values ​​from the 1:1 kinetic fit model are reported. KD values ​​using the steady-state fit model were reported for the four FVII(a) variants.Furthermore, capture signals are reported for all FVII(a) variants. An amino acid residue is considered a hotspot residue if its substitution with alanine results in a 10-fold or greater affinity reduction relative to the wild type. Based on the data presented in Table 19, it is concluded that amino acid residues H115, T130, V131, and R392 are hotspots. [Table 28]

[0218] Example 18: Pharmacokinetics of recombinant FVIIa in co-formulation with humanized 11F2 antibody in cynomolgus monkeys FVIIa plasma activity time profiles were estimated in cynomolgus monkey studies following IV or SC administration of either recombinant FVIIa (rFVIIa) alone or co-formulated with monovalent one-armed 11F2 mAb0705(OA) in a 1:3 molar ratio. Formulations were administered as a single dose of 5.4 nmol / kg FVIIa (including 16.2 nmol / kg mAb0705(OA) for the co-formulation) and blood samples were collected over a 3-week period.

[0219] During the experiment, animals were housed and handled according to standard procedures of the local health authorities and had free access to food and water. FVIIa plasma activity was measured using the FVIIa activity assay described in Example 8. Endogenous cynomolgus FVIIa was below the LLOQ (0.1 nM) before dosing and was therefore ignored.

[0220] Pharmacokinetic analysis of FVIIa plasma activity time profile was performed by non-compartmental methods using Phoenix WinNonlin 6.4. The following parameters were estimated from the data: clearance (CL), mean residence time (MRT), and SC bioavailability (F). The parameters are listed in Table 20 and show a substantial prolongation of FVIIa activity by co-formulation with mAb0705(OA) both after IV and SC administration compared to FVIIa in the absence of antibody. [Table 29]

[0221] Example 19: Humanization and optimization of murine anti-TLT-1 antibody mAb0012 Humanization The murine anti-human TLT-1 antibody mAb0082 disclosed in WO2012 / 117091 was used as the starting point for the humanization process. mAb0082 is derived from mAb0012 by two point mutations incorporating C41A in VL and T61A in VH, which remove the unpaired cysteine ​​in FR1 of VL and the N-glycosylation site in CDR2 of VH, respectively. The humanization process was based on standard molecular biology methods known to those skilled in the art.

[0222] Briefly, the CDRH1, CDRH2, and CDRH3 sequences of mAb0082 were grafted onto human germline sequences based on the VH3_74 / JH1 sequence defined in the IMGT database. In addition, three VH3_74 / JH1 human amino acid substitutions were introduced into the grafted CDRH2 sequence to further humanize this CDR sequence: P62D, L64V, and D66G. To obtain a binding affinity comparable to that of mAb0082, three back mutations were introduced into the VL sequence at positions S49G, D62P, and R98S. Regarding VL humanization, the CDRL1, CDRL2, and CDRL3 sequences of mAb0082 were grafted onto human germline sequences based on the VKII_A23 / JK2 sequence defined in the IMGT database. The VL sequence contains a potential deamidation hotspot (NG motif) in CDR1. Using saturation mutagenesis at this position, it was found that the NG motif could be removed by an N33Q substitution without compromising affinity for TLT-1.

[0223] The final humanized and optimized variant of mAb0082 corresponds to SEQ ID NOs: 934 (VL) and 938 (VH) and is designated mAb1076.

[0224] SPR analysis of humanized TLT1 variants Binding of sTLT1 (corresponding to SEQ ID NO: 3 with six histidine residues added to the C-terminus) from (Example 4) to the biAb was probed by surface plasmon resonance (Biacore T200) at 25°C. Anti-human IgG was immobilized on a CM5 sensor chip (both supplied by GE Healthcare) using standard amine coupling chemistry. Purified biAb according to Table 21 (1 nM) was injected for 1 min at a flow rate of 10 μl / min. Subsequently, sTLT1 between 0 and 60 μM was injected for 3 min at a flow rate of 30 μl / min to allow binding to the biAb, followed by a 3 min buffer injection to allow dissociation from the biAb. Running buffer was prepared by ten-fold dilution of 10xHBS-P buffer (provided by GE Healthcare) and supplemented with 1 mg / ml BSA and 5 mM CaCl2 to obtain 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 7.4, 5 mM CaCl2, 1 mg / ml bovine serum albumin (BSA). Running buffer was also used for dilution of biAb and sTLT1 samples. Chip regeneration was achieved using the recommended regeneration buffer consisting of 3 M MgCl2 (provided by GE Healthcare). Binding data were analyzed by a 1:1 model using BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala, Sweden). Analysis showed that the binding constants reported in Table 21 indicated a range of affinities from 2.9 nM to 320 nM for sTLT1 binding by the biAbs. [Table 30]

[0225] Example 20: Crystal structure of Hz-TLT1 and TLT-1 peptide complex preparation The Fab fragment used for crystallization in complex with the TLT-1 stalk peptide contains VL and VH domain sequences corresponding to mAb1076 (SEQ ID NOs: 854 and 858, respectively), a human IgG4 CH1 domain, and a human kappa CL with a single point mutation (G157C). The G to C substitution is within the constant domain of the Fab fragment, i.e., far from the antigen binding site, and does not affect binding to TLT-1. A 37-mer stalk peptide, EEEEETHKIGSLAENAFSDPAGSANPLEPSQDEKSIP (SEQ ID NO: 13), corresponding to residues 111-147 of SEQ ID NO: 2, was prepared by standard peptide synthesis methods known to those skilled in the art. The Fab and stalk peptides were mixed in a 1:2 molar ratio in Hepes buffer (20 mM Hepes (pH 7.3), 150 mM NaCl). The 1:1 Fab:peptide complex was isolated using gel filtration on a superdex200 column eluted with hepes buffer, then concentrated to approximately 11 mg / mL and used for crystallization.

[0226] Crystallization Crystals of gel filtered 1:1 molar Fab / peptide complex were grown using the sitting drop vapor diffusion method at 18° C. 150 nl of a 10.8 mg / ml protein solution of Fab:peptide complex in 20 mM Hepes, pH 7.3 and 150 mM NaCl was mixed with 50 nl of 1 M LiCl, 0.1 M Na citrate-citric acid, pH 4 and 20% (w / v) PEG 6000 as precipitant and incubated on 60 μl of precipitant.

[0227] Diffraction data collection Crystals were cryoprotected by the addition of 1 μl of precipitant, 20% ethylene glycol, to the crystallization drop before flash cooling in liquid nitrogen. Diffraction data were collected at 100 K at the BioMAX beamline of the MAX IV synchrotron (Lund, Sweden) using an Eiger 16M Hybrid-pixel detector from Dectris. Automatic indexing, integration, and scaling of the data were performed using programs from the XDS package (diffraction data statistics are summarized in Table 22).

[0228] Structure determination and refinement The asymmetric unit contains two Fab:peptide complexes as judged from Matthews coefficient analysis. The structure was determined by molecular replacement. Phaser implemented in the program suite Phenix was used with the heavy and light chains of protein data bank entry 5KMV as search models to localize the two Fabs. These were built with the correct amino acid sequence using COOT and then refined using Phenix refinement. Amino acids 7-21 from the peptide are clearly seen in the difference electron density map and can be modeled manually using COOT. The model was refined in COOT using Phenix refinement and manual rebuild steps. Refinement statistics are shown in Table 22. [Table 31]

[0229] Fab / peptide complex epitopes and paratopes The epitope is defined as the residues of the TLT-1 stalk peptide that are characterized by having a heavy atom (i.e., a non-hydrogen atom) within 4.0 Å of a heavy atom of the Fab in both complexes of the asymmetric unit. Similarly, the paratope is defined as the residues of the Fab fragment that are characterized by having a heavy atom within 4.0 Å of a heavy atom of the TLT-1 stalk peptide in both complexes of the asymmetric unit, and the epitope was found to include the following residues from the 37aa TLT-1 peptide according to SEQ ID NO: 13: K8 I9 G10 S11 L12 A13 N15 A16 F17 S18 D19 P20 A21 (This corresponds to K118, I119, G120, S121, L122, A123, N125, A126, F127, S128, D129, P130 and A131 in SEQ ID NOs: 2 and 3). The paratope comprises the following residues from the heavy chain variable domain (SEQ ID NO:938): V2 F27 R31 Y32 W33 E50 T57 N59 S98 G99 V100 T102 S103 and from the light chain variable domain (SEQ ID NO:934): H31 Y37 H39 Y54 F60 S61 S96 T97 V99 Y101, included.

[0230] Example 21: Effect of affinity on anti-FVII(a) / anti-TLT-1 bispecific antibody stimulatory activity To determine the effect of affinity on bispecific antibody activity, several anti-FVII(a) and anti-TLT-1 mAbs with varying affinities for FVIIa and TLT-1, respectively, from humanization of 11F2 mAb0005 (see Example 14) and mAb0012 (see Example 19) were tested in a bispecific format in a FXa generation assay using lipidated TLT-1 as described in WO2011 / 023785.

[0231] In a first step, FX activation was measured in the presence of 4 nM recombinant TLT-1, 2.5 nM FVIIa and bispecific antibodies (biAbs) incorporated in 10:90 phosphatidylserine:phosphatidylcholine vesicles (WO2011023785) in a concentration series from 0 to 300 nM. After a 10 min preincubation at room temperature in assay buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.3 + 1 mg / mL BSA and 0.1% PEG8000), 150 nM plasma-derived FX (Heamatologic Technologies) was added to obtain a total volume of 50 μl and activated for 20 min. Activation was then terminated by the addition of 25 μl of quench buffer (50 mM HEPES, 100 mM NaCl, 80 mM EDTA, pH 7.3) and the generated FXa was quantified from its ability to hydrolyze 0.5 mM S-2765 (Chromogenix) chromogenic substrate (added as a 2 mM stock in a volume of 25 μl) and followed at 405 nm for 5 min on a SPECTRAmax Plus384 plate reader. The slope of the linear absorbance increase was used to determine the activity (A) normalized for each biAb at a concentration of 100 nM by subtraction of background activity in the absence of biAb and division by the FVIIa concentration in the assay. biAb ) was calculated.

[0232] In a second step, the same assay was performed replacing the biAb with assay buffer and a concentration series of FVIIa from 0 to 80 nM. The slope of the linear relationship between background-subtracted FXa generation and the concentration of FVIIa in the assay was indicative of the free FVIIa (A) under the assay conditions employed. FVIIa ) gave a measure of the specific activity.

[0233] Based on the measured activity, the stimulatory activity of each biAb at 100 nM was calculated as A biAb / A FVIIa Stimulatory activity was calculated as a ratio of fold increase in FXa produced by FVIIa upon addition of 100 nM of biAb.

[0234] The stimulatory activity is provided in Table 23 and shows the dependence of biAb stimulation on the strength with which FVIIa and TLT-1 are bound, respectively (expressed as dissociation constant (KD) values). Among the biAbs tested, biAb0001 shows the highest stimulatory activity. [Table 32]

[0235] Example 22: Effect of epitope location on anti-FVII(a) / anti-TLT-1 bispecific antibody stimulatory activity To determine the effect of epitope location on bispecific antibody activity, several anti-TLT-1 and anti-FVIIa mAbs that bind to different epitopes on TLT-1 and FVIIa, respectively, were tested in a bispecific format in a FXa generation assay, as performed in Example 21.

[0236] The results are presented in Table 24 and show the dependence of biAb stimulatory activity on epitope location. Notably, anti-TLT-1 mAbs mAb1076, mAb0023, mAb0051 and mAb0062 in combination with anti-FVIIa mAb mAb0865 exhibit comparable stimulatory activity. [Table 33]

[0237] Example 23: Antigen assay for human IgG (LOCI) The presence of human IgG (hIgG) in cynomolgus monkey plasma was measured by Luminescent Oxygen Channelling Immunoassay (LOCI). Briefly, the LOCI reagent contained two latex bead reagents (donor and acceptor beads) and a biotinylated monoclonal antibody against hIgG (Biosite, catalog number AFC4249). The donor bead reagent, which contains a light-sensitive dye, was coated with streptavidin. The second bead reagent, the acceptor bead, was conjugated with an in-house monoclonal antibody against hIgG (0421) constituting a sandwich. During the assay, the three reactants were combined with hIgG in the plasma to form a bead-aggregated immune complex. Excitation of the complex released singlet oxygen molecules from the donor bead and delivered to the acceptor bead, eliciting a chemiluminescent response, which was then measured on an EnVision plate reader. The amount of light produced was reported as counts per second (cps) and was proportional to the concentration of hIgG. Samples were diluted at least 100-fold in assay buffer and a standard curve was prepared based on hIgG spiked into 1% cynomolgus monkey plasma.

[0238] Example 24: Antigen Assay for FVII(a) (LOCI) FVII(a) antigen, including FVII zymogen, FVIIa, and FVIIa:antithrombin (FVIIa:AT) complex, was measured by the LOCI assay described in Example 23, except that the assay for FVII(a) consisted of an in-house anti-FVII(a) antibody (4F9) and acceptor beads coated with an in-house biotinylated anti-FVII monoclonal antibody (4F7). Samples were diluted at least 100-fold in assay buffer and a standard curve was prepared by adding known amounts of human rFVIIa to the assay buffer.

[0239] Example 25: Antigen assay for FVIIa:AT (antithrombin) (EIA) FVIIa:AT (antithrombin) complexes were measured using enzyme immunoassay (EIA) as described by Agersφ H et al, J Thromb Haemost 2011;9:333-8. A monoclonal anti-FVIIa antibody (Dako Denmark A / S, Glostrup, Denmark, product code O9572) that binds to the N-terminal EGF domain and does not block antithrombin binding was used to capture the FVIIa:AT complex. A preformed complex of human FVIIa and cynomolgus monkey antithrombin (FVIIa:AT) was prepared by incubating FVIIa with a two-fold molar excess of antithrombin in the presence of 10 μM low molecular weight heparin (enoxaparin). The residual amidolytic activity of FVIIa after overnight incubation at room temperature (see Example 11) was verified to be less than 10% of the initial FVIIa activity. The complexes were used to construct an EIA standard curve. Polyclonal anti-human antithrombin antibody peroxidase conjugate (Siemens Healthcare Diagnostics ApS, Ballerup, Denmark, product code OWMG15) was used for detection. TMB was added and color development was allowed to proceed until sufficient color development was achieved, stopped by adding H2SO4, and absorbance was measured at 450 nm with a reference at 650 nm using an absorbance plate reader (BioTek). Color intensity is proportional to the concentration of FVIIa:AT.

[0240] Example 26: Preparation of human FVIIa, FVII (zymogen), and FVIIa:AT (antithrombin) complex Preparation of human FVIIa (activated FVII) Unless otherwise stated, recombinant human activated FVII (FVIIa) was prepared as described in Thim et al. (1988) Biochemistry 27:7785-7793 and Persson et al. (1996) FEBS Lett 385:241-243.

[0241] Preparation of human FVII (enzyme precursor FVII) Recombinant human FVII produced in CHO cells was purified by single-step calcium-dependent affinity chromatography as described by Thim et al. (1988) Biochemistry 27:7785-7793. After purification, zymogen FVII was dialyzed into a buffer of 10 mM MES, 100 mM NaCl, 10 mM CaCl2, pH 6.0. The level of activated FVII (FVIIa) in the preparation was determined by measuring the amidolytic activity in the presence of 1 mM chromogenic substrate S-2288 and 200 nM sTF (see Example 11). By relating this to a standard curve prepared with known concentrations of FVIIa, the measured activity can be converted to the molar concentration of FVIIa in the zymogen FVII preparation.

[0242] Preparation of human FVIIa:AT (antithrombin) complex FVIIa:AT (antithrombin) complex was prepared by incubating equimolar concentrations of human recombinant FVIIa, human plasma-derived AT (Baxter), and low molecular weight heparin (enoxaparin sodium) for 16 h at 4 °C. To remove contaminating excipients, AT was repurified on a Heparin Sepharose 6 Fast Flow (GE Healthcare) column applying a sodium chloride gradient before use. Eluted AT was concentrated by ultrafiltration to obtain the final preparation in 10 mM HEPES, 25 mM NaCl, pH 7.3 containing 50% glycerol. FVIIa:AT complex was purified by SEC chromatography in 20 mM MES, 100 mM NaCl, 1 mM EDTA, pH 5.5 at 4 °C to maximize the stability of the complex. Residual levels of FVIIa in the preparation were determined as described above. To minimize degradation of the complex, the preparation was stored in aliquots at −80°C and immediately thawed and kept on ice before use.

[0243] Example 27: Single-dose pharmacokinetics of anti-FVII(a) / anti-TLT-1 biAb in cynomolgus monkeys BiAb0001 and the corresponding YTE variant, biAb0352, in which three additional half-life extending substitutions (M252Y, S254T and T256E) have been introduced into the constant heavy domain, were administered intravenously (iv) at 3.0, 9.49 or 30 nmol / kg or subcutaneously (sc) at 9.49 nmol / kg to cynomolgus monkeys. Each group consisted of two monkeys (one male and one female). 1 mL sodium citrate stabilized blood samples were collected pre-dose and for the iv group at 0.5, 2.5, 6, 12, 24, 48, 72 hours and on days 8, 10 and 15 after dosing. For the sc group, blood samples were taken pre-dose and 0.5, 3, 6, 12, 16, 24, 30, 38, 48, 54, 72, 78, 96, 120 hours post-dose, and on days 8, 10, and 15. Blood samples were centrifuged at 2000 g for 10 minutes, plasma removed, divided into aliquots, and stored at −80° C. until analysis of hIgG (see Example 23), total FVII(a) antigen (see Example 24), FVIIa activity (see Example 8), and FVIIa:AT complex (Example 25). Pharmacokinetic (PK) analysis of the concentration versus time profile of hIgG was performed by non-compartmental methods using Phoenix WinNonlin 6.4. The following PK parameters are shown in Table 24: half-life (t1 / 2), clearance (Cl), volume of distribution (Vz), mean residence time (MRT) and SC bioavailability (F). [Table 34]

[0244] Accumulation of endogenous FVII(a) was observed in monkeys administered BiAb0001 or the corresponding YTE variant, biAb0352. Table 25 shows pre-dose levels of FVII(a) antigen, FVIIa activity, and FVII:AT and the mean cumulative levels measured between 72 and 240 hours post-dose. Antibody dose-dependent accumulation of FVII(a) antigen, FVIIa activity, and FVIIa:AT was observed. FVII(a) antigen increased up to 3-fold compared to pre-dose levels, and FVIIa and FVIIa:AT increased up to 5-fold. The data demonstrate that administration of a single dose iv or sc of biAb0001 or biAb0352, with the YTE mutation, leads to accumulation of endogenous FVII(a) antigen, FVIIa activity, and FVIIa:AT complex in vivo. [Table 35]

[0245] Example 28: Single- and multiple-dose pharmacokinetics of mAb0705(OA), a monovalent 11F2 anti-FVII(a) antibody, in cynomolgus monkeys In vivo accumulation of FVII, FVIIa, and FVIIa:AT was analyzed by administering the monovalent 11F2 anti-FVII(a) antibody mAb0705(OA) to cynomolgus monkeys, followed by measurement of FVII antigen, FVIIa activity, and FVIIa:AT in plasma samples. Two male cynomolgus monkeys weighing approximately 2.5 kg were administered 40 nmol / kg mAb0705(OA) iv into the saphenous, cephalic, or lateral tail vein of the tail, and three male cynomolgus monkeys were administered 20 nmol / kg one-armed anti-FVIIa antibody subcutaneously (sc) in the thighs (alternating between the left and right thighs) every other day for 2 weeks (i.e., antibody was administered on days 1, 3, 5, 7, 9, 11, 13, and 15). Blood was collected into 3.8% trisodium citrate from the cephalic or femoral vein up to 21 days after dosing.

[0246] Blood was centrifuged at 2300 g for 10 min and plasma was aliquoted and stored at −80° C. until analysis of hIgG (see Example 23), total FVII(a) antigen (see Example 24), FVIIa activity (see Example 8), and FVIIa:AT complex (see Example 25). PK analysis of concentration versus time profiles was performed by non-compartmental methods using Phoenix WinNonlin 6.4. PK parameters for iv administration are shown in Table 26. The half-life (t1 / 2) of the one-armed antibody averaged 116 hours (4.8 days) after a single IV dose. The estimated half-life of the sc-administered antibody was 181±20 hours (mean and SD of n=3). The antibody accumulated over the course of 2 weeks of dosing, resulting in a maximum level of 1179±140 nM at a time point between 336 and 366 hours after the first dose (mean and SD of data from n=3 animals and 3 time points). [Table 36]

[0247] Single IV or repeated sc administration of mAb0705(OA) resulted in accumulation of endogenous FVII(a). Total FVII(a) antigen, FVIIa:AT and FVIIa after a single dose of 40 nmol / kg are shown in Table 27. Total FVII antigen increased from 7.0 nM pre-dose to 35.0±4.7 nM on days 7-14. Similarly, FVIIa increased from below detection limit (0.009 nM) to 2.3±0.7 nM and FVIIa:AT increased from 1.0 to 6.3±0.8 nM on days 7-14. Zymogen FVII levels after administration of 40 nmol / kh one-armed antibody were 26.4 nM, calculated by subtracting FVIIa and FVIIa:AT from total FVII(a) antigen. [Table 37]

[0248] Steady-state levels of total FVII(a), FVIIa, and FVIIa:AT after multiple sc doses are shown in Table 28. Total FVII(a) antigen increased from 6.5±1.5 nM pre-dose to 36.9±9.8 nM on days 12-21. Similarly, FVIIa increased from below the limit of detection (0.009 nM) to 3.9±1.6 nM on days 12-21, and FVIIa:AT increased from 1.0±0.3 to 9.1±0.6 nM on days 12-21. The steady-state zymogen FVII level, calculated by subtracting FVIIa and FVIIa:AT from total FVII(a) antigen, was 24 nM.

[0249] The data demonstrate that administration of the one-armed anti-FVII(a) antibody mAb0705(OA) resulted in accumulation of endogenous FVII(a), FVIIa and FVIIa:AT in vivo. The clearance of the one-armed anti-FVII(a) antibody (0.42-0.49 mL / kg×kg, Table 26) was comparable to that of biAb0001 after intravenous administration (0.33-0.64 mL / kg×kg, Example 27, Table 24). Thus, the steady-state levels of FVII(a) antigen, FVIIa and FVIIa:AT measured after repeated administration of the one-armed anti-FVII(a) antibody are expected to represent the levels that would be achieved at steady state after repeated administration of a biAb with the same FVII(a) binding arm. [Table 38]

[0250] Example 29: Thromboelastography in hemophilia A-like states in human whole blood supplemented with anti-FVII(a) / anti-TLT-1 biAb0001 and steady-state levels of zymogens FVII, FVIIa and FVIIa:AT The effect of bispecific anti-FVII(a) / anti-TLT-1 antibody biAb0001 (parent anti-FVII(a) is mAb0865, parent anti-TLT-1 antibody is mAb1076) and accumulated levels of zymogens FVII, FVIIa and FVIIa:AT from Example 28 was assessed by thromboelastography in human whole blood in a hemophilia A-like state. Thromboelastography analysis was performed using a TEG® instrument (Thrombelastograph Coagulation Analyzer, Haemoscope Corp.) essentially as described in Viuff D, et al. Thromb Res 2010; 126:144-9. Citrate-stabilized whole blood from healthy donors was incubated with 1 mg / mL neutralizing anti-FVIII sheep polyclonal antibody (Haematological Technologies Inc, Cat. No. PAHFVIII-SC) and 5 μg / mL neutralizing anti-TF mouse monoclonal antibody (1F44, prepared in-house) for 30 min. biAb0001 at a final plasma concentration of 100 nM in HBS / BSA buffer (20 mM Hepes, 140 mM NaCl, pH 7.4, 2% BSA) was mixed with rFVIIa (Novo Seven®, Novo Nordisk, final plasma concentration 3.9 nM), zymogen FVII (as prepared in Example 26), final plasma concentration 24 nM) and FVIIa:AT complex (as prepared in Example 26), final plasma concentration 9 nM) and added to the blood sample. Predilutions of FVIIa:AT were made in cold 20 mM MES, 100 mM NaCl, 1 mM EDTA, pH 5.5 + 2% BSA and added to the remaining protein immediately before starting the assay. Because the FVII zymogen and FVIIa:AT preparations contained trace amounts of FVIIa, a corresponding lower amount of FVIIa was added (0.1 nM, Morrissey JH et al Blood, 1993;81:734-44) to compensate for this and the expected plasma concentration of FVIIa in donor blood. Similarly, the amount of zymogen FVII added compensated for the expected plasma concentration of 10 nM zymogen FVIIa in donor blood.Another sample contained 25 nM rFVIIa, corresponding to the theoretical maximum plasma concentration after administration of 90 μg / kg rFVIIa (NovoSeven®) to human subjects with hemophilia A (Lindley CM et al. Clin Pharmacol Ther 1994;55:638-48). Controls included biAb only and FVII / FVIIa / FVIIa:AT mix without BiAb. Platelets were maximally activated by adding the PAR1 agonist peptide SFLLRN (Tocris Biosciences Cat. No. 3497) to a final concentration of 30 μM and the GPVI agonist convulxin (5-Diagnostics Cat. No. 5D-1192-50UG) to a final concentration of 10 ng / mL. A volume of 20 μl of 0.2 M CaCl2 in 20 mM Hepes, pH 7.4 was added to the TEG cup, followed by the addition of 340 μl of blood sample, and analysis was started immediately. Clotting time (R-time), defined as the time to 2 mm amplitude of the TEG trace, was calculated by the software (TEG® Analytical Software, version 4.1.73). Data from four donors are shown in Table 29. Clotting time was delayed from 290 ± 12 seconds in normal blood to 3506 ± 1561 seconds after inducing a hemophilia A-like state by neutralizing FVIII. Addition of 25 nM rFVIIa reduced the clotting time of simulated hemophilia A blood to 694 ± 158 seconds. Addition of 100 nM biAb to the blood slightly reduced the clotting time to 2198 ± 712 seconds, possibly due to an enhanced potentiation of the effect of endogenous FVIIa in the blood. Addition of steady-state levels of FVII / FVIIa / FVIIa:AT reduced the clotting time to 1440 ± 275 seconds. Combining 100 nM biAb and steady-state levels of FVII / FVIIa / FVIIa:AT reduced the clotting time to 495 ± 39 seconds, i.e., to a level similar to or slightly lower than that after addition of 25 nM FVIIa.The data show that the biAb potentiates the effect of accumulated levels of FVII / FVIIa / FVIIa:AT, resulting in clotting time reductions similar or slightly better than those achieved with therapeutically effective concentrations of rFVIIa. [Table 39]

[0251] Example 30: Thromboelastography in hemophilia A-like states in human whole blood supplemented with bispecific anti-FVII(a) / anti-TLT-1 antibodies with different TLT-1 affinities and steady-state levels of zymogens FVII, FVIIa and FVIIa:AT In this example, the following bispecific anti-FVII(a) / anti-TLT-1 antibodies with different affinities for TLT-1 were tested: [Table 40]

[0252] Bispecific antibodies at a concentration of 100 nM were evaluated by thromboelastography as described in Example 29. Clotting times (R-times) are listed in Table 30. Upon induction of hemophilia A (HA), the clotting time was delayed from 340 to 5433 seconds by the addition of anti-FVIII antibodies. The presence of the biAb with the highest affinity for TLT-1 (biAb0001) reduced the clotting time to 2465 seconds, i.e., greater than the reduction in clotting time observed with only the three other biAbs (biAb0015 reduced the clotting time to 3645 seconds, -biAb0090 to 4335 seconds, and biAb0095 to 4110 seconds). Similarly, combination of biAbs with steady-state levels of FVII, FVIIa, and FVII:AT resulted in a more pronounced decrease in clotting time for biAb0001 (to 540 seconds) than the three other biAbs (i.e., to 1100 seconds for biAb0015, to 1040 seconds for biAb0090, and to 815 seconds for biAb0095). The data indicate that biAb0001, which has the highest affinity for TLT-1 (i.e., lowest KD), is the most effective in shortening clotting time. [Table 41]

[0253] Example 31: In vivo efficacy of anti-FVII(a) / anti-TLT-1 bispecific antibody biAb0001 in a tail vein section model in FVIII-deficient transgenic human TLT-1 mice The in vivo efficacy of anti-FVII(a) / anti-TLT-1 bispecific antibody biAb0001 was determined using a tail vein transection (TVT) model in transgenic FVIII knockout (i.e., hemophilia A), mouse TLT-1 knockout, and human knock-in mice. Because anti-FVII(a) does not recognize mouse FVII(a), the biAb was co-administered with human FVIIa, FVII, and FVII:AT to obtain target plasma levels of these components in mice (3.8, 26.2, and 9.0 nM, respectively) that mimic their expected clinical steady-state plasma levels according to Example 28. The concentration of biAb was either 40 or 100 nM. Briefly, mice were anesthetized with isoflurane and placed on a heating pad with the tail immersed in saline (37°C), with the animal's body temperature set to be maintained at 37°C. Dosing was performed into the right tail vein 5 min prior to injury. In this TVT model (Johansen et al., Haemophilia, 2016, 625-31), the lateral vein is cut. When bleeding stopped at 10, 20, or 30 minutes, the tail was removed from the saline and the wound was gently wiped with a saline-moistened gauze swab. Total blood loss was determined 40 minutes after administration by quantifying the amount of hemoglobin in the saline. Blood samples were taken from the orbital plexus in 3.8% trisodium citrate 40 minutes after administration. Blood was centrifuged at 4000g for 5 minutes and plasma was aliquoted and stored at -80°C until analysis of hIgG (see Example 23), total FVII(a) antigen (see Example 24), and FVIIa activity (see Example 8).

[0254] As shown in Table 31, all combinations of FVIIa and biAb resulted in a significant reduction in blood loss compared to biAb alone or FVII(a) administered without biAb. For all combinations, platelet counts measured 45 minutes after treatment were comparable to those observed for the vehicle group.

[0255] In conclusion, these data demonstrate significant in vivo hemostatic effects of biAb0001 in the presence of expected steady-state levels of FVIIa, FVII, and FVII:AT. Table 31. Blood loss following tail vein transection (TVT) in FVIII knockout / mouse TLT-1 knockout / human TLT-1 knockin mice administered combinations of biAb0001, FVIIa, FVIIa, and FVIIa:AT as indicated. Administered doses, expected and measured plasma concentrations, and determined blood loss are shown as mean ± SEM (n=10). Using one-way ANOVA followed by Dunnett's multiple comparison test, blood loss in groups 3-5 was found to be significantly different from groups 1 and 2. Total FVII(a) antigen concentrations (measured according to Example 23) are listed in the row labeled "FVII" and values ​​are indicated with an asterisk. [Table 42]

[0256] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes that fall within the true spirit of the invention.

[0257] Example 32: Identification of antibodies that compete with anti-TLT-1 mAb 1076 for binding to TLT-1 in a competitive ELISA The Fab fragment (anti-TLT-1 Fab) used for the competition experiments contains the VH and VL domain sequences corresponding to mAb1076 (SEQ ID NOs: 938 and 934, respectively), a human IgG4 CH1 domain, and a human kappa CL with a single point mutation (G157C). The G to C substitution is within the constant domain of the Fab fragment, i.e., far from the antigen binding site, and does not affect binding to TLT-1 (see Example 20). Recombinant is produced as TLT-1 as described in WO2011 / 023785. Anti-TLT-1 Fab is biotinylated using standard methods, including application of a biotinylation kit (EZ-link, Thermo) according to the manufacturer's instructions.

[0258] To determine whether anti-TLT-1 antibodies compete with anti-TLT-1 Fab and antibodies derived therefrom, competition studies for binding to TLT-1 were performed. Recombinant TLT-1 is immobilized on NUNC maxisorp plates overnight at 4°C in dilution buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, pH 7.2). Plates are washed for 15 min in wash buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, 0.5 mL / L Tween 20, pH 7.2) and blocked. For competition studies, biotinylated anti-TLT-1 Fab at final immobilized concentration is combined with a dilution series of anti-TLT-1 antibodies to give final concentrations ranging from 0 up to 100 mg / ml in dilution buffer. The mixtures are added to the wells of the plate and incubated for 1 h. Plates are then washed and HRP-labeled streptavidin-HRPO (1:2000 in dilution buffer, Kirkegaard & Perry Labs) is added and incubated for 1 hour. Finally, plates are washed and developed with TMB ONE (KEMENTEC) for 10 minutes. The reaction is stopped by adding H3PO4 (4M) and plates are read on a FLUOStar Optima plate reader at 450 nm with subtraction of the background signal measured at 620 nm. Unless otherwise specified, all incubations are performed at room temperature and plates are washed five times using wash buffer.

[0259] The concentration of recombinant TLT-1 immobilized on the NUNC maxisorp plate, as well as the immobilization concentration of biotinylated anti-TLT-1 Fab mixed with the competing antibody for competition studies, is determined by individual titration of the two components with the goal of giving a sufficient signal to allow detection of competition (i.e., reduction of signal) by the competing antibody. The concentration of TLT-1 for immobilization is typically in the range of 0-1 mg / ml, such as 125 ng / ml. The concentration of biotinylated anti-TLT-1 Fab is typically in the range of 0-1 mg / ml, such as 10 ng / ml.

[0260] From the measured signal (in OD units), the competition at any given antibody concentration is Percent inhibition = (1-(OD units-100% inhibition) / (0% inhibition-100% inhibition))*100 where 0% inhibition is determined from the signal in wells with no competing anti-TLT-1 antibody and 100% inhibition is determined as the signal from wells with no biotinylated anti-TLT-1 Fab (i.e., corresponding to the assay background). An antibody is considered to compete with anti-TLT-1 Fab for binding to TLT-1 if there is at least 50% observed inhibition (% inhibition) when concentrations of antibody are tested up to a 10,000-fold excess of biotinylated anti-TLT-1 Fab.

Claims

1. A bispecific antibody, (i) a first antigen-binding site capable of binding to Factor VII(a); and (ii) a second antigen-binding site capable of binding to TREM-like transcript 1 (TLT-1); and A bispecific antibody comprising:

2. The bispecific antibody of claim 1 , wherein the antibody comprises an Fc region.

3. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding site competes for binding to FVII(a) with any one of the anti-FVII(a) antibodies comprising the light chain variable domain (VL) and heavy chain variable domain (VH) shown below: mAb0522 (VL: SEQ ID NO: 846 and VH: SEQ ID NO: 850), Fab0883 (VL: SEQ ID NO: 814 and VH: SEQ ID NO: 818), mAb0005 (VL: SEQ ID NO: 750 and VH: SEQ ID NO: 754), mAb0004 (VL: SEQ ID NO: 14 and VH: SEQ ID NO: 18), mAb0013 (VL: SEQ ID NO: 46 and VH: SEQ ID NO: 50), mAb0018 (VL: SEQ ID NO: 62 and VH: SEQ ID NO: 66), mAb0544 (VL: SEQ ID NO: 694 and VH: SEQ ID NO: 698), mAb0552 (VL: SEQ ID NO: 702 and VH: SEQ ID NO: 706), mAb0001 (VL: SEQ ID NO: 710 and VH: SEQ ID NO: 714), mAb0007 (VL: SEQ ID NO: 718 and VH: SEQ ID NO: 722), mAb0578 (VL: SEQ ID NO: 726 and VH: SEQ ID NO: 730), mAb0701 (VL: SEQ ID NO: 734 and VH: SEQ ID NO: 738), and - mAb0587 (VL: SEQ ID NO: 742 and VH: SEQ ID NO: 746).

4. 3. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding site is capable of binding to an epitope comprising amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO:1).

5. 3. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding site is capable of binding an epitope comprising the following amino acid residues of FVII(a) (SEQ ID NO: 1): R113, C114, H115, E116, G117, Y118, S119, L120, T130, V131, N184, T185, P251, V252, V253, Q388, M391, and R392.

6. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding site comprises: - CDRL1 represented by amino acid residues RASQGISDYLH (SEQ ID NO: 847), having 0, 1, 2 or 3 amino acid substitutions; - a CDRL2 represented by amino acid residues YTSQPAT (SEQ ID NO:848), having 0, 1, 2 or 3 amino acid substitutions; - a CDRL3 represented by amino acid residues QNGHSFPLT (SEQ ID NO: 849), having 0, 1 or 2 amino acid substitutions; - CDRH1 represented by amino acid residues SDSAWS (SEQ ID NO: 851), - a CDRH2 represented by amino acid residues YIQYSGST NYNPSLKS (SEQ ID NO: 852), with 0 or 1 amino acid substitutions; - CDRH3 represented by amino acid residues SVNYYGNSFAVGY (SEQ ID NO:853), with 0, 1, 2 and 3 amino acid substitutions.

7. 3. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO:846 and a heavy chain variable domain defined by SEQ ID NO:

850.

8. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding site competes for binding to TLT-1 with any one of the anti-TLT-1 antibodies comprising the light chain variable domain (VL) and heavy chain variable domain (VH) shown below: mAb0524 (VL: SEQ ID NO: 854 and VH: SEQ ID NO: 858), mAb0012 (VL: SEQ ID NO: 862 and VH: SEQ ID NO: 866), mAb0023 (VL: SEQ ID NO: 870 and VH: SEQ ID NO: 874), mAb0051 (VL: SEQ ID NO: 878 and VH: SEQ ID NO: 882), and - mAb0062 (VL: SEQ ID NO: 894 and VH: SEQ ID NO: 898).

9. 3. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding site is capable of binding to an epitope comprising the following amino acid residues of TLT-1 (SEQ ID NO: 13): K8, I9, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, A21.

10. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding site comprises: CDRL1 represented by SEQ ID NO: 855, - CDRL2 represented by SEQ ID NO: 856, - CDRL3 represented by SEQ ID NO: 857, - CDRH1 represented by SEQ ID NO: 859, - CDRH2 represented by SEQ ID NO: 860, - CDRH3 represented by SEQ ID NO:

861.

11. 3. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding site is constituted by a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO:

858.

12. 2. The bispecific antibody of claim 1, wherein the first antigen-binding site comprises a light chain variable domain defined by SEQ ID NO: 846 and a heavy chain variable domain defined by SEQ ID NO: 850, and the second antigen-binding site is constituted by a light chain variable domain defined by SEQ ID NO: 854 and a heavy chain variable domain defined by SEQ ID NO:

858.

13. 2. The bispecific antibody of claim 1, wherein the first antigen-binding site comprises a first light chain variable domain defined by SEQ ID NO: 846 and a first heavy chain variable domain defined by SEQ ID NO: 850, the second antigen-binding site is constituted by a second light chain variable domain defined by SEQ ID NO: 854 and a second heavy chain variable domain defined by SEQ ID NO: 858, the heavy chain constant domains associated with the first and second heavy chain variable domains are defined by SEQ ID NO: 943 and 942, respectively, and the light chain constant domains associated with the first and second light chain variable domains are both defined by SEQ ID NO:

12.

14. 14. A pharmaceutical formulation comprising a bispecific antibody according to any one of claims 1 to 13 and a pharma- ceutically acceptable carrier.

15. 15. The bispecific antibody of any one of claims 1 to 13 or the pharmaceutical formulation of claim 14 for use in the treatment of a coagulation disorder, wherein said coagulation disorder is selected from the group consisting of hemophilia A with or without inhibitors, or hemophilia B with or without inhibitors, FVII(a) deficiency and Glanzmann thrombasthenia.

Images