Dual-specific 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, such as pharmaceutical and therapeutic uses.

[0002] Sequence List This application is filed together with an electronic sequence listing. The entire contents of the sequence listing are incorporated herein by reference. [Background technology]

[0003] The blood coagulation process involves several proteins that work together after vascular injury to form a thrombus that prevents severe fluid loss and / or pathogenic invasion. The cascade of events leading to thrombus formation can be initiated via two pathways known as the intrinsic (contact) and extrinsic (tissue factor) pathways. Each pathway consists of a series of enzyme precursor activation steps in which newly activated enzymes catalyze the activation of the next enzyme precursor in a series until prothrombin is converted to thrombin. Thrombin converts fibrinogen into a fibrin network structure, activates platelets to form a platelet thrombus, and together they form a stable thrombus. The initiation of the extrinsic coagulation pathway is mediated by the formation of a complex between membrane connective tissue factor (TF), which is exposed as a result of injury to the vascular 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 initial stages, low concentrations of FXa produce trace amounts of thrombin that can activate factor XI and cofactors VIII and V. During the proliferation stage, procoagulant complexes are assembled, which activate tenases (FIXa, FVIIIa, Ca, F 2+ , phospholipids) and prothrombinase (FXa, FVa, Ca 2+ It significantly enhances the production of FXa and thrombin by the phospholipid complex.

[0004] In patients with hemophilia A and B (HA and HB, respectively), various steps of the coagulation cascade become dysfunctional due to the absence or insufficient presence of functional FVIII and FIX, respectively. This leads to blood clotting 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) bleeding treatment in hemophilia (A and B) patients with inhibitors (HwI). When administered intravenously (IV), rFVIIA has a short systemic half-life of 2-3 hours, and when administered subcutaneously (SC), it has low bioavailability. The short systemic half-life of rFVIIA is thought to be due to 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, among other mechanisms. 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 administration and with SC administration.

[0007] Roche recently launched emicizumab, a bispecific antibody, which is indicated for routine prophylaxis administered once weekly via SC to individuals with HA and inhibitor-carrying HA (HAwI). Nevertheless, the development of safe and effective molecules with alternative mechanisms of action remains a key area of ​​interest for improving and complementing standard care for hemophilia patients. [Overview of the project]

[0008] The present invention relates to bispecific antibodies that exhibit improved pharmaceutical properties, and particularly to bispecific antibodies that can be used for the treatment of subjects with congenital and / or acquired coagulation disorders, for example, the treatment of people with inhibitor-bearing or inhibitor-free hemophilia A or B. Further, the present invention particularly relates to bispecific antibodies that can bind coagulation factor VII (FVII(a)) and triggering receptor expressed on myeloid cells-like transcript 1 (TLT-1).

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

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

[0011] In one embodiment of the present invention, the bispecific antibody comprises an Fc region. The Fc region mediates recycling of the bispecific antibody and extends its half-life in circulation. In one embodiment of the present 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) 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: Sequence ID 710 and VH: Sequence ID 714), • mAb0007 (VL: Sequence ID 718 and VH: Sequence ID 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 present invention, the first antigen-binding site of the bispecific antibody can bind an epitope comprising amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO: 1).

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

[0014] In one aspect of the present invention, the bispecific antibody is administered parenterally, such as intravenously, intramuscularly, or subcutaneously. In one aspect of the present invention, the bispecific antibody enables prophylactic treatment of subjects with congenital and / or acquired coagulation disorders such as hemophilia A, hemophilia B, inhibitor-possessed hemophilia A, or inhibitor-possessed hemophilia B. Therefore, the bispecific antibody is designed to provide a hemostatic scope for bleeding. In one aspect of the present invention, the bispecific antibody is designed to be suitable for administration once a week, once a month, or less frequently.

[0015] Therapies using the bispecific antibodies of the present invention may offer many advantages, including longer duration of action between injections, easier administration, and potentially improved hemostatic protection between injections. Therefore, the bispecific antibodies described herein may have a substantial impact on the quality of life of individuals with inhibitor-bearing or inhibitor-free hemophilia A or B.

[0016] A brief explanation of sequence listings Table 1 is a summary of the antibodies and their corresponding sequence numbers for the corresponding VL and VL domain sequences. The heavy chain constant domain types are as defined in Table 2a ("M" indicates the mouse IgG1 constant domain).

[0017] Table 2a outlines the various formats used for recombinant expression of bivalent, monovalent (OA) antibodies, and bispecific antibodies. Sequence IDs corresponding to the first heavy chain (HC-1) and the second heavy chain (HC-2, or, for OA antibodies, the truncated heavy chain (trHC)) are listed. In all cases, the constant light chain domain was human kappa, corresponding to Sequence ID 12.

[0018] Table 2b provides an overview of the mouse anti-FVII(a) antibody of the present invention. It includes the clone name, the complete mouse antibody (hybridoma-derived, excluding recombinantly expressed mAb0765), and the correspondence between the mouse / human chimeric variant (mouse variable domain, human IgG4 S228P constant domain).

[0019] Table 2c provides an overview of antibodies in mouse and humanized 11F2 strains.

[0020] Table 2d is an overview of the anti-TLT-1 mAb0012 antibody lineage. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 2] [Table 3] [Table 4] [Table 5]

[0021] In this invention, all recombinant 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, rapidly cleaved in vivo; see Cai et al. Biotechnol. Bioeng. 2011 vol. 108, pp. 404-412) was omitted (referred to as delta-lys). As shown in Table 2a, additional substitutions were introduced into the heavy chain constant domain to ensure bispecificity and desired chain pairing in monovalent antibodies. (Duobody mutation F405L R409K (Labrijn et al. PNAS 2013, vol.110, pp.5145-5150) and knob-in-hole mutation (see Carter et al. J.Imm.Methods 2001, vol.248, pp.7-15) T366W (knob) and T366S L368A Y407V (hole)), or extending the in vivo half-life (YTE mutation M252Y S254T T256E (Dall'Acqua et al. J.Biol.Chem. 2006, vol.18, pp.23514-23524)). All recombinant antibodies produced in this invention have a human kappa light chain constant domain (SEQ ID NO: 12). [Modes for carrying out the invention]

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

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

[0024] Of particular interest for therapeutic antibodies are the IgG subclasses, which in humans are classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequences of their heavy chain constant regions. The light chains can be divided into two types, kappa chains and lambda chains, based on differences in their sequence compositions. An IgG molecule consists 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. The IgG heavy chain contains a heavy chain variable domain (V H ) and up to three heavy chain constant (C H ) domains: C H 1, C H 2, and C H 3. The light chain can contain a light chain variable domain (V L ) and a light chain constant domain (C L ). The V H and V L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The V H and V[[ID=Z]] L domains typically consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable domain and the light chain variable domain containing the hypervariable regions (CDRs) form a structure that can interact with an antigen, while the constant regions of the antibody can mediate binding to Fc receptors and C1q, the first component of the classical complement system's C1 complex.

[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 the antibody that retains the ability to bind to its own 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 V H Domain or V L Domains are an example (but are not limited to these).

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

[0028] As used herein, the term “single-specific” antibody refers to an antibody (including, but not limited to, bivalent antibodies) that can bind to one specific epitope.

[0029] In this specification, the terms “bispecific antibody” and “biAb” refer to antibodies that can bind to two different antigens, such as FVII(a) and TLT-1, or to two different epitopes on the same antigen.

[0030] The bispecific antibodies of the present invention are derived from an antibody or its antigen-binding fragment. The bispecific antibodies of the present invention may be fusions or conjugates of an antibody and an antigen-binding fragment of the antibody, such as Fab, Fab', Fab2, Fab'2, or scFv. The bispecific antibodies of the present invention may be fusions or conjugates of antibody fragments. A wide range of molecular forms 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 prepared by various methods 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, desired heavy chain pair formation can be achieved by manipulating the dimerization interface of the Fc region to promote heterodimerization. One example of this is the so-called knob-in-hole mutation, in which a sterically bulky side chain (knob) is introduced into one Fc that matches a sterically smaller side chain (hole) on the opposing Fc, thereby creating steric complementarity that promotes heterodimerization. Other methods for manipulating the heterodimerization Fc interface include electrostatic complementarity, fusion to a non-IgG heterodimerization domain, or in vitro heterodimerization utilizing the natural Fab-arm exchange phenomenon of human IgG4. Examples of heterodimerized bispecific antibodies are described in detail in the literature, e.g., (Klein C, et al.; MAbs. 2012 4, pp653-663). Special attention must be paid to the light chain of heterodimerized antibodies. Correct pairing between LC and HC can be achieved by using a common light chain. In this case as well, manipulation of the LC / HC interface can be used to facilitate heterodimerization or light chain crossover operations, as in the case of CrossMab. In vitro reconstruction of antibodies from two individual IgGs containing the appropriate mutations under mild reduction conditions can also be used to generate bispecific antibodies (e.g., Labrijnet al., PNAS, 110(2013), pp5145-5150). Methods for exchanging natural Fab arms to ensure correct light chain pairing have also been reported.

[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. Therefore, multispecific antibodies include bispecific antibodies.

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

[0034] In one embodiment, the antibody of the present invention is a chimeric antibody, a human antibody, or a humanized antibody. Such antibodies can be produced, for example, by using a suitable antibody display or immunization platform, or 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 is also derived from a human germline immunoglobulin sequence. The human antibody of the present invention may contain amino acid residues not encoded by a human germline immunoglobulin sequence (for example, mutations introduced by random or site-directed mutagenesis in vitro, or somatic mutations in vivo).

[0036] Human antibodies may also be isolated from sequence libraries constructed based on the selection of human germline sequences, and are further diversified by natural and synthetic sequence diversity. Human antibodies can 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] As used herein, the term “humanized antibody” refers to a human / non-human antibody containing a sequence (CDR region or portion thereof) derived from a non-human immunoglobulin. Thus, a humanized antibody is a human immunoglobulin (recipient antibody) in which at least residues from 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. Examples of such modifications include the introduction of one or more so-called reverse mutations, which are typically amino acid residues derived from the donor antibody. Antibody humanization may be carried out using recombination techniques known to those skilled in the art (see, e.g., Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. Lo). A suitable human recipient framework for both the light-chain 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 its structure, biophysical properties, and biochemical properties. The recipient framework can be germline-derived or mature antibody sequence-derived. The CDR region from the donor antibody can be transferred by CDR transplantation. CDR-transplanted humanized antibodies can be further optimized in terms of affinity, functionality, and biophysical properties, for example, by identifying key framework locations where the reintroduction of amino acid residues from the donor antibody (reverse mutation) has a beneficial effect on the properties of the humanized antibody. In addition to reverse mutation from the donor antibody, humanized antibodies can be manipulated by introducing germline residues into the CDR or framework region, removing immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0038] Furthermore, humanized antibodies may contain residues not found in recipient or donor antibodies. These modifications are made to further refine antibody performance. Humanized antibodies may also optionally contain at least a portion of the constant region (Fc) of an immunoglobulin, typically that portion of a human immunoglobulin.

[0039] As used herein, the term "chimeric antibody" refers to an antibody that contains antibody portions derived from two or more species. For example, the gene encoding such an antibody includes a gene encoding a variable domain and a gene encoding a constant domain, both originating from two different species. For instance, the gene encoding the variable domain of a mouse monoclonal antibody may be bound to the gene encoding the constant domain of a human-derived antibody.

[0040] Antibodies or fragments thereof can be defined in terms of their complementarity-determining regions (CDRs). As used herein, the term “complementarity-determining region” refers to the region of an antibody where amino acid residues involved in antigen binding are typically located. A CDR can be identified as the region with the highest variability among the antibody's variable domains. Databases such as the Kabat database can be used for CDR identification, and this CDR is defined, for example, as containing amino acid residues 24–34 (L1), 50–56 (L2), and 89–97 (L3) in the light chain variable domain, and amino acid residues 31–35 (H1), 50–65 (H2), and 95–102 (H3) in the heavy chain variable domain (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). Typically, amino acid residue numbering in this region is performed by the method described in Kabat et al. above. In this specification, terms such as “Kabat position,” “Kabat residue,” and “according to Kabat” refer to this numbering system for heavy-chain or light-chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to shortenings of the variable domain framework (FR) or CDR, or insertions into them. For example, a heavy-chain variable domain may include amino acid insertions after residue 52 of the CDR H2 (residues 52a, 52b, and 52c according to Kabat) and residues inserted after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a homology region of a “standard” Kabat numbering sequence. Numbering follows Kabat only if specifically stated; otherwise, numbering proceeds sequentially according to the specified array index.

[0041] The terms “framework region” or “FR” residues are defined herein as these V not located within the CDR. H or V L This refers to an amino acid residue.

[0042] The antibody of the present invention may comprise a CDR region from one or more of the specific antibodies disclosed herein.

[0043] The term “antigen” (Ag) refers to a molecular entity used for immunization of immune-responsive vertebrates to produce an antibody (Ab) that recognizes Ag. In this specification, Ag is a broader term and is generally intended to include target molecules recognized by Ab.

[0044] The present invention comprises variants of the antibody or its antigen-binding fragments, which may include one, two, three, four, or five amino acid substitutions and / or deletions and / or insertions in the specific sequences disclosed herein.

[0045] A "substitution" variant preferably involves substituting one or more amino acids with an equal number of amino acids.

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

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

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

[0049] An epitope on an antigen may contain one or more hotspot residues, i.e., residues particularly important for interaction with a congener antibody, and the interaction mediated by the side chain of the hotspot residue significantly contributes to the binding energy of the antibody / antigen interaction (Peng et al. PNAS 111, (2014), E2656-E2665). Hotspot residues can be identified by testing for antigen variants, where a single epitope residue is substituted, for example, with alanine for binding to a congener antibody. If the substitution of an epitope residue with alanine has a strong effect on binding to the antibody, the epitope residue is considered a hotspot residue and is therefore particularly important for antibody binding to the antigen.

[0050] Antibodies that bind to the same antigen can be characterized in terms of their ability to bind to that common antigen simultaneously, 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 the competitive binding of two antibodies to their common antigen in assays based on standard techniques. Antibody “bins” are defined using a reference antibody. If a 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 secondary antibody competitively bind to the same portion of the antigen and are called “competitive antibodies.” If a secondary antibody can bind to the antigen simultaneously with the reference antibody, the secondary antibody is said to belong to a separate “bin.” In this case, the reference antibody and secondary antibody do not competitively bind to the same portion of the antigen and are called “non-competitive antibodies.”

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

[0052] Typically, competitive assays involve 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, while the test antibody is not. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cell in the presence of the test antibody. Typically, 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., competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, or to a duplicate epitope, and antibodies that bind to an adjacent epitope sufficiently proximal to the epitope to which the reference antibody binds due to steric hindrance.

[0053] In an exemplary competitive assay, the reference anti-FVII or anti-FVIIa antibody is biotinylated using a commercially available reagent. The biotinylated reference antibody is mixed with serial dilutions of the test antibody or unlabeled reference antibody (self-competitive control) to obtain mixtures of the test antibody (or unlabeled reference antibody) at various molar ratios relative to the labeled reference antibody (e.g., 1, 5, 10, 20, 100, 1000, 10000 or 100000 times). The antibody mixture is added to an FVII or FVIIa polypeptide-coated ELISA plate. The plate is then washed, and horseradish peroxidase (HRP)-streptavidin is added to the plate as the detection reagent. The amount of labeled reference antibody bound to the target antigen is detected after the addition of a chromogenic substrate 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 performed using a spectrometer (e.g., SpectraMax® M2 spectrometer (Molecular Devices)). The response (OD units) corresponding to zero percent inhibition is determined from wells without competing antibodies. The response (OD units) corresponding to 100% inhibition, i.e., the assay background, is determined from wells without labeled reference antibody or test antibody. The inhibition rate of the labeled reference antibody against FVII or FVIIa by the 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 a binding region, bin, and / or competitively bind to the antigen. Those skilled in the art will also understand that competitive assays can be performed using various detection systems known in the art.

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

[0056] Unless otherwise specified, competition is determined using the competitive ELISA assay described above and 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, between an antibody or a fragment thereof and an antigen. The term "binding affinity" is used to describe monovalent interactions.

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

[0059] According to the definition above, binding affinity, which is related to different molecular interactions such as the comparison of binding affinities of different antibodies to a given antigen, is the K of individual antibody / antigen complexes. D The comparison may be made by comparing the values.

[0060] The K of the antibody of the present invention against that target D This can be less than 1 pM, for example less than 10 pM, for example less than 100 pM, for example less than 200 pM, for example less than 400 pM, for example less than 600 pM, for example less than 1 nM, for example less than 5 nM, for example less than 10 nM, for example less than 20 nM, for example less than 50 nM, for example 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, and so on.

[0061] In this embodiment, the antibody is K for FVIIa at concentrations such as less than 1 pM, e.g. less than 10 pM, e.g. less than 100 pM, e.g. less than 200 pM, e.g. less than 400 pM, e.g. less than 600 pM, e.g. less than 1 nM, e.g. less than 5 nM, e.g. less than 10 nM, e.g. less than 20 nM, e.g. less than 50 nM, e.g. less than 100 nM, e.g. less than 200 nM, e.g. less than 400 nM, e.g. less than 600 pM, e.g. less than 800 pM. D An anti-FVII(a) arm having and K against TLT-1 such as less than 1 pM, e.g. less than 10 pM, e.g. less than 100 pM, e.g. less than 200 pM, e.g. less than 400 pM, e.g. less than 600 pM, e.g. less than 1 nM, e.g. less than 5 nM, e.g. less than 10 nM, e.g. less than 20 nM, e.g. less than 50 nM, e.g. less than 100 nM, e.g. less than 200 nM, e.g. less than 400 nM, e.g. less than 600 nM, e.g. less than 800 nM D It is a bispecific antibody comprising a second anti-TLT-1 arm having [specific characteristic].

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

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

[0064] The antibodies of the present invention may include an Fc region that may have a wild-type amino acid sequence, or may include 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 in which binding to the Fcγ receptor is reduced. One specific example of such a variant is IgG1 containing L234A, L235E, G237A, A330S and P331S (residue numbering by EU index) substitutions that reduce affinity for certain Fcγ receptors and C1q.

[0065] bispecific molecules The term “bispecific molecule” as used herein refers to a molecule capable of binding to different targets, such as FVII(a) and TLT-1. The binding portion of a bispecific molecule may be antibody-derived or non-antibody-derived. One specific example of a bispecific molecule is a bispecific antibody.

[0066] In one aspect of the present invention, the bispecific molecule includes 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 molecule of the present invention may include a non-antibody-derived binding site, also known as an alternative scaffold. The bispecific molecule of the present invention may be a fusion or conjugate of an alternative scaffold. The bispecific molecule of the present invention may be a fusion or conjugate of an antibody and an alternative scaffold. The bispecific molecule of the present invention may also be a fusion or conjugate of an antibody fragment and an alternative scaffold.

[0068] Numerous 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 include Adnectin, Affilin, Anticalin, Avimer, Atrimer, FN3 scaffold, Fynomer, Obody, Kringle domain, Kunitz domain, Knottin, Affibody, DARPin, bicyclic peptides, and Cys-knot.

[0070] Factor VII(a) The terms “Factor VII” and “FVII” as used herein refer to the enzyme precursor of coagulation factor VII. The terms “Factor VIIa” and “FVIIa” as used herein refer to activated coagulation factor VII, which is a serine protease. The terms “Factor VII(a)” and “FVII(a)” as used herein encompass both factor VII (FVII), which is the uncleaved enzyme precursor, and factor VIIa (FVIIa), which is the cleaved and activated protease. The terms “Factor VII(a)” and “FVII(a)” as used herein include possible native allele variants of FVII(a). One wild-type human factor VII(a) sequence is presented in Sequence 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 composed of four domains. It has an N-terminal gamma-carboxyglutamate-rich (Gla) domain, in which 10 glutamate residues (highlighted in bold in the sequence above) may be gamma-carboxylated. The gla domain is followed by two epidermal growth factor (EGF)-like domains and a C-terminal serine protease domain. Both FVII and FVIIa are present in circulation, but FVIIa is present in small amounts (approximately 1% of the total FVII(a) pool, see Morrissey JH, Broze Jr GJ, in *Morrissey JH, Broze Jr GJ, in *Tissue factor and the initiation and regulation (TFPI) of coagulation*, 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 Gla and EGF-like domains, while the heavy chain contains a protease domain. FVIIa requires binding to its cell surface cofactor tissue factor (TF) to achieve its full biological activity.

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

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

[0074] Factor VIIa can be found in various conformations. Factor VIIa circulates in the blood in an inactive conformation or form. This conformation does not possess catalytic activity. FVIIa may also be found in an active conformation or form, which is also referred to herein as fully active or fully activated FVIIa. The term FVIIa encompasses FVIIa in its inactive form or conformation, and FVIIa in its active form or conformation. For example, the active conformation of FVIIa may include a complex between FVIIa and tissue factor (FVIIa / TF) in the form of FVIIa / sTF(1-219), where sTF(1-219) are cleaved and soluble forms 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 treatment with dansyl-Glu-Gly-Arg chloromethyl ketone or Phe-Phe-Arg-chloromethyl ketone (FFR-chloromethyl ketone) (Wildgoose et al (1990) Biochemistry 29:3413-3420 and Shφrensen et al (1997) J Biol Chem 272:11863-11868). FVIIai is thought to retain its affinity for TF and adopt the same conformation that is induced in FVIIa by TF binding. Therefore, FVIIai has an activated conformation, and the binding of the 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-style transfer 1 (TLT-1) Trigger receptors expressed on myeloid cells (TREMs) have well-established roles in the biology of various myeloid genes and play a crucial 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 monophyletic cells, namely megakaryocytes and platelets, and is found only in alpha granules of megakaryocytes and platelets. TLT-1 is a transmembrane protein exposed on the surface of activated platelets during alpha granule release. To date, TLT-1 has not been found on the surface of resting platelets or other cell types.

[0077] TLT-1 contains an extracellular spherical head, a stalk region, a transmembrane domain, and an intracellular domain containing an immunoreceptor tyrosine system inhibitory motif (Washington et al. Blood, 2002;100:3822-3824). The extracellular spherical head of human TLT-1 (hTLT-1) is a single immunoglobulin-like (Ig-like) domain. This 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 a cytoplasmic immunoreceptor tyrosine system inhibitory motif (ITIM) that can function as an intracellular signaling motif.

[0079] A small portion of TLT-1 is detached upon platelet activation, forming 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. Shorter isoforms of TLT-1 with cleaved intracellular domains are also present in platelets.

[0080] TLT-1 is involved in controlling coagulation and possibly inflammation at injury sites. TLT-1 plays a role in platelet aggregation in response to suboptimal concentrations of several 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 also been shown to bind to 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 sepsis-related bleeding by attenuating leukocyte activation and modulating platelet-neutrophil crosstalk (Derive, J Immunol 2012,188:5585-5592).

[0081] In relation to the present invention, TLT-1 may be derived from any vertebrate, such as rodents (mice, rats, or guinea pigs, etc.), lagomorphs (rabbits, etc.), artiodactyls (pigs, cattle, sheep, or camels, etc.), or any mammal such as primates (monkeys or humans, etc.). TLT-1 is preferably human TLT-1. TLT-1 may be translated from any naturally occurring genotype or allele that produces a functional TLT-1 protein. A 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 TLT-1 molecule described herein.

[0083] Anti-FVII(a) antibody The term “anti-FVII(a) antibody” as used herein refers to an antibody that has FVII(a) as its target. Anti-FVII(a) antibodies can bind to the FVII(a) molecules described herein, and these include, but are 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] Anti-FVII(a) antibodies can be monoclonal, monospecific antibodies that target FVII(a). Monospecific anti-FVII(a) antibodies typically contain two identical antigen-binding sites that bind to FVII(a), and in non-limiting examples, are monoclonal IgG4 anti-FVII(a) antibodies.

[0085] A suitable anti-FVII(a) antibody is, but is not limited to, one of the anti-FVII(a) antibodies listed in Table 3. [Table 6]

[0086] An anti-FVII(a) antibody may compete for binding to FVII(a) with any of the antibodies listed in Table 3. Whether an anti-FVII(a) antibody competes with any of the antibodies listed in Table 3 for binding to FVII(a) can be determined using well-known methods such as surface plasmon resonance (SPR), ELISA, or flow cytometry (i.e., competitive binding assays). Example 7 describes how competitive binding to FVII(a) can be determined using a 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 of less than 1 pM, e.g., less than 10 pM, e.g., less than 100 pM, e.g., less than 200 pM, e.g., less than 400 pM, e.g., less than 600 pM, e.g., less than 1 nM, e.g., less than 5 nM, e.g., less than 10 nM, e.g., less than 20 nM, e.g., less than 50 nM, e.g., less than 100 nM, e.g., less than 200 nM, e.g., less than 400 nM, e.g., less than 600 nM, e.g., less than 800 nM, etc. D In vivo, high-affinity anti-FVII(a) antibodies reduce the clearance of FVII(a) by forming a complex with it. This extends the half-life of FVII(a) and causes it to accumulate in circulation. In this way, the antibody can lead to 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., to autoactivate) while bound to TF (as described in Example 12). It is also preferable that the anti-FVII(a) antibody does not prevent FVII(a) from forming a so-called initiation complex with tissue factor (TF) (as described in Example 10) and does not prevent it 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" as used herein refers to an antibody having TLT-1 as its target. Anti-TLT-1 antibodies can bind to the TLT-1 molecule as described herein. TLT-1 antibodies may be monoclonal, single-specific antibodies.

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

[0091] Anti-TLT-1 antibodies may compete for binding to TLT-1 with any of the antibodies listed in Table 4. Whether an anti-TLT-1 antibody competes for binding to TLT-1 with any of the antibodies listed in Table 4 can be determined using well-known methods such as surface plasmon resonance (SPR), ELISA, or flow cytometry (i.e., competitive binding assays). 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] Anti-TLT-1 antibodies have K against their target at concentrations such as less than 1 pM, e.g., less than 10 pM, e.g., less than 100 pM, e.g., less than 200 pM, e.g., less than 400 pM, e.g., less than 600 pM, e.g., less than 1 nM, e.g., less than 5 nM, e.g., less than 10 nM, e.g., less than 20 nM, e.g., less than 50 nM, e.g., less than 100 nM, e.g., less than 200 nM, e.g., less than 400 nM, e.g., less than 600 nM, e.g., less than 800 nM. D It may have.

[0093] It is preferable 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 one preferred embodiment, the anti-TLT-1 antibody can bind to TLT-1 without interfering with platelet aggregation.

[0095] In another preferred embodiment, the anti-TLT-1 antibody can bind 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 elimination of TLT-1.

[0097] It is preferable that the anti-TLT-1 antibody does not bind to any trigger receptor (TREM) expressed on any other bone marrow cells besides TLT-1, or to any other receptor on resting or activated platelets, or shows little affinity to them.

[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 antibody of the present invention comprises a first antigen-binding site capable of binding to FVII(a) and a second antigen-binding site capable of binding to TLT-1.

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

[0101] In some embodiments of the present invention, the first antigen-binding site of the bispecific antibody competes with any one of the anti-FVII(a) antibodies identified in Table 3 for binding to FVII(a), has the same epitope as any one of the antibodies identified in Table 3, has the same CDR region 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 present invention includes a second antigen-binding site capable of binding to TLT-1.

[0103] In some embodiments of the present invention, the second antigen-binding site of the bispecific antibody competes with any one of the anti-TLT-1 antibodies identified in Table 4 for binding to TLT-1, has the same epitope as any one of the antibodies identified in Table 4, has the same CDR region 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] Modification effect function The bispecific antibodies of the present invention may contain an Fc region that may have a wild-type amino acid sequence, or may contain 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 in which binding to the Fcγ receptor is reduced. One specific example of such a variant is IgG1 containing substitutions L234A, L235E, G237A, A330S and P331S (residue numbering by EU index) with reduced affinity for certain Fcγ receptors and C1q.

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

[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-lives. Another approach is the attachment of polymers such as XTEN or PEG (insert reference). Furthermore, in vivo half-life extension can be achieved by attachment of 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 circulating half-life of FVII(a) present in circulation, and by binding to TLT-1, the bispecific antibody and bound FVII(a) are directed to the surface of activated platelets. This then leads to an increase in the accumulation of FVII(a) on activated platelets, and thus enhances the procoagulant activity of FVII(a) 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] To extend the activity 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 the recycling of the complex and rescues it from degradation in endosomes via binding to FcRn. Furthermore, the high-affinity anti-FVII arm of the bispecific antibody protects endogenous FVIIa from α2M inhibition and renal clearance because it increases the 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 prolonged endogenous FVIIa to activated platelets. This localization of FVIIa to activated platelets enhances FVIIa activity without increasing sensitivity to AT inhibition.

[0110] The bispecific antibodies of the present invention can increase the mean residence time (MRT) of endogenous or exogenous FVII(a). Preferably, the bispecific antibodies can enhance 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 as a function of steady-state volume of distribution (Vss) divided by systemic clearance (CL) according to Equation 1. MRT=Vss / CL Equation 1

[0112] The result is expressed in time. MRT is calculated according to Equation 2, where the effective plasma half-life (t 1 / 2 It correlates with ). MRT = ln(2) * t 1 / 2 formula 2

[0113] The ability of an antibody to increase the functional MRT of FVII(a) can be determined by well-known methods, such as those described in *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 experimental animals, such as mice, rats, or monkeys. 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), measured by assays such as the FVIIa activity assay described in Example 8, but is not limited to these.

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

[0115] In some embodiments, the bispecific antibodies of the present invention can increase 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) (FVII(a) polypeptide alone) in the absence of the antibody of the present invention (FVII(a)).

[0116] In some embodiments, the bispecific antibodies of the present invention can increase 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 antibodies of the present invention, as measured in the FVIIa activity assay described in Example 8.

[0117] Accumulation of endogenous FVIIa The ability of the bispecific antibodies of the present 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 antibodies to experimental animals, such as mice, rats, or monkeys, using assays such as the FVIIa activity assay described in Example 8, for example.

[0118] The ability of an antibody to increase the level of circulating endogenous functional activity FVIIa can be determined, for example, as described in Examples 27 and 28.

[0119] In some embodiments, the bispecific antibodies of the present invention can increase 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, and at least 640-fold compared to the level of circulating endogenous FVIIa in the absence of the administered bispecific antibodies.

[0120] TLT-1 and TF-independent thrombin generation In one embodiment, the bispecific antibody of the present invention can maintain or increase the TLT-1 and TF-independent ability of factor VIIa to generate thrombin.

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

[0122] In some embodiments, the bispecific antibodies of the present invention can maintain (neutralize) / increase (stimulate) the thrombin-producing ability of the VII(a) polypeptide, as measured by a thrombin-producing assay, compared to FVII(a) in the absence of the antibody.

[0123] In some embodiments, the bispecific antibodies of the present invention can increase the ability of factor VII(a) to generate thrombin, as measured by a thrombin production assay, by at least 20% compared to FVII(a) in the absence of the antibody.

[0124] Inhibition by antithrombin and / or alpha-2-macroglobulin In one embodiment, the bispecific antibody of the present invention can reduce the sensitivity 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 present invention can reduce 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 (stimulus activity assay) In one embodiment, the bispecific antibody of the present invention can maintain or increase the ability of the FVIIa polypeptide to promote FX activation in the presence of a TLT-1-containing procoagulation membrane surface.

[0128] The ability of the bispecific antibody of the present invention to increase the ability of the FVIIa polypeptide to promote FX activation can be determined by methods well known in the art, such as 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 the anti-FVII(a) / anti-TLT-1 bispecific antibody. The so-called stimulatory activity (expressed as a multiplier) of the bispecific antibody is the amount of FXa produced in the presence of 100 nM of the bispecific antibody compared to the amount produced by FVIIa in the absence of the bispecific antibody.

[0129] In some embodiments, the stimulating activity of the bispecific antibody of the present 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 antibody of the present invention can promote thrombus formation under hemophilia A conditions at a concentration similar to or better than the therapeutically effective concentration of recombinant FVIIa.

[0131] The ability of bispecific antibodies to improve whole blood thrombosis can be determined by methods well known in the art, such as the thromboelastography assay described in Example 29. In this assay, thrombosis is measured in human hemophilia-A induced whole blood, where bispecific antibodies are added along with FVII, FVIIa, and FVIIa:AT at concentrations mimicking in vivo steady-state plasma levels of the corresponding endogenous factors during repeated administration of the bispecific antibodies as described in Examples 27, 28, and 29. Coagulation is induced by the addition of the PAR1 agonist peptide SFLLRN and calcium. The coagulation time under these conditions is compared to the coagulation time achieved by the addition of 25 nM FVIIa in the absence of the antibody.

[0132] In some embodiments, the bispecific antibodies of the present invention can reduce the clotting time in human hemophilia A-induced whole blood to a level similar to or lower than that achieved by the addition of 25 nM FVIIa. In some embodiments, the bispecific antibodies of the present invention can reduce the clotting time in human hemophilia A-induced whole blood to a level similar to or lower than 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 embodiment, the present invention provides compositions and formulations comprising bispecific antibodies as described herein. For example, the present invention provides a pharmaceutical composition comprising a bispecific antibody formulated with a pharmaceutically acceptable carrier.

[0134] In one embodiment of the present invention, the pharmaceutical formulation contains a bispecific antibody present at a concentration of 80 mg / mL to 200 mg / mL, for example, 100 to 180 mg / mL, and the formulation has a pH of 2.0 to 10.0. The formulation may further contain one or more of a buffer system, a preservative, an isotonic agent, a chelating agent, a stabilizer, or a surfactant, or various combinations thereof. The use of preservatives, isotonic 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 The 1995 edition may be referenced.

[0135] In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersants, emulsions, and multiphase materials. The term “aqueous formulation” is defined as a formulation containing at least 50% w / w water. Similarly, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water.

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

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

[0138] The composition of the present invention may be administered parenterally, for example intravenously, intramuscularly, subcutaneously, and preferably subcutaneously. The composition of the present invention may also be administered prophylactically.

[0139] The pharmaceutical compositions of the present invention may be used to treat subjects having coagulation disorders. 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 any medical therapy for any human or other animal subject in need of it. 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 such particular treatment would be beneficial to the health of the human or other animal subject. The timing and purpose of such treatment may vary from person to person depending on the subject's current health condition. Prophylactic or preventative administration of the coagulation-promoting compounds of the present invention is also intended, with prevention defined as delaying or avoiding the onset or worsening of one or more symptoms of a disease or disorder. Thus, such treatments may be prophylactic, palliative, symptomatic ("on-demand"), and / or curative.

[0141] With respect to the present invention, preventive, palliative, and / or symptomatic treatments may represent distinct aspects of the present invention.

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

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

[0144] A non-limited example of acquired coagulation disorder is serine protease deficiency caused by vitamin K deficiency, which may be caused by the administration of vitamin K antagonists such as warfarin. Acquired coagulation disorders can also occur after extensive trauma. In this case, also known as the "bloody vicious cycle," it is characterized by hemodilution (dilutional thrombocytopenia and dilution of coagulation factors), hypothermia, consumption of coagulation factors, and metabolic abnormalities (acidosis). Fluid therapy and increased fibrinolysis can worsen this situation. The bleeding may originate from any part of the body.

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

[0146] Non-limited cases of iatrogenic coagulation disorders include those induced by excessive and / or inappropriate fluid therapy, such as those that can be triggered by blood transfusions.

[0147] In one preferred embodiment of the present invention, bleeding is associated with hemophilia A. In another preferred embodiment of the present invention, bleeding is associated with hemophilia B. In another preferred embodiment, bleeding is associated with hemophilia A or B with acquired inhibitors. In another preferred embodiment, bleeding is associated with FVII deficiency. In another preferred embodiment, bleeding is associated with Glanzmann thrombasthenia. In another embodiment, bleeding is associated with von Willebrand disease. In another embodiment, bleeding is associated with severe tissue injury. In another embodiment, bleeding is associated with severe trauma. In another embodiment, bleeding is associated with surgery. In another embodiment, bleeding is associated with hemorrhagic gastritis and / or enteritis. In another embodiment, bleeding is massive uterine bleeding, such as placental abruption. In another embodiment, bleeding occurs in organs where mechanical hemostasis is limited, such as intracranial, intraaural, or intraocular. In another embodiment, bleeding is associated with anticoagulant therapy.

[0148] In further embodiments, bleeding may be associated with thrombocytopenia. In individuals with thrombocytopenia, the bispecific antibodies described herein can be administered 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] Administration regimen The bioavailability and half-lives of the bispecific antibodies described herein make them particularly attractive 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 without bleeding. 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 administered co-administered with exogenous FVIIa, such as plasma-derived or recombinant FVIIa, for on-demand or prophylactic treatment of subjects with coagulation disorders or who have experienced bleeding episodes.

[0152] The dose administered to patients with coagulation disorders depends on the route of administration, whether it is administered prophylactically or on demand, and individual differences. Subcutaneous administration requires a higher dose 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 specified herein, terms presented in the singular form also include plural states.

[0155] List of embodiments The present invention is further described by the following non-limiting list of embodiments. 1. A bispecific antibody, (i) A first antigen-binding site capable of binding factor VII(a), (ii) A bispecific antibody comprising a second antigen-binding site capable of binding to TREM-like transcript 1 (TLT-1). 2. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site competes with any one of the anti-FVII(a) antibodies in Table 3 for binding to FVII(a). [Table 8] 3. The bispecific antibody according to Embodiment 1, wherein the antibody competes in a competitive ELISA assay such as that described in Example 7. 4. The bispecific antibody according to Embodiment 1, 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. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site can bind to an epitope containing one or more amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO: 1). 6. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site can bind to 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. 7. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site can bind to an epitope containing one or more of 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. 8. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site includes the following: CDRL1, represented by Sequence ID No. 847, having 0, 1, 2, or 3 amino acid substitutions. CDRL2, represented by Sequence ID No. 848, having 0, 1, 2, or 3 amino acid substitutions. CDRL3, represented by Sequence ID No. 849, has 0, 1, or 2 amino acid substitutions. CDRH1, represented by Sequence ID No. 851, has 0 or 1 amino acid substitutions. CDRH2, represented by Sequence ID No. 852, has 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 according to Embodiment 1, wherein the first antigen-binding site includes the following: • CDRL1 represented by sequence number 847, • CDRL2 represented by sequence number 848, • CDRL3 represented by sequence number 849, • CDRH1 represented by sequence number 851, • CDRH2 represented by sequence number 852, • CDRH3 represented by sequence number 853 10. The first antigen-binding site includes a variable heavy chain domain sequence and a variable light chain domain sequence of the antibody listed in Table 13 or 14, and has affinity (K D A bispecific antibody according to Embodiment 1, wherein the concentration is 1 nM or less. 11. The first antigen-binding site includes a variable heavy chain domain sequence and a variable light chain domain sequence of the antibody listed in Table 13 or 14, and has affinity (K D A bispecific antibody according to Embodiment 1, wherein the concentration is 5 nM or less. 12. The first antigen-binding site includes a variable heavy chain domain sequence and a variable light chain domain sequence of the antibody listed in Table 13 or 14, and has affinity (K D A bispecific antibody according to Embodiment 1, wherein the concentration is 10 nM or less. 13. The first antigen-binding site includes a variable heavy chain domain sequence and a variable light chain domain sequence of the antibody listed in Table 13 or 14, and the affinity (K D A bispecific antibody according to Embodiment 1, wherein the concentration is 25 nM or less. 14. The antibody has an affinity (K) for FVII(a) of less than 1 pM, e.g., less than 10 pM, e.g., less than 100 pM, e.g., less than 200 pM, e.g., less than 400 pM, e.g., less than 600 pM, e.g., less than 1 nM, e.g., less than 5 nM, e.g., less than 10 nM, e.g., less than 20 nM, e.g., less than 50 nM, e.g., less than 100 nM, e.g., less than 200 nM, e.g., less than 400 nM, e.g., less than 600 nM, e.g., less than 800 nM. D A bispecific antibody according to any one of the prior embodiments, having ) 15. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850. 16. The bispecific antibody according to Embodiment 15, wherein the light chain variable domain and / or 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 according to Embodiment 1, wherein the second antigen-binding site competes with any one of the anti-TLT-1 antibodies in Table 4 for binding to TLT-1. [Table 9] 18. The bispecific antibody according to Embodiment 17, wherein the antibody competes in a competitive ELISA assay such as that described in Example 32. 19. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding site can bind to an epitope containing 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 according to Embodiment 1, wherein the second antigen-binding site includes the following: CDRL1, represented by Sequence ID No. 855, having 0, 1, 2, or 3 amino acid substitutions. CDRL2, represented by Sequence ID No. 856, having 0, 1, or 2 amino acid substitutions. CDRL3, represented by Sequence ID No. 857, having 0, 1, or 2 amino acid substitutions. CDRH1, represented by Sequence ID No. 859, has 0 or 1 amino acid substitutions. CDRH2, represented by Sequence ID No. 860, has 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 according to Embodiment 1, wherein the second antigen-binding site includes the following: • CDRL1 represented by sequence number 855, • CDRL2 represented by sequence number 856, • CDRL3 represented by sequence number 857, • CDRH1 represented by sequence number 859, • CDRH2 represented by sequence number 860, • CDRH3 represented by sequence number 861 22. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding site includes the following: • CDRL1 represented by sequence number 871, • CDRL2 represented by sequence number 872, • CDRL3 represented by sequence number 873, • CDRH1 represented by sequence number 875, • CDRH2 represented by sequence number 876, and • CDRH3 represented by sequence number 877 23. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding site includes the following: • CDRL1 represented by sequence number 879, • CDRL2 represented by sequence number 880, • CDRL3 represented by sequence number 881, • CDRH1 represented by sequence number 883, • The CDRH2 represented by sequence number 884, and • CDRH3 represented by sequence number 885 24. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding site includes the following: • CDRL1 represented by sequence number 895, • CDRL2 represented by sequence number 896, • CDRL3 represented by sequence number 897, • CDRH1 represented by sequence number 899, • CDRH2 represented by sequence number 900, and • CDRH3 represented by sequence number 901 25. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858. 26. The bispecific antibody according to Embodiment 25, wherein the light chain variable domain and / or 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, e.g., less than 10 pM, e.g., less than 100 pM, e.g., less than 200 pM, e.g., less than 400 pM, e.g., less than 600 pM, e.g., less than 1 nM, e.g., less than 5 nM, e.g., less than 10 nM, e.g., less than 20 nM, e.g., less than 50 nM, e.g., less than 100 nM, e.g., less than 200 nM, e.g., less than 400 nM, e.g., less than 600 nM, e.g., less than 800 nM. D A bispecific antibody according to any one of the prior embodiments, having ) 28. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850, and the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858. 29. A bispecific antibody according to any one of the prior embodiments, wherein the antibody comprises an Fc region. 30. The bispecific antibody according to Embodiment 29, wherein the Fc region is an Fc variant of the IgG1 Fc region, including substitutions of L234A, L235E, G237A, A330S, and P331S. 31. A bispecific antibody according to any one of the prior embodiments, wherein the antibody is a fusion or conjugate of antigen-binding fragments of the antibody. 32. A bispecific antibody according to Embodiment 31, wherein one or more of the binding fragments are selected from the group Fab, Fab', Fab2, Fab'2, and scFv. 33. A bispecific antibody according to any one of the prior 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 identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are identified by SEQ ID NOs: 5 and 4, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both identified 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 identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are identified by SEQ ID NOs: 943 and 942, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both identified 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 identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are identified by SEQ ID NOs: 7 and 6, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both identified 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 identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are identified by SEQ ID NOs: 4 and 5, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both identified 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 identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are identified by SEQ ID NOs: 942 and 943, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both identified 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 identified by SEQ ID NO: 846 and a heavy chain variable domain identified by SEQ ID NO: 850, the second antigen-binding site comprises a light chain variable domain identified by SEQ ID NO: 854 and a heavy chain variable domain identified by SEQ ID NO: 858, the heavy chain constant domains attached to the first and second heavy chain variable domains are identified by SEQ ID NOs: 6 and 7, respectively, and the light chain constant domains attached to the first and second light chain variable domains are both identified by SEQ ID NO: 12. 40. The bispecific antibody according to 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 listed in Table 5 (abbreviations for constant domains are defined according to Table 2a). [Table 10] 41. The bispecific antibody according to Embodiment 38, wherein the antibody competes for binding to FVII(a) in a competitive ELISA and competes for binding to TLT-1 with one or more bispecific antibodies as listed in Table 5. 42. The bispecific antibody according to Embodiment 38, wherein the antibody competes in competitive ELISA assays such as those described in Examples 7 and 32. 43. The bispecific antibody according to Embodiment 1, wherein the first antigen-binding site can bind an epitope containing the amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO: 1), and the second antigen-binding site can bind an epitope containing the following amino acid residues K8, I9, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, and A21 of TLT-1 (SEQ ID NO: 13). 44. The first antigen-binding site includes the following: CDRL1 represented by an amino acid residue (SEQ ID NO: 847) having 0, 1, 2, or 3 amino acid substitutions. CDRL2 represented by an amino acid residue (SEQ ID NO: 848) having 0, 1, 2, or 3 amino acid substitutions. CDRL3 represented by an amino acid residue (SEQ ID NO: 849) having 0, 1, or 2 amino acid substitutions. • The aforementioned CDRH1, represented by an amino acid residue (SEQ ID NO: 851), CDRH2, represented by an amino acid residue (SEQ ID NO: 852) having 0 or 1 amino acid substitutions, CDRH3, represented by an amino acid residue (SEQ ID NO: 853) having 0, 1, 2, or 3 amino acid substitutions. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding site comprises the following: CDRL1 represented by Sequence ID No. 855, having 0, 1, 2, or 3 amino acid substitutions. CDRL2 represented by Sequence ID No. 856, having 0, 1, or 2 amino acid substitutions. CDRL3 represented by Sequence ID No. 857 has 0, 1, or 2 amino acid substitutions. CDRH1 represented by SEQ ID NO: 859, having 0 or 1 amino acid substitutions. CDRH2 represented by Sequence 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. A bispecific antibody according to any one of the preceding embodiments, wherein the antibody increases the functional MRT of FVII(a) as measured by the FVIIa activity assay described in Example 8. 46. ​​The bispecific antibody according to 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 present invention. 47. A bispecific antibody according to any one of the preceding embodiments, which can increase 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 functional activity FVIIa by at least twofold, at least fourfold, at least tenfold, at least twentyfold, at least fortyfold, at least eightyfold, at least 160fold, at least 320fold, and at least 640fold. 49. A bispecific antibody according to any one of the prior embodiments, wherein the antibody can increase the ability of the FVIIa polypeptide to promote FX activation as determined by the stimulating activity assay described in Example 21. 50. A bispecific antibody according to 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 a bispecific antibody and a pharmaceutically acceptable carrier as described in any one of the prior embodiments. 52. A bispecific antibody according to Embodiments 1 to 48, wherein the pharmaceutical formulation described in Embodiment 49 is administered by subcutaneous injection. 53. A bispecific antibody according to Embodiments 1 to 48, or a pharmaceutical formulation according to Embodiment 49, for use as a pharmaceutical agent. 54. A bispecific antibody according to any one of Embodiments 1 to 48 and 50 to 51, or a pharmaceutical formulation according to Embodiment 49, for use in the treatment of coagulation disorders, wherein the coagulation disorder is congenital and / or acquired. 55. The bispecific antibody according to Embodiment 52, wherein the coagulation disorder is selected from the group consisting of inhibitor-possessing or inhibitor-free hemophilia A, inhibitor-possessing or inhibitor-free hemophilia B, FVII(a) deficiency, and Glanzmann thrombasthenia. 56. A 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, inhibitor-carrying hemophilia A, inhibitor-carrying hemophilia B, or factor VII(a) deficiency. 57. The use according to any one of Embodiments 1 to 48, wherein the bispecific antibody or pharmaceutical formulation described in Embodiment 49 is administered parenterally, such as intravenously, intramuscularly, or subcutaneously. [Examples]

[0156] Example 1: Generation of anti-FVII(a) mouse monoclonal antibody using hybridoma technology The monoclonal antibody was prepared by immunization of NMRCF1 mice (Charles River) using the FVIIa Q64C-sTF(1-219)G109C disulfide bond complex, as described in the international patent application, publication number WO07 / 115953.

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

[0158] Cells were seeded in microtiter plates and cultured at 37°C and 5% CO2. The tissue culture medium containing HAT(Sigma) for selection (RPMI 1640 + 10% fetal bovine serum) 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 1 μg / mL (HEPES buffer containing 5 mM CaCl2), 50 μl / well, with either FVIIa Q64C-sTF(1-219)G109C disulfide-bonded 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) and incubated overnight at 4°C. The plate was washed five times and blocked for 15 minutes in washing buffer (HEPES buffer, 5 mM CaCl2, 0.05% TWEEN 20). 50 μL of supernatant was transferred to each well and incubated for 1 hour. The plate was washed five times and 50 μL of HRP-labeled goat anti-mouse ointment was added (Fc gamma fragment specific, Jackson, working dilution 1 / 10000). The plate was incubated for 1 hour, washed five times, and chromogenically treated with 50 μL of TMB (ready-to-use TMB ONE, Kem-En-Tec) for 10 minutes. The reaction was stopped by adding 50 μL of 4 M H3PO4 and read at 450 and 620 nm using a FLUOStar Optima plate reader.

[0159] Hybridoma cells that yielded positive results were subcloned at least twice using 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 to T flasks or shaking flasks in RPMI1640 + 10% FBS medium. The conditional medium was collected by centrifugation, and the antibodies were purified by protein A affinity chromatography and then desalted.

[0161] Example 2: Cloning and sequencing of mouse anti-FVII(a) antibody This example describes the cloning and sequencing of mouse heavy chain (HC) and light chain (LC) cDNA sequences encoding the variable domain 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 via a 5'-RACE reaction using the SMARTer RACE cDNA amplification kit from Clontech. Subsequent targeted amplification of the variable domains of the HC (designated VH) sequence and 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 primers. The reverse primer sequence used for VH amplification was 5'agctgggaaggtgtgcacac3'. The reverse primer sequence used for VL amplification was 5'gctctagactaacactcattcctgttgaagctcttg3'.

[0163] PCR products were separated by gel electrophoresis, extracted using the GE Healthcare Bio-Sciences GFX PCR DNA & Gel Band Purification Kit, and cloned for sequencing using the Zero Blunt TOPO PCR Cloning Kit and chemically responsive 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 manufacturer's instructions.

[0164] Example 3: Recombinant expression of bivalent antibody, monovalent antibody (OA antibody), and antibody Fab fragment Bivalent antibodies, monovalent antibodies (referred to as one-armed or OA antibodies), and antibody Fab fragments were expressed using transient transfection of HEK293 suspension cells (293Expi, Invitrogen), essentially following the manufacturer's instructions. 293Expi cells were passaged every 3–4 days in Expi293F expression medium (Invitrogen, catalog number A1435104) supplemented with 1% P / S (GIBCO catalog number 15140-122). Expi293F cells were transfected with Expifectamine at a cell density of 2.5 million–3 million cells / mL. Transfection was performed by diluting a total of 1 mg of plasmid DNA in 50 mL of Optimem (GIBCO, catalog number 51985-026, dilution A) per 1 liter of Expi293F cells, and by diluting 2.7 mL of Expifectamine in 50 mL of Optimem (dilution B). Bivalent antibodies were produced by co-transfecting with 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 OA antibody production, cells were transfected with three plasmids: LC plasmid, HC plasmid, and a third plasmid encoding cleaved HC (trHC). The HC of the OA antibody contained hole mutations (T366S, L368A, Y407V), and the trHC contained a knob mutation (T366W), although the knob and hole could also be reversed. Knob / hole mutations are described in international patent EP0979281B1 and are introduced to optimize the pairing of desired heterodimers, i.e., HC and trHC, and to suppress the undesirable formation of homodimers, i.e., pairing of trHC and trHC and HC and HC. Dilutions A and B were mixed and incubated at room temperature for 10-20 minutes. 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. The cell culture supernatant was collected 4–5 days after transfection by centrifugation followed by filtration.

[0165] The 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. The Fab fragment was purified by affinity chromatography using an affinity resin that recognizes the kappa chain of Fab.

[0166] Example 4: Preparation of bispecific antibodies and OA antibodies by in vitro assembly Bispecific antibodies prepared by in vitro assembly The bispecific antibody was generated by in vitro assembly of two parent antibodies using a method similar to that described in Labrijn et al. PNAS 2013, vol. 110, pp. 5145-5150, and a method known as DuoBody® technology (Genmab). IgG4 was used in this invention instead of the IgG1 subtype used by Labrijn et al. The exchange reaction was carried out at 30°C for 4 hours in the presence of 75 mM 2-mercaptoethylamine (2-MEA). The resulting bispecific antibody was purified by ion-exchange chromatography, and the residual parent antibody was separated from the bispecific antibody. 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 parental antibodies were produced as described in Example 3. A bispecific anti-FVII(a) / anti-TLT-1 antibody may be constructed from a set of parental antibodies in which 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 in the same manner as described above for bispecific antibodies, except that (1) instead of combining two antibodies to form a bispecific antibody, a monovalent antibody was formed by combining an antibody with a trHC dimer. In this example, the trHC was IgG4 S228P F405L R409K and the HC was IgG4 S228P (both using EU numbering). The light chain constant region was human kappa. Typically, to minimize the amount of bivalent antibody in the reaction mixture, the arm exchange reaction was carried out using trHC dimers in a 20-50% molar excess. The monovalent antibodies were 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 a functional assay. To promote the accumulation of endogenous FVIIa and enable it to exert its procoagulation activity, the anti-FVII(a) antibody of the present invention preferably does not impair the activity of FVIIa, and similarly preferably does not promote the inactivation of FVIIa by antithrombin, its major plasma inhibitor (Agersφ H, et al. (2011) J Thromb Haemost 9:333-338). To explore these aspects, the effects of the anti-FVII(a) antibody on the procoagulation activity of FVIIa and the inactivation of FVIIa by antithrombin were determined in vitro using the assays described below.

[0169] Effect of anti-FVII(a) antibodies on thrombin production in hemophilia A-induced human plasma The effect of bivalent or monovalent anti-FVII(a) antibodies on thrombin generation was measured using a 96-well setup kaolin-triggered thrombin generation assay (TGT). Briefly, hemophilia A-inducible plasma was prepared by adding anti-hFVIII antibody 4F30 (described in the international patent application, publication number WO2012 / 035050) to a final concentration of 37.5 μg / ml. Phospholipid at a final concentration of 10 μM (Rossix) was added to the 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 on an EnVision multi-label reader for 2 hours.

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

[0171] Effect of anti-FVII(a) antibodies on FVII(a) 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 antibody (200-1000 nM) for 10 minutes in 50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 0.1% PEG8000, 1 mg / ml BSA, pH 7.3. Next, AT (5 μM) was added, and at 10, 20, 30, 40, 60, and 80 minutes, the sample was transferred to a new microtiter plate where the residual activity was measured for 5 minutes at 405 nm using 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 polyblen (0.5 mg / ml). Samples using buffer instead of AT provided uninhibited FVIIa activity.

[0172] Residual amid degradation activity was determined as a function of time relative to activity in the absence of the inhibitor. The inhibition constant (kinh) was estimated by fitting the data to a one-phase decay model. The kinh value in the presence of the FVII(a) antibody (as a percentage of the value determined in the absence of the antibody) is denoted as kinh%, and is 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 AT-mediated inhibition of FVIIa. Antibodies from all three categories were identified, as reported in Table 6. Preferred antibodies were either neutral or protected FVIIa from AT inhibition.

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

[0175] Example 6: SPR analysis of antibodies binding to FVIIa and the effects of pH and calcium The binding of antibodies from Example 5 to FVIIa was probed by surface plasmon resonance (Biacore T200). Anti-mouse IgG (GE Healthcare) was immobilized on a CM4 sensor chip using a standard amine coupling chemistry kit (both supplied by GE Healthcare). Purified anti-FVII(a) antibody (0.25 nM) according to Table 7 was injected at a flow rate of 10 μl / min for 1 minute. Subsequently, 5, 15, 45, and 135 nM FVIIa were injected at a flow rate of 30 μl / min for 7 minutes to enable binding to the anti-FVII(a) antibody, followed by dissociation from the anti-FVII(a) antibody by buffer injection for 9 minutes. Running buffer was prepared by diluting 10x HBS-P buffer (provided by GE Healthcare) tenfold 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, and 1 mg / ml bovine serum albumin (BSA). The running buffer was also used to dilute anti-FVII(a) antibody and FVII samples. Chip regeneration 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 a 1:1 model using BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala, Sweden). Analysis revealed the binding constants reported in Table 7, and several antibodies, including 11F2 (mAb005 and mAb0048, corresponding to complete mouse and mouse / human chimera, respectively), showed high affinity binding to FVIIa. [Table 12]

[0176] The effects of pH and CaCl2 on the binding of selected antibodies from Example 5 to FVIIa and cFVIIa-chimeric molecules were probed by surface plasmon resonance (Biacore T200) at 37°C. The cFVIIa-chimeric sequences are shown in the section titled Detailed Description of the Invention and are represented as outlined in Examples 16 and 26. Anti-mouse IgG (GE Healthcare) was immobilized on a CM4 sensor chip using a standard amine coupling chemistry kit (both supplied by GE Healthcare). A pre-equalized 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 at a flow rate of 10 μl / min for 1 minute. Next, anti-FVII(a) antibodies of 540, 180, 60, 20, 6.66, 2.22, 0.74, and 0.25 nM were injected at a flow rate of 30 μl / min for 7 minutes to enable binding to FVIIa, followed by dissociation from FVIIa by buffer injection for 9 minutes. Two running buffers were prepared. Buffer 1 was prepared by diluting 10×HBS-P+ (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, pH 7.4, 5 mM CaCl2, and 1 mg / ml bovine serum albumin (BSA). Buffer 2 was prepared by diluting 10x HBS-P+ (provided by GE Healthcare) tenfold, 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, and 1 mg / mL bovine serum albumin (BSA). FVIIa, anti-FVII antibody 4F9, and anti-FVII(a) antibody were diluted separately in both running buffers. Chip regeneration 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, provided by the manufacturer (Biacore AB, Uppsala, Sweden). The results of the analysis yielded the binding constants reported in Table 7b, indicating sustained high-affinity binding between FVIIa and the anti-FVII(a) antibody. Table 7b shows the estimated binding constants and fold-differences for the interaction between the FVIIa antibody and the anti-FVII(a) antibody 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 competitive ELISA. To determine whether the anti-FVII(a) antibody with the desired in vitro properties from Example 5 competes with mAb0005(11F2) and antibodies derived therefrom for binding to FVIIa(a), a competitive study was performed using the corresponding Fab fragment, Fab0076. FVIIa, HD-Phe-Phe-Arg chloromethyl ketone (FFR-cmk, Bachem, Switzerland) active site inhibited FVIIa (FVIIai, see Example 9), was fixed overnight at 4°C on a NUNC maxisorp plate at a concentration of 125 ng / ml in dilution buffer (20 mM HEPES, 5 mM CaCl2, NaCl, pH 7.2). The plate was washed and blocked for 15 minutes with washing buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, 0.5 mL / L Tween 20, pH 7.2). 11F2-Fab0076 was biotinylated using a standard biotinylation kit (EZ-Link, Thermo) as directed by the manufacturer. In the competing study, biotinylated Fab0076 with a final fixed concentration of 10 ng / ml was combined with a series of dilutions of anti-FVIIa(a) antibodies. 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 a plate and incubated for 1 hour. The plate was then washed, and HRP-labeled streptavidin-HRPO (1:2000 in dilution buffer, Kirkegaard & Perry Labs) was added and incubated for 1 hour. Finally, the plate was washed and colored with TMB ONE (KEMENTEC) for 10 minutes. The reaction was stopped by adding H3PO4 (4M), and the plate was read at 450 nm with background signal subtracted, measured at 620 nm, using a FLUOStar Optima plate reader. Unless otherwise specified, all incubations were performed at room temperature, and plates were washed five times with washing buffer.

[0178] From the measured signal (in OD units), the competition at any given antibody concentration is calculated as follows: Inhibition rate % = (1 - (OD unit - 100% inhibition) / (0% inhibition - 100% inhibition)) * 100 In the formula, 0% inhibition was determined from the signal in wells containing no competing anti-FVII(a) antibody, and 100% inhibition was determined from the signal in wells containing no biotinylated Fab0076 (i.e., corresponding to the assay background). When the antibody concentration was tested in up to 10,000 times excess of biotinylated Fab0076, an antibody was considered to compete with 11F2(mAb0005) for binding to FVII(a) if at least 50% inhibition (inhibition%) was observed. 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), primarily as described in Morrissey JH et al Blood, 1993;81:734-44. The assay was performed using an ACLTOP500 automated coagulation system from Instrumentation Laboratory. The assay consisted of either a 40 μl diluted animal plasma sample, a calibrator (recombinant FVIIa (rFVIIa) standard calibrated against international WHO standards), or a quality control (QC) sample consisting of 40 μl of FVII-deficient plasma and 40 μl of soluble tissue factor (sTF) mixed with phospholipids. To initiate the reaction, 40 μl of 25 mM CaCl2 was added, and the coagulation time was measured by the instrument. The coagulation times of the samples and QC were compared to an rFVIIa calibration curve using plasma of the same concentration as the diluted samples to reduce plasma interference. The calibration curve range was 5–1000 mIU / mL and fitted using a cubic polynomial. QC and sample results were calculated using software on the ACLTOP500 instrument. The IU / mL results were converted to nM using the specific activity (IU / Pmol) of the administered compound.

[0180] Example 9: Pharmacokinetics of recombinant FVIIa in rats in co-formulation with anti-FVII(a) antibody A monoclonal anti-FVII(a) antibody with the desired in vitro properties from Example 5 was intravenously administered to male Sprague Dolly rats in a co-formulation with 20 nmol / kg of human FVIIa. As shown in Table 9, the molar ratio of FVIIa to antibody was 1:1 or 1:5. During the experiment, the animals were given free access to feed and water. FVIIa plasma activity was measured using the FVIIa activity assay described in Example 8.

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

[0182] Among the antibodies possessing the desired properties of Example 5, mAb0005 (11F2) and mAb0001 (11F26) induced the longest average residence times for human FVIIa, at 7.9 and 7.5 hours, respectively, compared to an average free residence time of 1.1 hours for free human FVIIa (see Table 9). mAb0005 and mAb0001 (mAb0759) are high-affinity antibodies (Example 6) that exhibit competition for binding to FVIIa (Example 7). [Table 15]

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

[0184] Effect of 11F2 antibody on FX activation by FVIIa in the presence of a phospholipid membrane 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.). The reaction was initiated by the addition of 0–300 nM human plasma-derived factor X (FX) and incubated in a 96-well plate (n=2) with a total reaction volume of 100 μl at room temperature for 20 minutes. After incubation, the reaction was quenched by adding 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. The conversion of the chromogenic substrate by the generated FXa was measured as the slope of linear absorbance increase at 405 nm for 10 minutes using a Spectramax microplate spectrophotometer. The initial rate of molar-based FXa production could be estimated by relating the measured slope to the slope produced under similar conditions using a known amount of plasma-derived human FXa. Enzyme reaction rate parameters were estimated by fitting nonlinear curves 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 fragments 1-244 incorporated into 25:75 phosphatidylserine:phosphatidylcholine (PS:PC) vesicles, as described in Smith and Morrissey (2005) J. Thromb. Haemost., 2:1155-1162. The reaction was initiated by the addition of 0-30 nM human plasma-derived factor X (FX) and incubated in a 96-well plate (n=2) at room temperature for 20 minutes in a total reaction volume of 100 μl. After incubation, the reaction was 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 circulation, partly due to its inactivation by the abundant plasma inhibitor antithrombin (AT). Similarly, in animal studies, another plasma inhibitor, alpha-2-macroglobulin, has been associated with the inactivation of FVIIa. Using purified plasma-derived inhibitors, we investigated the effect of a monovalent 11F2 antibody on the inactivation of FVIIa by these inhibitors.

[0189] Effect of 11F2 antibody on antithrombin-mediated inhibition of FVIIa Inhibition of FVIIa by human plasma-derived AT in the presence of monovalent 11F2 antibodies (mAb0048, mAb0077(OA)) was performed under pseudo-primary conditions. The assay was performed at room temperature in 200 μl volumes of 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, refer to the European Pharmacopoeia, code E0180000 batch 5.0, Id 00CK18), and either 0 or 1000 nM of antibody. After a 10-minute pre-incubation, the reaction was initiated by adding 5 μM AT (Antithrombin III, Baxter, Lot VNB1M007; re-purified on a heparin-Sepharose column to remove serum albumin from the formulation). At the selected point, a 10 μL aliquot was transferred to a total volume of 200 μL containing 0.5 mg / ml Polybrene (Hexadimethrin bromide, Sigma, catalog no. H9268, lot SLBC8683V), 200 nM sTF, and 1 mM S-2288 (Chromogenix). The reaction was quenched to saturate FVIIa with sTF, allowing for the measurement of residual FVIIa activity by hydrolysis of the chromogenic substrate, monitored at 405 nm for 5 minutes. The residual amide decomposition activity was determined as the slope of the linear progression curve after blank subtraction. These were 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 by dividing kapp by the AT concentration.

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

[0191] Effect of 11F2 antibody on alpha-2 macroglobulin-mediated inhibition of FVIIa The inhibition of FVIIa by human plasma-derived alpha-2-macroglobulin was carried out under pseudo-first-order conditions in the presence of 0 or 1000 nM of the monovalent 11F2 antibody (mAb0077(OA)). The assay was performed 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 of FVIIa and either 0 or 1000 nM of the antibody. The reaction was initiated by the addition of 0 or 1000 nM of 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 of sTF and 1 mM of S-2288 (Chromogenix), which allowed the measurement of residual FVIIa activity by hydrolysis of the chromogenic substrate monitored at 405 nm for 5 min. The residual amidolytic activity was determined as the slope of the linear progress curve after blank subtraction, and these were 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 the antibody, the apparent second-order rate constant for the inhibition of FVIIa by alpha-2-macroglobulin was found to be 475 ± 21 M-1s-1. However, in the presence of the monovalent 11F2 antibody (mAb0077(OA)), no significant inhibition of FVIIa was observed up to the last time point at 125 min. From these studies, it was 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 proteolytic cleavage. Activation is thought to occur as a result of TF-mediated FVIIa-FVII transactivation, also called autoactivation. To determine the effect of the 11F2 antibody on FVII autoactivation, activation of FVII was measured in the presence of lipidated TF, FVII, limiting concentrations of FVIIa, and the monovalent ǀ1F2 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, and 0 or 200 nM monovalent 11F2 antibody (mAb0077(OA)). Reactions were initiated by the addition of 2 nM lipidated Escherichia coli-derived TF fragment 1-244 incorporated into 25:ǀ5 PS:PC vesicles as described by Smith and Morrissey (2005) J. Thromb Haemost., 2:1155-1162. The total reaction volume was 100 μl. FVIIa generated at selected time points (typically 5, 10, 15, 20, 30, 40, 50, and 60 minutes) 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 it to 140 μl of 5 mM EDTA containing 50 mM HEPES, 100 mM NaCl, 0.1% PEG8000, 1 mg / ml BSA, and 215 nM soluble tissue factor (sTF). After collection of all samples, the FVIIa chromogenic activity was measured by adding 50 μl of S-2288 (4 mM) to 60 mM CaCl2. The conversion of the chromogenic substrate by the generated FVIIa was measured as the slope of the linear absorbance increase over 10 minutes at 405 nm using a Spectramax plate reader. The molar concentration of FVIIa could be estimated by relating the measured slope to the slope generated under similar conditions for a known amount of FVIIa.

[0196] FVIIa self-activation was found to be TF-dependent, and the measured FVIIa activity in Table 11 shows that it is not impaired by the presence of a monovalent 11F2 antibody. [Table 17]

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

[0198] crystallization Crystals of Fab0076, mixed with the FVIIai / sTF complex in a 1:1 molar ratio, were grown using the hanging-drop vapor diffusion technique at 18°C. A protein solution of 1 μl of the 4.6 mg / ml protein complex in 10 mM HEPES, 50 mM NaCl, 5 mM CaCl2, pH 7.0 was mixed with 0.5 μl of 100 mM sodium citrate, pH 6.2, and 20% PEG 6000 as a precipitant. The mixture was incubated with 1 ml of the precipitate solution at 18°C ​​to obtain crystals of the complex.

[0199] Diffraction data collection The 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 100K at MAX-lab (Lund, Sweden) beamline I911-3 using a MAR Research marCCD225 detector. Automated indexing, merging, and scaling of the data were performed using a program from the XDS package (diffraction data statistics are summarized in Table 12).

[0200] Determination and refinement of the structure The structure was determined by molecular substitution using Phaser, implemented in the Phenix program suite, with the A and B chains from protein databank entries 1YY8 and 3ELA. The asymmetric unit contains two Fab:FVIIai / sTF complexes. The model was refined in COOT using Phenix refinement and manual reconstruction steps. Elaboration statistics are shown in Table 12. [Table 18]

[0201] An epitope, defined as the residues in FVIIai having non-hydrogen atoms positioned within 4 Å of the non-hydrogen atoms of Fab0076, was found to include the following residues according to SEQ ID NO: 1: R113 C114 H115 E116 G117 Y118 S119 [[ID=1,6]]L120 T130 V131 N184 T,-185 I186 P251 V252[[ID=3,1]] E265 M39,1 R392 E394 A paratope, defined as the residues in Fab0076 having non-hydrogen atoms positioned within 4 Å of the non-hydrogen atoms of FVIIai, includes the following light chain residues according to SEQ ID NO: 64: Q27 G28 S30 D31 Y32 [[ID=5,2]]K49 Y50 Q53 [[ID=5,8]]H92 S93 F94 and was found to include the following heavy chain residues according to SEQ ID NO: 63. D32<00009,75>Y54 N59 N101 Y102<00,00979>Y103 G104 N105

[0202] Example 14: Humanized Mouse 11F2 Note: There seem to be some potential errors or unclear parts in the original text, such as "T,-185" and "M39,1" which might need further clarification in the source material. The translation is done based on the provided text as accurately as possible.To humanize the mouse antibody 11F2 (mAb0005) with the VH and VL domain sequences corresponding to SEQ ID NOs. 754 and 750, respectively, we combined information from human germline-related 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, while preserving high binding affinity to FVII(a). First, we used the blast algorithm to search human germline databases for human VH, VL, and VJ sequences (for both heavy chain (HC) and light chain (LC)) that have high sequence identity to the mouse variable domain sequence of 11F2. The sequences with the highest sequence identity between HC and VH are IGHV4-30-4*01, IGHV4-28*01, IGHV4-28*06, and IGHV459*01, and in the VJ segment of the HC upper germline: IGHJ5*01 and IGHJ4*01. For LC, the upper VL sequences are IGKV6D-41*01 and IGKV3-11*01, and in the VJ segment: IGKJ2*01 and IGKJ2*02 (Table 12). Next, the differences between the human germline and the mouse VH and VL sequences were mapped onto the crystal structure of the Fab0076 / FVIIa complex. Residues in the mouse variable domain that are more than 10 Å away from the residues in the epitope were expected to have little or no effect on binding affinity and were replaced with the corresponding human germline amino acid entities. Next, it was considered more problematic to substitute the residues constituting the paratope without affecting affinity, thus conserving the mouse amino acid entity. A subset of residues near the binding interface that could potentially affect binding affinity was identified. Humanized variants were generated by mutating the distal residues of the epitope with human amino acid entities from germline alignment. Furthermore, residues close to the paratope were mutated with human entities from the subset to bring the variant as close to the human germline as possible. The variants included in the set were those in which mouse CDRs were transplanted into fully human germlines of HC and LC.According to Table 13, the initial analysis generated 12 LCs and 13 HCs, which were paired into 23 variants.

[0203] The affinity of humanized antibodies for binding 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 at a concentration of 10 μg / ml on an anti-human tip (AHC, Fortebio) for 3 minutes. A 3-minute incubation was then performed to establish a baseline. Subsequently, association was monitored for 3 minutes using four different concentrations of FVIIa (25 nm, 50 nM, 100 nM, and 200 nM), followed by a 3-minute dissociation. Sensograms were analyzed using Fortebio data analysis software. Because Fortebio data was used for ranking, absolute affinity values ​​may deviate from the values ​​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 that of the parental mouse antibody were identified, and mutations that were found to maintain or improve binding affinity were used to design variants for the second round. From this series of mutations, variants for the second round were generated by inserting many of these mutations into 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. Experimental testing of all VL sequences in combination with VL sequences yielded a total of 475 combinations. As described above, the dissociation constant (KD) values ​​for binding to FVIIa were measured for 475 humanized antibodies obtained 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 the selected humanized 11F2 variant was determined by SPR analysis, as detailed in Example 6. The measured dissociation constants are listed in Table 15, demonstrating that the variant retains high affinity binding to human FVIIa. [Table 24]

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

[0207] Pharmacokinetics of FVIIa in rats in co-formulation with humanized 11F2 variant The humanized 11F2 variant family was administered intravenously to male Sprague Dolly rats in a co-formulation with 20 nmol / kg of FVIIa, as detailed in Example 9.

[0208] The results are shown in Table 17, indicating that some humanized 11F2 variants impart the same long half-life to FVIIa as the parent antibody. [Table 26]

[0209] Example 15: Crystallization and epitope mapping of 11F2Fab0883 complexed with active site inhibitor 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 fragment Fab0883 was crystallized by complexing it with HD-Phe-Phe-Arg chloromethyl ketone (FFR-cmk; Bachem, Switzerland) active site inhibitor FVIIa (FVIIai) and soluble tissue factor fragments 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 Fab's SEC-purified complex, which had formed a complex with FVIIai / sTF, were grown using the sitting drop vapor diffusion technique at 18°C. A protein solution of 360 nl 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 precipitate solution containing 0.15 M CsCl and 15% (w / v) polyethylene glycol 3350, and 360 nl of water, and equilibrated against 80 μl of precipitate solution. Crystals grew within 6 weeks.

[0211] Diffraction data collection The 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 using a Rigaku FRX rotating anode generator with a Dectris Pilatus 1M detector. Data reduction was performed using a program from the XDS package (diffraction data statistics are summarized in Table 18).

[0212] Determination and refinement of the structure All crystallographic calculations were performed using the Phenix suite of crystallographic programs. The structure was determined by molecular substitution using the Phaser program, which has coordinates for the complex structure obtained as described in Example 13 as the search model. The asymmetric unit contains four Fab:FVIIai / sTF complexes. The final model was obtained by iterative cycles of manual reconstruction using COOT and Phenix modifications (Table 18). [Table 27]

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

[0214] Paratope confirmed If all four independently determined Fab molecules in the crystal have a non-hydrogen atom of the residue located within a distance of 4 Å or less from the non-hydrogen atom in FVIIa, then the residue is considered part of a paratope. The paratope is the following light chain residue (according to Sequence ID No. 814): Q27 G28 Y32 Y50 H92 S93 F94 It was also found to contain 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 the alanine variant of FVIIa The hFVII alanine variant was generated using the stable episome expression system QMCF technology (Icosagen). CHOEBNALT85 cells were cultured in Qmix1 medium (1 L = 1:1 CD-CHO and SFM II (NVO11514701) + 10 ml Pen / Strep (Gibco, 15140-122) + 2 ml puromycin (Gibco, A11138-03)) in an E125 flask in a CO2 shaking incubator. On the day of transfection, 1 × 10 e7 CHOEBNALT85 cells were transfected with 2 μg of hFVII variant coding plasmid and 50 μg of salmon sperm DNA using electroporation (Bio-Rad Gene Pulser Xcell Electroporation System, 300 V, 900 μF, 4 mm cuvette). One day after transfection, G418 selection was initiated by transferring the cells to Qmix2 medium (1 L = 1:1 CD-CHO and SFM II (NVO11514701) + 10 ml Pen / Strep (Gibco, 15140-122) + 1 ml K-vitamin (K.vit 13A 01311) + 14 ml G418 (Gibco, 10131-027)). After 10–14 days, G418-selected cells reached a viability of >95% (Vi-Cell XR cell counter). The cells were divided into 2 × E1000 flasks from 0.4 × 10⁶ cells / ml to 2 × 250 ml of Qmix2. After 3–4 days, the cells reached a density of approximately 4–5 × 10⁶ 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 culture was harvested and spun down (200 g, 5 min). The supernatant was collected and supplemented with 15 mM HEPES (Gibco, 15630) and 5 mM CaCl2 (Sigma, 21115). 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 generated using an expression system similar to that outlined above. As described in Example 26, the enzyme precursor 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. The anti-FVII(a) antibody F1A2 binds to the Gla domain of FVII(a) in a Ca++-dependent manner. The enzyme precursor 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 the monovalent humanized antibody mAb0842(OA) was performed by a coupling study with a panel of 19 FVII(a) variants at 25°C using surface plasmon resonance (Biacore T200). A 25 μg / ml 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). As shown in Table 19, FVII(a) variants in cell culture supernatant (as described above) were diluted in running buffer and injected at a flow rate of 10 μl / min for 1 minute to achieve capture levels of 5–55 RU. Each FVII(a) variant was captured by the immobilized anti-FVII(a)gla Ab. Next, 540 nM (3-fold dilution) mAb0842(OA) was infused at a flow rate of 30 μl / min for 7 minutes to bind to the captured FVII(a) variant, followed by 9 minutes of buffer infusion to allow dissociation of the one-armed anti-FVII(a) antibody. Running buffer was prepared by diluting 10x HBS-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, and 1 mg / ml bovine serum albumin (BSA). The running buffer was also used to dilute 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, and pH 7.4. The binding data was analyzed using a 1:1 kinetic model and steady-state analysis with BiaEvaluation 4.1 provided by the manufacturer (Biacore AB, Uppsala, Sweden). Wherever possible, ka, kd, and KD values ​​from the 1:1 kinetic model are reported. KD values ​​using the steady-state model are reported for four FVII(a) variants.Furthermore, capture signals are reported for all FVII(a) variants. An amino acid residue is considered a hotspot residue if substitution of that residue with alanine results in a more than 10-fold decrease in affinity compared to the wild type. Based on the data presented in Table 19, amino acid residues H115, T130, V131, and R392 are concluded to be hotspots. [Table 28]

[0218] Example 18: Pharmacokinetics of recombinant FVIIa in co-formulation with humanized 11F2 antibody in cynomolgus monkeys The FVIIa plasma activity time profile was estimated in cynomolgus monkey studies after IV or SC administration of either recombinant FVIIa (rFVIIa) alone or a co-formulation in a 1:3 molar ratio with monovalent one-armed 11F2 mAb0705(OA). The formulation was 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 3 weeks.

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

[0220] Pharmacokinetic analysis of the FVIIa plasma activity time profile was performed using a non-compartmental method with 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, showing a substantial extension of FVIIa activity with co-formulation with mAb0705(OA) both after IV administration and SC administration, compared to FVIIa in the absence of the antibody. [Table 29]

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

[0222] In short, the CDRH1, CDRH2, and CDRH3 sequences of mAb0082 were transplanted onto human germline sequences based on the VH3_74 / JH1 sequence defined in the IMGT database. Furthermore, three VH3_74 / JH1 human amino acid substitutions were introduced into the transplanted CDRH2 sequence to further humanize this CDR sequence: P62D, L64V, and D66G. Three revertant mutations were introduced into the VL sequence at positions S49G, D62P, and R98S to obtain binding affinity comparable to that of mAb0082. Regarding the humanization of the VL, the CDRL1, CDRL2, and CDRL3 sequences of mAb0082 were transplanted 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. It was found that using saturated mutagenesis at this location allows for the removal of the NG motif by N33Q substitution without compromising affinity for TLT-1.

[0223] The final humanized and optimized variant of mAb0082 corresponds to sequence numbers 934(VL) and 938(VH) and is designated mAb1076.

[0224] SPR analysis of humanized TLT1 variants The binding of sTLT1 (corresponding to Sequence ID No. 3 with 6 histidine residues added to the C-terminus) from (Example 4) to biAb was probed by surface plasmon resonance (Biacore T200) at 25°C. Anti-human IgG was immobilized on CM5 sensor chips (both supplied by GE Healthcare) using standard amine coupling chemistry. BiAb purified according to Table 21 (1 nM) was injected at a flow rate of 10 μl / min for 1 minute. Subsequently, sTLT1 between 0 and 60 μM was injected at a flow rate of 30 μl / min for 3 minutes to enable binding to biAb, followed by 3 minutes of buffer injection to enable dissociation from biAb. Running buffer was prepared by diluting 10x HBS-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, pH 7.4, 5 mM CaCl2, and 1 mg / ml bovine serum albumin (BSA). The running buffer was also used to dilute 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 using a 1:1 model with BiaEvaluation 4.1 supplied by the manufacturer (Biacore AB, Uppsala, Sweden). Analysis revealed that the binding constants reported in Table 21 ranged in affinity from 2.9 nM to 320 nM for sTLT1 binding by biAb. [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 mAb1076 (sequence numbers 854 and 858, respectively), the human IgG4 CH1 domain, and VL and VH domain sequences corresponding to 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. The 37-mer stalk peptide, EEEEETHKIGSLAENAFSDPAGSANPLEPSQDEKSIP (sequence number 13), corresponding to residues 111-147 of sequence number 2, was prepared by a standard peptide synthesis method known to those skilled in the art. The Fab peptide and the stalk peptide 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 by gel filtration on a Superdex200 column eluted with hepes buffer, then concentrated to approximately 11 mg / mL for crystallization.

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

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

[0228] Determination and refinement of the structure The asymmetric unit contains two Fab:peptide complexes, as determined from Matthews coefficient analysis. The structure was determined by molecular substitution. Phaser, implemented in the Phenix program suite, was used in conjunction with the H and L chains of protein databank entry 5KMV as a search model to localize the two Fabs. These were constructed using COOT with the correct amino acid sequence and then refined using Phenix refinement. Amino acids 7–21, derived from the peptide, are clearly visible in the differential electron density map and can be considered a model manually constructed using COOT. The model was refined in COOT using the steps of Phenix refinement and manual reconstruction. Elaboration statistics are shown in Table 22. [Table 31]

[0229] Fab / peptide complex epitopes and paratopes An epitope is defined as a residue of the TLT-1 stalk peptide characterized by having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4.0 Å from the heavy atom of Fab in both complexes of the asymmetric units. Similarly, a paratope is defined as a residue of the Fab fragment characterized by having a heavy atom within a distance of 4.0 Å from the heavy atom of the TLT-1 stalk peptide in both complexes of the asymmetric units, and it has been found that the epitope contains the following residues from the 37aa TLT-1 peptide according to Sequence 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 of sequence numbers 2 and 3). The paratope contains 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 Includes Y101.

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

[0231] In the first step, FX activation was measured in a concentration series from 0 to 300 nM in the presence of 4 nM recombinant TLT-1 incorporated into 10:90 phosphatidylserine:phosphatidylcholine vesicles (WO2011023785), 2.5 nM FVIIa, and a bispecific antibody (biAb). After pre-incubation for 10 minutes 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, which was activated for 20 minutes. Next, activation was terminated by adding 25 μl of quench buffer (50 mM HEPES, 100 mM NaCl, 80 mM EDTA, pH 7.3), and the resulting FXa was quantified by its ability to hydrolyze 0.5 mM S-2765 (Chromogenix) chromogenic substrate (added as 2 mM stock in 25 μl volume) and tracked for 5 minutes at 405 nm with a SPECTRAmax Plus 384 plate reader. The normalized activity (A) for each biAb at a concentration of 100 nM was calculated from the slope of the linear absorbance increase by subtracting the background activity in the absence of biAb and dividing by the FVIIa concentration during the assay. biAb ) was calculated.

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

[0233] Based on the measured activity, the stimulating activity of each biAb at 100 nM is determined to be A biAb / A FVIIa The ratio was calculated. Stimulatory activity provides a measure of the multiplicative increase in FXa generated by FVIIa when 100 nM biAb is added.

[0234] Table 23 provides the stimulating activity, showing the dependence of biAb stimulation on the binding intensity (expressed as a dissociation constant (KD) value) of FVIIa and TLT-1, respectively. Among the biAbs tested, biAb0001 showed the highest stimulating activity. [Table 32]

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

[0236] The results are presented in Table 24, showing the dependence of biAb stimulating activity on epitope location. In particular, the anti-TLT-1 mAbs mAb1076, mAb0023, mAb0051, and mAb0062, when combined with the anti-FVIIa mAb mAb 0865, exhibited comparable stimulating 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 Channeling Immunoassay (LOCI). Briefly, the LOCI reagent consisted of two latex bead reagents (donor and acceptor beads) and a biotinylated monoclonal antibody against hIgG (Biosite, catalog number AFC4249). The donor bead reagent, containing a photosensitive dye, was coated with streptavidin. The second bead reagent, the acceptor bead, was conjugated with an in-house monoclonal antibody against hIgG (0421), which formed the sandwich. During the assay, the three reactants combined with hIgG in the plasma to form a bead-aggregated immunocomplex. Excitation of the complex released singlet oxygen molecules from the donor bead, which were then transferred to the acceptor bead, inducing a chemiluminescent response. This was then measured using an EnVision plate reader. The amount of light generated was reported as counts per second (cps) and was proportional to the hIgG concentration. Samples were diluted at least 100-fold in assay buffer, and a calibration curve was prepared based on hIgG added to 1% cynomolgus monkey plasma.

[0238] Example 24: Antigen assay (LOCI) against FVII(a) The FVII(a) antigen, including the FVII enzyme precursor, FVIIa, and the FVIIa:antithrombin (FVIIa:AT) complex, was measured by the LOCI assay described in Example 23 (except that the FVII(a) assay consisted of acceptor beads coated with in-house anti-FVII(a) antibody (4F9) and in-house biotinylated anti-FVII monoclonal antibody (4F7)). Calibration curves were prepared by diluting samples at least 100-fold in assay buffer and adding known amounts of human rFVIIa to the assay buffer.

[0239] Example 25: FVIIa: Antigen assay for AT (antithrombin) (EIA) The FVIIa:AT (antithrombin) complex was measured using an enzyme immunoassay (EIA) as described in 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. Pre-formed complexes of human FVIIa and cynomolgus monkey antithrombin (FVIIa:AT) were prepared by incubating FVIIa with a 2x molar excess of antithrombin in the presence of 10 μM low molecular weight heparin (enoxaparin). The residual amide degradation activity of FVIIa after overnight incubation at room temperature (see Example 11) was verified to be less than 10% of the initial FVIIa activity. An EIA calibration curve was constructed using the complexes. A polyclonal anti-human antithrombin antibody peroxidase conjugate (Siemens Healthcare Diagnostics ApS, Ballerup, Denmark, product code OWMG15) was used for detection. TMB was added, and the color development was allowed to proceed until sufficient color development was achieved. H2SO4 was then added to stop the process, and the absorbance at 450 nm was measured using an absorbance plate reader (BioTek), with 650 nm as the reference. Color intensity is proportional to the concentration of FVIIa:AT.

[0240] Example 26: Preparation of human FVIIa, FVII (enzyme precursor), and FVIIa:AT (antithrombin) complex Preparation of human FVIIa (activated FVII) Unless otherwise noted, 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, the enzyme precursor FVII was dialyzed to 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 amide solubility 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 using known concentrations of FVIIa, the measured activity can be converted to the molar concentration of FVIIa in the enzyme precursor FVII preparation.

[0242] Preparation of the human FVIIa:AT (antithrombin) complex The 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) at 4°C for 16 hours. To remove contaminating excipients, the AT was re-purified before use by applying a sodium chloride gradient using a heparin Sepharose 6 fast-flow (GE Healthcare) column. The eluted AT was concentrated by ultrafiltration to obtain the final preparation in 10 mM HEPES containing 50% glycerol, 25 mM NaCl, and pH 7.3. The FVIIa:AT complex was purified by SEC chromatography at 4°C in 20 mM MES, 100 mM NaCl, 1 mM EDTA, and pH 5.5 to maximize the stability of the complex. The residual level of FVIIa in the preparation was determined as described above. To minimize the degradation of the complex, the preparations were stored in aliquots at -80°C, thawed immediately before use, and kept on ice.

[0243] Example 27: Single-dose pharmacokinetics of anti-FVII(a) / anti-TLT-1 biAb in cynomolgus monkeys biAb0001 and its corresponding YTE variant, biAb0352, in which three additional half-life-extending substitutions (M252Y, S254T, and T256E) are introduced into the constant heavy chain 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 before administration, and for the iv group, at 0.5 hours, 2.5 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, and on days 8, 10, and 15 after administration. For the sc group, blood samples were collected before administration and at 0.5 hours, 3 hours, 6 hours, 12 hours, 16 hours, 24 hours, 30 hours, 38 hours, 48 ​​hours, 54 hours, 72 hours, 78 hours, 96 hours, 120 hours, and on days 8, 10, and 15 after administration. Blood samples were centrifuged at 2000 g for 10 minutes to remove plasma, 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 (see Example 25). Pharmacokinetic (PK) analysis of the hIgG concentration-versus-time profile was performed using a non-compartmental method with 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] Endogenous FVII(a) accumulation was observed in monkeys administered with BiAb0001 or the corresponding YTE variant, biAb0352. Table 25 shows the pre-administration levels of FVII(a) antigen, FVIIa activity, and FVII:AT, and the mean cumulative levels measured between 72 and 240 hours post-administration. 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-administration levels, and FVIIa and FVIIa:AT increased up to 5-fold. The data demonstrate that a single dose of biAb0001 or biAb0352 administered intravenously or sc-mediated leads to in vivo accumulation of endogenous FVII(a) antigen, FVIIa activity, and FVIIa:AT complexes in monkeys with YTE mutations. [Table 35]

[0245] Example 28: Single-dose and repeated-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 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 each received IV administration of 40 nmol / kg mAb0705(OA) via the saphenous vein, cephalic vein, or lateral tail vein of the tail. Three male cynomolgus monkeys received 20 nmol / kg of one-armed anti-FVIIa antibody subcutaneously (sc) in the thigh (alternating between the left and right thighs) every other day for two weeks (i.e., antibody was administered on days 1, 3, 5, 7, 9, 11, 13, and 15). Blood samples were collected in 3.8% trisodium citrate from the cephalic vein or femoral vein up to 21 days after administration.

[0246] Blood was centrifuged at 2300g for 10 minutes, 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 using a non-compartmental method with Phoenix WinNonlin 6.4. PK parameters for IV administration are shown in Table 26. The half-life (t1 / 2) of the one-armed antibody was a mean 116 hours (4.8 days) after a single IV administration. The estimated half-life of the sc-administered antibody was 181 ± 20 hours (mean and SD for n=3). Antibodies accumulated over two weeks of administration, reaching a peak level of 1179 ± 140 nM at a time between 336 and 366 hours after the initial administration (mean and SD for n=3 animals and data at three time points). [Table 36]

[0247] Single IV or repeated sc administration of mAb0705(OA) resulted in the accumulation of endogenous FVII(a). Table 27 shows the total FVII(a) antigen, FVIIa:AT, and FVIIa after a single 40 nmol / kg administration. Total FVII antigen increased from 7.0 nM pre-administration to 35.0 ± 4.7 nM on days 7–14. Similarly, FVIIa increased from below the 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. The enzyme precursor FVII level after administration of a 40 nmol / kg one-armed antibody was 26.4 nM, calculated by subtracting FVIIa and FVIIa:AT from total FVII(a) antigen. [Table 37]

[0248] Table 28 shows the steady-state levels of total FVII(a), FVIIa, and FVIIa:AT after multiple sc administrations. Total FVII(a) antigen increased from 6.5 ± 1.5 nM before administration to 36.9 ± 9.8 nM on days 12–21. Similarly, FVIIa increased from below the detection limit (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 enzyme precursor 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 single-armed anti-FVII(a) antibody mAb0705(OA) resulted in the accumulation of endogenous FVII(a), FVIIa, and FVIIa:AT in vivo. The clearance of the single-armed anti-FVII(a) antibody (0.42–0.49 mL / kg×kg, Table 26) was comparable to the clearance of biAb0001 after intravenous administration (0.33–0.64 mL / kg×kg, Example 27, Table 24). Therefore, the steady-state levels of FVII(a) antigen, FVIIa, and FVIIa:AT measured after repeated administration of the single-armed anti-FVII(a) antibody are expected to represent the levels that would be achieved in steady state after repeated administration of biAb with the same FVII(a) binding arm. [Table 38]

[0250] Example 29: Thromboelastography of hemophilia A-like conditions in human whole blood with added steady-state levels of anti-FVII(a) / anti-TLT-1 biAb0001 and enzyme precursors FVII, FVIIa, and FVIIa:AT. The effects of the bispecific anti-FVII(a) / anti-TLT-1 antibody biAb0001 (parent anti-FVII(a) is mAb0865 and parent anti-TLT-1 antibody is mAb1076) and accumulated levels of enzyme precursors FVII, FVIIa, and FVIIa:AT from Example 28 were evaluated 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.) in principle as described in Viuff D, et al. Thromb Res 2010; 126:144-9. Citrate-stabilized whole blood from healthy donors was incubated for 30 minutes with 1 mg / mL neutralizing anti-FVIII sheep polyclonal antibody (Haematological Technologies Inc, catalog number, PAHFVIII-SC) and 5 μg / mL neutralizing anti-TF mouse monoclonal antibody (1F44, prepared in-house). 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), enzyme precursor 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. Pre-dilution of FVIIa:AT was performed in cold 20 mM MES, 100 mM NaCl, 1 mM EDTA, and pH 5.5+ 2% BSA, and added to the remaining protein immediately before initiating the assay. Since the FVII enzyme precursor and FVIIa:AT preparations contained trace amounts of FVIIa, a corresponding lower amount of FVIIa was added to compensate for this and the expected plasma concentration of FVIIa in the donor blood (0.1 nM, Morrissey JH et al Blood, 1993;81:734-44). Similarly, the amount of added enzyme precursor FVII compensated for the expected plasma concentration of 10 nM enzyme precursor FVIIa in the 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 alone and a biAb-free FVII / FVIIa / FVIIa:AT mixture. Platelets were maximally activated by adding the PAR1 agonist peptide SFLLRN (Tocris Biosciences catalog no. 3497) to a final concentration of 30 μM and the GPVI agonist conbruxin (5-Diagnostics, catalog no. 5D-1192-50UG) to a final concentration of 10 ng / mL. 20 μl of 0.2 M CaCl2 in 20 mM Hepes, pH 7.4 was added to a TEG cup, followed by the addition of 340 μl of blood sample, and analysis was immediately initiated. Coagulation 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. Coagulation 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 shortened the coagulation time of pseudo-hemophilia A blood to 694 ± 158 seconds. Addition of 100 nM biAb to the blood slightly shortened the coagulation time to 2198 ± 712 seconds, which is likely due to an enhanced enhancement of the effect of endogenous FVIIa in the blood. Adding a steady-state level of FVII / FVIIa / FVIIa:AT reduced the coagulation time to 1440 ± 275 seconds. Combining 100 nM biAb with a steady-state level of FVII / FVIIa / FVIIa:AT reduced the coagulation time to 495 ± 39 seconds, which is equivalent to or slightly lower than the coagulation time after adding 25 nM FVIIa.The data show that biAb enhances the effect of accumulated levels of FVII / FVIIa / FVIIa:AT, resulting in a reduction in clotting time similar to or slightly better than that achieved with therapeutically effective concentrations of rFVIIa. [Table 39]

[0251] Example 30: Thromboelastography of human whole blood in hemophilia A-like conditions after addition of bispecific anti-FVII(a) / anti-TLT-1 antibodies having different TLT-1 affinity and steady-state levels for enzyme precursors FVII, FVIIa, and FVIIa:AT. In this example, the following bispecific anti-FVII(a) / anti-TLT-1 antibodies, each having different affinities to TLT-1, were tested. [Table 40]

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

[0253] Example 31: In vivo effect of anti-FVII(a) / anti-TLT-1 bispecific antibody biAb0001 in a tail vein transection model in FVIII-deficient transgenic human TLT-1 mice. The in vivo efficacy of the anti-FVII(a) / anti-TLT-1 bispecific antibody biAb0001 was determined using transgenic FVIII knockout (i.e., hemophilia A), mouse TLT-1 knockout, and tail vein transection (TVT) models in human knock-in mice. Since anti-FVII(a) does not recognize mouse FVII(a), biAb was administered 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), mimicking their expected clinical steady-state plasma levels according to Example 28. The concentration of biAb was either 40 or 100 nM. In short, mice were anesthetized with isoflurane, placed on a warming pad, and their tails were immersed in saline (37°C) to maintain their body temperature at 37°C. Administration was performed into the right tail vein 5 minutes prior to injury. In this TVT model (Johansen et al., Haemophilia, 2016, 625-31), the lateral vein was severed. If bleeding stopped after 10, 20, or 30 minutes, the tail was removed from the saline solution and the wound was gently wiped with a saline-soaked gauze swab. The total blood loss was determined at 40 minutes by quantifying the amount of hemoglobin in the saline solution. Forty minutes after administration, a blood sample was collected from the orbital plexus with 3.8% trisodium citrate. The blood was centrifuged at 4000g for 5 minutes, the 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 FVII(a) administered with biAb alone or without biAb. For all combinations, platelet counts measured 45 minutes after treatment were comparable to those observed in the vehicle group.

[0255] In conclusion, these data demonstrate a significant in vivo hemostatic effect of biAb0001 in the presence of expected steady-state levels of FVIIa, FVII, and FVII:AT. Table 31 shows blood loss after tail vein transection (TVT) in FVIII knockout / mouse TLT-1 knockout / human TLT-1 knock-in mice administered with biAb0001, FVIIa, FVIIa, and FVIIa:AT combinations. Administered dose, 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 that of groups 1 and 2. Total FVII(a) antigen concentrations (measured according to Example 23) are listed in rows labeled "FVII," with values ​​indicated by asterisks. [Table 42]

[0256] While certain features of the present invention are illustrated and described herein, many modifications, substitutions, alterations, and equivalents will be conceivable to those skilled in the art. It should therefore be understood that the appended claims are intended to encompass all such modifications and alterations that fall within the true spirit of the invention.

[0257] Example 32: Identification of antibodies that compete with anti-TLT-1 mAb1076 for binding to TLT-1 in competitive ELISA. The Fab fragment used in the competitive experiment (anti-TLT-1 Fab) contains VH and VL domain sequences corresponding to mAb1076 (sequence numbers 938 and 934, respectively), a human IgG4 CH1 domain, and 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). The recombinant is produced as TLT-1 as described in WO2011 / 023785. The anti-TLT-1 Fab is biotinylated using standard methods, including the application of a biotinylation kit (EZ-link, Thermo) according to the manufacturer's instructions.

[0258] A competition study for binding to TLT-1 was performed to determine whether the anti-TLT-1 antibody competed with anti-TLT-1Fab and antibodies derived from it. Recombinant TLT-1 was immobilized on a NUNC maxisorp plate overnight at 4°C in dilution buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, pH 7.2). The plate was washed and blocked for 15 minutes in wash buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, 0.5 mL / L Tween 20, pH 7.2). In the competition study, biotinylated anti-TLT-1 Fab at the final immobilization concentration was combined with dilutions of a series of anti-TLT-1 antibodies to obtain final concentrations ranging from 0 to a maximum of 100 mg / ml in dilution buffer. The mixture was added to the plate wells and incubated for 1 hour. Next, the plate is washed, HRP-labeled streptavidin-HRPO (1:2000 in dilution buffer, Kirkegaard & Perry Labs) is added, and incubated for 1 hour. Finally, the plate is washed and colored with TMB ONE (KEMENTEC) for 10 minutes. The reaction is stopped by adding H3PO4 (4M), and the plate is read at 450 nm with a FLUOStar Optima plate reader, subtracting the background signal measured at 620 nm. Unless otherwise specified, all incubations are performed at room temperature, and the plate is washed five times with washing buffer.

[0259] The fixation concentration of recombinant TLT-1 immobilized on NUNC maxisorp plates, as well as the fixation concentration of biotinylated anti-TLT-1 Fab mixed with competitive antibodies for competitive studies, is determined by the individual titration of the two components, aiming to provide sufficient signal to allow for the detection of competition by the competitive antibody (i.e., signal reduction). 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 (OD units), competition at any given antibody concentration is: Inhibition rate = (1 - (OD unit - 100% inhibition) / (0% inhibition - 100% inhibition)) * 100 In the formula, 0% inhibition is determined from the signal in the well without competing anti-TLT-1 antibody, and 100% inhibition is determined from the signal in the well without biotinylated anti-TLT-1 Fab (i.e., corresponding to the assay background). If there is at least 50% observed inhibition (inhibition%) when the antibody concentration is tested in up to 10,000 times excess of biotinylated anti-TLT-1 Fab, the antibody is considered to compete with anti-TLT-1 Fab for binding to TLT-1.

Claims

1. It is a bispecific antibody, (i) A first antigen-binding site capable of binding to factor VII(a) (FVII(a)), A first light chain variable domain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 846, comprising CDRL1 represented by SEQ ID NO: 847, CDRL2 represented by SEQ ID NO: 848, and CDRL3 represented by SEQ ID NO:

849. A first heavy chain variable domain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 850, comprising CDRH1 represented by SEQ ID NO: 851, CDRH2 represented by SEQ ID NO: 852, and CDRH3 represented by SEQ ID NO:

853. The first antigen-binding site includes, (ii) A second antigen-binding site that can bind to TREM-like transcript 1 (TLT-1), A second light chain variable domain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 854, and comprising CDRL1 represented by SEQ ID NO: 855, CDRL2 represented by SEQ ID NO: 856, and CDRL3 represented by SEQ ID NO:

857. A second heavy chain variable domain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 858, and comprising CDRH1 represented by SEQ ID NO: 859, CDRH2 represented by SEQ ID NO: 860, and CDRH3 represented by SEQ ID NO:

861. A second antigen-binding site, (iii) An Fc region comprising a first heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 943 and a second heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 942, wherein the first heavy chain constant domain is bound to the first heavy chain variable domain and the second heavy chain constant domain is bound to the second heavy chain variable domain, and Includes, The first and second light chain variable domains each bind to a light chain constant domain, and each light chain constant domain contains the amino acid sequence of SEQ ID NO:

12. Bispecific antibodies.

2. The bispecific antibody according to claim 1, wherein the first antigen-binding site comprises a light chain variable domain which is at least 99% identical to the amino acid sequence specified by SEQ ID NO: 846 and a heavy chain variable domain which is at least 99% identical to the amino acid sequence specified by SEQ ID NO: 850, and the second antigen-binding site comprises a light chain variable domain which is at least 99% identical to the amino acid sequence specified by SEQ ID NO: 854 and a heavy chain variable domain which is at least 99% identical to the amino acid sequence specified by SEQ ID NO:

858.

3. A pharmaceutical formulation comprising a bispecific antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. A bispecific antibody according to claim 1 or 2, or a pharmaceutical formulation according to claim 3, for use in the treatment of coagulation disorders, wherein the coagulation disorder is selected from the group consisting of inhibitor-possessing or inhibitor-free hemophilia A, inhibitor-possessing or inhibitor-free hemophilia B, FVII(a) deficiency, and Glanzmann thrombasthenia.

Images