Bispecific antibodies

Abstract

FIELD: biotechnology.SUBSTANCE: bispecific antibodies are proposed that specifically bind to factor VII(a) and TREM-like transcript 1 (TLT-1), pharmaceutical compositions containing such bispecific antibodies, and their use for the treatment of coagulopathy.EFFECT: production of bispecific antibodies with improved pharmaceutical properties that can be used in the treatment of subjects with coagulopathies.20 cl, 38 tbl, 31 ex

Description

[0001] This application is separated from application No. 2021132993 for a patent for an invention, filed on 15.04.2020, claiming priority based on the filing date of applications No. 19169704.4 and 19213867.5, filed on 17.04.2019 and 05.12.2019, respectively, with the European Patent Office.

[0002] FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0003] The present invention relates to bispecific antibodies exhibiting improved pharmaceutical properties, to compositions containing such antibodies, and to uses of such antibodies and compositions, such as pharmaceutical and therapeutic uses.

[0004] SEQUENCE LISTING

[0005] This application is filed with a sequence listing in electronic form. The entire contents of the sequence listing are incorporated herein by reference.

[0006] BACKGROUND OF THE INVENTION

[0007] Blood coagulation involves several proteins that act together after vascular injury to form a thrombus, which prevents severe fluid loss and / or pathogen invasion. The cascade of events leading to thrombus formation can be initiated by two pathways, known as the intrinsic (contact) and extrinsic (tissue factor) pathways. Each pathway consists of a series of zymogen activation steps, where the newly activated enzyme catalyzes the activation of the next zymogen in the series until prothrombin is converted to thrombin. Thrombin converts fibrinogen into a fibrin network and activates platelets to form a platelet plug, which collectively leads to the formation of a stable thrombus.Initiation of the extrinsic pathway of thrombus formation is mediated by the formation of a complex between membrane-bound tissue factor (TF), which is exposed as a result of vessel wall injury, 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 coagulation factor X (FX). During the initial phase, low concentrations of FXa produce trace amounts of thrombin, which can activate factor XI and cofactors VIII and V. During the propagation phase, procoagulant complexes assemble and significantly enhance the formation of FXa and thrombin via tenase (FIXa, FVIIIa, Ca). 2+ , phospholipids) and prothrombinase (FXa, FVa, Ca 2+ , phospholipids) of the complex, respectively.

[0008] In people with hemophilia A and B (HA and HB, respectively), the function of various stages of the blood coagulation cascade is impaired due to the absence or insufficient presence of functional FVIII and FIX, respectively. This leads to impaired or insufficient blood clotting and the development of potentially life-threatening bleeding or damage to internal organs, such as joints.

[0009] Recombinant FVIIa (rFVIIa) is widely used as a bypass agent for the treatment of on-demand (OD) bleeding in people with hemophilia (A and B) who have inhibitors (HwI). rFVIIa has a short systemic half-life of 2-3 hours when administered intravenously (IV) and low bioavailability when administered subcutaneously (SC). The short systemic half-life of rFVIIa is thought to be due to several mechanisms, including inhibition by plasma inhibitors antithrombin III (AT) (Agerso H et al. (2010) J. Thromb. Haemost. 9: 333-8.) and alpha-2-macroglobulin (a2M), as well as renal clearance. The short systemic half-life and low bioavailability of rFVIIa SC complicate the use of rFVIIa for prophylactic treatment. Furthermore, the low intrinsic activity of rFVIIa requires higher doses of rFVIIa.

[0010] Therefore, there is a need for improved compounds that can support prophylactic treatment with less frequent dosing and SC administration.

[0011] Roche recently launched a bispecific antibody, emicizumab, which is indicated for routine prophylaxis and administered once weekly via SC injection for people with HA and HA with inhibitors (HAwI).

[0012] Nevertheless, the development of safe, effective molecules with an alternative mechanism of action remains an area of ​​key interest to improve and complement standard treatment for people with hemophilia.

[0013] BRIEF DESCRIPTION OF THE INVENTION

[0014] The present invention relates to bispecific antibodies exhibiting improved pharmaceutical properties, in particular, it relates to bispecific antibodies that can be used in the treatment of subjects with congenital and / or acquired coagulopathies, for example, in the treatment of people with hemophilia A or B with or without inhibitors. Furthermore, the present invention, in particular, relates to bispecific antibodies that are capable of binding blood coagulation factor VII (FVII(a)) and TREM-like transcript 1 (TLT-1).

[0015] In one aspect, the bispecific antibodies of the present invention comprise (i) a first antigen-binding region that binds FVII(a) and (ii) a second antigen-binding region that binds TLT-1.

[0016] In one aspect of the present invention, the bispecific antibody prolongs the active half-life of endogenous FVIIa in the bloodstream without losing the activity of endogenous FVIIa and stimulates the activity of endogenous FVIIa by selectively localizing it to activated platelets.

[0017] In one embodiment of the present invention, the bispecific antibody comprises an Fc region. The Fc region mediates the recycling of the bispecific antibody, prolonging its half-life in the bloodstream.

[0018] In one embodiment of the present invention, the first antigen-binding region competes for binding to FVII(a) in a competitive ELISA assay with any of the FVII(a) antibodies comprising a light chain variable domain (VL) and a heavy chain variable domain (VH), as follows:

[0019] • mAb0522 (VL: SEQ ID NO: 846 and VH: SEQ ID NO: 850),

[0020] • Fab0883 (VL: SEQ ID NO: 814 and VH: SEQ ID NO: 818),

[0021] • mAb0005 (VL: SEQ ID NO: 750 и VH: SEQ ID NO: 754),

[0022] • mAb0004 (VL: SEQ ID NO: 14 и VH: SEQ ID NO: 18),

[0023] • mAb0013 (VL: SEQ ID NO: 46 и VH: SEQ ID NO: 50),

[0024] • mAb0018 (VL: SEQ ID NO: 62 и VH: SEQ ID NO: 66),

[0025] • mAb0544 (VL: SEQ ID NO: 694 и VH: SEQ ID NO: 698),

[0026] • mAb0552 (VL: SEQ ID NO: 702 и VH: SEQ ID NO: 706),

[0027] • mAb0001 (VL: SEQ ID NO: 710 и VH: SEQ ID NO: 714),

[0028] • mAb0007 (VL: SEQ ID NO: 718 и VH: SEQ ID NO: 722),

[0029] • mAb0578 (VL: SEQ ID NO: 726 и VH: SEQ ID NO: 730),

[0030] • mAb0701 (VL: SEQ ID NO: 734 и VH: SEQ ID NO: 738) и

[0031] • mAb0587 (VL: SEQ ID NO: 742 и VH: SEQ ID NO: 746).

[0032] In a further embodiment of the present invention, the first antigen-binding portion of the bispecific antibody is capable of binding an epitope comprising amino acid residues H115, T130, V131, and R392 of FVII(a) (SEQ ID NO: 1).

[0033] In one aspect of the present invention, the bispecific antibodies are formulated into pharmaceutical compositions comprising the bispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0034] In one aspect of the present invention, the bispecific antibodies are administered parenterally, such as intravenously, intramuscularly, or subcutaneously. In one aspect of the present invention, the bispecific antibodies enable prophylactic treatment of subjects with congenital and / or acquired coagulopathy, such as hemophilia A, hemophilia B, hemophilia A with inhibitors, or hemophilia B with inhibitors. Therefore, the bispecific antibodies are designed to provide hemostatic protection against bleeding. In one aspect of the present invention, the bispecific antibodies are designed such that they can be administered once a week, once a month, or less frequently.

[0035] Conservative treatment with the bispecific antibodies of the present invention may have several advantages, such as a longer interval between injections, more convenient administration, and potentially improved hemostatic protection between injections. Therefore, the bispecific antibodies described herein may have a significant impact on the quality of life of individuals with hemophilia A or B, with or without inhibitors.

[0036] BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0037] Table 1 provides an overview of the antibodies and their corresponding SEQ ID NOs for the corresponding VL and VL domain sequences. The heavy chain constant domain type is as defined in Table 2a (“M” denotes the mouse IgG1 constant domain).

[0038] Table 2a provides an overview of the various formats used for recombinant expression of bivalent antibodies, monovalent (OA) antibodies, and bispecific antibodies. The SEQ ID NOs corresponding to the first heavy chain (HC-1) and second heavy chain (HC-2, or for OA antibodies, the truncated heavy chain (trHC) are listed. The light chain constant domain in all cases was the human kappa chain, corresponding to SEQ ID NO: 12.

[0039] Table 2b is an overview of the mouse anti-FVII(a) antibodies of the present invention. Correspondence between the clone name, the abbreviation for the fully mouse (hybridoma-derived, except for mAb0765, which was expressed recombinantly) antibodies and the corresponding mouse / human chimeric variants (mouse variable domains, human IgG4 S228P constant domains).

[0040] Table 2c provides an overview of antibodies in the murine and humanized 11F2 line.

[0041] Table 2d is an overview of the mAb0012 antibody line to TLT-1.

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] In the present invention, all recombinantly produced antibodies were expressed based on human IgG4 and all contained the standard hinge-stabilizing substitution S228P (EU numbering). In some embodiments, the C-terminal lysine of the heavy chain (K447, which is rapidly cleaved in vivo; see Cai et al. Biotechnol. Bioeng. 2011 vol. 108, pp. 404-412) was omitted (referred to as delta-lys). According to Table 2a, additional substitutions were introduced into the constant domain of the heavy chain to ensure the required chain pairing in bispecific and monovalent antibodies (Duobody mutations F405L R409K (Labrijn et al. PNAS 2013, vol. 110, pp. 5145-5150) and knob-in-hole mutations (see Carter et al. J. Imm. Methods 2001, vol. 248, pp. 7-15) T366W (knob) and T366S L368A Y407V (hole)) or to extend the half-life in vivo (YTE mutations M252Y S254T T256E (Dall'Acqua et al. J. Biol. Chem. 2006, vol. 18, pp. 23514-23524)).All recombinantly produced antibodies of the present invention contain the human kappa light chain constant domain (SEQ ID NO: 12).

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention relates to the design and use of antibody-based compositions exhibiting improved pharmaceutical properties. In particular, it relates to bispecific antibodies that are capable of binding the blood coagulation factor FVII(a) and TLT-1.

[0054] Antibodies

[0055] The term "antibody" as used herein refers to a protein derived from an immunoglobulin sequence that is capable of binding to an antigen or a portion thereof. The term "antibody" includes, but is not limited to, full-length antibodies of any class (or isotype), i.e., IgA, IgD, IgE, IgG, IgM, and / or IgY.

[0056] Of particular interest from a therapeutic antibody perspective is the IgG subclass, which in humans is divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the sequence of their heavy chain constant regions. Light chains can be divided into two types, kappa and lambda chains, based on differences in their sequence compositions. IgG molecules consist 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 may contain a heavy chain variable domain (V H ) and no more than three constant domains of the heavy chain (C H ): WITH H 1, C H 2 and C H 3. The light chain may contain a light chain variable domain (V L ) and the light chain constant domain (C L ). Regions V H and V Lcan be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Domains V H and V L Typically, antibodies consist of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable domains of the heavy and light chains, containing the hypervariable regions (CDRs), form a structure that is capable of interacting with an antigen, while the constant region of the antibody can mediate binding of the antibody to Fc receptors and the first component, C1q, of the C1 complex of the classical complement system.

[0057] The term "antigen-binding site" or "binding portion" refers to the part of an antibody that mediates antigen binding.

[0058] The term "antigen-binding fragment" of an antibody refers to a fragment of an antibody that retains the ability to bind to its cognate antigen, such as FVII(a), TLT-1, or another target molecule, as described herein. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', Fab2, Fab'2, Fv, single-chain Fv (scFv), or single V domain H or V L .

[0059] As used herein, the term "single-armed antibody" refers to a specific type of monovalent antibody fragment consisting of an antibody heavy chain, a truncated heavy chain lacking the Fab region, and one light chain.

[0060] As used herein, the term "monospecific" antibody refers to an antibody that is capable of binding alone to one specific epitope (including, but not limited to, bivalent antibodies).

[0061] As used herein, the terms “bispecific antibody” and “biAb” refer to an antibody that is capable of binding to two different antigens, such as FVII(a) and TLT-1, or two different epitopes on the same antigen.

[0062] The bispecific antibodies of the present invention are derived from antibodies or an antigen-binding fragment thereof. The bispecific antibodies of the present invention may be fusions or conjugates of antibodies and antigen-binding fragments of antibodies, such as, for example, Fab, Fab', Fab2, Fab'2, or scFv. The bispecific antibodies of the present invention may also be fusions or conjugates of antibody fragments. A wide range of molecular formats for 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).

[0063] Bispecific antibodies can be produced by various methods described in the prior art, see, for example, (Spiess et al.: Molecular Immunology 67, (2015), pp. 95-106) and (Brinkmann and Kontermann: MABS, 9 (2017), pp. 182-212). For example, the desired pairing of heavy chains can be achieved by engineering the dimerization interface in the Fc region to promote heterodimerization. One example of this is the so-called knob-into-hole mutations, where sterically bulky side chains ("knobs") are introduced into one Fc, matching sterically small side chains ("holes") on the opposite Fc, thereby creating steric complementarity that promotes heterodimerization.Other ways to engineer Fc interfaces for heterodimerization include electrostatic complementarity, hybridization with heterodimerization domains other than IgG, or exploiting the natural phenomenon of Fab-arm exchange in human IgG4 to mediate heterodimerization in vitro. Examples of heterodimerized bispecific antibodies are well described in the literature, e.g., (Klein C, et al.; MAbs. 2012 4, pp 653-663). Particular attention should be paid to the light chains in heterodimeric antibodies. Proper pairing of LC and HC can be achieved by using a common light chain. Engineering of the LC / HC interface can be used to promote heterodimerization or to engineer a light chain crossover, as in CrossMabs. Reassembly of antibodies in vitro under mildly reducing conditions from two separate IgGs containing the appropriate mutations can also be used to generate bispecific antibodies (e.g., Labrijn et al., PNAS, PO (2013), pp 5145 5150). It is also reported that the natural method of Fab-arm exchange ensures correct pairing of light chains.

[0064] As used herein, the term "polyspecific" antibody refers to an antibody that is capable of binding to two or more different antigens or two or more different epitopes on the same antigen. Thus, polyspecific antibodies include bispecific antibodies.

[0065] The antibodies of the present invention can be combined with other antibodies and antibody fragments known in the art to form bispecific, trispecific or polyspecific antibody molecules.

[0066] In one aspect, the antibody of the present invention is a chimeric antibody, a human antibody, or a humanized antibody. Such an antibody can be produced using, for example, suitable antibody display or immunization platforms or other suitable platforms or methods known in the art.

[0067] Furthermore, if the antibody comprises a constant region, the constant region or a portion thereof is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo).

[0068] Human antibodies can be isolated from sequence libraries constructed from human germline sequence samples further modified by introducing natural and synthetic sequence diversity.

[0069] Human antibodies can be produced by immunizing human lymphocytes in vitro, followed by transformation of the lymphocytes using the Epstein-Barr virus. Human antibodies can be produced by recombinant methods known in the art.

[0070] The term "humanized antibody" as used herein refers to a human / non-human antibody that comprises a sequence (CDR regions or portions thereof) derived from a non-human immunoglobulin. Thus, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from at least the hypervariable region of the recipient are replaced by residues from the hypervariable region of an antibody of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate, which has the required specificity, affinity, composition, and sequence functionality. In some cases, the framework (FR) residues of the human immunoglobulin are replaced with the corresponding non-human residues.An example of such a modification is the introduction of one or more so-called back mutations, which typically represent amino acid residues derived from the donor antibody. Humanization of an antibody can be accomplished using recombinant techniques known to those skilled in the art (see, for example, Antibody Engineering, Methods in Molecular Biology, vol. 248, edited by Benny K. Lo). A suitable human recipient scaffold for the variable domain of both the light and heavy chains can be identified, for example, by sequence or structure homology. Alternatively, established recipient scaffolds can be used, for example, based on knowledge of the structure, biophysical, and biochemical properties. Recipient scaffolds can be germline-derived or can be derived from the sequence of a mature antibody. The CDR regions from the donor antibody can be transferred by CDR grafting.A CDR-grafted humanized antibody can be further optimized, for example, with respect to affinity, functionality, and biophysical properties, by identifying essential framework positions where the reintroduction (backmutation) of an amino acid residue from the donor antibody has a beneficial effect on the properties of the humanized antibody. In addition to backmutations derived from donor antibodies, a humanized antibody can be constructed by introducing germline residues into CDRs or framework regions, removing immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.

[0071] Furthermore, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further improve the antibody's characteristics. A humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0072] As used herein, the term "chimeric antibody" refers to an antibody that contains antibody portions derived from two or more species. For example, the genes encoding such an antibody include genes encoding variable domains and genes encoding constant domains derived from two different species. For example, genes encoding the variable domains of a mouse monoclonal antibody can be fused to genes encoding the constant domains of an antibody of human origin.

[0073] 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 regions of an antibody where the amino acid residues involved in antigen binding are typically located. CDRs can be identified as the regions with the highest variability among the variable domains of antibodies. Databases such as the Kabat database can be used to identify CDRs, where CDRs are defined, for example, as containing amino acid residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain (Kabat et al. 1991; Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). Typically, amino acid residue numbering in this region is performed using the method described in Kabat et al.Above. Phrases such as "Kabat position," "Kabat residue," and "according to Kabat" as used herein refer to this numbering system for heavy chain variable domains or light chain variable domains. When using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a truncation or insertion into the framework (FR) or CDR of the variable domain. For example, a heavy chain variable domain may include amino acid insertions (residues 52a, 52b, and 52c according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82 of the heavy chain FR. Kabat residue numbering can be determined for a given antibody by aligning the regions of homology of the antibody sequence with a "standard" Kabat-numbered sequence.The numbering is in accordance with Kabat only if specifically stated, otherwise the numbering is sequential in accordance with the specified SEQ ID NO.

[0074] The term "framework region" or "FR" residues refers to those amino acid residues V H or V L , which are not in the CDR as defined in this document.

[0075] The antibody of the present invention may comprise a CDR region from one or more specific antibodies disclosed herein.

[0076] The term "antigen" (Ag) refers to a molecular entity used to immunize an immunocompetent vertebrate animal to produce an antibody (Ab) that recognizes the Ag. In this document, the term "Ag" is broader and generally includes target molecules recognized by the Ab.

[0077] The present invention encompasses variants of the antibodies or antigen-binding fragments thereof of the present invention, which may contain 1, 2, 3, 4 or 5 amino acid substitutions and / or deletions and / or insertions in the individual sequences disclosed herein.

[0078] Variants with "replacement" preferably involve the replacement of one or more amino acids with the same number of amino acids.

[0079] The term "epitope" as used herein is defined in the context of the molecular interaction between an "antigen-binding polypeptide," such as an antibody (Ab), and its corresponding antigen (Ag). Typically, "epitope" refers to a region or site on Ag to which Ab binds, i.e., an area or site of physical contact with Ab. In the present invention, epitopes are defined using a crystal structure obtained by X-ray crystallography, determining the spatial coordinates of the complex between Ab, such as a Fab fragment, and its Ag. The term epitope as used herein, unless otherwise indicated or contradicted by the context, is defined as residues of Ag (herein FVII(a) or TLT-1) characterized by the presence of a heavy atom (i.e., an atom other than hydrogen) at a distance of 4 A from the heavy atom in Fab.

[0080] Epitopes described at the amino acid level, such as those determined by X-ray structure, are considered identical if they contain the same set of amino acid residues. Epitopes are considered overlapping if at least one amino acid residue is common to both epitopes. Epitopes are considered distinct (unique) if no amino acid residues are common to both epitopes.

[0081] The definition of the term "paratope" is derived from the above definition of "epitope" by changing the perspective. Thus, the term "paratope" refers to the zone or region on an antibody or antibody fragment to which an antigen binds, i.e., through which it comes into physical contact with the antigen. The term "paratope" in this document, unless otherwise specified or unless inconsistent with the context, is defined as Ab residues characterized by the presence of a heavy atom (i.e., an atom other than hydrogen) within 4 Å of the heavy atom in FVII(a) or TLT-1.

[0082] Epitopes on an antigen may contain one or more “hotspot” residues, i.e., residues that are particularly important for interaction with a cognate antibody and where interactions mediated by the side chain of the said “hotspot” residue contribute significantly to the binding energy of the antibody / antigen interaction (Peng et al. PNAS 111 (2014), E2656-E2665). Hotspot residues can be identified by testing antigen variants where individual epitope residues have been replaced, for example, with alanine, for binding to the cognate antibody. If the replacement of an epitope residue with alanine has a strong effect on binding to the antibody, then the said epitope residue is considered a “hotspot” residue and, therefore, has particular importance for binding of the antibody to the antigen.

[0083] Antibodies that bind to the same antigen can be characterized in terms of their ability to bind to their common antigen simultaneously and can be subject to "competitive binding" / "categorization." In the context of the present invention, the term "categorization" refers to a method of grouping antibodies that bind to the same antigen. "Categorization" of antibodies can be based on the competitive binding of two antibodies to their common antigen in assays using standard methods. The "category" of an antibody is determined using a reference antibody. If a second antibody is unable to bind to the antigen at the same time as the reference antibody, the second antibody is said to belong to the same "category" as the reference antibody. In this case, the reference and second antibodies competitively bind the same portion of the antigen and constitute "competing antibodies."If a second antibody is capable of binding to an antigen simultaneously with the reference antibody, the second antibody is said to belong to a separate "category." In this case, the reference and second antibodies do not competitively bind the same portion of the antigen and are considered "non-competing antibodies."

[0084] Competitive assays for determining whether an antibody competes for binding with the anti-FVII(a) antibody or anti-TLT-1 antibody disclosed herein are known in the art. Illustrative competitive assays include immunoassays (e.g., ELISA assays, RTA assays), surface plasmon resonance assays (e.g., using a BIAcore™ instrument), biolayer interferometry (ForteBio®), and flow cytometry.

[0085] Typically, a competitive assay involves the use of an antigen bound to a solid surface or expressed on a cell surface, a test antibody that binds FVII or FVIIa, and a reference antibody. The reference antibody is labeled, and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody bound to the solid surface or cells in the presence of the test antibody. The test antibody is typically present in excess (e.g., 1-fold, 5-fold, 10-fold, 20-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold). Antibodies identified as competitive in a competition assay (i.e., competing antibodies) include antibodies that bind to the same epitope or overlapping epitopes as the reference antibody and antibodies that bind to an adjacent epitope that is sufficiently proximal to the epitope bound by the reference antibody to cause steric hindrance.

[0086] In an exemplary competition assay, an anti-FVII or anti-FVIIa reference antibody is biotinylated using commercially available reagents. The biotinylated reference antibody is mixed with serial dilutions of the test antibody or an unlabeled reference antibody (self-competition control), resulting in a mixture of various molar ratios (e.g., 1, 5, 10, 20, 100, 1,000, 10,000, or 100,000-fold) of the test antibody (or unlabeled reference antibody) to the labeled reference antibody. The antibody mixture is added to an ELISA plate coated with the FVII or FVIIa polypeptide. The plate is then washed, and horseradish peroxidase (HRP)-strepavidin is added to the plate as a detection reagent. The amount of labeled reference antibody bound to the target antigen is determined after addition of a chromogenic substrate (e.g., TMB (3,3',5,5'-tetramethylbenzidine) or ABTS (2,2''-azino-di-(3-ethylbenzthiazoline-6-sulfonate)), which are known in the art.Optical density readings (OD units) are obtained using a spectrometer (e.g., SpectraMax® M2 spectrometer (Molecular Devices)). The response (OD units) corresponding to zero percent inhibition is determined in wells without any competing antibody. The response (OD units) corresponding to 100% inhibition, i.e., the assay background, is determined in wells without any labeled reference antibody or test antibody. The percent inhibition of labeled FVII or FVIIa reference antibody by test antibody (or unlabeled reference antibody) at each concentration is calculated as follows: % inhibition = (1 - (OD units - 100% inhibition) / (0% inhibition - 100% inhibition)) * 100.

[0087] One skilled in the art will appreciate that similar assays can be performed to determine whether two or more anti-TLT-1 antibodies share a common binding region, category, and / or competitively bind an antigen. Those skilled in the art will also appreciate that competitive assays can be performed using various detection systems known in the art.

[0088] A test antibody competes with a reference antibody for binding to an antigen if an excess of one antibody (e.g., 1, 5, 10, 20, 100, 1000, 10,000, or 100,000-fold) inhibits the binding of the other antibody by, e.g., at least 50%), 75%, 90%o, 95%, or 99%, as measured in a competitive binding assay.

[0089] Unless otherwise stated, competition is determined using the competitive ELISA assay described above and presented in Example 7 and Example 31.

[0090] The term "binding affinity" is used in this document to measure the strength of a non-covalent interaction between two molecules, such as an antibody or its fragment and an antigen. The term "binding affinity" is used to describe monovalent interactions.

[0091] The binding affinity between two molecules, such as an antibody or its fragment and an antigen, through a monovalent interaction can be quantitatively determined by determining the equilibrium dissociation constant (K D ). K D can be determined by measuring the kinetics of formation and dissociation of the complex, for example, by surface plasmon resonance (SPR), as was done in Examples 6 and 16, or by other methods known in the art. The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as the association rate constant k а (or k оn ) and the dissociation rate constant kd (or k off ) respectively. K D correlates with k а and k d through equation K D = k d / k a .

[0092] Following the above definition, binding affinity indices associated with different molecular interactions, such as comparing the binding affinity of different antibodies for a given antigen, can be compared by comparing the K values D for individual antibody / antigen complexes.

[0093] K D The antibody of the present invention with respect to its target may be less than 1 pM, such as less than 10 pM, such as less than 100 pM, such as less than 200 pM, such as less than 400 pM, such as less than 600 pM, such as less than 1 nM, such as less than 5 nM, such as less than 10 nM, such as less than 20 nM, such as less than 50 nM, such as less than 100 nM, such as less than 200 nM, such as less than 400 nM, such as less than 600 nM, such as less than 800 nM.

[0094] In one such embodiment, the antibody is a bispecific antibody comprising an arm to FVII(a) with a K D in relation to FVIIa, which is less than 1 pM, such as less than 10 pM, such as less than 100 pM, such as less than 200 pM, such as less than 400 pM, such as less than 600 pM, such as less than 1 nM, such as less than 5 nM, such as less than 10 nM, such as less than 20 nM, such as less than 50 nM, such as less than 100 nM, such as less than 200 nM, such as less than 400 nM, such as less than 600 nM, such as less than 800 nM, and a second arm to TLT-1 with K D in relation to TLT-1, which is 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.

[0095] The term "identity," as known in the art, refers to the relationship between the sequences of two or more polypeptides, determined by sequence comparison. In this art, "identity" also refers to the degree of sequence relatedness between polypeptides, determined by the number of matches between the chains of two or more amino acid residues. "Identity" is measured as the percentage of identical matches between the smaller of the two or more sequences, with gap alignments (if any), using a specific mathematical model or computer program (i.e., "algorithms"). The identity of related polypeptides can be readily calculated using known methods. In the present invention, similarity and identity were determined using the Needleman algorithm (Needleman et al. J. Mol. Biol. 1970; 48:443-453) from EMBOSS-6.6.0 using parameters 10 and 0.5 for gap introduction and extension, respectively (gapopen = 10, gapextend = 0.5).

[0096] The crystallizable fragment region (“Fc region” / “Fc domain”) of an antibody is the C-terminal region of the antibody, which contains the hinge and C constant domains H 2 and C H 3.

[0097] The antibodies of the present invention may comprise an Fc region, which may have a wild-type amino acid sequence or may comprise amino acid substitutions that modulate the effector functions of the antibody, see, for example, (Wang et al.: Protein Cell. 9 (2018), pp. 63-73). Specific examples of Fc variants with modified effector functions are variants in which binding to Fcγ receptors is reduced. One specific example of such a variant is IgG1 containing the substitutions L234A, L235E, G237A, A330S, and P331S (residue numbering according to the EU index), with reduced affinity for certain Fcγ receptors and C1q.

[0098] Bispecific molecules

[0099] The term "bispecific molecules" as used herein refers to molecules capable of binding to different targets, such as FVII(a) and TLT-1. The binding moieties of a bispecific molecule may be derived from an antibody or may be of non-antibody origin. One specific example of a bispecific molecule is a bispecific antibody.

[0100] In one aspect of the present invention, the bispecific molecule comprises a first antigen-binding region capable of binding factor VII(a) and a second antigen-binding region capable of binding TREM-like transcript 1 (TLT-1).

[0101] The bispecific molecules of the present invention may comprise binding fragments that are not derived from antibodies, also referred to as alternative scaffolds. The bispecific molecules of the present invention may be fusions or conjugates of alternative scaffolds. The bispecific molecules of the present invention may be fusions or conjugates of antibodies and alternative scaffolds. The bispecific molecules of the present invention may also be fusions or conjugates of antibody fragments and alternative scaffolds.

[0102] A large number and variety of 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).

[0103] Specific examples of alternative scaffolds are adnectins, affilins, anticalins, avimers, atrimers, FN3 scaffolds, finomers, OBodys, kringle domains, Kunitz domains, knottins, affibodies, DARPin, bicyclic peptides, and cis-knots.

[0104] Factor VII(a)

[0105] The terms "factor VII" and "FVII" as used herein refer to the zymogen 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 the uncleaved zymogen, factor VII (FVII), as well as the cleaved and thus activated protease, factor VIIa (FVIIa). The terms "factor VII(a)" and "FVII(a)" as used herein include naturally occurring allelic variants of FVII(a) that may exist. One sequence of wild-type human factor VII(a) is provided as SEQ ID NO: 1.

[0106] Human coagulation factor VII(a) wild type (SEQ ID NO: 1):

[0107] ANAFLEELRPGSLERECKEEQCSFEEAREIFKDAERTKLFWISYSDGDQCASS PCQNGGSCKDQLQSYICFCLPAFEGRNCETHKDDQLICVNENGGCEQYCSDHTGTK RSCRCHEGYSLLADGVSCTPVEYPCGQQQGGGGCWGCPGC QVLLLVNGAQLCGGTLINTIWVVSAAHCFDKIKNWRNLIAVLGEHDLSEHDGDEQS RRVAQVIIPSTYVPGTTNHDIALLRLHQPWLTDHVVPLCLPERTFSERTLAFVRFSL VSGWGQLLDRGATALELMVLNVPRLMTQDCLQQSDRGTALELMVLNVPRLMTQDCLQQSDRGNIGSPYGFCYMS GSKDSCKGDSGGPHATHYRGTWYLTGIVSWGQGCATVGHFGVYTRVSQYIEWLQ KLMRSEPRPGVLLRAPFP Wild-type FVII(a) contains 406 amino acid residues and consists of four domains. There is an N-terminal gamma-carboxyglutamic acid (Gla)-rich domain, where ten glutamate residues (shown in bold in the sequence above) can 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 the bloodstream, but FVIIa is present only in small amounts (approximately 1% of the total pool; Morrissey JH, Broze Jr GJ. Tissue factor and the initiation and regulation (TFPI) of coagulation. In: Marder VJ, Aird WC, Bennett JS, Schulman S, White II GC, editors. Hemostasis and thrombosis: basic principles and clinical practice. 6th ed. Wolters Kluwer & Lippincott Williams & Wilkins: Philadelphia; 2013. p. 163–78).FVII can be activated to FVIIa via proteolytic cleavage between residues Arg152 and Ile153, yielding 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, and the heavy chain contains a protease domain. FVIIa requires binding to its cell surface cofactor, tissue factor (TF), to achieve its full biological activity.

[0109] The predicted full-length X1 isoform of cynomolgus monkey (Macaca fascicularis) FVII consists of 406 amino acids with the reference sequence in NCBI under the ID XP_015295043.1. The term "cFVIIa chimera" in this document refers to the chimeric construct of cynomolgus monkey FVIIa. The amino acid sequence of the cFVIIa chimera is such that the Gla domain and the first EGF-like domain (amino acids 1-88 when aligned with the human FVIIa sequence) are composed of the human FVII sequence (Uniprot ID P08709); whereas the second EGF-like and protease domains (amino acids 89–106 when aligned with the human FVIIa sequence) are composed of the sequence of the cynomolgus monkey FVII isoform X1 (NCBI reference sequence ID XP_015295043.1)

[0110] The active half-life of recombinant FVIIa (and endogenous FVIIa) in humans is approximately 2-3 hours when administered intravenously. Factor VII(a) can be endogenous, obtained from blood plasma, or can be produced recombinantly using well-known production and purification methods. The degree and location of glycosylation, gamma-carboxylation, and other post-translational modifications can vary depending on the host cell and its growth conditions.

[0111] Factor VIIa can exist in different conformations. Factor VIIa circulates in the blood in an inactive conformation or inactive form. This conformation does not have catalytic activity. FVIIa can also exist in an active conformation or active form; it is also referred to herein as fully active or fully activated FVIIa. The term FVIIa encompasses FVIIa in an inactive form or conformation and FVIIa in an active form or conformation. For example, the active conformation of FVIIa may involve a complex between FVIIa and tissue factor (FVIIa / TF), such as FVIIa / sTF(1-219), where sTF(1-219) is a truncated and soluble form of tissue factor, or the active conformation of FVIIa may involve FVIIa with an inhibited active site (FVIIai).FVIIai is a catalytically inert form of FVIIa that can be generated by treating FVIIa with dansyl-Glu-Glu-Arg-chloromethyl ketone or Phe-Phe-Arg-chloromethyl ketone (FFR-chloromethyl ketone) (Wildgoose et al. (1990) Biochemistry 29: 3413 3420 and Sørensen et al. (1997) J Biol Chem 272: 11863–11868). FVIIai retains its affinity for TF and is thought to adopt the same conformation as the conformation induced in FVIIa by TF binding. Thus, FVIIai has an activated conformation, and binding of the test compound to FVIIai indicates that the test compound will also bind to the activated form of wild-type FVIIa.

[0112] The target molecule for the FVII(a) antibody may be any FVII(a) molecule as described herein.

[0113] TREM-like transcript 1 (TLT-1)

[0114] Triggering receptors expressed on myeloid cells (TREMs) have a well-established role in the biology of various myeloid lineages, including important roles in the regulation of innate and adaptive immunity. TREM-like transcript (TLT)-1 belongs to this family of proteins, although the TLT-1 gene is expressed only in one lineage, megakaryocytes and platelets, and is located exclusively in the alpha granules of megakaryocytes and platelets. TLT-1 is a transmembrane protein that is exposed on the surface of activated platelets upon release of alpha granules. To date, TLT-1 has not been detected on the surface of resting platelets or on the surface of any other cell type.

[0115] TLT-1 contains an extracellular globular head, a stalk region, a transmembrane domain, and an intracellular domain containing immunoreceptor tyrosine-based inhibitory motifs (Washington et al. Blood, 2002; 100: 3822 3824). The extracellular globular head of human TLT-1 (hTLT-1) is a distinct immunoglobulin-like (Ig-like) domain. It is connected to the platelet membrane by a 37-amino acid linker region called the stalk (Gattis et al., Jour Biol Chem, 2006, 281, 19, 13396-13403).

[0116] The predicted length of the hTLT-1 transmembrane segment is 20 amino acids. TLT-1 also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM), which may function as an intracellular signal transduction motif.

[0117] A portion of TLT-1 is cleaved during platelet activation, forming a soluble form (sTLT-1) (Gattis et al., Jour Biol Chem, 2006, 281, 19, 13396 13403). The cleavage site is located in close proximity to the platelet membrane. A shorter TLT-1 isoform with a truncated intracellular domain also exists in platelets.

[0118] TLT-1 is involved in the regulation of blood coagulation and possibly inflammation at the site of injury. TLT-1 plays a role in platelet aggregation in response to suboptimal concentrations of some platelet agonists (Giomarelli et al., Thromb Haemost 2007; 97: 955-963). The best-described TLT-1 ligand is fibrinogen (Washington et al. J Clin Invest 2009; 119: 1489 3824). Recently, TLT-1 has been shown to also bind von Willebrand factor (Doerr A et al., abstract PB359, 2019 ISH meeting). sTLT-1 is proposed to play a role in sepsis-associated bleeding by suppressing leukocyte activation and modulating platelet-neutrophil crosstalk (Derive, J Immunol 2012, 188: 5585-5592).

[0119] Within the context of the present invention, TLT-1 may be derived from any vertebrate animal, such as any mammal, such as a rodent (e.g., a mouse, rat, or guinea pig), a member of the order Lagomorpha (e.g., a rabbit), a member of the order Artiodactyla (e.g., a pig, cow, sheep, or camel), or a primate (e.g., a monkey or a human). TLT-1 is preferably human TLT-1. TLT-1 may be translated from any naturally occurring genotype or allele that gives rise to a functional TLT-1 protein. A non-limiting example of one human TLT-1 is the polypeptide sequence of SEQ ID NO: 2.

[0120] The target molecule for the TLT-1 antibody may be any TLT-1 molecule as described herein.

[0121] Antibodies to FVII(a)

[0122] The term "anti-FVII(a) antibody" as used herein refers to an antibody that targets FVII(a). The anti-FVII(a) antibody is capable of binding to FVII(a) molecules as described herein; including, but not limited to, endogenous FVII(a) found in human plasma, exogenous FVII(a) such as wild-type recombinant human FVII(a), and endogenous FVII(a) found in the plasma of animals such as rabbits, mice, rats, dogs, or monkeys.

[0123] An "anti-FVII(a) antibody" may be a monoclonal monospecific antibody that targets FVII(a). A monospecific antibody to FVII(a) typically contains two identical antigen-binding sites that bind FVII(a); non-limiting examples include IgG4 monoclonal antibodies to FVII(a).

[0124] Suitable antibodies to FVII(a) include, but are not limited to, any of the antibodies to FVII(a) shown in Table 3.

[0125]

[0126]

[0127] An anti-FVII(a) antibody may have the ability to compete with any of the antibodies shown in Table 3 for binding to FVII(a). The presence or absence of competition between the anti-FVII(a) antibody and any of the antibodies shown in Table 3 for binding to FVII(a) can be determined by methods (i.e., by competitive binding assays) that are well known, such as surface plasmon resonance (SPR), ELISA, or flow cytometry. Example 7 describes how competitive binding to FVII(a) can be determined using a competitive ELISA.

[0128] In one embodiment, the anti-FVII(a) antibody binds to FVII(a) with high affinity. The anti-FVII(a) antibody may have a K Dwith respect to its target, which is less than 1 pM, such as less than 10 pM, such as less than 100 pM, such as less than 200 pM, such as less than 400 pM, such as less than 600 pM, such as less than 1 nM, such as less than 5 nM, such as less than 10 nM, such as less than 20 nM, such as less than 50 nM, such as less than 100 nM, such as less than 200 nM, such as less than 400 nM, such as less than 600 nM, such as less than 800 nM. In vivo, the high-affinity antibody to FVII(a) reduces the clearance of FVII(a) by forming a complex with it. It thereby prolongs the half-life of FVII(a) and causes its accumulation in the bloodstream. Thus, the antibody may provide an increased steady-state concentration of FVII(a).

[0129] 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 the activation of endogenous FVII to FVIIa. For example, the anti-FVII(a) antibody does not impair the ability of FVII to convert to FVIIa (i.e., autoactivate) while it is bound to TF (as described in Example 12). It is also preferred that the anti-FVII(a) antibody does not prevent the formation of the so-called initiation complex of FVII(a) with tissue factor (TF) and the activation of factor X (FX) in a TF-dependent or -independent manner (as described in Example 10). In one embodiment, the anti-FVII(a) antibody does not compete with FVII(a) substrates or cofactors. However, anti-FVII(a) antibody can compete with FVIIa inhibitors such as antithrombin (AT) and alpha-2-macroglobulin (as described in Example 11).

[0130] Antibodies to TLT-1

[0131] The term "anti-TLT-1 antibody" as used herein refers to an antibody that targets TLT-1. The anti-TLT-1 antibody is capable of binding to TLT-1 molecules as described herein. The anti-TLT-1 antibody may be a monoclonal, monospecific antibody.

[0132] Suitable TLT-1 antibodies include, but are not limited to, the TLT-1 antibodies shown in Table 4

[0133]

[0134]

[0135] An anti-TLT-1 antibody may have the ability to compete with any of the antibodies shown in Table 4 for binding to TLT-1. The presence or absence of competition between an anti-TLT-1 antibody and any of the antibodies shown in Table 4 for binding to TLT-1 can be determined by known methods (i.e., by competitive binding assays), such as surface plasmon resonance (SPR), ELISA, or flow cytometry. Competitive binding to TLT-1 can be determined using a competitive ELISA. Example 31 describes how competitive binding to TLT-1 can be determined using a competitive ELISA.

[0136] Anti-TLT-1 antibody can be characterized by K Din relation to its target, which is 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.

[0137] It is preferable that the TLT-1 antibody does not interfere with the functions of TLT-1 and, in particular, does not suppress platelet aggregation.

[0138] In one preferred embodiment, the TLT-1 antibodies are capable of binding to TLT-1 without interfering with platelet aggregation.

[0139] In another preferred embodiment, the TLT-1 antibodies are capable of binding to TLT-1 without competing with fibrinogen for binding to TLT-1.

[0140] In another preferred embodiment, the antibodies to TLT-1 do not interfere with the release of TLT-1.

[0141] It is preferred that the TLT-1 antibodies do not bind to or exhibit low affinity for any other trigger receptor expressed on myeloid cells (TREM) other than TLT-1, or for any other receptor on resting or activated platelets.

[0142] In one embodiment, the TLT-1 antibody binds to the TLT-1 stalk.

[0143] Bispecific antibodies to FVII(a) / TLT-1

[0144] The bispecific antibodies of the present invention comprise a first antigen-binding region capable of binding FVII(a) and a second antigen-binding region capable of binding TLT-1.

[0145] Antigen-binding site to FVII(a)

[0146] In one aspect, the bispecific antibodies of the present invention comprise a first antigen-binding region capable of binding FVII(a).

[0147] In some embodiments of the present invention, the first antigen-binding region of the bispecific antibody competes for binding to FVII(a) with any of the FVII(a) antibodies listed in Table 3; has an epitope that is identical to any of the antibodies listed in Table 3; comprises CDR regions that are identical to any of the antibodies listed in Table 3; and comprises VL and VH regions that are identical to any of the antibodies listed in Table 3.

[0148] TLT-1 antigen-binding region

[0149] In one aspect, the bispecific antibodies of the present invention comprise a second antigen-binding region capable of binding TLT-1.

[0150] In some embodiments of the present invention, the second antigen-binding region of the bispecific antibody competes for binding to TLT-1 with any of the TLT-1 antibodies listed in Table 4; has an epitope that is identical with respect to any of the antibodies listed in Table 4; comprises CDR regions that are identical with respect to any of the antibodies listed in Table 4; and has VL and VH regions that are identical with respect to any of the antibodies listed in Table 4.

[0151] Modified effector functions

[0152] The bispecific antibodies of the present invention may comprise an Fc region, which may have a wild-type amino acid sequence or may comprise amino acid substitutions that modulate the effector functions of the antibody, see, for example, (Wang et al.: Protein Cell. 9 (2018), pp. 63-73). Specific examples of Fc variants with modified effector functions are variants in which binding to Fcγ receptors is reduced. One specific example of such a variant is IgG1 containing the substitutions L234A, L235E, G237A, A330S, and P331S (residue numbering according to the EU index), with reduced affinity for certain Fcγ receptors and C1q.

[0153] A desired property of the bispecific antibodies of the present invention is a long half-life in vivo. Bispecific antibodies containing an Fc region can be recycled and stored via the FcRn receptor, which in turn can lead to the desired prolongation of the half-life. For bispecific antibodies of the present invention that lack an Fc region, the half-life can be extended by other methods. Various methods and principles for obtaining an extended half-life of polypeptides and antibodies are known in the art, see, for example, Kontermann: Expert Opinion on Biological Therapy, 16 (2016), pp. 903-915) and references therein.

[0154] In addition to Fc-based half-life extension of polypeptides and antibodies, hybridization or conjugation with albumin or albumin variants has proven effective in prolonging half-life. Another approach is the attachment of polymers such as XTEN or PEG (insert link). Furthermore, in vivo half-life extension can be achieved by attaching an albumin-binding moiety, see, e.g., Tan et al.: Current Pharmaceutical Design 24 (2018), pp. 4932–1946; Kontermann: Expert Opinion on Biological Therapy, 16 (2016), pp. 903–915) and Kontermann: Current Opinion in Biotechnology 22 (2011), pp. 868–876).

[0155] Functional features

[0156] By binding to cFVII(a), the bispecific antibodies of the present invention can prolong the active half-life of FVIIa present in the bloodstream; by binding to TLT-1, the bispecific antibodies and bound FVII(a) are targeted to the surface of activated platelets. This, in turn, leads to increased accumulation of FVIIa on activated platelets and, thus, to enhanced procoagulant activity of FVIIa at the site of vascular injury. Thus, the bispecific antibodies described herein are capable of providing the endogenous pool of FVII(a) with improved pharmacokinetic (PK) and pharmacodynamic (PD) properties.

[0157] The active half-life of administered recombinant FVIIa in humans is known to be approximately 2-3 hours. The short half-life of recombinant FVIIa is believed to be due to several mechanisms, including inhibition by antithrombin III (AT), inhibition by alpha-2-macroglobulin (a2M), and renal clearance. Similar mechanisms are believed to apply to endogenous FVIIa, thereby contributing to its similarly short half-life.

[0158] To extend the active half-life of FVIIa, including the half-life of endogenous FVIIa, the bispecific antibodies described herein aim to intercept one or more of these clearance mechanisms via their FVII arm, or so-called "first antigen-binding site," without losing endogenous FVIIa activity. The Fc portion of the biAb:FVIIa complex will mediate recycling of the complex in the endosome and by binding to FcRn, preserving it from degradation. Furthermore, the high-affinity FVII arm of the bispecific antibodies protects endogenous FVIIa from inhibition by α2M and renal clearance due to the increased molecular size of the biAb:FVIIa complex compared to the size of free endogenous FVIIa. The TLT-1 bispecific antibody arm selectively localizes endogenous FVIIa with prolonged action on activated platelets.This localization of FVIIa on activated platelets enhances FVIIa activity without increasing its sensitivity to AT inhibition.

[0159] The bispecific antibodies of the present invention can increase the mean retention time (MRT) of endogenous or exogenous FVII(a). Preferably, the bispecific antibodies are capable of enhancing FVII(a) activity in vivo.

[0160] Average retention time

[0161] The mean residence time (MRT) is the average time a molecule remains in the body available for therapeutic activity. MRT is calculated using equation 1 as a function of the volume of distribution at steady state (Vss) divided by the systemic clearance (CL).

[0162] MRT = Vss / CL Lv. 1

[0163] Results are expressed in units of time. MRT correlates with the effective plasma half-life (t 1 / 2 ) in accordance with equation 2.

[0164] MRT = ln(2)*t 1 / 2 Level 2

[0165] The ability of an antibody to increase the 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 analyzing the plasma concentration or activity profiles of FVII(a) after intravenous or subcutaneous administration to experimental animals, such as mice, rats, or monkeys. The ability of an antibody to increase the functional MRT for FVII(a) can be determined by analyzing the plasma FVII(a) activity profiles measured using assays such as, but not limited to, the FVIIa activity assay described in Example 8.

[0166] The ability of an antibody to increase MRT and functional MRT for FVII(a) can, for example, be determined as described in Examples 9 and 17.

[0167] In some embodiments, the bispecific antibodies of the present invention are capable of increasing the MRT for 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 antibody of the present invention (FVII(a) polypeptide only).

[0168] In some embodiments, the bispecific antibodies of the present invention are capable of increasing the functional MRT for FVII(a), measured using the FVIIa activity assay described in Example 8, 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.

[0169] Accumulation of endogenous FVIIa

[0170] The ability of the bispecific antibodies of the present invention to increase the level of endogenous functionally active FVIIa in the bloodstream can, for example, be determined by measuring the level of endogenous FVIIa before and after administration of the antibody to experimental animals, such as mice, rats, or monkeys, as measured by assays such as, but not limited to, the FVIIa activity assay described in Example 8.

[0171] The ability of an antibody to increase the level of endogenous functionally active FVIIa in the bloodstream can, for example, be determined as described in Examples 26 and 27.

[0172] In some embodiments, the bispecific antibodies of the present invention are capable of increasing the level of endogenous FVIIa in the bloodstream by at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 80-fold, at least 160-fold, at least 320-fold, at least 640-fold compared to the level of endogenous FVIIa in the bloodstream in the absence of the administered bispecific antibody.

[0173] TLT-1- and TF-independent thrombin formation

[0174] In one aspect, the bispecific antibodies of the present invention are capable of maintaining or increasing the TLT-1- and TF-independent ability of factor VIIa to generate thrombin.

[0175] The ability of an antibody to increase thrombin generation using a FVII(a) polypeptide can be determined by methods well known in the art, such as, for example, the thrombin generation assay described in Example 5. In this assay, thrombin generation is measured in human plasma with induced hemophilia A in the presence of phospholipids, 25 nM FVIIa, and an antibody at a concentration approaching saturation of the added FVIIa. Saturation occurs if > 90% of FVIIa is bound by the antibody in accordance with the measured dissociation constant determined for the FVIIa-antibody interaction, for example, using SPR, as illustrated in Example 6. Based on the ratio of the peak value of thrombin generation in the presence and absence of antibodies, antibodies are classified as stimulatory (> 120%), inhibitory (< 90%), or neutral (90-120%).

[0176] In some embodiments, the bispecific antibodies of the present invention are capable of maintaining (neutral) / enhancing (stimulating) the thrombin-generating ability of a factor VII(a) polypeptide, as measured in a thrombin-generating assay, compared to FVII(a) in the absence of the antibody.

[0177] In some embodiments, the bispecific antibodies of the present invention are capable of increasing the thrombin-generating capacity of factor VII(a), as measured in a thrombin generation assay, by at least 20% compared to FVII(a) in the absence of the antibody.

[0178] Inhibition by antithrombin and / or alpha-2-macroglobulin

[0179] In one aspect, the bispecific antibodies of the present invention are capable of reducing the sensitivity of FVIIa to inhibition by antithrombin (AT) and / or alpha-2-macroglobulin.

[0180] 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 the methods described in Examples 5 and 11.

[0181] In some embodiments, the bispecific antibodies of the present invention are capable of reducing the inhibition of FVIIa by antithrombin (AT) and / or alpha-2-macroglobulin compared to the inhibition of FVIIa in the absence of the antibody.

[0182] TLT-1-dependent FXa formation (stimulatory activity assay)

[0183] In one aspect, the bispecific antibodies of the present invention are capable of maintaining or increasing the ability of a FVIIa polypeptide to stimulate FX activation in the presence of a TLT-1-containing procoagulant membrane surface.

[0184] The ability of the bispecific antibody of the present invention to enhance the ability of a FVIIa polypeptide to stimulate FX activation can be determined by methods well known in the art, such as, for example, the TLT-1-dependent stimulatory activity assay described in Example 20. In this assay, FX activation is measured in the presence of FX (150 nM), FVIIa (2.5 nM), phospholipid membranes containing TLT-1 (4 nM), and an anti-FVII(a) / TLT-1 bispecific antibody. The so-called stimulatory activity of the bispecific antibody (expressed as a fold increase) is the amount of FXa formed in the presence of 100 nM of the bispecific antibody compared to the amount formed by FVIIa in the absence of the bispecific antibody.

[0185] In some embodiments, the stimulatory activity of the bispecific antibodies 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.

[0186] TLT-1-dependent thrombus formation in whole blood

[0187] In one aspect, the bispecific antibodies of the present invention are capable of stimulating thrombus formation in a manner similar to or better than a therapeutically effective concentration of recombinant FVIIa in hemophilia A conditions.

[0188] The ability of a bispecific antibody to improve thrombus formation in whole blood can be determined by methods well known in the art, such as, for example, thromboelastography, as described in Example 28. In this assay, thrombus formation is measured in whole blood of a human with induced hemophilia A, to which bispecific antibodies are added together with FVII, FVIIa and FVIIa:AT at concentrations that mimic the levels of the corresponding endogenous factors in blood plasma at steady state in vivo upon repeated administration of the bispecific antibody, as described in Examples 26, 27 and 28. Blood coagulation is induced by the addition of the PAR1 agonist peptide SFLLRN and calcium. The clotting time under these conditions is compared with the clotting time achieved by the addition of 25 nM FVIIa in the absence of antibodies.

[0189] In some embodiments, the bispecific antibodies of the present invention are capable of reducing the clotting time in human hemophilia A-induced whole blood to a level similar to or lower than that achieved by adding 25 nM FVIIa. In some embodiments, the bispecific antibodies of the present invention are capable of reducing the clotting time in human hemophilia A-induced whole blood to a level similar to or lower than that achieved by adding 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.

[0190] Pharmaceutical compositions

[0191] In one aspect, the present invention provides compositions and formulations comprising the bispecific antibodies described herein. For example, the present invention provides a pharmaceutical composition that comprises a bispecific antibody formulated together with a pharmaceutically acceptable carrier.

[0192] In one embodiment of the present invention, the pharmaceutical formulation comprises a bispecific antibody that is present at a concentration of 80 mg / ml to 200 mg / ml, such as 100-180 mg / ml, and wherein said formulation has a pH of 2.0 to 10.0. The formulation may further comprise one or more of a buffer system, a preservative, a tonicity adjusting agent, a chelating agent, a stabilizer, or a surfactant, as well as 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. Reference may be made to Remington: The Science and Practice of Pharmacy, 19 th edition, 1995.

[0193] In one embodiment, the pharmaceutical formulation is an aqueous formulation. Such a formulation is typically a solution or suspension, but may also contain colloids, dispersions, 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.

[0194] In another embodiment, the pharmaceutical formulation is a freeze-dried formulation to which solvents and / or diluents are added by the physician or patient prior to use.

[0195] In a further aspect, the pharmaceutical composition comprises an aqueous solution of a FVII(a) polypeptide, a bispecific antibody described herein, and a buffer, wherein the antibody is present at a concentration of 1 mg / ml or higher and wherein said composition has a pH of about 2.0 to about 10.0.

[0196] The composition of the present invention can be administered parenterally, for example intravenously, for example intramuscularly, for example subcutaneously; preferably subcutaneously. The composition of the present invention can be administered prophylactically.

[0197] The pharmaceutical composition of the present invention can be used to treat a subject with coagulopathy. As used herein, the term "subject" includes any human patient or non-human vertebrate animal with coagulopathy.

[0198] Medical Applications

[0199] The term "treatment" as used herein refers to the medical therapy of any human or other animal subject in need thereof. It is assumed that said subject has undergone a physical examination by a practicing physician who has made a preliminary or final diagnosis indicating that the use of said specific treatment is beneficial for the health of said human or other animal subject. The timing and purpose of said treatment may vary from one individual to another depending on the health condition of the subject. Preventive or prophylactic administration of the procoagulant compounds of the present invention is also contemplated, where prevention is defined as delaying or preventing the onset or exacerbation of one or more symptoms of a disease or disorder. Thus, said treatment may be prophylactic, palliative, symptomatic ("on-demand") and / or curative.

[0200] Within the framework of the present invention, prophylactic, palliative and / or symptomatic treatment may constitute separate aspects of the present invention.

[0201] A coagulopathy that leads to increased bleeding tendency may be caused by any qualitative or quantitative deficiency of any procoagulant component of the normal coagulation cascade or any activation of fibrinolysis. Such coagulopathies may be congenital, acquired, and / or iatrogenic and are determined by a specialist in the field.

[0202] Non-limiting examples of congenital hypocoagulopathies are hemophilia A, hemophilia B, factor VII deficiency, factor X deficiency, factor XI deficiency, von Willebrand disease, and types of thrombocytopenia such as Glanzmann thrombasthenia and Bernard-Soulier syndrome.

[0203] A non-limiting example of acquired coagulopathy is serine protease deficiency caused by vitamin K deficiency; such vitamin K deficiency can be induced by the administration of a vitamin K antagonist, such as warfarin. Acquired coagulopathy can also occur after major trauma. In this case, also known as the "vicious cycle of bleeding," it is characterized by hemodilution (thrombocytopenia and dilution of clotting factors), hypothermia, depletion of clotting factors, and metabolic disturbances (acidosis). Fluid therapy and increased fibrinolysis can exacerbate this situation. This bleeding can occur anywhere in the body.

[0204] Hemophilia A with "inhibitors" (i.e., with alloantibodies to factor VIII) and hemophilia B with "inhibitors" (i.e., with alloantibodies to factor IX) are non-limiting examples of coagulopathies that are partly congenital and partly acquired.

[0205] A non-limiting example of iatrogenic coagulopathy is coagulopathy that is caused by excessive and / or inappropriate fluid therapy, such as coagulopathy that may be caused by blood transfusion.

[0206] In one preferred embodiment of the present invention, the bleeding is associated with hemophilia A. In another preferred embodiment of the present invention, the bleeding is associated with hemophilia B. In another preferred embodiment, the bleeding is associated with hemophilia A or B with the presence of acquired inhibitors. In another preferred embodiment, the bleeding is associated with FVII deficiency. In another preferred embodiment, the bleeding is associated with Glanzmann thrombasthenia. In another embodiment, the bleeding is associated with von Willebrand disease. In another embodiment, the bleeding is associated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is associated with surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis and / or enteritis.In another embodiment, the bleeding is profuse uterine bleeding, such as placental abruption. In another embodiment, the bleeding occurs in organs with limited mechanical hemostasis, such as within the skull, ear, or eye. In another embodiment, the bleeding is associated with anticoagulant therapy.

[0207] In a further embodiment, the bleeding may be associated with thrombocytopenia. For patients with thrombocytopenia, the bispecific antibodies described herein may be administered in conjunction with platelets.

[0208] Routes of administration

[0209] 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.

[0210] Dosage regimens

[0211] The bioavailability and half-life of the bispecific antibodies described herein make them particularly attractive as prophylactic treatments via subcutaneous administration to subjects with acquired and / or congenital coagulopathy. The bispecific antibodies described herein can be administered once a week, such as once every two weeks, preferably once a month, to subjects with acquired and / or congenital coagulopathy but who do not have bleeding. The bispecific antibodies described herein can be administered prior to surgery to subjects with coagulopathy who are to undergo an invasive procedure, such as surgery. The bispecific antibodies described herein can be administered to subjects with coagulopathy and to subjects undergoing an invasive procedure, such as surgery.

[0212] The bispecific antibodies described herein can also be co-administered with exogenous FVIIa, such as plasma-derived or recombinantly produced FVIIa, for on-demand or prophylactic treatment of subjects with coagulopathy or experiencing a bleeding episode.

[0213] The dose administered to subjects with coagulopathy will depend on the route of administration, whether it is administered prophylactically or on demand, and individual characteristics. Subcutaneous administration will require higher doses than intravenous administration.

[0214] In one embodiment of the present invention, the bispecific antibody is administered subcutaneously at a dose of 1.0 nmol / kg to 30.0 nmol / kg.

[0215] Unless otherwise specified in the description, terms given in the singular also include the plural.

[0216] LIST OF IMPLEMENTATION OPTIONS

[0217] The present invention is further described by means of the following non-limiting list of embodiments of the present invention:

[0218] 1. Bispecific antibody containing

[0219] (i) a first antigen-binding region capable of binding factor VII(a), and

[0220] (ii) a second antigen-binding site capable of binding TREM-like transcript 1 (TLT-1).

[0221] 2. The bispecific antibody of embodiment 1, wherein the first antigen-binding region competes for binding to FVII(a) with any one of the FVII(a) antibodies from Table 3:

[0222]

[0223]

[0224] 3. The bispecific antibody according to embodiment 1, wherein the antibody competes according to the competitive ELISA assay described in Example 7.

[0225] 4. The bispecific antibody according to embodiment 1, wherein the first antigen-binding region is capable of binding an epitope comprising amino acid residues H115, T130, V131 and R392 of FVII(a) (SEQ ID NO: 1).

[0226] 5. The bispecific antibody according to embodiment 1, wherein the first antigen-binding region is capable of binding an epitope comprising one or more of amino acid residues H115, T130, V131 and R392 of FVII(a) (SEQ ID NO: 1).

[0227] 6. The bispecific antibody of embodiment 1, wherein the first antigen-binding portion is capable of binding an epitope comprising the following amino acid residues: R113, C114, H115, E116, G117, Y118, S119, L120, ISO, V131, N184, T185, P251, V252, V253, Q388, M391 and R392 of FVII(a) (SEQ ID NO: 1).

[0228] 7. The bispecific antibody of embodiment 1, wherein the first antigen-binding region is capable of binding an epitope comprising one or more of the following amino acid residues: R113, C114, NH3, E116, G117, Y118, S119, L120, ISO, V131, N184, T185, P251, V252, V253, Q388, M391 and R392 of FVII(a) (SEQ ID NO: 1).

[0229] 8. The bispecific antibody according to embodiment 1, wherein the first antigen-binding region comprises:

[0230] • CDRL1, presented under SEQ ID NO: 847 with 0, 1, 2 or 3 amino acid substitutions,

[0231] • CDRL2, presented under SEQ ID NO: 848 with 0, 1, 2 or 3 amino acid substitutions,

[0232] • CDRL3, presented under SEQ ID NO: 849 with 0, 1 or 2 amino acid substitutions,

[0233] • CDRH1 shown under SEQ ID NO: 851 with 0 or 1 amino acid substitution,

[0234] • CDRH2, presented under SEQ ID NO: 852 with 0 or 1 amino acid substitution,

[0235] • CDRH3 shown as SEQ ID NO: 853 with 0, 1, 2 and 3 amino acid substitutions.

[0236] 9. The bispecific antibody according to embodiment 1, wherein the first antigen-binding region comprises:

[0237] • CDRL1, represented by SEQ ID NO: 847,

[0238] • CDRL2, represented by SEQ ID NO: 848,

[0239] • CDRL3, represented by SEQ ID NO: 849,

[0240] • CDRH1, presented under SEQ ID NO: 851,

[0241] • CDRH2, presented under SEQ ID NO: 852, and

[0242] • CDRH3, shown under SEQ ID NO: 853.

[0243] 10. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises heavy chain variable domain and light chain variable domain sequences according to the antibody listed in Table 13B or 14, wherein the affinity (K D ) is 1 nM or less.

[0244] 11. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises heavy chain variable domain and light chain variable domain sequences according to an antibody listed in Table 13B or 14, wherein the affinity (K D ) is 5 nM or less.

[0245] 12. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises heavy chain variable domain and light chain variable domain sequences according to an antibody listed in Table 13B or 14, wherein the affinity (K D ) is 10 nM or less.

[0246] 13. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises heavy chain variable domain and light chain variable domain sequences according to an antibody listed in Table 13B or 14, wherein the affinity (K D) is 25 nM or less.

[0247] 14. The bispecific antibody of any of the previous embodiments, wherein the antibody is characterized by an affinity (K D ) in relation to FVII(a), which is less than 1 pM, such as less than 10 pM, such as less than 100 pM, such as less than 200 pM, such as less than 400 pM, such as less than 600 pM, such as less than 1 nM, such as less than 5 nM, such as less than 10 nM, such as less than 20 nM, such as less than 50 nM, such as less than 100 nM, such as less than 200 nM, such as less than 400 nM, such as less than 600 nM, such as less than 800 nM.

[0248] 15. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises a light chain variable domain as defined by SEQ ID NO: 846 and a heavy chain variable domain as defined by SEQ ID NO: 850.

[0249] 16. The bispecific antibody of embodiment 15, wherein the sequences of the light chain variable domain and / or the heavy chain variable domain comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions.

[0250] 17. The bispecific antibody of embodiment 1, wherein the second antigen-binding region competes for binding to TLT-1 with any one of the TLT-1 antibodies from Table 4:

[0251]

[0252] 18. The bispecific antibody of embodiment 17, wherein the antibody competes according to the competitive ELISA assay described in Example 31.

[0253] 19. The bispecific antibody of embodiment 1, wherein the second antigen-binding region is capable of binding an epitope comprising the following amino acid residues: K8, 19, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, A21 of TLT-1 (SEQ ID NO: 13).

[0254] 20. The bispecific antibody according to embodiment 1, wherein the second antigen-binding region comprises:

[0255] • CDRL1, presented under SEQ ID NO: 855 with 0, 1, 2 or 3 amino acid substitutions,

[0256] • CDRL2, presented under SEQ ID NO: 856 with 0, 1 or 2 amino acid substitutions,

[0257] • CDRL3, presented under SEQ ID NO: 857 with 0, 1 or 2 amino acid substitutions,

[0258] • CDRH1 shown as SEQ ID NO: 859 with 0 or 1 amino acid substitution,

[0259] • CDRH2 shown under SEQ ID NO: 860 with 0, 1, 2 or 3 amino acid substitutions,

[0260] • CDRH3 shown as SEQ ID NO: 861 with 0 or 1 amino acid substitution.

[0261] 21. The bispecific antibody according to embodiment 1, wherein the second antigen-binding region comprises:

[0262] • CDRL1, represented by SEQ ID NO: 855,

[0263] • CDRL2, represented by SEQ ID NO: 856,

[0264] • CDRL3, represented by SEQ ID NO: 857,

[0265] • CDRH1, presented under SEQ ID NO: 859,

[0266] • CDRH2, presented under SEQ ID NO: 860,

[0267] • CDRH3, presented under SEQ ID NO: 861.

[0268] 22. The bispecific antibody according to embodiment 1, wherein the second antigen-binding region comprises:

[0269] • CDRL1, presented under SEQ ID NO: 871,

[0270] • CDRL2, represented by SEQ ID NO: 872,

[0271] • CDRL3, represented by SEQ ID NO: 873,

[0272] • CDRH1, presented under SEQ ID NO: 875,

[0273] • CDRH2, presented under SEQ ID NO: 876, and

[0274] • CDRH3, presented under SEQ ID NO: 877.

[0275] 23. The bispecific antibody according to embodiment 1, wherein the second antigen-binding region comprises:

[0276] • CDRL1, represented by SEQ ID NO: 879,

[0277] • CDRL2, represented by SEQ ID NO: 880,

[0278] • CDRL3, represented by SEQ ID NO: 881,

[0279] • CDRH1, presented under SEQ ID NO: 883,

[0280] • CDRH2, presented under SEQ ID NO: 884, and

[0281] • CDRH3, represented by SEQ ID NO: 885.

[0282] 24. The bispecific antibody according to embodiment 1, wherein the second antigen-binding region comprises:

[0283] • CDRL1, represented by SEQ ID NO: 895,

[0284] • CDRL2, represented by SEQ ID NO: 896,

[0285] • CDRL3, represented by SEQ ID NO: 897,

[0286] • CDRH1, presented under SEQ ID NO: 899,

[0287] • CDRH2, presented under SEQ ID NO: 900, and

[0288] • CDRH3, shown under SEQ ID NO: 901.

[0289] 25. The bispecific antibody of embodiment 1, wherein the second antigen-binding region consists of a light chain variable domain as defined by SEQ ID NO: 854 and a heavy chain variable domain as defined by SEQ ID NO: 858.

[0290] 26. The bispecific antibody of embodiment 25, wherein the sequences of the light chain variable domain and / or the heavy chain variable domain comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions.

[0291] 27. The bispecific antibody of any of the previous embodiments, wherein the antibody is characterized by an affinity (K D) in relation to TLT-1, which is 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.

[0292] 28. The bispecific antibody according to embodiment 1, wherein the first antigen-binding region comprises the light chain variable domain defined by SEQ ID NO: 846 and the heavy chain variable domain defined by SEQ ID NO: 850, and wherein the second antigen-binding region consists of the light chain variable domain defined by SEQ ID NO: 854 and the heavy chain variable domain defined by SEQ ID NO: 858.

[0293] 29. The bispecific antibody of any of the previous embodiments, wherein the antibody comprises an Fc region.

[0294] 30. The bispecific antibody of embodiment 29, wherein the Fc region is an Fc variant of an IgG1 Fc region comprising the substitutions L234A, L235E, G237A, A330S and P331S.

[0295] 31. A bispecific antibody according to any of the previous embodiments, wherein the antibody is a hybrid molecule or a conjugate of antigen-binding fragments of antibodies.

[0296] 32. The bispecific antibody of embodiment 31, wherein one or more binding fragments are selected from the group of Fab, Fab', Fab2, Fab'2 and scFv.

[0297] 33. The bispecific antibody of any of the previous embodiments, wherein the antibody is a multispecific antibody.

[0298] 34. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises a first light chain variable domain defined by SEQ ID NO: 846 and a first heavy chain variable domain defined by SEQ ID NO: 850, and wherein the second antigen-binding region consists of a second light chain variable domain defined by SEQ ID NO: 854 and a second heavy chain variable domain defined by SEQ ID NO: 858, and wherein the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 5 and 4, respectively, and wherein both light chain constant domains attached to the first and second light chain variable domains are defined by SEQ ID NO: 12.

[0299] 35. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises a first light chain variable domain defined by SEQ ID NO: 846 and a first heavy chain variable domain defined by SEQ ID NO: 850, and wherein the second antigen-binding region consists of a second light chain variable domain defined by SEQ ID NO: 854 and a second heavy chain variable domain defined by SEQ ID NO: 858, and wherein the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 943 and 942, respectively, and wherein both light chain constant domains attached to the first and second light chain variable domains are defined by SEQ ID NO: 12

[0300] 36. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises a first light chain variable domain defined by SEQ ID NO: 846 and a first heavy chain variable domain defined by SEQ ID NO: 850, and wherein the second antigen-binding region consists of a second light chain variable domain defined by SEQ ID NO: 854 and a second heavy chain variable domain defined by SEQ ID NO: 858, and wherein the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 7 and 6, respectively, and wherein both light chain constant domains attached to the first and second light chain variable domains are defined by SEQ ID NO: 12.

[0301] 37. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises a first light chain variable domain defined by SEQ ID NO: 846 and a first heavy chain variable domain defined by SEQ ID NO: 850, and wherein the second antigen-binding region consists of a second light chain variable domain defined by SEQ ID NO: 854 and a second heavy chain variable domain defined by SEQ ID NO: 858, and wherein the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 4 and 5, respectively, and wherein both light chain constant domains attached to the first and second light chain variable domains are defined by SEQ ID NO: 12.

[0302] 38. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises a first light chain variable domain defined by SEQ ID NO: 846 and a first heavy chain variable domain defined by SEQ ID NO: 850, and wherein the second antigen-binding region consists of a second light chain variable domain defined by SEQ ID NO: 854 and a second heavy chain variable domain defined by SEQ ID NO: 858, and wherein the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 942 and 943, respectively, and wherein both light chain constant domains attached to the first and second light chain variable domains are defined by SEQ ID NO: 12.

[0303] 39. The bispecific antibody of embodiment 1, wherein the first antigen-binding region comprises a first light chain variable domain defined by SEQ ID NO: 846 and a first heavy chain variable domain defined by SEQ ID NO: 850, and wherein the second antigen-binding region consists of a second light chain variable domain defined by SEQ ID NO: 854 and a second heavy chain variable domain defined by SEQ ID NO: 858, and wherein the heavy chain constant domains attached to the first and second heavy chain variable domains are defined by SEQ ID NO: 6 and 7, respectively, and wherein both light chain constant domains attached to the first and second light chain variable domains are defined by SEQ ID NO: 12.

[0304] 40. The bispecific antibody of embodiment 1, wherein the antibody competes for binding to FVII(a) and competes for binding to TLT-1 with one or more of the bispecific antibodies set forth in Table 5 (wherein the abbreviation for the constant domain is defined in accordance with Table 2a):

[0305]

[0306]

[0307] 41. The bispecific antibody of 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 of the bispecific antibodies set forth in Table 5.

[0308] 42. The bispecific antibody of embodiment 38, wherein the antibody competes according to the competitive ELISA assay described in Example 7 and Example 31.

[0309] 43. The bispecific antibody of embodiment 1, wherein the first antigen-binding region is capable of binding an epitope comprising amino acid residues H115, T130, V131 and R392 of FVII(a) (SEQ ID NO: 1), and wherein the second antigen-binding region is capable of binding an epitope comprising the following amino acid residues: K8, 19, G10, S11, L12, A13, N15, A16, F17, S18, D19, P20, A21 of TLT-1 (SEQ ID NO: 13).

[0310] 44. The bispecific antibody according to embodiment 1, wherein the first antigen-binding region comprises:

[0311] • CDRL1, represented by amino acid residues (SEQ ID NO: 847) with 0, 1, 2 or 3 amino acid substitutions,

[0312] • CDRL2, represented by amino acid residues (SEQ ID NO: 848) with 0, 1, 2 or 3 amino acid substitutions,

[0313] • CDRL3, represented by amino acid residues (SEQ ID NO: 849) with 0, 1 or 2 amino acid substitutions,

[0314] • CDRH1, represented by amino acid residues (SEQ ID NO: 851),

[0315] • CDRH2, represented by amino acid residues (SEQ ID NO: 852) with O or 1 amino acid substitution,

[0316] • CDRH3, represented by amino acid residues (SEQ ID NO: 853) with 0, 1, 2 and 3 amino acid substitutions, and

[0317] wherein the second antigen-binding region comprises:

[0318] • CDRL1, presented under SEQ ID NO: 855 with 0, 1, 2 or 3 amino acid substitutions,

[0319] • CDRL2, presented under SEQ ID NO: 856 with 0, 1 or 2 amino acid substitutions,

[0320] • CDRL3, presented under SEQ ID NO: 857 with 0, 1 or 2 amino acid substitutions,

[0321] • CDRH1 shown as SEQ ID NO: 859, containing 0 or 1 amino acid substitution

[0322] • CDRH2 shown under SEQ ID NO: 860 with 0, 1, 2 or 3 amino acid substitutions,

[0323] • CDRH3 shown as SEQ ID NO: 861 with 0 or 1 amino acid substitution.

[0324] 45. The bispecific antibody of any of the previous embodiments, wherein the antibody increases the functional MRT for FVII(a), as measured by the FVIIa activity assay described in Example 8.

[0325] 46. The bispecific antibody of embodiment 41, wherein the antibody increases the functional MRT for 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.

[0326] 47. The bispecific antibody of any of the previous embodiments, wherein the antibody is capable of increasing the level of endogenous functionally active FVIIa in the bloodstream, as determined according to Examples 26 and 27, compared to the level of endogenous FVIIa in the bloodstream in the absence of the administered bispecific antibody.

[0327] 48. The bispecific antibody of embodiment 45, wherein the antibody increases the level of endogenous functionally active FVIIa in the bloodstream by at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 80-fold, at least 160-fold, at least 320-fold, at least 640-fold.

[0328] 49. The bispecific antibody of any of the previous embodiments, wherein the antibody is capable of increasing the ability of a FVIIa polypeptide to stimulate FX activation, as determined by the stimulatory activity assay described in Example 20.

[0329] 50. The bispecific antibody of embodiment 43, wherein the stimulatory activity is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 60-fold, at least 80-fold, at least 100-fold, or at least 150-fold.

[0330] 51. A pharmaceutical composition comprising a bispecific antibody according to any of the previous embodiments and a pharmaceutically acceptable carrier.

[0331] 52. The bispecific antibody of embodiments 1-48, wherein the pharmaceutical formulation of embodiment 49 is administered subcutaneously.

[0332] 53. The bispecific antibody according to embodiments 1 to 48 or the pharmaceutical formulation according to embodiment 49 for use as a medicament.

[0333] 54. A bispecific antibody according to any one of embodiments 1-48 and 50-51 or a pharmaceutical composition according to embodiment 49 for use in the treatment of coagulopathy, wherein said coagulopathy is congenital and / or acquired.

[0334] 55. The bispecific antibody of embodiment 52, wherein said coagulopathy is selected from the group consisting of hemophilia A with or without inhibitors or hemophilia B with or without inhibitors, FVII(a) deficiency, and Glanzmann's thrombasthenia.

[0335] 56. The bispecific antibody of embodiments 1-48 and 50-51 for the treatment of bleeding, wherein the bleeding is caused by congenital or acquired hemophilia A, congenital or acquired hemophilia B, hemophilia A with inhibitors, hemophilia B with inhibitors, or factor VII(a) deficiency.

[0336] 57. The use according to any one of embodiments 1-48, wherein said bispecific antibody or pharmaceutical formulation according to embodiment 49 is administered parenterally, for example intravenously, intramuscularly or subcutaneously.

[0337] EXAMPLES

[0338] Example 1. Production of mouse monoclonal antibodies to FVII(a) using hybridoma technology

[0339] Monoclonal antibodies were generated by immunizing NMRCF1 mice (Charles River) with the disulfide-linked FVIIa Q64C-sTF (1-219) G109C complex described in International Patent Application Publication Number WO 07 / 115953.

[0340] Mice received an initial subcutaneous injection of 20 μg in FCA (complete Freund's adjuvant), followed by booster intraperitoneal injections of 20 μg of antigen in IFA (incomplete Freund's adjuvant). Spleens were removed under aseptic conditions and dispersed into single-cell suspensions. Splenocytes were fused with X63Ag8.653 myeloma cells by electrofusion.

[0341] Cells were seeded in microtiter plates and cultured at 37°C, 5% CO2. Tissue culture medium (RPMI 1640 + 10% fetal bovine serum) containing HAT (Sigma) for selection was changed twice. After 10 days of growth, hybridoma clones producing specific antibodies were identified by ELISA screening using the following protocol. NUNC Maxisorb plates were coated with disulfide-linked FVIIa Q64C-sTF(1 219) G109C complex or FVIIa (expressed and purified as described by Thim et al. (1988) Biochemistry 27:7785–7793 and Persson et al. (1996) FEBS Lett 385:241–243) at 50 μl / well at 1 μg / ml (HEPES buffer containing 5 mM CaCl2) and incubated overnight at 4°C. Plates were washed 5 times and blocked with wash buffer for 15 min (HEPES buffer, 5 mM CaCl2, 0.05% TWEEN 20). 50 μl of supernatant was transferred to each well and incubated for 1 h.The plates were washed five times, and 50 μl of HRP-labeled goat anti-mouse IgG (specific for the Fc-gamma fragment; Jackson, working dilution 1 / 10,000) were added. The plates were incubated for 1 h, washed five times, and developed with 50 μl of TMB (TMB ONE, ready-to-use; Kem-En-Tec) for 10 min. The reaction was stopped by adding 50 μl of 4 M H3PO4 and read on a FLUOStar Optima microplate reader at 450 nM and 620 nM.

[0342] Hybridoma cells that yielded positive results were subcloned at least twice by limiting dilution to maintain monoclonality. Antibody purification was performed using standard protein A purification.

[0343] Hybridoma cells producing antibodies for use in RK studies were grown in T-flasks or shake flasks in RPMI1640 medium + 10% FBS. The conditioned medium was collected by centrifugation, and the antibodies were purified by protein A-based affinity chromatography followed by desalting.

[0344] Example 2. Cloning and sequencing of mouse antibodies to FVII(a)

[0345] This example describes the cloning and sequencing of the murine heavy chain (HC) and light chain (LC) cDNA sequences encoding the variable domains of an antibody to FVII(a).

[0346] Total RNA was extracted from hybridoma cells using the RNeasy-Mini Kit from Qiagen and used as a template for cDNA synthesis. cDNA was synthesized in a 5'-RACE reaction using the SMARTer RACE cDNA Amplification Kit from Clontech. Subsequent targeted amplification of the HC variable domain (designated VH) and LC variable domain (designated VL) sequences was performed by PCR using Phusion Hot Start polymerase (Finnzymes) and the universal primer mix (UPM) included in the SMARTer™ RACE kit as the forward primer. The reverse primer sequence used for VH amplification was 5' agctgggaaggtgtgcacac 3'. The reverse primer sequence used for VL amplification was 5' gctctagactaacactcattcctgttgaagctcttg 3'.

[0347] PCR products were separated by gel electrophoresis, extracted using the GFX PCR DNA & Gel Band Purification Kit from GE Healthcare Bio-Sciences, and cloned for sequencing using the Zero Blunt TOPO PCR Cloning Kit and chemically competent E. coli TOPO (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.

[0348] Example 3. Recombinant expression of bivalent antibodies, monovalent antibodies (OA antibodies) and Fab fragments of antibodies

[0349] Bivalent antibodies, monovalent antibodies (single-armed, called OA antibodies), and Fab fragments of antibodies were expressed by transient transfection of a HEK293 cell suspension (293Expi, Invitrogen), essentially following the manufacturer's instructions. 293Expi cells were passaged every 3-4 days in Expi293F expression medium (Invitrogen, cat. no. A1435104) supplemented with 1% P / S (GIBCO, cat. no. 15140-122). Expi293F cells were transfected at a cell density of 2.5-3 million / mL using Expifectamine. For each liter of Expi293F cells, transfection was performed by diluting a total of 1 mg of plasmid DNA in 50 ml of Optimem (GIBCO, cat. no. 51985-026, dilution A) and by diluting 2.7 ml of Expifectamine in 50 ml of Optimem (dilution B). Bivalent antibodies were generated by cotransfection of VH-CH1-CH2-CH3 (HC) and VL-CL (LC) plasmids (in a 1:1 ratio), and for Fab fragments, the plasmids were VH-CH1 and LC (in a 1:1 ratio).To produce OA antibodies, cells were transfected with three plasmids: HC and LC plasmids and a third plasmid encoding a truncated HC (trHC). HC from OA antibodies contained hole mutations (T366S, L368A, Y407V), and trHC contained a knob mutation (T366W), but knobs and holes can also be reversible. Knob-in-hole mutations are described in international patent EP0979281B1 and were introduced to optimize the formation of the desired heterodimer, i.e., the pairing of HC and trHC, and to suppress the undesirable formation of homodimers, i.e., the pairing of trHC with trHC and HC with HC. Dilutions A and B were mixed and incubated at room temperature for 10-20 minutes. The transfection mixture was then added to Expi293F cells, and the cells were incubated at 37°C in a humidified rotary incubator (85-125 rpm). One day after transfection, 5 ml of ExpiFectamine 293 Transfection Enhancer 1 and 50 ml of ExpiFectamine 293 Transfection Enhancer 2 were added to the transfected cells.The supernatant of cell cultures was collected 4-5 days after transfection by centrifugation followed by filtration.

[0350] Bivalent and monovalent antibodies were purified using standard Protein A-based affinity chromatography known to those skilled in the art, with additional purification steps such as gel filtration or ion exchange chromatography where necessary. Fab fragments were purified by affinity chromatography using an affinity resin that recognizes the Fab kappa chain.

[0351] Example 4. Production of bispecific antibodies and OA antibodies by in vitro assembly

[0352] Bispecific antibodies produced by in vitro assembly

[0353] Bispecific antibodies were produced by in vitro assembly of two parental antibodies using a method similar to that described in Labrijn et al. PNAS 2013, vol. 110, pp. 5145-5150, known as DuoBody® technology (Genmab). Instead of the IgG1 subtype used by Labrijn et al., IgG4 was used in the present invention. The exchange reaction was performed at 30°C for four hours in the presence of 75 mM 2-mercaptoethylamine (2-MEA). The resulting bispecific antibody was purified by ion exchange chromatography to separate residual parental antibodies from the bispecific antibody. In this example, the heavy chain constant region of the first parental antibody (anti-FVII(a)) was IgG4 S228P F405L R409K, and the heavy chain constant region of the second parental antibody (anti-TLT-1) was IgG4 S228P (EU numbering was used in both cases). The light chain constant domains were human kappa chain.Two parent antibodies were prepared as described in Example 3. Bispecific antibodies to FVII(a) / TLT-1 can also be assembled from a set of parent antibodies, where the constant domain of the antibody to FVII(a) is IgG4 S228P and the constant domain of the antibody to TLT-1 is IgG4 S228P F405L R409K.

[0354] Monovalent antibodies produced by in vitro assembly

[0355] Monovalent antibodies were prepared by in vitro assembly as described above for bispecific antibodies, except that (1) instead of combining two antibodies to form a bispecific antibody, an antibody and a trHC dimer were combined to form a monovalent antibody. In this example, trHC was IgG4 S228P F405L R409K, and HC was IgG4 S228P (EU numbering was used in both cases). The light chain constant domain was the human kappa chain. Typically, the arm exchange reaction was performed using the trHC dimer in a 20–50% molar excess to minimize the amount of bivalent antibody in the reaction mixture. The monovalent antibody was purified by size exclusion chromatography, optionally with additional purification steps such as ion exchange chromatography.

[0356] Example 5. Characterization of FVIIa(a) antibody in in vitro functional assays

[0357] In order to facilitate the accumulation of endogenous FVIIa and allow it to exhibit procoagulant activity, the anti-FVII(a) antibodies of the present invention should preferably not impair the activity of FVIIa and similarly should preferably not promote the inactivation of FVIIa by its primary plasma inhibitor antithrombin (Agersø H, et al. (2011) J Thromb Haemost 9:333 338). To examine these aspects, the effects of anti-FVII(a) antibodies on the procoagulant activity of FVIIa and the inactivation of FVIIa by antithrombin were determined in vitro using the assays described below.

[0358] The effect of antibodies to FVII(a) on thrombin formation in human blood plasma with induced hemophilia A

[0359] The effect of bivalent or monovalent anti-FVII(a) antibodies on thrombin generation was measured using a kaolin-activated thrombin generation (TGT) assay in a 96-well format. Briefly, hemophilia A-induced plasma was prepared by adding the anti-hFVIII antibody 4F30 (described in International Patent Application Publication Number WO 2012 / 035050) to a final concentration of 37.5 μg / mL. Phospholipids at a final concentration of 10 μM (Rossix) were added to the hemophilia A plasma and incubated at 37°C for 15 min. Purified antibodies (100 nM) and FVIIa (25 nM) were added to the mixture, and the mixture was incubated for 10 min at room temperature. Antibodies to FVII(a) were prepared as described in Examples 1, 2 and 3.Stimulation was performed by adding 10 µl kaolin (Haemonetics) followed by 10 µl FIIa FluCa-kit (Thrombinoscope BV) and fluorescence was measured (excitation at 390 nM and emission at 460 nM) on an EnVision multichannel reader for two hours.

[0360] Thrombograms were calculated as the first derivative of the measured fluorescence. The thrombin peak height was calculated as the maximum value in the thrombogram. It was subsequently normalized and expressed as a percentage by dividing the observed peak height by the peak height corresponding to the height observed for 25 nM FVIIa in the absence of antibody. Based on this, antibodies were classified as stimulatory (>120%), inhibitory (<90%), or neutral (90-120%). As shown in Table 6, antibodies of all categories were determined. Stimulatory or neutral antibodies were preferred during the TOT assay.

[0361] The effect of antibodies to FVII(a) on the inactivation of FVIIa by antithrombin from blood plasma

[0362] The inhibition of FVIIa activity by plasma antithrombin (AT) in the presence of bivalent or monovalent anti-FVII(a) antibody was measured by incubating FVIIa (200 nM) with low molecular weight heparin (Enoxaparin, 12 μM) and antibody (200-1000 nM) for 10 min in 50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 0.1% PEG8000, 1 mg / ml BSA, pH 7.3. Then, AT (5 μM) was added, and at time points of 10, 20, 30, 40, 60, and 80 min. The sample was transferred to a new microtiter plate, where residual activity was measured in the presence of 1 mM chromogenic substrate S-2288 (Chromogenix), 200 nM soluble tissue factor (sTF; prepared as described in Freskgard et al. (1996) Protein Sci 5: 1531 1540), and polybrene (0.5 mg / mL) in a Spectramax instrument (Molecular Devices) at 405 nM for 5 min. A sample containing buffer instead of AT provided FVIIa activity without inhibition.

[0363] Residual amidolytic activity was determined as a function of time relative to activity in the absence of inhibitor. The inhibition constant (kinh) was estimated by fitting the data to a single-phase decay model. The kinh value in the presence of an anti-FVII(a) antibody as a percentage of the value determined in the absence of the antibody is called kinh% and is given for each antibody in Table 6.

[0364] Antibodies with a calculated kinh% value < 60% were classified as protecting FVIIa from inhibition by AT. Antibodies with a calculated kinh% value 60% ≤ kinh% ≤ 150% were classified as neutral, while antibodies with a calculated kinh% value > 150% were classified as accelerating FVIIa inhibition by AT. As shown in Table 6, antibodies from all three categories were determined. Neutral antibodies or antibodies that protect FVIIa from inhibition by AT were preferred.

[0365] Among the antibodies tested, a subgroup of antibodies including 11F2 (mAb005 and mAb0048, corresponding to the full mouse and mouse / human chimera, respectively) was found to have the required in vitro properties as described above.

[0366] Table 6. Functional characterization of antibodies to FVII(a) in thrombin generation (TGT) and antithrombin inhibition (AT) assays as described in Example 2

[0367]

[0368] Example 6. SPR analysis of antibody binding to FVIIa and the effect of pH and calcium

[0369] The binding of the antibodies from Example 5 to FVIIa was studied using surface plasmon resonance (Biacore T200). Anti-mouse IgG (GE Healthcare) was immobilized on the CM4 sensor chip using a standard amine immobilization chemistry kit (both from GE Healthcare). Purified anti-FVII(a) antibodies according to Table 7 (0.25 nM) were injected at 10 μl / min for 1 min. Subsequently, 5 nM, 15 nM, 45 nM, and 135 nM FVIIa were injected at 30 μl / min for 7 min. to allow binding to the anti-FVII(a) antibody, followed by a 9-min injection of buffer to allow dissociation from the anti-FVII(a) antibody. The working buffer was prepared by 10-fold dilution of 10x HBS-P buffer (supplied by GE Healthcare) with 1 mg / mL BSA and 5 mM CaCl2 to obtain 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20 (P20), pH 7.4, 5 mM CaCl2, 1 mg / mL bovine serum albumin (BSA).The running buffer was also used to dilute the anti-FVII(a) antibody samples and the FVII samples. The chip was regenerated using a regeneration buffer consisting of 10 mM Gly-HCl, pH 1.7 (supplied by GE Healthcare). Binding data were analyzed according to a 1:1 model using BiaEvaluation 4.1, provided by the manufacturer (Biacore AB, Uppsala, Sweden). This analysis yielded the binding constants presented in Table 7, demonstrating high-affinity binding of several antibodies to FVIIa, including 11F2 (mAb005 and mAb0048, corresponding to the fully murine antibody and the murine / human chimeric antibody, respectively).

[0370] Table 7A. Calculated binding constants for the interaction of FVII(a) antibodies with FVIIa, determined by surface plasmon resonance (SPR) according to Example 6

[0371]

[0372]

[0373] The effect of pH and CaCl2 on the binding of selected antibodies from Example 5 to FVIIa and cFVIIa chimera was investigated by surface plasmon resonance (Biacore T200) at 37°C. The sequence of the cFVIIa chimera is described in the section entitled Detailed Description of the Invention and was expressed as described in Examples 16 and 25. Anti-mouse IgG (from GE Healthcare) was immobilized on a CM4 sensor chip using a standard amine immobilization chemistry kit (both from GE Healthcare). A pre-equilibrated mixture of FVIIa at a concentration of 1.2 nM and 4F9 (a new internal name for a mouse anti-FVII antibody that binds to the EGF1 domain of FVIIa) at a concentration of 0.5 nM was injected at a rate of 10 μl / min for 1 min. Then 540 nM, 180 nM, 60 nM, 20 nM, 6.66 nM, 2.22 nM, 0.74 nM, 0.25 nM anti-FVII(a) antibody were injected at a rate of 30 μl / min for 7 min.to allow binding to FVIIa, followed by a 9-min injection of buffer to allow dissociation from FVIIa. Two working buffers were prepared. Buffer 1 was prepared by 10-fold dilution with 10x HBS-P+ (supplied by GE Healthcare) and addition of 1 mg / mL BSA and 5 mM CaCl2 to yield 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 7.4, 5 mM CaCl2, 1 mg / mL bovine serum albumin (BSA). Buffer 2 was prepared by 10-fold dilution with 10x HBS-P+ (supplied by GE Healthcare) and addition of 1 mg / ml BSA, 5 μM CaCl2, and adjusted to pH 6.0 (with 4 M HCl) to obtain 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 6.0, 5 μM CaCl2, 1 mg / ml bovine serum albumin (BSA). FVIIa, anti-FVII antibody 4F9, and anti-FVII(a) antibody were diluted in both working buffers separately. Chip regeneration was performed using regeneration buffer consisting of 10 mM Gly-HCl, pH 1.7 (supplied by GE Healthcare).Binding data were analyzed according to a 1:1 model using BiaEvaluation 4.1, provided by the manufacturer (Biacore AB, Uppsala, Sweden). This analysis yielded the binding constants presented in Table 7B, demonstrating maintained high-affinity binding between FVIIa and the anti-FVII(a) antibody.

[0374] Table 7B Calculated binding constants and fold differences for the interaction of FVIIa and anti-FVII(a) antibodies in two different buffers, determined by surface plasmon resonance (SPR) according to Example 6

[0375]

[0376] Example 7. Determination of antibodies competing with mAb0005 (11F2) for binding to FVII(a) in a competitive ELISA

[0377] To determine whether anti-FVII(a) antibodies with the desired properties from Example 5 compete with mAb0005 (11F2) and antibodies derived from it for binding to FVIIa(a) in vitro, competition studies were performed with the corresponding Fab fragment, Fab0076. FVIIa, FVIIa with the active site inhibited by HD-Phe-Phe-Arg-chloromethyl ketone (FFR-cmk; Bachem, Switzerland) (FVIIai; see Example 9), was immobilized on a NUNC maxisorp plate at a concentration of 125 ng / mL in dilution buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, pH 7.2) overnight at 4°C. Plates were washed and blocked with wash buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, 0.5 ml / l Tween 20, pH 7.2) for 15 min. 11F2-Fab0076 was biotinylated using a standard biotinylation kit (EZ-Link, Thermo) applied according to the manufacturer's instructions.For the competition assay, biotinylated Fab0076 at a final fixed concentration of 10 ng / mL was combined with a dilution series of anti-FVIIa(a) antibody to obtain final concentrations ranging from 100 mg / mL to 9.5 ng / mL in dilution buffer. The mixture was added to the wells of the 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 treated with TMB ONE (KEMENTEC) for 10 min. The reaction was stopped by adding H3P04 (4 M) and the plate was read on a FLUOStar Optima plate reader at 450 nM with background signal subtraction measured at 620 nM. Unless otherwise stated, all incubations were performed at room temperature, and the plates were washed 5 times with wash buffer.

[0378] From the measured signals (optical density [OD] units), competition at any given antibody concentration was calculated as % inhibition = (1 - (OD units - 100% inhibition value) / (0% inhibition value - 100% inhibition value)) * 100, where 0% inhibition was defined as the signal in wells without any competing FVII(a) antibody, and 100% inhibition was defined as the signal from wells without biotinylated Fab0076 (i.e., corresponding to the assay background). Antibodies were considered to compete with 11F2 (mAb0005) for binding to FVII(a) if at least 50% inhibition (% inhibition) was observed when antibody concentrations were tested in up to a 10,000-fold excess of biotinylated Fab0076. Results are shown in Table 8.

[0379] Table 8. Competition with 11F2-Fab0076 (mAb0005) for binding to FVII(a) As described in Example 7, a cutoff value of 50% was used to classify antibodies into competitor or non-competitor categories.

[0380]

[0381] Example 8. FVIIa activity assay

[0382] FVIIa activity was measured using the reagents from the STAclot®VIIa-rTF kit (Diagnostica Stago), essentially as described by Morissey JH et al. Blood, 1993; 81: 734-44. The assay was performed on an ACLTOP500 automated coagulometer from Instrumentation Laboratory. The assay used 40 µl of diluted animal plasma, a calibrator (recombinant FVIIa reference material (rFVIIa), calibrated against the WHO international standard) or a quality control (QC) sample mixed with 40 µl of FVII-deficient plasma and 40 µl of soluble tissue factor (sTF) with phospholipids. To initiate the reaction, 25 mM CaCl2 was added in a volume of 40 µl, and the clotting time was measured on the instrument. Sample clotting time and QC were compared with rFVIIa calibration curves with the same plasma concentration as in the diluted sample to reduce the influence of plasma.The calibration curve range was 5–1000 mIU / mL and was approximated using a third-degree polynomial equation. Results for quality control and samples were calculated using software on the ACLTOP500 instrument. Results expressed in IU / mL were converted to nM using the specific activity (IU / pmol) of the injected compounds.

[0383] Example 9. Pharmacokinetics in rats of recombinant FVIIa in a co-formulation with antibodies to FVII(a)

[0384] The anti-FVII(a) monoclonal antibodies from Example 5 with the desired in vitro properties were administered intravenously to male Sprague-Dawley rats in combination with 20 nmol / kg human FVIIa. The molar ratio of FVIIa to antibody was 1:1 or 1:5, as indicated in Table 9. Animals were given free access to food and water throughout the experiment. Plasma FVIIa activity was measured using the FVIIa activity assay described in Example 8.

[0385] Pharmacokinetic analysis was performed using noncompartmental methods using WinNonlin. The mean residence time (MRT) was calculated from the data. The results are presented in Table 9.

[0386] Among the antibodies with the desired properties in Example 5, mAb0005 (11F2) and mAb0001l (11F26) conferred the longest average retention time to human FVIIa, at 7.9 and 7.5 hours, respectively, compared to the average free retention time of 1.1 hours for free human FVIIa (see Table 9). MAb0005 and mAb0001 (mAb0759) are high-affinity antibodies (Example 6) that compete for binding to FVIIa (Example 7).

[0387] Table 9. Mean retention time (MRT) of FVIIa activity in plasma of Sprague-Dawley rats after i.v. dose of 20 nmol / kg FVIIa at a molar ratio of 1:1 or 1:5 with antibody, as indicated. Vehicle medium indicates that FVIIa was administered in combination with buffer instead of antibody.

[0388]

[0389] Example 10. Effect of 11F2 antibody to FVII(a) on FX activation by free or TF-bound FVIIa

[0390] Preferably, the anti-FVII(a) antibodies of the present invention do not interfere with the pharmacological activity of FVIIa on the membrane surface or with the initiation of blood coagulation, i.e., the activation of FX by the FVIIa-TF complex. To investigate these aspects, the effect of 11F2 on the TF-independent and TF-dependent proteolytic activity of FVIIa was determined in the absence and presence of lipidated TF, respectively. The single-arm monovalent antibody format was used to prevent avidity effects arising from the simultaneous binding of two FVIIa molecules to a normal bivalent antibody.

[0391] Effect of antibody 11F2 on FX activation by FVIIa in the presence of a phospholipid membrane

[0392] 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, O, or 200 nM antibody (see Table 10) and 25 μM 25:75 phosphatidylserine:phosphatidylcholine vesicles (Haematologic Technologies Inc.). The reaction was initiated by adding 0-300 nM factor X (FX) from human plasma and incubated for 20 min at room temperature in a total reaction volume of 100 μL in a 96-well plate (n = 2). After incubation, the reaction was quenched by adding 50 μl of quenching buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 80 mM EDTA, pH 7.3), followed by 50 μl of 1 mM chromogenic substrate S-2765 (Chromogenix) in water. The conversion of the chromogenic substrate by the formed FXa was measured as the slope of the linear increase in absorbance at 405 nM over 10 min using a Spectramax microplate reader.By comparing the measured slopes with those obtained under similar conditions with known amounts of human FXa from plasma, the initial rates of FXa formation can be estimated on a molar basis. The kinetic parameters of the enzyme were estimated by nonlinear curve fitting to the data using the Michaelis-Menten equation (v = kcat * [FX] * [FVIIa-TF] / (Km + [FX])) using GraphPad Prism.

[0393] Table 10 shows that monovalent 11F2 did not affect the rate of FX activation by free FVIIa in the presence of phospholipid vesicles.

[0394] Effect of 11F2 antibody on FX activation by FVIIa in the presence of TF

[0395] 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 antibodies (see Table 10), and 2 pM of the E. coli-derived TF 1-244 fragment incorporated into 25:75 phosphatidylserine:phosphatidylcholine (PS:PC) vesicles as described by Smith and Morrissey (2005) J. Thromb. Haemost, 2:1155–1162. The reaction was initiated by adding 0.30 nM factor X (FX) from human plasma and incubated for 20 min at room temperature in a total reaction volume of 100 µl in a 96-well plate (n = 2). After incubation, the reaction was quenched and FXa was quantified as described above.

[0396] Table 10 shows that monovalent 11F2 (mAb0077(OA)) did not affect the activation rate of FX by the FVIIa / TF complex.

[0397] Table 10. Effect of 200 nM 11F2 in monovalent format on the kinetic parameters of FX activation by the FVIIa-TF complex according to Example 10 Parameters presented as mean ± standard deviation (n = 3) were estimated by fitting to the Michaelis-Menten equation. k cat / K M shown relative to the value determined in the absence of antibody

[0398]

[0399] Example 11. Effect of antibody 11F2 on FVIIa inhibition by plasma inhibitors FVIIa exhibits a relatively short half-life in the bloodstream, partly due to its inactivation by the abundant plasma inhibitor antithrombin (AT). Similarly, animal studies have shown that another plasma inhibitor, alpha-2-macroglobulin, is involved in FVIIa inactivation. Using purified inhibitors obtained from plasma, the effect of the monovalent antibody 11F2 on FVIIa inactivation by these inhibitors was investigated.

[0401] Effect of 11F2 antibody on FVIIa inhibition by antithrombin

[0402] FVIIa inhibition by human plasma-derived Ab in the presence of monovalent antibody 11F2 (mAb0048, mAb0077(OA)) was performed under apparent first-order conditions. The assay was performed in a volume of 200 μl in assay buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, 0.1% PEG8000, 1 mg / ml BSA, pH 7.3) at room temperature containing 200 nM FVIIa, 12 μM low molecular weight heparin (Enoxaparin, European Pharmacopoeia standard, code E0180000, lot 5.0, identification number 00CK18) and 0 or 1000 nM antibody. After a 10-min 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 selected time points, 10 μl aliquots were transferred to a total volume of 200 μl containing 0.5 mg / ml polybrene (hexadimethrine bromide, Sigma, cat.(number H9268, lot SLBC8683V), 200 nM sTF, and 1 mM S-2288 (Chromogenix) were used to quench the reaction and saturate FVIIa with sTF, allowing the measurement of residual FVIIa activity by hydrolysis of the chromogenic substrate, read at 405 nM for 5 min. Residual amidolytic activity was determined as the slope of the linear reaction curves after subtraction of the blank, which were subsequently fitted to a first-order exponential decay function using GraphPad Prism software to obtain apparent first-order reaction rate constants (cAβ). The apparent second-order rate constant (kIβ) was defined as cAβ divided by the AT concentration.

[0403] Based on this assay, the rate of FVIIa inhibition in the presence of monovalent 11F2 (mAb0077(OA)) was found to be 133 ± 10 M-1 s-1 compared to 124 ± 7 M-1 s-1 for inhibition of free FVIIa.

[0404] The effect of antibody 11F2 on the inhibition of FVIIa by alpha-2-macroglobulin

[0405] Inhibition of FVIIa by human plasma-derived alpha-2-macroglobulin in the presence of 0 or 1000 nM monovalent antibody 11F2 (mAb0077(OA)) was performed under apparent first-order conditions. The assay was performed in a 100 μl volume in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% PEG8000, 0.01% Tween 80, pH 7.3) containing 200 nM FVIIa and 0 or 1000 nM antibody at room temperature. The reaction was initiated by adding 0 or 1000 nM alpha-2-macroglobulin isolated from human plasma according to Banbula et al. (2005) J. Biochem., 138:527–537. At selected time points, 10 μl aliquots were transferred to 190 μl of buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.1% PEG8000, 1 mg / ml BSA, pH 7.3) containing 200 nM sTF and 1 mM S-2288 (Chromogenix), which allowed measurement of residual FVIIa activity by hydrolysis of the chromogenic substrate, read at 405 nM for 5 min.Residual amidolytic activity was determined as the slope of the linear reaction curves after subtracting the blank, which were subsequently fitted to a first-order exponential decay function using GraphPad Prism software to obtain the apparent first-order reaction rate constants (cAr). The apparent second-order rate constant (kInh) was determined as cAr divided by the alpha-2-macroglobulin concentration.

[0406] In the absence of antibody, the apparent second-order rate constant for FVIIa inhibition by alpha-2-macroglobulin was 475±21 M-1s-1. However, in the presence of the monovalent antibody 11F2 (mAb0077(OA)) no visible inhibition of FVIIa was observed until the final time point of 125 min. These studies suggest that 11F2 does not affect FVIIa inhibition by antithrombin but protects FVIIa from inhibition by alpha-2-macroglobulin.

[0407] Example 12. Effect of 11F2 antibody on FVII autoactivation

[0408] During vascular injury, endogenous FVII binds with high affinity to its cofactor tissue factor (TF), which is found on cells surrounding the vascular endothelium. During this process, FVII is converted to FVIIa through limited proteolysis. Activation is thought to occur through TF-mediated FVIIa-FVII transactivation, also known as autoactivation. To determine the effect of the 11F2 antibody on FVII autoactivation, FVII activation was measured in the presence of lipidated TF, FVII, a limiting concentration of FVIIa, and the monovalent 11F2 antibody.

[0409] Activity measurements were performed in assay buffer (50 mM HEPES, 100 mM NaCl, 5 mM CaCl12, pH 7.3, containing 0.1% PEG8000 and 1 mg / ml BSA) containing 2 nM FVIIa, 145 nM FVII, 0 or 200 nM monovalent antibody 11F2 (mAb0077(OA)). The reaction was initiated by adding 2 nM lipidated fragment of E. coli-derived TF 1-244 incorporated into vesicles from a 25:75 PS:PC mixture as described by Smith and Morrissey (2005) J. Thromb. Haemost, 2:1155–1162. The total reaction volume was 100 μL. At selected time points (typically 5, 10, 15, 20, 30, 40, 50, and 60 min), the FVIIa formed was quantified according to the subsampling procedure described below.

[0410] FVIIa quantification by subsampling: At selected time points, 10 μL of samples were quenched by transferring them to 140 μL of a mixture of 50 mM HEPES, 100 mM NaCl, 5 mM EDTA containing 0.1% PEG8000, 1 mg / mL BSA, and 215 nM soluble tissue factor (sTF). After collecting all samples, FVIIa chromogenic activity was measured by adding 50 μL of S-2288 (4 mM) in 60 mM CaCl2. The conversion of the chromogenic substrate by the formed FVIIa was measured as the slope of the linear increase in absorbance at 405 nM over 10 min using a Spectramax plate reader. By comparing the measured slopes with slopes obtained under similar conditions with known amounts of FVIIa, the molar concentrations of FVIIa can be estimated.

[0411] FVIIa autoactivation was found to be TF dependent and, based on the measured FVIIa activities in Table 11, it was not impaired in the presence of monovalent antibody 11F2.

[0412] Table 11. TF-mediated autoactivation of FVIIa according to Example 12 in the presence or absence of 200 nM monovalent antibody 11F2 mAb0077(OA)

[0413]

[0414] Example 13. Crystal structure of 11F2-Fab0076 in complex with FVIIa with inhibited active site and soluble tissue factor

[0415] To determine the epitope recognized by the mouse antibody 11F2 on FVII(a), the corresponding Fab fragment (Fab0076) was crystallized in complex with FVIIa with the active center inhibited by HD-Phe-Phe-Arg-chloromethyl ketone (FFR-cmk; Bachem, Switzerland) (FVIIai) and the soluble fragment of tissue factor 1-219 (sTF) using the hanging drop method according to Kirchhofer D., et al., Proteins Structure Function and Genetics. (1995), 22, 419-425.

[0416] Crystallization

[0417] Fab0076 crystals mixed at a 1:1 molar ratio with the FVIIai / sTF complex were grown using the hanging drop modification of the protein vapor diffusion method at 18°C. A protein solution of 1 μL of 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 and incubated at 18°C ​​over 1 mL of the precipitant solution to obtain crystals of the complex.

[0418] Diffraction data collection

[0419] The crystal was 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 in liquid nitrogen. Diffraction data were acquired at SHOCK in MAX-lab (Lund, Sweden) in channel I911-3 using a marCCD225 detector from MAR Research. Auto-indexing, integration, and scaling of the data were performed using programs from the XDS package (the statistical values ​​of the diffraction data are summarized in Table 12).

[0420] Definition and clarification of structure

[0421] The structure was solved by molecular replacement using Phaser, implemented in the Phenix software suite, with chains A and B from the 1YY8 entry in the Protein Structure Database and the 3ELA entry in the Protein Structure Database. The asymmetric unit contains two Fab:FVIIai / sTF complexes. The model was refined using refinement steps in Phenix and manual rebuilding in COOT. The refinement statistics are presented in Table 12.

[0422] Table 12. Statistical characteristics of data collection and refinement from X-ray crystallographic analysis of the crystallographic structure of the complex between FVIIa with inhibited active site, soluble tissue factor (sTF), and Fab0076. Statistical values ​​for the highest-resolution shell are shown in parentheses.

[0423]

[0424]

[0425] The epitope (defined as residues in FVIIai having a non-hydrogen atom located at a distance of less than or equal to 4 Å from the non-hydrogen atom in Fab0076) was found to include the following residues according to SEQ ID NO: 1:

[0426] R113

[0427] C114

[0428] HI 15

[0429] E116

[0430] G117

[0431] Y118

[0432] S119

[0433] L120

[0434] T130

[0435] V131

[0436] N184

[0437] T185

[0438] 1186

[0439] P251

[0440] V252

[0441] E265

[0442] M391

[0443] R392

[0444] E394

[0445] The paratope (defined as residues in Fab0076 having a non-hydrogen atom located at a distance less than or equal to 4 Å from a non-hydrogen atom in FVIIai) was found to include the following light chain residues:

[0446] Q27

[0447] G28

[0448] S30

[0449] D31

[0450] Y32

[0451] K49

[0452] Y50

[0453] Q53

[0454] H92

[0455] S93

[0456] F94

[0457] according to SEQ ID NO: 64

[0458] and heavy chain residues:

[0459] D32

[0460] Y54

[0461] N59

[0462] N101

[0463] Y102

[0464] Y103

[0465] G104

[0466] N105

[0467] according to SEQ ID NO: 63.

[0468] Example 14. Humanization of mouse 11F2

[0469] To humanize the murine antibody 11F2 (mAb0005) with the VH and VL domain sequences corresponding to SEQ ID NO: 754 and SEQ ID NO: 750, respectively, while maintaining its high binding affinity to FVII(a), we combined sequence identity information relative to human germlines, the crystal structure of the complex of Fab0076 (Example 12) and FVIIa with inhibited active site (Example 9), and in vitro binding data. We initially searched the human germline database for VH, VL, and VJ sequences (for both the heavy chain (HC) and light chain (LC)) with high sequence identity to the variable domain sequences of murine 11F2 using the Blast algorithm. The sequences with the highest sequence identity for the VH HC were: IGHV4-30-4*01, IGHV4-28*01, IGHV4-28*06 and IGHV459*01, and for the VJ segment of HC the priority germlines were: IGHJ5*01, IGHJ4*01.For LC, the priority sequences for VL were IGKV6D-41*01 and IGKV3-11*01, and for the VJ segment: IGKJ2*01 and IGKJ2*02 (Table 13A). The differences between the human germline sequences and the mouse VH and VL were then mapped on the crystal structure of the Fab0076 / FVIIa complex. Residues in the mouse variable domains separated by a distance of greater than or equal to 10 Å from residues in the epitope were expected to have little effect on binding affinity, so they were replaced with the corresponding amino acid element of the human germline. In turn, residues constituting the paratope were considered more problematic to replace without affecting affinity, so in this case the mouse amino acid element was retained. A subset of residues near the binding surface were identified that could potentially influence binding affinity.Humanized variants were generated by mutating residues distant from the epitope with a human amino acid element identified through germline alignment. Additionally, residues close to the paratope were mutated with a human element from a subset to make the variants as close as possible to the human germline. The set included variants in which mouse CDRs were grafted onto fully human germline HC and LC. Based on the initial analysis, 12 LC and 13 HC were obtained, which were combined into 23 variants according to Table 13B.

[0470] FVIIa binding affinity values ​​(listed in Table 13B) were determined for humanized antibodies using biolayer interferometry (Fortebio). All steps were performed 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) at 30°C.

[0471] Antibodies were immobilized on anti-human antibody (AHC, Fortebio) tips for 3 min at a concentration of 10 μg / mL. This was followed by a 3-min incubation to establish baseline. Association was then monitored for 3 min using four different concentrations of FVIIa (25 nM, 50 nM, 100 nM, and 200 nM), followed by 3-min dissociation. Sensorgrams were analyzed using Fortebio data analysis software. Fortebio data were used for ranking, and absolute affinity values ​​may deviate from those determined by SPR (Example 6, Table 7A).

[0472] Table 13. Human germline sequences used for humanization of mouse 11F2. Numbers in parentheses reflect the identity: ((n / m) denotes n identical positions out of a total of m positions) between the mouse variable domains of Fab0076 and the indicated human germline sequence

[0473]

[0474] Table 14. VH and VL pairing in the first round of humanization of mouse HF2-mAb0048 with measured affinities of the corresponding antibodies to FVIIa (determined as described in this document). For some variants, no binding was detected in the measurement (nb)

[0475]

[0476]

[0477] Variants from the first round of humanization were identified with affinity equal to or greater than that of the parent murine antibody. Therefore, those mutations found to maintain or improve binding affinity were used for the second round of variant construction. Using this set of mutations, a second round of variant construction was performed by inserting as many of these mutations as possible onto humanized HC and LC that already possessed the required affinity.Based on 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 combined by combining all VHs with VLs, yielding a total of 475 combinations. Dissociation constant (KD) values ​​for binding to FVIIa were measured for the resulting 475 humanized antibodies using biolayer interferometry (Fortebio) as described above. The measured KD values ​​are presented in Table 15.

[0478] Table 15. Dissociation constant values ​​(M, nb for no binding) for humanized variants of the murine antibody 11F2 to FVII(a). Antibodies are identified by their respective SEQ ID numbers for VH and VL:

[0479]

[0480]

[0481]

[0482] SPR analysis of humanized 11F2 variants

[0483] The affinity of selected humanized 11F2 variants was determined using the SPR assay detailed in Example 6. The measured dissociation constants are listed in Table 15 and show continued high-affinity binding of the variants to human FVIIa.

[0484] Table 16. Calculated binding constants (K D ) for the interaction of humanized 11F2 antibodies in monovalent and biAb format with FVIIa, determined by surface plasmon resonance (SPR) in accordance with example 6

[0485]

[0486]

[0487] Functional characteristics of humanized variants of 11F2

[0488] The effect of selected humanized 11F2 variants on FVIIa activity and sensitivity to antithrombin inhibition was determined as detailed in Example 5. The results are listed in Table 17 and show that the humanized variants retain the desired properties with respect to these parameters.

[0489] Table 17. Functional characterization of antibodies to FVII(a) in thrombin generation assays (TTA) and antithrombin inhibition assays (ATI) as described in Example 5

[0490]

[0491] Pharmacokinetics of FVIIa in rats coformulated with humanized 11F2 variants

[0492] The humanized 11F2 family of variants was administered intravenously to male Sprague-Dawley rats in combination with 20 nmol / kg FVIIa as detailed in Example 9.

[0493] The results are presented in Table 18 and show that several humanized 11F2 variants confer FVIIa a half-life as long as the parent antibody.

[0494] Table 18. Mean retention time (MRT) of FVIIa activity in plasma of Sprague-Dawley rats after i.v. administration of 20 nmol / kg FVIIa at different molar ratios to antibody. Values ​​represent mean ± standard deviation, n = 3. N.p.: not applicable

[0495]

[0496] Example 15. Crystallization and epitope mapping of 11F2 Fab0883 in complex with FVIIa with inhibited active site and soluble tissue factor

[0497] To determine the epitope on FVII(a) recognized by humanized 11F2, i.e., mAb0705 (OA) and mAb0842 (OA), the corresponding Fab fragment (Fab0883) was crystallized in complex with FVIIa inhibited by HD-Phe-Phe-Arg chloromethyl ketone (FFR-cmk; Bachem, Switzerland)

[0498] active site (FVIIai) and soluble fragment of tissue factor 1-219 (sTF) using the hanging drop method according to [Kirchhofer. D., et al., Proteins Structure Function and Genetics. (1995), 22, 419-425.

[0499] Crystallization

[0500] Crystals of the SEC-purified Fab complex with FVIIai / sTF were grown using a modified sessile drop vapor diffusion method 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 a precipitant solution containing 0.15 M CsCl and 15% (w / v) polyethyleneglycol 3350 and 360 nL of water and equilibrated with 80 μL of the precipitant solution. The crystals were grown for 6 weeks.

[0501] Diffraction data collection

[0502] The crystal was cryoprotected in a solution consisting of 0.15 M CsCl, 15% (w / v) polyethyleneglycol 3350, and 20% (v / v) glycerol before flash cooling in liquid nitrogen. Diffraction data were collected at 100 K on a Rigaku FRX rotating anode generator equipped with a Dectris Pilatus 1M detector. Data processing was performed using programs from the XDS package (the statistical values ​​of the diffraction data are summarized in Table 19).

[0503] Definition and clarification of the structure

[0504] All crystallographic calculations were performed using the Phenix crystallographic software suite. The structure was solved by molecular replacement using the Phaser program with the coordinates of the complex structure, obtained as described in Example 13, as the search model. The asymmetric unit contains four Fab:FVIIai / sTF complexes. Iterative cycles of manual rearrangement using COOT and refinement in Phenix yielded the final model (Table 19).

[0505] Table 19. Statistical characteristics of data collection and refinement from X-ray crystallographic analysis of the crystallographic structure of the complex between FVIIa with inhibited active site, soluble tissue factor (sTF), and Fab0883. Statistical values ​​for the highest-resolution shell are shown in parentheses.

[0506]

[0507]

[0508] Epitope mapping

[0509] A residue is considered part of an epitope if all four independently determined FVIIa molecules in the crystal contain a non-hydrogen atom of the specified residue at a location less than or equal to 4 Å from a non-hydrogen atom in the Fab. Thus, the epitope was found to contain the following residues according to SEQ ID NO: 1:

[0510] R113

[0511] C114

[0512] H115

[0513] E116

[0514] G117

[0515] Y118

[0516] S119

[0517] L120

[0518] T130

[0519] V131

[0520] N184

[0521] T185

[0522] P251

[0523] V252

[0524] V253

[0525] Q388

[0526] M391

[0527] R392

[0528] Definition of paratope

[0529] A residue is considered part of a paratope if all four independently determined Fab molecules in the crystal contain a non-hydrogen atom of the specified residue at a location less than or equal to 4 Å from a non-hydrogen atom in FVII(a). The paratope was found to contain the following light chain residues (according to SEQ ID NO: 814):

[0530] Q27

[0531] G28

[0532] Y32

[0533] Y50

[0534] H92

[0535] S93

[0536] F94

[0537] and heavy chain residues (according to SEQ ID NO: 818):

[0538] D32 Y54 Y103 N105

[0539] Example 16. Hot spot analysis of 11F2 mAb0842 (OA) using SPR

[0540] Expression of alanine variants of FVIIa

[0541] Alanine variants of hFVII were produced using QMCF technology, a stable episomal expression system (Icosagen). CHOEBNALT85 cells were cultured in Qmixl medium (1 L = 1:1 CD-CHO and SFM II (NVO11514701) + 10 mL penicillin / streptococcus (Gibco, 15140-122) + 2 mL puromycin (Gibco, A11138-03)) in E125 flasks in a CO2 incubator with shaking. On the day of transfection, 1 × 10e7 CHOEBNALT85 cells were transfected with 2 μg of plasmid encoding the hFVII variant and 50 μg of salmon milt 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 cells to Qmix2 medium (1 L = 1:1 CD-CHO and SFM II (NVO11514701) + 10 ml penicillin / streptococcus (Gibco, 15140-122) + 1 ml vitamin K (K.vit 13A 01311) + 14 ml G418 (Gibco, 10131-027)). After 10-14 days of selection, G418 cells reached >95% viability (Vi-Cell XR cell counter).Cells were split at 0.4 x 10e6 cells / ml in 2 x 250 ml Qmix2 in 2 x E1000 flasks. After 3-1 days, the cells reached a density of approximately 4-5 x 10e6 cells / ml. Expression was initiated by adding 20% ​​CHO CD Efficient Feed B (Gibco A10240) + 6 mM GlutaMax (Gibco, 35050). After 4 days of initiation, another 10% CHO CD Efficient Feed B + 6 mM GlutaMAX were added. On day 6 after initiation, the cultures were harvested and centrifuged (200 g, 5 min). The supernatants were collected and supplemented with 15 mM HEPES (Gibco, 15630) and 5 mM CaCl2 (Sigma, 21115). The supernatants were sterilized by filtration using a 0.22 μm filter pad (Corning, CLS430049).

[0542] Expression and purification of cFVIIa chimera (22017-051)

[0543] cFVIIa chimera was produced using a similar expression system described above. The cFVII chimera zymogen was isolated from the medium using an affinity column prepared by coupling an in-house anti-FVII(a) antibody (F1A2) to Sepharose beads as described in Example 25. The F1A2 anti-FVII(a) antibody binds to the Gla domain of FVII(a) in a Ca++-dependent manner. The cFVII chimera zymogen was activated using human FIXa and re-purified using F1A2 affinity purification to yield the final cFVIIa chimera.

[0544] Hot Spot Analysis

[0545] Hot spot analysis using the monovalent humanized antibody mAb0842(OA) was performed by binding studies with a panel of 19 FVII(a) variants using surface plasmon resonance (Biacore T200) at 25°C. Anti-FVII(a) antibody (internal Ab 4F6 (Nielsen AL et al., PNAS 114 (47) 12454–12459, 2017)) targeting the gla domain of FVII(a) was immobilized at a concentration of 25 μg / mL on the CM4 sensor chip using a standard amine coupling chemistry kit (both supplied by GE Healthcare). FVII(a) variants according to Table 19 in cell culture supernatants (as described above) were diluted in running buffer and injected at 10 μl / min for 1 min to achieve a capture level of 5-55 units. Capture of each FVII(a) variant was achieved using immobilized antibody to gla FVII(a).Next, 540 nM (3x diluted) mAb0842(OA) was injected at 30 μl / min for 7 min to allow binding to the captured FVII(a) variant, followed by a 9-min injection of buffer to allow dissociation of the single-armed anti-FVII(a) antibody. The running buffer was prepared by 10-fold dilution of 10x HBS-P buffer (supplied by GE Healthcare) with 1 mg / ml BSA and 5 mM CaCl2 to obtain 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 7.4, 5 mM CaCl2, 1 mg / ml bovine serum albumin (BSA). The running buffer was also used to dilute the anti-FVII(a) antibody samples and FVII(a) samples. The chip was regenerated using 10 mM HEPES, 150 mM NaCl, 20 mM EDTA, 0.05 v / v polysorbate 20, pH 7.4. Binding data were analyzed according to the 1:1 kinetic model and steady-state analysis using BiaEvaluation 4.1, provided by the manufacturer (Biacore AB, Uppsala, Sweden).Where possible, ka, kd, and KD values ​​from a 1:1 kinetic approximation model are reported. KD values ​​using a steady-state approximation model were reported for the four FVII(a) variants. In addition, the capture signal is reported for all FVII(a) variants. An amino acid residue is considered a hotspot residue if substitution of that amino acid residue with alanine results in a tenfold (10) or greater decrease in affinity compared to the wild-type. Based on the data presented in Table 20, amino acid residues H115, T130, V131, and R392 are hotspots.

[0546] Table 20. Binding interaction of FVII(a) variants with monovalent humanized antibody mAb0842(OA) determined by surface plasmon resonance (SPR) analysis according to Example 16

[0547]

[0548]

[0549] Example 17. Pharmacokinetics of recombinant FVIIa in combination with humanized antibody 11F2 in cynomolgus macaques

[0550] The activity-time profiles of FVIIa in plasma were assessed in studies in cynomolgus monkeys following intravenous or subcutaneous administration of recombinant FVIIa (rFVIIa) alone or in a co-formulation with monovalent single-arm 11F2 mAb0705(OA) at a 1:3 molar ratio. The formulations were administered as a single dose of 5.4 nmol / kg FVIIa (containing 16.2 nmol / kg mAb0705(OA) for the co-formulation) with blood samples collected over a three-week period.

[0551] During the animal experiment, animal care and handling were performed in accordance with standard procedures approved by local health authorities. Animals had free access to food and water. Plasma FVIIa activity was measured using the FVIIa activity assay described in Example 8. Endogenous FVIIa levels in cynomolgus monkeys were below the LLOQ (0.1 nM) before administration and were therefore not measured.

[0552] Pharmacokinetic analysis of the activity-time profiles for FVIIa in plasma was performed using non-compartmental methods using Phoenix WinNonlin 6.4. The following parameters were estimated from the data: clearance (CL), mean residence time (MRT), and subcutaneous bioavailability (F). The parameters are listed in Table 21 and show a significant increase in FVIIa activity when co-formulated with mAb0705(OA) after both intravenous and subcutaneous administration compared to FVIIa in the absence of antibody.

[0553] Table 21. Clearance (CL), mean residence time (MRT), and subcutaneous bioavailability (F) of plasma FVIIa activity in cynomolgus monkeys following intravenous or subcutaneous administration of 5.4 nmol / kg FVIIa alone or coformulated with mAb0705(OA) at a molar ratio of 1:3. Values ​​represent mean (SD), n = 3

[0554]

[0555] Example 18. Humanization and optimization of mouse anti-TLT-1 antibody mAb0012

[0556] Humanization

[0557] The murine anti-human TLT-1 antibody mAb0082 disclosed in WO2012 / 117091 was used as the starting point for the humanization process. MAb0082 was derived from mAb0012 by introducing two point mutations, C41A in VL and T61A in VH, which removed the unpaired cysteine ​​in FR1 of VL and the N-glycosylation site in CDR2 of VH, respectively. The humanization process was based on standard molecular biology techniques known to those skilled in the art.

[0558] Briefly, the CDRH1, CDRH2, and CDRH3 sequences of mAb0082 were grafted onto the human germline sequence based on the VH3_74 / JH1 sequence identified in the IMGT database. In addition, three amino acid substitutions from human VH3_74 / JH1 were introduced into the grafted CDRH2 sequence to further humanize this CDR sequence: P62D, L64V, and D66G. To achieve binding affinity comparable to that of mAb0082, three backmutations were introduced into the VL sequence at positions S49G, D62P, and R98S. For VL humanization, the CDRL1, CDRL2, and CDRL3 sequences of mAb0082 were grafted onto the human germline sequence based on the VKH A23 / JK2 sequence identified in the IMGT database. The VL sequence contains a potential deamidation hotspot (NG motif) in CDR1.Using saturation mutagenesis at this position, it was found that the NG motif could be removed by a N33Q substitution without compromising affinity for TLT-1.

[0559] The final humanized and optimized version of mAb0082 is designated as mAb1076, which corresponds to SEQ ID NO: 934 (VL) and 938 (VH).

[0560] SPR analysis of humanized TLT1 variants

[0561] Binding of sTLT1 (corresponding to SEQ ID NO:3 with six histidine residues added at the C-terminus) to biAb (from Example 4) was studied by surface plasmon resonance (Biacore T200) at 25°C. The anti-human IgG antibody was immobilized on a CM5 sensor chip (both from GE Healthcare) using standard amine coupling chemistry. Purified biAb according to Table 21 (1 nM) was injected at 10 μL / min for 1 min. sTLT1 was then injected in the range of 0 to 60 μM at 30 μL / min for 3 min to ensure binding to biAb, followed by a 3-min buffer injection to ensure dissociation from biAb. The working buffer was prepared by 10-fold dilution of 10x HBS-P buffer (supplied by GE Healthcare) with 1 mg / ml BSA and 5 mM CaCl2 to obtain 10 mM HEPES, 150 mM NaCl, 0.05% v / v polysorbate 20, pH 7.4, 5 mM CaCl2, 1 mg / ml bovine serum albumin (BSA).The working buffer was also used to dilute the biAb and sTLT1 samples. Chip regeneration was performed using the recommended regeneration buffer consisting of 3 M MgCl2 (supplied by GE Healthcare). Binding data were analyzed according to the 1:1 model using BiaEvaluation 4.1, provided by the manufacturer (Biacore AB, Uppsala, Sweden). This analysis yielded the binding constants presented in Table 22, demonstrating a range of affinities from 2.9 nM to 320 nM for sTLT1 binding by biAb.

[0562] Table 22. Calculated binding constants for the interaction of sTLTl with biAb determined using surface plasmon resonance (SPR) analysis according to Example X

[0563]

[0564] Example 19. Crystal structure of a drug based on the peptide complex Hz-TLT1 and TLT-1

[0565] The Fab fragment used for crystallization in complex with the TLT-1 core peptide contained VL and VH domain sequences corresponding to mAL1076 (SEQ ID NOs: 854 and 858, respectively), the CH1 domain of human IgG4, and human CL-kappa with one point mutation (G157C). The G to C substitution is located in the constant domain of the Fab fragment, i.e., away from the antigen-binding site, and does not affect binding to TLT-1. The 37-membered core peptide, EEEEETHKIGSLAENAFSDPAGSANPLEPSQDEKSIP (SEQ ID NO: 13), corresponding to residues 111-147 of SEQ ID NO: 2, was prepared by standard peptide synthesis methods known to one skilled in the art. Fab and core peptide were mixed at a 1:2 molar ratio in Hepes buffer (20 mM Hepes (pH 7.3), 150 mM NaCl). The 1:1 Fab:peptide complex was isolated using gel filtration on a Superdex 200 column eluted with Hepes buffer, then concentrated to -11 mg / ml and used for crystallization.

[0566] Crystallization

[0567] Fab / peptide complex crystals at a molar ratio of 1:1 after gel filtration were grown using a modified sitting drop vapor diffusion method at 18°C. A protein solution of 150 nL of 10.8 mg / mL Fab:peptide complex in 20 mM Hepes, pH 7.3, and 150 mM NaCl was mixed with 50 nL of 1 M LiCl, 0.1 M Na citrate-citric acid, pH 4, and 20% (w / v) PEG 6000 as a precipitant and incubated over 60 μL of the precipitant.

[0568] Diffraction data collection

[0569] The crystal was cryoprotected by adding 1 μL of precipitating agent containing 20% ​​ethylene glycol to the crystallization droplet before flash cooling in liquid nitrogen. Diffraction data were collected at 100 K in the BioMAX synchrotron beamline at the MAX IV synchrotron (Lund, Sweden) using an Eiger 16M hybrid pixel detector from Dectris. Auto-indexing, integration, and data scaling were performed using programs from the XDS package (the statistical values ​​of the diffraction data are summarized in Table 22).

[0570] Definition and clarification of structure

[0571] The asymmetric unit contains two Fab:peptide complexes based on Matthews coefficient analysis. The structure was solved by molecular replacement. Phaser, implemented in the Phenix suite of programs, was used with the H and L chains from the 5KMV protein structure database entry as a search model localizing the two Fabs. They were modeled using the correct amino acid sequence using COOT and then refined using refinement in Phenix. Amino acids 7–21 of the peptide were clearly visible in the difference electron density maps and could be modeled manually using COOT. The model was further refined using refinement steps in Phenix and manual rearrangement in COOT. The refinement statistics are presented in Table 23.

[0572]

[0573]

[0574]

[0575] Statistical values ​​for the highest resolution shell are shown in parentheses

[0576] Epitope and paratope of the Fab / nenmud complex

[0577] The epitope is defined as residues in the TLT-1 core peptide characterized by the presence of a heavy atom (i.e., an atom other than hydrogen) within 4.0 Å of the heavy atom in the Fab in both complexes in an asymmetric unit. Similarly, the paratope is defined as residues in the Fab fragment characterized by the presence of a heavy atom within 4.0 Å of the heavy atom in the TLT-1 core peptide in both complexes in an asymmetric unit. The following residues of the 37aa TLT-1 peptide were found to contain the epitope according to SEQ ID NO: 13:

[0578] K8

[0579] I9

[0580] G10

[0581] S11

[0582] L12

[0583] A13

[0584] N15

[0585] A16

[0586] F17 S18 D19 P20

[0587] A21,

[0588] corresponding to K118, 1119, G120, S121, L122, A123, N125, A126, F127, S128, D129, P130 and A131 of SEQ ID NO:2 and 3).

[0589] Paratop contains the following residues of the heavy chain variable domain (SEQ ID NO: 938):

[0590] V2

[0591] F27

[0592] R31

[0593] Y32

[0594] W33

[0595] E50

[0596] T57

[0597] N59

[0598] S98

[0599] G99

[0600] V100

[0601] T102

[0602] S103

[0603] and light chain variable domain (SEQ ID NO: 934):

[0604] H31

[0605] Y37

[0606] H39

[0607] Y54

[0608] F60

[0609] S61

[0610] S96

[0611] T97

[0612] V99

[0613] Y101.

[0614] Example 20. Effect of affinity on the stimulatory activity of a bispecific antibody to FVII(a) / TLT-1

[0615] To determine the effect of affinity on the activity of the bispecific antibody, a series of humanized FVII(a) and TLT-1 mAbs, mAb0005 11F2 (see Example 14) and mAb0012 (see Example 18), and with different affinities for FVIIa and TLT-1, respectively, were tested in a bispecific format in an FXa formation assay using lipidated TLT-1, as described in WO2011 / 023785.

[0616] In the first step, FX activation was measured in the presence of 4 nM recombinant TLT-1 incorporated into 10:90 phosphatidylserine:phosphatidylcholine vesicles (WO 2011023785), 2.5 nM FVIIa, and a bispecific antibody (biAb) at a concentration series from 0 to 300 nM. After 10 min pre-incubation in assay buffer (50 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.3 + 1 mg / mL BSA, and 0.1% PEG8000) at room temperature, 150 nM plasma-derived FX (Haematologic Technologies) was added to a total volume of 50 μL, and activation was continued for 20 min. Activation was then stopped by adding 25 μl quench buffer (50 mM HEPES, 100 mM NaCl, 80 mM EDTA, pH 7.3) and FXa was obtained, quantified by its ability to hydrolyze 0.5 mM chromogenic substrate S-2765 (Chromogenix) (added as a 2 mM stock solution in 25 μl volume), which was monitored at 405 nM for 5 min on a SPECTRAmax Plus384 plate reader.The normalized activity (A) was calculated from the slope of the linear increase in absorption. biAb ) for each biAb at a concentration of 100 nM by subtracting the background activity in the absence of biAb and dividing by the concentration of FVIIa in the assay.

[0617] In a second step, the same assay was performed with biAb replaced by assay buffer and a series of FVIIa concentrations from 0 to 80 nM. The slope of the linear relationship between background-subtracted FXa formation and the FVIIa concentration in the assay provides a measure of the specific activity of free FVIIa (A FVIIa ) under the analytical conditions used.

[0618] Based on the measured activity values, the stimulating activity of each biAb at a concentration of 100 nM was calculated as the ratio A biAb / A FVIIa Stimulating activity allows one to measure the fold increase in FXa produced by FVIIa upon addition of 100 nM biAb.

[0619] The stimulating activity is presented in Table 24 and shows the dependence of biAb stimulation on the binding forces (expressed as dissociation constant (KD) values) of FVIIa and TLT-1, respectively. Among the biAbs tested, biAb0001 exhibited the highest stimulating activity.

[0620] Table 24. Stimulatory activity of the bispecific antibodies in the presence of FX, FVIIa, and lipidated TLT-1 described in Example 20. For each bispecific antibody, the measured stimulatory activity (mean ± standard deviation, n = 2) is indicated along with the dissociation constants for interaction with FVIIa and TLT-1, respectively.

[0621]

[0622]

[0623] Example 21. Effect of epitope location on the stimulatory activity of bispecific antibodies to FVII(a) / TLT-1

[0624] To determine the effect of epitope location on the activity of the bispecific antibody, a series of TLT-1 and FVIIa mAbs binding to different epitopes on TLT-1 and FVIIa, respectively, were tested in a bispecific format in the FXa formation assay as performed in Example 20.

[0625] The results are presented in Table 25 and demonstrate the dependence of biAb stimulatory activity on epitope location. Specifically, anti-TLT-1 mAbs mAb 1076, mAb0023, mAb0051, and mAb0062 in combination with anti-FVIIa mAb mAb0865 exhibit comparable stimulatory activity.

[0626] Table 25. Stimulatory activity of the bispecific antibodies in the presence of FX, FVIIa, and lipidated TLT-1 described in Example 21. For each bispecific antibody, the measured stimulatory activity (mean ± standard deviation, n = 2) is indicated along with the dissociation constants for interaction with FVIIa and TLT-1, respectively.

[0627]

[0628]

[0629] Example 22. Human IgG Antigen Assay (LOCI)

[0630] The presence of human IgG (hIgG) in cynomolgus macaque plasma was measured using a luminescent oxygen channeling immunoassay (LOCI). Briefly, the LOCI reagents included two latex bead reagents (donor and acceptor beads) and a biotinylated monoclonal antibody to hIgG (Biosite, cat. no. AFC4249). The donor bead reagent, containing a light-sensitive dye, was coated with streptavidin. The second bead reagent, acceptor beads, was conjugated with a proprietary monoclonal antibody (0421) to hIgG, resulting in a sandwich structure. During the assay, the three reagents combined with hIgH in plasma to form a bead-aggregate immune complex. Excitation of the complex released singlet oxygen molecules from the donor granules, which channeled into the acceptor granules and initiated a chemiluminescent response. This response was then measured in the EnVision plate reader.The amount of light generated, expressed as counts per second (cps), was proportional to the hIgG concentration. Samples were diluted at least 100x in assay buffer, and a calibration curve was constructed using hIgG added to 1% cynomolgus monkey plasma.

[0631] Example 23. FVII(a) antigen assay (LOCI)

[0632] FVII(a) antigens, including FVII zymogen, FVIIa, and FVIIa:antithrombin (FVIIa:AT) complexes, were measured using the LOCI assay described in Example 22, except that the FVII(a) assay consisted of acceptor beads coated with an in-house anti-FVII(a) antibody (4F9) and an in-house biotinylated anti-FVII(a) monoclonal antibody (4F7). Samples were diluted at least 100x in assay buffer, and a calibration curve was generated by adding known amounts of human rFVIIa to the assay buffer.

[0633] Example 24. FVIIa:AT (antithrombin) antigen assay (EIA)

[0634] FVIIa:AT (antithrombin) complexes were measured by enzyme-linked immunosorbent assay (EIA) as described by Agersoø H et al, J Thromb Haemost 2011; 9: 333–8. An anti-FVIIa monoclonal antibody (Dako Denmark A / S, Glostrup, Denmark, product code 09572), which binds to the N-terminal EGF domain and does not block antithrombin binding, was used to capture the FVIIa:AT complex. A preformed complex of human FVIIa and cynomolgus monkey antithrombin (FVIIa:AT) was prepared by incubating FVIIa with a 2-fold molar excess of antithrombin in the presence of 10 μM low molecular weight heparin (enoxaparin). The residual amidolytic activity of FVIIa (see Example 11) after overnight incubation at room temperature was confirmed to be less than 10% of the initial FVIIa activity. The complex was used to construct EIA calibration curves.A conjugate of peroxidase and a polyclonal antibody to human antithrombin (Siemens Healthcare Diagnostics ApS, Ballerup, Denmark, product code OWMG15) was used for detection. TMB was added, the reaction was continued until sufficient color developed, stopped by adding H2SO4, and the absorbance was measured at 450 nM with 650 nM as a reference on a BioTek plate spectrophotometer. Color intensity is proportional to the concentration of FVIIa:AT.

[0635] Example 25. Preparation of human FVIIa, FVII (zymogen), and FVIIa:AT (antithrombin) complex

[0636] Production of human FVIIa (activated FVII)

[0637] Unless otherwise stated, recombinant activated human FVII (FVIIa) was prepared as described by Thim et al. (1988) Biochemistry 27:7785–7793 and Persson et al. (1996) FEBS Lett 385:241–243.

[0638] Production of human FVII (FVII zymogen)

[0639] Recombinant human FVII produced in CHO cells was purified by one-step calcium-dependent affinity chromatography as described by Thim et al. (1988) Biochemistry 27:7785–7793. After purification, the FVII zymogen was dialyzed against 10 mM MES, 100 mM NaCl, 10 mM CaCl2, pH 6.0. The level of activated FVII (FVIIa) in the preparation was determined by measuring the amidolytic activity in the presence of 1 mM chromogenic substrate S-2288 and 200 nM sTF (see Example 11). By relating this to a standard curve constructed with a known concentration of FVIIa, the measured activity can be converted to the molar concentration of FVIIa in the FVII zymogen preparation.

[0640] Preparation of human FVIIa:AT (antithrombin) complex

[0641] The FVIIa:AT (antithrombin) complex was prepared by incubating equimolar concentrations of recombinant human FVIIa derived from human plasma AT (Baxter) and low molecular weight heparin (enoxaparin sodium) for 16 hours at 4°C. To remove contaminating excipients, AT was re-purified before use on a heparin-Sepharose 6 Fast Flow column (GE Healthcare) using a sodium chloride gradient. The eluted AT was concentrated by ultrafiltration to obtain the final preparation in 10 mM HEPES, 25 mM NaCl, pH 7.3, containing 50% glycerol. The FVIIa:AT complex was purified by SEC chromatography at 4°C in 20 mM MES, 100 mM NaCl, 1 mM EDTA, pH 5.5 to maximize complex stability. Residual FVIIa levels in the preparation were determined as described above. To minimize complex degradation, preparations were stored in aliquots at -80°C, then immediately thawed and kept on ice before use.

[0642] Example 26. Pharmacokinetics of biAb to FVII(a) / TLT-1 after single dose administration in cynomolgus monkeys

[0643] biAb0001 and the corresponding YTE variant, biAb0352, in which three additional half-life-prolonging substitutions (M252Y, S254T, and T256E) were introduced into the heavy chain constant domain, were administered to cynomolgus monkeys intravenously (iv) at concentrations of 3.0, 9.49, or 30 nmol / kg or subcutaneously (s.c.) at a concentration of 9.49 nmol / kg. Each group consisted of two monkeys (male and female). Sodium citrate-stabilized blood samples of 1 mL were collected before dosing and, for the i.v. groups, at 0.5 h; 2.5 h; 6 h; 12 h; 24 h; 48 h; 72 h and on days 8, 10, and 15 post-dose. For the s.c. groups, blood samples were collected pre-dose and at 0.5 h; 3 h; 6 h; 12 h; 16 h; 24 h; 30 h, 38 h; 48 h; 54 h; 72 h; 78 h; 96 h; 120 h and on days 8, 10, and 15 post-dose. Blood samples were centrifuged for 10 min at 2000 g, plasma was removed, aliquoted, and stored at 80°C until analysis for hIgG (see Example 22), total FVII(a) antigen (seeExample 23), FVIIa activity (see Example 8), and FVIIa:AT complexes (Example 24). Pharmacokinetic (PK) analysis of hlgG concentration-time profiles was performed by non-compartmental methods using Phoenix WinNonlin 6.4. The following PK parameters are shown in Table 26A: half-life (tVi), clearance (C1), volume of distribution (Vz), mean residence time (MRT), and subcutaneous bioavailability (F).

[0644] Table 26A. PK parameters for biAb with and without the YTE mutation analyzed in cynomolgus macaque plasma samples*

[0645] *) Each line represents data for one cynomolgus macaque, **) C1 is for the IV administration groups, and C1 / F is for the SC administration groups. F corresponds to the bioavailability after SC administration.

[0646] Endogenous FVII(a) accumulation was observed in monkeys administered BiAb0001 or the corresponding YTE variant, biAb0352. Table 26B shows pre-dose FVII(a) antigen levels, FVIIa, and FVII:AT activities, along with mean accumulated levels measured between 72 and 240 h post-dose. A dose-dependent antibody accumulation of FVII(a) antigen and increases in FVIIa and FVIIa:AT activities were observed. FVII(a) antigen increased 3-fold compared to pre-dose levels, and FVIIa and FVIIa:AT increased up to 5-fold. The data demonstrate that IV or SC administration of a single dose of biAb0001 or biAb0352 with the YTE mutation results in accumulation of endogenous FVII(a) antigen, increased FVIIa activity, and FVIIa:AT complexes in vivo.

[0647] Table 26B. FVII(a), FVIIa, and FVIIa:Ab antigen accumulation after a single intravenous or subcutaneous administration of biAb with or without the YTE mutation in cynomolgus monkeys

[0648]

[0649] *) Data represent the mean and SD of measurements from n=2 animals per group and 3 time points for the IV groups and 6 time points for the SC groups. Pre-dose values ​​are based on a single time point (pre-dose) for all 16 animals.

[0650] Example 27. Pharmacokinetics of the monovalent antibody 11F2 to FVII(a) mAb0705(OA) after single and multiple dose administration in cynomolgus macaques

[0651] The in vivo accumulation of FVII, FVIIa, and FVIIa:AT was analyzed by administering the monovalent anti-FVII(a) antibody 11F2 mAb0705(OA) to cynomolgus monkeys, followed by measuring FVII antigen, FVIIa, and FVIIa:AT activity in plasma samples. Two male cynomolgus macaques, weighing approximately 2.5 kg, were injected intravenously with 40 nmol / kg mAb0705(OA) into the saphenous, cephalic, or lateral tail vein, and three male cynomolgus macaques were injected subcutaneously (SC) into 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 at time points up to 21 days post-dose.

[0652] Blood was centrifuged for 10 min at 2300 g, and plasma aliquots were stored at 80°C until analysis for hIgG (see Example 22), total FVII(a) antigen (see Example 23), FVIIa activity (see Example 8), and FVIIa:AT complexes (Example 24). PK analysis of concentration-time profiles was performed by non-compartmental methods using Phoenix WinNonlin 6.4. PK parameters for intravenous administration are shown in Table 27. Half-life (t 1 / 2) of the single-arm antibody averaged 116 h (4.8 days) after a single i.v. administration. The estimated half-life for the subcutaneous antibody was 181 ± 20 h (mean and SD, n = 3). Antibodies accumulated over two weeks of administration, resulting in a maximum level of 1179 ± 140 nM at time points between 336 and 366 h after the initial dose (mean and SD, n = 3 animals, 3 time points).

[0653] Table 27. PK parameters of mAb0705(OA) administered intravenously to cynomolgus macaques at a dose of 40 nmol / kg

[0654]

[0655]

[0656] A single intravenous or repeated subcutaneous administration of mAb0705(OA) resulted in the accumulation of endogenous FVII(a). Total FVII(a) antigen, FVIIa:AT, and FVIIa after a single intravenous dose of 40 nmol / kg are shown in Table 28. Total FVII antigen increased from 7.0 nM before dosing to 35.0 ± 4.7 nM on days 7-14. Similarly, FVIIa increased from the lower limit of detection (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 FVII zymogen level after administration of 40 nmol / kg single-arm antibody was 26.4 nM, calculated by subtracting FVIIa and FVIIa:AT from total FVII(a) antigen.

[0657] Table 28. FVII(a) levels after a single 40 nmol / kg IV dose of mAb0705(OA), measured 7–14 days post-dose

[0658]

[0659] The steady-state level of total FVII(a), FVIIa, and FVIIa:AT after multiple SC dosing is shown in Table 29. Total FVII(a) antigen increased from 6.5 ± 1.5 nM before dosing to 36.9 ± 9.8 nM on days 12-21. Similarly, FVIIa increased from the lower limit of detection (0.009 nM) to 3.9 ± 1.6 nM on days 12-21, and FVIIa:AT increased from 1.0 ± 0.3 to 9.1 ± 0.6 nM on days 12-21. The steady-state level of FVII zymogen, calculated by subtracting FVIIa and FVIIa:AT from total FVII(a) antigen, was 24 nM.

[0660] 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:Ab in vivo. The clearance of the single-armed anti-FVII(a) antibody (0.42–0.49 mL / kg × kg, Table 26) was comparable to that of biAb0001 after intravenous administration (0.33–0.64 mL / kg × kg, Example 26, Table 25A). Therefore, the steady-state levels of FVII(a) antigen, FVIIa, and FVIIa:Ab measured after repeated administration of the single-armed anti-FVII(a) antibody are expected to be representative of the levels that can be achieved at steady-state after repeated administration of biAb with the same FVII(a) binding arm.

[0661] Table 29. Steady-state levels of FVII(a) after multiple subcutaneous administration of 20 nmol / kg mAb0705(OA), measured on days 12–21 after the initial dose

[0662]

[0663] Example 28. Thromboelastography in hemophilia A-like conditions of human whole blood with biAb0001 added to FVII(a) / TLT-1 and steady-state levels of FVII zymogen, FVIIa, and FVIIa:AT

[0664] The effect of the FVII(a) / TLT-1 bispecific antibody biAb0001 (where the parental anti-FVII(a) antibody is mAb0865 and the parental anti-TLT-1 antibody is mAb1076) and the accumulated levels of FVII zymogen, FVIIa, and FVIIa:AT from Example 27 were assessed by thromboelastography of human whole blood in hemophilia A-like conditions and compared with the effect of adding rFVIIa to the blood. In general, thromboelastography analysis was performed using TEG® (Thromboelastography Coagulograph, Haemoscope Corp.) instruments as described in Viuff D, et al. Thromb Res 2010; 126: 144-9. Citrate-stabilized whole blood from healthy donors was incubated for 30 min with 0.1 mg / ml neutralizing sheep polyclonal antibody to FVIII (Haematological Technologies Inc, cat. no. PAHFVIII-SC) and 5 μg / ml neutralizing mouse monoclonal antibody to TF (1F44, self-synthesized).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), FVII zymogen (prepared in Example 25, final plasma concentration 24 nM), and FVIIa:AT complexes (prepared in Example 25, final plasma concentration 9 nM) and added to the blood sample. FVIIa:AT predilutions were prepared in the cold in 20 mM MES, 100 mM NaCl, 1 mM EDTA, pH 5.5 + 2% BSA and added to the remaining proteins immediately before the assay. The FVII zymogen and FVIIa:AT preparations contained trace amounts of FVIIa, so a correspondingly smaller amount of FVIIa was added to compensate for this and for the expected FVIIa concentration in donor plasma (0.1 nM, Morissey JH et al. Blood, 1993; 81: 734–44). Similarly, the amount of added FVII zymogen was compensated for by the expected 10 nM FVIIa zymogen concentration in donor plasma.A single sample contained 25 nM rFVIIa, corresponding to the theoretical maximum plasma concentration after administration of 90 μg / kg rFVIIa (Novo Seven®) to human subjects with hemophilia A (Lindley CM et al. Clin Pharmacol Ther 1994; 55: 638-48). Control samples included biAb alone and a FVII / FVIIa / FVIIa:AT mixture without biAb. Platelets were maximally activated by the addition of the PAR1 agonist peptide SFLLRN (Tocris Biosciences, cat. no. 3497) to a final concentration of 30 μM and the GPVI agonist convulxin (5-Diagnostics, cat. no. 5D-1192-50UG) to a final concentration of 10 ng / mL. A 20 µl volume of 0.2 M CaCl2 in 20 mM Hepes, pH 7.4, was added to the TEG plate, followed by 340 µl of blood sample, and the analysis was started immediately. Clotting time (R-time), defined as the time to 2 mm of TEG curve amplitude, was calculated using software (TEG® Analytical Software, version 4.1.73). Data from 4 donors are shown in Table 30.The clotting time increased from 290 ± 12 s in normal blood to 3506 ± 1561 s after induction of hemophilia A-like conditions by neutralization of FVIII. The addition of 25 nM rFVIIa resulted in a reduction in the clotting time of hemophilia A-mimicking blood to 694 ± 158 s. The addition of 100 nM biAb to blood resulted in a moderate reduction in clotting time to 2198 ± 712 s, most likely due to potentiation of the action of endogenous FVIIa in blood. The addition of steady-state levels of FVII / FVIIa / FVIIa:AT reduced the clotting time to 1440 ± 275 s. The combination of 100 nM biAb and steady-state levels of FVII / FVIIa / FVIIa:AT reduced the clotting time to 495 ± 39 s, i.e., to a level comparable to or slightly lower than the clotting time after the addition of 25 nM FVIIa.The data demonstrate that biAb enhances the effect of cumulative levels of FVII / FVIIa / FVIIa:AT, resulting in a reduction in clotting time similar to or slightly better than the reduction in clotting time achieved using a therapeutically effective concentration of rFVIIa.

[0665] Table 30. Thromboelastography analysis of the effect of biAb0001 (100 nM) in human whole blood with or without steady-state levels of FVII / FVIIa / FVIIa:AT

[0666]

[0667] Example 29. Thromboelastography in hemophilia A-like conditions of human whole blood with the addition of bispecific antibodies to FVII(a) / TLT-1 with different affinities to TLT-1 and steady-state levels of FVII zymogen, FVIIa, and FVIIa:AT

[0668] In this example, the following bispecific antibodies to FVII(a) / TLT-1 with different affinities to TLT-1 were tested:

[0669]

[0670] Bispecific antibodies at a concentration of 100 nM were assessed by thromboelastography as described in Example 28. The clotting time (R-time) is shown in Table 32. The clotting time increased from 340 s to 5433 s after the induction of hemophilia A (HA) by the addition of antibodies to FVIII. The presence of biAb with the highest affinity to TLT-1 (biAb0OO1) shortened the clotting time to 2465 s, i.e., to a greater extent than the reduction in clotting time observed for the other three biAbs individually: biAb0015 shortened the clotting time to 3645 s; biAb0090 to 4335 s; and biAb0095 to 4110 s. Similarly, the combination of biAb with steady-state levels of FVII, FVIIa, and FVILAT resulted in a more pronounced reduction in clotting time for biAb0001 (up to 540 s) than for the other three biAbs; i.e., up to 1100 s for biAb0015, up to 1040 s for biAb0090, and up to 815 s for biAb0095. The data demonstrate that biAb0001 with the highest affinity values ​​(i.e.,(lowest KD) for TLT-1 was most effective in reducing clotting time.

[0671] Table 32. Thromboelastography analysis of the effect of biAb0001, biAb0015, or biAb0090(100 nM) in human whole blood with or without steady-state levels of FVII / FVIIa / FVIIa:AT

[0672]

[0673] Example 30. In vivo effect of biAb0001 bispecific antibody to FVII(a) / TLT-1 on the tail vein dissection bleeding model in transgenic FVIII-deficient mice with human TLT-1 knockout

[0674] The in vivo efficacy of the FVII(a) / TLT-1 bispecific antibody biAb0001 was determined using a tail vein transection (TVT) bleeding model in FVIII knockout (i.e., hemophilia A), mouse TLT-1 knockout, and human TLT-1 knockin transgenic mice. Since the anti-FVII(a) antibody does not recognize mouse FVII(a), biAb was co-administered with human FVIIa, FVII, and FVII:AT to obtain the target levels of these components in mouse plasma (3.8, 26.2, and 9.0 nM, respectively), simulating their expected clinical steady-state plasma levels according to Example 27. The concentration of biAb was 40 or 100 nM. Briefly, mice were anesthetized with isoflurane and placed on a heating pad set to maintain body temperature at 37°C, with their tails immersed in saline (37°C). The dose was administered into the right lateral tail vein 5 minutes before injury. In the present TVT model (Johansen et al., Haemophilia, 2016, 625-31) the lateral vein was dissected. If bleeding ceased after 10, 20, or 30 min, the tail was removed from the saline solution, and the wound was gently wiped with a gauze pad moistened with saline. Total blood loss was determined after 40 min by determining the amount of hemoglobin in the saline solution. Forty minutes after administration, a blood sample was collected from the orbital plexus in 3.8% trisodium citrate. Blood was centrifuged for 5 min at 4000 g, and plasma aliquots were stored at 80°C until analysis for hIgG (see Example 22), total FVII(a) antigen (see Example 23), and FVIIa activity (see Example 8).

[0675] Table 33 shows that all combinations of FVIIa and biAb resulted in a significant reduction in blood loss compared to biAb alone or FVII(a) administration without biAb. For all combinations, platelet counts measured 45 minutes after treatment were comparable to those observed in the vehicle-only group.

[0676] In conclusion, these data demonstrate a significant hemostatic effect of biAbOOOl in vivo in the presence of expected steady-state levels of FVIIa, FVII and FVIFAT.

[0677] Table 33. Blood loss after tail vein transection (TVT) in FVIII knockout / mouse TLT-1 knockout / human TLT-1 knockin mice administered with biAb0001, FVIIa, FVII, and FVIIa:AT combinations as indicated. Administered doses, expected and measured plasma concentrations, and determined blood loss are shown as mean ± SEM (n = 10). Using one-way analysis of variance followed by Dunnett's multiple comparison test, blood loss in groups 3 and 5 was found to be significantly different from that in groups 1 and 2. Total FVII(a) antigen concentrations (measured according to Example 22) are listed in the rows labeled "FVII", and values ​​are marked with asterisks

[0678]

[0679]

[0680] Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, variations, and equivalents will be apparent to those of ordinary skill in the art. It is therefore intended that the appended claims cover all such modifications and variations that fall within the true spirit of the present invention.

[0681] Example 31. Identification of antibodies that compete with the anti-TLT-1 antibody mAb1076 for binding to TLT-1 in a competitive ELISA

[0682] The Fab fragment (Fab to TLT-1), which will be used in competition experiments, contains the VH and VL domain sequences corresponding to mAbl076 (SEQ ID NOs: 938 and 934, respectively), the CH1 domain of human IgG4, and human CL-kappa with one point mutation (G157C). The G to C substitution is located in the constant domain of the Fab fragment, i.e., away from the antigen-binding site, and does not affect binding to TLT-1 (see Example 19). The recombinant product was prepared as TLT-1 as described in WO2011 / 023785. Fab to TLT-1 was biotinylated using standard methods, including the use of a biotinylation kit (EZ-link, Thermo), according to the manufacturer's instructions.

[0683] To determine whether anti-TLT-1 antibodies compete with anti-TLT-1 Fab and its derivatives for binding to TLT-1, competition studies were performed. 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 plates were washed and blocked with wash buffer (20 mM HEPES, 5 mM CaCl2, 150 mM NaCl, 0.5 ml / L Tween 20, pH 7.2) for 15 min. For the competition assay, biotinylated anti-TLT-1 Fab at a fixed final concentration was combined with a serial dilution of anti-TLT-1 antibody to obtain final concentrations ranging from 0 to 100 mg / mL in dilution buffer. The mixture was added to the wells of the 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 treated with TMB ONE (KEMENTEC) for 10 min. The reaction was stopped by adding H3PO4 (4 M) and the plate was read on a FLUOStar Optima plate reader at 450 nM with background signal subtraction measured at 620 nM. Unless otherwise stated, all incubations were performed at room temperature, and the plates were washed five times with wash buffer.

[0684] The concentration of recombinant TLT-1 to be immobilized in the NUNC maxisorp plate, as well as a fixed concentration of biotinylated anti-TLT-1 Fab mixed with the competing antibody for the competition assay, were determined by separate titrations of the two components to obtain a sufficient signal to detect competition (i.e., signal reduction) from the competing antibodies. The concentration of TLT-1 for immobilization is typically in the range of 0 to 1 mg / mL, for example, 125 ng / mL. The concentration of biotinylated anti-TLT-1 Fab is typically in the range of 0 to 1 mg / mL, for example, 10 ng / mL.

[0685] From the measured signals (optical density [OD] units), competition at any given antibody concentration was calculated as % inhibition = (1 - (OD units - 100% inhibition value) / (0% inhibition value - 100% inhibition value)) * 100, where 0% inhibition is defined as the signal in wells without any competing anti-TLT-1 antibody and 100% inhibition is defined as the signal in wells without biotinylated anti-TLT-1 Fab (i.e., relevant background to the assay). Antibodies were considered to compete with anti-TLT-1 Fab for binding to TLT-1 if at least 50% inhibition (% inhibition) was observed when the antibody was tested at a concentration with a 10,000-fold excess of biotinylated anti-TLT-1 Fab.

[0686] --->

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[0688] <110> Novo Nordisk A / S

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[0704] Asp Ala Glu Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser Asp Gly Asp

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[0712] Gly Cys Glu Gln Tyr Cys Ser Asp His Thr Gly Thr Lys Arg Ser Cys

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[0714] Arg Cys His Glu Gly Tyr Ser Leu Leu Ala Asp Gly Val Ser Cys Thr

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[0716] Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile Pro Ile Leu Glu Lys Arg

[0717] 130 135 140

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[0765] Thr Asp Leu Gly Gly Gly Leu Leu Gln Val Glu Met Val Thr Leu Gln

[0766] 65 70 75 80

[0767] Glu Glu Asp Ala Gly Glu Tyr Gly Cys Met Val Asp Gly Ala Arg Gly

[0768] 85 90 95

[0769] Pro Gln Ile Leu His Arg Val Ser Leu Asn Ile Leu Pro Pro Glu Glu

[0770] 100 105 110

[0771] Glu Glu Glu Thr His Lys Ile Gly Ser Leu Ala Glu Asn Ala Phe Ser

[0772] 115 120 125

[0773] Asp Pro Ala Gly Ser Ala Asn Pro Leu Glu Pro Ser Gln Asp Glu Lys

[0774] 130 135 140

[0775] Ser Ile Pro Leu Ile Trp Gly Ala Val Leu Leu Val Gly Leu Leu Val

[0776] 145 150 155 160

[0777] Ala Ala Val Val Leu Phe Ala Val Met Ala Lys Arg Lys Gln Gly Asn

[0778] 165 170 175

[0779] Arg Leu Gly Val Cys Gly Arg Phe Leu Ser Ser Arg Val Ser Gly Met

[0780] 180 185 190

[0781] Asn Pro Ser Ser Val Val His His Val Ser Asp Ser Gly Pro Ala Ala

[0782] 195 200 205

[0783] Glu Leu Pro Leu Asp Val Pro His Ile Arg Leu Asp Ser Pro Pro Ser

[0784] 210 215 220

[0785] Phe Asp Asn Thr Thr Tyr Thr Ser Leu Pro Leu Asp Ser Pro Ser Gly

[0786] 225 230 235 240

[0787] Lys Pro Ser Leu Pro Ala Pro Ser Ser Leu Pro Pro Leu Pro Pro Lys

[0788] 245 250 255

[0789] Val Leu Val Cys Ser Lys Pro Val Thr Tyr Ala Thr Val Ile Phe Pro

[0790] 260 265 270

[0791] Gly Gly Asn Lys Gly Gly Gly Thr Ser Cys Gly Pro Ala Gln Asn Pro

[0792] 275 280 285

[0793] Pro Asn Asn Gln Thr Pro Ser Ser

[0794] 290 295

[0795] <210> 3

[0796] <211> 147

[0797] <212> БЕЛОК

[0798] <213> Homo sapiens

[0799] <400> 3

[0800] Gln Gly Ile Val Gly Ser Leu Pro Glu Val Leu Gln Ala Pro Val Gly

[0801] 1 5 10 15

[0802] Ser Ser Ile Leu Val Gln Cys His Tyr Arg Leu Gln Asp Val Lys Ala

[0803] 20 25 30

[0804] Gln Lys Val Trp Cys Arg Phe Leu Pro Glu Gly Cys Gln Pro Leu Val

[0805] 35 40 45

[0806] Ser Ser Ala Val Asp Arg Arg Ala Pro Ala Gly Arg Arg Thr Phe Leu

[0807] 50 55 60

[0808] Thr Asp Leu Gly Gly Gly Leu Leu Gln Val Glu Met Val Thr Leu Gln

[0809] 65 70 75 80

[0810] Glu Glu Asp Ala Gly Glu Tyr Gly Cys Met Val Asp Gly Ala Arg Gly

[0811] 85 90 95

[0812] Pro Gln Ile Leu His Arg Val Ser Leu Asn Ile Leu Pro Pro Glu Glu

[0813] 100 105 110

[0814] Glu Glu Glu Thr His Lys Ile Gly Ser Leu Ala Glu Asn Ala Phe Ser

[0815] 115 120 125

[0816] Asp Pro Ala Gly Ser Ala Asn Pro Leu Glu Pro Ser Gln Asp Glu Lys

[0817] 130 135 140

[0818] Ser Ile Pro

[0819] 145

[0820] <210> 4

[0821] <211> 327

[0822] <212> PROTEIN

[0823] <213> Artificial sequence

[0824] <220>

[0825] <223> Recombinant protein

[0826] <400> 4

[0827] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

[0828] 1 5 10 15

[0829] Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

[0830] 20 25 30

[0831] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

[0832] 35 40 45

[0833] Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

[0834] 50 55 60

[0835] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

[0836] 65 70 75 80

[0837] Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

[0838] 85 90 95

[0839] Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

[0840] 100 105 110

[0841] Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

[0842] 115 120 125

[0843] Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

[0844] 130 135 140

[0845] Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

[0846] 145 150 155 160

[0847] Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

[0848] 165 170 175

[0849] Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

[0850] 180 185 190

[0851] Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

[0852] 195 200 205

[0853] Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

[0854] 210 215 220

[0855] Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

[0856] 225 230 235 240

[0857] Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

[0858] 245 250 255

[0859] Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

[0860] 260 265 270

[0861] Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

[0862] 275 280 285

[0863] Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

[0864] 290 295 300

[0865] Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

[0866] 305 310 315 320

[0867] Leu Ser Leu Ser Leu Gly Lys

[0868] 325

[0869] <210> 5

[0870] <211> 327

[0871] <212> PROTEIN

[0872] <213> Artificial sequence

[0873] <220>

[0874] <223> Recombinant protein

[0875] <400> 5

[0876] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

[0877] 1 5 10 15

[0878] Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

[0879] 20 25 30

[0880] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

[0881] 35 40 45

[0882] Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

[0883] 50 55 60

[0884] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

[0885] 65 70 75 80

[0886] Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

[0887] 85 90 95

[0888] Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

[0889] 100 105 110

[0890] Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

[0891] 115 120 125

[0892] Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

[0893] 130 135 140

[0894] Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

[0895] 145 150 155 160

[0896] Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

[0897] 165 170 175

[0898] Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

[0899] 180 185 190

[0900] Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

[0901] 195 200 205

[0902] Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

[0903] 210 215 220

[0904] Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

[0905] 225 230 235 240

[0906] Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

[0907] 245 250 255

[0908] Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

[0909] 260 265 270

[0910] Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser

[0911] 275 280 285

[0912] Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

[0913] 290 295 300

[0914] Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

[0915] 305 310 315 320

[0916] Leu Ser Leu Ser Leu Gly Lys

[0917] 325

[0918] <210> 6

[0919] <211> 327

[0920] <212> PROTEIN

[0921] <213> Artificial sequence

[0922] <220>

[0923] <223> Recombinant protein

[0924] <400> 6

[0925] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

[0926] 1 5 10 15

[0927] Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

[0928] 20 25 30

[0929] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

[0930] 35 40 45

[0931] Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

[0932] 50 55 60

[0933] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

[0934] 65 70 75 80

[0935] Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

[0936] 85 90 95

[0937] Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

[0938] 100 105 110

[0939] Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

[0940] 115 120 125

[0941] Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val

[0942] 130 135 140

[0943] Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

[0944] 145 150 155 160

[0945] Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

[0946] 165 170 175

[0947] Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

[0948] 180 185 190

[0949] Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

[0950] 195 200 205

[0951] Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

[0952] 210 215 220

[0953] Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

[0954] 225 230 235 240

[0955] Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

[0956] 245 250 255

[0957] Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

[0958] 260 265 270

[0959] Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

[0960] 275 280 285

[0961] Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

[0962] 290 295 300

[0963] Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

[0964] 305 310 315 320

[0965] Leu Ser Leu Ser Leu Gly Lys

[0966] 325

[0967] <210> 7

[0968] <211> 327

[0969] <212> PROTEIN

[0970] <213> Artificial sequence

[0971] <220>

[0972] <223> Recombinant protein

[0973] <400> 7

[0974] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

[0975] 1 5 10 15

[0976] Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

[0977] 20 25 30

[0978] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

[0979] 35 40 45

[0980] Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

[0981] 50 55 60

[0982] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

[0983] 65 70 75 80

[0984] Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

[0985] 85 90 95

[0986] Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

[0987] 100 105 110

[0988] Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

[0989] 115 120 125

[0990] Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val

[0991] 130 135 140

[0992] Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

[0993] 145 150 155 160

[0994] Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

[0995] 165 170 175

[0996] Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

[0997] 180 185 190

[0998] Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

[0999] 195 200 205

[1000] Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

[1001] 210 215 220

[1002] Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

[1003] 225 230 235 240

[1004] Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

[1005] 245 250 255

[1006] Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

[1007] 260 265 270

[1008] Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser

[1009] 275 280 285

[1010] Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

[1011] 290 295 300

[1012] Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

[1013] 305 310 315 320

[1014] Leu Ser Leu Ser Leu Gly Lys

[1015] 325

[1016] <210> 8

[1017] <211> 230

[1018] <212> PROTEIN

[1019] <213> Artificial sequence

[1020] <220>

[1021] <223> Recombinant protein

[1022] <400> 8

[1023] Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

[1024] 1 5 10 15

[1025] Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

[1026] 20 25 30

[1027] Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

[1028] 35 40 45

[1029] Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

[1030] 50 55 60

[1031] Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

[1032] 65 70 75 80

[1033] Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

[1034] 85 90 95

[1035] Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

[1036] 100 105 110

[1037] Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

[1038] 115 120 125

[1039] Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

[1040] 130 135 140

[1041] Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

[1042] 145 150 155 160

[1043] Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

[1044] 165 170 175

[1045] Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys

[1046] 180 185 190

[1047] Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

[1048] 195 200 205

[1049] Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

[1050] 210 215 220

[1051] Ser Leu Ser Leu Gly Lys

[1052] 225 230

[1053] <210> 9

[1054] <211> 327

[1055] <212> PROTEIN

[1056] <213> Artificial sequence

[1057] <220>

[1058] <223> Recombinant protein

[1059] <400> 9

[1060] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

[1061] 1 5 10 15

[1062] Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

[1063] 20 25 30

[1064] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

[1065] 35 40 45

[1066] Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

[1067] 50 55 60

[1068] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

[1069] 65 70 75 80

[1070] Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

[1071] 85 90 95

[1072] Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

[1073] 100 105 110

[1074] Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

[1075] 115 120 125

[1076] Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

[1077] 130 135 140

[1078] Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

[1079] 145 150 155 160

[1080] Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

[1081] 165 170 175

[1082] Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

[1083] 180 185 190

[1084] Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

[1085] 195 200 205

[1086] Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

[1087] 210 215 220

[1088] Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

[1089] 225 230 235 240

[1090] Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp

[1091] 245 250 255

[1092] Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

[1093] 260 265 270

[1094] Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser

[1095] 275 280 285

[1096] Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

[1097] 290 295 300

[1098] Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

[1099] 305 310 315 320

[1100] Leu Ser Leu Ser Leu Gly Lys

[1101] 325

[1102] <210> 10

[1103] <211> 229

[1104] <212> PROTEIN

[1105] <213> Artificial sequence

[1106] <220>

[1107] <223> Recombinant protein

[1108] <400> 10

[1109] Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

[1110] 1 5 10 15

[1111] Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

[1112] 20 25 30

[1113] Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

[1114] 35 40 45

[1115] Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

[1116] 50 55 60

[1117] Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

[1118] 65 70 75 80

[1119] Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

[1120] 85 90 95

[1121] Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

[1122] 100 105 110

[1123] Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

[1124] 115 120 125

[1125] Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

[1126] 130 135 140

[1127] Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

[1128] 145 150 155 160

[1129] Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

[1130] 165 170 175

[1131] Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

[1132] 180 185 190

[1133] Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

[1134] 195 200 205

[1135] Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

[1136] 210 215 220

[1137] Leu Ser Leu Gly Lys

[1138] 225

[1139] <210> 11

[1140] <211> 98

[1141] <212> PROTEIN

[1142] <213> Artificial sequence

[1143] <220>

[1144] <223> Recombinant protein

[1145] <400> 11

[1146] Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

[1147] 1 5 10 15

[1148] Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

[1149] 20 25 30

[1150] Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

[1151] 35 40 45

[1152] Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

[1153] 50 55 60

[1154] Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

[1155] 65 70 75 80

[1156] Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

[1157] 85 90 95

[1158] Arg Val

[1159] <210> 12

[1160] <211> 107

[1161] <212> PROTEIN

[1162] <213> Artificial sequence

[1163] <220>

[1164] <223> Recombinant protein

[1165] <400> 12

[1166] Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

[1167] 1 5 10 15

[1168] Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

[1169] 20 25 30

[1170] Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

[1171] 35 40 45

[1172] Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

[1173] 50 55 60

[1174] Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

[1175] 65 70 75 80

[1176] Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

[1177] 85 90 95

[1178] Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

[1179] 100 105

[1180] <210> 13

[1181] <211> 37

[1182] <212> PROTEIN

[1183] <213> Artificial sequence

[1184] <220>

[1185] <223> Recombinant protein

[1186] <400> 13

[1187] Glu Glu Glu Glu Glu Thr His Lys Ile Gly Ser Leu Ala Glu Asn Ala

[1188] 1 5 10 15

[1189] Phe Ser Asp Pro Ala Gly Ser Ala Asn Pro Leu Glu Pro Ser Gln Asp

[1190] 20 25 30

[1191] Glu Lys Ser Ile Pro

[1192] 35

[1193] <210> 14

[1194] <211> 107

[1195] <212> PROTEIN

[1196] <213> Mus musculus

[1197] <400> 14

[1198] Asp Ile Val Met Thr Gln Ser His Lys Ile Met Ser Thr Leu Val Gly

[1199] 1 5 10 15

[1200] Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Pro Ala

[1201] 20 25 30

[1202] Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Tyr Pro Lys Leu Leu Ile

[1203] 35 40 45

[1204] Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

[1205] 50 55 60

[1206] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

[1207] 65 70 75 80

[1208] Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Lys Tyr Pro Tyr

[1209] 85 90 95

[1210] Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

[1211] 100 105

[1212] <210> 15

[1213] <211> 11

[1214] <212> БЕЛОК

[1215] <213> Mus musculus

[1216] <400> 15

[1217] Lys Ala Ser Gln Asp Val Asp Pro Ala Val Ala

[1218] 1 5 10

[1219] <210> 16

[1220] <211> 7

[1221] <212> БЕЛОК

[1222] <213> Mus musculus

[1223] <400> 16

[1224] Trp Ala Ser Thr Arg His Thr

[1225] 1 5

[1226] <210> 17

[1227] <211> 9

[1228] <212> WHITE

[1229] <213> Mus musculus

[1230] <400> 17

[1231] Gln Gln Tyr Ser Lys Tyr Pro Tyr Thr

[1232] 1 5

[1233] <210> 18

[1234] <211> 118

[1235] <212> WHITE

[1236] <213> Mus musculus

[1237] <400> 18

[1238] Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

[1239] 1 5 10 15

[1240] Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr

[1241] 20 25 30

[1242] Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

[1243] 35 40 45

[1244] Gly Val Ile Trp Gly Gly Gly Asn Thr Tyr Asn Asn Ser Ala Leu Lys

[1245] 50 55 60

[1246] Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

[1247] 65 70 75 80

[1248] Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Met Tyr Phe Cys Ala

[1249] 85 90 95

[1250] Lys His Gly Tyr Tyr Gly Tyr Val Met Asp Tyr Trp Gly Gln Gly Thr

[1251] 100 105 110

[1252] Ser Val Thr Val Ser Ser

[1253] 115

[1254] <210> 19

[1255] <211> 5

[1256] <212> WHITE

[1257] <213> Mouse musculus

[1258] <400> 19

[1259] Asp Tyr Gly Val Ser

[1260] 1 5

[1261] <210> 20

[1262] <211> 16

[1263] <212> WHITE

[1264] <213> Mouse musculus

[1265] <400> 20

[1266] Val Ile Trp Gly Gly Gly Asn Thr Tyr Asn Asn Ser Ala Leu Lys Ser

[1267] 1 5 10 15

[1268] <210> 21

[1269] <211> 10

[1270] <212> WHITE

[1271] <213> Mouse musculus

[1272] <400> 21

[1273] His Gly Tyr Tyr Gly Tyr Val Met Asp Tyr

[1274] 1 5 10

[1275] <210> 22

[1276] <211> 107

[1277] <212> PROTEIN

[1278] <213> Artificial sequence

[1279] <220>

[1280] <223> Recombinant protein

[1281] <400> 22

[1282] Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Ala Gly

[1283] 1 5 10 15

[1284] Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[1285] 20 25 30

[1286] Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

[1287] 35 40 45

[1288] Lys Tyr Thr Ser Gln Pro Val Ser Gly Ile Pro Ser Arg Phe Ser Gly

[1289] 50 55 60

[1290] Ser Gly Ser Gly Ser Glu Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

[1291] 65 70 75 80

[1292] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[1293] 85 90 95

[1294] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[1295] 100 105

[1296] <210> 23

[1297] <211> 11

[1298] <212> PROTEIN

[1299] <213> Artificial sequence

[1300] <220>

[1301] <223> Recombinant protein

[1302] <400> 23

[1303] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[1304] 1 5 10

[1305] <210> 24

[1306] <211> 7

[1307] <212> PROTEIN

[1308] <213> Artificial sequence

[1309] <220>

[1310] <223> Recombinant protein

[1311] <400> 24

[1312] Tyr Thr Ser Gln Pro Val Ser

[1313] 1 5

[1314] <210> 25

[1315] <211> 9

[1316] <212> PROTEIN

[1317] <213> Artificial sequence

[1318] <220>

[1319] <223> Recombinant protein

[1320] <400> 25

[1321] Gln Asn Gly His Ser Phe Pro Leu Thr

[1322] 1 5

[1323] <210> 26

[1324] <211> 122

[1325] <212> PROTEIN

[1326] <213> Artificial sequence

[1327] <220>

[1328] <223> Recombinant protein

[1329] <400> 26

[1330] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[1331] 1 5 10 15

[1332] Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

[1333] 20 25 30

[1334] Cys Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

[1335] 35 40 45

[1336] Met Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[1337] 50 55 60

[1338] Lys Ser Arg Ile Ser Val Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

[1339] 65 70 75 80

[1340] Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

[1341] 85 90 95

[1342] Thr Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[1343] 100 105 110

[1344] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[1345] 115 120

[1346] <210> 27

[1347] <211> 6

[1348] <212> PROTEIN

[1349] <213> Artificial sequence

[1350] <220>

[1351] <223> Recombinant protein

[1352] <400> 27

[1353] Ser Asp Cys Ala Trp Ser

[1354] 1 5

[1355] <210> 28

[1356] <211> 16

[1357] <212> PROTEIN

[1358] <213> Artificial sequence

[1359] <220>

[1360] <223> Recombinant protein

[1361] <400> 28

[1362] Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[1363] 1 5 10 15

[1364] <210> 29

[1365] <211> 13

[1366] <212> PROTEIN

[1367] <213> Artificial sequence

[1368] <220>

[1369] <223> Recombinant protein

[1370] <400> 29

[1371] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[1372] 1 5 10

[1373] <210> 30

[1374] <211> 107

[1375] <212> PROTEIN

[1376] <213> Artificial sequence

[1377] <220>

[1378] <223> Recombinant protein

[1379] <400> 30

[1380] Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Ala Gly

[1381] 1 5 10 15

[1382] Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[1383] 20 25 30

[1384] Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

[1385] 35 40 45

[1386] Lys Tyr Thr Ser Gln Pro Val Ser Gly Ile Pro Ser Arg Phe Ser Gly

[1387] 50 55 60

[1388] Ser Gly Ser Gly Ser Glu Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

[1389] 65 70 75 80

[1390] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[1391] 85 90 95

[1392] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[1393] 100 105

[1394] <210> 31

[1395] <211> 11

[1396] <212> PROTEIN

[1397] <213> Artificial sequence

[1398] <220>

[1399] <223> Recombinant protein

[1400] <400> 31

[1401] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[1402] 1 5 10

[1403] <210> 32

[1404] <211> 7

[1405] <212> PROTEIN

[1406] <213> Artificial sequence

[1407] <220>

[1408] <223> Recombinant protein

[1409] <400> 32

[1410] Tyr Thr Ser Gln Pro Val Ser

[1411] 1 5

[1412] <210> 33

[1413] <211> 9

[1414] <212> PROTEIN

[1415] <213> Artificial sequence

[1416] <220>

[1417] <223> Recombinant protein

[1418] <400> 33

[1419] Gln Asn Gly His Ser Phe Pro Leu Thr

[1420] 1 5

[1421] <210> 34

[1422] <211> 122

[1423] <212> PROTEIN

[1424] <213> Artificial sequence

[1425] <220>

[1426] <223> Recombinant protein

[1427] <400> 34

[1428] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[1429] 1 5 10 15

[1430] Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

[1431] 20 25 30

[1432] Cys Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

[1433] 35 40 45

[1434] Met Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[1435] 50 55 60

[1436] Lys Ser Arg Ile Ser Val Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

[1437] 65 70 75 80

[1438] Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

[1439] 85 90 95

[1440] Thr Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[1441] 100 105 110

[1442] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[1443] 115 120

[1444] <210> 35

[1445] <211> 6

[1446] <212> PROTEIN

[1447] <213> Artificial sequence

[1448] <220>

[1449] <223> Recombinant protein

[1450] <400> 35

[1451] Ser Asp Cys Ala Trp Ser

[1452] 1 5

[1453] <210> 36

[1454] <211> 16

[1455] <212> PROTEIN

[1456] <213> Artificial sequence

[1457] <220>

[1458] <223> Recombinant protein

[1459] <400> 36

[1460] Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[1461] 1 5 10 15

[1462] <210> 37

[1463] <211> 13

[1464] <212> PROTEIN

[1465] <213> Artificial sequence

[1466] <220>

[1467] <223> Recombinant protein

[1468] <400> 37

[1469] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[1470] 1 5 10

[1471] <210> 38

[1472] <211> 107

[1473] <212> PROTEIN

[1474] <213> Artificial sequence

[1475] <220>

[1476] <223> Recombinant protein

[1477] <400> 38

[1478] Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Ala Gly

[1479] 1 5 10 15

[1480] Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[1481] 20 25 30

[1482] Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

[1483] 35 40 45

[1484] Lys Tyr Thr Ser Gln Pro Val Ser Gly Ile Pro Ser Arg Phe Ser Gly

[1485] 50 55 60

[1486] Ser Gly Ser Gly Ser Glu Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

[1487] 65 70 75 80

[1488] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[1489] 85 90 95

[1490] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[1491] 100 105

[1492] <210> 39

[1493] <211> 11

[1494] <212> PROTEIN

[1495] <213> Artificial sequence

[1496] <220>

[1497] <223> Recombinant protein

[1498] <400> 39

[1499] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[1500] 1 5 10

[1501] <210> 40

[1502] <211> 7

[1503] <212> PROTEIN

[1504] <213> Artificial sequence

[1505] <220>

[1506] <223> Recombinant protein

[1507] <400> 40

[1508] Tyr Thr Ser Gln Pro Val Ser

[1509] 1 5

[1510] <210> 41

[1511] <211> 9

[1512] <212> PROTEIN

[1513] <213> Artificial sequence

[1514] <220>

[1515] <223> Recombinant protein

[1516] <400> 41

[1517] Gln Asn Gly His Ser Phe Pro Leu Thr

[1518] 1 5

[1519] <210> 42

[1520] <211> 122

[1521] <212> PROTEIN

[1522] <213> Artificial sequence

[1523] <220>

[1524] <223> Recombinant protein

[1525] <400> 42

[1526] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[1527] 1 5 10 15

[1528] Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

[1529] 20 25 30

[1530] Cys Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

[1531] 35 40 45

[1532] Met Gly Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[1533] 50 55 60

[1534] Lys Ser Arg Ile Ser Val Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

[1535] 65 70 75 80

[1536] Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

[1537] 85 90 95

[1538] Thr Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[1539] 100 105 110

[1540] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[1541] 115 120

[1542] <210> 43

[1543] <211> 6

[1544] <212> PROTEIN

[1545] <213> Artificial sequence

[1546] <220>

[1547] <223> Recombinant protein

[1548] <400> 43

[1549] Ser Asp Cys Ala Trp Ser

[1550] 1 5

[1551] <210> 44

[1552] <211> 16

[1553] <212> PROTEIN

[1554] <213> Artificial sequence

[1555] <220>

[1556] <223> Recombinant protein

[1557] <400> 44

[1558] Tyr Ile Asn Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[1559] 1 5 10 15

[1560] <210> 45

[1561] <211> 13

[1562] <212> PROTEIN

[1563] <213> Artificial sequence

[1564] <220>

[1565] <223> Recombinant protein

[1566] <400> 45

[1567] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[1568] 1 5 10

[1569] <210> 46

[1570] <211> 113

[1571] <212> WHITE

[1572] <213> Mus musculus

[1573] <400> 46

[1574] Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

[1575] 1 5 10 15

[1576] Glu Lys Val Thr Val Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

[1577] 20 25 30

[1578] Gly Asp Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

[1579] 35 40 45

[1580] Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

[1581] 50 55 60

[1582] Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

[1583] 65 70 75 80

[1584] Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

[1585] 85 90 95

[1586] Asp Phe Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

[1587] 100 105 110

[1588] Light

[1589] <210> 47

[1590] <211> 17

[1591] <212> WHITE

[1592] <213> Mus musculus

[1593] <400> 47

[1594] Light Ser Ser Gln Ser Leu Leu Asn Ser Gly Asp Gln Lys Asn Tyr Leu

[1595] 1 5 10 15

[1596] Thr

[1597] <210> 48

[1598] <211> 7

[1599] <212> WHITE

[1600] <213> Mus musculus

[1601] <400> 48

[1602] Trp Ala Ser Thr Arg Glu Ser

[1603] 1 5

[1604] <210> 49

[1605] <211> 9

[1606] <212> WHITE

[1607] <213> Mus musculus

[1608] <400> 49

[1609] Gln Asn Asp Phe Ser Tyr Pro Tyr Thr

[1610] 1 5

[1611] <210> 50

[1612] <211> 118

[1613] <212> WHITE

[1614] <213> Mus musculus

[1615] <400> 50

[1616] Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

[1617] 1 5 10 15

[1618] Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

[1619] 20 25 30

[1620] Ser Ile Gln Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

[1621] 35 40 45

[1622] Gly Trp Ile Asn Thr Glu Thr Gly Lys Pro Ser Tyr Ala Asp Asp Leu

[1623] 50 55 60

[1624] Lys Gly Arg Ser Ala Phe Ser Leu Glu Thr Ser Gly Ser Thr Ala Tyr

[1625] 65 70 75 80

[1626] Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys

[1627] 85 90 95

[1628] Val Arg Asp Asp Tyr Tyr Gly Arg Glu Asp Tyr Trp Gly Gln Gly Thr

[1629] 100 105 110

[1630] Thr Leu Thr Val Ser Ser

[1631] 115

[1632] <210> 51

[1633] <211> 5

[1634] <212> БЕЛОК

[1635] <213> Mus musculus

[1636] <400> 51

[1637] Asp Tyr Ser Ile Gln

[1638] 1 5

[1639] <210> 52

[1640] <211> 17

[1641] <212> PROTEIN

[1642] <213> Mus musculus

[1643] <400> 52

[1644] Trp Ile Asn Thr Glu Thr Gly Lys Pro Ser Tyr Ala Asp Asp Leu Lys

[1645] 1 5 10 15

[1646] Gly

[1647] <210> 53

[1648] <211> 9

[1649] <212> PROTEIN

[1650] <213> Mus musculus

[1651] <400> 53

[1652] Asp Asp Tyr Tyr Gly Arg Glu Asp Tyr

[1653] 1 5

[1654] <210> 54

[1655] <211> 107

[1656] <212> PROTEIN

[1657] <213> Artificial sequence

[1658] <220>

[1659] <223> Recombinant protein

[1660] <400> 54

[1661] Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Ala Gly

[1662] 1 5 10 15

[1663] Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[1664] 20 25 30

[1665] Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

[1666] 35 40 45

[1667] Lys Tyr Thr Ser Gln Pro Val Ser Gly Ile Pro Ser Arg Phe Ser Gly

[1668] 50 55 60

[1669] Ser Gly Ser Gly Ser Glu Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

[1670] 65 70 75 80

[1671] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[1672] 85 90 95

[1673] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[1674] 100 105

[1675] <210> 55

[1676] <211> 11

[1677] <212> PROTEIN

[1678] <213> Artificial sequence

[1679] <220>

[1680] <223> Recombinant protein

[1681] <400> 55

[1682] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[1683] 1 5 10

[1684] <210> 56

[1685] <211> 7

[1686] <212> PROTEIN

[1687] <213> Artificial sequence

[1688] <220>

[1689] <223> Recombinant protein

[1690] <400> 56

[1691] Tyr Thr Ser Gln Pro Val Ser

[1692] 1 5

[1693] <210> 57

[1694] <211> 9

[1695] <212> PROTEIN

[1696] <213> Artificial sequence

[1697] <220>

[1698] <223> Recombinant protein

[1699] <400> 57

[1700] Gln Asn Gly His Ser Phe Pro Leu Thr

[1701] 1 5

[1702] <210> 58

[1703] <211> 122

[1704] <212> PROTEIN

[1705] <213> Artificial sequence

[1706] <220>

[1707] <223> Recombinant protein

[1708] <400> 58

[1709] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[1710] 1 5 10 15

[1711] Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

[1712] 20 25 30

[1713] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

[1714] 35 40 45

[1715] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[1716] 50 55 60

[1717] Lys Ser Arg Ile Ser Val Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

[1718] 65 70 75 80

[1719] Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

[1720] 85 90 95

[1721] Thr Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[1722] 100 105 110

[1723] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[1724] 115 120

[1725] <210> 59

[1726] <211> 6

[1727] <212> PROTEIN

[1728] <213> Artificial sequence

[1729] <220>

[1730] <223> Recombinant protein

[1731] <400> 59

[1732] Ser Asp Ser Ala Trp Ser

[1733] 1 5

[1734] <210> 60

[1735] <211> 16

[1736] <212> PROTEIN

[1737] <213> Artificial sequence

[1738] <220>

[1739] <223> Recombinant protein

[1740] <400> 60

[1741] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[1742] 1 5 10 15

[1743] <210> 61

[1744] <211> 13

[1745] <212> PROTEIN

[1746] <213> Artificial sequence

[1747] <220>

[1748] <223> Recombinant protein

[1749] <400> 61

[1750] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[1751] 1 5 10

[1752] <210> 62

[1753] <211> 113

[1754] <212> PROTEIN

[1755] <213> Mus musculus

[1756] <400> 62

[1757] Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

[1758] 1 5 10 15

[1759] Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

[1760] 20 25 30

[1761] Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln His Lys Pro Gly Gln

[1762] 35 40 45

[1763] Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

[1764] 50 55 60

[1765] Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

[1766] 65 70 75 80

[1767] Ile Ser Asn Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln

[1768] 85 90 95

[1769] Tyr Tyr Asn Tyr Leu Leu Thr Phe Gly Pro Gly Thr Lys Leu Glu Leu

[1770] 100 105 110

[1771] Light

[1772] <210> 63

[1773] <211> 17

[1774] <212> WHITE

[1775] <213> Mus musculus

[1776] <400> 63

[1777] Light See See Green See Leu Leu Bull See See Donkey Green Light Donkey See Leu

[1778] 1 5 10 15

[1779] Ala

[1780] <210> 64

[1781] <211> 7

[1782] <212> WHITE

[1783] <213> Mus musculus

[1784] <400> 64

[1785] Trp Ala Ser Thr Arg Glu Ser

[1786] 1 5

[1787] <210> 65

[1788] <211> 9

[1789] <212> БЕЛОК

[1790] <213> Mus musculus

[1791] <400> 65

[1792] His Gln Tyr Tyr Asn Tyr Leu Leu Thr

[1793] 1 5

[1794] <210> 66

[1795] <211> 123

[1796] <212> БЕЛОК

[1797] <213> Mus musculus

[1798] <400> 66

[1799] Gln Ile Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln

[1800] 1 5 10 15

[1801] Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

[1802] 20 25 30

[1803] Gly Leu Gly Val Gly Trp Ile Arg Gln Thr Ser Gly Lys Gly Leu Glu

[1804] 35 40 45

[1805] Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Tyr Pro Pro

[1806] 50 55 60

[1807] Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val

[1808] 65 70 75 80

[1809] Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

[1810] 85 90 95

[1811] Cys Ala Arg Arg Ala Phe Tyr Asp Gly Tyr Tyr Gly Pro Met Glu Tyr

[1812] 100 105 110

[1813] Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[1814] 115 120

[1815] <210> 67

[1816] <211> 7

[1817] <212> WHITE

[1818] <213> Mus musculus

[1819] <400> 67

[1820] Thr Ser Gly Leu Gly Val Gly

[1821] 1 5

[1822] <210> 68

[1823] <211> 16

[1824] <212> WHITE

[1825] <213> Mus musculus

[1826] <400> 68

[1827] His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Tyr Pro Pro Leu Lys Ser

[1828] 1 5 10 15

[1829] <210> 69

[1830] <211> 13

[1831] <212> WHITE

[1832] <213> Mus musculus

[1833] <400> 69

[1834] Arg Ala Phe Tyr Asp Gly Tyr Tyr Gly Pro Met Glu Tyr

[1835] 1 5 10

[1836] <210> 70

[1837] <211> 107

[1838] <212> WHITE

[1839] <213> Artificial sequence

[1840] <220>

[1841] <223> Recombinant protein

[1842] <400> 70

[1843] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[1844] 1 5 10 15

[1845] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Tyr

[1846] 20 25 30

[1847] Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

[1848] 35 40 45

[1849] Lys Tyr Ala Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[1850] 50 55 60

[1851] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[1852] 65 70 75 80

[1853] Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Gly His Ser Phe Pro Tyr

[1854] 85 90 95

[1855] Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

[1856] 100 105

[1857] <210> 71

[1858] <211> 11

[1859] <212> PROTEIN

[1860] <213> Artificial sequence

[1861] <220>

[1862] <223> Recombinant protein

[1863] <400> 71

[1864] Arg Ala Ser Gln Gly Val Ser Ser Tyr Leu His

[1865] 1 5 10

[1866] <210> 72

[1867] <211> 7

[1868] <212> PROTEIN

[1869] <213> Artificial sequence

[1870] <220>

[1871] <223> Recombinant protein

[1872] <400> 72

[1873] Tyr Ala Ser Gln Pro Ala Thr

[1874] 1 5

[1875] <210> 73

[1876] <211> 9

[1877] <212> PROTEIN

[1878] <213> Artificial sequence

[1879] <220>

[1880] <223> Recombinant protein

[1881] <400> 73

[1882] Gln Ser Gly His Ser Phe Pro Tyr Thr

[1883] 1 5

[1884] <210> 74

[1885] <211> 122

[1886] <212> PROTEIN

[1887] <213> Artificial sequence

[1888] <220>

[1889] <223> Recombinant protein

[1890] <400> 74

[1891] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[1892] 1 5 10 15

[1893] Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

[1894] 20 25 30

[1895] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

[1896] 35 40 45

[1897] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[1898] 50 55 60

[1899] Lys Ser Arg Ile Ser Val Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

[1900] 65 70 75 80

[1901] Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

[1902] 85 90 95

[1903] Thr Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[1904] 100 105 110

[1905] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[1906] 115 120

[1907] <210> 75

[1908] <211> 6

[1909] <212> PROTEIN

[1910] <213> Artificial sequence

[1911] <220>

[1912] <223> Recombinant protein

[1913] <400> 75

[1914] Ser Asp Ser Ala Trp Ser

[1915] 1 5

[1916] <210> 76

[1917] <211> 16

[1918] <212> PROTEIN

[1919] <213> Artificial sequence

[1920] <220>

[1921] <223> Recombinant protein

[1922] <400> 76

[1923] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[1924] 1 5 10 15

[1925] <210> 77

[1926] <211> 13

[1927] <212> PROTEIN

[1928] <213> Artificial sequence

[1929] <220>

[1930] <223> Recombinant protein

[1931] <400> 77

[1932] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[1933] 1 5 10

[1934] <210> 78

[1935] <211> 107

[1936] <212> PROTEIN

[1937] <213> Artificial sequence

[1938] <220>

[1939] <223> Recombinant protein

[1940] <400> 78

[1941] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[1942] 1 5 10 15

[1943] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[1944] 20 25 30

[1945] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[1946] 35 40 45

[1947] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[1948] 50 55 60

[1949] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[1950] 65 70 75 80

[1951] Glu Asp Val Gly Val Tyr Tyr Cys Gln Ser Gly His Ser Phe Pro Leu

[1952] 85 90 95

[1953] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[1954] 100 105

[1955] <210> 79

[1956] <211> 11

[1957] <212> PROTEIN

[1958] <213> Artificial sequence

[1959] <220>

[1960] <223> Recombinant protein

[1961] <400> 79

[1962] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[1963] 1 5 10

[1964] <210> 80

[1965] <211> 7

[1966] <212> PROTEIN

[1967] <213> Artificial sequence

[1968] <220>

[1969] <223> Recombinant protein

[1970] <400> 80

[1971] Tyr Thr Ser Gln Pro Ala Thr

[1972] 1 5

[1973] <210> 81

[1974] <211> 9

[1975] <212> PROTEIN

[1976] <213> Artificial sequence

[1977] <220>

[1978] <223> Recombinant protein

[1979] <400> 81

[1980] Gln Ser Gly His Ser Phe Pro Leu Thr

[1981] 1 5

[1982] <210> 82

[1983] <211> 122

[1984] <212> PROTEIN

[1985] <213> Artificial sequence

[1986] <220>

[1987] <223> Recombinant protein

[1988] <400> 82

[1989] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[1990] 1 5 10 15

[1991] Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

[1992] 20 25 30

[1993] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

[1994] 35 40 45

[1995] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[1996] 50 55 60

[1997] Lys Ser Arg Ile Ser Val Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

[1998] 65 70 75 80

[1999] Leu Arg Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys

[2000] 85 90 95

[2001] Thr Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[2002] 100 105 110

[2003] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2004] 115 120

[2005] <210> 83

[2006] <211> 6

[2007] <212> PROTEIN

[2008] <213> Artificial sequence

[2009] <220>

[2010] <223> Recombinant protein

[2011] <400> 83

[2012] Ser Asp Ser Ala Trp Ser

[2013] 1 5

[2014] <210> 84

[2015] <211> 16

[2016] <212> PROTEIN

[2017] <213> Artificial sequence

[2018] <220>

[2019] <223> Recombinant protein

[2020] <400> 84

[2021] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2022] 1 5 10 15

[2023] <210> 85

[2024] <211> 13

[2025] <212> PROTEIN

[2026] <213> Artificial sequence

[2027] <220>

[2028] <223> Recombinant protein

[2029] <400> 85

[2030] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[2031] 1 5 10

[2032] <210> 86

[2033] <211> 107

[2034] <212> PROTEIN

[2035] <213> Artificial sequence

[2036] <220>

[2037] <223> Recombinant protein

[2038] <400> 86

[2039] Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Ala Gly

[2040] 1 5 10 15

[2041] Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2042] 20 25 30

[2043] Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

[2044] 35 40 45

[2045] Lys Tyr Thr Ser Gln Pro Val Ser Gly Ile Pro Ser Arg Phe Ser Gly

[2046] 50 55 60

[2047] Ser Gly Ser Gly Ser Glu Phe Thr Leu Ser Ile Asn Ser Val Glu Pro

[2048] 65 70 75 80

[2049] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2050] 85 90 95

[2051] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2052] 100 105

[2053] <210> 87

[2054] <211> 11

[2055] <212> PROTEIN

[2056] <213> Artificial sequence

[2057] <220>

[2058] <223> Recombinant protein

[2059] <400> 87

[2060] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2061] 1 5 10

[2062] <210> 88

[2063] <211> 7

[2064] <212> PROTEIN

[2065] <213> Artificial sequence

[2066] <220>

[2067] <223> Recombinant protein

[2068] <400> 88

[2069] Tyr Thr Ser Gln Pro Val Ser

[2070] 1 5

[2071] <210> 89

[2072] <211> 9

[2073] <212> PROTEIN

[2074] <213> Artificial sequence

[2075] <220>

[2076] <223> Recombinant protein

[2077] <400> 89

[2078] Gln Asn Gly His Ser Phe Pro Leu Thr

[2079] 1 5

[2080] <210> 90

[2081] <211> 122

[2082] <212> PROTEIN

[2083] <213> Artificial sequence

[2084] <220>

[2085] <223> Recombinant protein

[2086] <400> 90

[2087] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

[2088] 1 5 10 15

[2089] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Asp

[2090] 20 25 30

[2091] Ser Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

[2092] 35 40 45

[2093] Ile Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2094] 50 55 60

[2095] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2096] 65 70 75 80

[2097] Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

[2098] 85 90 95

[2099] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Trp Val Asp Pro Trp

[2100] 100 105 110

[2101] Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[2102] 115 120

[2103] <210> 91

[2104] <211> 6

[2105] <212> PROTEIN

[2106] <213> Artificial sequence

[2107] <220>

[2108] <223> Recombinant protein

[2109] <400> 91

[2110] Ser Asp Ser Ala Trp Ser

[2111] 1 5

[2112] <210> 92

[2113] <211> 16

[2114] <212> PROTEIN

[2115] <213> Artificial sequence

[2116] <220>

[2117] <223> Recombinant protein

[2118] <400> 92

[2119] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2120] 1 5 10 15

[2121] <210> 93

[2122] <211> 13

[2123] <212> PROTEIN

[2124] <213> Artificial sequence

[2125] <220>

[2126] <223> Recombinant protein

[2127] <400> 93

[2128] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Trp Val Asp Pro

[2129] 1 5 10

[2130] <210> 94

[2131] <211> 107

[2132] <212> PROTEIN

[2133] <213> Artificial sequence

[2134] <220>

[2135] <223> Recombinant protein

[2136] <400> 94

[2137] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2138] 1 5 10 15

[2139] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Tyr

[2140] 20 25 30

[2141] Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

[2142] 35 40 45

[2143] Lys Tyr Ala Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2144] 50 55 60

[2145] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2146] 65 70 75 80

[2147] Glu Asp Phe Ala Val Tyr Tyr Cys Gln Ser Gly His Ser Phe Pro Tyr

[2148] 85 90 95

[2149] Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

[2150] 100 105

[2151] <210> 95

[2152] <211> 11

[2153] <212> PROTEIN

[2154] <213> Artificial sequence

[2155] <220>

[2156] <223> Recombinant protein

[2157] <400> 95

[2158] Arg Ala Ser Gln Gly Val Ser Ser Tyr Leu His

[2159] 1 5 10

[2160] <210> 96

[2161] <211> 7

[2162] <212> PROTEIN

[2163] <213> Artificial sequence

[2164] <220>

[2165] <223> Recombinant protein

[2166] <400> 96

[2167] Tyr Ala Ser Gln Pro Ala Thr

[2168] 1 5

[2169] <210> 97

[2170] <211> 9

[2171] <212> PROTEIN

[2172] <213> Artificial sequence

[2173] <220>

[2174] <223> Recombinant protein

[2175] <400> 97

[2176] Gln Ser Gly His Ser Phe Pro Tyr Thr

[2177] 1 5

[2178] <210> 98

[2179] <211> 122

[2180] <212> PROTEIN

[2181] <213> Artificial sequence

[2182] <220>

[2183] <223> Recombinant protein

[2184] <400> 98

[2185] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

[2186] 1 5 10 15

[2187] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Asp

[2188] 20 25 30

[2189] Ser Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

[2190] 35 40 45

[2191] Ile Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2192] 50 55 60

[2193] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2194] 65 70 75 80

[2195] Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

[2196] 85 90 95

[2197] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Trp Val Asp Pro Trp

[2198] 100 105 110

[2199] Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[2200] 115 120

[2201] <210> 99

[2202] <211> 6

[2203] <212> PROTEIN

[2204] <213> Artificial sequence

[2205] <220>

[2206] <223> Recombinant protein

[2207] <400> 99

[2208] Ser Asp Ser Ala Trp Ser

[2209] 1 5

[2210] <210> 100

[2211] <211> 16

[2212] <212> PROTEIN

[2213] <213> Artificial sequence

[2214] <220>

[2215] <223> Recombinant protein

[2216] <400> 100

[2217] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2218] 1 5 10 15

[2219] <210> 101

[2220] <211> 13

[2221] <212> PROTEIN

[2222] <213> Artificial sequence

[2223] <220>

[2224] <223> Recombinant protein

[2225] <400> 101

[2226] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Trp Val Asp Pro

[2227] 1 5 10

[2228] <210> 102

[2229] <211> 107

[2230] <212> PROTEIN

[2231] <213> Artificial sequence

[2232] <220>

[2233] <223> Recombinant protein

[2234] <400> 102

[2235] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2236] 1 5 10 15

[2237] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2238] 20 25 30

[2239] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2240] 35 40 45

[2241] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2242] 50 55 60

[2243] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2244] 65 70 75 80

[2245] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2246] 85 90 95

[2247] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2248] 100 105

[2249] <210> 103

[2250] <211> 11

[2251] <212> PROTEIN

[2252] <213> Artificial sequence

[2253] <220>

[2254] <223> Recombinant protein

[2255] <400> 103

[2256] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2257] 1 5 10

[2258] <210> 104

[2259] <211> 7

[2260] <212> PROTEIN

[2261] <213> Artificial sequence

[2262] <220>

[2263] <223> Recombinant protein

[2264] <400> 104

[2265] Tyr Thr Ser Gln Pro Ala Thr

[2266] 1 5

[2267] <210> 105

[2268] <211> 9

[2269] <212> PROTEIN

[2270] <213> Artificial sequence

[2271] <220>

[2272] <223> Recombinant protein

[2273] <400> 105

[2274] Gln Asn Gly His Ser Phe Pro Leu Thr

[2275] 1 5

[2276] <210> 106

[2277] <211> 122

[2278] <212> PROTEIN

[2279] <213> Artificial sequence

[2280] <220>

[2281] <223> Recombinant protein

[2282] <400> 106

[2283] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[2284] 1 5 10 15

[2285] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2286] 20 25 30

[2287] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[2288] 35 40 45

[2289] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2290] 50 55 60

[2291] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2292] 65 70 75 80

[2293] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[2294] 85 90 95

[2295] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Trp Val Gly Tyr Trp

[2296] 100 105 110

[2297] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2298] 115 120

[2299] <210> 107

[2300] <211> 6

[2301] <212> PROTEIN

[2302] <213> Artificial sequence

[2303] <220>

[2304] <223> Recombinant protein

[2305] <400> 107

[2306] Ser Asp Ser Ala Trp Ser

[2307] 1 5

[2308] <210> 108

[2309] <211> 16

[2310] <212> PROTEIN

[2311] <213> Artificial sequence

[2312] <220>

[2313] <223> Recombinant protein

[2314] <400> 108

[2315] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2316] 1 5 10 15

[2317] <210> 109

[2318] <211> 13

[2319] <212> PROTEIN

[2320] <213> Artificial sequence

[2321] <220>

[2322] <223> Recombinant protein

[2323] <400> 109

[2324] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Trp Val Gly Tyr

[2325] 1 5 10

[2326] <210> 110

[2327] <211> 107

[2328] <212> PROTEIN

[2329] <213> Artificial sequence

[2330] <220>

[2331] <223> Recombinant protein

[2332] <400> 110

[2333] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2334] 1 5 10 15

[2335] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2336] 20 25 30

[2337] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2338] 35 40 45

[2339] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2340] 50 55 60

[2341] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2342] 65 70 75 80

[2343] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2344] 85 90 95

[2345] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2346] 100 105

[2347] <210> 111

[2348] <211> 11

[2349] <212> PROTEIN

[2350] <213> Artificial sequence

[2351] <220>

[2352] <223> Recombinant protein

[2353] <400> 111

[2354] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2355] 1 5 10

[2356] <210> 112

[2357] <211> 7

[2358] <212> PROTEIN

[2359] <213> Artificial sequence

[2360] <220>

[2361] <223> Recombinant protein

[2362] <400> 112

[2363] Tyr Thr Ser Gln Pro Ala Thr

[2364] 1 5

[2365] <210> 113

[2366] <211> 9

[2367] <212> PROTEIN

[2368] <213> Artificial sequence

[2369] <220>

[2370] <223> Recombinant protein

[2371] <400> 113

[2372] Gln Asn Gly His Ser Phe Pro Leu Thr

[2373] 1 5

[2374] <210> 114

[2375] <211> 122

[2376] <212> PROTEIN

[2377] <213> Artificial sequence

[2378] <220>

[2379] <223> Recombinant protein

[2380] <400> 114

[2381] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

[2382] 1 5 10 15

[2383] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2384] 20 25 30

[2385] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[2386] 35 40 45

[2387] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2388] 50 55 60

[2389] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2390] 65 70 75 80

[2391] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[2392] 85 90 95

[2393] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[2394] 100 105 110

[2395] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2396] 115 120

[2397] <210> 115

[2398] <211> 6

[2399] <212> PROTEIN

[2400] <213> Artificial sequence

[2401] <220>

[2402] <223> Recombinant protein

[2403] <400> 115

[2404] Ser Asp Ser Ala Trp Ser

[2405] 1 5

[2406] <210> 116

[2407] <211> 16

[2408] <212> PROTEIN

[2409] <213> Artificial sequence

[2410] <220>

[2411] <223> Recombinant protein

[2412] <400> 116

[2413] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2414] 1 5 10 15

[2415] <210> 117

[2416] <211> 13

[2417] <212> PROTEIN

[2418] <213> Artificial sequence

[2419] <220>

[2420] <223> Recombinant protein

[2421] <400> 117

[2422] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[2423] 1 5 10

[2424] <210> 118

[2425] <211> 107

[2426] <212> PROTEIN

[2427] <213> Artificial sequence

[2428] <220>

[2429] <223> Recombinant protein

[2430] <400> 118

[2431] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2432] 1 5 10 15

[2433] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2434] 20 25 30

[2435] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2436] 35 40 45

[2437] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2438] 50 55 60

[2439] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2440] 65 70 75 80

[2441] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2442] 85 90 95

[2443] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2444] 100 105

[2445] <210> 119

[2446] <211> 11

[2447] <212> PROTEIN

[2448] <213> Artificial sequence

[2449] <220>

[2450] <223> Recombinant protein

[2451] <400> 119

[2452] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2453] 1 5 10

[2454] <210> 120

[2455] <211> 7

[2456] <212> PROTEIN

[2457] <213> Artificial sequence

[2458] <220>

[2459] <223> Recombinant protein

[2460] <400> 120

[2461] Tyr Thr Ser Gln Pro Ala Thr

[2462] 1 5

[2463] <210> 121

[2464] <211> 9

[2465] <212> PROTEIN

[2466] <213> Artificial sequence

[2467] <220>

[2468] <223> Recombinant protein

[2469] <400> 121

[2470] Gln Asn Gly His Ser Phe Pro Leu Thr

[2471] 1 5

[2472] <210> 122

[2473] <211> 122

[2474] <212> PROTEIN

[2475] <213> Artificial sequence

[2476] <220>

[2477] <223> Recombinant protein

[2478] <400> 122

[2479] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[2480] 1 5 10 15

[2481] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2482] 20 25 30

[2483] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[2484] 35 40 45

[2485] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2486] 50 55 60

[2487] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2488] 65 70 75 80

[2489] Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

[2490] 85 90 95

[2491] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[2492] 100 105 110

[2493] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2494] 115 120

[2495] <210> 123

[2496] <211> 6

[2497] <212> PROTEIN

[2498] <213> Artificial sequence

[2499] <220>

[2500] <223> Recombinant protein

[2501] <400> 123

[2502] Ser Asp Ser Ala Trp Ser

[2503] 1 5

[2504] <210> 124

[2505] <211> 16

[2506] <212> PROTEIN

[2507] <213> Artificial sequence

[2508] <220>

[2509] <223> Recombinant protein

[2510] <400> 124

[2511] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2512] 1 5 10 15

[2513] <210> 125

[2514] <211> 13

[2515] <212> PROTEIN

[2516] <213> Artificial sequence

[2517] <220>

[2518] <223> Recombinant protein

[2519] <400> 125

[2520] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[2521] 1 5 10

[2522] <210> 126

[2523] <211> 107

[2524] <212> PROTEIN

[2525] <213> Artificial sequence

[2526] <220>

[2527] <223> Recombinant protein

[2528] <400> 126

[2529] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2530] 1 5 10 15

[2531] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2532] 20 25 30

[2533] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2534] 35 40 45

[2535] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2536] 50 55 60

[2537] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2538] 65 70 75 80

[2539] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2540] 85 90 95

[2541] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2542] 100 105

[2543] <210> 127

[2544] <211> 11

[2545] <212> PROTEIN

[2546] <213> Artificial sequence

[2547] <220>

[2548] <223> Recombinant protein

[2549] <400> 127

[2550] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2551] 1 5 10

[2552] <210> 128

[2553] <211> 7

[2554] <212> PROTEIN

[2555] <213> Artificial sequence

[2556] <220>

[2557] <223> Recombinant protein

[2558] <400> 128

[2559] Tyr Thr Ser Gln Pro Ala Thr

[2560] 1 5

[2561] <210> 129

[2562] <211> 9

[2563] <212> PROTEIN

[2564] <213> Artificial sequence

[2565] <220>

[2566] <223> Recombinant protein

[2567] <400> 129

[2568] Gln Asn Gly His Ser Phe Pro Leu Thr

[2569] 1 5

[2570] <210> 130

[2571] <211> 122

[2572] <212> PROTEIN

[2573] <213> Artificial sequence

[2574] <220>

[2575] <223> Recombinant protein

[2576] <400> 130

[2577] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[2578] 1 5 10 15

[2579] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2580] 20 25 30

[2581] Ser Ala Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp

[2582] 35 40 45

[2583] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2584] 50 55 60

[2585] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2586] 65 70 75 80

[2587] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[2588] 85 90 95

[2589] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[2590] 100 105 110

[2591] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2592] 115 120

[2593] <210> 131

[2594] <211> 6

[2595] <212> PROTEIN

[2596] <213> Artificial sequence

[2597] <220>

[2598] <223> Recombinant protein

[2599] <400> 131

[2600] Ser Asp Ser Ala Trp Ser

[2601] 1 5

[2602] <210> 132

[2603] <211> 16

[2604] <212> PROTEIN

[2605] <213> Artificial sequence

[2606] <220>

[2607] <223> Recombinant protein

[2608] <400> 132

[2609] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2610] 1 5 10 15

[2611] <210> 133

[2612] <211> 13

[2613] <212> PROTEIN

[2614] <213> Artificial sequence

[2615] <220>

[2616] <223> Recombinant protein

[2617] <400> 133

[2618] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[2619] 1 5 10

[2620] <210> 134

[2621] <211> 107

[2622] <212> PROTEIN

[2623] <213> Artificial sequence

[2624] <220>

[2625] <223> Recombinant protein

[2626] <400> 134

[2627] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2628] 1 5 10 15

[2629] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2630] 20 25 30

[2631] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2632] 35 40 45

[2633] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2634] 50 55 60

[2635] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2636] 65 70 75 80

[2637] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2638] 85 90 95

[2639] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2640] 100 105

[2641] <210> 135

[2642] <211> 11

[2643] <212> PROTEIN

[2644] <213> Artificial sequence

[2645] <220>

[2646] <223> Recombinant protein

[2647] <400> 135

[2648] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2649] 1 5 10

[2650] <210> 136

[2651] <211> 7

[2652] <212> PROTEIN

[2653] <213> Artificial sequence

[2654] <220>

[2655] <223> Recombinant protein

[2656] <400> 136

[2657] Tyr Thr Ser Gln Pro Ala Thr

[2658] 1 5

[2659] <210> 137

[2660] <211> 9

[2661] <212> PROTEIN

[2662] <213> Artificial sequence

[2663] <220>

[2664] <223> Recombinant protein

[2665] <400> 137

[2666] Gln Asn Gly His Ser Phe Pro Leu Thr

[2667] 1 5

[2668] <210> 138

[2669] <211> 122

[2670] <212> PROTEIN

[2671] <213> Artificial sequence

[2672] <220>

[2673] <223> Recombinant protein

[2674] <400> 138

[2675] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[2676] 1 5 10 15

[2677] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2678] 20 25 30

[2679] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[2680] 35 40 45

[2681] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2682] 50 55 60

[2683] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2684] 65 70 75 80

[2685] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[2686] 85 90 95

[2687] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Asp Tyr Trp

[2688] 100 105 110

[2689] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2690] 115 120

[2691] <210> 139

[2692] <211> 6

[2693] <212> PROTEIN

[2694] <213> Artificial sequence

[2695] <220>

[2696] <223> Recombinant protein

[2697] <400> 139

[2698] Ser Asp Ser Ala Trp Ser

[2699] 1 5

[2700] <210> 140

[2701] <211> 16

[2702] <212> PROTEIN

[2703] <213> Artificial sequence

[2704] <220>

[2705] <223> Recombinant protein

[2706] <400> 140

[2707] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2708] 1 5 10 15

[2709] <210> 141

[2710] <211> 13

[2711] <212> PROTEIN

[2712] <213> Artificial sequence

[2713] <220>

[2714] <223> Recombinant protein

[2715] <400> 141

[2716] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Asp Tyr

[2717] 1 5 10

[2718] <210> 142

[2719] <211> 107

[2720] <212> PROTEIN

[2721] <213> Artificial sequence

[2722] <220>

[2723] <223> Recombinant protein

[2724] <400> 142

[2725] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2726] 1 5 10 15

[2727] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2728] 20 25 30

[2729] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2730] 35 40 45

[2731] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2732] 50 55 60

[2733] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2734] 65 70 75 80

[2735] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2736] 85 90 95

[2737] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2738] 100 105

[2739] <210> 143

[2740] <211> 11

[2741] <212> PROTEIN

[2742] <213> Artificial sequence

[2743] <220>

[2744] <223> Recombinant protein

[2745] <400> 143

[2746] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2747] 1 5 10

[2748] <210> 144

[2749] <211> 7

[2750] <212> PROTEIN

[2751] <213> Artificial sequence

[2752] <220>

[2753] <223> Recombinant protein

[2754] <400> 144

[2755] Tyr Thr Ser Gln Pro Ala Thr

[2756] 1 5

[2757] <210> 145

[2758] <211> 9

[2759] <212> PROTEIN

[2760] <213> Artificial sequence

[2761] <220>

[2762] <223> Recombinant protein

[2763] <400> 145

[2764] Gln Asn Gly His Ser Phe Pro Leu Thr

[2765] 1 5

[2766] <210> 146

[2767] <211> 122

[2768] <212> PROTEIN

[2769] <213> Artificial sequence

[2770] <220>

[2771] <223> Recombinant protein

[2772] <400> 146

[2773] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[2774] 1 5 10 15

[2775] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2776] 20 25 30

[2777] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

[2778] 35 40 45

[2779] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2780] 50 55 60

[2781] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2782] 65 70 75 80

[2783] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[2784] 85 90 95

[2785] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[2786] 100 105 110

[2787] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2788] 115 120

[2789] <210> 147

[2790] <211> 6

[2791] <212> PROTEIN

[2792] <213> Artificial sequence

[2793] <220>

[2794] <223> Recombinant protein

[2795] <400> 147

[2796] Ser Asp Ser Ala Trp Ser

[2797] 1 5

[2798] <210> 148

[2799] <211> 16

[2800] <212> PROTEIN

[2801] <213> Artificial sequence

[2802] <220>

[2803] <223> Recombinant protein

[2804] <400> 148

[2805] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2806] 1 5 10 15

[2807] <210> 149

[2808] <211> 13

[2809] <212> PROTEIN

[2810] <213> Artificial sequence

[2811] <220>

[2812] <223> Recombinant protein

[2813] <400> 149

[2814] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[2815] 1 5 10

[2816] <210> 150

[2817] <211> 107

[2818] <212> PROTEIN

[2819] <213> Artificial sequence

[2820] <220>

[2821] <223> Recombinant protein

[2822] <400> 150

[2823] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2824] 1 5 10 15

[2825] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2826] 20 25 30

[2827] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2828] 35 40 45

[2829] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2830] 50 55 60

[2831] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2832] 65 70 75 80

[2833] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2834] 85 90 95

[2835] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2836] 100 105

[2837] <210> 151

[2838] <211> 11

[2839] <212> PROTEIN

[2840] <213> Artificial sequence

[2841] <220>

[2842] <223> Recombinant protein

[2843] <400> 151

[2844] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2845] 1 5 10

[2846] <210> 152

[2847] <211> 7

[2848] <212> PROTEIN

[2849] <213> Artificial sequence

[2850] <220>

[2851] <223> Recombinant protein

[2852] <400> 152

[2853] Tyr Thr Ser Gln Pro Ala Thr

[2854] 1 5

[2855] <210> 153

[2856] <211> 9

[2857] <212> PROTEIN

[2858] <213> Artificial sequence

[2859] <220>

[2860] <223> Recombinant protein

[2861] <400> 153

[2862] Gln Asn Gly His Ser Phe Pro Leu Thr

[2863] 1 5

[2864] <210> 154

[2865] <211> 122

[2866] <212> PROTEIN

[2867] <213> Artificial sequence

[2868] <220>

[2869] <223> Recombinant protein

[2870] <400> 154

[2871] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[2872] 1 5 10 15

[2873] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2874] 20 25 30

[2875] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[2876] 35 40 45

[2877] Ile Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2878] 50 55 60

[2879] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2880] 65 70 75 80

[2881] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[2882] 85 90 95

[2883] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[2884] 100 105 110

[2885] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[2886] 115 120

[2887] <210> 155

[2888] <211> 6

[2889] <212> PROTEIN

[2890] <213> Artificial sequence

[2891] <220>

[2892] <223> Recombinant protein

[2893] <400> 155

[2894] Ser Asp Ser Ala Trp Ser

[2895] 1 5

[2896] <210> 156

[2897] <211> 16

[2898] <212> PROTEIN

[2899] <213> Artificial sequence

[2900] <220>

[2901] <223> Recombinant protein

[2902] <400> 156

[2903] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[2904] 1 5 10 15

[2905] <210> 157

[2906] <211> 13

[2907] <212> PROTEIN

[2908] <213> Artificial sequence

[2909] <220>

[2910] <223> Recombinant protein

[2911] <400> 157

[2912] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[2913] 1 5 10

[2914] <210> 158

[2915] <211> 107

[2916] <212> PROTEIN

[2917] <213> Artificial sequence

[2918] <220>

[2919] <223> Recombinant protein

[2920] <400> 158

[2921] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[2922] 1 5 10 15

[2923] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[2924] 20 25 30

[2925] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[2926] 35 40 45

[2927] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[2928] 50 55 60

[2929] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[2930] 65 70 75 80

[2931] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[2932] 85 90 95

[2933] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[2934] 100 105

[2935] <210> 159

[2936] <211> 11

[2937] <212> PROTEIN

[2938] <213> Artificial sequence

[2939] <220>

[2940] <223> Recombinant protein

[2941] <400> 159

[2942] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[2943] 1 5 10

[2944] <210> 160

[2945] <211> 7

[2946] <212> PROTEIN

[2947] <213> Artificial sequence

[2948] <220>

[2949] <223> Recombinant protein

[2950] <400> 160

[2951] Tyr Thr Ser Gln Pro Ala Thr

[2952] 1 5

[2953] <210> 161

[2954] <211> 9

[2955] <212> PROTEIN

[2956] <213> Artificial sequence

[2957] <220>

[2958] <223> Recombinant protein

[2959] <400> 161

[2960] Gln Asn Gly His Ser Phe Pro Leu Thr

[2961] 1 5

[2962] <210> 162

[2963] <211> 122

[2964] <212> PROTEIN

[2965] <213> Artificial sequence

[2966] <220>

[2967] <223> Recombinant protein

[2968] <400> 162

[2969] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[2970] 1 5 10 15

[2971] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[2972] 20 25 30

[2973] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[2974] 35 40 45

[2975] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[2976] 50 55 60

[2977] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[2978] 65 70 75 80

[2979] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[2980] 85 90 95

[2981] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[2982] 100 105 110

[2983] Gly Gln Gly Thr Leu Val Thr Val Ser Ser

[2984] 115 120

[2985] <210> 163

[2986] <211> 6

[2987] <212> PROTEIN

[2988] <213> Artificial sequence

[2989] <220>

[2990] <223> Recombinant protein

[2991] <400> 163

[2992] Ser Asp Ser Ala Trp Ser

[2993] 1 5

[2994] <210> 164

[2995] <211> 16

[2996] <212> PROTEIN

[2997] <213> Artificial sequence

[2998] <220>

[2999] <223> Recombinant protein

[3000] <400> 164

[3001] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3002] 1 5 10 15

[3003] <210> 165

[3004] <211> 13

[3005] <212> PROTEIN

[3006] <213> Artificial sequence

[3007] <220>

[3008] <223> Recombinant protein

[3009] <400> 165

[3010] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3011] 1 5 10

[3012] <210> 166

[3013] <211> 107

[3014] <212> PROTEIN

[3015] <213> Artificial sequence

[3016] <220>

[3017] <223> Recombinant protein

[3018] <400> 166

[3019] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3020] 1 5 10 15

[3021] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3022] 20 25 30

[3023] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3024] 35 40 45

[3025] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3026] 50 55 60

[3027] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3028] 65 70 75 80

[3029] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3030] 85 90 95

[3031] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3032] 100 105

[3033] <210> 167

[3034] <211> 11

[3035] <212> PROTEIN

[3036] <213> Artificial sequence

[3037] <220>

[3038] <223> Recombinant protein

[3039] <400> 167

[3040] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3041] 1 5 10

[3042] <210> 168

[3043] <211> 7

[3044] <212> PROTEIN

[3045] <213> Artificial sequence

[3046] <220>

[3047] <223> Recombinant protein

[3048] <400> 168

[3049] Tyr Thr Ser Gln Pro Ala Thr

[3050] 1 5

[3051] <210> 169

[3052] <211> 9

[3053] <212> PROTEIN

[3054] <213> Artificial sequence

[3055] <220>

[3056] <223> Recombinant protein

[3057] <400> 169

[3058] Gln Asn Gly His Ser Phe Pro Leu Thr

[3059] 1 5

[3060] <210> 170

[3061] <211> 122

[3062] <212> PROTEIN

[3063] <213> Artificial sequence

[3064] <220>

[3065] <223> Recombinant protein

[3066] <400> 170

[3067] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3068] 1 5 10 15

[3069] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser Asp

[3070] 20 25 30

[3071] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3072] 35 40 45

[3073] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3074] 50 55 60

[3075] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3076] 65 70 75 80

[3077] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3078] 85 90 95

[3079] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3080] 100 105 110

[3081] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3082] 115 120

[3083] <210> 171

[3084] <211> 6

[3085] <212> PROTEIN

[3086] <213> Artificial sequence

[3087] <220>

[3088] <223> Recombinant protein

[3089] <400> 171

[3090] Ser Asp Ser Ala Trp Ser

[3091] 1 5

[3092] <210> 172

[3093] <211> 16

[3094] <212> PROTEIN

[3095] <213> Artificial sequence

[3096] <220>

[3097] <223> Recombinant protein

[3098] <400> 172

[3099] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3100] 1 5 10 15

[3101] <210> 173

[3102] <211> 13

[3103] <212> PROTEIN

[3104] <213> Artificial sequence

[3105] <220>

[3106] <223> Recombinant protein

[3107] <400> 173

[3108] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3109] 1 5 10

[3110] <210> 174

[3111] <211> 107

[3112] <212> PROTEIN

[3113] <213> Artificial sequence

[3114] <220>

[3115] <223> Recombinant protein

[3116] <400> 174

[3117] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3118] 1 5 10 15

[3119] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3120] 20 25 30

[3121] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3122] 35 40 45

[3123] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3124] 50 55 60

[3125] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3126] 65 70 75 80

[3127] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3128] 85 90 95

[3129] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3130] 100 105

[3131] <210> 175

[3132] <211> 11

[3133] <212> PROTEIN

[3134] <213> Artificial sequence

[3135] <220>

[3136] <223> Recombinant protein

[3137] <400> 175

[3138] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3139] 1 5 10

[3140] <210> 176

[3141] <211> 7

[3142] <212> PROTEIN

[3143] <213> Artificial sequence

[3144] <220>

[3145] <223> Recombinant protein

[3146] <400> 176

[3147] Tyr Thr Ser Gln Pro Ala Thr

[3148] 1 5

[3149] <210> 177

[3150] <211> 9

[3151] <212> PROTEIN

[3152] <213> Artificial sequence

[3153] <220>

[3154] <223> Recombinant protein

[3155] <400> 177

[3156] Gln Asn Gly His Ser Phe Pro Leu Thr

[3157] 1 5

[3158] <210> 178

[3159] <211> 122

[3160] <212> PROTEIN

[3161] <213> Artificial sequence

[3162] <220>

[3163] <223> Recombinant protein

[3164] <400> 178

[3165] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3166] 1 5 10 15

[3167] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3168] 20 25 30

[3169] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3170] 35 40 45

[3171] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3172] 50 55 60

[3173] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3174] 65 70 75 80

[3175] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3176] 85 90 95

[3177] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Pro Trp

[3178] 100 105 110

[3179] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3180] 115 120

[3181] <210> 179

[3182] <211> 6

[3183] <212> PROTEIN

[3184] <213> Artificial sequence

[3185] <220>

[3186] <223> Recombinant protein

[3187] <400> 179

[3188] Ser Asp Ser Ala Trp Ser

[3189] 1 5

[3190] <210> 180

[3191] <211> 16

[3192] <212> PROTEIN

[3193] <213> Artificial sequence

[3194] <220>

[3195] <223> Recombinant protein

[3196] <400> 180

[3197] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3198] 1 5 10 15

[3199] <210> 181

[3200] <211> 13

[3201] <212> PROTEIN

[3202] <213> Artificial sequence

[3203] <220>

[3204] <223> Recombinant protein

[3205] <400> 181

[3206] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Pro

[3207] 1 5 10

[3208] <210> 182

[3209] <211> 107

[3210] <212> PROTEIN

[3211] <213> Artificial sequence

[3212] <220>

[3213] <223> Recombinant protein

[3214] <400> 182

[3215] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3216] 1 5 10 15

[3217] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3218] 20 25 30

[3219] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3220] 35 40 45

[3221] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3222] 50 55 60

[3223] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3224] 65 70 75 80

[3225] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3226] 85 90 95

[3227] Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys

[3228] 100 105

[3229] <210> 183

[3230] <211> 11

[3231] <212> PROTEIN

[3232] <213> Artificial sequence

[3233] <220>

[3234] <223> Recombinant protein

[3235] <400> 183

[3236] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3237] 1 5 10

[3238] <210> 184

[3239] <211> 7

[3240] <212> PROTEIN

[3241] <213> Artificial sequence

[3242] <220>

[3243] <223> Recombinant protein

[3244] <400> 184

[3245] Tyr Thr Ser Gln Pro Ala Thr

[3246] 1 5

[3247] <210> 185

[3248] <211> 9

[3249] <212> PROTEIN

[3250] <213> Artificial sequence

[3251] <220>

[3252] <223> Recombinant protein

[3253] <400> 185

[3254] Gln Asn Gly His Ser Phe Pro Leu Thr

[3255] 1 5

[3256] <210> 186

[3257] <211> 122

[3258] <212> PROTEIN

[3259] <213> Artificial sequence

[3260] <220>

[3261] <223> Recombinant protein

[3262] <400> 186

[3263] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3264] 1 5 10 15

[3265] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3266] 20 25 30

[3267] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3268] 35 40 45

[3269] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3270] 50 55 60

[3271] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3272] 65 70 75 80

[3273] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3274] 85 90 95

[3275] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3276] 100 105 110

[3277] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3278] 115 120

[3279] <210> 187

[3280] <211> 6

[3281] <212> PROTEIN

[3282] <213> Artificial sequence

[3283] <220>

[3284] <223> Recombinant protein

[3285] <400> 187

[3286] Ser Asp Ser Ala Trp Ser

[3287] 1 5

[3288] <210> 188

[3289] <211> 16

[3290] <212> PROTEIN

[3291] <213> Artificial sequence

[3292] <220>

[3293] <223> Recombinant protein

[3294] <400> 188

[3295] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3296] 1 5 10 15

[3297] <210> 189

[3298] <211> 13

[3299] <212> PROTEIN

[3300] <213> Artificial sequence

[3301] <220>

[3302] <223> Recombinant protein

[3303] <400> 189

[3304] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3305] 1 5 10

[3306] <210> 190

[3307] <211> 107

[3308] <212> PROTEIN

[3309] <213> Artificial sequence

[3310] <220>

[3311] <223> Recombinant protein

[3312] <400> 190

[3313] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3314] 1 5 10 15

[3315] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr

[3316] 20 25 30

[3317] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3318] 35 40 45

[3319] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3320] 50 55 60

[3321] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3322] 65 70 75 80

[3323] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3324] 85 90 95

[3325] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3326] 100 105

[3327] <210> 191

[3328] <211> 11

[3329] <212> PROTEIN

[3330] <213> Artificial sequence

[3331] <220>

[3332] <223> Recombinant protein

[3333] <400> 191

[3334] Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu His

[3335] 1 5 10

[3336] <210> 192

[3337] <211> 7

[3338] <212> PROTEIN

[3339] <213> Artificial sequence

[3340] <220>

[3341] <223> Recombinant protein

[3342] <400> 192

[3343] Tyr Thr Ser Gln Pro Ala Thr

[3344] 1 5

[3345] <210> 193

[3346] <211> 9

[3347] <212> PROTEIN

[3348] <213> Artificial sequence

[3349] <220>

[3350] <223> Recombinant protein

[3351] <400> 193

[3352] Gln Asn Gly His Ser Phe Pro Leu Thr

[3353] 1 5

[3354] <210> 194

[3355] <211> 122

[3356] <212> PROTEIN

[3357] <213> Artificial sequence

[3358] <220>

[3359] <223> Recombinant protein

[3360] <400> 194

[3361] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3362] 1 5 10 15

[3363] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3364] 20 25 30

[3365] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3366] 35 40 45

[3367] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3368] 50 55 60

[3369] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3370] 65 70 75 80

[3371] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3372] 85 90 95

[3373] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3374] 100 105 110

[3375] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3376] 115 120

[3377] <210> 195

[3378] <211> 6

[3379] <212> WHITE

[3380] <213> Artificial sequence

[3381] <220>

[3382] <223> Recombinant protein

[3383] <400> 195

[3384] Ser Asp Ser Ala Trp Ser

[3385] 1 5

[3386] <210> 196

[3387] <211> 16

[3388] <212> PROTEIN

[3389] <213> Artificial sequence

[3390] <220>

[3391] <223> Recombinant protein

[3392] <400> 196

[3393] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3394] 1 5 10 15

[3395] <210> 197

[3396] <211> 13

[3397] <212> PROTEIN

[3398] <213> Artificial sequence

[3399] <220>

[3400] <223> Recombinant protein

[3401] <400> 197

[3402] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3403] 1 5 10

[3404] <210> 198

[3405] <211> 107

[3406] <212> PROTEIN

[3407] <213> Artificial sequence

[3408] <220>

[3409] <223> Recombinant protein

[3410] <400> 198

[3411] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3412] 1 5 10 15

[3413] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3414] 20 25 30

[3415] Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

[3416] 35 40 45

[3417] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3418] 50 55 60

[3419] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3420] 65 70 75 80

[3421] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3422] 85 90 95

[3423] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3424] 100 105

[3425] <210> 199

[3426] <211> 11

[3427] <212> PROTEIN

[3428] <213> Artificial sequence

[3429] <220>

[3430] <223> Recombinant protein

[3431] <400> 199

[3432] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3433] 1 5 10

[3434] <210> 200

[3435] <211> 7

[3436] <212> PROTEIN

[3437] <213> Artificial sequence

[3438] <220>

[3439] <223> Recombinant protein

[3440] <400> 200

[3441] Tyr Thr Ser Gln Pro Ala Thr

[3442] 1 5

[3443] <210> 201

[3444] <211> 9

[3445] <212> PROTEIN

[3446] <213> Artificial sequence

[3447] <220>

[3448] <223> Recombinant protein

[3449] <400> 201

[3450] Gln Asn Gly His Ser Phe Pro Leu Thr

[3451] 1 5

[3452] <210> 202

[3453] <211> 122

[3454] <212> PROTEIN

[3455] <213> Artificial sequence

[3456] <220>

[3457] <223> Recombinant protein

[3458] <400> 202

[3459] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3460] 1 5 10 15

[3461] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3462] 20 25 30

[3463] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3464] 35 40 45

[3465] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3466] 50 55 60

[3467] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3468] 65 70 75 80

[3469] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3470] 85 90 95

[3471] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3472] 100 105 110

[3473] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3474] 115 120

[3475] <210> 203

[3476] <211> 6

[3477] <212> PROTEIN

[3478] <213> Artificial sequence

[3479] <220>

[3480] <223> Recombinant protein

[3481] <400> 203

[3482] Ser Asp Ser Ala Trp Ser

[3483] 1 5

[3484] <210> 204

[3485] <211> 16

[3486] <212> PROTEIN

[3487] <213> Artificial sequence

[3488] <220>

[3489] <223> Recombinant protein

[3490] <400> 204

[3491] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3492] 1 5 10 15

[3493] <210> 205

[3494] <211> 13

[3495] <212> PROTEIN

[3496] <213> Artificial sequence

[3497] <220>

[3498] <223> Recombinant protein

[3499] <400> 205

[3500] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3501] 1 5 10

[3502] <210> 206

[3503] <211> 107

[3504] <212> PROTEIN

[3505] <213> Artificial sequence

[3506] <220>

[3507] <223> Recombinant protein

[3508] <400> 206

[3509] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3510] 1 5 10 15

[3511] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3512] 20 25 30

[3513] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3514] 35 40 45

[3515] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3516] 50 55 60

[3517] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3518] 65 70 75 80

[3519] Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3520] 85 90 95

[3521] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3522] 100 105

[3523] <210> 207

[3524] <211> 11

[3525] <212> PROTEIN

[3526] <213> Artificial sequence

[3527] <220>

[3528] <223> Recombinant protein

[3529] <400> 207

[3530] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3531] 1 5 10

[3532] <210> 208

[3533] <211> 7

[3534] <212> PROTEIN

[3535] <213> Artificial sequence

[3536] <220>

[3537] <223> Recombinant protein

[3538] <400> 208

[3539] Tyr Thr Ser Gln Pro Ala Thr

[3540] 1 5

[3541] <210> 209

[3542] <211> 9

[3543] <212> PROTEIN

[3544] <213> Artificial sequence

[3545] <220>

[3546] <223> Recombinant protein

[3547] <400> 209

[3548] Gln Asn Gly His Ser Phe Pro Leu Thr

[3549] 1 5

[3550] <210> 210

[3551] <211> 122

[3552] <212> PROTEIN

[3553] <213> Artificial sequence

[3554] <220>

[3555] <223> Recombinant protein

[3556] <400> 210

[3557] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3558] 1 5 10 15

[3559] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3560] 20 25 30

[3561] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3562] 35 40 45

[3563] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3564] 50 55 60

[3565] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3566] 65 70 75 80

[3567] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3568] 85 90 95

[3569] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3570] 100 105 110

[3571] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3572] 115 120

[3573] <210> 211

[3574] <211> 6

[3575] <212> PROTEIN

[3576] <213> Artificial sequence

[3577] <220>

[3578] <223> Recombinant protein

[3579] <400> 211

[3580] Ser Asp Ser Ala Trp Ser

[3581] 1 5

[3582] <210> 212

[3583] <211> 16

[3584] <212> PROTEIN

[3585] <213> Artificial sequence

[3586] <220>

[3587] <223> Recombinant protein

[3588] <400> 212

[3589] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3590] 1 5 10 15

[3591] <210> 213

[3592] <211> 13

[3593] <212> PROTEIN

[3594] <213> Artificial sequence

[3595] <220>

[3596] <223> Recombinant protein

[3597] <400> 213

[3598] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3599] 1 5 10

[3600] <210> 214

[3601] <211> 107

[3602] <212> PROTEIN

[3603] <213> Artificial sequence

[3604] <220>

[3605] <223> Recombinant protein

[3606] <400> 214

[3607] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3608] 1 5 10 15

[3609] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Asp Tyr

[3610] 20 25 30

[3611] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3612] 35 40 45

[3613] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3614] 50 55 60

[3615] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3616] 65 70 75 80

[3617] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3618] 85 90 95

[3619] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3620] 100 105

[3621] <210> 215

[3622] <211> 11

[3623] <212> PROTEIN

[3624] <213> Artificial sequence

[3625] <220>

[3626] <223> Recombinant protein

[3627] <400> 215

[3628] Arg Ala Ser Gln Gly Val Ser Asp Tyr Leu His

[3629] 1 5 10

[3630] <210> 216

[3631] <211> 7

[3632] <212> PROTEIN

[3633] <213> Artificial sequence

[3634] <220>

[3635] <223> Recombinant protein

[3636] <400> 216

[3637] Tyr Thr Ser Gln Pro Ala Thr

[3638] 1 5

[3639] <210> 217

[3640] <211> 9

[3641] <212> PROTEIN

[3642] <213> Artificial sequence

[3643] <220>

[3644] <223> Recombinant protein

[3645] <400> 217

[3646] Gln Asn Gly His Ser Phe Pro Leu Thr

[3647] 1 5

[3648] <210> 218

[3649] <211> 122

[3650] <212> PROTEIN

[3651] <213> Artificial sequence

[3652] <220>

[3653] <223> Recombinant protein

[3654] <400> 218

[3655] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3656] 1 5 10 15

[3657] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3658] 20 25 30

[3659] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3660] 35 40 45

[3661] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3662] 50 55 60

[3663] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3664] 65 70 75 80

[3665] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3666] 85 90 95

[3667] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3668] 100 105 110

[3669] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3670] 115 120

[3671] <210> 219

[3672] <211> 6

[3673] <212> PROTEIN

[3674] <213> Artificial sequence

[3675] <220>

[3676] <223> Recombinant protein

[3677] <400> 219

[3678] Ser Asp Ser Ala Trp Ser

[3679] 1 5

[3680] <210> 220

[3681] <211> 16

[3682] <212> PROTEIN

[3683] <213> Artificial sequence

[3684] <220>

[3685] <223> Recombinant protein

[3686] <400> 220

[3687] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3688] 1 5 10 15

[3689] <210> 221

[3690] <211> 13

[3691] <212> PROTEIN

[3692] <213> Artificial sequence

[3693] <220>

[3694] <223> Recombinant protein

[3695] <400> 221

[3696] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3697] 1 5 10

[3698] <210> 222

[3699] <211> 107

[3700] <212> PROTEIN

[3701] <213> Artificial sequence

[3702] <220>

[3703] <223> Recombinant protein

[3704] <400> 222

[3705] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3706] 1 5 10 15

[3707] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3708] 20 25 30

[3709] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3710] 35 40 45

[3711] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3712] 50 55 60

[3713] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3714] 65 70 75 80

[3715] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Tyr

[3716] 85 90 95

[3717] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3718] 100 105

[3719] <210> 223

[3720] <211> 11

[3721] <212> PROTEIN

[3722] <213> Artificial sequence

[3723] <220>

[3724] <223> Recombinant protein

[3725] <400> 223

[3726] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3727] 1 5 10

[3728] <210> 224

[3729] <211> 7

[3730] <212> PROTEIN

[3731] <213> Artificial sequence

[3732] <220>

[3733] <223> Recombinant protein

[3734] <400> 224

[3735] Tyr Thr Ser Gln Pro Ala Thr

[3736] 1 5

[3737] <210> 225

[3738] <211> 9

[3739] <212> PROTEIN

[3740] <213> Artificial sequence

[3741] <220>

[3742] <223> Recombinant protein

[3743] <400> 225

[3744] Gln Asn Gly His Ser Phe Pro Tyr Thr

[3745] 1 5

[3746] <210> 226

[3747] <211> 122

[3748] <212> PROTEIN

[3749] <213> Artificial sequence

[3750] <220>

[3751] <223> Recombinant protein

[3752] <400> 226

[3753] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3754] 1 5 10 15

[3755] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3756] 20 25 30

[3757] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3758] 35 40 45

[3759] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3760] 50 55 60

[3761] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3762] 65 70 75 80

[3763] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3764] 85 90 95

[3765] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3766] 100 105 110

[3767] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3768] 115 120

[3769] <210> 227

[3770] <211> 6

[3771] <212> PROTEIN

[3772] <213> Artificial sequence

[3773] <220>

[3774] <223> Recombinant protein

[3775] <400> 227

[3776] Ser Asp Ser Ala Trp Ser

[3777] 1 5

[3778] <210> 228

[3779] <211> 16

[3780] <212> PROTEIN

[3781] <213> Artificial sequence

[3782] <220>

[3783] <223> Recombinant protein

[3784] <400> 228

[3785] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3786] 1 5 10 15

[3787] <210> 229

[3788] <211> 13

[3789] <212> PROTEIN

[3790] <213> Artificial sequence

[3791] <220>

[3792] <223> Recombinant protein

[3793] <400> 229

[3794] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3795] 1 5 10

[3796] <210> 230

[3797] <211> 107

[3798] <212> PROTEIN

[3799] <213> Artificial sequence

[3800] <220>

[3801] <223> Recombinant protein

[3802] <400> 230

[3803] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Ala Gly

[3804] 1 5 10 15

[3805] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3806] 20 25 30

[3807] Leu His Trp Tyr Gln Gln Lys Ser Gly Glu Ala Pro Arg Leu Leu Ile

[3808] 35 40 45

[3809] Lys Tyr Thr Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3810] 50 55 60

[3811] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3812] 65 70 75 80

[3813] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3814] 85 90 95

[3815] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3816] 100 105

[3817] <210> 231

[3818] <211> 11

[3819] <212> PROTEIN

[3820] <213> Artificial sequence

[3821] <220>

[3822] <223> Recombinant protein

[3823] <400> 231

[3824] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3825] 1 5 10

[3826] <210> 232

[3827] <211> 7

[3828] <212> PROTEIN

[3829] <213> Artificial sequence

[3830] <220>

[3831] <223> Recombinant protein

[3832] <400> 232

[3833] Tyr Thr Ser Gln Pro Ala Thr

[3834] 1 5

[3835] <210> 233

[3836] <211> 9

[3837] <212> PROTEIN

[3838] <213> Artificial sequence

[3839] <220>

[3840] <223> Recombinant protein

[3841] <400> 233

[3842] Gln Asn Gly His Ser Phe Pro Leu Thr

[3843] 1 5

[3844] <210> 234

[3845] <211> 122

[3846] <212> PROTEIN

[3847] <213> Artificial sequence

[3848] <220>

[3849] <223> Recombinant protein

[3850] <400> 234

[3851] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3852] 1 5 10 15

[3853] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3854] 20 25 30

[3855] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3856] 35 40 45

[3857] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3858] 50 55 60

[3859] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3860] 65 70 75 80

[3861] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3862] 85 90 95

[3863] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3864] 100 105 110

[3865] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3866] 115 120

[3867] <210> 235

[3868] <211> 6

[3869] <212> PROTEIN

[3870] <213> Artificial sequence

[3871] <220>

[3872] <223> Recombinant protein

[3873] <400> 235

[3874] Ser Asp Ser Ala Trp Ser

[3875] 1 5

[3876] <210> 236

[3877] <211> 16

[3878] <212> PROTEIN

[3879] <213> Artificial sequence

[3880] <220>

[3881] <223> Recombinant protein

[3882] <400> 236

[3883] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3884] 1 5 10 15

[3885] <210> 237

[3886] <211> 13

[3887] <212> PROTEIN

[3888] <213> Artificial sequence

[3889] <220>

[3890] <223> Recombinant protein

[3891] <400> 237

[3892] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3893] 1 5 10

[3894] <210> 238

[3895] <211> 107

[3896] <212> PROTEIN

[3897] <213> Artificial sequence

[3898] <220>

[3899] <223> Recombinant protein

[3900] <400> 238

[3901] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

[3902] 1 5 10 15

[3903] Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Ile Ser Asp Tyr

[3904] 20 25 30

[3905] Leu His Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile

[3906] 35 40 45

[3907] Lys Tyr Ala Ser Gln Pro Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

[3908] 50 55 60

[3909] Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

[3910] 65 70 75 80

[3911] Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Leu

[3912] 85 90 95

[3913] Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[3914] 100 105

[3915] <210> 239

[3916] <211> 11

[3917] <212> PROTEIN

[3918] <213> Artificial sequence

[3919] <220>

[3920] <223> Recombinant protein

[3921] <400> 239

[3922] Arg Ala Ser Gln Gly Ile Ser Asp Tyr Leu His

[3923] 1 5 10

[3924] <210> 240

[3925] <211> 7

[3926] <212> PROTEIN

[3927] <213> Artificial sequence

[3928] <220>

[3929] <223> Recombinant protein

[3930] <400> 240

[3931] Tyr Ala Ser Gln Pro Ala Thr

[3932] 1 5

[3933] <210> 241

[3934] <211> 9

[3935] <212> PROTEIN

[3936] <213> Artificial sequence

[3937] <220>

[3938] <223> Recombinant protein

[3939] <400> 241

[3940] Gln Asn Gly His Ser Phe Pro Leu Thr

[3941] 1 5

[3942] <210> 242

[3943] <211> 122

[3944] <212> PROTEIN

[3945] <213> Artificial sequence

[3946] <220>

[3947] <223> Recombinant protein

[3948] <400> 242

[3949] Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Glu Pro Ser Gln

[3950] 1 5 10 15

[3951] Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

[3952] 20 25 30

[3953] Ser Ala Trp Ser Trp Ile Arg Gln Phe Pro Gly Lys Lys Leu Glu Trp

[3954] 35 40 45

[3955] Met Gly Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

[3956] 50 55 60

[3957] Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

[3958] 65 70 75 80

[3959] Leu Lys Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys

[3960] 85 90 95

[3961] Ala Arg Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr Trp

[3962] 100 105 110

[3963] Gly Gln Gly Thr Ser Val Thr Val Ser Ser

[3964] 115 120

[3965] <210> 243

[3966] <211> 6

[3967] <212> PROTEIN

[3968] <213> Artificial sequence

[3969] <220>

[3970] <223> Recombinant protein

[3971] <400> 243

[3972] Ser Asp Ser Ala Trp Ser

[3973] 1 5

[3974] <210> 244

[3975] <211> 16

[3976] <212> PROTEIN

[3977] <213> Artificial sequence

[3978] <220>

[3979] <223> Recombinant protein

[3980] <400> 244

[3981] Tyr Ile Gln Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

[3982] 1 5 10 15

[3983] <210> 245

[3984] <211> 13

[3985] <212> PROTEIN

[3986] <213> Artificial sequence

[3987] <220>

[3988] <223> Recombinant protein

[3989] <400> 245

[3990] Ser Val Asn Tyr Tyr Gly Asn Ser Phe Ala Val Gly Tyr

[3991] 1 5 10

[3992] <210> 246

[3993] <211> 107

[3994] <212> PROTEIN

[3995] <213> Artificial sequence

[3996] <220>

[3997] <223> Recombinant protein

[3998] <400> 246

[3999] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser ...

Claims

1. A bispecific antibody that specifically binds to factor VII(a) and TREM-like transcript 1 (TLT-1), comprising: (i) a first antigen-binding site capable of binding factor VII(a); and (ii) a second antigen-binding site capable of binding TREM-like transcript 1 (TLT1); wherein said first antigen-binding region comprises a light chain variable domain that is at least 80% identical to the amino acid sequence of SEQ ID NO: 846, provided that said light chain variable domain comprises: CDRL1, represented by SEQ ID NO: 847, CDRL2, represented by SEQ ID NO: 848, and CDRL3, represented by SEQ ID NO: 849, and a heavy chain variable domain that is at least 80% identical to the amino acid sequence of SEQ ID NO: 850, provided that said heavy chain variable domain comprises: CDRH1, represented by SEQ ID NO: 851, CDRH2 represented by SEQ ID NO: 852, and CDRH3 represented by SEQ ID NO: 853; wherein said second antigen-binding region comprises a light chain variable domain that is at least 80% identical to the amino acid sequence of SEQ ID NO: 854, provided that said light chain variable domain comprises: CDRL1, represented by SEQ ID NO: 855, CDRL2, represented by SEQ ID NO: 856, and CDRL3, represented by SEQ ID NO: 857, and a heavy chain variable domain that is at least 80% identical to the amino acid sequence of SEQ ID NO: 858, provided that said heavy chain variable domain comprises: CDRH1 represented by SEQ ID NO: 859, CDRH2 represented by SEQ ID NO: 860, and CDRH3 represented by SEQ ID NO: 861; and (iii) Fc region.

2. The bispecific antibody of claim 1, wherein the first antigen-binding region comprises a light chain variable domain that is at least 85% identical to the amino acid sequence of SEQ ID NO: 846 and a heavy chain variable domain that is at least 85% identical to the amino acid sequence of SEQ ID NO: 850, and wherein the second antigen-binding region comprises a light chain variable domain that is at least 85% identical to the amino acid sequence of SEQ ID NO: 854 and a heavy chain variable domain that is at least 85% identical to the amino acid sequence of SEQ ID NO:

858.

3. The bispecific antibody of claim 1, wherein the first antigen-binding region comprises a light chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 846 and a heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 850, and wherein the second antigen-binding region comprises a light chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO: 854 and a heavy chain variable domain that is at least 90% identical to the amino acid sequence of SEQ ID NO:

858.

4. The bispecific antibody of claim 1, wherein the first antigen-binding region comprises a light chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 846 and a heavy chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 850, and wherein the second antigen-binding region comprises a light chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO: 854 and a heavy chain variable domain that is at least 95% identical to the amino acid sequence of SEQ ID NO:

858.

5. The bispecific antibody of claim 1, wherein the first antigen-binding region comprises a light chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 846 and a heavy chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 850, and wherein the second antigen-binding region comprises a light chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO: 854 and a heavy chain variable domain that is at least 99% identical to the amino acid sequence of SEQ ID NO:

858.

6. A bispecific antibody according to claim 1, comprising: (i) a first heavy chain constant domain that is at least 80% identical to the amino acid sequence of SEQ ID NO: 943, linked to a heavy chain variable domain of a first antigen-binding region, and a second heavy chain constant domain that is at least 80% identical to the amino acid sequence of SEQ ID NO: 942, linked to a heavy chain variable domain of a second antigen-binding region; and (ii) first and second light chain constant domains, each independently at least 80% identical to the amino acid sequence of SEQ ID NO: 12, wherein the first light chain constant domain is linked to the light chain variable domain of the first antigen-binding region, and the second light chain constant domain is linked to the light chain variable domain of the second antigen-binding region.

7. Bispecific antibody according to claim 6, where: (i) the first heavy chain constant domain is at least 85% identical to the amino acid sequence of SEQ ID NO: 943, and the second heavy chain constant domain is at least 85% identical to the amino acid sequence of SEQ ID NO: 942; and (ii) each of the first and second light chain constant domains is independently at least 85% identical to the amino acid sequence of SEQ ID NO:

12.

8. Bispecific antibody according to claim 6, where: (i) the first heavy chain constant domain is at least 90% identical to the amino acid sequence of SEQ ID NO: 943, and the second heavy chain constant domain is at least 90% identical to the amino acid sequence of SEQ ID NO: 942; and (ii) each of the first and second light chain constant domains is independently at least 90% identical to the amino acid sequence of SEQ ID NO:

12.

9. Bispecific antibody according to claim 6, where: (i) the first heavy chain constant domain is at least 95% identical to the amino acid sequence of SEQ ID NO: 943, and the second heavy chain constant domain is at least 95% identical to the amino acid sequence of SEQ ID NO: 942; and (ii) each of the first and second light chain constant domains is independently at least 95% identical to the amino acid sequence of SEQ ID NO:

12.

10. Bispecific antibody according to claim 6, where: (i) the first heavy chain constant domain is at least 99% identical to the amino acid sequence of SEQ ID NO: 943, and the second heavy chain constant domain is at least 99% identical to the amino acid sequence of SEQ ID NO: 942; and (ii) each of the first and second light chain constant domains is independently at least 99% identical to the amino acid sequence of SEQ ID NO:

12.

11. A pharmaceutical composition for the treatment of coagulopathy, comprising a bispecific antibody according to any one of claims 1-10 and a pharmaceutically acceptable carrier.

12. A method for treating coagulopathy in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a bispecific antibody according to any one of claims 1-10.

13. The bispecific antibody of any one of claims 1-10 or the pharmaceutical composition of claim 11 for use in the treatment of a coagulopathy including hemophilia A with or without inhibitors, hemophilia B with or without inhibitors, factor VII(a) deficiency and Glanzmann thrombasthenia, or a combination thereof.

14. Use of a bispecific antibody according to any one of claims 1-10 or a pharmaceutical composition according to claim 11 in the manufacture of a medicament for the treatment of coagulopathy including hemophilia A with or without inhibitors, hemophilia B with or without inhibitors, factor VII(a) deficiency and Glanzmann thrombasthenia, or a combination thereof.

15. A bispecific antibody that specifically binds to factor VII(a) and TREM-like transcript 1 (TLT-1), comprising: (i) a first antigen-binding site that binds factor VII(a); and (ii) a second antigen-binding site that binds TREM-like transcript 1 (TLT1); wherein said first antigen-binding region comprises: CDRL1, represented by SEQ ID NO: 847, CDRL2, represented by SEQ ID NO: 848, CDRL3, represented by SEQ ID NO: 849, CDRH1, represented by SEQ ID NO: 851, CDRH2 represented by SEQ ID NO: 852, and CDRH3 represented by SEQ ID NO: 853; wherein said second antigen-binding region comprises: (a) CDRL1, represented by SEQ ID NO: 855, CDRL2, represented by SEQ ID NO: 856, CDRL3, represented by SEQ ID NO: 857, CDRH1 represented by SEQ ID NO: 859, CDRH2 represented by SEQ ID NO: 860, and CDRH3 represented by SEQ ID NO: 861; (b) CDRL1, represented by SEQ ID NO: 879, CDRL2, represented by SEQ ID NO: 880, CDRL3, represented by SEQ ID NO: 881, CDRH1, represented by SEQ ID NO: 883, CDRH2 represented by SEQ ID NO: 884, and CDRH3 represented by SEQ ID NO: 885; (c) CDRL1, represented by SEQ ID NO: 895, CDRL2, represented by SEQ ID NO: 896, CDRL3, represented by SEQ ID NO: 897, CDRH1 represented by SEQ ID NO: 899, CDRH2 represented by SEQ ID NO: 900, and CDRH3 represented by SEQ ID NO: 901; or (d) CDRL1, represented by SEQ ID NO: 871, CDRL2, represented by SEQ ID NO: 872, CDRL3, represented by SEQ ID NO: 873, CDRH1, represented by SEQ ID NO: 875, CDRH2 represented by SEQ ID NO: 876, and CDRH3, represented by SEQ ID NO:

877.

16. A bispecific antibody that specifically binds to factor VII(a) and TREM-like transcript 1 (TLT-1), comprising: (i) a first antigen-binding site that binds factor VII(a); and (ii) a second antigen-binding site that binds TREM-like transcript 1 (TLT1), (a) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 774 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 778, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 934 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 938; (b) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 830 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 834, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 854 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 858; (c) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 774 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 778, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 910 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 914; (d) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 774 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 778, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 926 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 930; (e) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 774 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 778, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 918 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 922; (f) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 846 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 850, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 854 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 858; (g) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 774 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 778, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 870 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 874; (h) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 774 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 778, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 878 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 882; (i) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 774 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 778, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 894 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 898; (j) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 766 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 770, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 934 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 938; (k) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 790 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 794, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 934 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 938; (l) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 806 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 810, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 934 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 938; (m) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 806 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 810, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 910 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 914; (n) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 806 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 810, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 918 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 922; (o) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 806 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 810, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 926 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 930; (p) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 782 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 786, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 934 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 938; (q) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 782 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 786, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 910 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 914; (r) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 782 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 786, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 918 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 922; (s) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 782 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 786, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 926 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 930; (t) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 798 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 802, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 934 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 938; (u) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 798 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 802, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 910 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 914; (v) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 798 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 802, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 918 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 922; (w) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 798 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 802, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 926 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 930; (x) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 790 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 794, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 934 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 938; (y) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 790 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 794, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 918 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 922; or (z) wherein the first antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 790 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 794, and wherein the second antigen-binding region comprises a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 926 and a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO:

930.

17. A pharmaceutical composition for the treatment of coagulopathy, comprising a bispecific antibody according to claim 15 or 16 and a pharmaceutically acceptable carrier.

18. The bispecific antibody of claim 15 or 16 or the pharmaceutical composition of claim 17 for use in the treatment of a coagulopathy including hemophilia A with or without inhibitors, hemophilia B with or without inhibitors, factor VII(a) deficiency and Glanzmann thrombasthenia, or a combination thereof.

19. Use of a bispecific antibody according to claim 15 or 16 or a pharmaceutical composition according to claim 17 for the treatment of coagulopathy including hemophilia A with or without inhibitors, hemophilia B with or without inhibitors, factor VII(a) deficiency and Glanzmann thrombasthenia, or a combination thereof.

20. Use of a bispecific antibody according to claim 15 or 16 or a pharmaceutical composition according to claim 17 in the manufacture of a medicinal product for the treatment of coagulopathy including hemophilia A with or without inhibitors, hemophilia B with or without inhibitors, factor VII(a) deficiency and Glanzmann thrombasthenia, or a combination thereof.

Images