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WO2016005564A2 - Anticorps dirigés contre le facteur v activé - Google Patents

Anticorps dirigés contre le facteur v activé Download PDF

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Publication number
WO2016005564A2
WO2016005564A2 PCT/EP2015/065848 EP2015065848W WO2016005564A2 WO 2016005564 A2 WO2016005564 A2 WO 2016005564A2 EP 2015065848 W EP2015065848 W EP 2015065848W WO 2016005564 A2 WO2016005564 A2 WO 2016005564A2
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WIPO (PCT)
Prior art keywords
seq
antibody
fva
antigen
binding fragment
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PCT/EP2015/065848
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English (en)
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WO2016005564A3 (fr
Inventor
Heidi Lindgreen Holmberg
Gustav RØDER
Kristoffer Winther BALLING
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Novo Nordisk A/S
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Publication of WO2016005564A2 publication Critical patent/WO2016005564A2/fr
Publication of WO2016005564A3 publication Critical patent/WO2016005564A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to antibodies, and compositions thereof, that are capable of binding to coagulation Factor V complex.
  • coagulation factor replacement therapy such as exogenous Factor VIII (FVIII) or Factor IX (FIX), respectively.
  • FVIII exogenous Factor VIII
  • FIX Factor IX
  • exogenous coagulation factors may only be administered intravenously, which is of considerable inconvenience and discomfort to patients.
  • infants and toddlers may have to have intravenous catheters surgically inserted into a chest vein, in order for venous access to be guaranteed. This leaves them at great risk of developing bacterial infections.
  • coagulopathy may only receive therapy after a bleed has commenced, rather than as a precautionary measure, which often impinges upon their general quality of life.
  • Activation of the blood coagulation system relies on a complex cascade of biological reactions.
  • tissue factor TF
  • FVII/FVIIa Factor VI l/activated Factor VII
  • FXa Factor VI l/activated Factor VII
  • FVa activated Factor V
  • Fl la thrombin
  • Small amounts of thrombin activate platelets, which results in surface exposure of phospholipids (PL) that supports the binding of the tenase complex consisting of activated FVIII/FIX
  • FVIIIa/FIXa generation of a thrombin burst is central for a stable clot formation after injury to the vessel wall.
  • FXa and its cofactor FVa.
  • FXa forms a complex with FVa
  • the FVa:FXa complex generates both the initial small amount of thrombin required for the first activation of platelets during the initiation phase of the coagulation process and the thrombin burst on the activated platelets during the coagulation propagation phase where large amounts of FXa are generated by the complex the tenase complex.
  • haemophilia A or B the propagation phase cannot take place and consequently insufficient thrombin is generated to form a stable clot.
  • Factor V is a pro-cofactor in the coagulation system. In it's activated form it has a procoagulant action that is associated with cofactor activity for FXa in the prothrombinase complex.
  • FV is activated by thrombin or FXa, where it is spliced in two chains which are noncovalently bound to each other by metal ions.
  • FVa is a cofactor of the prothrombinase complex and catalyses the conversion of prothrombin to thrombin on a phospholipid (PL) surface.
  • Blood coagulation factor V is a single-chain 330 kDa glycoprotein produced by hepatocytes and present in both plasma and platelets (Chesney, C. et al. (1981 ) Natl Acad Sci USA 78:5180). It is composed of six domains A1 (AA 29-329), A2 (AA 348-684), B (AA 692-1573), A3 (AA 1578-1907), C1 (AA 1907-2061 ), and C2 (AA2066-2221 ) (SEQ ID NO: 36). The A and C domains of the two chains are approximately 40% homologous with the equivalent domains of FVIII, but the B domains are not conserved.
  • the mature Factor V molecule consists of 2196 residues and has a domain structure that comprises three A-domains, one B-domain and two C-domains organized as follows: NH2-A1 -A2-B-A3-C1-C2-COOH.
  • FV Activation of FV is catalysed by thrombin or activated factor X (FXa) and occurs via cleavages at residues within and flanking the B-domain (Arg 709, Arg 1018 and Arg 1545) (Tans, G. et al. (1994) J Biol Chem 269:15969).
  • Activation of FV to FVa causes dissociation of the B-domain from the remaining FVa heavy chain (HC, A1-A2, SEQ ID NO 37) and light chain (LC, A3-C1-C2, SEQ ID 38).
  • the two chains of the FVa hetero-dimer remain associated in a bivalent metal-ions dependent fashion (Hibbard, L. and Mann, K. (1980) J Biol Chem 255:638).
  • FVa is a co-factor to the enzyme FXa in the activation of prothrombin to thrombin.
  • the prothrombinase complex (FVa:FXa complex) is responsible for the thrombin burst which occurs during normal haemostasis. Complete deficiency of either FV or FX has been shown to cause fatal embryonic or postnatal bleeding in mice (Cui J et al.
  • APC activated protein C
  • APC inactivates FVa via two pathways: one involves a kinetically favoured cleavage at Arg 506 in FVa, which yields a partially active FVa intermediate that is subsequently inactivated completely by cleavage at Arg 306 which leads to dissociation of the A2 domain from the rest of the molecule; the alternative inactivation pathway involves initial cleavage at Arg 306 in FVa (Nicolaes, GA et al. (1995) J Biol Chem 270:21 158).
  • FV Leiden (Arg 506 Gin substitution), FV Hong-Kong (Arg 306 Gly substitution) and FV Cambridge (Arg 306 Thr) (Bertina, R. et al. (1994) Nature 369:64, Chan, WP. et al. (1998) Blood 91 :1 135, Williamson, D. et al. (1998) Blood 91 : 1 140).
  • FV Leiden is a defect in the rapid phase of FVa inactivation, causes APC resistance and is a risk factor for venous thrombosis.
  • FV Cambridge and FV Hong Kong are not associated with APC resistance or an increased risk of venous thrombosis.
  • FV Leiden also shows impaired anti-coagulant co-factor activity in APC-mediated inactivation of FVIIIa (Thorelli E. et al. (1998) J Biol Chem 273:16140). Similar features to the FV Leiden gene mutation have been described for FV Nara gene mutation (Trp 1920 Arg substitution) (Nogami et al., (2014) Blood 123:2420).
  • the FV Leiden mutation has been shown to cause increased thrombin generation in normal plasma in which the APC system was activated either by the including thrombomodulin or by direct addition of APC (Castoldi et al. (2004) Blood 103:4173).
  • FV Leiden has been shown to improve the haemophilia A and B phenotype in mice, especially in the microcirculation (Schlachterman, A et al. (2005) J Thromb Haemost 3:2730).
  • FV Leiden has also been suggested that prevention of the APC-mediated inactivation of FVa by using an antibody directed towards APC (Patent application number: 200901 10683) would improve the haemophilia A and B phenotypes.
  • FVa levels include direct supplementation of exogenous FVa (Schlachterman A et al., (2005) J Throm Haemost, 3:2730), FVa variants (von Drygalski, A. et al., (2012) Blood 120, 17) or interference with FVa inactivation by activated protein C (APC; Patent application number: WO200901 10683).
  • Protein C also known as autoprothrombin IIA and blood coagulation factor XIV, is a vitamin K-dependent glycoprotein structurally similar to other vitamin K-dependent proteins affecting blood clotting, such as prothrombin, FVII, FIX and FX.
  • the activation of protein C is strongly promoted by thrombomodulin and endothelial protein C receptor (EPCR), the latter of which is found primarily on endothelial cells (cells on the inside of blood vessels).
  • EPCR endothelial protein C receptor
  • the presence of thrombomodulin accelerates activation by several orders of magnitude, and EPCR speeds up activation by a factor of 20.
  • APC On the endothelium, APC performs a major role in regulating blood clotting, inflammation, and cell death (apoptosis).
  • APC proteolytically inactivates proteins FVa and FVIIIa APC inactivates FVa by catayzing three cleavages (Arg 306 , Arg 506 , Arg 679 ). The cleavages at both Arg 306 and Arg 506 diminish the molecule's affinity to FXa, and though the first of these sites is cleaved at the slowest rate, it is entirely necessary to the functioning of FV. Protein S aids this process by catalyzing the proteolysis at Arg 306 , in which the A2 domain of FV dissociates from the rest of the protein. Protein S also binds to FXa, inhibiting the latter from diminishing APC's inactivation of Factor Va. Protein S
  • Protein S is a vitamin K-dependent plasma glycoprotein with a molecular weight of approximately 70 kDa synthesized predominantly within the liver. However a significant amount is also synthesized in endothelial cells. Protein S is a cofactor for APC. Mature Protein S comprises 5 distinct structural domains, including an N-terminal gamma- carboxylation (Gla) domain and aromatic stack, a so-called "thrombin-sensitive region” , 4 EGF-like domains and EGF-4, and a large C-terminal region of 393 amino acids referred to as a sex-hormone binding globulin (SHBG)-like domain the structure of which represents two laminin G-type domains.
  • Gla N-terminal gamma- carboxylation
  • aromatic stack a so-called "thrombin-sensitive region”
  • 4 EGF-like domains and EGF-4 and a large C-terminal region of 393 amino acids referred to as a sex-hormone binding globulin
  • the plasma concentration of Protein S is -350 nM and roughly 60% is bound to the complement 4 binding protein (C4b-BP), while the remaining fraction circulates as "free" Protein S.
  • the complex bound Protein S has approximately 40% anti-coagulant activity compared to that of free Protein S.
  • the half-life in plasma is 48-60 hours.
  • FVIII Factor VIII
  • HC heavy chain
  • LC light chain
  • thrombin and von Willebrand factor (vWF or VWF)
  • vWF or VWF von Willebrand factor
  • Endogenous FVIII molecules circulate in vivo as a pool of molecules with B domains of various sizes, the shortest having C-terminal at position 740, i.e. at the C-terminal of A2- a2. These FVIII molecules with B-domains of different length all have full procoagulant activity.
  • FVIII Upon activation with thrombin, FVIII is cleaved C-terminal of A1 -a1 at position 372, C-terminal of A2-a2 at position 740, and between a3 and A3 at position 1689, the latter cleavage releasing the a3 region with concomitant loss of affinity for VWF.
  • the activated FVIII molecule is termed FVIIIa.
  • the activation allows interaction of FVIIIa with phospholipid surfaces like activated platelets and activated factor IX (FIXa), i.e. the tenase complex is formed, allowing efficient activation of factor X (FX).
  • FIXa
  • the B domain is cleaved at several different positions, generating large
  • Thrombin also known as activated blood-coagulation factor II, is a serine protease that in humans is encoded by the F2 gene.
  • Thrombin is produced by the enzymatic cleavage of two sites on prothrombin by activated Factor X (Xa).
  • the activity of factor Xa is greatly enhanced by binding to activated Factor V (Va), termed the prothrombinase complex.
  • Va activated Factor V
  • Prothrombin is produced in the liver and is post-translationally modified in a vitamin K-dependent reaction that converts ten glutamic acids on prothrombin to gamma-carboxyglutamic acid (Gla). In the presence of calcium, the Gla residues promote the binding of prothrombin to phospholipid bilayers
  • Prothrombin (coagulation factor II) is proteolytically cleaved to form thrombin in the coagulation cascade, which ultimately results in the reduction of blood loss.
  • thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.
  • the prothrombinase complex consists of the serine protease, Factor Xa, and the protein cofactor, Factor Va.
  • the complex assembles on negatively charged phospholipid membranes in the presence of calcium ions.
  • the prothrombinase complex catalyzes the conversion of prothrombin (Factor II), an inactive zymogen, to thrombin (Factor lla), an active serine protease.
  • the activation of thrombin is a critical reaction in the coagulation cascade, which functions to regulate hemostasis in the body. To produce thrombin, the
  • prothrombinase complex cleaves two peptide bonds in prothrombin, one after Arg271 and the other after Arg320. Although it has been shown that Factor Xa can activate prothrombin when unassociated with the prothrombinase complex, the rate of thrombin formation is severely decreased under such circumstances.
  • the prothrombinase complex can catalyze the activation of prothrombin at a rate 10 5 -fold faster than can Factor Xa alone. Thus, the prothrombinase complex is required for the efficient production of activated thrombin and also for adequate hemostasis.
  • the present invention relates to antibodies or fragments thereof, that bind to activated Factor V (FVa).
  • the invention describes a group of anti-FVa antibodies that display a pro-coagulant effect. The effect has been demonstrated in various setups to show that the antibodies are capable of increasing thrombin generation compared to set-ups where the antibody is not included. Different parameters in the thrombin generation test have been measured showing that antibodies of the invention increase or stimulate one or more of; peak thrombin, velocity index and/or endogenous thrombin potential.
  • One aspect of this invention relates to a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa, that when bound to FVa protects it from inactivation by activated protein C (APC).
  • APC activated protein C
  • the antibodies of the invention display the pro-coagulant effect also in the presence of activated protein C (APC).
  • APC activated protein C
  • the antibodies of the invention display the pro-coagulant effect in normal plasma.
  • the antibodies of the invention display the pro-coagulant effect in haemophilia plasma, such as in, haemophilia A plasma which is factor VIII deficient.
  • the antibodies of the invention display the pro-coagulant effect under haemophilia A like conditions, which may also include the ability to reverse the inhibitory effect of thrombomodulin on thrombin
  • the antibodies of the invention display the pro-coagulant effect in haemophilic platelet poor plasma (PRP) in the presence of thrombomodulin.
  • PRP haemophilic platelet poor plasma
  • the antibodies of the invention display the pro-coagulant effect by stimulation of the thromboelastographic response, such as by increasing the elastic properties of blood during thrombus formation measured by thrombelastography using a TEG® hemostasis analyzer.
  • the antibody dosage dependency decrease Clot time (R) in the absence of APC.
  • the antibody dosage dependency decrease Clot time (R) in the presence of APC.
  • the antibody dosage dependency increase MTG in the absence of APC.
  • the antibody dosage dependency increase MTG in the presence of APC.
  • Another aspect of this invention relates to a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa, that when bound to FVa improves or stabilizes FVa co-factor activity to the enzyme FXa.
  • Another aspect of this invention relates to a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa that displays pro-coagulant effect in haemophilia.
  • One aspect of this invention regards a use of a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa as a pro-coagulant in prophylactic treatment of haemophilia.
  • Another aspect of this invention relates to the use of a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa, for the prophylactic treatment of haemophilia, such as haemophilia A and B and patients with inhibitors.
  • haemophilia such as haemophilia A and B
  • a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa for use in a method of treatment.
  • Said treatment may be prophylactic treatment of haemophilia, such as haemophilia A.
  • SEQ ID NO: 1 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0161 monoclonal antibody.
  • SEQ ID NO: 2 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0161 monoclonal antibody.
  • SEQ ID NO: 3 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0006 monoclonal antibody.
  • SEQ ID NO: 4 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0006 monoclonal antibody.
  • SEQ ID NO: 5 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0005 monoclonal antibody.
  • SEQ ID NO: 6 gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0005 monoclonal antibody.
  • SEQ ID NO: 7 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0028 monoclonal antibody.
  • SEQ ID NO: 8 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0028 monoclonal antibody.
  • SEQ ID NO: 9 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0128 monoclonal antibody.
  • SEQ ID NO: 10 gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0128 monoclonal antibody.
  • SEQ ID NO: 1 1 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0302 monoclonal antibody.
  • SEQ ID NO: 12 gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0302 monoclonal antibody.
  • SEQ ID NO: 13 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0337 monoclonal antibody.
  • SEQ ID NO: 14 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0337 monoclonal antibody.
  • SEQ ID NO: 15 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0293 monoclonal antibody.
  • SEQ ID NO: 16 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0293 monoclonal antibody.
  • SEQ ID NO: 17 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0327 monoclonal antibody.
  • SEQ ID NO: 18 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0327 monoclonal antibody.
  • SEQ ID NO: 19 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0296 monoclonal antibody.
  • SEQ ID NO: 20 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0296 monoclonal antibody.
  • SEQ ID NO: 21 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0426 monoclonal antibody.
  • SEQ ID NO: 22 gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0426 monoclonal antibody.
  • SEQ ID NO: 23 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-01 10 monoclonal antibody.
  • SEQ ID NO: 24 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-01 10 monoclonal antibody.
  • SEQ ID NO: 25 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0222 monoclonal antibody.
  • SEQ ID NO: 26 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0222 monoclonal antibody.
  • SEQ ID NO: 27 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0143 monoclonal antibody.
  • SEQ ID NO: 28 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0143 monoclonal antibody.
  • SEQ ID NO: 29 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0271 monoclonal antibody.
  • SEQ ID NO: 30 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0271 monoclonal antibody.
  • SEQ ID NO: 31 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0263 monoclonal antibody.
  • SEQ ID NO: 32 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0263 monoclonal antibody.
  • SEQ ID NO: 33 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0270 monoclonal antibody.
  • SEQ ID NO: 34 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0270 monoclonal antibody.
  • SEQ ID NO: 35 - gives the amino acid sequences of human coagulation factor V precursor y.
  • SEQ ID NO: 36 - gives the amino acid sequences of human coagulation factor V.
  • SEQ ID NO: 37 - gives the amino acid sequences of the heavy chain of activated human coagulation factor V.
  • SEQ ID NO: 38 - gives the amino acid sequences of the light chain of activated human coagulation factor V.
  • SEQ ID NO: 39 gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-000-0008 monoclonal antibody.
  • SEQ ID NO: 40 gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-000-0008 monoclonal antibody.
  • SEQ ID NO: 41 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0418 monoclonal antibody.
  • SEQ ID NO: 42 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0418 monoclonal antibody.
  • SEQ ID NO: 43 - gives the amino acid sequences of the light chain variable domain (VL) of the NNC 0233-0000-0422 monoclonal antibody.
  • SEQ ID NO: 44 - gives the amino acid sequences of the heavy chain variable domain (VH) of the NNC 0233-0000-0422 monoclonal antibody.
  • Coagulopathy/Haemophilia and treatment Currently, the gold standard in treatment of haemophilia is prophylactic replacement therapy, wherein treatment has to be administered intravenously 2-3 times weekly causing a significant burden to the patient. Furthermore, approximately 30% of the patients treated with factor VIII develop inhibitors which reduce the possibilities for an effective prophylactic treatment.
  • Antibodies according to the present invention or a pharmaceutical formulation comprising such antibodies may be used to treat a subject with a coagulopathy.
  • subject includes any human patient, or non-human vertebrate.
  • coagulopathy refers to an increased haemorrhagic tendency which may be caused by any qualitative or quantitative deficiency of any pro- coagulative component of the normal coagulation cascade, or any up-regulation of fibrinolysis.
  • Such coagulopathies may be congenital and/or acquired and/or iatrogenic and are identified by a person skilled in the art.
  • Non-limiting examples of congenital hypocoagulopathies are haemophilia A, haemophilia B, Factor VII deficiency, Factor X deficiency, Factor XI deficiency, von
  • haemophilia A or B may be severe, moderate or mild.
  • the clinical severity of haemophilia is determined by the concentration of functional units of FIX/FVI 11 in the blood and is classified as mild, moderate, or severe.
  • Severe haemophilia is defined by a clotting factor level of ⁇ 0.01 U/ml corresponding to ⁇ 1 % of the normal level, while moderate and mild patients have levels from 1-5% and >5%, respectively.
  • Haemophilia A with “inhibitors” that is, neutralizing allo-antibodies against factor VIII
  • haemophilia B with “inhibitors” that is, neutralizing allo-antibodies against factor IX
  • coagulopathies that are partly congenital and partly acquired.
  • treatment refers to the medical therapy of any human or other animal subject in need thereof. Said subject is expected to have undergone physical examination by a medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other animal subject.
  • the timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject's health.
  • said treatment may be prophylactic, palliative and/or symptomatic.
  • prophylactic, palliative and symptomatic may represent separate aspects of the invention.
  • a non-limiting example of an acquired coagulopathy is serine protease deficiency caused by vitamin K deficiency; such vitamin K-deficiency may be caused by administration of a vitamin K antagonist, such as warfarin.
  • Acquired coagulopathy may also occur following extensive trauma. In this case otherwise known as the "bloody vicious cycle", it is
  • haemodilution diastolic thrombocytopaenia and dilution of clotting factors
  • hypothermia a substance that influences the rate of clotting factors
  • metabolic derangements acidosis. Fluid therapy and increased fibrinolysis may exacerbate this situation.
  • Said haemorrhage may be from any part of the body.
  • a non-limiting example of an iatrogenic coagulopathy is an over dosage of anticoagulant medication - such as heparin, aspirin, warfarin and other platelet aggregation inhibitors - that may be prescribed to treat thromboembolic disease.
  • anticoagulant medication - such as heparin, aspirin, warfarin and other platelet aggregation inhibitors - that may be prescribed to treat thromboembolic disease.
  • iatrogenic coagulopathy is that which is induced by excessive and/or
  • inappropriate fluid therapy such as that which may be induced by a blood transfusion.
  • haemorrhage is associated with haemophilia A or B. In another embodiment, haemorrhage is associated with haemophilia A or B with acquired inhibitors. In another embodiment, haemorrhage is associated with thrombocytopenia. In another embodiment, haemorrhage is associated with von Willebrand's disease. In another embodiment, haemorrhage is associated with severe tissue damage. In another embodiment, haemorrhage is associated with severe trauma. In another
  • haemorrhage is associated with surgery. In another embodiment, haemorrhage is associated with haemorrhagic gastritis and/or enteritis. In another embodiment, the haemorrhage is profuse uterine bleeding, such as in placental abruption. In another embodiment, haemorrhage occurs in organs with a limited possibility for mechanical haemostasis, such as intracranially, intraaurally or intraocularly. In another embodiment, haemorrhage is associated with anticoagulant therapy.
  • antibody herein refers to a protein, derived from an immunoglobulin sequence, which is capable of specifically 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), that is, IgA, IgD, IgE, IgG, IgM, and/or IgY.
  • the term may also include one or more antigen- binding fragments of full length antibodies.
  • An antibody that specifically binds to an antigen, or a portion thereof may bind exclusively to that antigen, or portion thereof, or it may bind to a limited number of homologous antigens, or portions thereof.
  • Natural full-length antibodies usually comprise at least four polypeptide chains: two heavy (H) chains and two light (L) chains that are connected by disulfide bonds. In some cases, natural antibodies comprise less than four chains, as in the case of the heavy chain only antibodies found in camelids (V H H fragments) and the IgNARs found in Chondrichthyes.
  • One class of immunoglobulins of particular pharmaceutical interest is the IgGs. In humans, the IgG class may be sub-divided into four sub-classes lgG1 , lgG2, lgG3 and lgG4, based on the sequence of their heavy chain constant regions.
  • the light chains can be divided into two types, kappa and lambda chains based on differences in their sequence composition.
  • IgG molecules are composed of two heavy chains, interlinked by two or more disulfide bonds, and two light chains, each attached to a heavy chain by a disulfide bond.
  • An IgG heavy chain may comprise a heavy chain variable region (VH) and up to three heavy chain constant (CH) regions: CH1 , CH2 and CH3.
  • a light chain may comprise a light chain variable region (VL) and a light chain constant region (CL).
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs) or hypervariable regions (HvRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • HvRs hypervariable regions
  • VH and VL regions are typically composed 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 with the hypervariable regions of the heavy and light chains form a domain that is capable of interacting with an antigen, whilst the constant region of an antibody may mediate binding of the immunoglobulin to host tissues or factors, including, but not limited to various cells of the immune system (effector cells), Fc receptors and the first component (C1 q) of the C1 complex of the classical complement system.
  • Antibodies of the invention may be monoclonal antibodies, in the sense that they represent a set of unique heavy and light chain variable domain sequences as expressed from a single B-cell or by a clonal population of B cells. Antibodies of the invention may be produced and purified using various methods that are known to the person skilled in the art. For example, antibodies may be produced from hybridoma cells. Antibodies may be produced by B-cell expansion. Antibodies or fragments thereof may be recombinantly expressed in mammalian or microbial expression systems, or by in-vitro translation.
  • Antibodies or fragments thereof may also be recombinantly expressed as cell surface bound molecules, by means of e.g. phage display, bacterial display, yeast display, mammalian cell display or ribosome or mRNA display. Once produced, antibodies may be screened for binding to activated Factor V (FVa).
  • FVa activated Factor V
  • Antibodies of the current invention may be isolated.
  • isolated antibody refers to an antibody that has been separated and/or recovered from (an)other component(s) in the environment in which it was produced and/or that has been purified from a mixture of components present in the environment in which it was produced.
  • antigen-binding fragments of antibodies may be suitable in the context of the current invention, as it has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • antigen-binding fragment refers to one or more fragment(s) of an antibody that retain(s) the ability to specifically bind to or recognise an antigen, such as activated Factor V (FVa) or another target molecule, as described herein.
  • antigen-binding fragments include Fab, Fab', Fab 2 , Fab' 2 , FabS, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv); see e.g. Bird et al. Science 1988; 242:423-426; and Huston et al.
  • dsFv, Fd typically the VH and CH1 domain
  • dAb typically a VH domain
  • VH, VL, VhH, and V-NAR domains monovalent molecules comprising a single VH and a single VL chain
  • minibodies, diabodies, triabodies, tetrabodies, and kappa bodies see, e.g. Ill et al.
  • Fab fragments of an antibody can be derived from said antibody by cleavage of the heavy chain in the hinge region on the N- terminal or C-terminal side of the hinge cysteine residues connecting the heavy chains of the antibody.
  • a “Fab” fragment includes the variable and constant domains of the light chain and the variable domain and the first constant domain (CH1 ) of the heavy chain.
  • "Fab' 2 " fragments comprise a pair of "Fab"' fragments that are generally covalently linked by their hinge cysteines.
  • a Fab' is formally derived from a Fab' 2 fragment by cleavage of the hinge disulfide bonds connecting the heavy chains in the Fab' 2 .
  • Fab fragments retains the ability of the parent antibody to bind to its antigen, potentially with a lower affinity.
  • Fab' 2 fragments are capable of divalent binding, whereas Fab and Fab' fragments can bind monovalently.
  • Fab fragments lack the constant CH2 and CH3 domains, i.e. the Fc part, where interaction with the Fc receptors would occur.
  • Fab fragments are in general devoid of effector functions.
  • Fab fragments may be produced by methods known in the art, either by enzymatic cleavage of an antibody, e.g. using papain to obtain the Fab or pepsin to obtain the Fab' 2 , Fab fragments including Fab, Fab', Fab' 2 may be produced recombinantly using techniques that are well known to the person skilled in the art.
  • an "Fv” fragment is an antibody fragment that contains a complete antigen recognition and binding site, and generally comprises a dimer of one heavy and one light chain variable domain in association that can be covalent in nature, for example in a single chain variable domain fragment (scFv). It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions or a subset thereof confer antigen binding specificity to the antibody.
  • variable domain comprising only three hypervariable regions specific for an antigen can retain the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site (Cai & Garen, PNAS 1996; 93:6280-6285).
  • VHH heavy chain variable domain
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, where these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, in which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH and VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH and VL polypeptide chain
  • linker that is too short to allow pairing between the two variable domains on the same chain, the variable domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites.
  • Diabodies are described more fully, for example, in EP 404,097; WO 93/1 1 161 ; and Hollinger et al. PNAS 1993; 90:6444-6448.
  • linear antibodies refers to antibodies as described in Zapata et al. Protein Eng.
  • these antibodies contain a pair of tandem Fd segments (VH-CH1 -VH-CH1 ) that, together with complementary light chain polypeptides, form a pair of antigen binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • a monobody can bind to an antigen in the absence of light chains and typically has three hypervariable regions, for example CDRs designated CDRH 1 , CDRH2, and CDRH3.
  • a heavy chain IgG monobody has two heavy chain antigen binding molecules connected by a disulfide bond.
  • the heavy chain variable domain comprises one or more hypervariable regions, preferably a CDRH3 or HVL- H3 region.
  • Antibody fragments may be obtained using conventional recombinant or protein engineering techniques and the fragments can be screened for binding to factor V or activated factor V, or another function, in the same manner as intact antibodies.
  • Antibody fragments of the invention may be made by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide. Fragments may also be generated by one or more internal deletions.
  • An antibody of the invention may be, or may comprise, an antigen binding portion of one of these antibodies, or variants thereof.
  • the antibody of the invention may be a Fab fragment of one of these antibodies or variants thereof, or it may be a single chain antibody derived from one of these antibodies, or a variant thereof.
  • binding affinity is herein used as a measure of the strength of a non- covalent interaction between two molecules, e.g. an antibody, or fragment thereof, and an antigen.
  • binding affinity is used to describe monovalent interactions (intrinsic activity).
  • Binding affinity between two molecules, e.g. an antibody, or fragment thereof, and an antigen, through a monovalent interaction may be quantified by determining the equilibrium dissociation constant (KD).
  • KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g. by the SPR method.
  • the rate constants corresponding to the association and the dissociation of a monovalent complex are referred to as the association rate constant k a (or k on ) and dissociation rate constant k d (or k off ), respectively.
  • binding affinities associated with different molecular interactions such as comparison of the binding affinity of different antibodies for a given antigen, may be compared by comparison of the KD values for the individual antibody/antigen complexes.
  • An antibody according to the current invention may be able to compete with another molecule, such as a naturally occurring ligand or receptor or another antibody, for binding to FVa or FV. Therefore, an antibody according to the current invention may be able to bind FVa or FV with a greater affinity that that of another molecule also capable of binding FVa or FV.
  • the ability of an antibody to compete with a natural ligand/receptor for binding to an antigen may be assessed by determining and comparing the KD value for the interactions of interest, such as a specific interaction between an antibody and an antigen, with that of the KD value of an interaction not of interest.
  • the KD for the antibody with respect to the target will be 2-fold, preferably 5-fold, and more preferably 10-fold less than KD with respect to the other, non-target molecule such as unrelated material or accompanying material in the environment. More preferably, the KD will be 50-fold less, such as 100-fold less, or 200-fold less; even more preferably 500-fold less, such as 1 ,000-fold less, or 10,000- fold less.
  • the value of this dissociation constant can be determined directly by well-known methods. Standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody also can be assessed by standard assays known in the art, such as SPR.
  • a competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another ligand of that target, such as another antibody.
  • An antibody of the invention may have a KD for its target of 1 x 10 "7 M or less, 1 x 10 " 8 M or less, or 1 x 10 "9 M or less, or 1 x 10 "10 M or less, 1 x 10 "11 M or less, or 1 x 10 "12 M or less.
  • the KD of an antibody of the current invention may be less than 5 nM, such as less than 4 nM, such as less than 3 nM, such as less than 2 nM, such as less than 1 nM, such as less than 0.8 nM, such as less than 0.7 nM, such as less than 0.6 nM, such as less than 0.5 nM, such as less than 0.4 nM, such as less than 0.3 nM, such as less than 0.2 nM, such as less than 0.1 nM, such as less than 0.05 nM, such as less than 0.025 nM, such as less than 0.015 nM, such as between 0.015 nM and 0 nM.
  • An antibody of the invention may be a human antibody or a humanised antibody.
  • the term "human antibody”, as used herein, is intended to include antibodies having variable regions in which at least a portion of a framework region and/or at least a portion of a CDR region are derived from human germline immunoglobulin sequences. (For example, a human antibody may have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences.) Furthermore, if the antibody contains a constant region, the constant region or a portion thereof is also derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences ⁇ e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • Such a human antibody may be a human monoclonal antibody.
  • Such a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising human immunoglobulin heavy and light chain gene segments repertoires, fused to an immortalised cell.
  • Human antibodies may be isolated from sequence libraries built on selections of human germline sequences, further diversified with natural and synthetic sequence diversity.
  • Human antibodies may be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus.
  • human antibody derivative refers to any modified form of the human antibody, such as a conjugate of the antibody and another agent or antibody.
  • humanised antibody refers to a human/non-human chimeric antibody that contains a sequence (CDR regions or parts thereof) derived from a non-human immunoglobulin.
  • a humanised antibody is, thus, a human immunoglobulin
  • recipient antibody in which at least residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of an antibody from a non-human species (donor antibody) such as from a mouse, rat, rabbit or non-human primate, which have the desired specificity, affinity, sequence composition and functionality.
  • donor antibody such as from a mouse, rat, rabbit or non-human primate
  • framework (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues.
  • An example of such a modification is the introduction of one or more so- called back-mutations, which are typically amino acid residues derived from the donor antibody.
  • Humanisation of an antibody may be carried out using recombinant techniques known to the person skilled in the art (see, e.g., Antibody Engineering, Methods in Molecular Biology, vol.
  • a suitable human recipient framework for both the light and heavy chain variable domain may be identified by, for example, sequence or structural homology.
  • fixed recipient frameworks may be used, e.g., based on knowledge of structure, biophysical and biochemical properties.
  • the recipient frameworks can be germline derived or derived from a mature antibody sequence.
  • CDR regions from the donor antibody can be transferred by CDR grafting.
  • the CDR grafted humanised antibody can be further optimised for e.g. affinity, functionality and biophysical properties by identification of critical framework positions where re-introduction (back mutation) of the amino acid residue from the donor antibody has beneficial impact on the properties of the humanised antibody.
  • the humanised antibody can be engineered by introduction of germline residues in the CDR or framework regions, elimination of immunogenic epitopes, site-directed mutagenesis, affinity maturation, etc.
  • humanised antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a humanised antibody will comprise at least one - typically two - variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and in which all or substantially all of the FR residues are those of a human immunoglobulin sequence.
  • the humanised antibody can, optionally, also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • humanised antibody derivative refers to any modified form of the humanised antibody, such as a conjugate of the antibody and another chemical agent or antibody or antibody fragment or polypeptide.
  • chimeric antibody refers to an antibody whose light and heavy chain genes have been constructed, typically by genetic engineering, from
  • variable segments of genes from a mouse monoclonal antibody may be joined to human constant regions.
  • the fragment crystallisable region (“Fc region'V'Fc domain”) of an antibody is the C- terminal region of an antibody, which comprises the constant CH2 and CH3 domains.
  • the Fc domain may interact with cell surface receptors called Fc receptors, as well as some proteins of the complement system.
  • Fc region enables antibodies to interact with the immune system.
  • antibodies may be engineered to include
  • an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • An lgG1 antibody may carry a modified Fc domain comprising one or more, and perhaps all of the following mutations that will result in decreased affinity to certain Fc receptors (L234A, L235E, and G237A) and in reduced C1 q- mediated complement fixation (A330S and P331 S), respectively (residue numbering according to the EU index).
  • the isotype of an antibody of the invention may be IgG, such as lgG1 , such as lgG2, such as lgG4.
  • the class of an antibody may be "switched" by known techniques.
  • an antibody that was originally produced as an IgM molecule may be class switched to an IgG antibody.
  • Class switching techniques also may be used to convert one IgG subclass to another, for example: from lgG1 to lgG2 or lgG4; from lgG2 to lgG1 or lgG4; or from lgG4 to lgG1 or lgG2.
  • Engineering of antibodies to generate constant region chimeric molecules, by combination of regions from different IgG subclasses, can also be performed.
  • the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • the constant region may be modified to stabilise the antibody, e.g., to reduce the risk of a bivalent antibody separating into two monovalent VH-VL fragments.
  • residue S228 (according to the EU numbering index and S241 according to Kabat) may be mutated to a proline (P) residue to stabilise inter heavy chain disulphide bridge formation at the hinge (see, e.g., Angal et al. Mol Immunol. 1993; 30:105- 8).
  • Antibodies or fragments thereof may be defined in terms of their complementarity- determining regions (CDRs).
  • CDRs complementarity-determining regions
  • hypervariable region refers to the regions of an antibody in which amino acid residues involved in antigen-binding are situated.
  • the region of hypervariability or CDRs can be identified as the regions with the highest variability in amino acid alignments of antibody variable domains.
  • Databases can be used for CDR identification such as the Kabat database, the CDRs e.g. being defined as comprising amino acid residues 24-34 (L1 ), 50-56 (L2) and 89-97 (L3) of the light-chain variable domain and 31 -35 (H1 ), 50-65 (H2) and 95- 102 (H3) in the heavy-chain variable domain; (Kabat et al. 1991 ; Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.
  • CDRs can be defined as those residues from a "hypervariable loop" (residues 26-33 (L1 ), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1 ), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987; 196:901 -917).
  • the numbering of amino acid residues in this region is performed by the method described in Kabat et al. supra.
  • phrases such as "Kabat position”, “Kabat residue”, and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains.
  • the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a framework (FR) or CDR of the variable domain.
  • a heavy chain variable domain may include amino acid insertions (residue 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 heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
  • framework region or "FR” residues refer to those VH or VL amino acid residues that are not within the CDRs, as defined herein.
  • anigen refers to the molecular entity used for immunisation of an immunocompetent vertebrate to produce the antibody (Ab) that recognizes the Ag.
  • Ag is termed more broadly and is generally intended to include target molecules that are specifically recognized by the Ab, thus including fragments or mimics of the molecule used in the immunisation process, or other process, e.g. phage display, used for generating the Ab.
  • epitope is defined in the context of a molecular interaction between an "antigen binding polypeptide”, such as an antibody (Ab), and its corresponding antigen (Ag).
  • an “antigen binding polypeptide” such as an antibody (Ab)
  • Ag antigen
  • epitopope refers to the area or region on an Ag to which an Ab specifically binds, i.e. the area or region in physical contact with the Ab.
  • a protein epitope may comprise amino acid residues in the Ag that are directly involved in binding to a Ab (also called the immunodominant component of the epitope) and other amino acid residues, which are not directly involved in binding, such as amino acid residues of the Ag which are effectively blocked by the Ab, i.e. amino acid residues within the "solvent-excluded surface" and/or the "footprint" of the Ab.
  • epitope herein comprises both types of binding region in any particular region of FVa that specifically binds to an anti-FVa antibody, or another FVa specific agent according to the invention, unless otherwise stated.
  • FVa may comprise a number of different epitopes, which may include, without limitation, (1 ) linear peptide epitopes (2) conformational epitopes which consist of one or more non-contiguous amino acids located near each other in the mature FVa conformation; and (3) post-translational epitopes which consist, either in whole or part, of molecular structures covalently attached to Fva, such as carbohydrate groups.
  • the epitope for a given antibody (Ab)/antigen (Ag) pair can be described and characterized at different levels of detail using a variety of experimental and computational epitope mapping methods.
  • the experimental methods include mutagenesis, X-ray crystallography, Nuclear Magnetic Resonance (NMR) spectroscopy, Hydrogen deuterium exchange Mass Spectrometry (HX-MS) and various competition binding methods; methods that are known in the art.
  • NMR Nuclear Magnetic Resonance
  • HX-MS Hydrogen deuterium exchange Mass Spectrometry
  • each method relies on a unique principle, the description of an epitope is intimately linked to the method by which it has been determined.
  • the epitope for a given Ab/Ag pair may be described differently.
  • the epitope for the interaction between the Ag and the Ab can be described by the spatial coordinates defining the atomic contacts present in the Ag- Ab interaction, as well as information about their relative contributions to the binding thermodynamics.
  • the epitope can be characterized by the spatial coordinates defining the atomic contacts between the Ag and Ab.
  • the epitope can be characterized by the amino acid residues that it comprises as defined by a specific criteria such as the distance between or solvent accessibility of atoms in the Ab:Ag complex.
  • the epitope can be characterized through function, e.g. by competition binding with other Abs.
  • the epitope can also be defined more generically as comprising amino acid residues for which substitution by another amino acid will alter the characteristics of the interaction between the Ab and Ag.
  • epitope In the context of an X-ray derived crystal structure defined by spatial coordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag, the term epitope is herein, unless otherwise specified or contradicted by context, specifically defined as activated Factor V (FVa) residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4 A, from a heavy atom in the Ab.
  • FVa activated Factor V
  • the definition of the term “paratope” is derived from the above definition of “epitope” by reversing the perspective.
  • the term “paratope” refers to the area or region on the Ab to which an Ag specifically binds, i.e. with which it makes physical contact to the Ag.
  • an X-ray derived crystal structure defined by spatial coordinates of a complex between an Ab, such as a Fab fragment, and its Ag
  • paratope is herein, unless otherwise specified or contradicted by context, specifically defined as Ag residues characterized by having a heavy atom (i.e. a non-hydrogen atom) within a distance of 4 A from a heavy atom in activated Factor V (FVa).
  • the epitope and paratope for a given antibody (Ab)/antigen (Ag) pair may be identified by routine methods. For example, the general location of an epitope may be determined by assessing the ability of an antibody to bind to different fragments or variant FVa polypeptides.
  • the specific amino acids within FVa that make contact with an antibody (epitope) and the specific amino acids in an antibody that make contact with FVa (paratope) may also be determined using routine methods.
  • the antibody and target molecule may be combined and the Ab:Ag complex may be crystallised.
  • the crystal structure of the complex may be determined and used to identify specific sites of interaction between the antibody and its target.
  • Antibodies that bind to the same antigen can be characterised with respect to their ability to bind to their common antigen simultaneously and may be subjected to "competition binding'Vbinning".
  • the term “binning” refers to a method of grouping antibodies that bind to the same antigen. “Binning” of antibodies may be based on competition binding of two antibodies to their common antigen in assays based on standard techniques such as surface plasmon resonance (SPR), ELISA or flow cytometry.
  • An antibody's "bin” is defined using a reference antibody. If a second antibody is unable to bind to an antigen at the same time as the reference antibody, the second antibody is said to belong to the same "bin” as the reference antibody. In this case, the reference and the second antibody competitively bind the same part of an antigen and are coined
  • Antibody "binning” does not provide direct information about the epitope. Competing antibodies, i.e. antibodies belonging to the same “bin” may have identical epitopes, overlapping epitopes or even separate epitopes. The latter is the case if the reference antibody bound to its epitope on the antigen takes up the space required for the second antibody to contact its epitope on the antigen ("steric hindrance"). Non-competing antibodies generally have separate epitopes.
  • the problem addressed in the present invention relates to methods for improved utilization of the endogenous FV pool by a method of increasing the level and/or improving the co-factor activity of FVa by a monoclonal antibody and/or fragments hereof. Furthermore the problem addressed in the present invention relates to methods for improved utilization of the endogenous FV pool by a method of increasing the level and/or improving the co-factor activity of FVa by a monoclonal antibody binding to FV and FVa and/or fragments hereof.
  • the present invention relates to antibodies that bind to Factor Va.
  • the invention relates to antibodies that modulate the function of FVa.
  • the invention relates to antibodies capable of improving the co-cofactor function of FVa.
  • a key functionality of FVa is in the generation of thrombin. As mentioned above FVa function is to stimulate thrombin generation working as a cofactor for FXa in the
  • prothrombinase and as described in the Examples herein anti-FVa antibodies capable of increasing thrombin generation has been identified.
  • the invention relates to monoclonal antibodies and/or fragments thereof capable of reducing inactivation of FVa.
  • the invention also relates to uses for such antibodies and fragments thereof, such as therapeutic and pharmaceutical uses.
  • An antibody according to the invention against FVa and/or FV may offer a prophylactic treatment option for haemophilia patients with inhibitors.
  • the present invention also provides a method for treatment of haemophilia patients in a FVIII and FIX independent manner. It has been found that monoclonal antibodies of the invention raised against human FVa and/or FV significantly improve the thrombin generation in haemophilic plasma as the addition of the antibody increases thrombin generation compared to the assay performed in the absence of the antibody e.g. the pro-coagulant effect of the antibody is to stimulate thrombin generation beyond the thrombin generation of FVa alone (e.g. in the absence of antibody).
  • an antibody of the invention may have the ability to improve the peak thrombin generation in human FVII l-deficient plasma (see example 3) or to reduce time to clot as measured in a thromboelastography (TEG) analysis of human FVII l-deficient whole blood (see example 4). Furthermore, the antibodies may provide a prophylactic treatment for patients with haemophilia.
  • the present invention relates to antibodies or fragments thereof, that bind to activated Factor V (FVa) and/or Factor V also refered to as anti-activated Factor V (FVa) and/or anti FV antibodies.
  • FVa activated Factor V
  • FVa anti-activated Factor V
  • the present invention relates to antibodies, or fragments thereof, that bind to Factor V and activated Factor V (FVa).
  • the invention describes anti-FV and/or anti-FVa antibodies that display a pro-coagulant effect and in particular antibodies that bind both Factor V and activated Factor V that display a pro-coagulant effect.
  • the effect has been demonstrated in various setups to show that the antibodies are capable of increasing thrombin generation. Different parameters have been measured showing that antibodies of the invention increase one or more of peak thrombin, velocity index and/or endogenous thrombin potential. Likewise the thromboelastographic response has been measured by thrombelastography using a TEG® hemostasis analyzer.
  • One aspect of this invention relates to a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa, that when bound to FVa protects it from inactivation by activated protein C (APC).
  • APC activated protein C
  • the antibodies of the invention display the pro-coagulant effect also in the presence of activated protein C (APC).
  • APC activated protein C
  • the antibodies of the invention display the pro-coagulant effect in normal plasma.
  • the antibodies of the invention display the pro-coagulant effect in haemophilia plasma, such as in, haemophilia A plasma which is factor VIII deficient.
  • the anti-FVa antibody or an antigen-binding fragment thereof increases thrombin generation in the presence of APC, such as in the presence of exogenously added APC in a human plasma-based thrombin generation assay.
  • the FVa antibody has an EC 50 below 150 nM in the thrombin generation assay employing haemophilia A plasma and added APC as described in Example 2 herein.
  • the EC50 is below 125 nM, such as below 100 nM, such as below 75 nM such as below 50 nM, such as below 40 nM.
  • the FVa antibody has EC 50 equal to or below the EC 50 for the 01 10 antibody (defined by SEQ ID NO 23 and 24) in the thrombin generation assay employing haemophilia A plasma and added APC as described in Example 2 herein. Equal here means that the EC50 ratio 01 10/FVa antibody is around 1 , such as 0.5-1 . 5, 0.6-1.4, 0.7-1.3, 0.8-1.2 or 0.9-1.1 .
  • the peak height, in the thrombin generation assay employing haemophilia A plasma and added APC as described in Example 2 herein, is equal to the peak height of the 01 10 antibody.
  • the peak height ratio for an FVa antibody/ the 01 10 antibody is at least 0.5, such as 0.6, such as 0.7 or such as 0.8.
  • the antibodies of the invention display the pro- coagulant effect under haemophilia A like conditions such as in the presence of an anti-FVIII antibody.
  • ability of the antibody to increase thrombin generation is measured by an ability to increase one or more of peak thrombin, the velocity index and/or endogenous thrombin potential as described in example 3 herein. The effect may be compared to the effect of Factor VIII.
  • the peak thrombin, the velocity index and/or endogenous thrombin potential is higher for 90 nM antibody than 10 % FVIII either in the presence or absence of APC.
  • the effect may be compared to one or more of the antibodies of the present invention.
  • the effect is equal to or higher than the effect of one or more of antibody 0005, 0028, 0159, 0056, 0008, 0150, 0161 and 01 10.
  • the effect is equal to or higher than the effect of antibody 0005, 0028, 0159, 0056, 0008, 0150, 0161 or 01 10.
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) is equal to or better than the effect of antibody 0005.
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) is equal to or better than the effect of antibody 0028.
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) is equal to or better than the effect of antibody 0159.
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) ise equal to or better than the effect of antibody 0056.
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) is equal to or better than the effect of antibody 0008.
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) is equal to or better than the effect of antibody fragment 0150.
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) is equal to or better than the effect of antibody 0161 .
  • the effect (one or more of peak thrombin, the velocity index and/or endogenous thrombin potential) is equal to or better than the effect of antibody 01 10.
  • Peak Thrombin in haemophilia A patient plasma in the presence of APC (5 nM) as measured after Thrombinoscope PPP triggering of thrombin generation is at least 10 nM when using 100 nM anti-FVa antibody. In one further embodiment Peak Thrombin is at least 25 nM, such as at least 50nM, such as at least 75 nM when using 100 nM anti-FVa antibody. In one further embodiment Peak Thrombin is at least 40 when using 500 nM anti-FVa antibody. In one further embodiment Peak Thrombin is at leastl OO, such as at least 125 when using 500 nM anti-FVa antibody.
  • the Velocity Index in haemophilia A patient plasma in the presence of APC (5 nM) as measured after Thrombinoscope PPP triggering of thrombin generation, is at least 1 .5 nM/min when using 20 nM anti-FVa antibody. In one further embodiment the Velocity Index is at least 2.5 nM/min, such as at least 15 nM/min, when using 50 nM anti-FVa antibody. In one further embodiment the Velocity Index is at least 3.5 nM/min, such as at least 10 nM/min, such as at least 25 nM/min when using 100 nM anti-FVa antibody. In one further embodiment the Velocity Index is at least 4 nM/min, such as at least 8, such as at least 50 nM/min when using 500 nM anti-FVa antibody.
  • an anti-FVa antibody with Velocity Index is at least 10 nM/min, such as at least 20, such as at least 25 nM/min or such as at least 40 nM/min when using 500 nM anti-FVa antibody in haemophilia
  • a patient plasma in the presence of APC (5 nM) and measured after Thrombinoscope PPP triggering of thrombin generation which is based on table 8 and the data obtained for antibodies 0159, 0056, 0008, 01 10 (and Fab 0150) and 0161.
  • the antibody stimulates the thromboelastographic response, such as by increasing the elastic properties of blood during thrombus formation measured by thrombelastography using a TEG® hemostasis analyzer.
  • the effect of the antibody may be measured as the clot time or maximum rate of Thrombus Generation (MTG) measuring the elastic properties of blood during thrombus formation as described in Example 4 herein.
  • the antibody dose dependency reduce clotting time.
  • a similar effect is observed in the presence of APC.
  • the antibody is capable of reducing clotting time equal to recombinant FVIIa.
  • the relative Clot time (R) is dosage dependency reduced by the anti-FVa antibody. It may be observed that the antibody clot time is increased by a low concentration of the antibody while increasing dosage reduces the clot time in a dosage dependent fashion.
  • MTG Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically Therapeutically.
  • the antibody dosage dependency increases MTG.
  • a similar effect is observed in the absence of APC.
  • a similar effect is observed in the presence of APC.
  • the antibodies of the invention display the pro-coagulant effect under haemophilia A like conditions, which may also include the ability to reverse the inhibitory effect of thrombomodulin on thrombin generation.
  • the antibodies of the invention display the pro-coagulant effect in haemophilic platelet rich plasma (PRP) in the presence of thrombomodulin.
  • the FVa antibody of the invention has a peak height above 20 % of the peak height for the 01 10 antibody in a thrombin generation assay in platelet rich plasma under haemophilia A like conditions in the presence of thrombomodulin as described in Example 5 herein.
  • the peak height is above 20 % of the 01 10 antibody when the antibody concentration is 500 nM. In even further embodiments the peak height is above 40 %, such as above 60 % of the peak height for the 01 10 antibody when the antibody concentration is or 500 nM.
  • the peak height is above 20 % of the peak height for the 01 10 antibody when the antibody concentration is or 50 nM. In even further embodiments the peak height is above 40 %, such as above 60 % of the peak height for the 01 10 antibody when the antibody concentration is or 50 nM.
  • Another aspect of this invention relates to a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa, that when bound to FVa improves or stabilizes FVa co-factor activity to the enzyme FXa.
  • Another aspect of this invention relates to a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa that displays pro-coagulant effect in haemophilia.
  • One aspect of this invention regards a use of a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa (as a pro-coagulant) in prophylactic treatment of haemophilia.
  • Another aspect of this invention relates to the use of a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa, for the prophylactic treatment of haemophilia.
  • One aspect of this invention relates to a monoclonal antibody or a fragment of said monoclonal antibody that binds to FVa for use in a method of treatment.
  • Said treatment may be prophylactic treatment of haemophilia, such as haemophilia A.
  • the antibodies identified binds to various regions of FV, and the invention thus includes anti-FVa antibodies or an antigen-binding fragments thereof that binds to a) the A3 domain of the FVa light chain, b) the C2 domain of the FVa light chain, c) the A1 domain the FVa heavy chain or d) the A2 domain of the FVa heavy chain.
  • the antibodies of the invention include one or more of an anti-FVa antibody or antigen-binding fragment thereof, wherein the CDRs of the light and heavy chain variable regions are as in SEQ ID NO 1 and SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 1 1 and SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24,SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34, SEQ ID NO 39
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 1 and SEQ ID NO 2, SEQ ID NO 7 and SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24,SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34, SEQ ID NO 39 and SEQ ID NO 40, SEQ ID NO 41 and SEQ ID NO 42 or SEQ ID NO 43 and SEQ ID NO 44.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 5 and SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24,SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34, SEQ ID NO 39 and SEQ ID NO 40 or SEQ ID NO 41 and SEQ ID NO 42.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24,SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32, SEQ ID NO 33 and SEQ ID NO 34, SEQ ID NO 39 and SEQ ID NO 40 or SEQ ID NO 41 and SEQ ID NO 42.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24,SEQ ID NO 25 and SEQ ID NO 26, SEQ ID NO 27 and SEQ ID NO 28, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32 or SEQ ID NO 41 and SEQ or ID NO 42.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24, SEQ ID NO 29 and SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32 or SEQ ID NO 41 and SEQ or ID NO 42.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24 or SEQ ID NO 41 and SEQ or ID NO 42.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24 or SEQ ID NO 41 and SEQ or ID NO 42.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24 or SEQ ID NO 41 and SEQ or ID NO 42.
  • the anti-FVa antibody or antigen-binding fragment thereof comprises the CDRs of the light and heavy chain variable regions are as in the light chain and heavy chains set fort in SEQ ID NO 9 and SEQ ID NO 10, SEQ ID NO 13 and SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16, SEQ ID NO 21 and SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24 or SEQ ID NO 41 and SEQ ID NO 42.
  • Antibodies of the invention include the human monoclonal antibodies 0233-0000- 0302, 0233-0000-0337, 0233-0000-0293, 0233-0000-0327, 0233-0000-0296, 0233-0000- 0418 and 0233-0000-0426. It further includes the preferred murine antibodies 0233-0000- 0028 and 0233-0000-0128. And current invention further includes the preferred rabbit antibodies 0233-0000-01 10, 0233-0000-0271 and 0233-0000-0270. Heavy and light CDRs of these antibodies are provided below and in tables 28-30.
  • An antibody of the invention may be, or may comprise, a fragment of the 0233-0000-
  • the antibody of the invention may be, or may comprise, a fragment of the 0233-0000-01 10 antibody, the 0233-0000-0128 antibody or the 0233-0000-0270 antibody, or a variant of any one of these antibodies.
  • the antibody of the invention may be, or may comprise, a fragment of the 0233-0000-0271 antibody, the 0233-0000-0293 antibody or the 0233-0000-0296 antibody, or a variant of any one of these antibodies.
  • the antibody of the invention may be, or may comprise, a fragment of the 0233-0000-0302 antibody, the 0233-0000-0327 antibody or the 0233-0000-0337 antibody, or a variant of any one of these antibodies.
  • the antibody of the invention may be, or may comprise, a fragment of the 0233-0000-0418 antibody, the 0233-0000-0422 antibody or the 0233-0000-0426 antibody, or a variant of any one of these antibodies.
  • the antibody of the invention may be, or may comprise, a fragment of the 0233-0000-0005 antibody or the 0233-0000-0006 antibody, or a variant of any one of these antibodies.
  • An antibody of the invention may comprise a CDR region from one or more of the specific antibodies disclosed herein.
  • the light chain variable region (SEQ ID NO:1 1 ) of human monoclonal antibody 0233-0000-0302 contains the following CDRs; LC-CDR1 : "RASQDISHWLA” corresponding to Kabat residues 24-34 of SEQ ID NO 1 1 ; LC-CDR2: " IASTLQT” corresponding to Kabat residues 50-56 of SEQ ID NO 1 1 and LC-CDR3 " QQSNSFPLT” corresponding to Kabat residues (89-97) of SEQ ID NO 1 1.
  • the heavy chain variable region (SEQ ID NO: 12) of human monoclonal antibody 0233-0000-0302 contains the following CDRs; HC-CDR1 "DYAMH” corresponding to Kabat residues 31-35 of SEQ ID NO 12; HC-CDR2
  • 0233-0000-0337 contains the following CDRs: LC-CDR1 "RASQDISNWLA” corresponding to Kabat residues 24-34 of SEQ ID NO: 13, LC-CDR2 "ITSTLHI” corresponding to Kabat residues 50-56 of SEQ ID NO: 13, LC-CDR3 "QQANSFPFT” corresponding to Kabat residues 89-97 of SEQ ID NO:13.
  • the heavy chain variable region (SEQ ID NO:14) of human monoclonal antibody 0233-0000-0337 contains the following CDRs: HC-CDR1 "DYAMH” corresponding to Kabat residues 31-35 of SEQ ID NO:14, HC-CDR2
  • the light chain variable region (SEQ ID NO:15) of human monoclonal antibody 0233-0000-0293 contains the following CDRs: LC-CDR1 " KSSQSVLYSSN N KN YLA” corresponding to Kabat residues 24-40 of SEQ ID NO:15, LC-CDR2 "WASTRES” corresponding to Kabat residues 56-62 of SEQ ID NO:15 and LC-CDR3 "QQYYSTPWT" corresponding to Kabat residues 95-103 of SEQ ID NO: 15.
  • the heavy chain variable region (SEQ ID NO: 16) of human monoclonal antibody 0233-0000-0293 contains the following CDRs: HC-CDR1 "SYDIN” corresponding to Kabat residues 31 -35 of SEQ ID NO:16; HC- CDR2 "WMNPNSGNTGYALKFQG” corresponding to Kabat residues 50-66 of SEQ ID NO: 16 and HC-CDR3 "RTYYDILTGSLGAFDI” corresponding to Kabat residues 99-1 14 of SEQ ID NO:16.
  • the light chain variable region (SEQ ID NO:17) of human monoclonal antibody 0233-0000-0327 contains the following CDRs: LC-CDR1 "RASQDISSWLA” corresponding to Kabat residues 24-34 of SEQ ID NO:17; LC-CDR2 "lASSLQS” corresponding to Kabat residues 50-56 of SEQ ID NO: 17 and LC-CDR3 "QQANSFPFT” corresponding to Kabat residues 89-97 of SEQ ID NO:17.
  • the heavy chain variable region (SEQ ID NO:18) of the human monoclonal antibody 0233-0000-0327 contains the following CDRs: HC-CDR1 "DYAMH" corresponding to Kabat residues 31-35 of SEQ ID NO:18, HC-CDR2
  • the light chain variable region (SEQ ID NO:19) of the monoclonal human antibody 0233-0000-0296 contains the following CDRs: LC-CDR1 "RASQDISTWLA” corresponding to Kabat residues 24-34 of SEQ ID NO:19, LC-CDR2 "ITSTLHI” corresponding to Kabat residues 50-56 of SEQ ID NO:19 and LC-CDR3 "QQAYSFPFT” corresponding to Kabat residues 89-98 of SEQ ID NO:19.
  • the heavy chain variable region (SEQ ID NO:20) of the human monoclonal antibody 0233-0000-0296 contains the following CDRs: HC-CDR1 "DYAMH" corresponding to Kabat residues 31-35 of SEQ ID NO:20, HC-CDR2
  • the light chain variable region (SEQ ID NO:21 ) of the monoclonal human antibody 0233-0000-0426 contains the following CDRs: LC-CDR1 "SGDILGDKYAC” corresponding to Kabat residues 23-33 of SEQ ID NO:21 , LC-CDR2 "QDIKRPS” corresponding to Kabat residues 49-55 of SEQ ID NO:21 and LC-CDR3 "QAWDSTTPVV" corresponding to Kabat residues 88-97 of SEQ ID NO:21 .
  • the heavy chain variable region (SEQ ID NO:22) of the monoclonal antibody 0233-0000-0426 contains the following CDRs: HC-CDR1 "SYDIN” corresponding to Kabat residues 31-35 of SEQ ID NO:22, HC-CDR2
  • 0233-0000-0418 contains the following CDRs: LC-CDR1 "RSSQSLLDSDDGNTYMD” corresponding to Kabat residues 24-40 of SEQ ID NO:41 , LC-CDR2 "MGFYRAS” corresponding to Kabat residues 56-62)of SEQ ID NO:41 and LC-CDR3 "MQRIEFPST” corresponding to Kabat residues 95-103 of SEQ ID NO:41 .
  • the heavy chain variable region (SEQ ID NO:42) of the monoclonal antibody 0233-0000-0418 contains the following CDRs: HC-CDR1 "TSGVGVG” corresponding to Kabat residues 31-37 of SEQ ID NO:42, HC-CDR2 "LIYWDDVKRYSPSLRR” corresponding to Kabat residues 52-67 of SEQ ID NO:42 and HC- CDR3 "YNWKMRVD” corresponding to Kabat residues 100-107 of SEQ ID NO:42.
  • 0233-0000-0028 contains the following CDRs: LC-CDR1 "KASQDVGTAVG” corresponding to Kabat residues 24-34 of SEQ ID NO:7, LC-CDR2 "WASTRHT” corresponding to Kabat residues 50-56 of SEQ ID NO:7 and LC-CDR3 "QQYSSNPT” corresponding to Kabat residues 89-96 of SEQ ID NO:7.
  • the heavy chain variable region (SEQ ID NO:8) of the monoclonal murine antibody 0233-0000-0028 contains the following CDRs: HC-CDR1 "NYGMN" corresponding to Kabat residues 31-35 of SEQ ID NO:8, HC-CDR2
  • 0233-0000-0128 contains the following CDRs: LC-CDR1 "KTSTDIDDDMN" corresponding to Kabat residues 24-34 of SEQ ID NO:9, LC-CDR2 "EGNTLRP” corresponding to Kabat residues 50-56 of SEQ ID NO:9 and LC-CDR3 "LQSANMPFT” corresponding to Kabat residues 89-97 of SEQ ID NO:9.
  • the heavy chain variable region (SEQ ID NO:10) of the monoclonal murine antibody 0233-0000-0128 contains the following CDRs: HC-CDR1 "SYAMS” corresponding to Kabat residues 31 -35 of SEQ ID NO: 10, HC-CDR2 "TISSGGSYTYYPDSVKG” corresponding to Kabat residues 50-66 of SEQ ID NO: 10 and HC-CDR3 " G P Y LTTAT PS FTY” corresponding to Kabat residues 99-1 1 1 of SEQ ID NO: 10.
  • 0000-01 10 contains the following CDRs: LC-CDR1 "QASESISSYLT” corresponding to Kabat residues 24-34 of SEQ ID NO:23, LC-CDR2 "YASTLAS” corresponding to Kabat residues 50-56 of SEQ ID NO:23 and LC-CDR3 "LGVYSYSRDDGIA” corresponding to Kabat residues 89-101 of SEQ ID NO:23.
  • the heavy chain variable region (SEQ ID NO:24) of rabbit monoclonal antibody 0233-0000-01 10 contains the following CDRs: HC-CDR1 "SSYYMC” corresponding to Kabat residues 31-36 of SEQ ID NO:24, HC-CDR2
  • 0000-0271 contains the following CDRs: LC-CDR1 "QASQSIGGNLA” corresponding to Kabat residues 24-34 of SEQ ID NO:29, LC-CDR2 "DASKLAS” corresponding to Kabat residues 50-56 of SEQ ID NO:29 and LC-CDR "QCTYGSSGNIGNG” corresponding to Kabat residues 89-101 of SEQ ID NO:29.
  • the heavy chain variable region (SEQ ID NO:30) of rabbit monoclonal antibody 0233-0000-0271 contains the following CDRs: HC-CDR1 "SYAMI” corresponding to Kabat residues 30-34 of SEQ ID NO:30, HC-CDR2
  • 0000-0270 contains the following CDRs: LC-CDR1 "QASQSISSYLS” corresponding to Kabat residues 24-34 of SEQ ID NO:33, LC-CDR2 "RTSTLES” corresponding to Kabat residues 50-56 of SEQ ID NO:33 and LC-CDR3 "RTSTLES” corresponding to Kabat residues 89-102 of SEQ ID NO:33.
  • the heavy chain variable region (SEQ ID NO:34) of rabbit monoclonal antibody 0233-0000-0270 contains the following CDRs: HC-CDR1 "SYYHIC” corresponding to Kabat residues 30-35 of SEQ ID NO:34, HC-CDR2 "C I YAAS G DTWYATWVN A” corresponding to Kabat residues 50-66 of SEQ ID NO:34 and HC-CDR3
  • the antibody comprises the light chain variable region of SEQ ID NO:1 1 and/ or the heavy chain variable region of SEQ ID NO:12 or fragments thereof.
  • the antibody comprises light chain CDR1 with residues 24-34 of SEQ ID NO 1 1 , and/or light chain CDR2: comprising residues 50-56 of SEQ ID NO 1 1 and/or light chain CDR comprising residues (89-97) of SEQ ID NO 1 1 and/or heavy chain CDR1 comprising residues 31-35 of SEQ ID NO 12 and/or HC- CDR2 comprising residues 50-66 of SEQ ID NO: 12 and HC-CDR3 comprising residues 99- 1 15 of SEQ ID NO:12.
  • the antibody comprises the light chain variable region of SEQ ID NO:13 and/or heavy chain variable region SEQ ID NO:14, or fragments thereof.
  • the antibody comprises light chain CDR1 with residues 24-34 of SEQ ID NO:13 and/or LC-CDR2 with residues 50-56 of SEQ ID NO:13 and/or LC-CDR3 with residues 89-97 of SEQ ID NO:13 and/or heavy chain CDR1 comprising residues 31-35 of SEQ ID NO:14 and/or HC-CDR2 "comprising residues 50-66 of SEQ ID NO:14 and/or HC-CDR3 comprising residues 90-1 15 of SEQ ID NO:14.
  • the antibody comprises the light chain variable region (SEQ ID NO:15) and/or heavy chain variable region (SEQ ID NO:16) or fragments thereof.
  • the antibody comprises light chain CDR1 comprising residues 24-40 of SEQ ID NO: 15 and/or LC-CDR2 comprising residues 56-62 of SEQ ID NO:15 and/or LC-CDR3 comprising 95-103 of SEQ ID NO:15 and/or heavy chain CDR1 comprising residues 31-35 of SEQ ID NO:16 and/or HC-CDR2 comprising residues 50-66 of SEQ ID NO:16 and/or HC-CDR3 comprising residues 99-1 14 of SEQ ID NO:16.
  • the antibody comprises light chain variable region of SEQ ID NO: 17 and/or heavy chain variable region of SEQ ID NO: 18 or fragment thereof.
  • the antibody comprises light chain CDR1 comprising residues 24-34 of SEQ ID NO: 17 and/or LC-CDR2 comprising residues 50-56 of SEQ ID NO:17 and/or LC-CDR3 comprising residues 89-97 of SEQ ID NO:17 and/or heavy chain CDR1 comprising residues 31-35 of SEQ ID NO:18 and/or HC-CDR2 comprising residues 50-66 of SEQ ID NO:18 and/or HC-CDR3 comprising residues 99-1 15 of SEQ ID NO:18.
  • the antibody comprises the light chain variable region of(SEQ ID NO: 19 and/or the heavy chain variable region of SEQ ID NO:20 or a fragment thereof.
  • the antibody comprises a light chain CDR1 comprising residues 24-34 of SEQ ID NO: 19 and/or LC-CDR2 comprising residues 50-56 of SEQ ID NO:19 and/or LC-CDR3 comprising residues 89-98 of SEQ ID NO:19 and/or heavy chain CDR1 comprising residues 31-35 of SEQ ID NO:20 and/or HC-CDR2 comprising residues 50-66 of SEQ ID NO:20 and/or HC-CDR3 comprising residues 99-1 15 of SEQ ID NO:20.
  • the antibody comprises the light chain variable region SEQ ID NO:21 and/or heavy chain variable region SEQ ID NO:22, or fragment thereof.
  • the antibody comprises light chain CDR1 comprising residues 23-33 of SEQ ID NO:21 and/or LC-CDR2 comprising residues 49-55 of SEQ ID NO:21 and/or LC-CDR3 comprising residues 88-97 of SEQ ID NO:21 and/or HC- CDR1 comprising residues 31-35 of SEQ ID NO:22 and/or HC-CDR2 comprising residues 50-66 of SEQ ID NO:22 and/or HC-CDR3 comprising residues 99-1 19 of SEQ ID NO:22.
  • the antibody comprises a light chain variable region of SEQ ID NO:41 and/or heavy chain variable region of SEQ ID NO:42, or a fragment thereof.
  • the antibody comprises a LC-CDR1 comprising residues 24-40 of SEQ ID NO:41 and/or LC-CDR2 comprising 56-62 of SEQ ID NO:41 and/or LC-CDR3 comprising residues 95-103 of SEQ ID NO:41 and/or heavy chain CDR1 comprising residues 31 -37 of SEQ ID NO:42 and/or HC-CDR2 comprising residues 52-67 of SEQ ID NO:42 and/or HC-CDR3 comprising residues 100-107 of SEQ ID NO 42.
  • the antibody comprises light chain variable region SEQ ID NO:7 and/or the heavy chain variable region of SEQ ID NO:8, or fragment thereof.
  • the antibody comprises light chain CDR1 comprising residues 24-34 of SEQ ID NO:7 and/or LC-CDR2 comprising residues 50-56 of SEQ ID NO:7 and/or LC-CDR3 comprising residues 89-96 of SEQ ID NO:7 and/or heavy chain CDR1 comprising residues 31 -35 of SEQ ID NO:8 and/or HC-CDR2 comprising residues 50-66 of SEQ ID NO:8 and/or HC-CDR3 comprising residues 99-1 1 1 of SEQ ID NO:8.
  • the antibody comprises light chain variable region of SEQ ID NO:9 and/or heavy chain variable region of SEQ ID NO:10.
  • the antibody comprises light chain CDR1 comprising residues 24-34 of SEQ ID NO:9 and/or LC-CDR2 comprising residues 50-56 of SEQ ID NO:9 and/or LC-CDR3 residues 89-97 of SEQ ID NO:9 and/or heavy chain CDR1.comprising residues 31-35 of SEQ ID NO:10 and/or HC-CDR2 comprising residues 50-66 of SEQ ID NO:10 and/or HC- CDR3 comprising residues 99-1 1 1 of SEQ ID NO:10.
  • the antibody comprises the light chain variable region of SEQ ID NO:23 and/or heavy chain variable region of SEQ ID NO:24, or fragment thereof.
  • the antibody comprises a light chain CDR1 comprising residues 24-34 of SEQ ID NO:23 and/or LC-CDR2 comprising 50-56 of SEQ ID NO:23 and/or LC-CDR3 comprising residues 89-101 of SEQ ID NO:23 and/or HC-CDR1 comprising residues 31 -36 of SEQ ID NO:24 and/or HC-CDR2 comprising residues 51 -67 of SEQ ID NO:24 and/or HC-CDR3 comprising residues 99-109 of SEQ ID NO:24.
  • the antibody comprises the light chain variable region of SEQ ID NO:29 and/or heavy chain variable region of SEQ ID NO:30, or a fragment thereof.
  • the antibody comprises a light chain CDR1 comprising residues 24-34 of SEQ ID NO:29 and/or LC-CDR2 comprising residues 50-56 of SEQ ID NO:29 and/or LC-CDR comprising residues 89-101 of SEQ ID NO:29 and/or HC- CDR1 comprising residues 30-34 of SEQ ID NO:30 and/or HC-CDR2 comprising residues 49-64 of SEQ ID NO:30 and/or HC-CDR3 comprising residues 95-108 of SEQ ID NO:30.
  • the antibody comprises a light chain variable region of SEQ ID NO:33 and/or heavy chain variable region of SEQ ID NO:34, or a fragment thereof.
  • the antibody comprises a LC-CDR1 comprising residues 24-34 of SEQ ID NO:33 and/or LC-CDR2 comprising 50-56 of SEQ ID NO:33 and/or LC-CDR3 comprising residues 89-102 of SEQ ID NO:33 and/or heavy chain CDR1 comprising residues 30-35 of SEQ ID NO:34 and/or HC-CDR2 comprising residues 50-66 of SEQ ID NO:34 and/or HC-CDR3 comprising residues 98-1 14 of SEQ ID NO:34.
  • VH and/or VL amino acid sequences may be 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth above.
  • epitopes described at the amino acid level e.g. determined from an X-ray structure, are said to be identical if they contain the same set of amino acid residues.
  • Epitopes are said to overlap if at least one amino acid is shared by the epitopes.
  • Epitopes are said to be separate (unique) if no amino acid residue is shared by the epitopes.
  • HX-MS Hydrogen exchange mass spectrometry
  • pro-coagulant anti-FVa antibodies of the inventions have been found to bind to various regions of the activated FV molecule.
  • the antibodies bind the activated human FV molecule.
  • An aspect of the invention relates to a monoclonal anti-FVa antibody or antigen- binding fragment thereof that displays a pro-coagulant effect, wherein the anti-FVa antibody or antigen-binding fragment thereof binds to one or more of
  • the antibody or fragment thereof binds one or more of aa K34- I54 and aa I 164-Q182 in the A1 domain of human FVa.
  • the antibody or fragment thereof binds one or more of aa 485 to 493, 532-542, aa 553-554, aa 577-580 and 588-600 in the C2 domain of human FVa. In one embodiment the antibody or fragment thereof binds two of aa 485 to 493, 532-542, aa 553- 554, aa 577-580 and 588-600 in the C2 domain of human FVa. In one embodiment the antibody or fragment thereof binds three of aa 485 to 493, 532-542, aa 553-554, aa 577-580 and 588-600 in the C2 domain of human FVa.
  • the antibody or fragment thereof binds to aa 532-542, aa 553-554, aa 577-580 and 588-600 in the C2 domain of human FVa. In one embodiment the antibody or fragment thereof binds all of aa 485 to 493, 532-542, aa 553-554, aa 577-580 and 588-600 in the C2 domain of human FVa. In one embodiment the antibody or fragment thereof binds to one or more regions including aa E330-V331 and N534-C539 in the A2 domain. In one embodiment the antibody or fragment thereof binds to at least two regions including aa E330-V331 and N534-C539 in the A2 domain.
  • the antibody or fragment thereof binds one or more of aa F325- V331 and N534-C539 in the A2 domain of human FVa.
  • the antibody or fragment thereof binds one or more of aa E330- Y335, S356-S376 and N534-F552 in the A2 domain of human FVa.
  • the antibody or fragment thereof binds two of aa E330-Y335,
  • the antibody or fragment thereof binds to all of aa E330-Y335, S356-S376 and N534-F552 in the A2 domain of human FVa
  • the antibody or fragment thereof binds to both the A2, and A3 domains, such as aa E330-Y335, S356-S376 and N534-F552 in the A2 domain of human FVa and Y211-M226 in the A3.
  • the antibody or fragment thereof binds to both the A2, A3 and C2 domains, such as aa E330-Y335, S356-S376 and N534-F552 in the A2 domain of human FVa and Y211-M226 in the A3 and aa F514-L530, aa D553-L554 and aa L555-S579 of the C2 domain.
  • the antibody or fragment thereof binds aa Y211-M226 in the A3 domain of human FVa.
  • the pro-coagulant anti-FVa antibody or antigen-binding fragment competes with a reference antibody for binding to human FVa.
  • Surface Plasmon resonance can be used to determine if an antibody in question can bind in the presence of another antibody. If both antibodies can bind the antibodies does not compete, whereas only one of two competing antibodies can bind at the same time.
  • the antibody or antigen binding fragment competes with an antibody disclosed herein as defined in table 27 and 28.
  • the antibody competes in binding to FVa with antibody 0233-0000-01 10 or a fragment thereof (such as 0233-0000-0150). In another embodiment of the invention, the antibody competes in binding to FVa with antibody 0233-0000-0028. In a further embodiment the antibody competes in binding to FVa with antibody 0233-0000-0128. In other embodiment the antibody competes in binding to FVa with antibody 0233-0000-0159. In other embodiment the antibody competes in binding to FVa with antibody 0233-0000-418.
  • the invention relates to a monoclonal anti-FVa antibody or antigen-binding fragment thereof that displays a pro-coagulant effect, wherein the anti-FVa antibody or antigen-binding fragment thereof binds to
  • the epitope of the antibody comprises residues of the heavy chain of FVa (SEQ ID NO:37) within the sequence from K34 to I54. In one embodiment the antibody or antibody fragment binds K34 to I54 of the heavy chain of FVa.
  • the epitope of the antibody comprises one or more of the residues K34, K35, I36, V37, Y38, R39, E40, Y41 , E42, P43, Y44, F45, K46, K47, E48, K49, P50, Q51 , S52, T53 and I54,
  • the epitope of the antibody comprises residues on the heavy chain of FVa (SEQ ID NO:37) within the sequence from 1164 to Q182. In one embodiment the antibody or antibody fragment binds 1164 to Q182 of the heavy chain of FVa.
  • the epitope of the antibody comprises one or more of the residues 1164, C165, K166, K167, G168, T169, L170, T171 , E172, G173, G174, T175, Q176, K177, T178, F179, D180, K181 and/or Q182 (SEQ ID NO:37).
  • the epitope of the antibody comprises residues of the heavy chain of FVa (SEQ ID NO:37) within the sequence from K34 to I54 and residues on the heavy chain of FVa (SEQ ID NO:37) within the sequence from 1164 to Q182.
  • the antibody or antibody fragment binds K34 to I54 and 1164 to Q182 and of the heavy chain of FVa.
  • the epitope of the antibody comprises one or more of the residues K34, K35, I36, V37, Y38, R39, E40, Y41 , E42, P43, Y44, F45, K46, K47, E48, K49, P50, Q51 , S52, T53, I54 and one or more of the residues 1164, C165, K166, K167, G168, T169, L170, T171 , E172, G173, G174, T175, Q176, K177, T178, F179, D180, K181 and/or Q182 (SEQ ID NO:37).
  • the epitope of an antibody comprises residues of the heavy chain of FVa (SEQ ID NO:37) within the sequence from F325 to V331 . In one embodiment the antibody or antibody fragment binds F325 to V331 of the heavy chain of FVa.
  • the epitope of an antibody comprises residues of the heavy chain of FVa (SEQ ID NO:37) within the sequence from N534 to C539. In one embodiment the antibody or antibody fragment binds N534 to C539 of the heavy chain of FVa.
  • the epitope of the antibody comprises one or more of the residues F325, I326, A327, A328, E329, E330, V331 and one or more of N534, I535, N536, K537, F538, and C539.
  • the epitope of an antibody comprises residues of the heavy chain of FVa (SEQ ID NO:37) within the sequence from E330 to Y335
  • the epitope of an antibody comprises residues of the heavy chain of FVa (SEQ ID NO:37) within the sequence from S356 to S376.
  • the epitope of an antibody comprises residues of the heavy chain of FVa (SEQ ID NO:37) within the sequence from N534 to F552.
  • the antibody binds FVa within one or more of the sequences from E330 to Y335, S356 to S376 and N534 to F552. In one embodiment the antibody binds FVa within two of the sequences from E330 to Y335, S356 to S376 and N534 to F552. In one embodiment the antibody binds FVa within the three sequences from E330 to Y335, S356 to S376 and N534 to F552.
  • the epitope of the antibody may thus comprise residues within the regions E330 to Y335, S356 to S376, and N534 to F552 of the FVa heavy-chain sequence and those residues are E330, V331 , I332, W333, D334, Y335 and S356, N357, Q358, I359, G360, K361 , H362, Y363, K364, K365, V366, M367, Y368, T369, Q370, Y371 , E372, D373, E374, S375, S376 and N534, I535, N536, K537, F538, C539, E540, N541 , P542, D543, E544, V545, K546, R547, D548, D549, P550, K551 , F552.
  • the epitope of an antibody comprises residues of the light chain of FVa (SEQ ID NO:38) within the sequence from F514 to L530, In one embodiment the epitope of an antibody comprises residues of the light chain of FVa (SEQ ID NO:38) within the sequence from D553 to L554
  • the epitope of an antibody comprises residues of the light chain of FVa (SEQ ID NO:38) within the sequence from L555 to S579
  • the epitope of an antibody comprises residues of the light chain of FVa (SEQ ID NO:38) within the sequence from F514 to L530 from D553 to L554 and from L555 to S579.
  • the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38) within the sequence from A532 to L554 (SEQ ID NO:38).
  • the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38) within the sequence from A532 to L554 (SEQ ID NO:38).
  • the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38) within the sequence from Y577 to Y580.
  • the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38) within the sequence from G588 to M600.
  • the antibody or antibody fragment binds one or more of N532 to L554, A532 to L554 and/or Y577 to Y580 of the heavy chain of FVa.
  • the epitope of the antibody comprises one or more of the residues A532, Q533, G534, R535, V536, N537, A538, W539, Q540, A541 , K542, A543, N544, N545, N546, K547, Q548, W549, L550, E551 , I552, D553, L554 and/or one or more of the residues K478, S479, YS480 and/or one or more of the residues G588, V589, E590, W591 , K592, P593, Y594, R595, L596, K597, S598, S599, and M600 (SEQ ID NO:38).
  • the epitope of the antibody comprises one or more of the residues A532, Q533, G534, R535, V536, N537, A538, W539, Q540, A541 , K542, A543, N544, N545, N546, K547, Q548, W549, L550, E551 , I552, D553, L554 and/or one or more of the residues K478, S479, YS480 and/or one or more of the residues G588, V589, E590, W591 , K592, P593, Y594, R595, L596, K597, S598, S599, and M600 (SEQ ID NO:38).
  • the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38), within the sequence from N532 to A543. In one embodiment of the current invention, the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38) within the sequence from A532 to L554 (SEQ ID NO:38). In another embodiment the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38) within the sequence from D553 to L554. In a further embodiment the epitope of the antibody comprises residues on the light chain of FVa (SEQ ID NO:38) within the sequence from G588 to M600.
  • the epitope of the antibody one or more of the residues A532, Q533, G534, R535, V536, N537, A538, W539, Q540, A541 , K542 and/or A543 and one or more of the residues D553 and/or L554 and/or one or more of the residues G588, V589, E590, W591 , K592, P593, Y594, R595, L596, K597, S598, S599 and/or M600.
  • the epitope of an antibody comprises residues of the light chain of FVa (SEQ ID NO:38) within the sequence from Y21 1 to M226.
  • the antibody or antibody fragment binds Y21 1 to M226 of the FVa light chain.
  • the epitope of the antibody comprises one or more of the residues Y21 1 , E212, K213, K214, S215, R216, S217, S218, W219, R220, L121 , T222, S223, S224, E225, and M226.
  • the epitope comprises at least 5 of the amino acid residues Y21 1 , E212, K213, K214, S215, R216, S217, S218, W219, R220, L1221 , T222, S223, S224, E225, and M226 and in further embodiments at least 8, 10, 12 or 14 amino acid residues.
  • the present invention provides compositions and formulations comprising molecules of the invention, such as the antibodies and fragments thereof, polynucleotides, vectors and cells described herein.
  • the invention provides a pharmaceutical composition that comprises one or more of the antibodies or antibody fragments of the invention, formulated together with a pharmaceutically acceptable carrier.
  • one object of the invention is to provide a pharmaceutical formulation comprising such an antibody or antibody fragment which is present in a concentration from 0.25 mg/ml to 250 mg/ml, and wherein said formulation has a pH from 2.0 to 10.0.
  • the formulation may further comprise one or more of a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, as well as various combinations thereof.
  • a buffer system a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, as well as various combinations thereof.
  • preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
  • the pharmaceutical formulation is an aqueous formulation.
  • aqueous formulation is typically a solution or a suspension, but may also include colloids, dispersions, emulsions, and multi-phase materials.
  • aqueous formulation is defined as a formulation comprising at least 50% w/w water.
  • aqueous solution is defined as a solution comprising at least 50 % w/w water
  • aqueous suspension is defined as a suspension comprising at least 50 %w/w water.
  • the pharmaceutical formulation is a freeze-dried formulation, to which the physician or the patient adds solvents and/or diluents prior to use.
  • the pharmaceutical formulation comprises an aqueous solution of such an antibody, and a buffer, wherein the antibody is present in a concentration from 1 mg/ml or above, and wherein said formulation has a pH from about 2.0 to about 10.0.
  • the antibodies of the invention are useful in treatment of diseases or disorder where an increase in thrombin is desired.
  • Hemophilia and in particular Hemophilia A is a key indication where stimulation of the blood coagulation cascade is useful
  • An aspect of the invention relates to an FVa antibody as described herein above for use in a method of treatment.
  • An aspect of the invention relates to the use of an FVa antibody as described herein above for use in the preparation of a pharmaceutical product for use in a method of treatment.
  • the method of treatment is in further embodiment related to the treatment of Coagulopathy, such as Haemophilia described herein.
  • the invention relates to a method of treatment of a coagulopathy comprising the steps of administering a therapeutically effect amount of an anti-FVa antibody to a subject in need and thereby treating coagulopathy.
  • treatment may be prophylactic, palliative and/or symptomatic.
  • Prophylactic treatment may also be termed prevention e.g. at treatment where the medicament is administered in the absence of symptoms to reduce the occurrence and/or severity of symptoms.
  • compounds of the inventions may be use both as prevention/prophylactic treatment and/or symptomatic treatment.
  • An antibody (or fragment thereof) of the invention may be administered parenterally, such as intravenously, such as intramuscularly, such as subcutaneously.
  • an antibody of the invention may be administered via a non-parenteral route, such as perorally or topically.
  • An antibody of the invention may be administered prophylactically.
  • An antibody of the invention may be administered therapeutically (on demand).
  • antibodies or fragments thereof may be any suitable immunoglobulin analogs.
  • the antibodies or fragments thereof may be any suitable immunoglobulin analogs.
  • Embodiments A monoclonal anti-activated Factor V (FVa) or an anti- Factor V (FV) antibody or an
  • APC activated protein C
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases thrombin generation in the presence of APC measured as thrombin generation parameter "Velocity Index in nM per minute", in haemophilia
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases thrombin generation in the presence of APC measured as thrombin generation parameter "endogenous thrombin potential (ETP) in nM x minute", in haemophilia A patient plasma based thrombin generation assay.
  • EDP endogenous thrombin potential
  • ETP Endogenous Thrombin Potential in nM minute
  • EMP Endogenous Thrombin Potential in nM minute
  • ETP Endogenous Thrombin Potential in nM minute
  • ETP Endogenous Thrombin Potential in nM ⁇ minute
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates the thromboelastographic response.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates the thromboelastographic response in normal blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates the thromboelastographic response in haemophilia A blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates thrombelastography, for prophylactic treatment of haemophilia.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates the thromboelastographic response in blood with added anti- Factor VIII antibody.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates the thromboelastographic response in haemophilia A blood measured as clot time (R) in seconds.
  • TM Thrombo-modulin
  • MMG Maximum Rate of Thrombin Generation
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates clot formation in normal blood. 43.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates clot formation in haemophilia A-like blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates clot formation in haemophilia A blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates clot formation, for prophylactic treatment of haemophilia.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates clot formation in blood with added anti-Factor VIII antibody.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa stimulates clot formation in blood in the presence of APC.
  • TM thrombomodulin
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in normal blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in haemophilia A-like blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in haemophilia A blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in haemophilia.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in blood with added anti-Factor VIII antibody.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in blood in the presence of APC.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in blood, for prophylactic treatment of haemophilia.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases clot formation in blood in the presence of TM.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa reduces clotting time.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa reduces clotting time in normal blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa reduces clotting time in haemophilia A blood.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa reduces clotting time in blood in the presence of APC.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa enhances thrombin generation in human platelet rich plasma (PRP) in haemophilia A-like conditions in the presence of TM.
  • PRP platelet rich plasma
  • PRP platelet rich plasma
  • PRP platelet rich plasma
  • PRP platelet rich plasma
  • PRP platelet rich plasma
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa reverses TM inhibiting effect on thrombin generation in human platelet rich plasma (PRP) in haemophilia A-like conditions measured as thrombin generation parameter "Peak Thrombin in nM" in thrombin generation assay.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that when bound to FVa increases thrombin generation in haemophilia, for prophylactic treatment of haemophilia.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 6, 20, 41 , 50, 59 or 73, that binds to the C2 domain of FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, that binds to the C2 domain of FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiment 84, wherein the binding region comprises one or more of the amino acid residues: N532, A532, Q533, G534, R535, V536, N537, A538, W539, Q540, A541 , K542, A543, D553, L554, G588, V589, E590, W591 , K592, P593, Y594, R595, L596 K597, S598, S599 or M600 of SEQ ID NO:38.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a CDR1 sequence comprising residues 24-34 SEQ ID NO: 23 (QASESISSYLT), and/or a CDR2 sequence comprising residues 50-56 of SEQ ID NO:
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90, wherein the heavy chain of said antibody or antigen- binding fragment comprises a CDR1 sequence comprising residues 31 -36 SEQ ID NO:
  • SEQ ID NO: 24 A monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-91 , wherein said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO: 23
  • YASTLAS and/or a LC-CDR3 sequence comprising residues 89-101 of SEQ ID NO: 23 (LGVYSYSRDDGIA), and/or a HC-CDR1 sequence comprising residues 31 -36 SEQ ID NO: 24 (SSYYMC), and/or a HC-CDR2 sequence comprising residues 51-67 of SEQ ID NO: 24 (CIYTAWDGASYANWAKG), and/or a HC-CDR3 sequence comprising residues 99-109 of SEQ ID NO: 24 (AMGSSDGANNL).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92, wherein the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 23, and/or the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 23.
  • a monoclonal anti-FVa/anti-FV antibody or antigen binding fragment thereof according to any of embodiments 1 -93, that competes with an antibody or antigen-binding fragment wherein the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 23, and/or the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 24, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 6, 20, 41 , 50, 59 or 73, that competes with an antibody or antigen-binding fragment wherein the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 23, and/or the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 24, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, that competes with an antibody or antigen-binding fragment wherein the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 23, and/or the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 24, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a CDR1 sequence comprising residues 23-33 SEQ ID NO:21 (SGDILGDKYAC), and/or a CDR2 sequence comprising residues 49-55 of SEQ ID NO:21 (QDIKRPS), and/or a CDR3 sequence comprising residues 88-97 of SEQ ID NO:21 (QAWDSTTPW).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 97, wherein the heavy chain of said antibody or antigen- binding fragment comprises a CDR1 sequence comprising residues 31 -35 SEQ ID NO:22 (SYDIN), and/or a CDR2 sequence comprising residues 50-66 of SEQ ID NO:22
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 97 or 98 wherein said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 23-33 SEQ ID NO:21
  • SGDILGDKYAC and/or a LC-CDR2 sequence comprising residues 49-55 of SEQ ID NO:21 (QDIKRPS), and/or a LC-CDR3 sequence comprising residues 88-97 of SEQ ID NO:21 (QAWDSTTPW), and/or a HC-CDR1 comprising residues 31-35 SEQ ID NO:22 (SYDIN), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:22 (WMNPNTDDTGYAQKFQG), and/or a HC-CDR3 sequence comprising residues 99-1 19 of SEQ ID NO:22 (YWSVTSWKWNDDHYYYYGMDV).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92 or 97-99 wherein the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 21 , and/or the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 22.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or antigen binding fragment thereof is provided.
  • the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 21
  • the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 22, in binding to the C2 domain of FVa. 102.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 6, 20, 41 , 50, 59 or 73, that competes with an antibody or antigen-binding fragment wherein the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 21 , and/or the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 22, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, that competes with an antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO: 21 , and/or a heavy chain variable domain (VH) comprising SEQ ID NO: 22, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 6, 20, 41 , 50, 59 or 73, that competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO: 23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO: 24 and/or an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, that competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO: 23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO: 24 and/or an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, in binding to the C2 domain of FVa. 107.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89 wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-40 SEQ ID NO:15 (KSSQSVLYSSNNKNYLA), and/or a LC-CDR2 sequence comprising residues 56-62 of SEQ ID NO:15 (WASTRES), and/or a LC-CDR3 sequence comprising residues 95-103 of SEQ ID NO:15 (QQYYSTPWT).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 107, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:16 (SYDIN), and/or a HC-CDR2 sequence comprising residues 50-66 of
  • SEQ ID NO:16 (WMNPNSGNTGYALKFQG), and/or a HC-CDR3 sequence comprising residues 99-1 14 of SEQ ID NO:16 (RTYYDILTGSLGAFDI).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 107 or 108, wherein said antibody or antigen- binding fragment comprises a LC-CDR1 sequence comprising residues 24-40 SEQ ID NO:15 (KSSQSVLYSSNNKNYLA), and/or a LC-CDR2 sequence comprising residues 56- 62 of SEQ ID NO: 15 (WASTRES), and/or a LC-CDR3 sequence comprising residues 95- 103 of SEQ ID NO:15 (QQYYSTPWT), and/or a HC-CDR1 comprising residues 31 -35 SEQ ID NO:16 (SYDIN), and/or a HC-CDR2 sequence comprising residues 50-66 of
  • SEQ ID NO:16 (WMNPNSGNTGYALKFQG), and/or a HC-CDR3 sequence comprising residues 99-1 14 of SEQ ID NO:16 (RTYYDILTGSLGAFDI).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92 or 107-109 wherein the light chain variable domain (VL) of said antibody or antigen-binding fragment comprises SEQ ID NO: 15, and/or the heavy chain variable domain (VH) of said antibody or antigen-binding fragment comprises SEQ ID NO: 16. .
  • a monoclonal anti-FVa/anti-FV antibody or antigen binding fragment thereof that competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO: 15, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, in binding to the C2 domain of FVa. .
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 6, 20, 41 , 50, 59 or 73 or 84-1 12 that competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:15, and/or a heavy chain variable domain (VH) comprising
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, that competes with an antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, in binding to the C2 domain of FVa. .
  • VL light chain variable domain
  • VH heavy chain variable domain
  • An monoclonal anti-FVa/anti-FV antibody or antigen binding fragment thereof that competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 , and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 84-1 14 that competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 , and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15, and/or a heavy chain variable domain (VL) comprising SEQ ID NO:15, and/or a heavy chain variable domain (VL) comprising SEQ ID NO:15, and/or a heavy chain variable domain (VL) comprising SEQ
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, that competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 , and a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15, and a heavy chain variable domain (VH) comprising SEQ ID NO:16, in binding to the C2 domain of FVa, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:19 (RASQDISTWLA), and/or a LC-CDR2 sequence comprising residues 50- 56 of SEQ ID NO:19 (ITSTLHI), and/or a LC-CDR3 sequence comprising residues 89-97 of SEQ ID NO:19 (QQAYSFPFT).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 1 17 wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35
  • SEQ ID NO:20 (DYAMH), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:20 (GISWNSGGIGYADSVQG), and/or a HC-CDR3 sequence comprising residues 99-1 15 of SEQ ID NO:20 (DARWLVEEDYYYYGMDV). 1 19.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 1 17 or 1 18 wherein said antibody or antigen- binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO: 19 (RASQDISTWLA), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:19 (ITSTLHI), and/or a LC-CDR3 sequence comprising residues 89-97 of SEQ ID NO:19 (QQAYSFPFT), and/or a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:20 (DYAMH), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:20 (GISWNSGGIGYADSVQG), and/or a HC-CDR3 sequence comprising residues 99-1 15 of SEQ ID NO:20 (DARWLVEEDYYYYGM
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92 or 1 17-1 19 with a light chain variable domain (VL) comprising SEQ ID NO:19, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:19, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79 or 84-121 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:19, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:19, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 84-124 that competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 , and a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15, and a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19, and a heavy chain variable domain (VH) comprising SEQ ID NO:
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, that competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 , and a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15, and a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19, and a heavy chain variable domain (VH) comprising S
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:1 1 (RASQDISHWLA), and/or a LC-CDR2 sequence comprising residues 50- 56 of SEQ ID NO:1 1 (IASTLQT), and/or a LC-CDR3 sequence comprising residues 89- 97 of SEQ ID NO:1 1 (QQSNSFPLT). 128.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 127, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:12 (DYAMH), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:12 (GISWNSGAIGYADSVKG), and/or a HC-CDR3 sequence comprising residues 99-1 15 of SEQ ID NO:12 (DARWLVEEDYQYYGLDV).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 127 or 128, wherein said antibody or antigen- binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:1 1 (RASQDISHWLA), and/or a LC-CDR2 sequence comprising residues 50-56 of
  • SEQ ID NO:1 1 (IASTLQT), and/or a LC-CDR3 sequence comprising residues 89-97 of SEQ ID NO:1 1 (QQSNSFPLT) and the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31-35 SEQ ID NO:12 (DYAMH), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:12 (GISWNSGAIGYADSVKG), and/or a HC-CDR3 sequence comprising residues 99-1 15 of
  • SEQ ID NO: 12 (DARWLVEEDYQYYGLDV).
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:1 1 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:12, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79 or 84-131 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:1 1 , and/or a heavy chain variable domain (VH) comprising SEQ ID NO:12, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:1 1 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:12, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20, and/or an antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:1 1 and/or a
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 84-134 wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or a heavy
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:17 (RASQDISSWLA), and/or a LC-CDR2 sequence comprising residues 50- 56 of SEQ ID NO:17 (IASSLQS), and/or a LC-CDR3 sequence comprising residues 89- 97 of SEQ ID NO:17 (QQANSFPFT).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 137, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:18 (DYAMH), and/or a HC-CDR2 sequence comprising residues 50-66 of
  • SEQ ID NO:18 (GVSWNSGAIGYADSVKG), and/or a HC-CDR3 sequence comprising residues 99-1 15 of SEQ ID NO:18 ( D ARWLVE E D YQYYG M D V) .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 137 or 139, wherein said antibody or antigen- binding fragment comprises a light chain LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:17 (RASQDISSWLA), and/or a LC-CDR2 sequence comprising residues 50- 56 of SEQ ID NO:17 (IASSLQS), and/or a LC-CDR3 sequence comprising residues 89- 97 of SEQ ID NO:17 (QQANSFPFT), and the heavy chain of said antibody or antigen- binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92 or 137-139, wherein said antibody or antigen- binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:17, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:18.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:17, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:18, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79 or 84-141 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:17, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:18, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:17, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:18, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and a heavy chain variable domain (VH) comprising SEQ ID NO:20, and/or an antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:1 1 and a heavy chain variable domain
  • VH comprising SEQ ID NO:12, and/or an antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:17, and a heavy chain variable domain (VH) comprising SEQ ID NO:18, in binding to the C2 domain of FVa. 145.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 84-144 wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and a heavy chain variable domain (VH) comprising
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and a heavy chain variable domain (VL
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-35 SEQ ID NO:39 (SASSSISSNYLH), and/or a LC-CDR2 sequence comprising residues 51- 57 of SEQ ID NO:39 (RTSNLAS), and/or a LC-CDR3 sequence comprising residues 90- 98 of SEQ ID NO:39 (QQGSSIPLT). .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 147 wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:40 (NYGMH), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:40 (MIYYDSSKMNYADTVKG), and/or a HC-CDR3 sequence comprising residues 99-107 of SEQ ID NO:40 (PTSHYVVDV). .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 147 or 148, wherein said antibody or antigen- binding fragment wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-35 SEQ ID NO:39
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92 or 146-149, wherein said antibody or antigen- binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:39, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:40. 151.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:39, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:40, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79 or 84-151 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:39, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:40, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:39, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:40, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20, and/or an antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:1 1 and/or
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 84-154, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen- binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID
  • VH heavy chain variable domain
  • VL heavy chain variable domain
  • VH antigen-binding fragment with light chain variable domain
  • VL antigen-binding fragment with light chain variable domain
  • VL antigen-binding fragment with light chain variable domain
  • VL comprising SEQ ID NO:19 and/or a heavy chain variable domain
  • VH heavy chain variable domain
  • VH antibody or antigen-binding fragment with light chain variable domain
  • VL comprising SEQ ID NO:1 1 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:12
  • VH antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:17, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:18, and/or an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:39 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:40, in binding to the C2 domain of FV
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:13 (RASQDISNWLA), and/or a LC-CDR2 sequence comprising residues 50- 56 of SEQ ID NO:13 (ITSTLHI), and/or a LC-CDR3 sequence comprising residues 89-97 of SEQ ID NO:13 (QQANSFPFT).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 157, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35
  • SEQ ID NO:14 (DYAMH), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:14 (GISWNSGSTGYADSVQG), and/or a HC-CDR3 sequence comprising residues 99-1 15 of SEQ ID NO:14 (DARWLVEEDYYYYGMDV). 159.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 157 or 158, wherein said antibody or antigen- binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:13 (RASQDISNWLA), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:13 (ITSTLHI), and/or a LC-CDR3 sequence comprising residues 89-97 of SEQ ID NO:13 (QQANSFPFT), and/or a HC-CDR1 sequence comprising residues 31-35 SEQ ID NO:14 (DYAMH), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:14 (GISWNSGSTGYADSVQG), and/or a HC-CDR3 sequence comprising residues 99-1 15 of SEQ ID NO:14 (DARWLVEEDYYYYGMDV
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92 or 157-159, wherein said antibody or antigen- binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:13, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:14. 161.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:13, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:14, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79 or 84-161 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:13, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:14, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:13, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:14, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20, and/or an antibody or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:1 1 and/or
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 84-164, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen- binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or
  • VL light chain variable domain
  • VH heavy chain variable domain
  • VH antibody or antigen-binding fragment with light chain variable domain
  • VL comprising SEQ ID NO:13 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:14, in binding to the C2 domain of FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 23-33 SEQ ID NO:43 (SGDKLESKYAC), and/or a LC-CDR2 sequence comprising residues 49-
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-90 or 167, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:44 (SYDIN), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:44 (WMNPNSGNTGYAQKFQG), and/or a HC-CDR3 sequence comprising residues 99-1 19 of SEQ ID NO:44 (YFSSTSWKWDDDYFYYYGMDV).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-89, 167 or 169, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 23-33 SEQ ID NO:43 (SGDKLESKYAC), and/or a LC-CDR2 sequence comprising residues 49-55 of SEQ ID NO:43 (HDDKRPS), and/or a LC-CDR3 sequence comprising residues 88-97 of SEQ ID NO:43 (QAWDSSTPVV), and the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:44 (SYDIN), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:44 (WMNPNSGNTGYAQKFQG), and/or a HC-CDR3 sequence comprising
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 84-92 or 167-169, wherein said antibody or antigen- binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO: 1
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79 or 84-171 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:43, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:44, in binding to the C2 domain of FVa. .
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:43, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:44, in binding to the C2 domain of FVa. 174.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or antigen binding fragment thereof wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:20, and/or an antibody or antigen-binding fragment with
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 84-174, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen- binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or a heavy chain variable
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:23 and a heavy chain variable domain (VH) comprising SEQ ID NO:24, and/or an antibody or an antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:21 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:22, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:15 and/or a heavy chain variable domain (VH) comprising SEQ ID NO:16, and/or antigen-binding fragment with light chain variable domain (VL) comprising SEQ ID NO:19 and/or a
  • a binding region on the C2 domain of FVa wherein said binding region comprises one or more of the amino acid residues A532 to A543, D553-L554, and G588 to M600 of SEQ ID NO:38.
  • a binding region on the C2 domain of FVa according to embodiment 177, 178 or 179, wherein said binding region comprises at least one of the amino acid residues ,A532, Q533, G534, R535, V536, N537, A538, W539, Q540, A541 , K542 and A543 of
  • SEQ ID NO:38 and at least one of the amino acid residues D553 or L554 of SEQ ID NO:38; and at least one of amino acid residues G588, V589, E590, W591 , K592, P593, Y594, R595, L596 K597, S598, S599 or M600 of SEQ ID NO:38. 181.
  • a binding region on the C2 domain of FVa according to embodiment 177, 178, 179 or 180, wherein said binding region comprises amino acid residues A532, Q533, G534, R535, V536, N537, A538, W539, Q540, A541 , K542, A543, D553, L554, G588, V589, E590, W591 , K592, P593, Y594, R595, L596 K597, S598, S599 and M600 of SEQ ID NO:38.
  • a monoclonal activated Factor V (FVa) or an anti- Factor V (FV) antibody or an antigen-binding fragment thereof, that when bound to the binding region of embodiments 177-181 displays pro-coagulant effect in haemophilia A.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 182-189, wherein said antibody competes with the antibody or antigen-binding fragments of embodiments 149 and/or 150 in binding to FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 149 and/or 150 in binding to the binding region of any of embodiments 177-181 .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 182-189, wherein said antibody competes with the antibody or antigen-binding fragments of embodiments 92 and/or 93 in binding to FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 92 and/or 93 in binding to the binding region of any of embodiments 177-181.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 109 and/or 1 10 in binding to the binding region of any of embodiments 177-181 .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 99 and/or 100 in binding to the binding region of any of embodiments 177-181.
  • 202. A monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 182-189, wherein said antibody is the antibody or antigen- binding fragment of embodiments 1 19 and/or 120.
  • 203. A monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 182-189, wherein said antibody competes with the antibody or antigen-binding fragments of embodiments 1 19 and/or 120 in binding to FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 1 19 and/or 120 in binding to the binding region of any of embodiments 177-181 .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 129 and/or 130 in binding to the binding region of any of embodiments 177-181 .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 139 and/or 140 in binding to the binding region of any of embodiments 177-181 .
  • 21 1.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 182-189, wherein said antibody competes with the antibody or antigen-binding fragments of embodiments 159 and/or 160 in binding to FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 159 and/or 160 in binding to the binding region of any of embodiments 177-181 .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 169 and/or 170 in binding to the binding region of any of embodiments 177-181 . 217.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiment 217, 218, 219 or 220, wherein the antibody binds to a binding region on activated FV and wherein the binding site comprises amino acid residues F325, I326, A327, A328, E329, E330, V331 , N534, I535, N536, K537, F538, and C539 of the
  • FVa heavy-chain sequence (SEQ ID NO:37).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 , wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34
  • SEQ ID NO:9 KTSTDIDDDMN
  • LC-CDR2 sequence comprising residues 50- 56 of SEQ ID NO:9
  • LQSANMPFT residues 89- 97 of SEQ ID NO:9
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-222, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO: 10 (SYAMS), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:10 (TISSGGSYTYYPDSVKG), and/or a HC-CDR3 sequence comprising residues 99-1 1 1 of SEQ ID NO:10 (GPYLTTATPSFTY).
  • SYAMS HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO: 10
  • HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:10
  • GPYLTTATPSFTY a HC-CDR3 sequence comprising residues 99-1 1 1 of SEQ ID NO:10
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-223, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprises residues 24-34 SEQ ID NO:9 (KTSTDIDDDMN), and/or a LC-CDR2 sequence comprising residues 50-
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-224, wherein said antibody or antigen-binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:9, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:10.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof
  • VL light chain variable domain
  • VH comprising SEQ ID NO:10, in binding to the C2 domain of FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 217, 218, 219, 220 or 221 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:9, and a heavy chain variable domain (VH) comprising SEQ ID NO:10, in binding to the C2 domain of FVa. 228.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:9, and a heavy chain variable domain (VH) comprising SEQ ID NO:10, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof
  • KTSTDIDDDMN LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:9
  • LQSANMPFT LC-CDR3 sequence comprising residues 89-97 of SEQ ID NO:9
  • a heavy chain comprising a HC-CDR1 sequence comprising residues 31 -35 SEQ ID NO:10 (SYAMS), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:10 (TISSGGSYTYYPDSVKG), and/or a HC-CDR3 sequence comprising residues 99-1 1 1 of SEQ ID NO:10 (GPYLTTATPSFTY), in binding to the C2 domain of FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 , wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34
  • SEQ ID NO:33 (QASQSISSYLS), and/or a LC-CDR2 sequence comprising residues 50- 56 of SEQ ID NO:33 (RTSTLES), and/or a LC-CDR3 sequence comprising residues 89- 102 of SEQ ID NO:33 (QSNYYSSGSSYENA). 231.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 or 230, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 30-35 SEQ ID NO:34 (SYYHIC), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:34 (CIYAASGDTWYATWVNA), and/or a HC-CDR3 sequence comprising residues 98-1 14 of SEQ ID NO:34 (GPRYVSSSGAGPYCLDL).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 , 230 or 231 , wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:33 (QASQSISSYLS), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:33 (RTSTLES), and/or a LC-CDR3 sequence comprising residues 89-102 of SEQ ID NO:33 (QSNYYSSGSSYENA) and the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 30-35 SEQ ID NO:34 (SYYHIC), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:34 (CIYAASGDTWYATWVNA), and/or a HC-
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 or 229-232, wherein said antibody or antigen- binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:33, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:34.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 217, 218, 219, 220 or 221 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:33, and a heavy chain variable domain (VH) comprising SEQ ID NO:34, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:33, and a heavy chain variable domain (VH) comprising SEQ ID NO:34, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof
  • a light chain comprising a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:33 (QASQSISSYLS), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:33 (RTSTLES), and/or a LC-CDR3 sequence comprising residues 89-102 of SEQ ID NO:33 (QSNYYSSGSSYENA) and/or a heavy chain comprising comprises a HC-CDR1 sequence comprising residues 30-35 SEQ ID NO:34 (SYYHIC), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:34 (CIYAASGDTWYATWVNA), and/or a HC-CDR3 sequence comprising residues 98-1 14 of SEQ ID NO:34
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof according to any of embodiments 1-84, 217-233, wherein said antibody or antigen- binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:33, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:34, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain (VL) comprising SEQ ID NO:9, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:10, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 217, 218, 219, 220 or 221 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:33, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:34, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain
  • VL light chain variable domain
  • VH heavy chain variable domain
  • VL comprising SEQ ID NO:9
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment thereof, with a light chain variable domain (VL) comprising SEQ ID NO:33, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:34, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain (VL) comprising SEQ ID NO:9, and/or a heavy chain variable domain (VH) comprising
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof
  • said antibody or antigen- binding fragment comprises a light chain comprising a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:33 (QASQSISSYLS), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:33 (RTSTLES), and/or a LC-CDR3 sequence comprising residues 89-102 of SEQ ID NO:33 (QSNYYSSGSSYENA) and/or a heavy chain comprising comprises a HC-CDR1 sequence comprising residues 30-35 SEQ ID NO:34 (SYYHIC), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:34 (CIYAASGDTWYATWVNA), and/or a HC-CDR3 sequence comprising residues 98-1 14 of SEQ ID NO:34 (GPRYVSSSGAGPYCLDL) or/and with an antibody or an anti
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 , wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:29 (QASQSIGGNLA), and/or a LC-CDR2 sequence comprising residues 50-
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 or 242, wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 30-34 SEQ ID NO:30 (SYAMI), and/or a HC-CDR2 sequence comprising residues 49-64 of SEQ ID NO:30 (FIDTGGSAYYASWAKG), and/or a HC-CDR3 sequence comprising residues 95-108of SEQ ID NO:30 (A LYVYS D VYTA F N I ) .
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 , 242 or 243, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:29 (QASQSIGGNLA), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:29 (DASKLAS), and/or a LC-CDR3 sequence comprising residues 89-10 of SEQ ID NO:29 (QCTYGSSGNIGNG) and the heavy chain of said antibody or antigen-binding fragment comprises a HC-CDR1 sequence comprising residues 30-34 SEQ ID NO:30 (SYAMI), and/or a HC-CDR2 sequence comprising residues 49-64 of SEQ ID NO:30 (FIDTGGSAYYASWAKG), and/or a HC- CDR3 sequence comprising residues 95-108 of
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 217-221 or 242-244, wherein said antibody or antigen- binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:29, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:30.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof
  • said antibody or antigen- binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:29, and a heavy chain variable domain (VH) comprising SEQ ID NO:30, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 217, 218, 219, 220 or 221 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:29, and a heavy chain variable domain (VH) comprising SEQ ID NO:30, in binding to the C2 domain of FVa. 248.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or antigen- binding fragment with light chain variable domain (VL) comprising SEQ ID NO:29, and a heavy chain variable domain (VH) comprising SEQ ID NO:30, in binding to the C2 domain of FVa.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof
  • said antibody competes with an antibody or an antigen-binding fragment thereof with a light chain comprising a LC- CDR1 sequence comprising residues 24-34 SEQ ID NO:29 (QASQSIGGNLA), and/or a
  • LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:29 (DASKLAS), and/or a LC-CDR3 sequence comprising residues 89-10 of SEQ ID NO:29 (QCTYGSSGNIGNG) and a heavy chain comprising a HC-CDR1 sequence comprising residues 30-34 SEQ ID NO:30 (SYAMI), and/or a HC-CDR2 sequence comprising residues 49-64 of SEQ ID NO:30 (FIDTGGSAYYASWAKG), and/or a HC-CDR3 sequence comprising residues 95-
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof according to any of embodiments 1-84, 217-249, wherein said antibody or antigen- binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:33, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:34, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain (VL) comprising SEQ ID NO:9, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:10, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain (VL) comprising SEQ ID NO:29, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:30, in binding to the
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 1 , 2, 3, 4, 5, 20, 41 , 50, 59, 73, 79, 217, 218, 219, 220 or 221 , wherein said antibody or antigen-binding fragment competes with an antibody or antigen-binding fragment with a light chain variable domain (VL) comprising SEQ ID NO:33, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:34, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain (VL) comprising SEQ ID NO:9, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:10, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain (VL) comprising SEQ ID NO:29, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:30, in binding to the C2
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 7, 8, 12, 16, 24, 32, 39, 45, 48, 54, 57, 63, 72, 78 or 80, wherein said antibody or antigen-binding fragment competes with an antibody or an antigen-binding fragment thereof, with a light chain variable domain (VL) comprising SEQ ID NO:33, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:34, and/or with an antibody or an antigen-binding fragment comprising a light chain variable domain (VL) comprising SEQ ID NO:9, and/or a heavy chain variable domain (VH) comprising
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody an antigen-binding fragment thereof according to any of embodiments 1-84 or 217-236, wherein said antibody or antigen- binding fragment comprises a light chain comprising a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:33 (QASQSISSYLS), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:33 (RTSTLES), and/or a LC-CDR3 sequence comprising residues 89-102 of SEQ ID NO:33 (QSNYYSSGSSYENA) and/or a heavy chain comprising comprises a HC-CDR1 sequence comprising residues 30-35 SEQ ID NO:34 (SYYHIC), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:34 (CIYAASGDTWYATWVNA), and/or a HC-CDR3 sequence comprising residues 98-1 14 of S
  • SYAMS and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:10 (TISSGGSYTYYPDSVKG), and/or a HC-CDR3 sequence comprising residues 99-1 1 1 of SEQ ID NO: 10 (GPYLTTATPSFTY), and/or with an antibody or an antigen-binding fragment thereof with a light chain comprising a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:29 (QASQSIGGNLA), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:29 (DASKLAS), and/or a LC-CDR3 sequence comprising residues 89-10 of SEQ ID NO:29 (QCTYGSSGNIGNG) and a heavy chain comprising a HC-CDR1 sequence comprising residues 30-34 SEQ ID NO:30 (SYAMI), and/or a HC- CDR2 sequence comprising residues 49-64 of SEQ ID NO:
  • binding region on the C2 domain of FVa according to embodiment 254, 255 or 256, wherein said binding region comprises at least one of the amino acid residues F325, 1326, A327, A328, E329, E330 or V331 (SEQ ID NO:37) and at least one of the amino acid residues N534, I535, N536, K537, F538 or C539 (SEQ ID NO:37).
  • antigen-binding fragment thereof that when bound to the binding region of embodiments 254-258, displays pro-coagulant effect in haemophilia A.
  • APC activated protein C
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 259-266, wherein said antibody competes with the antibody or antigen-binding fragments of embodiments 224 and/or 225 in binding to FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 224 and/or 225 in binding to the binding region of any of embodiments 254-258.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 259-266, wherein said antibody is the antibody or antigen- binding fragment of embodiments 232 and/or 233.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to embodiments 259-266, wherein said antibody competes with the antibody or antigen-binding fragments of embodiments 232 and/or 233 in binding to FVa.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 232 and/or 233 in binding to the binding region of any of embodiments 254-258.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment of embodiments 244 and/or 245 in binding to the binding region of any of embodiments 254-258.
  • CDR1 sequence comprising residues 24-34 SEQ ID NO:7 (KASQDVGTAVG), and/or a LC-CDR2 sequence comprising residues 50-56 of SEQ ID NO:7 (WASTRHT), and/or a LC-CDR3 sequence comprising residues 89-96 of SEQ ID NO:7 (QQYSSNPT). 277.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof wherein the heavy chain of said antibody or antigen-binding fragment comprises a HC- CDR1 sequence comprising residues 31 -35 SEQ ID NO:8 (NYGMN), and/or a HC-CDR2 sequence comprising residues 50-66 of SEQ ID NO:8 (WINTYTGEPTYADDFKG), and/or a HC-CDR3 sequence comprising residues 99-1 1 1 of SEQ ID NO:10
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 276-277, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-34 SEQ ID NO:7 (KASQDVGTAVG), and/or a LC-CDR2 sequence comprising residues 50-
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 276-278, wherein said antibody or antigen-binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:7, and/or a heavy chain variable domain (VH) comprising SEQ ID NO:8.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 281 or 282, wherein the light chain of said antibody or antigen-binding fragment comprises a LC-CDR1 sequence comprising residues 24-40 SEQ ID NO:41 (RSSQSLLDSDDGNTYMD), and/or a LC-CDR2 sequence comprising residues 56-62 of SEQ ID NO:41 (MGFYRAS), and/or a LC-CDR3 sequence comprising residues 95-103 of SEQ ID NO:41 (MQRIEFPST) and the heavy chain of said antibody comprises a HC-CDR1 sequence comprising residues 31-37 SEQ ID NO:42
  • TSGVGVG TSGVGVG
  • HC-CDR2 sequence comprising residues 52-67 of SEQ ID NO:42
  • LIYWDDVKRYSPSLRR LIYWDDVKRYSPSLRR
  • HC-CDR3 sequence comprising residues 100-107 of
  • SEQ ID NO:42 (YNWKMRVD).
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 281-283, wherein said antibody or antigen-binding fragment comprises a light chain variable domain (VL) comprising SEQ ID NO:41 , and/or a heavy chain variable domain (VH) comprising SEQ ID NO:42.
  • VL light chain variable domain
  • VH heavy chain variable domain
  • antigen-binding fragment thereof according to any of embodiments 276, 277, 278, 279 or 280, that when bound to the C2 domain of FVa displays pro-coagulant effect in haemophilia A.
  • a monoclonal activated Factor V (FVa) or an anti- Factor V (FV) antibody or an antigen-binding fragment thereof according to any of embodiments 281 , 282, 283, 284 or 285, that when bound to the C2 domain of FVa displays pro-coagulant effect in haemophilia A.
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 281 , 282, 283, 284 or 285, that when bound to the C2 domain of FVa protects FVa from inactivation by activated protein C (APC).
  • APC activated protein C
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 281 , 282, 283, 284 or 285, that when bound to the C2 domain of FVa increases thrombin generation.
  • a monoclonal activated Factor V (FVa) or an anti- Factor V (FV) antibody or an antigen-binding fragment thereof according to any of embodiments 276, 277, 278, 279 or 280, that when bound to the C2 domain of FVa displays pro-coagulant effect in haemophilia A.
  • FVa monoclonal activated Factor V
  • FV anti- Factor V
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 281 , 282, 283, 284 or 285, that when bound to the C2 domain of FVa protects FVa from inactivation by activated protein C (APC).
  • APC activated protein C
  • a monoclonal anti-FVa/anti-FV antibody or an antigen-binding fragment thereof according to any of embodiments 281 , 282, 283, 284 or 285, that when bound to the C2 domain of FVa increases thrombin generation.
  • Use according to embodiments 302 or 303, wherein the monoclonal anti-FVa/anti- FV antibody or antigen-binding fragment thereof, is the antibody of any of embodiments 224, 225, 232, 233, 244 and/or 245. 307.
  • Use according to embodiments 302 or 303, wherein the monoclonal anti-FVa/anti- FV antibody or antigen-binding fragment thereof, is the antibody of embodiments 278 and/or 279.
  • a pharmaceutical composition comprising the antibody of embodiments 92 and/or 93.
  • a pharmaceutical composition comprising the antibody of any of embodiments 92,
  • a pharmaceutical composition comprising the antibody of any of embodiments 224, 225, 232, 233, 244 and/or 245. .
  • a pharmaceutical composition comprising the antibody of embodiments 278 and/or 279. .
  • a pharmaceutical composition comprising the antibody of embodiments 283 and/or 284. .
  • An antigen-binding fragment of the antibody of any of embodiments 1 -176 or 182- 301 which is a Fab, Fab', F(ab)2, F(ab')2, Fv, single-chain Fv, dsFv, Fd or a dAb fragment, a VH, VL, VhH, or V-NAR domains, a monovalent molecule, minibody, diabody, triabody, tetrabody or kappa body, or an IgNAR.
  • a variant of the antibody of any of embodiments 1 -176 or 182-301 which is a deletion variant or an insertion variant.
  • a recombinant vector that comprises the nucleic acid construct of embodiment 316. An isolated cell that expresses the antibody of any of embodiments 1-176 or182- 301. . An isolated cell that comprises the nucleic acid construct of embodiment 316 or the vector of embodiment 318.
  • mice Conventional mice (RBF, NMRCF1 ) and HK or HL -Kymice (Kymab, Cambridge,
  • Serum was added with a starting dilution of 1 :50 and followed by threefold serial dilution and the plates were incubated for 1 hour at room temperature. After another wash, HRP labelled gt anti-mouse Fc-HRP was added at a concentration of 1 ⁇ g/ml, and incubated for 1 hour. After washing, plates were developed with TMB-substrate (Kem- EN-Tec) as described by the manufacturer. Absorbance at 450 nm was measured on an ELISA-reader. Only mice, which showed a specific response towards human FVa were considered for fusions and mice with the highest titer were selected. Mice with positive titer were boosted i.v.
  • spleenocytes were removed aseptically and dispersed to a single cell suspension. Fusion of spleenocytes with myeloma cells (FOX-Ny or Ag8-X63) was done by standard electrofusion. Cells were seeded in microtiter plates and cultured for 13 days under selection with HAT/HT. Supernatants were screened in a direct ELISA on human FVa. Nunc immunoplates were coated with 1 ⁇ g/ml of peptide of and incubated overnight at 4°C. Plates were blocked with blocking buffer (PBS with 0.05% Tween20) for 15 min and were washed with PBS/0.05%Tween20.
  • blocking buffer PBS with 0.05% Tween20
  • Rabbits (New Zealand White) were immunized biweekly four times with 50 ⁇ g human FVa (SEQ ID NO: 37 and 38) using emulsified in Sigma Adjuvant System® a standard protocol.
  • the hFVa immunized rabbits were sacrificed, the spleen removed and passed through a nylon mesh. After thorough wash, the spleen cells were frozen in aliquots in liquid N 2 .
  • EL4.B5 mouse thymoma cells expressing the CD40-ligand crucial for B-cell proliferation, differentiation and Ig-production.
  • the cells were cultured at 37 ° C and 5% C0 2 for 24 to 48 hours in medium supplemented with 100ng/ml mBAFF, 600ng/ml RblL-2 and 5% of a PMA- activated splenic supernatant.
  • Frozen spleen cells were thawed, washed in FACS buffer (PBS + 1 % BSA) and 2 vials of 15x10 6 rabbit splenocytes were resuspended in 200 ⁇ FACS buffer. To block unspecific binding, 500 nM FVIII was added to the cells. The suspension was incubated for 10 min, hFVa biotin (10 ⁇ g/ml) was added and the cells incubated for further 30 min on ice.
  • Facs buffer was added up to 1 ml with 1 ⁇ SYTOX Red dead cell stain and the cells incubated further 20 min on ice. The cells were then washed twice in Facs Buffer, resuspended in a desired volume and filtered through a 30 ⁇ filcon.
  • Cells were acquired on a FACSAria Sorter. Gates were initially set on lymphocytes, then live lymphocytes and finally on single live lymphocytes. This final gate was used when sorting gate was set on the antigen specific B-cells identified as those simultaneously binding to both anti rabbit IgG and biotinylated hFVa. Cells were sorted as 1 cell/well into 384-well plates already seeded with the EL4B5 cells. The cells were then kept in the incubator at 37 ° C and 5% C0 2.
  • Example 2 Identification of pro-coagulant anti-Factor Va antibodies in human haemophilic plasma by thrombin generation assay
  • anti-FVa antibodies were identified, that were capable of increasing thrombin generation in the presence of exogenously added APC in a human plasma-based thrombin generation assay.
  • the purified test antibodies were tested in the final assay at 0 nM - 500 nM, and the assay was run at room temperature.
  • human haemophilia A (HA) (factor VIII deficient) plasma (Georg King Medical, #0800) stored at -80C was thawed in water at 37C for 5 min, and then stored at room temperature until use.
  • thrombogram was calculated as the first derivative of the integral fluorescence curve, and the ETP and peak thrombin parameters were calculated from the thrombogram, and used in the evaluation of thrombin generation.
  • Antibodies that were capable of increasing thrombin generation in the presence of exogenously added APC in a human plasma-based thrombin generation assay were categorized as hits. During the screening of 500 antibodies, 35 antibodies were categorized as hits. Table 1 lists 21 antibodies out of the 35 identified as hits, including three low and non-functioning antibodies determined by the plateau from the concentration-response curve.
  • the amount of thrombin generated in plasma was measured by Calibrated Automated Thrombography (Hemker et al., "Calibrated Automated Thrombin Generation Measurement in Clotting Plasma,” Pathophysiol Haemost Thromb. 33:4-15 (2003); Hemker et al., "Thrombin Generation in Plasma: Its Assessment via the Endogenous Thrombin Potential,” Thromb Haemost. 74:134-138 (1995)).
  • factor VIII deficient plasma pool ( ⁇ 1 % residual activity, platelet-poor) from severe haemophilia A patients lacking factor VIII inhibitor (George King Bio-Medical, Overland Park, Kans.) was incubated with 8 ⁇ _ of antibody (or antibody fragment, HEPES-BSA buffer or recombinant Factor FVIIa) for 10 minutes at 37° C.
  • Thrombinoscope PPP 5 pM tissue-factor and 4 ⁇ phospholipid
  • Thrombinoscope PPP LOW 1 pM tissue-factor and 4 ⁇ phospholipid
  • All reagents were pre- warmed to 37° C.
  • the development of a fluorescent signal at 37° C was monitored at 20 second intervals using a Fluoroskan Ascent reader (Thermo Labsystems OY, Helsinki, Finland). Fluorescent signals were corrected by the reference signal from the thrombin calibrator samples (Hemker et al., "Calibrated Automated Thrombin Generation
  • Tables 2, 3 and 4 below set forth the thrombin generation parameters peak thrombin, velocity index and endogenous thrombin potential, respectively, determined in haemophilia A plasma in the presence of either Buffer, Factor VI 11 (1 % or 10%), or antibody 0233-0000-0005 (90 nM). Both in the absence and presence of APC or thrombo-modulin (TM), antibody 0233-0000-0005 yields a stronger stimulation of thrombin generation than 10% Factor VIII.
  • Table 2 lists Thrombin generation as a thrombin generation parameter Peak
  • Thrombin (in nM) from thrombin generation in haemophilia A patient plasma in the presence of various concentrations of Activated Protein C (APC) or thrombo-modulin (TM) and either Buffer, 1 % Factor VIII (0.01 lll/mL), 10% Factor VIII (0.1 IU/ml_) or 90 nM NNC-0233-0000- 0005. Last column indicates the number of independent experiments (n). Thrombin generation was triggered by Thrombinoscope PPP LOW (1 pM tissue-factor and 4 ⁇ phospholipid). NS indicates no thrombin generation signal.
  • Table 3 lists Thrombin generation as thrombin generation parameter Velocity Index (in nM per minute) from thrombin generation in haemophilia
  • a patient plasma in the presence of various concentrations of Activated Protein C (APC) or thrombo-modulin (TM) and either Buffer, 1 % Factor VIII (0.01 lll/mL), 10% Factor VIII (0.1 lll/mL) or 90 nM NNC-0233-0000- 0005.
  • Last column indicates the number of independent experiments (n).
  • Thrombin generation was triggered by Thrombinoscope PPP LOW (1 pM tissue-factor and 4 ⁇ phospholipid).
  • NS indicates no thrombin generation signal.
  • Table 3 Thrombin generation in haemophilia A patient plasma with different concentration of
  • Thrombin generation is show as thrombin generation parameter endogenous thrombin potential (ETP, in nM minute) from thrombin generation in haemophilia
  • ETP endogenous thrombin potential
  • a patient plasma in the presence of various concentrations of Activated Protein C (APC) or thrombo-modulin (TM) and either Buffer, 1 % Factor VIII (0.01 lU/mL), 10% Factor VIII (0.1 lU/mL) or 90 nM NNC-0233-0000-0005.
  • Last column indicates the number of independent experiments (n).
  • Thrombin generation was triggered by Thrombinoscope PPP LOW (1 pM tissue-factor and 4 ⁇ phospholipid).
  • NS indicates no thrombin generation signal.
  • Tables 5, 6 and 7 below set forth the thrombin generation parameters peak thrombin, velocity index and endogenous thrombin potential, respectively, determined haemophilia A plasma in the presence of activated protein C (APC, 5 nM) and increasing concentrations (5 nM to 500 nM) of three antibodies (0233-0000-0005, 0233-0000-0028, 0233-0000-01 10). All three antibodies stimulated thrombin generation (as judged by either of the parameters) but to different extent, with 0233-0000-01 10 providing the most pronounced stimulation.
  • APC activated protein C
  • Table 5 shows the thrombin generation parameter Peak Thrombin (in nM) from thrombin generation in haemophilia A patient plasma in the presence of Activated Protein C (5 nM) and various antibody concentrations (5 nM to 500 nM). Last column indicates the number of independent experiments (n). Thrombin generation was triggered by
  • Thrombinoscope PPP (5 pM tissue-factor and 4 ⁇ phospholipid). NS indicates no thrombin generation signal. In the absence of antibody, no thrombin generation was observed under these conditions. Table 5. Thrombin generation in haemophilia A patient plasma in the presence of APC (5 nM) and various antibody concentrations (5 nM to 500 nM)
  • Table 6 shows thrombin generation parameter Velocity Index (in nM per minute) from thrombin generation in haemophilia A patient plasma in the presence of Activated Protein C (5 nM) and various antibody concentrations (5 nM to 500 nM). Last column indicates the number of independent experiments (n). Thrombin generation was triggered by Thrombinoscope PPP (5 pM tissue-factor and 4 ⁇ phospholipid). NS indicates no thrombin generation signal. In the absence of antibody, no thrombin generation was observed under these conditions. Table 6. Thrombin generation in haemophilia A patient plasma in the presence of APC (5 nM) and various antibody concentrations (5 nM to 500 nM).
  • Table 7 lists the thrombin generation parameter endogenous thrombin potential (ETP, in nM minute) from thrombin generation in haemophilia
  • ETP endogenous thrombin potential
  • a patient plasma in the presence of Activated Protein C (5 nM) and various antibody concentrations (5 nM to 500 nM) is demonstrated in table 7.
  • Last column indicates the number of independent experiments (n).
  • Thrombin generation was triggered by Thrombinoscope PPP (5 pM tissue- factor and 4 ⁇ phospholipid).
  • NS indicates no thrombin generation signal. In the absence of antibody, no thrombin generation was observed under these conditions.
  • Table 8 shows the thrombin generation parameters peak thrombin, velocity index and endogenous thrombin potential determined in haemophilia A plasma in the absence or presence of activated protein C (APC, 5 nM) and various concentrations (250 nM or 500 nM) of six antibodies or antibody fragments. Last column indicates the number of independent experiments (n). Thrombin generation was triggered by Thrombinoscope PPP (5 pM tissue- factor and 4 ⁇ phospholipid). NS indicates no thrombin generation signal.
  • Tables 9, 10 and 1 1 below set forth the thrombin generation parameters peak thrombin, velocity index and endogenous thrombin potential, respectively, determined in normal human plasma or protein C deficient plasma in the absence or presence of a neutralising anti-Factor VIII polyclonal antibody (0.1 mg/mL) and increasing concentrations of 0233-0000-0005 (0 nM to 500 nM). In both normal and protein C-deficient plasma 0233- 0000-0005 dose-dependently stimulated thrombin generation as judged by peak thrombin and velocity index both in the absence and presence of a neutralising anti-Factor VIII antibody.
  • Table 9 shows thrombin generation parameter Peak Thrombin (in nM) from thrombin generation in human normal plasma or protein C deficient plasma (PC-def.) in the presence various concentrations of 0233-0000-0005 (0 nM to 500 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (0.1 mg/mL). Last column indicates the number of independent experiments (n). Thrombin generation was triggered by Thrombinoscope PPP (5 pM tissue-factor and 4 ⁇ phospholipid). NS indicates no thrombin generation signal. Table 9. Thrombin generation in normal or PC-def. plasma with 0233-0000-0005 (0 nM to 500 nM) with or without anti-Factor VIII antibody (0.1 mg/mL)
  • Table 10 shows the thrombin generation parameter Velocity Index (in nM per minute) from thrombin generation in human normal plasma or protein C deficient plasma (PC-def.) in the presence various concentrations of 0233-0000-0005 (0 nM to 500 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (0.1 mg/mL). Last column indicates the number of independent experiments (n). Thrombin generation was triggered by Thrombinoscope PPP (5 pM tissue-factor and 4 ⁇ phospholipid). NS indicates no thrombin generation signal.
  • Thrombin generation parameter Endogenous Thrombin Potential EPP, in nMxminute
  • EPP Endogenous Thrombin Potential
  • PC-def. protein C deficient plasma
  • 0233-0000-0005 0. nM to 500 nM
  • a neutralising polyclonal anti-Factor VIII antibody 0.1 mg/mL
  • Last column indicates the number of independent experiments (n).
  • Thrombin generation was triggered by Thrombinoscope PPP (5 pM tissue-factor and 4 ⁇
  • NS indicates no thrombin generation signal.
  • Table 1 Thrombin generation in normal or PC-def. plasma with 0233-0000-0005 (0 nM to 500 nM) with or without anti-Factor VIII antibody (0.1 mg/mL)
  • the elastic properties of blood during thrombus formation were measured by thrombelastography using a TEG® hemostasis analyzer (U.S. Pat. No. 5,223,227, and Luddington, RJ, "Thrombelastography/thromboelastometry.,” Clin Lab Haematol. 27:81 -90 (2005)).
  • the TEG® hemostasis analyzer monitors the elastic properties of blood as it is induced to clot under a low shear environment resembling sluggish venous blood flow.
  • the patterns of changes in shear elasticity of the developing clot enable the determination of the kinetics of clot formation, as well as the strength and stability of the formed clot; in short, the mechanical properties of the developing clot.
  • a total volume of 340 ⁇ _ of preheated (37°C) human whole blood (that had been incubated with combinations of compound, neutralising polyclonal anti-Factor VIII antibody, activated protein C or thrombo-modulin) was recalcified using 20 ⁇ _ calcium chloride (0.2 M) and the TEG analysis was initiated.
  • the clotting time (R) was defined as the time from initiation to amplitude had reached 2 mm
  • Maximum Rate of Thrombus Generation (MTG) was defined as the global maximum of the first derivative of amplitude in time.
  • Tables 12A and 12B demonstrate the thrombelastography parameters R (clot time) and Maximum Rate of Thrombin Generation (MTG), respectively, determined in normal and haemophilia A-like human blood (i.e. in the absence or presence of a neutralising anti-Factor VIII polyclonal antibody, 0.1 mg/mL) and increasing concentrations of antibodies 0233-0000- 01 10 and 0233-0000-0150 (0 nM to 400 nM).
  • R clot time
  • MMG Maximum Rate of Thrombin Generation
  • Table 12A shows the thrombelastography parameter clot time (R, in seconds) from thrombelastography in normal or haemophilia A-like whole blood (i.e. in the absence or presence of a neutralising anti-Factor VIII polyclonal antibody, 0.1 mg/mL) in the presence of various concentrations of antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL).
  • Last column indicates the number of independent experiments (n). Thrombelastography was initiated using 40,000- fold diluted Innovin. ND indicates not determined.
  • Table 12B shows the thrombelastography parameter Maximum Rate of Thrombus Generation (MTG, in 100xmm per second) from thrombelastography in normal or
  • haemophilia A-like whole blood (i.e. in the absence or presence of a neutralising anti-Factor VIII polyclonal antibody, 0.1 mg/mL) in the presence of various concentrations of antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL).
  • Last column indicates the number of independent experiments (n). Thrombelastography was initiated using 40,000-fold diluted Innovin. ND indicates not determined.
  • Tables 13A and 13B below set forth the thrombelastography parameters R (clot time) and Maximum Rate of Thrombin Generation (MTG), respectively, determined in normal and haemophilia A-like human blood (i.e. in the absence or presence of a neutralising anti- Factor VIII polyclonal antibody, 0.1 mg/mL) in the presence of activated protein C (APC, 5 nM) and increasing concentrations of 0233-0000-01 10 and 0233-0000-0150 (0 nM to 400 nM). In normal and haemophilia A-like blood both 0233-0000-01 10 and 0233-0000-0150 stimulated thrombelastography by dose-dependently reducing the clotting time in the presence of APC.
  • R clot time
  • MTG Maximum Rate of Thrombin Generation
  • Table 13A shows thrombelastography parameter clot time (R, in seconds) from thrombelastography in normal or haemophilia A-like whole blood in the presence of Activated Protein C (5 nM) and various concentrations of antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL).
  • Last column indicates the number of independent experiments (n). Thrombelastography was initiated using 40,000-fold diluted Innovin. ND indicates not determined.
  • Table 13B shows thrombelastography parameter Maximum Rate of Thrombus Generation (MTG, in 100xmm per second) from thrombelastography in normal or haemophilia A-like whole blood (i.e. in the absence or presence of a neutralising anti-Factor VIII polyclonal antibody, 0.1 mg/mL) in the presence of Activated Protein C (5 nM) and various concentrations of antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL). Last column indicates the number of independent experiments (n). Thrombelastography was initiated using 40,000- fold diluted Innovin. ND indicates not determined. Table 13B. TMG in normal or haemophilia A-like whole blood with ACP (5 nM) and various concentrations of antibody or antibody fragment (0 nM to 400 nM)
  • Tables 14A and 14B below set forth the thrombelastography parameters R (clot time) and Maximum Rate of Thrombin Generation (MTG), respectively, determined in normal and haemophilia A-like human blood (i.e. in the absence or presence of a neutralising anti- Factor VIII polyclonal antibody, 0.1 mg/mL) in the presence of Thrombo-modulin (TM, 5 nM) and increasing concentrations of 0233-0000-01 10 and 0233-0000-0150 (0 nM to 400 nM).
  • TM Thrombo-modulin
  • 0233-0000-01 10 and 0233-0000-0150 stimulated thrombelastography by dose-dependently reducing the clotting time in the presence of TM.
  • both antibodies also increased the MTG in the presence of TM.
  • Table 14A shows thrombelastography parameter clot time (R, in seconds) from thrombelastography in normal or haemophilia A-like whole blood in the presence of
  • Thrombo-modulin (5 nM) and various concentrations of antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL) is set forth in table 16.
  • Last column indicates the number of independent
  • Table 14B shows hrombelastography parameter Maximum Rate of Thrombus Generation (MTG, in 100xmm per second) from thrombelastography in normal or haemophilia A-like whole blood in the presence of Thrombo-modulin (5 nM) and various concentrations of antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL). Last column indicates the number of independent experiments (n). Thrombelastography was initiated using 40,000- fold diluted Innovin. ND indicates not determined. Table 14B. MTG in normal or haemophilia A-like whole blood with Thrombo-modulin (5 nM) and various concentrations of antibody or antibody fragment (0 nM to 400 nM)
  • Table 15A and 15B show the thrombelastography parameters R (clot time) and Maximum Rate of Thrombin Generation (MTG), respectively, determined in normal and haemophilia A-like human blood (i.e. in the absence or presence of a neutralising anti-Factor VIII polyclonal antibody, 0.1 mg/mL) in the presence of activated protein C (APC, 5 nM) and increasing concentrations of recombinant activated Factor VII (rFVIIa), 0233-0000-01 10 or 0233-0000-0150 (0 nM to 400 nM).
  • R clot time
  • MMG Maximum Rate of Thrombin Generation
  • Table 15A shows thrombelastography parameter clot time (R, in seconds) from thrombelastography in normal or haemophilia A-like whole blood in the absence and presence of Activated Protein C (APC, 5 nM) and various concentrations of recombinant activated Factor VII (rFVIIa) or antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL).
  • Last column indicates the number of independent experiments (n). Thrombelastography was initiated using kaolin. ND indicates not determined.
  • Table 15B shows thrombelastography Maximum Rate of Thrombus Generation (MTG, in 100xmm per second) from thrombelastography in normal or haemophilia A-like whole blood in the absence and presence of Activated Protein C (APC, 5 nM) and various concentrations of recombinant activated Factor VII (rFVIIa) or antibody or antibody fragment (0 nM to 400 nM) with or without a neutralising polyclonal anti-Factor VIII antibody (a-FVIII, 0.1 mg/mL).
  • MTG Maximum Rate of Thrombus Generation
  • Thrombelastography was initiated using kaolin. ND indicates not determined.
  • Table 15B MTG in normal or haemophilia A-like whole blood with or without 5 nM APC and various concentrations of rFVIIa or antibody or antibody fragment (0 nM to 400 nM)
  • Table 16 shows thrombelastography parameters clot time (R, in seconds) and MTG from thrombelastography in normal blood and blood made haemophilia A-like by incubation with a neutralising polyclonal anti-FVIII antibody (0.1 mg/mL). Various concentrations of antibody or antibody fragment (0 nM to 1000 nM) was tested in the absence of APC.
  • Thrombelastography was initiated with 40000-fold diluted Innovin®. All compounds dose-dependently shortened the clotting time and increased the MTG under haemophilia A-like conditions in human whole blood.
  • Table 17 shows relative thrombelastography parameters clot time , R (in seconds) and MTG from thrombelastography in normal blood and blood made haemophilia A-like by incubation with a neutralising polyclonal anti-FVI 11 antibody (0.1 mg/mL). Various concentrations of antibody or antibody fragment (0 nM to 1000 nM) was tested in the absence of APC.
  • Thrombelastography was initiated with 40000-fold diluted Innovin®.
  • the measured clotting time was expressed relative to a signal window ranging from clot time determined under normal conditions (0%) to clot time determined under haemophilia A-like conditions (100%) in the same donors in the absence of drug (Buffer).
  • MTG was expressed relative to a signal window ranging from MTG determined under under haemophilia A-like conditions (0%) to MTG determined under normal conditions (100%). All compounds dose-dependently shortened the relative clotting time under haemophilia A-like conditions in human whole blood. Compounds 0233-0000- 0150, 0233-0000-0270 and 0233-0000-0271 dose-dependently stimulated the relative MTG.
  • Thrombelastography was initiated with 40000-fold diluted Innovin®. All compounds dose-dependently shortened the clotting time and increased the MTG under haemophilia A-like conditions in human whole blood in the presence of APC (5 nM).
  • Table 19 shows relative thrombelastography parameters clot time, R (in seconds) and MTG from thrombelastography in normal blood and blood made haemophilia A-like by incubation with a neutralising polyclonal anti-FVIII antibody (0.1 mg/mL).
  • Example 5 Enhancement of thrombin generation by anti-FVa antibodies in platelet rich plasma under haemophilia A like conditions in the presence of thrombomodulin
  • Anti-FVa antibodies were tested for their capacity to reverse the inhibitory effect of thrombomodulin in a thrombin generation assay including platelets.
  • Generated thrombin was measured using a fluorogenic method from Thrombinoscope®.
  • human platelet rich plasma (PRP) was obtained by centrifugation of citrated human whole blood at 220g for 20 min. The upper phase containing platelets was collected and the remaining sample was centrifuged at 2500g for 10 min to obtain platelet poor plasma (PPP) used to adjust the platelet concentration to a final concentration of 150000 plts/ ⁇ .
  • PRP human platelet rich plasma
  • PPP platelet poor plasma
  • the PRP was made haemophilic by 30 min incubation with a sheep anti-human FVIII polyclonal antibody (0.1 mg/ml) (HTI #Z0429).
  • the haemophilic-like PRP were let to incubate with anti-FVa antibodies together with thrombomodulin (25 nM) (HTI#4202) and Innovin (1 pM) (Siemens #B4212-50) for 30 min (RT) before the reaction was started by addition of a combination of protease activated recertor-1 (PAR-1 ) activation peptide (30 ⁇ ) (SFLLRN; Bachem #H- 2936) and the GPVI activating Convulxin (100 ng/ml) (Pentapharm #1 19-02) together with the FluCa reagent to the samples in 96-well Nunc Microwell round bottom well plates (Nunc #268152).
  • the fluorescent signal from the substrate was detected in a ThermoFisher Fluoroskan plate
  • thrombomodulin TM
  • thrombomodulin TM
  • Table 20 The results showed a dose dependent reversal of the thrombomodulin (TM) inhibiting effect on thrombin generation by antibody 0233-0000-01 10, as shown in table 20.
  • Thrombin generation was measured under induced haemophilia A conditions (sheep anti- human FVIII polyclonal antibodies) with a fixed platelet concentration of 150000 plts/ ⁇ and with added thrombomodulin (25 nM) and innovin (1 pM).
  • the reaction was started with platelet activation through the addition of PAR-1 activating peptide SFLLRN (30 ⁇ ) and the GPIV agonist Convulxin (100 ng/ml). Peak thrombin generation (nM) is shown as mean values. NA indicates not applicable.
  • Example 6 Evaluating prior-art anti-FVa monoclonal antibodies in human haemophilic plasma by thrombin generation assay
  • HA human haemophilia A
  • Fact VIII deficient plasma (Georg King Medical, #0800) stored at -80C was thawed in water at 37C for 5 min, and then stored at room temperature until use.
  • 18 ⁇ _ plasma was added to a 384-well microtiter plate (Perkin Elmer), and then 2 ⁇ _ antibody solution (in 20 nM Tris, pH 7.4) was added, and the antigen binding was allowed to proceed for 20 min.
  • the thrombogram was calculated as the first derivative of the integral fluorescence curve, and the ETP and peak thrombin parameters were calculated from the thrombogram, and used in the evaluation of thrombin generation.
  • Table 22 lists the results of all the antibodies tested, along with the background (APC only) and the 0233-0000-01 10 antibody disclosed in this invention. The result value is the peak height relative to the 0233-0000-01 10 antibody.
  • GMA-044 GMA Antibodies GMA-044 0,0
  • Biacore T200 Surface plasmon resonance
  • the antibodies that were found not to compete with 0233-0000-01 10 for binding to FVa was further subdivided by direct coating of the antibodies on a CM5 chip and measuring binding of FVa alone or incubated with the respective antibodies. Based on these results the antibodies were divided into additional four bins.
  • This example identifies the interaction surface on human plasma FVa when binding the antibodies 0233-0000-0005, 0233-0000-0006, 0233-0000-01 10, 0233-0000-0128, 0233- 0000-0271 , 0233-0000-0453 (Fab of 0233-0000-0418), and 0233-0000-0302, respectively.
  • Residues in the sequence for FVa light chain (SEQ ID NO 38) used in this example are numbered as the first amino acid residue being no. 1546. The numbering can be converted to the sequence numbering for SEQ ID NO 38 by subtracting 1545 from the residue number used in this example.
  • the residue S1546 used in this example corresponds to residue S1 in SEQ ID NO 38.
  • Hydrogen exchange mass spectrometry exploits that the change in mass when hydrogen is exchanged with deuterium can be followed over time using mass spectrometry.
  • HX-MS Hydrogen exchange mass spectrometry
  • an antibody binds to FVa
  • amino acid residues located in the binding interface are protected from hydrogen exchange with the solvent.
  • Pepsin digestion allows identification of peptides that are part of the epitope that show slower rates of hydrogen exchange when the antibody is bound.
  • the borders of an epitope can be further defined by using information from overlapping peptides. If a peptide that is not protected from hydrogen- deuterium exchange is partially overlapping with a protected peptide, the common sequence between the two peptides will not be part of the epitope.
  • 0000-0005, 0233-0000-0006, 0233-0000-01 10, 0233-0000-0128, 0233-0000-0271 , 0233- 0000-0453, or 0233-0000-0302 were diluted 25-fold in 95% deuterated imidazole buffer (20 mM imidazole, 150 mM sodium chloride, 10 mM calcium chloride, pH 7.4). Non-deuterated controls were prepared by diluting into protiated imidazole buffer. The hydrogen exchange experiments were performed on a nanoAcquity UPLC system with HDX technology (Waters Corporation, Milford, MA, USA) which includes the HD-x PAL auto sampler (LEAP
  • UPLC ultra-high performance liquid chromatography
  • a volume containing 100 pmol of FVa with or without 120 pmol of one of the antibodies or antibody fragments was diluted into deuterated imidazole buffer. At the time intervals from 15 seconds to 4 hours 50 ⁇ of the sample was quenched in 50 ⁇ 1 .35 mM Tris (2-carboxyethyl) phosphine adjusted to pH 2.7 and held at 3°C.
  • the peptides were separated on a Waters UPLC BEH C18 1.7 ⁇ (1 .0 mm x 100 mm) column using a 9 min 10 - 40 % acetonitrile gradient containing 0.1 % formic acid at a 40 ⁇ /min flow-rate.
  • the mass spectrometer was run in positive ion mode with ion mobility separation enabled.
  • the instrument parameters used were 3.2 kV capillary, 30 V sample cone, and 4 V extraction cone offsets, 850 ml/min flow of desolvation gas and 25 ml/min cone gas flow.
  • the source block was heated to 120°C and the desolvation gas to 350°C.
  • Lock-mass correction data using acquired using the 1 + ion of Leucine-enkephalin (m/z 556.2771 ) as reference compound and applied during data analysis.
  • the ions were separated by ion mobility in 3 mbar pressure of nitrogen maintained by a 90 ml/min gas flow.
  • the trap and transfer regions were flushed with a 2 ml/min gas flow of argon gas to trap the ions before and after ion mobility separation and for collision induced dissociation in MSe-type experiments.
  • the MSE-data was analysed using Waters ProteinLynx Global Server 2.5 and peptides of FVa were identified that covered 82 % (0233-0000-0005 and 0233-0000-0006, see table 25), 87 % (0233-0000-01 10, 0233-0000-0128, and 0233-0000-0302, see table 26), and 83 % (0233-0000-0271 or 0233-0000-0453, see table 27) of the protein sequence, respectively.
  • the HX-MS data files were analysed using Waters DynamX 2.0 that automatically applies lock-mass correction and determines the degree of deuterium incorporation in each peptide. In addition, all data was manually inspected to ensure correct peak assignment and calculation of deuterium incorporation.
  • Table 25 Epitope mapping of mAbs 0233-0000-0005 and 0233-0000-0006 on human plasma FVa

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Abstract

La présente invention concerne des anticorps monoclonaux ou des fragments de ceux-ci, qui se lient au Facteur V activé (FVa), qu présentent un effet pro-coagulant, ainsi que leurs utilisations thérapeutiques.
PCT/EP2015/065848 2014-07-11 2015-07-10 Anticorps dirigés contre le facteur v activé WO2016005564A2 (fr)

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WO2019071116A1 (fr) * 2017-10-05 2019-04-11 Epivax, Inc. Épitopes de lymphocytes t régulateurs

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DK0807691T3 (da) * 1994-02-14 2002-02-04 Univ Leiden Fremgangsmåde til screening for tilstedeværelse af en genetisk defekt forbundet med trombose og/eller lav antikoagulant reaktion på aktiveret protein C
US20060014934A1 (en) * 2004-05-18 2006-01-19 Everse Stephen J Crystal structure of factor Vai and method for identifying blood factor Va modulators
US8236764B2 (en) * 2006-02-23 2012-08-07 The Children's Hospital Of Philadelphia Methods for treating a hemostasis related disorder using activated forms of Factor V
EP2091564A4 (fr) * 2006-10-16 2010-09-01 Novelmed Therapeutics Inc Procede d'inhibition de la coagulation avec des anticorps anti-facteur va humains et utilisation de ceux-ci

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019071116A1 (fr) * 2017-10-05 2019-04-11 Epivax, Inc. Épitopes de lymphocytes t régulateurs
EP3691673A4 (fr) * 2017-10-05 2021-06-09 Epivax, Inc. Épitopes de lymphocytes t régulateurs
US11441122B2 (en) 2017-10-05 2022-09-13 Epivax Inc. Regulatory T cell epitopes
US12275956B2 (en) 2017-10-05 2025-04-15 Epivax Inc. Regulatory T cell epitopes

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