WO2008059009A2 - Factor v mutants for hemostasis in hemophilia - Google Patents

Abstract

The invention provides a method for preventing or treating bleeding in joints, muscle and/or soft tissues in a hemophilia patient, comprising administering to the patient an effective hemostatic amount of APC-resistant Factor V.

Description

TITLE FACTOR V MUTANTS FOR HEMOST ASIS IN HEMOPHILIA

[0001] The invention relates to the field of pharmaceutical products, in particular blood clotting factors and use thereof for hemostasis.

BACKGROUND OF THE INVENTION

[0002] Blood coagulation is a highly regulated process required to prevent blood loss in response to vascular injury. It should be triggered immediately upon injury and switched off as soon as the vasculature is intact. When this balance between activation (coagulation) and inactivation (anti-coagulation) is disturbed, a bleeding disorder or thrombotic disease may ensue. A typical example of a bleeding disorder is hemophilia.

[0003] The coagulation response leads to the conversion of soluble fibrinogen into insoluble fibrin and is mediated by the clotting factor proteins. These circulate in plasma as inactive cofactors and pro-enzymes. Inactive cofactors and pro-enzymes are sequentially activated in a cascade-like fashion. At each step, the newly formed protease complex catalyzes the activation of the next factor. Activation is initiated upon contact of blood with the membrane protein tissue factor (TF). TF is normally not expressed by endothelial cells but is abundantly present on sub-endothelial cells such as smooth muscle cells. As a result, disruption of the endothelial cell layer leading to vascular injury triggers blood coagulation by exposure of constitutively expressed TF.

[0004] A simplified view of the coagulation system is shown in Fig. 12. Activation of the coagulation system is initiated by the formation of the TF-FVIIa complex and propagated by the action of the FVIIIa-FIXa complex. TF-FVIIa complex activates FX as well as FIX to generate FXa and FIXa, respectively. The FVIIIa-FIXa complex, similarly as the TF-FVIIa, also activates FX. Thus by activating FIX the action of TF-FVIIa on FX is amplified (Fig. 12). Under physiological conditions most hemostatic responses need this FIX- and FVIII-dependent amplification to ensure sufficient activation of FX (and hence thrombin generation). Lack of this amplification loop manifests itself in the bleeding disorders hemophilia A (FVIII deficiency) or B (FIX deficiency). [0005] FV plays a central role in the coagulation cascade. Upon activation, FVa acts as a cofactor for FXa, and increases the rate of FXa-induced thrombin generation by 300,000 times compared to FXa alone (Mann and Kalafatis 2003). Factor V (FV) in its activated form thus has a critical procoagulant function.

[0006] An anti-coagulant system regulates the pro-coagulant functions of the clotting cascade. This anti-coagulant system involves activated protein C (APC), which inactivates FVIII and FV. A simplified representation of the APC system is shown in Fig. 13. Activation of protein C is mediated by thrombin when it becomes bound to thrombomodulin (TM) (Fig. 13) (Esmon, 1989). Upon binding to TM the substrate specificity of thrombin changes from fibrinogen into protein C. Activation of protein C by TM-bound thrombin is enhanced by the endothelial protein C receptor (EPCR). The activity of APC to cleave and inactivate activated FVIII and FV is further increased by its cofactor, protein S. Overall therefore, APC constitutes a major anticoagulant protein with a significant impact on regulating the clotting system. FV also has anticoagulant effects since it can act as a cofactor for APC to assist in inactivating FVIIIa (Thorelli et al, 1999; for review see Mann et al, 2003).

[0007] APC-resistant FV mutants have been described including FV-Leiden (Arg506Gln), FV-Cambridge (Arg306Thr) and FV- Hong Kong (Arg306Gly) (Bertina et al, 1994, Svensson et al 1994, Williamson et al, 1998 and Chan, 1998). These mutants are inactivated more slowly by APC and hence prolong the activity of factor Xa. Thus, via their effect on FXa these FV mutants enhance thrombin formation. Indeed, these FV mutants are all to a variable degree associated with thrombosis. It has additionally been found that hemophilia patients carrying the FV-Leiden mutation demonstrate a milder form of hemophilia with less severe bleeding (van Dijk et al 2004), suggesting that APC- resistant FV can mitigate the clinical symptoms of severe hemophilia. APC-resistant FV cannot act as a cofactor for APC and thus lacks the anti-coagulant effect of its wild type counterpart.

[0008] Hemophilia is typically managed by replacement therapy, which is based on the complementation of the patient's defective coagulation system with the deficient coagulation factor. Thus, hemophilia A and B patients are infused with Factor VIII and Factor IX concentrates to treat or to prevent bleeding episodes. Usually bleeding episodes in hemophilia patients resolve upon one or two infusions of Factor VIII at concentrations of 25-50 U per kg body weight, although notably a fraction of the patients (7-29%) poorly respond to this therapy (see e.g. Ananyeva et al, 2004).

[0009] Hemophilia patients treated with preparations of the deficient factor are at risk to develop neutralizing antibodies (inhibitors) to the missing factor. This occurs in 15-30% of severe hemophilia A patients and 1-3% of hemophilia B patients. Particularly patients with severe gene defects that lead to FVIII plasma levels <1% of normal, such as gene deletion or inversion, nonsense and frameshift mutations, may develop inhibitors. When circulating levels of inhibitors are high, patients may require very high and expensive amounts of the deficient factor in order to overcome the neutralizing effects of the inhibitors. As an alternative, FVIII or FIX bypass therapy has been developed. The principle underlying these treatments is that the defect in the FVIII/FIX complex is bypassed by the use of activated forms of FVII, IX and X. Examples of such therapies are activated prothrombin-complex concentrates and recombinant FVIIa. FVIIa stops bleeding in 70-75% of the patients with inhibitors. Recombinant FVIIa is expensive and not applicable as prophylactic therapy due to its short half-life of 2-3 hours.

[0010] There is a need for alternative and possibly improved treatment and/or prophylaxis of hemophilia. Recently, APC-resistant recombinant FV has been suggested as a therapeutic option to increase thrombin generation in hemophilia patients (EP 0756638 Bl; Van 't Veer et al, 1997; Bos et al, 2005).

[0011] Experimental data also confirm that APC-resistant FV added to FVIII- or FIX-deficient plasma can increase thrombin generation. In EP 0756638 Bl, Mertens and Voorberg have shown in example 5 that APC-resistant FV added to a plasma from a hemophilia A patient results in an increased thrombin generation. This effect was surprisingly not observed with wild-type FV, which suggests that APC had been present in their assay, despite the fact that the APC system was not triggered through the addition of thrombomodulin, nor by addition of APC.

[0012] In later studies, Mertens and coworkers (Bos et al., 2005) reported an important finding, which could explain why APC-resistant FV can increase thrombin generation in a FVIII- or FIX-deficient plasma. In fact, Bos et al. (2005) have discovered that the impaired thrombin generation in hemophilia A and B not only is the result of a lack of FVIII and FIX, but also is due to the inactivation of FV by APC. In other words, hemophilic patients display a dual deficiency, i.e., a FVIII or FIX deficiency and an acquired, APC-induced FV deficiency. In addition to what Voorberg and Mertens have shown in EP 0756638 Bl, Bos et al. (2005) have demonstrated that APC-resistant FV, in contrast to wild-type FV, could support a sustained thrombin generation in human plasma immune -depleted of both FV and FIX under conditions in which the APC system is triggered with thrombomodulin. Based on these data, Bos et al. (2005) discussed the potential therapeutic use of APC-resistant FV to correct the APC-induced FV deficiency in hemophilia patients.

[0013] Although these data have shown that APC-resistant FV can increase thrombin generation in hemophilic plasma, the data do not at all predict whether APC- resistant FV is sufficient to replace FVIII or FIX and whether it can convert a hemophilic phenotype into a less severe or normal phenotype. Thus, these earlier studies (EP 0 756 638, Bos et al., 2005) have not addressed the question whether APC-resistant FV can drive thrombin generation in plasma of a hemophilia patient beyond the level that is needed to correct the APC-induced FV deficiency and, hence, whether it can replace FVIII or FIX as well. Moreover, neither the data of Bos et al. (2005) nor those of EP 0 756 638 Bl provide a clue on the relationship between the potency of APC-resistant FV to increase thrombin generation in hemophilia patient plasma and the concentration of APC.

[0014] Taken together, three critical questions remained unanswered in the above-mentioned studies, i.e., (1) can APC-resistant FV restore the defect in thrombin generation caused by a FVIII or FIX deficiency, (2) how much APC-resistant FV is needed to by-pass a FVIII or FIX deficiency in order to prevent hemophilic patients from spontaneous bleedings, and (3) how is the potency of APC-resistant FV influenced by the APC-system?

BRIEF SUMMARY OF THE INVENTION

[0015] The present invention discloses for the first time that APC-resistant Factor V can replace Factor VIII and restore clotting in plasma that is deficient in Factor VIII, such as plasma from hemophilia A patients. Wild-type FV cannot bypass the requirement for FVIII in such plasma. It is shown that APC-resistant Factor V can restore clotting (measured by thrombin/fibrin formation) in FVIII-deficient plasma in the absence of added activated protein C (APC) or thrombomodulin. Importantly, it is demonstrated that the potency of APC-resistant FV to bypass FVIII requirement is strongly increased in the presence of elevated APC concentrations, and surprisingly that the potency per Unit of APC-resistant FV can be higher than that of FVIII in the presence of APC. These surprising findings imply that APC-resistant FV can be used to treat and/or prevent bleeding in hemophilia patients, and is especially suited for treatment or prevention of bleedings under conditions where high APC concentrations are present, such as bleedings in the joints, muscle and soft tissue (bleedings in microcapillaries).

[0016] The invention provides a method for preventing spontaneous bleeding in a hemophilia patient, comprising administering to the patient APC-resistant Factor V. The invention provides a method for preventing bleeding in the microcapillaries in a hemophilia patient, comprising administering to the patient APC-resistant Factor V. The invention provides a method for preventing bleeding in a hemophilia patient at sites with an increased concentration of activated protein C (as compared to the (averaged) concentration thereof in the total blood plasma of the patient), comprising administering to the patient APC-resistant Factor V. The invention provides a method for preventing bleeding in joints, muscle and/or soft tissues in a hemophilia patient, comprising administering to the patient APC-resistant Factor V. In certain embodiments, the hemophilia patient is deficient in Factor VIII activity and/or the patient's Factor VIII activity is inhibited. In other embodiments, the hemophilia patient is deficient in Factor IX activity and/or the patient's Factor DC activity is inhibited. The invention also provides a method for reducing or preventing the possibility of generating inhibitors to Factor VIII in a hemophilia A patient, comprising administering APC-resistant Factor V to the patient. The invention further provides a method for reducing or preventing the possibility of generating inhibitors to Factor IX in a hemophilia B patient, comprising administering APC-resistant Factor V to the patient.

[0017] According to the aspects of the invention described above, a hemostatic amount of APC-resistant Factor V is administered to said patient. Said amount preferably is an effective hemostatic amount. The invention thus also provides a method for treatment or prophylaxis of a patient having a clotting factor deficiency or inhibitor, comprising administering to the patient an effective hemostatic amount of APC-resistant Factor V.

[0018] In certain embodiments, APC-resistant Factor V is administered to obtain a plasma concentration thereof of about between 0.01 and 5, preferably of about between 0.05 and 2, more preferably of about between 0.1 and 1.0 Units/ml.

[0019] In certain embodiments, the APC-resistant Factor V has a mutation of Arg306, Arg506 or both Arg 306 and Arg506 as compared to the wild type Factor V sequence (SEQ. ID. NO. 1). In one embodiment, the APC-resistant Factor V has a mutation of both Arg306 and Arg506 as compared to the wild type Factor V sequence (SEQ. ID. NO. 1).

[0020] In certain embodiments, the APC-resistant Factor V is free from other clotting factors.

[0021] In certain embodiments, said patient is a hemophilia A patient that has not been diagnosed with inhibitors against Factor VIII. In other embodiments, said patient is a hemophilia B patient not diagnosed with inhibitors against Factor IX. In further embodiments, said patient is a hemophilia A patient that has been diagnosed with inhibitors against FVIII. In other embodiments, said patient is a hemophilia B patient not diagnosed with inhibitors against Factor IX. In further embodiments, said patient is a hemophilia B patient that has been diagnosed with inhibitors against FIX.

[0022] In one aspect, the invention provides a method for treating a patient having a clotting factor deficiency or inhibitor, comprising administering to the patient APC-resistant Factor V to to obtain a plasma concentration thereof of between 0.01 and 5, preferably between 0.05 and 2, more preferably between 0.1 and 1 Units/ml. In certain embodiments, said patient is a hemophilia A patient.

[0023] In one aspect, the invention provides in a method for treating a patient having a Factor VIII deficiency wherein an effective hemostatic amount of Factor VIII is administered to the patient, the improvement comprising administering an effective hemostatic amount of APC-resistant Factor V to the patient. In certain embodiments, Factor VIII is not administered or administered at a level sufficiently low to not provoke an immune response in the patient to F VIII. It is also an aspect of the invention to provide the use of APC-resistant Factor V for the manufacture of a medicament for preventing spontaneous bleeding, and/or bleeding in the microcapillaries, and/or at sites with an increased concentration of activated protein C, and/or in joints, muscle and/or soft tissues, in a hemophilia patient. The treatment comprises administering a hemostatic amount of APC-resistant Factor V to said patient, all as described in the embodiments relating to treatment provided supra.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0024] FIG. 1. Scheme of assay for Chromogenic activity of Factor V preparations.

[0025] FIG. 2. Scheme of assay for clot activity of Factor V preparations, using a Prothrombin Time (PT) assay in Factor V-deficient human plasma.

[0026] FIG. 3. Scheme of assay for APC-resistance of Factor V preparations, using an APTT assay in Factor V-deficient human plasma with and without Activated Protein C (APC).

[0027] FIG. 4. Outline of Fibrin Generation Time (FGT) assay used to determine the ability of APC-resistant Factor V preparations to restore clotting in FVIII-deficient human plasma.The end point of the assay is Fibrin formation, which is recorded over time by measurements at an optical density of 405nm. The FGT (Ti/2inax) is then calculated. The assay is designed to be critically dependent on FVIII through (A) the addition low TF concentrations and (B) the addition of APC. Using this assay, the effect of addition of APC-resistant FV can be tested and compared to addition of FVIII.

[0028] FIG. 5. Influence of Factor VIII on fibrin generation time at low TF concentration. Arrow 1 shows the curve of Fibrin generation measured in FVIII-depleted plasma. Arrow 2 indicates the curve of fibrin generation measured in FVIII-depleted plasma spiked with 10% (0.1 U/ml) FVIII. In further experiments shown below, preparations of FV-L/C were tested in a similar assay to evaluate whether they could shorten (restore) the clotting times and thus bypass or replace FVIII.

[0029] FIG. 6. Titration of FVIII in FVIII-immune depleted plasma, at a TF dilution of 1 :40,000. [0030] FIG. 7. FV-L/C restores clotting in FVIII-depleted plasma in the absence of added APC. Conditions: TF dilution 1:40,000, no APC added.

[0031] FIG. 8. FV-L/C potency increased in the presence of APC. Conditions: A: TF dilution 1 :40,000, 30 nM APC. B: TF dilution: 1 :40,000, 60 nM APC.

[0032] FIG. 9. FV-L/C potency in the presence of APC is maintained at higher TF concentrations. Conditions: TF dilution: 1 :20,000, 30 nM APC.

[0033] FIG. 10. FV-L/C restores clotting in hemophilic plasma (of hemophilia A patients). Six different hemophilic plasmas were tested (A-F), in A-C rFV-L/C was used, in D-F rFV-L/C-ST. Conditions: TF dilution 1:40,000, 30 nM APC.

[0034] FIG. 11. Potency of FV-L/C to generate thrombin in hemophilic plasma is confirmed using a different assay. Thrombin generation is triggered by the addition of TF in the presence of (A) 16 nm Thrombomodulin and (B) 8 nM APC. Thrombin generation was recorded using a Thrombogram (Thrombinoscope) according to manufacturers instructions.

[0035] FIG. 12. Simplified scheme of coagulation. Upon endothelial injury tissue factor (TF) becomes exposed to blood. FVII interacts with TF and becomes activated to activate in its turn FX. FXa in presence of its cofactor FVa converts prothrombin into thrombin, which in its turn generates fibrin. The system is amplified by 2 loops, one involving FVIII and FIX, and the other FXI. Activated protein C (APC) acts as an anticoagulant by inactivating FVa and FVIIIa.

[0036] FIG. 13. Schematic representation of protein C activation. Thrombin (tr) generated during coagulation binds to the endothelial membrane protein thrombomodulin (TM) and obtains a different substrate specificity. TM-bound thrombin activates protein C to generate APC, which process is accelerated in presence of endothial protein C receptor. APC inactivates the cofactors FVa and FVIIIa.

[0037] Fig. 14. FV-L/C restores clotting in FIX-depleted (A) and FXI-depleted (B) human plasma in the absence of added APC. Conditions: TF dilution 1 :40,000, no APC added.

[0038] Fig 15. Potency of FV-L/C in FIX-depleted (A) and FXI-depleted (B) human plasma is increased compared to rFIX and pFXI in the presence of APC. Conditions: TF dilution 1 :40,000, 30nm APC. [0039] Fig. 16. The potency of rFV-L/C and rFVIII to reduce clotting times in FVIII-immune depleted human plasma are increased when added in combination. Conditions: TF dilution 1 :40,000, no APC added.

[0040] Fig. 17. rFV-L/C converts a sub-effective dose of rFVIII to an effective treatment that completely stops bleeding in a tail clip bleeding model in FVIII knockout mice.

[0041] Fig. 18. rFV-L/C-ST restores clotting in plasmas from 3 different hemophilia B patients.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Hemophilia is an X-linked congenital bleeding disorder generally resulting from a deficiency of coagulation FVIII (hemophilia A) or FIX (hemophilia B). The frequency of the disease is about one in 10,000 births. Worldwide approximately 400,000 males are affected. The prevalence of hemophilia A is about 5-6 times that of hemophilia B. Persons with hemophilia often have a history of easy bruising in early childhood, spontaneous bleeding (particularly into the joints and muscles/soft tissue), and excessive bleeding following trauma or surgery. Appropriate clotting tests will confirm the diagnosis and reveal whether a patient suffers from hemophilia A or B.

[0043] The severity of bleeding manifestations in hemophilia is generally correlated with the clotting factor level as shown in Table 1.

Table 1: Relation between severity of bleeding tendency and plasma level of deficient clotting factor.

*1 International Unit is equal to the amount of clotting factor in 1 ml of normal pooled human plasma.

[0045] The practice of primary prophylaxis to prevent debilitating repeated hemarthroses, as these may result in hemophilic arthropathy, is based on the notion that hemophilia patients with clotting factor level > 1% seldom have spontaneous bleeding (Table 1) and have much better preservation of joint function. Hence, prophylactic replacement of clotting factor aims at keeping circulating levels of the deficient factor above 1% at all times. Primary treatment of bleeding episodes is the administration of FVIII (for hemophilia A) or FIX (for hemophilia B), either plasma derived or recombinant.

[0046] Hemostasis refers to the processes, such as coagulation activation, involved in stopping bleeding. Accordingly, a "hemostatic amount" as used herein is thus defined as an amount (of a clotting factor, e.g. APC-resistant FV) sufficient to restore thrombin generation or fibrin formation up to levels sufficient to support coagulation necessary for stopping or preventing bleeding. This can for instance normally be reached by adding the amount of the lacking clotting factor (e.g. FVIII in hemophilia A plasma) which would normally prevent and/or stop bleeding (e.g. by addition of the deficient clotting factor to the deficient plasma to reach a concentration of about between 1 and 100%, see Table 1)

[0047] The present invention discloses that hemostatic amounts can be reached with APC-resistant Factor V in hemophilic plasma as well, e.g. by addition of this molecule to reach concentrations of between about 0.01 and 5 U/ml plasma. In certain embodiments, said hemostatic amounts in hemophilic plasma are obtained by concentrations of about between 0.05 and 2 U/ml plasma, and in certain embodiments of about between 0.1 and 2 Units/ml plasma, e.g. at about between 0.2 and 1 Units/ml plasma.

[0048] One unit of a blood clotting factor in general is defined as the amount that is present in 1 ml pooled normal human plasma. One unit of Factor V activity or antigen corresponds to the amount of Factor V in 1 ml of normal plasma, which is about 5-10 μg/ml. For APC-resistant Factor V, one unit is thus defined as having the same amount of Factor V antigen as present in pooled human plasma. Accordingly, 1 U of FV-L/C corresponds to about 5-10 μg/ml.

[0049] The present invention surprisingly discloses that the potency of FV-L/C is comparable to that of FVIII to restore thrombin and fibrin generation in hemophiliacs, in particular under conditions with high APC levels. The required level of APC-resistant FV as disclosed in the present application (e.g. 0.01-5 U/ml plasma) can be obtained by administration of APC-resistant FV at a frequency and dosage (per kg of body weight) that will be dependent on pharmacokinetics, in vivo recovery and potency of the APC- resistant FV preparation, as is well known and can be routinely determined by the skilled person. It is therefore within the skill of the artisan to determine the dose and frequency to obtain the desired levels of APC-resistant Factor V in the plasma of the subject. The plasma volume is typically about 50 ml per kg body weight. Thus the dose and frequency can be varied by the clinician to arrive at the optimum therapy. According to the present invention, generally a dose of about from 0.01 to 5, preferably about from 0.05 to 2, more preferably about between 0.1 and 1 Units/ml plasma is preferred. The frequency of dosing will ordinarily be every 1 to 7 days for prophylaxis.

[0050] One embodiment of therapeutic treatment is the administration of a therapeutically effective dose of an aqueous composition to obtain a plasma concentration of about from 0.01 to 5 units of APC-resistant FV/ml to a hemophilia patient exhibiting or not exhibiting a clotting factor inhibitor.

[0051] The APC-resistant Factor V can be used according to the invention for prophylaxis, meaning that it is used for prevention of bleeding, i.e. at times when no bleeding occurs. It can also be used for treatment of bleeding, i.e. at times when bleeding already started, to stop the bleeding.

[0052] Moreover, the present invention surprisingly discloses that the potency of APC-resistant Factor V is actually significantly higher in a plasma that is deficient in Factor VIII than the potency of the Factor VIII protein itself upon addition to that plasma, under conditions where APC is added to the plasma. Elevated concentrations of APC occur for instance in capillary blood vessels (Esmon, 1989). Even under conditions where no APC is added to the plasma, APC-resistant Factor V potency is in a similar range of that of Factor VIII. [0053] Hemophilic patients may bleed spontaneously from many sites. Most frequent sites however are the joints (70%-80%) particularly knees and elbows, and muscle/soft tissue (10%-20%). Other major bleeds (5%-10%), and sometimes central nervous system bleeding (< 5%) also occur. Notably, spontaneous bleeding typically results from capillary damage.

[0054] Human plasma from normal persons contain low but measurable amounts of activated protein C (Gruber et al, 1992). The concentration of APC depends on the levels of Thrombomodulin (TM). TM is located on the endothelium. TM concentration in the microcirculation (capillaries) has been shown to be particularly high (100-500 nmol/L; Esmon, 1989). Therefore, most thrombin in the microvascular bed will be bound to TM and activate protein C. Hence, the concentration of APC is the highest in the microcirculation. This may have implications to hemophilia as it is the microcirculation which is a major site for spontaneous bleeding episodes in hemophilia patients. The present finding that the potency of APC-resistant FV is improved at elevated APC levels thus unexpectedly suggests that APC-resistant FV is particularly suitable to treat or prevent spontaneous bleeding in hemophilia, e.g. to treat or prevent bleedings in the joints, muscles or soft tissues. The better efficacy of APC-resistant FV to restore fibrin generation in FVIII-deficient plasma as compared to FVIII in presence of APC, reveals that APC-resistant FV may even be the preferred product for prophylaxis for spontaneous bleeding in (severe and moderate) hemophiliacs.

[0055] The APC-resistant Factor V can be used according to the invention for prophylaxis to prevent hemarthroses, for instance especially in joints, muscles and soft tissues. It thus can be used to prevent hemophilic arthropathy.

[0056] The Factor V (FV) molecule as present in blood of normal individuals is composed of three A domains, one B domain, and two C domains. This structure resembles that of factor VIII (Jenny et al, 1987). Upon synthesis in the liver, the FV molecule undergoes multiple posttranslational alterations, including sulfation, phosphorylation and glycosylation. FV in plasma is a single-chain protein with MW 33OkD. During activation by thrombin, FV undergoes several proteolytic cleavages, i.e. at Arg709, Argl018 and Argl545, and moreover, the large connecting B domain is released from the molecule. As a result its cofactor activity for FXa is enhanced by several orders of magnitude. The resulting FVa is composed of the noncovalently associated heavy (A1-A2) and light (A3-C1-C2) chains. By serving as an essential cofactor of FXa, FVa has a clear procoagulant effect. The activity of activated FV is tightly regulated by activated protein C (APC), which inactivates the molecule by cleavage at one or more of several residues to yield FVi (for review see Mann et al, 2003).

[0057] Human Factor V contains cleavage sites for activated protein C (APC), to be cleaved between Arg³⁰⁶-Asn³⁰⁷, Arg⁵⁰⁶-Gly⁵⁰⁷, Arg⁶⁷⁹-Lys⁶⁸⁰ and Arg¹⁷⁶⁵-Leu¹⁷⁶⁶ (EP 0756638). "APC- resistant Factor V" as used in the present invention is a Factor V (FV) molecule having a modification at or near a cleavage site for APC so as to reduce or abolish the activity of APC to cleave at the original cleavage site, i.e. to induce APC resistance. An APC-resistant Factor V molecule as used herein will result in a clotting time (e.g. in an APTT test) that is less than 150%, typically less than 120% (e.g. 90- 110%), of the clotting time in the absence of added APC, under conditions where APC is present at a concentration such that wt Factor V (from pooled human plasma) has a clotting time that is at least 150% (typically at least 180%, e.g. 200-300%) of the clotting time in the absence of added APC. Preferably the APC resistant FV is derived from the human FV sequence, but it could also be derived from FV from another species, e.g. monkey, bovine, porcine etc. In certain preferred embodiments the APC resistant FV is derived from human FV and has a modification at amino acid position Arg (one possible mutation is into Thr which yields a FV molecule referred to as 'Factor V- Cambridge' or 'FV-C), or Arg (one possible mutation is into GIn which yields a FV molecule referred to as 'Factor V-Leiden' or 'FV-L'), or Arg⁶⁷⁹, or both Arg³⁰⁶ and Arg (e.g. mutations of Arg306 into Thr and Arg506 into GIn yielding a molecule also referred to as 'Factor V-Leiden/Cambridge' or 'FV-L/C'), or other combinations thereof (all as compared to the mature sequence disclosed in Jenny et al, 1987, or to the amino acid sequence as present in Swissprot entry P 12259, which both represent wild type human Factor V sequences; SEQ. ID. NO. 1 in the present disclosure provides a wild- type mature human Factor V sequence). The modification in certain embodiments is an amino acid substitution. In certain embodimens, the Arginine residue that precedes the APC-cleavage site is changed into a GIn, He, Thr, GIy, or any other amino acid. In certain embodiments, Arg³⁰⁶ is replaced by Thr ('FV-R306T'). In other embodiments, Arg⁵⁰⁶ is replaced by GIn ('FV-R506Q'). In certain embodiments, Arg³⁰⁶ is replaced by Thr and Arg⁵⁰⁶ is replaced by GIn ('FV-R306T/R506Q'). It will be clear to the skilled person that further amino acid additions, deletions and/or substitutions could be present in the molecule without further affecting the biological activity of APC-resistant FV for the purpose of the present invention, e.g. the B-domain could be deleted (Pittman et al 1994), or allelic variants could be used, and it will be understood that such molecules are included within the definition of APC-resistant Factor V according to the present invention. Preparation of such variants can be done by routine molecular biology methods. It is preferred that APC-resistant FV is in non-activated form for use according to the present invention, but it could also be wholly or in part in its activated form (APC- resistant Factor Va) for use according to the present invention, and thus APC-resistant Factor Va is included within the scope of the term APC-resistant Factor V according to the present invention. Activation of FV during purification or storage in vitro may be prevented by the addition of thrombin inhibitors, and/or storage at pH lower than 7.4, etc. The APC-resistant FV molecules used herein will include variants, fragments, functional equivalents, derivatives, homo logs and fusions of the native APC-resistant FV molecule so long as the product retains the APC resistance and Factor V procoagulant property. Useful derivatives generally have substantial sequence similarity (at the amino acid level) in regions or domains of the APC resistant FV molecules as identified above (FV-R306T, FV-F506Q, FV-R306T/R506Q), e.g. are at least 50%, at least 60%, preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, still more preferably at least 95% identical in amino acid sequence with the APC-resistant Factor V molecules identified above.

[0058] Preparations containing APC-resistant FV may be obtained by purification from plasma of patients that have a mutation in the FV gene leading to APC-resistance (see e.g. EP0756638), e.g. having a FV-L or FV-C mutation. Preferably however, the APC-resistant FV molecules are produced through recombinant DNA technology involving expression of the molecules in cells, preferably eukaryotic cells, e.g. Chinese hamster ovary (CHO) cells, HEK293 cells, BHK cells, PER.C6 cells (as deposited at the ECACC under no. 96022940; for recombinant expression of proteins in PER.C6 cells see e.g. US patent 6,855,544), yeast, fungi, insect cells, and the like, or prokaryotic cells, or transgenic animals or plants. In certain embodiments, recombinant expression is achieved in PER.C6 cells that further over-express a sialyltransferase, e.g. human α-2,3- sialyltransferase (see e.g. WO 2006/070011). Methods for recombinant expression of desired proteins are known in the art, and recombinant production of APC-resistant FV has been described (e.g. EP 0756638 Bl; Egan et al, 1997; Bos et al, 2005). In general, the production of a recombinant protein, such as APC-resistant FV of the invention, in a host cell comprises the introduction of nucleic acid encoding the protein in expressible format into the host cell, culturing the cells under conditions conducive to expression of the nucleic acid and allowing expression of the said nucleic acid in said cells. Nucleic acid encoding a protein in expressible format may be in the form of an expression cassette, and usually requires sequences capable of bringing about expression of the nucleic acid, such as enhancer(s), promoter, polyadenylation signal, and the like. Several promoters can be used for expression of recombinant nucleic acid, and these may comprise viral, mammalian, synthetic promoters, and the like. In certain embodiments, a promoter driving the expression of the nucleic acid of interest is the CMV immediate early promoter, for instance comprising nt. -735 to +95 from the CMV immediate early gene enhancer/promoter. The nucleic acid of interest may be a genomic DNA, a cDNA, synthetic DNA, a combination of these, etc. Cell culture media are available from various vendors, and a suitable medium can be routinely chosen for a host cell to express the protein of interest, here APC-resistant Factor V. The suitable medium may or may not contain serum.

[0059] Harvesting and purification of the protein of interest can be done according to methods routinely available to the skilled person, e.g. employing chromatography such as affinity chromatography, ion-exchange chromatography, size- exclusion chromatography, and the like. Protocols for purification of APC-resistant Factor V from blood or plasma of patients with a FV-L mutation have been described (EP 0756638). Protocols for purification of APC-resistant Factor V from recombinant cell culture have also been described (e.g. Bos et al, 2005, who describe a procedure based on affinity chromatography with a monoclonal antibody) and are thus available for the skilled person. [0060] For administering to humans, the invention may employ pharmaceutical compositions comprising the APC-resistant Factor V and a pharmaceutically acceptable carrier or excipient. In the present context, the term "Pharmaceutically acceptable" means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the patients to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The APC-resistant FV of this invention preferably is formulated and administered as a sterile solution although it is within the scope of this invention to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g pH 5.0 to 7.5. The protein typically is in a solution having a suitable pharmaceutically acceptable buffer, and the solution of protein may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. For use in this invention APC-resistant FV may be formulated into an injectable preparation. Parenteral formulations are suitable for use in the invention, preferably for intravenous administration. These formulations contain therapeutically effective amounts of APC-resistant FV, are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.

[0061] APC-resistant FV may be administered by injection intravenously, or by other administration routes and/or sites, at a hemostatic amount, which thus is sufficient to correct a bleeding disorder, for example a FVIII or FIX deficiency.

[0062] The APC-resistant Factor V administered to the patients according to the invention may be free from other blood clotting factors. It is shown herein that APC- resistant FV can be administered to restore hemostasis in hemophilic plasmas, without addition of the clotting factor that is deficient in said plasmas. In other embodiments, the APC-resistant Factor V may be combined with other blood clotting factors, e.g. one or more of Factor VIII, Factor Vila, Factor IX, and the like. In certain embodiments it is free of (APC-resistant and/or wild-type) Factor Va.

[0063] Although APC-resistant Factor V has been suggested as a possible candidate for treatment of haemophilia patients having inhibitors to Factor VIII (EP 0756638), the present results surprisingly show that APC-resistant Factor V might be a better therapeutic than Factor VIII itself, and hence could also be used to replace treatment with Factor VIII in the group of haemophilia patients that do not suffer from inhibitors against Factor VIII, thereby providing an improved treatment for a new group of patients (whether inhibitors to FVIII are present can be routinely determined for instance using a Bethesda assay, wherein normal plasma is incubated with patient plasma and residual activity of FVIII is measured with a specific clotting assay). The improvement involves similar potency with lower risk of immunogenicity (and hence less chance of developing inhibitors), and possibly advantages in pharmacokinetics. It is therefore an aspect of the present invention to provide an improvement in a method for treating a patient having a Factor VIII deficiency wherein an effective hemostatic amount of Factor VIII is administered to the patient, the improvement comprising administering an effective hemostatic amount of APC-resistant Factor V to the patient.

[0064] According to the present invention hemophilia A patients are treated with APC-resistant Factor V. In preferred aspects, Factor VIII needs no longer to be administered, or administering of Factor VIII can be diminished to much lower levels, for instance to levels sufficiently low to not provoke an immune response in the patient to F VIII. For instance, it is possible to administer to hemophilia patients an amount of FVIII to reach a concentration thereof of 0.001-0.02, e.g. 0.01 U/ml plasma, and also treat the patient with APC-resistant FV according to the present invention (e.g. to reach plasma levels thereof of 0.01-5 U/ml).

[0065] In certain embodiments, the invention provides a method for prevention or treatment according to the invention, wherein the hemostatic level of APC-resistant Factor V is determined in an in vitro assay comprsing: a) providing plasma from said patient with (a dilution of) tissue factor, Ca ⁺, and optionally activated protein C or thrombomodulin at concentrations where clotting time (or fibrin/thrombin formation) is dependent from addition of the clotting factor that is deficient in said plasma, b) measuring fibrin or thrombin generation in the absence of said clotting factor, c) measuring fibrin or thrombin generation in the presence of a dose between 0.01 and 5 U/ml of said clotting factor, and d) measuring fibrin or thrombin generation in the absence of said clotting factor in the presence of APC-resistant Factor V, to determine a hemostatic level of said APC-resistant Factor V to replace the clotting factor that is deficient in said plasma. The invention further provides a method for testing the capacity of APC-resistant Factor V to bypass a clotting factor deficiency in a plasma, comprising: a) providing plasma which has a deficiency in a clotting factor (e.g. FVIII or FIX) with (a dilution of) tissue factor, Ca²⁺, and optionally activated protein C and/or thrombomodulin at concentrations where clotting time (or fibrin/thrombin generation) is dependent from addition of the clotting factor that is deficient in said plasma; b) measuring fibrin or thrombin generation in the absence of said clotting factor; c) measuring fibrin or thrombin generation in the presence of a dose between 0.01 and 5 U/ml of said clotting factor; and d) measuring fibrin or thrombin generation in the absence of said clotting factor in the presence of APC-resistant Factor V, to establish the capacity of APC- resistant Factor V to replace the clotting factor that is deficient in said plasma. Preferably the assay is performed under conditions with APC (or thrombomodulin, which induces APC), and in certain embodiments, the effect of APC is tested at different concentrations.

[0066] It is another aspect of the invention to provide a method for treatment or prevention of bleeding in a subject not having a (congenital or other) clotting factor deficiency, the method comprising administering to said subject a pharmaceutical composition comprising APC-resistant Factor V. Thus, the use of APC-resistant Factor V outside the patient group of hemophilia patients is envisaged, based on the data disclosed herein. Amenable subjects may for instance have a vitamin K deficiency, or be subject to surgery, or have trauma, etc. Thus in these aspects of the invention, APC-resistant Factor V is used as a hemostatic agent to treat (e.g. with trauma) or prevent/diminish (e.g. with surgery) bleeding, e.g. in normal plasmas. In these aspects it is used in a manner similar to FVIIa.

[0067] In another aspect the invention provides a method for prevention or treatment of bleeding in a patient with a FXI-defϊciency (hemophilia C), comprising administering to said patient APC-resistant FV. [0068] In yet another aspect the invention further provides method for treatment and/or prevention of bleeding in a subject having a deficiency or defect in Factor VIII, the method comprising administering to said subject Factor VIII and APC-resistant Factor V. A reduction of spontaneous bleeding events, in number and/or severity, is achieved. In certain embodiments thereof, no other blood clotting factors are administered. In certain embodiments, FVIII and APC-resistant FV are administered essentially simultaneously. In other embodiments, FVIII and APC-resistant FV are administered separately. In certain embodiments, Factor VIII is administered at levels sufficiently low to not provoke an immune response in the patient to F VIII, e.g. to obtain plasma concentrations of about between 0.001-0.02 Units FVIII per ml plasma, and APC- resistant Factor V is administered in a hemostatic amount, e.g. to reach plasma concentrations of about between 0.01 and 5 Units of APC-resistant FV per ml plasma. In another aspect, the invention provides a pharmaceutical composition comprising Factor VIII and APC-resistant Factor V. In certain embodiments, said composition is substantially free from other blood clotting factors. In another aspect, the invention provides a kit of parts comprising two pharmaceutical compositions, comprising: a first pharmaceutical compostion comprising Factor VIII, and a second pharmaceutical composition comprising APC-resistant Factor V. In certain embodiments, said kit of parts is substantially free from other blood clotting factors. In certain embodiments, said Factor VIII and said APC-resistant Factor V are present in said pharmaceutical composition or in said kit of parts in a ratio of 1 Unit Factor VIII to about between 10-5000 Units APC- resistant Factor V. Preferably, both clotting factors have been obtained by recombinant production means.

[0069] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology and the like, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Molecular Cloning: A Laboratory Manual, (J. Sambrook et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubel et al., eds., 1987 updated); Essential Molecular Biology (T. Brown ed., IRL Press 1991); Gene Expression Technology (Goeddel ed., Academic Press 1991); Methods for Cloning and Analysis of Eukaryotic Genes (A. Bothwell et al. eds., Bartlett Publ. 1990); Gene Transfer and Expression (M. Kriegler, Stockton Press 1990); Recombinant DNA Methodology (R. Wu et al. eds., Academic Press 1989); PCR: A Practical Approach (M. McPherson et al., IRL Press at Oxford University Press 1991); Oligonucleotide Synthesis (M. Gait ed., 1984); Cell Culture for Biochemists (R. Adams ed., Elsevier Science Publishers 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian Cell Biotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard et al. eds., Humana Press 1990); Culture of Animal Cells, 2.sup.nd Ed. (R. Freshney et al. eds., Alan R. Liss 1987); Flow Cytometry and Sorting (M. Melamed et al. eds., Wiley-Liss 1990); the series Methods in Enzymology (Academic Press, Inc.); and Animal Cell Culture (R. Freshney ed., IRL Press 1987); and Wirth M. and Hauser H. (1993) Genetic Engineering of Animal Cells, In: Biotechnology Vol. 2 Puhler A (ed.) VCH, Weinhcim 663-744.

EXAMPLES

Example 1: Recombinant production and testing of APC-resistant Factor V

[0070] Factor V mutants have been described, which are resistant to cleavage by APC. One of these mutants is the Factor V Leiden variant (Bertina et al. 1994), containing an Arg to GIn mutation at amino acid position 506. Another mutant is the Cambridge variant (Williamson et al. 1998), which contains a Thr residue instead of an Arg at positon 306.

[0071] Using routine molecular biology methods, three expression vectors were constructed, one containing the wild-type Factor V coding region, one containing a point mutation at amino acid position 506 (Arg506 to GIn, Factor V Leiden), and one containing a double mutation (Arg506 to GIn, and Arg306 to Thr). The factor V coding regions were inserted behind a CMV promoter into expression vector pcDNA2001Neo(- ), resulting in pCP-FV-wt (containing wild-type Factor V coding sequence), pCP-FV-Ll (containing the factor V coding sequence but with a mutation resulting in the R506Q mutation in the protein; Leiden mutant), and pCP-FV-LCl (containing the factor V coding sequence but with a mutation resulting in the R506Q and the R306T mutation in the protein; Leiden/Cambridge double mutant). [0072] The factor V sequence used (Bos et al., 2005) encoded the Factor V amino acid sequence as present in Swissprot entry P12259. Amino acid positions are according to the Factor V coding sequence, but after processing of the 28 amino acid leader peptide.

[0073] Similar expression plasmids with the same Factor V sequences, but with in addition a human α-2,3-sialyltransferase cDNA (Genbank accession number L23767, see also US patent 5,494,790) under control of a separate CMV promoter, were also constructed and used for obtaining clones expressing APC-resistant Factor V.

[0074] For the following, expression vector pCP-FV-LCl (encoding FV- R306T/R506Q, further called FV-L/C) was used.

[0075] Stable PER.C6 cell lines expressing rFV-L/C were generated using standard molecular biology and cell culture techniques (e.g. US patent 6,855,544, WO 2006/070011). Cell lines that were transfected with the expression vector containing only the Factor V-L/C cDNA were termed PER. C6 -FV-L/C. Cell lines that were transfected with the expression vector containing the Factor V-L/C and the human α-2,3- sialyltransferase cDNA were termed PER.C6-FV-L/C-ST. The products produced by these cells are referred to as rFV-L/C and rFV-L/C-ST, respectively.

[0076] Cell lines were tested for production of recombinant protein by measuring FV levels in culture supernatant with an ELISA using polyclonal sheep anti-human FV IgG antibodies (sheep a-human Factor V; Kordia, Leiden, the Netherlands). Cell-lines producing the highest amounts of factor V were used for production of recombinant factor V.

[0077] Cell culture supernatants were produced from these cell lines in roller bottles in serum-containing culture media (e.g. DMEM with 2.5% FCS), using standard cell culture techniques. FV-L/C was purified using standard chromatography techniques, including immuno-affinity and ion-exchange chromatography (see e.g. Bos et al, 2005).

[0078] Purified samples were stored in a buffer containing 50 mM Tris/HCl (pH 7.4), 100 mM NaCl , 5 mM CaCl₂ and 50% glycerol (v/v). FV-L/C is stable (SDS-PAGE, Western blot, chromogenic assay) for >6 months in this formulation.

[0079] Plasma FV was obtained using the same procedure. Normal human plasma (Sanquin Plasma Products, Amsterdam, the Netherlands) was used as a source of FV. [0080] On a 5% SDS-PAGE gel stained with silver, all FV species displayed a predominant band at 330 kDa, and a secondary band at 220 kDa. By immunoblotting using polyclonal anti-FV-IgG, the bands were identified as FV.

[0081] The activity of the preparations was tested for specific chromogenic and clot activity as well as for APC-rsistance.

[0082] Chromogenic activity was tested in the following assay.

[0083] Each sample (12.5 μl ) was added to 50 μl of an activation mix containing 2 nM FXa (Kordia), 20 μM PTT reagents (Roche), CaCl₂ in a buffer containing 0.1 M NaCl, 0.05 M TRIS and 0.1% (w/v) HSA (Sigma) and 12.5 μl of Prothrombin (Kordia) and the plate incubated for 5 minutes at 37⁰C. The reaction was then stopped by the addition of 12.5 μl of 0.1M EDTA in 0.1 M NaCl and 0.05 M TRIS buffer. A chromogenic substrate (S2238, Chromogenix) was added (12.5 μl) and the reaction read at 405nm. Fig. 1 shows a schematic view of the chromogenic assay. Pooled plasma was used as a standard (FV concentration = 1 U/ml). The specific chromogenic activity was calculated from the chromogenic activity (U ) divided by the Antigen concentration (U^Ag). All FV-L/C preparations prepared as described above showed specific chromogenic activity of at least 90% of that of plasma Factor V (Table 2).

Table 2. Activity of FV-L/C.

[0084] Clot activity was tested in a prothrombin time (PT) assay performed using FV-deflcient human plasma. Fig. 2 shows a schematic view of the clot activity assay. Briefly, purified preparations were added to FV-deficient plasma (Dade Behring, Liederbach, Germany) employing normal human plasma as reference. Clotting was induced with Innovin^®; Dade Behring. Pooled plasma was again used as a standard. One unit of factor V activity or antigen is similar to the amount of FV in 1 mL of normal plasma (± 8 μg/mL). The specific clot activity was calculated from the clot activity (U ) divided by the Antigen concentration (U^Ag). The results confirm that the produced FV- L/C has clot activity (Table 2). In fact, the somewhat higher specific clot activity of FV- L/C compared to wild type plasma derived FV may be due to the APC-resistance of FV- L/C.

[0085] APC-resistance was tested in an Activated Partial Thromboplastin Time (APTT) assay in FV-deficient human plasma with and without APC (Kordia, Leiden, The Netherlands). Fig.3 shows a schematic view of this assay. The results confirm that the produced FV-L/C is fully APC-resistant (Table 3).

Table 3. APC-resistance of FV-L/C.

[0086] In conclusion, the biochemical characterisation of the produced FV-L/C demonstrates that we were able to obtain a preparation with a purity of over 90% at a concentration of more than 1 mg/ml, which has a specific cofactor activity that is at least 90% of that of plasma Factor V, has clot activity and is fully APC-resistant. Example 2: FV-L/C restores clotting in FVIII-depleted plasma in the absence of added APC

[0087] Purified rFV-L/C molecules were tested using a Fibrin Generation Time (FGT) assay (schematically shown in Fig. 4), performed in both FVIII immune depleted human plasma and FVIII deficient plasma obtained from patients with severe haemophilia A (<0.01 U/ml FVIII).

[0088] The assay was established using FVIII-immune depleted plasma. Tissue Factor (TF) and Activated Protein C (APC) concentrations were titrated to give a dose response for Factor VIII. Thrombin formation was triggered by the addition of TF in the presence of APC. As controls, recombinant Factor VIII was added to 1% (0.01 U/ml; severe hemophilia), 3% (0.03 U/ml; moderate hemophilia), 10% (0.1 U/ml; mild hemophilia), 25% and 100% (0.25 and 1 U/ml, resp; normal). The endpoint of the assay is clotting time (or thrombin generation time). Suitable TF concentrations were determined in FVIII immune depleted plasma, using 1:2,500-1 :80,000 TF dilutions. Two TF concentrations (1:40,000 and 1:20,000 dilutions; Innovin^R [Dade Behring, Germany] was used in the assays in the following examples) that resulted in a dose-response curve of FVIII versus Ti_/2inax over the range of 0-25% FVIII were chosen. A typical example of the thrombin generation time curves is shown in Fig. 5. Using FVIII immune-depleted plasma reconstituted with 0, 1, 3, 10, 25 and 100% FVIII, the clotting time was determined in the presence of 0, 30 and 60 nM added APC (Fig. 6). TF/APC concentrations were selected that resulted in an optimal dose response of FVIII versus Tiβmax over the range 0-25% FVIII. In further experiments FV-L/C was added instead of FVIII to evaluate whether APC-resistant FV could shorten Ti^max to that obtained with 0.1 U/ml FVIII or more, since in that case APC-resistant FV is capable of restoring a severe hemophilia phenotype to a mild hemophilia phenotype or better. The experiments shown below demonstrate for the first time that APC-resistant Factor V, here FV-L/C, can be used to obtain this effect, and surprisingly that it can do so in a highly efficacious manner.

[0089] One hundred microliters of either FVIII-immune depleted human plasma (Dade Behring, OTXW 175) or FVIII-deficient plasma obtained from haemophilia A patients was introduced in duplicate into microtiter plates (low binding, flat bottom). Recombinant FVIII (Recombinate 500 U, Baxter, WD-060-352), Activated Protein C (APC, Kordia) and recombinant FV-L/C (see example 1) was added at concentrations indicated in the Figs. After addition of 75 μl of HEPES buffer (25 mM HEPES (Boehringer Mannheim), 137 mM NaCl (Merck) and 0.1% Ovalbumin (Sigma, A-5503), pH 7.4), the samples were incubated for 5 min. at 37 ⁰C. Then, 75 μl of a preheated (37 ⁰C) dilution of TF (Innovin, Dade Behring, B4212-50) was added. Dilutions of TF were made in HEPES calcium buffer: 25 mM HEPES (Boehringer Mannheim), 137 mM NaCl (Merck), 0.1% Ovalbumin (Sigma, A-5503), 38 mM CaCl₂, pH 7.4. After mixing, the samples were immediately analyzed for fibrin generation. Fibrin generation was measured in time by use of the SpectraMax microtiterplate reader and Softmax pro software.

[0090] The effect of FV-L/C was tested in FVIII-depleted plasma without addition of APC. TF was used at dilution 1:40,000. Fig. 7 shows that in the absence of added APC, the addition of 0.5 U/ml rFV-L/C restores the clotting time equivalent to 0.1 U/ml of FVIII. Fig. 7 shows that clotting times equivalent to 1 U/ml of FVIII can be achieved by the addition of 2 U/ml of rFV-L/C. The addition of plasma FV (pFV) at up to 2 U/ml does not restore clotting and results in clotting times equivalent to that observed in plasma where no FVIII was added. Thus, APC-resistant Factor V can compensate for absence of FVIII under conditions where no APC is added to plasma. Similar data were obtained using rFV-L/C-ST.

[0091] In similar experiments, FV-L/C was able to reduce clotting time in FDC- immune depleted and in FXI-immune depleted plasma in the absence of added APC (Fig. 14).

[0092] In FDC-immune depleted human plasma, addition of 1 U/ml of rFV-L/C restores the clotting time equivalent to approximately 0.25 U/ml of the commercially available recombinant FIX preparation (Benefix, Wyeth; Fig. 14A). Similar data were obtained using rFV-L/C-ST.

[0093] In FXI-immune depleted human plasma, rFV-L/C restores the clotting time equivalent to approximately 0.1 U/ml of the commercially available plasma derived FXI concentrate (Hemoleven, LFB, France; Fig. 14B). Thus, rFV-L/C can highly surprisingly restore or maintain hemostasis in FXI-deficient plasma. [0094] It may therefore be considered that APC-resistant Factor V is suitable for restoring or maintaining hemostasis in FVIII-, FIX- and FXI-deficient plasma at low or absent APC levels (endogenous APC concentrations in human plasma are typically in the 60-80 pM range). A hemostatic level may be obtained (in Units/ml plasma) by providing FV-L/C to a level of about 0.5-2 U/ml, which may lead to a phenotype that would be similar to mild hemophilia or better.

Example 3: FV-L/C potency increased in the presence of APC

[0095] The previous example showed that FV-L/C can restore clotting in FVIII- depleted plasma when no APC is added. The effect of APC addition is tested in this example, since APC plays an important role in the regulation of blood coagulation under physiological conditions. Methods were as described in example 2.

[0096] Fig 8A and 8B show that at 1 :40,000 times diluted TF and in the presence of APC (both at 30 nM and at 60 nM), the addition of 0.5 U/ml of rFV-L/C to FVIII immune depleted human plasma restores clotting to a greater extent than 1 U/ml of FVIII. The addition of up to 2 U/ml of plasma derived FV (pFV) does not restore clotting and results in similar clotting times to plasma where no FVIII was added. These data surprisingly demonstrate that FV-L/C may be more efficacious than FVIII in restoring hemostasis in situations where APC is present. In other words, APC-resistant Factor V may fully replace Factor VIII in therapy of FVIII-deficiency. Similar data were obtained using rFV-L/C-ST. The intruiging and unexpected implication of these results is that APC-resistant FV, such as FV-L/C, is particularly effective in case bleeding in hemophiliacs occur at sites where protein C is activated. Based on the distribution and concentration of TM in the circulation, this will be particularly the case for (spontaneous) bleedings in hemophiliacs such as in the joints, the muscles, soft tissues, and other sites, since these are capillary bleedings and the concentration of TM in capillaries can be as high as 500 nM (Esmon, 1989).

[0097] Fig 15 shows that in similar experiments the potency of rFV-L/C is also increased in the presence of APC when compared to rFIX (Benefix) and pFXI (Hemoleven) in FIX- and FXI-immune depleted human plasma respectively. The addition of 0.25 U/ml of rFV-L/C restores the clotting time of FIX-immune depleted human plasma to a greater extent than 1 U/ml of rFDC, Benefix, while the addition of 1 U/ml of rFV-L/C restored the clotting time of FXI-immune depleted human plasma to a similar extent as lU/ml of pFXI (Hemoleven). Similar data were obtained using rFV- L/C-ST (data not shown).

Example 4: FV-L/C potency in the presence of APC is maintained at higher TF concentrations

[0098] At increased TF concentrations, the effect of FVIII-deficiency is lower, because direct activation of FX (i.e. without the FIX/VIII amplification loop, see Fig 4) can take place via FVII. We analysed the effect FV-L/C at a higher TF concentration (1 :20,000 dilution, vs. 1 :40,000 in previous examples), at an APC concentration of 30 nM. Fig 9 shows that the same effects of rFV-L/C on clotting times are observed at higher TF concentrations. At 20,000 times diluted TF and in the presence of APC, the addition of 0.5 U/ml of rFV-L/C again restored clotting time to a greater extent than 1 U/ml of FVIII. Clotting times equivalent to 1 U/ml of FVIII could be achieved by the addition of 0.06 U/ml of rFV-L/C. Again, the addition of up to 2 U/ml of pFV was not capable of restoring clotting. Thus, the potency of FV-L/C to restore clotting in the absence of FVIII and in the presence of APC as demonstrated in the previous example, was maintained at higher TF concentrations. The data show that addition of 0.06 U/ml of FV-L/C results in fibrin generation times that are similar to those obtained by addition of 1 U/ml of FVIII under these conditions. Hence, these data confirm the strong potency of APC-resistant Factor V as a hemostatic agent in FVIII-deficient plasma.

Example 5: FV-L/C restores clotting in hemophilic plasma

[0099] The previous examples employed FVIII-immuno depleted human plasma. It was shown that FV-L/C can restore clotting in those plasmas in a very potent manner. In this example, the addition of FV-L/C to different independent plasmas obtained from hemophilia A patients (i.e. having a deficiency in FVIII) was tested. In contrast to the plasmas of the previous examples therefore, the plasmas in this example were not immuno depleted. The plasmas were obtained from hemophilia patients that did not have inhibitors to FVIII. The experiments with these hemophilia plasmas were performed in the presence of 30 nM APC, clotting was initiated using 1:40,000 times diluted TF. Fig 1OA, 1OB and 1OC shows that the addition of 0.5 U/ml rFV-L/C to plasma collected from three hemophilia A patients (plasma 1, 2 and 3, <0.02 U/ml FVIII in each plasma), could restore clotting to level similar to between 0.25 and 1.0 U/ml FVIII.

[00100] Fig 10 A, B and C show that the addition of 0.5 U/ml rFV-L/C to plasma collected from three hemophilia A patients (plasma 1, 2 and 3, <0.02 U/ml FVIII in each plasma), could restore clotting to level similar to between 0.25 and 1.0 U/ml FVIII.

[00101] Fig 10 D, E and F show that the addition of 0.25U/ml rFV-L/C-ST to plasma collected from three hemophilia A patients (plasma 4, 5 and 6, <0.02 U/ml FVIII in each plasma) could restore clotting to a similar extent as 1.0 U/ml FVIII.

[00102] Fig 18 A, B and C show that the addition of 0.25 U/ml rFV-L/C-

ST to plasma collected from three hemophilia B patients (plasma 1, 2 and 3, <0.02 U/ml FIX) could restore clotting to a similar extent as 1.0 U/ml FIX.

[00103] These data demonstrate that APC-resistant Factor V can restore clotting in plasma from patients with hemophilia A. They thus confirm and extend the data obtained with the FVIII-depleted normal plasmas (examples 2-4). Importantly, fibrin (clot) formation in hemophilic plasmas can be restored by addition of APC-resistant FV to the same range as obtained with current FVIII replacement therapy.

Example 6. Generation of thrombin in Hemophilic human plasma by FV-L/C measured by Thrombogram.

[00104] Experiments were also performed testing FV-L/C in plasma from a severe Hemophilia patient (<0.01 U/ml FVIII) using the Thrombogram technology (Thrombinoscope). Thrombin generation in these experiments was initiated using 1 pM TF (1:6,000 times diluted Innovin, Dade Behring). The data is shown in Fig. 11 and demonstrates that in the presence of 16 nM Thrombomodulin (TM, Fig 1 Ia), as well as 8 nM APC (Fig 1 Ib), 0.5 U/ml of FV-L/C can generate thrombin to a greater extent than 1 U/ml FVIII. The data generated using the Thrombogram technology and using Thrombomodulin to activate endogenous protein C as well as by addition of APC therefore supports the experiments described in example 5 that FV-L/C can bypass FVIII and restore clotting in severe hemophilic human plasma.

Example 7: Enhanced potency of combination of rFV-L/C and rFVIII in FVIII-immune depleted human plasma.

[00105] Experiments were performed in FVIII-immune depleted human plasma to determine whether rFV-L/C and rFVIII act to reduce the clotting time in a more effective manner than each of these clotting factors alone. In other words, to determine whether through the addition of rFV-L/C it would be possible to reduce the effective dose of rFVIII. These experiments were performed in the absence of added APC and using 1 :40,000 TF to trigger clotting.

[00106] Fig. 16 shows that rFV-L/C combination of 0.5 U/ml rFV-L/C and

0.01 U/ml rFVIII leads to a greater reduction in the clotting time than either 0.5 U/ml rFV-L/C or 0.01 U/ml rFVIII alone. Similar data were obtained using rFV-L/C-ST.

Example 8: rFV-L/C reduces the effective dose of rFVIII required to stop bleeding in a tail clip bleeding model of hemophilia in Factor VIII deficient mice.

[00107] A tail clip bleeding model has been established in FVIII-knock-out mice. These mice have a neo gene insertion in exon 16 of the FVIII gene and as a result are FVIII-deficient (Bi et al, 1995). These mice demonstrate a severe hemophilia A phenotype and when they are subject to a tail clip such that the end section of the tail is removed, they suffer a severe blood loss unless treated with high dose FVIII. By contrast, wild type mice subject to the same tail clip suffer relatively minor blood loss.

[00108] Experimental design. Hemophilia A mice are weighed and anaesthetized. Injections of rFVIII (Kogenate-FS, Bayer), rFV-L/C-ST or the appropriate vehicle are performed in the femoral vein. A transverse incision is then made at the end of the tail such that the end section of the tail is completely removed. Blood loss is quantified over 40 minutes using the Drabkin method (with Drapkin's reagent, Sigma, Saint Louis, Missouri, USA, product code D 5941).

[00109] Fig. 17 shows the results. Injection of the FV- and FVIII-vehicle

(veh) had no effect on blood loss. Injection of 200U/kg rFVIII (Kogenate-FS, Bayer) led to a significant but not complete reduction in the mean blood loss. However, administration of both 200 U/kg rFVIII and 280 U/kg rFV-L/C-ST resulted in a complete reduction in mean blood loss, comparable to levels seen in wild type mice. Thus, administration of 280 U/kg rFV-L/C-ST was able to convert a sub-effective dose of rFVIII to a fully effective treatment, reducing blood loss to wild type levels. A subgroup of FVIII knockout mice showed considerable blood loss (comparable to the non-treated group) even after treatment with rFVIII at 200 U/kg, whereas no such subgroup was observed when both 200 U/kg rFVIII and 280 U/kg rFV-L/C-ST were administered. In addition, these experiments demonstrate that the rFV-L/C-ST as produced is active in vivo.

[00110] Summarized, the data in the examples above for the first time and surprisingly demonstrate that: a) APC-resistant FV, in contrast to wtFV, can bypass FVIII and FIX deficiency; b) APC-resistant FV restores clotting in the absence of APC; c) The potency of APC-resistant FV is increased in the presence of APC; and d) APC-resistant FV restores clotting in hemophilic plasma. e) the potency of FVIII combined with APC-resistant FV is increased compared to the individual clotting factors

[00111] The data also strongly suggest that APC-resistant Factor V may be a powerful hemostatic agent, suitable for replacing FVIII, not only in patients with inhibitors to FVIII, but also in patients that did not develop inhibitors to FVIII.

REFERENCES

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Table 4. Sequence of mature wild-type Factor V (SEQ ID NO:1).

1 AQLRQFYVAA QGISWSYRPE PTNSSLNLSV TSFKKIVYRE YEPYFKKEKP QSTISGLLGP

61 TLYAEVGDII KVHFKNKADK PLSIHPQGIR YSKLSEGASY LDHTFPAEKM DDAVAPGREY

121 TYEWΞIΞEDΞ GPTHDDPPCL THIYYΞHENL IEDFNΞGLIG PLLICKKGTL TEGGTQKTFD

181 KQIVLLFAVF DESKSWSQSS ΞLMYTVNGYV NGTMPDITVC AHDHIΞWHLL GMΞΞGPELFΞ

241 IHFNGQVLEQ NHHKVΞAITL VΞATΞTTANM TVGPEGKWII ΞΞLTPKHLQA GMQAYIDIKN

301 CPKKTRNLKK ITREQRRHMK RWEYFIAAEE VIWDYAPVIP ANMDKKYRΞQ HLDNFΞNQIG

361 KHYKKVMYTQ YEDEΞFTKHT VNPNMKEDGI LGPIIRAQVR DTLKIVFKNM AΞRPYΞIYPH

421 GVTFΞPYEDE VNΞΞFTΞGRN NTMIRAVQPG ETYTYKWNIL EFDEPTENDA QCLTRPYYΞD

481 VDIMRDIAΞG LIGLLLICKΞ RΞLDRRGIQR AADIEQQAVF AVFDENKΞWY LEDNINKFCE

541 NPDEVKRDDP KFYEΞNIMΞT INGYVPEΞIT TLGFCFDDTV QWHFCΞVGTQ NEILTIHFTG

601 HΞFIYGKRHE DTLTLFPMRG EΞVTVTMDNV GTWMLTΞMNΞ ΞPRΞKKLRLK FRDVKCIPDD

661 DEDSYEIFEP PEΞTVMATRK MHDRLEPEDE EΞDADYDYQN RLAAALGIRΞ FRNΞΞLNQEE

721 EEFNLTALAL ENGTEFVΞΞN TDIIVGΞNYΞ ΞPΞNIΞKFTV NNLAEPQKAP SHQQATTAGS

781 PLRHLIGKNΞ VLNΞΞTAEHΞ SPYSEDPIED PLQPDVTGIR LLSLGAGEFK SQEHAKHKGP

841 KVERDQAAKH RFSWMKLLAH KVGRHLΞQDT GΞPΞGMRPWE DLPΞQDTGΞP SRMRPWKDPP

901 ΞDLLLLKQΞN SSKILVGRWH LASEKGSYEI IQDTDEDTAV NNWLISPQNA SRAWGESTPL

961 ANKPGKQSGH PKFPRVRHKΞ LQVRQDGGKΞ RLKKΞQFLIK TRKKKKEKHT HHAPLΞPRTF

1021 HPLRΞEAYNT FΞERRLKHΞL VLHKΞNETΞL PTDLNQTLPΞ MDFGWIAΞLP DHNQNΞΞNDT

1081 GQASCPPGLY QTVPPEEHYQ TFPIQDPDQM HΞTΞDPΞHRΞ SSPELSEMLE YDRΞHKΞFPT

1141 DIΞQMΞPΞΞE HEVWQTVISP DLSQVTLSPE LSQTNLSPDL SHTTLSPELI QRNLSPALGQ

1201 MPISPDLSHT TLΞPDLΞHTT LSLDLSQTNL ΞPELΞQTNLΞ PALGQMPLΞP DLΞHTTLΞLD

1261 FΞQTNLΞPEL SHMTLSPELS QTNLSPALGQ MPISPDLSHT TLΞLDFΞQTN LΞPELΞQTNL

1321 SPALGQMPLS PDPΞHTTLΞL DLΞQTNLΞPE LSQTNLSPDL ΞEMPLFADLΞ QIPLTPDLDQ

1381 MTLΞPDLGET DLSPNFGQMS LSPDLSQVTL SPDISDTTLL PDLSQISPPP DLDQIFYPSE

1441 SSQSLLLQEF NEΞFPYPDLG QMPΞPΞΞPTL NDTFLΞKEFN PLVIVGLΞKD GTDYIEIIPK

1501 EEVQΞΞEDDY AEIDYVPYDD PYKTDVRTNI NΞΞRDPDNIA AWYLRΞNNGN RRNYYIAAEE

1561 IΞWDYΞEFVQ RETDIEDΞDD IPEDTTYKKV VFRKYLDΞTF TKRDPRGEYE EHLGILGPII

1621 RAEVDDVIQV RFKNLASRPY ΞLHAHGLΞYE KSSEGKTYED DΞPEWFKEDN AVQPNΞΞYTY

1681 VWHATERΞGP EΞPGΞACRAW AYYΞAVNPEK DIHSGLIGPL LICQKGILHK DSNMPVDMRE

1741 FVLLFMTFDE KKSWYYEKKS RΞΞWRLTΞΞE MKKSHEFHAI NGMIYΞLPGL KMYEQEWVRL

1801 HLLNIGGΞQD IHWHFHGQT LLENGNKQHQ LGVWPLLPGΞ FKTLEMKAΞK PGWWLLNTEV

1861 GENQRAGMQT PFLIMDRDCR MPMGLΞTGII ΞDΞQIKAΞEF LGYWEPRLAR LNNGGSYNAW

1921 ΞVEKLAAEFA ΞKPWIQVDMQ KEVIITGIQT QGAKHYLKSC YTTEFYVAYS SNQINWQIFK

1981 GNSTRNVMYF NGNΞDAΞTIK ENQFDPPIVA RYIRISPTRA YNRPTLRLEL QGCEVNGCΞT

2041 PLGMENGKIE NKQITAΞΞFK KΞWWGDYWEP FRARLNAQGR VNAWQAKANN NKQWLEIDLL

2101 KIKKITAIIT QGCKΞLΞΞEM YVKΞYTIHYΞ EQGVEWKPYR LKSSMVDKIF EGNTNTKGHV

2161 KNFFNPPIIΞ RFIRVIPKTW NQΞIALRLEL FGCDIY

Claims

1. A method for preventing or treating bleeding in a joints, muscle and/or soft tissue in a hemophilia patient, comprising administering to the patient APC-resistant Factor V.

2. A method for preventing or treating spontaneous bleeding in a hemophilia patient, comprising administering to the patient APC-resistant Factor V.

3. A method for preventing or treating bleeding in the microcapillaries in a hemophilia patient, comprising administering to the patient APC-resistant Factor V.

4. A method for preventing or treating bleeding in a hemophilia patient at sites with an increased concentration of activated protein C, comprising administering to the patient APC-resistant Factor V.

5. A method according to any one of the preceding claims, wherein the hemophilia patient is deficient in Factor VIII activity or wherein the patient's Factor VIII activity is inhibited.

6. A method for reducing or preventing the possibility of generating inhibitors to Factor VIII in a hemophilia A patient, comprising administering an effective hemostatic amount of APC-resistant Factor V to the patient.

7. A method according to any one of the preceding claims, wherein APC- resistant Factor V is administered to obtain a plasma concentration thereof of between 0.01 and 5 Units/ml.

8. A method according to claim 7, wherein said obtained plasma concentration is between 0.05 and 2 Units/ml.

9. A method according to claim 8, wherein said obtained plasma concentration is between 0.1 and 1 Unit/ml.

10. A method according to any one of the preceding claims, wherein the APC- resistant Factor V has a mutation of Arg306, Arg506 or both Arg 306 and Arg506 as compared to the wild type Factor V sequence.

11. A method according to claim 10, wherein the APC-resistant Factor V has a mutation of Arg306 and Arg506 as compared to the wild type Factor V sequence.

12. A method according to any one of the preceding claims, wherein the APC- resistant Factor V is free from other clotting factors.

13. A method according to any one of the preceding claims, wherein said patient is a hemophilia A patient not diagnosed with inhibitors against Factor VIII.

14. A method for treatment or prophylaxis of a patient having a clotting factor deficiency or inhibitor, comprising administering to the patient an effective hemostatic amount of APC-resistant Factor V.