MUTATED FACTOR X POLYPEPTIDES AND USES THEREOF FOR THE TREATMENT OF HAEMOPHILIA

Abstract

The present invention relates to mutated factor (FX) polypeptides and uses thereof for the treatment of haemophilia. In particular, the present invention relates to a mutated factor X (FX) polypeptide wherein the heavy chain comprises at least one mutation selected from the group consisting of: the mutation which consists of the substitution of the glutamic acid residue (E) at position 215 by a glutamine residue (Q), an asparagine residue (N), a serine residue (S), an alanine residue (A), or a tyrosine residue (Y); the mutation which consists of the substitution of the glutamic acid residue (E) at position 216 by a glutamine residue (Q); and the mutation which consists of the substitution of the glutamic acid residue (E) at position 218 by a glutamine residue (Q);

Claims

1-9. (canceled)

10. A nucleic acid molecule which encodes for a mutated factor X (FX) polypeptide comprising the amino acid sequence of SEQ ID NO. 1 from the amino acid residue at position 235 to the amino acid residue at position 488 of SEQ ID NO: 1, wherein the heavy chain comprises at least one mutation selected from the group consisting of: a mutation comprising substitution of a glutamic acid residue (E) at position 255 by a glutamine residue (Q), a serine residue (S), an alanine residue (A), or a tyrosine residue (Y); a mutation comprising substitution of a glutamic acid residue (E) at position 256 by a glutamine residue (Q); and a mutation comprising substitution of a glutamic acid residue (E) at position 258 by a glutamine residue (Q).

11. A vector which comprises the nucleic acid molecule of claim 10.

12. A host cell which is transformed with the nucleic acid molecule of claim 10 or a vector comprising the nucleic acid.

13. (canceled)

14. A method of treating haemophilia in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a mutated factor X (FX) polypeptide comprising the amino acid sequence of SEQ ID NO. 1 from the amino acid residue at position 235 to the amino acid residue at position 488 of SEQ ID NO: 1, wherein the heavy chain comprises at least one mutation selected from the group consisting of: a mutation comprising substitution of a glutamic acid residue (E) at position 255 by a glutamine residue (Q), a serine residue (S), an alanine residue (A), or a tyrosine residue (Y); a mutation comprising substitution of a glutamic acid residue (E) at position 256 by a glutamine residue (Q); and a mutation comprising substitution of a glutamic acid residue (E) at position 258 by a glutamine residue (Q), or a nucleic acid molecule encoding the mutated factor X (FX) polypeptide.

15. A pharmaceutical composition which comprises the nucleic acid molecule of claim 10.

16. The method of claim 14, wherein the nucleic acid molecule is present in a vector.

17. The method of claim 14, wherein the nucleic acid molecule is not present in a vector.

18. The nucleic acid of claim 10 wherein the at least one mutation is a glutamine residue at position 255, 256, or 289.

19. The nucleic acid of claim 10 wherein the mutated FX polypeptide comprises an activation peptide and where a fibrinopeptide A is inserted between the activation peptide and the heavy chain.

20. A pharmaceutical composition which comprises the nucleic acid molecule of claim 18.

21. A pharmaceutical composition which comprises the nucleic acid molecule of claim 19.

Description

FIGURES

[0037] FIG. 1: Schematic representation of different parts of factor X zymogen amino acid sequence. The pre-peptide (or signal peptide) is defined by the amino acid sequence between the positions 40 to 18 and the pro-peptide by the amino acid sequence between the positions 17 to 1. The corresponds to the sequence between the amino acid positions 1 to 142 and the heavy chain between amino acid positions 195 to 448. The activation peptide (positions 143 to 194) is boxed and N-glycosylation sites of interest are tagged by an *. The numbering system used appears on the same line as the sequence and the other reference system appears in grey on the line under the sequence.

EXAMPLE

[0038] Material & Methods

[0039] Engineering and Production of Recombinant FX and FX Derivatives

[0040] cDNAs encoding wild-type (wt)-human FX (wt-hFX), and its variants FXE215Q, FXE216Q and FXE218Q (FIG. 1) were synthetically synthesized (Eurofins, Ebersberg Germany) and cloned in expression vectors using standard molecular biological protocols. Constructs were cloned into the pLIVE- and pNUT-plasmids for in vivo expression and in vitro expression in stably transfected in BHK-21 cells, respectively (23, 24, 25). All constructs contained a DNA sequence encoding the epitope recognized by antibody HPC4 (Roche, Meylan, France) that was added at the 3 end of the FX sequence. Stable BHK-21 cell lines producing FX or variants thereof were established as described (25) and detailed in the following section.

[0041] Obtention of Cell Lines Expressing the Recombinant Derivatives

[0042] The pNUT-constructs were transfected into Baby hamster kidney cells (BHK) using the jetPEI reactant (Qbiogen, Ozyme, France) as specified by the provider. After selection of transfected cells with medium containing methotrexate (Sigma) at a concentration of 100 M, single clones were picked and propagated in selective medium to obtain stable cell lines. Production of factor X antigen was assayed by enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies against factor X conjugated and not with horseradish peroxidase obtained from Cederlane (Cederlane Laboratories, Burlington, Canada). Purified human plasma derived factor X (pd-FX) from Cryopep (Montepellier, France) was used as reference.

[0043] Production and Purification of Recombinant Factor X and Derivatives

[0044] Stable cell lines producing recombinant factor X, and were maintained in 300 cm2 flasks for protein production in DMEM/F-12 supplemented with 10% FCS, 50 M methotrexate, 100 U/ml penicillin, 100 g/ml streptomycin, and 5 g/ml vitamin K1. Protein of interest containing medium was harvested every 48 hours. Benzamidine and PMSF were added to a final concentration of 10 and 2 mM, respectively, and the medium centrifuged (6 000 g), passed over cellulose acetate membranes (0.45 m) to eliminate cell debris, and stored at 20 C. until use. Conditioned medium was thawed at 37 C. EDTA was added to a final concentration of 5 mM. The medium was diluted in distilled water and in Tris (pH 7.4), to bring the final Tris and NaCl concentration to 25 and 60 mM, respectively. The mixture was then stirred at room temperature for 30 mM with QAE Sephadex A-50 beads to achieve a final concentration of 0.25% (wt/v). Beads were washed before elution with 50 mM Tris (pH 7.4), 500 mM NaCl, and 10 mM benzamidine. Recombinant proteins contained in the eluted fractions (ELISA) were immediately dialyzed against 25 mM Tris (pH 7.4), and 100 mM NaCl, containing 10 mM benzamidine, and stored at 20 C. before use. Concentrated proteins were thawed at 37 C. Calcium was added to a final concentration of 5 mM. Purification of recombinant proteins was performed by affinity-chromatography using HPC-4-agarose (Roche, Meylan, France) as instructed by the provider. 1 h prior to use as a zymogen, factor X derivatives were incubated with 1 mM PMSF to neutralize any trace of activated factor X that may have been generated during production or purification of the recombinant protein. Control experiments indicated that after 30 min in Tris-HCl buffer, PMSF was fully hydrolyzed and would not interfere with other reactions. Protein purity was assessed using 10% SDS-polyacrylamide gel electrophoresis analysis of the recombinant proteins under reducing (100 mM dithiothreitol, final concentration) and non-reducing conditions followed by staining with Coomassie Brilliant Blue R-250. Factor X identification was carried out after the purified recombinant proteins were reduced and loaded onto a 10% SDS-polyacrylamide gel. The resolved proteins were transferred to an Immobilon membrane and blotted using polyclonal antibodies against factor X conjugated with horseradish peroxydase (Cederlane). The purified derivatives were aliquoted and stored at 80 C. until use. The concentration of the aliquot is estimated by its absorbance at 280 nm, taking 1.16 to be the extinction coefficient (E280 nm 0.1%) of factor X.

[0045] Thrombin Generation Assay

[0046] Thrombin generation was measured according to the method described by Hemker et al (26), in a Fluoroscan Ascent fluorometer (Thermolabsystems OY, Helsink, Finland) equipped with a dispenser. Briefly, 80 l of plasma supplemented with either saline (control) or with indicated concentration of recombinant factor X derivatives were dispensed into round-bottom 96-well microtiter plates. Twenty l of a mixture containing TF (recombinant lipidated human tissue factor, Innovin, obtained from Dade Behring) and phospholipids (PL) vesicles was added to the plasma sample to obtain a final concentration of 1 pM TF and 4 M PL vesicles. PL vesicles prepared from L--Phosphatidyl-L-serine (PS) L--phosphatidylethanolamine (PE) and L--phosphatidylcholine (PC) (Avanti Polarlipids, Alabaster, Ala., USA) and of nominal 100 nm-diameter (PC:PE:PS, 3:1:1) were synthesized by the method of membrane extrusion (27). Phospholipid concentration was determined by phosphate analysis. Finally, thrombin generation was triggered by adding 20 l of starting reagent containing fluorogenic substrate and CaCl.sub.2. Fluorogenic substrate 1-1140 (Z-Gly-Gly-Arg-AMC) was from Bachem AG (Bubendorf, Switzerland). Kinetics of thrombin generation in clotting plasma was monitored for 60 min at 37 C. using a calibrated automated thrombogram and analyzed using the Thrombinoscope software (Thrombinoscope B.V., Maastricht, the Netherlands). Four wells were needed for each experiment, two wells to measure thrombin generation of a plasma sample and two wells for calibration. All experiments were carried out in triplicate and the mean value was reported. Endogenous thrombin potential (ETP), i.e. area under the curve, peak thrombin, and lag time for thrombin detection determined. In some experiments, immunodepleted FVIII-plasma (Diagnostica Stago, Asnieres, France) was supplemented with FX variants (60, 150 and 300 nM final concentrations) or recombinant purified FVIII (0.025, 0.1, and 1 U/ml Kogenate FS, Bayer HealthCare, Puteaux, France). In other experiments, immunodepleted FVIII-deficient human plasma was supplemented with mouse monoclonal anti-FVIII antibody D4H1 to create FVIII-inhibitor plasma (28). D4H1 at a final concentration of 10 g/ml or 50 g/ml corresponds to 30-40 Bethesda Units (BU)/ml and 150-200 BU, respectively. Subsequently, FVIII inhibitor plasma was supplemented in vitro with various concentrations FX derivatives. Finally, experiments were performed using immunodepleted FIX-plasma (Diagnostica Stago, Asnieres, France).

[0047] Results:

[0048] Thrombin Generation in FVIII and FIX-Deficient Plasmas

[0049] In a first series of experiments, we assessed the potential of different concentrations of FVIII, 1, 0.1, and 0.025 U/ml corresponding to a normal individual (control), a mild and a moderate hemophilia, respectively, to compensate for the absence of FVIII in the generation of thrombin. To this end, coagulation in immunodepleted FVIII-deficient human plasma was initiated by the addition of TF (1 pM) and phospholipids (4 M), and relevant thrombin generation parameters such as ETP and peak thrombin generation were determined. In the absence of any added coagulation factor, this resulted in an ETP and a peak thrombin generation (for summary see Table 1). Both values are significantly reduced compared to normal plasma (ETP: 1200 nM.Math.min; peak thrombin generation: 150-174 nM). As expected, the addition of FX derivatives (60, 150, 200 and 300 nM final concentrations) resulted in restoration of thrombin generation in FVIII-deficient plasma (Table 1). Also the addition of FXE215Q, FXE216Q or FXE218Q to a concentration of 300 nM resulted in normalization of thrombin generation, with both ETP and peak thrombin generation being within the same range as found for normal plasma (Table 1). A similar correction of the coagulation defect was observed when tested in immunodepleted FIX-deficient plasma (Table 2). Furthermore, no correction of thrombin generation was observed by the addition of wt-FX up to the highest concentration tested (0.5 M). These data indicate that under the conditions employed, the presence of the mutation gives the capacity to FX to overcome the absence of FVIII or FIX for efficient thrombin generation.

[0050] Thrombin Generation in FVIII-Deficient Inhibitor Plasma

[0051] We next evaluated FX derivatives for their ability to correct the coagulation deficiency in FVIII-deficient plasma in the presence of anti-FVIII antibody 4D1, dosed at a concentration of 150 BU/ml. The addition of increasing concentration FX derivatives resulted in normalization of the total thrombin generation (Table 3). Thus, the FXE215Q, FXE216Q or FXE218Q appears to be an efficient pro-coagulant agent to correct thrombin generation in FVIII-inhibitor plasma.

[0052] Tables:

TABLE-US-00001 TABLE 1 Thrombin generation test in FVIII-deficient plasma (Cryopep, Montpellier). Parameters for measuring thrombin generation (ETP, thrombin peak) were measured in immunodepleted FVIII- deficient plasma in the presence of tissue factor (1 pM) and phospholipids (4 M) with or without FVIII, FXE215Q, FXE216Q or FXE218Q. Data are presented as mean SD. Added coagulation factor in FVIII-deficient ETP Thromin Peak plasma n (nM .Math. min) (nM) FVIII (1 U/ml) 7 815 52 179 8 FVIII (0.1 U/ml) 6 634 31 56 5 FVIII (0.025 U/ml) 6 441 38 28 3 None 7 213 27 11 1 FXE215Q (300 nM) 3 835 35 156 7 FXE215Q (150 nM) 3 762 96 66 5 FXE215Q (60 nM) 3 558 43 39 2 FXE216Q (200 nM) 3 795 25 119 5 FXE218Q (300 nM) 3 856 40 139 4 FXE218Q (150 nM) 3 771 45 58 4 FXE218Q (60 nM) 3 297 28 18 1

TABLE-US-00002 TABLE 2 Thrombin generation test in FIX-immunodepleted plasma (Stago, France). Parameters for measuring thrombin generation (ETP, thrombin peak) were measured in immunodepleted FIX-deficient plasma in the presence of tissue factor (1 pM) and phospholipids (4 M) with or without FIX, FXE215Q, FXE216Q or FXE218Q. Data are presented as mean SD. Added coagulation factor in FIX-deficient ETP Thromin Peak plasma n (nM .Math. min) (nM) FIX (1 U/ml) 3 1356 30 295 8 FIX (0.1 U/ml) 3 802 19 81 5 FIX (0.025 U/ml) 3 424 18 29 1 None 3 239 68 10 3 FXE215Q (300 nM) 3 1019 17 223 3 FXE215Q (150 nM) 3 892 30 49 4 FXE215Q (60 nM) 3 467 12 21 4 FXE216Q (200 nM) 3 787 22 121 5 FXE218Q (300 nM) 3 1188 17 294 8 FXE218Q (150 nM) 3 993 23 81 4 FXE218Q (60 nM) 3 491 20 29 4

TABLE-US-00003 TABLE 3 Thrombin generation test in FVIII-deficient plasma (Cryopep, Montpellier) in the presence of inhibitor. Parameters for measuring thrombin generation (ETP, thrombin peak) were measured in FVIII-deficient plasma supplemented with mouse monoclonal anti-FVIII antibody D4H1 to create FVIII-inhibitor plasma in the presence of tissue factor (1 pM) and phospholipids (4 M) with or without FVIII, FXE215Q, FXE216Q or FXE218Q. Data are presented as mean SD. Added coagulation factor in FVIII-deficient plasma supplemented with ETP Thromin Peak inhibitor n (nM .Math. min) (nM) FXE215Q (300 nM) 3 978 41 101 6 FXE215Q (150 nM) 3 536 20 48 4 FXE215Q (60 nM) 3 306 15 17 4 FXE215Q (30 nM) 3 208 11 8 4 FXE216Q (200 nM) 3 685 20 54 4 FXE218Q (300 nM) 3 949 41 113 4 FXE218Q (150 nM) 3 643 11 64 4 FXE218Q (60 nM) 3 418 9 24 2 FXE218Q (30 nM) 3 276 13 14 1

[0053] Sequences:

TABLE-US-00004 SEQIDNO:1:FactorX(homosapiens) MGRPLHLVLLSASLAGLLLLGESLFIRREQANNILARVTR ANSFLEEMKKGHLERECMEETCSYEEAREVFEDSDKTNEF WNKYKDGDQCETSPCQNQGKCKDGLGEYTCTCLEGFEGKN CELFTRKLCSLDNGDCDQFCHEEQNSVVCSCARGYTLADN GKACIPTGPYPCGKQTLERRKRSVAQATSSSGEAPDSITW KPYDAADLDPTENPFDLLDFNQTQPERGDNNLTRIVGGQE CKDGECPWQALLINEENEGFCGGTILSEFYILTAAHCLYQ AKRFKVRVGDRNTEQEEGGEAVHEVEVVIKHNRFTKETYD FDIAVLRLKTPITFRMNVAPACLPERDWAESTLMTQKTGI VSGFGRTHEKGRQSTRLKMLEVPYVDRNSCKLSSSFIITQ NMFCAGYDTKQEDACQGDSGGPHVTRFKDTYFVTGIVSWG EGCARKGKYGIYTKVTAFLKWIDRSMKTRGLPKAKSHAPE VITSSPLK SEQIDNO:2:fibrinopeptideA(homosapiens) ADSGEGDFLAEGGGVR

REFERENCES

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