A non-enzymatic recombinant lytic agent

Abstract

There is provided a recombinant clotting factor X protein with a serine protease catalytic domain having at least 90 % sequence identity to SEQ ID NO:6 while still retaining X136, and wherein X is Q, G, A, V, P, S, N, F, Y, C, T, M, L, W, I, E or D. There is also provided a pharmaceutical composition containing the recombinant clotting factor X protein. The recombinant is useful for dissolving a blood clot in a subject in need thereof, reducing coagulation in a subject in need thereof, and / or treating heart attack, stroke, pulmonary embolism or deep vein thrombosis in a subject in need thereof.

Description

A NON-ENZYMATIC RECOMBINANT LYTIC AGENTCROSS REFERENCE TO A RELATED APPLICATION

[0001] This disclosure claims priority from U. S. Provisional Application No. 63 / 580,012 filed on September 1 , 2023, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of clot dissolution agents for dissolving clots and methods of using same.BACKGROUND OF THE ART

[0003] Clot formation limits or prevents the flow of blood and aberrant persistence and is one of the major causes of heart disease and stroke. To restore the flow of blood, the predominant clot busting medicine is tissue plasminogen activator (tPA) and recombinant derivatives thereof. The main medical and commercial problems with tPA are that: 1) it may cause hemorrhage because it is a functional enzyme exhibiting detrimental systemic effects; 2) about half of patients' clots are resistant to tPA and 3) it has only about a 4% penetrance into the target market because of a finite time of efficacy (3-5 hours after the onset of symptoms).

[0004] It would be highly desirable to be provided with a safer therapeutic agent capable of accelerating the dissolution of a clot and / or preventing the formation of a clot. When used alone, the safer therapeutic agent would preferably have reduced undesirable systemic effect (such as bleeding for example). When used in combination with a known clot-busting medicine, the safer therapeutic agent would preferably increase the thrombolytic potential of the combined known clot-busting medicine, reduce the dose required of the known clot-busting medicine to observe beneficial therapeutic effects and ultimately limit the side effects associated with known clotbusting medicine.

[0005] A plasma-derived clot-dissolving therapeutic protein with a reduced hemorrhage risk is described in US9579367. US9579367 describes a clotting factor Xa that has been chemically modified to contain a C-terminally-tethered amino acid linked via a tetra-ethylene glycol spacer to the active site. Unfortunately, this therapeutic agent has production limitations because it is derived from plasma, requires multiple process steps, and requires complex enzymatic conversion and purification. Accordingly, improvements in the therapeutic agents for blood clot clearance are still desired.SUMMARY

[0006] In one aspect, there is provided a recombinant clotting factor X protein comprising a serine protease catalytic domain having at least 90 % sequence identity to SEQ ID NO:6 while still retaining X136, and wherein X is Q, G, A, V, P, S, N, F, Y, C, T, M, L, W, I, E or D. X is preferably Q, F or W and more preferably is Q. In some embodiments, the serine protease catalytic domain is an inactive catalytic site. The recombinant clotting factor X protein can further comprise a heavy chain having at least 90 % sequence identity to SEQ ID NO:7 while still retaining X188, which comprises the serine protease catalytic domain. Optionally, the recombinant clotting factorX protein further comprises a light chain. With the light chain, the recombinant clotting factor X protein can have at least 90 % sequence identity to SEQ ID NO:8 while still retaining X330.

[0007] In some embodiments, the 10% variation in SEQ ID NO:6 includes S185 and the serine protease catalytic domain has at least 90 % sequence identity to SEQ ID NO:9 while retaining X136 and A185. In further embodiment, the 10% variation in SEQ ID NO:7 includes S237 and wherein the heavy chain has at least 90 % sequence identity to SEQ ID NQ:10 while still retaining X188 and A237 which comprises the serine protease catalytic domain. In yet further embodiments, the 10% variation in SEQ ID NO:8 includes S379 and the recombinant clotting factor X protein has at least 90 % sequence identity to SEQ ID NO:11 while still retaining X330 and A379.

[0008] In a further aspect, there is provided a pharmaceutical composition comprising a recombinant clotting factor X protein as described in the present disclosure. The pharmaceutical composition optionally further contains a thrombolytic agent and / or an anticoagulant (e.g. heparin). In some embodiments, the thrombolytic is a tissue plasminogen activator, a tissue plasminogen activator variant, a urokinase and / or a streptokinase, and preferably the tissue plasminogen activator is tenecteplase.

[0009] In still a further aspect there is provided a method of dissolving a blood clot in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant clotting factor X protein or the pharmaceutical composition of the present disclosure.

[0010] In an additional aspect, there is provided a method of reducing coagulation in a subject in need thereof, the method comprising administering to the subject a therapeutically effectiveamount of the recombinant clotting factor X protein or the pharmaceutical composition of the present disclosure.

[0011] In yet an additional aspect, there is provided a method of treating heart attack, stroke, pulmonary embolism or deep vein thrombosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant clotting factor X protein or the pharmaceutical of the present disclosure.

[0012] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is an image of a non-reduced gel electrophoresis showing expression of clotting factor X (1 .0 pM) fragments that are formed after treatment with purified plasmin (10 nM) for 60 minutes at room temperature. Non-fragmented factorX (FXa), and subsequent cleavage products factor Xp (FXp) and a ~46 kDa species were generated. Purified plasma-derived factor X (pFX), wild type (wt) recombinant (r) factor X, having the same amino acid sequence as the normal plasma derived analogue (rFX-wt), recombinant factor X (rFXc) with substitution K330Q, recombinant FX with substitution S379A (rFXi) and recombinant factor X with both substitutions S379A and K330Q (rFXic) are shown (nomenclature according to the numbering of the factor X amino acids after excision of the signal- and pro-peptides).

[0014] FIG. 2 is a graph showing plasmin generation as a function of time for rFX-wt, rFXc, rFXi, rFXic and a negative control (none).

[0015] FIG. 3 is a graph showing the turbidity of over time which indicates plasma clot formation and fibrinolysis.DETAILED DESCRIPTION

[0016] Clot formation is initiated by thrombin (Ila), which has a fibrin molecular scaffolding. Once the clot has served its purpose to seal leaky vasculature, the fibrinolysis pathway dissolves it. The prevailing “classical” model of fibrinolysis is that fibrin controls clot-busting by accelerating tissue plasminogen activator (tPA). This cofactor function of fibrin has two chemically distinct phases. In the first (slow) phase, binding sites on intact fibrin bring together tPA and plasminogen (Pg) resulting in the first molecules of plasmin (Pn). Plasmin cuts the clot, but this initial plasminproduction is generally inadequate to overcome the normal level of plasma inhibitors of fibrinolysis. Nevertheless, this low amount of plasmin slowly cleaves the fibrin, and primes it for participating in the second (fast) phase of tPA cofactor function by exposing C-terminal lysines (or CTK, where K is the conventional single letter abbreviation for lysine) on the cleaved fibrin. These CTK provide new binding sites for tPA and Pg activation. Thus, fibrin that has exposed CTKs is primed with enhanced tPA cofactor function that ultimately increases plasmin generation beyond the intrinsic anti-fibrinolysis threshold, enabling the clot to dissolve. Based on the general understanding in the art that the vast concentration of fibrin would overwhelm the potential contribution of any other protein in the vicinity of a clot, it is thus believed that fibrin is the only required tPA cofactor.

[0017] In blood coagulation, FX occupies a central position in the coagulation system and is an important driver of thrombin generation. FX is converted to activated FX (FXa) by either the extrinsic (tissue factor (TF)-FVIIa) or intrinsic (FVIIIa-FIXa) pathway. In the common pathway, FXa reversibly associates with its cofactor FVa on an anionic phospholipid-containing membrane surface in the presence of calcium ions to form prothrombinase, the physiologic activator of prothrombin. Due to its direct impact on thrombin generation, the regulation of prothrombinase or its individual components (FXa and FVa), has a major impact on blood clot formation.

[0018] FX is synthesized in the liver as a pre-pro protein of 488 amino acids (SEQ ID NO:1). Prior to secretion, the signal sequence and propeptide are removed as is the Arg-Lys-Arg tripeptide sequence separating the heavy and light chains. The mature protein (SEQ ID NO:2) has an N-terminal light chain of 139 amino acids comprised of the vitamin K-dependent Gia domain (10 Gia residues) and two EGF domains. The heavy chain (306 amino acids) is comprised of the glycosylated activation peptide (52 amino acids) and the serine protease domain also called the catalytic domain. The heavy and light chains are held together by a disulfide bond. The FX serine protease domain is homologous to other chymotrypsin-like enzymes with the catalytic triad residues His236, Asp282, and Ser379 (SEQ ID NOs:3-5). This domain also has calcium and sodium binding sites that are important to the function of the active enzyme.Table 1 . Sequences for pre-pro FX, matured FX, and recombinants thereof

[0019] There is provided a recombinant variant of clotting factor X (rFX) that has clotdissolving activity. The recombinant factor X has a mutation that allows the recombinant to block the active site of FXa and thereby lead to clot dissolution (SEQ ID NOs:6-1 1). The mutation is a mutation at the amino acid position Lys330 of FX to prevent proteolysis of FX (SEQ ID NO:8). The proteolysis of FX would render FX ineffective as a thrombolytic agent in plasma. The mutation is a substitution of Lys330 to a non-positively charged amino acid. The non-positively charged amino acid can be a non-natural or a natural amino acid such as glutamine, glycine, alanine, valine, proline, serine, asparagine, phenylalanine, tyrosine, cysteine, threonine, methionine, leucine, tryptophan, isoleucine, glutamic acid and aspartic acid. In preferred embodiments, the substitution is from Ly330 to glutamine, phenylalanine, or tryptophan, and most particularly glutamine. Glutamine is most preferred because it has the most similar three dimensional conformation to lysine without being positively charged thereby achieving the desired effect while minimizing gross conformational changes and immune recognition.

[0020] In preferred embodiments, the rFX is further modified to have an inactive serine protease catalytic site to improve and accelerate its clot dissolution activity. The protease catalytic site can be rendered inactive by mutating one or more of the three catalytic triad residues as shown in SEQ ID NO:5 in Table 1. This is because all three residues are necessary for the catalytic function and mutating any of the catalytic triad residue would suffice to block enzymatic activity.

[0021] In one example, the Ser379 can be substituted for Ala379 as exemplified in SEQ ID NOs:9-11 , any amino acid other than Ser in this position will prevent the coagulation protease activity and in this context would have the desired effect. The reason Ala is preferred is because it has a three dimensional conformation that is most similar to Ser and this conservative substitution is least likely to influence the gross structure of FX. Accordingly, other mutations are also contemplated herein to deactivate the catalytic site. By analogy, this has been done by chemical modification of the active site His236 by chloromethylketone incorporation in US9579367. The rFX with an inactive protease catalytic site acts as a tPA cofactor and, in the vicinity of a clot, accelerates clot dissolution, mediates tPA cofactor activity, and can bind to plasminogen, tPA or other fibrinolytic constituents.

[0022] In some embodiments, the rFX has a serine protease catalytic domain which has at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO:6 or 9. In some embodiments, the rFX has aheavy chain which has at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO:7 or 10. In some embodiments, the rFX has at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 97 %, at least 98 %, or at least 99 % sequence identity to SEQ ID NO:8 or 11 . When referring to sequence identity percentages, regardless of the recited percentage, the sequences necessarily include the mutations shown in bold and highlight in SEQ ID NO:6-11 of Table 1 .

[0023] One of the advantages of the rFX described herein, when compared to tissue plasminogen accelerator (tPA), is that it does not involve administration of a proteolytically functional enzyme, thus limiting systemic effects, such as those observed with tPA or its variants. Furthermore, currently, tPA must be administered within a short period after the onset of symptoms (3 to 5 hours), possibly because maturation of the clot may prevent it from undergoing proteolysis to expose C-terminal amino acids (or CTAA such as, for example, CTK), thereby it cannot easily be “primed” to become a “fast” cofactor. Many patients who could benefit from clotbusting therapy are excluded from treatment due to this limited timeframe. The present rFX is an effective fibrinolysis cofactor, which can be used to treat a subject with a therapeutic thrombolytic agent after the onset of symptoms. Without wishing to be bound to theory, the present disclosure suggests an “auxiliary cofactor” model of fibrinolysis in which the initial phase of plasmin production is augmented by the modified blood coagulation protein exhibiting increased and constitutive tPA cofactor activity. The CTAA-modified blood coagulation protein is more susceptible than fibrin to “priming” by plasmin and consequently it acquires additional CTK more quickly than fibrin to initially accelerate tPA. Therefore, the rFX is useful in the treatment of heart attack, stroke, pulmonary embolism and deep vein thrombosis.

[0024] The present disclosure provides a pharmaceutical composition comprising the rFX described herein and a pharmaceutically acceptable excipient. In an embodiment, the pharmaceutical composition further comprises a thrombolytic agent (such as, for example, tissue plasminogen activator, a tissue plasminogen activator variant, urokinase and / or streptokinase). In yet a further embodiment, the tissue plasminogen variant activator is tenecteplase. In another embodiment, the pharmaceutical composition further comprises an anticoagulant, such as, for example, heparin.

[0025] There is further provided a method for dissolving a clot in a subject in need thereof. Broadly, the method comprises administering a therapeutic effective amount of the rFX describedherein, or the pharmaceutical composition described herein to the subject a subject, such as, for example a mammalian subject (e.g., a human), so as to dissolve the blood clot.

[0026] There is further provided a method of improving the therapeutic property of a thrombolytic agent. Broadly, the method comprises administering a therapeutic effective amount of the rFX described herein or the pharmaceutical composition described herein with the thrombolytic agent to the subject. In an embodiment, the conventional thrombolytic agent is administered at a dose too low to be therapeutic but at this dose alleviates the potential hemorrhagic risk of the higher therapeutic dose. When combined with the rFX or the pharmaceutical composition comprising said rFX, the low dose of conventional thrombolytic therapeutic would have an adjunctive therapeutic efficacy. In another embodiment, the conventional thrombolytic agent is administered at a timing considered sub-therapeutic when used in the absence of the rFX or the pharmaceutical composition. In an embodiment, the conventional thrombolytic agent is, for example, tissue plasminogen activator or a tissue plasminogen activator variant such as tenecteplase.

[0027] In some embodiments, the treatment can comprise administering to a subject in need thereof the rFX or the pharmaceutical composition in a therapeutically effective amount. A “therapeutically effective amount” as used herein refers to an amount (dose) effective in mediating a therapeutic benefit (for example reducing, dissolve or preventing a blood clot or coagulation). An exemplary dose may be in the range of 0.5 mg / kg to 2 mg / kg. It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents. In some embodiments, the agent and the other therapeutic agent(s) are administered at the same time or within a predetermined time interval (ranging from a minute, an hour, a day, or a week for example). The therapeutic effect includes but is not limited to the prevention, treatment and / or alleviation of symptoms of blood coagulation and / or blood clots.EXAMPLESite-directed mutagenesis

[0028] For initial experiments, mutations were inserted into a previously generated F10 genecontaining plasmid (Camire, R. M., Larson, P. J., Stafford, D. W., & High, K. A. (2000). Enhanced y-carboxylation of recombinant factor X using a chimeric construct containing the prothrombinpropeptide. Biochemistry, 39(46), 14322-14329), pCMV4-ss-pro-ll-FX in which the signal sequence and propeptide of FX were substituted with those of prothrombin to increase expression of functional recombinant protein. Mutagenesis was facilitated using the Quikchange™ kit according to the manufacturer’s protocol. The lysine residue at position 330 was mutated to glutamine to preserve the general size of the side chain while neutralizing the positive charge. Complementary primers containing the desired mutation(s) were designed using the software Oligo and generated by Integrated DNA Technologies (Table 2).Table 2. Primer sequences for mutagenesis

[0029] Following polymerase chain reaction (PCR) amplification of the pCMV4-ss-pro-ll-FX (wtFX) plasmid using the mutant primers, each PCR product was digested with the restriction enzyme Dpn I (10 U) to digest parental DNA, transformed into XL10-Gold ultracompetent cells in the presence of p-mercaptoethanol and plated onto ampicillin (10 pg / mL)-containing Luria-Bertani (LB) agar plates. Six colonies per mutant were then selected and grown in LB media supplemented with ampicillin (10 pg / mL). DNA was extracted using a mini-prep kit (Qiagen), quantified and fully sequenced to confirm both successful mutation and the fidelity of the entire F10 gene. Aliquots of cells in LB-ampicillin media were also stored in 15 % glycerol at -80 °C for future use.

[0030] For additional experiments, plasmids were also purchased with the desired sequence F10 and mutants, and VKOR and PACE / Furin insertions from Twist Bioscience (San Francisco, USA).Stable expression of recombinant factor X

[0031] FW-containing plasmids were co-transfected with the selectable marker plasmid pcDNA3.1 into HEK293 cells using Lipofectamine™ 2000 according to the manufacturer’s protocol. Briefly, both plasmids and the transfection reagent were combined in Opti-MEM medium and incubated for 20 mins at room temperature. This mixture was then used to transfect HEK293 cells (at approximately 85 % confluency) in 6-well plates. After 6-8 hours, Opti-MEM was replaced with Dulbecco's Modified Eagle Medium (DMEM) F / 12 supplemented with 5 % fetal bovine serum (FBS), 1 % L-glutamine, and 1 % penicillin / streptomycin and cells were allowed to grow overnight at 37 °C, 5 % carbon dioxide (CO2). The following day, adherent cells were trypsinized (0.25 % trypsin, 1 mM ethylenediaminetetraacetic acid (EDTA)) and a range of cell dilutions were cultured in 6-well plates containing selection media (DMEM-F / 12 as described above further supplemented with 6 pg / mL vitamin K and 450 pg / mL geneticin). After 14-21 days, colonies were selected for expansion into T150 flasks (Corning CellBIND™) which were allowed to reach approximately 90 % confluency prior to serum deprivation and removal of small aliquots of conditioned media to be assayed for FX production by both western blot and clotting assay.

[0032] To increase production of functional recombinant FX, the vitamin K epoxide reductase (VKOR) gene-containing plasmid VKOR-pIRES was stably transfected into recombinant wtFX and Lys330Gln mutant FX-expressing HEK293 cells (SEQ ID NO:8 where X is Q). Selection media for these doubly transfected cells was supplemented with both geneticin and 1.75 pg / mL puromycin, the selection reagent for the VKOR-pIRES plasmid. At least 2-3 vials of cells from each clone were frozen in selection media containing 5 % DMSO and stored in liquid nitrogen for large-scale growth after clone selection.

[0033] Selected clones were thawed and expanded into triple flasks (Nunclon™) and after reaching 80-90 % confluency, selection media was replaced with expression media (DMEM-F / 12 supplemented with insulin-transferrin-selenium (ITS), 1 % L-glutamine, 1 % penicillin / streptomycin, 1.75 pg / mL puromycin, 450 pg / mL G418, and 6 pg / mL vitamin K). Conditioned media was collected daily for 5-14 days and stored at -80 °C in the presence of the protease inhibitor benzamidine (10 mM). Small aliquots of uninhibited conditioned media were also stored at -80 °C for use in western blots and activity assays.Purification of recombinant factor X

[0034] Conditioned media from large-scale protein expression (10-20 L) was thawed at 37 °C then immediately stored at 4 °C or on ice for the duration of the purification process (except whenbound to columns at room temperature). The conditioned media was centrifuged at 15,000 rpm for 30 mins to remove cell debris and then concentrated in a stirred cell concentrator under nitrogen using a regenerated cellulose ultrafiltration membrane with a 10 kDa molecular weight cut-off limit (Millipore). The concentrated media was then dialyzed overnight against a loading buffer (20 mM tris(hydroxymethyl)aminomethane (Tris) - HCI, 150 mM NaCI, 5 mM EDTA, pH 7.2) and then loaded onto a Q-Sepharose™ fast flow column equilibrated with loading buffer at a flow rate of 2 mL / min. The column was then washed with five column volumes of loading buffer prior to linear gradient elution with NaCI (150-750 mM). Fraction volumes ranged from 10 mL (during sample loading) to 1 mL (during elution).

[0035] Collected fractions were assayed for FX activity by chromogenic assay using a tripeptide-based substrate designed for FXa recognition, S-2765™. Small samples of each fraction were incubated for 20 min. at room temperature with Russell’s viper venom FX activator (RVV-X, 125 nM) and CaCh (2 mM) in a 96-well microplate to generate FXa. S-2765 was diluted in HBS / EDTA (20 mM) to a final concentration of 200 pM (HBS = HEPES buffer saline where HEPES is (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid)). Diluted substrate (150 pL) was added to each reaction well and FXa activity was monitored kinetically at 405 nm using a Spectramax190TMmicroplate reader (Molecular Devices). FX-containing fractions were pooled and dialysed overnight against loading buffer (8 mM Tris-HCI, 60 mM NaCI, pH 7.4). In the case of active-site mutated FX variants, fractions were pooled based on western blot analysis as described below.

[0036] The second purification step involved a Ca2+-sensitive conformation-specific FX antibody (4G3) linked to cyanogen bromide (CNBr)-activated Sepharose 4B according to the resin manufacturer’s protocol (GE Healthcare). The use of this antibody to separate partially y-glutamyl carboxylated FX from the fully modified protein has been previously described. Because the ability of the 4G3 antibody to bind FX is calcium-dependent, the column was equilibrated immediately prior to sample application with loading buffer containing 2.5 mM CaCh. The dialysate was also spiked with CaCh (2.5 mM) and loaded. After binding, the column was washed with loading buffer and then eluted with a linear gradient of EDTA (0-8 mM). Fraction volumes were collected as described above. Fractions were assayed for FXa activity as previously explained except higher concentrations of CaC (up to 25 mM) were required for EDTA-containing fractions. Some initial fractions collected during sample loading on the 4G3-Sepharose column (“flow-through”) were also found to contain FX. These fractions were pooled and, after the column was re-equilibrated with loading buffer and calcium, were loaded onto the column and eluted with EDTA again. Thisprocess was repeated until the flow-through contained insignificant amounts of optical density at A405 nm. All FX-containing fractions were then pooled and dialysed overnight against the third loading buffer (1 mM Na2HPO4 / NaH2PO4, pH 6.8).

[0037] The final pooled and dialysed protein sample from the 4G3-Sepharose column was loaded onto the third and final column, hydroxyapatite pre-equilibrated with loading buffer. FX was eluted from the column using a linear gradient of Na2HPO4 / NaH2PC>4 (1-400 mM) with 0.5 mL fractions. FX-containing fractions (as assessed by chromogenic assay, described above) were pooled and concentrated at 13,000 rpm in Microcon™ centrifugal filter devices with a 10 kDa molecular weight cut off. Buffer exchange to HBS was also carried out in these microtubes. Purified recombinant FX was stored in 50 % glycerol at -20 °C. Protein concentrations were determined by bicinchoninic acid assay (BCA) against bovine serum albumin (BSA) standards and protein concentrations were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10 % acrylamide gels) and Coomassie staining with plasma- derived commercially available human FX as a standard.Immunoblot detection of Factor X

[0038] 12% acrylamide SDS-PAGE was transferred to polyvinylidene difluoride and probed for FX. The monoclonal antibody specific for human FX(a) heavy chain was purchased from Green Mountain Antibodies (Vermont, USA). Peroxidase-conjugated goat anti-mouse IgG used for detection of FX(a) and derivatives by western blot was purchased from Jackson ImmunoResearch Laboratories (Pennsylvania, USA) and used in combination with the chemiluminescent ECL-Plus detection system. Purified plasmin used to treat the FX in this experiment was from Haemtech (Vermont, USA).Cleavage of rFX and variants by plasmin is protected by mutation at Lys330

[0039] FX undergoes the same cleavages as FXa by plasmin. Therefore, Lys330 was substituted to Gin (rFX-K330Q) to prevent the loss of clot-dissolving function. One of the three critical active site amino acids was also modified, Ser379 to Ala (rFX-S379A), to prevent clotforming activity due to the potential of conversion to FXa in vivo or in plasma. Both single point mutants and a double mutant combining both mutations were produced and purified.

[0040] Fig. 1 shows purified plasma-derived, or recombinant wild type (wt) and variant FX (1 pM) treated with plasmin (0.01 pM) in the presence of small unilamellar vesicles composed of75% phosphatidylcholine and 25% phosphatidylserine (50 pM), and calcium (5 mM) at room temperature. Cleavage from intact FX to FXp was observed for all forms of FX. Further cleavage of FXp by plasmin to smaller fragments was blocked by mutation of Lys330 to Gin. Purified protein (0.5 pg) was applied to a non-reduced, 12% SDS-PAGE, Coomassie blue stained gel. rFX and variants enhance tPA-mediated plasmin generation in vitro

[0041] Fig. 2 shows tPA (10 nM)-mediated activation of plasminogen (0.5 pM) based on optical density due to chromogenic substrate cleavage as plasmin is generated in the presence of various forms of recombinant (r) FX (0.1 pM), including: wild type FX (rFX-WT) having the same sequence as normal plasma-derived FX; FX K330Q (rFXc,) mutated at a critical plasmin cleavage site; FX S379A (rFXi) mutated within the active site to prevent clotting activity; the double mutant of both K330Q and S379A (rFXic); and the negative control in the absence of rFX (none). Each rFX version enhanced plasminogen activation to plasmin in this purified protein assay (n=3 ± standard deviation). rFX variants enhance plasma clot lysis in vitro

[0042] Fig. 3 shows clot formation initiated in normal plasma by thrombin (10 nM) supplemented with tPA (35 pM) and the effect of the indicated form of FX (0.1 pM) on clot lysis: rFX-WT; rFXc; plasma-derived (p) FX (pFX); or vehicle. The amount of clot formation and subsequent fibrinolysis were followed by turbidity analysis (n=3). rFX-K330Q accelerates clot dissolution formed in plasma compared to wild type recombinant FX, plasma-derived FX or the buffer vehicle.

Claims

WHAT IS CLAIMED IS:1 . A recombinant clotting factor X protein comprising a serine protease catalytic domain having at least 90 % sequence identity to SEQ ID NO:6 while still retaining X136, and wherein X is Q, G, A, V, P, S, N, F, Y, C, T, M, L, W, I, E or D.

2. The recombinant clotting factor X protein of claim 1 , wherein the serine protease catalytic domain is an inactive catalytic site.

3. The recombinant clotting factor X protein of claim 1 or 2, further comprising a heavy chain having at least 90 % sequence identity to SEQ ID NO:7 while still retaining X188, which comprises the serine protease catalytic domain.

4. The recombinant clotting factor X protein of any one of claims 1 to 3, further comprising a light chain.

5. The recombinant clotting factor X protein of claim 4, wherein the recombinant clotting factor X protein has at least 90 % sequence identity to SEQ ID NO:8 while still retaining X330.

6. The recombinant clotting factor X protein of any one of claims 1 to 5, wherein X is Q, F or W.

7. The recombinant clotting factor X protein of any one of claims 1 to 6, wherein X is Q.

8. The recombinant clotting factor X protein of claim 1 , wherein the 10% variation in SEQ ID NO:6 includes S185 and wherein the serine protease catalytic domain has at least 90 % sequence identity to SEQ ID NO:9 while retaining X136 and A185.

9. The recombinant clotting factor X protein of claim 3, wherein the 10% variation in SEQ ID NO:7 includes S237 and wherein the heavy chain has at least 90 % sequence identity to SEQ ID NQ:10 while still retaining X188 and A237 which comprises the serine protease catalytic domain.

10. The recombinant clotting factor X protein of claim 5, wherein the 10% variation in SEQ ID NO:8 includes S379 and wherein the recombinant clotting factor X protein has at least 90 % sequence identity to SEQ ID NO:11 while still retaining X330 and A379.1 1. A pharmaceutical composition comprising a recombinant clotting factor X protein as defined in any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

12. The pharmaceutical composition of claim 11 , further comprising a thrombolytic agent.

13. The pharmaceutical composition of claim 12, wherein the thrombolytic is a tissue plasminogen activator, a tissue plasminogen activator variant, a urokinase and / or a streptokinase.

14. The pharmaceutical composition of claim 13, wherein the tissue plasminogen activator is tenecteplase.

15. The pharmaceutical composition of any one of claims 11 to 14, further comprising an anticoagulant.

16. The pharmaceutical composition of claim 15, wherein the anticoagulant is heparin.

17. A method of dissolving a blood clot in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant clotting factor X protein as defined in any one of claims 1 to 10 or the pharmaceutical composition as defined in any one of claims 1 1 to 16.

18. A method of reducing coagulation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant clotting factor X protein as defined in any one of claims 1 to 10 or the pharmaceutical composition as defined in any one of claims 1 1 to 16.

19. A method of treating heart attack, stroke, pulmonary embolism or deep vein thrombosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the recombinant clotting factor X protein as defined in any one of claims 1 to 10 or the pharmaceutical composition as defined in any one of claims 11 to 16.

20. Use of the recombinant clotting factor X protein as defined in any one of claims 1 to 10 or the pharmaceutical composition as defined in any one of claims 1 1 to 16, for treating heart attack, stroke, pulmonary embolism or deep vein thrombosis, for reducing coagulation, or for dissolving a blood clot.

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