Non-enzymatic recombinant solvents

Abstract

The recombinant coagulation factor X protein is provided, having a serine protease catalytic domain that retains X136 while having at least 90% sequence identity with SEQ ID NO: 6, wherein X is Q, G, A, V, P, S, N, F, Y, C, T, M, L, W, I, E, or D. The pharmaceutical composition containing the recombinant coagulation factor X protein is also provided. The recombination is useful for dissolving blood clots in subjects where it is needed, reducing coagulation in subjects where it is needed, and / or treating heart attacks, strokes, pulmonary embolisms, or deep vein thrombosis in subjects where it is needed.

Description

Technical Field

[0001] Cross-Reference to Related Applications The present disclosure claims priority to U.S. Provisional Patent Application No. 63 / 580,012, filed September 1, 2023, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the field of clot lysing agents for dissolving clots and methods of use thereof.

Background Art

[0003] Clot formation restricts or obstructs blood flow, and abnormal persistence of clots is one of the leading causes of heart disease and stroke. To restore blood flow, the main thrombolytic agents are tissue plasminogen activator (tPA) and recombinant derivatives thereof. The main medical and commercial problems with tPA are: 1) tPA is a functional enzyme that exhibits adverse systemic effects, and therefore may cause bleeding; 2) clots in approximately half of patients are resistant to tPA, and 3) due to the limited window of efficacy (3 to 5 hours after symptom onset), penetration into the target market is only about 4%.

[0004] It would be highly desirable to provide safer therapeutic agents that are capable of accelerating clot dissolution and / or preventing clot formation. When used alone, such safer therapeutic agents would preferably have reduced undesirable systemic effects (e.g., bleeding, etc.). When used in combination with known thrombolytic agents, such safer therapeutic agents would preferably increase the thrombolytic potential of the combined known thrombolytic agents, reduce the required dose of the known thrombolytic agent to observe a beneficial therapeutic effect, and ultimately limit the side effects associated with the known thrombolytic agent.

[0005] U.S. Patent No. 9,579,367 describes a plasma-derived thrombolytic therapeutic protein with reduced bleeding risk. Patent No. 9,579,367 describes coagulation factor Xa containing a C-terminally tethered amino acid that is chemically modified and linked to the active site via a tetraethylene glycol spacer. Unfortunately, this therapeutic agent has production limitations due to its plasma origin, the need for multiple process steps, and the complex enzymatic conversion and purification required. Therefore, improvements to therapeutic agents for blood clot clearance remain desirable. [Overview of the project]

[0006] In one embodiment, a recombinant coagulation factor X protein is provided, comprising a serine protease catalytic domain having at least 90% sequence identity with SEQ ID NO: 6 while retaining X136, 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 Q. In some embodiments, the serine protease catalytic domain is an inactive catalytic site. The recombinant coagulation factor X protein may further comprise a heavy chain having at least 90% sequence identity with SEQ ID NO: 7 while retaining X188, which comprises the serine protease catalytic domain. Optionally, the recombinant coagulation factor X protein may further comprise a light chain. Together with the light chain, the recombinant coagulation factor X protein may have at least 90% sequence identity with SEQ ID NO: 8 while retaining X330.

[0007] In some embodiments, 10% of the mutations in SEQ ID NO: 6 include S185, and the serine protease catalytic domain has at least 90% sequence identity with SEQ ID NO: 9 while retaining X136 and A185. In further embodiments, SEQ ID NO: 7 In this embodiment, 10% of the mutations in SEQ ID NO: 8 include S237, and the heavy chain retains X188 and A237 while having at least 90% sequence identity with SEQ ID NO: 10, and contains a serine protease catalytic domain. In a further embodiment, 10% of the mutations in SEQ ID NO: 8 include S379, and the recombinant coagulation factor X protein retains X330 and A379 while having at least 90% sequence identity with SEQ ID NO: 11.

[0008] In a further embodiment, the present disclosure provides a pharmaceutical composition comprising the recombinant coagulation factor X protein described herein. The pharmaceutical composition optionally further comprises a thrombolytic agent and / or an anticoagulant (e.g., heparin). In some embodiments, the thrombolytic agent is tissue plasminogen activator, tissue plasminogen activator variant, urokinase and / or streptokinase, and preferably the tissue plasminogen activator is tenecteplase.

[0009] In a further embodiment, a method for dissolving a blood clot in a subject as needed is provided, comprising administering to the subject a therapeutically effective amount of the recombinant coagulation factor X protein or pharmaceutical composition of this disclosure.

[0010] In an additional embodiment, the present invention provides a method for reducing coagulation in a subject in need, comprising administering to the subject a therapeutically effective amount of the recombinant coagulation factor X protein or pharmaceutical composition of the present disclosure.

[0011] In a further additional embodiment, the present invention provides a method for treating a heart attack, stroke, pulmonary embolism, or deep vein thrombosis in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of the recombinant coagulation factor X protein or pharmaceutical of the present disclosure.

[0012] Many further features relating to this improvement, and combinations thereof, will be apparent to those skilled in the art after reading the present disclosure. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is an image of non-reducing gel electrophoresis showing the expression of coagulation factor X (1.0 μM) fragments formed after treatment with purified plasmin (10 nM) at room temperature for 60 minutes. Unfragmented factor X (FXα), followed by degradation products factor Xβ (FXβ) and approximately 46 kDa species were produced. The image shows purified plasma-derived factor X (pFX), wild-type (wt) recombinant (r) factor X (rFX-wt) with the same amino acid sequence as the normal plasma-derived analog, recombinant factor X (rFXc) with substitution K330Q, recombinant FX (rFXi) with substitution S379A, and recombinant factor X (rFXic) with substitution S379A and K330Q (named by the numbering of factor X amino acids after excision of signal- and propeptides). [Figure 2] Figure 2 is a graph showing plasmin production as a function of time for rFX-wt, rFXc, rFXi, rFXic, and negative control (none). [Figure 3] Figure 3 is a graph showing turbidity over time, illustrating the formation of coagulated plasma and fibrin lysis. [Modes for carrying out the invention]

[0014] The formation of blood clots is initiated by thrombin (Ila), which has a fibrin molecular scaffold. Once the clot has fulfilled its purpose of sealing the leaky vascular system, the fibrinolytic pathway dissolves it. The current "traditional" model of fibrinolysis is that fibrin controls thrombolysis by accelerating tissue plasminogen activator (tPA). This cofactor function of fibrin has two chemically distinct phases. In the first (slow) phase, the binding site in intact fibrin brings together tPA and plasminogen (Pg) This results in the production of the first molecule of plasmin (Pn). Plasmin cleaves the blood clot, but this initial plasmin production is generally insufficient to overcome the normal level of plasma inhibitors of fibrinolysis. Nevertheless, this low amount of plasmin slowly cleaves and primers fibrin, participating in the second (rapid) phase of tPA cofactor function by exposing C-terminal lysine (or CTK, where K is the conventional single-letter abbreviation for lysine) in the cleaved fibrin. These CTKs confer new binding sites for the activation of tPA and Pg. Thus, CTK-exposed fibrin is primed by enhanced tPA cofactor function, which ultimately increases plasmin production beyond the endogenous anti-fibrinolysis threshold, allowing the blood clot to dissolve. Therefore, based on the common understanding in the field that enormous concentrations of fibrin would render any potential contribution of any other protein in the vicinity of the blood clot impossible, fibrin is considered the only required tPA cofactor.

[0015] In blood coagulation, FX occupies a central position in the coagulation system and is a crucial promoter of thrombin formation. FX is converted to activated FX (FXa) via either an exogenous (tissue factor (TF)-FVIIa) or endogenous (FVIIIa-FIXa) pathway. In the common pathway, FXa reversibly associates with its cofactor FVa on the surface of an anionic phospholipid-containing membrane in the presence of calcium ions to form prothrombinase, the physiological activator of prothrombin. Due to its direct influence on thrombin formation, the regulation of prothrombinase or its individual components (FXa and FVa) significantly affects blood clot formation.

[0016] FX is synthesized in the liver as a pre-proprotein of 488 amino acids (SEQ ID NO: 1). Prior to secretion, the signal sequence and propeptide are removed, leaving the Arg-Lys-Arg tripeptide sequence separating the heavy chain and light chain. The mature protein (SEQ ID NO: 2) has a 139-amino acid N-terminal light chain composed of a vitamin K-dependent Gla domain (10 Gla residues) and two EGF domains. The heavy chain (306 amino acids) consists of a glycosylated activation peptide (52 amino acids) and a serine protease domain, also referred to as the catalytic domain. The heavy chain and light chain are held together by disulfide bonds. The FX serine protease domain is homologous to other chymotrypsin-like enzymes that have the three catalytic residues His236, Asp282, and Ser379 (SEQ ID NOs: 3 to 5). This domain also has calcium and sodium binding sites that are important for the function of the active enzyme.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0017] Provided is a recombinant variant of coagulation factor X (rFX) having clot lysing activity. The recombinant factor X has a mutation that leads to clot lysis by recombinantly blocking the active site of FXa (SEQ ID NOs: 6 to 11). The mutation is a mutation at amino acid position Lys330 of FX, which prevents proteolysis of FX (SEQ ID NO: 8). Proteolysis of FX can render FX ineffective as a thrombolytic agent in plasma. The mutation is a substitution of Lys330 with an amino acid that is not positively charged. The non-positively charged amino acid may be an unnatural or natural amino acid, for example, glutamine, glycine, alanine, valine, proline, serine, asparagine, phenylalanine, tyrosine, cysteine, threonine, methionine, leucine, tryptophan, isoleucine, glutamic acid and aspartic acid. In a preferred embodiment, the substitution is from Lys330 to glutamine, phenylalanine or tryptophan, most specifically glutamine. Glutamine is most preferred because it has a three-dimensional structure most similar to lysine, and achieves the desired effect while minimizing overall structural changes and immune recognition due to being not positively charged.

[0018] In a preferred embodiment, rFX has an inactive serine protease catalytic site and is further modified to improve and accelerate its clot lysing activity. The protease catalytic site can be inactivated by mutating one or more of the three catalytic residues set forth in SEQ ID NO: 5 in Table 1. This is because all three residues are required for catalytic function, and mutation of any of the three catalytic residues may be sufficient to block enzymatic activity.

[0019] In one example, Ser379 may be substituted with Ala379, as exemplified in SEQ ID NOs: 9-11, and any amino acid other than Ser at this position may inhibit coagulation protease activity, thereby producing the desired effect. Ala is preferred because it has the three-dimensional structure most similar to Ser, and this conservative substitution has the least impact on the overall structure of FX. Therefore, other mutations are also intended to inactivate the catalytic site. By analogy, this relates to the chloromethyl chemoacrylate in U.S. Patent No. 9,579,367. This is achieved through chemical modification of the active site His236 by tonification. rFX, which has an inactive protease active site, acts as a tPA cofactor, accelerating the dissolution of blood clots in the vicinity of the clot, mediating tPA cofactor activity, and potentially binding to plasminogen, tPA, or other fibrinolytic components.

[0020] In some embodiments, rFX has a serine protease catalytic domain having 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 with SEQ ID NO: 6 or 9. In some embodiments, rFX has a heavy chain having 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 with SEQ ID NO: 7 or 10. In some embodiments, 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 with SEQ ID NO: 8 or 11. Referring to the sequence identity percentages, regardless of the listed percentages, the sequences necessarily include the mutations shown in bold and highlighted in SEQ ID NOs: 6-11 in Table 1.

[0021] One of the advantages of the rFX described herein is that, compared to tissue plasminogen enhancers (tPAs), it does not involve the administration of proteolytic functional enzymes, and therefore does not limit systemic effects, such as those observed with tPA or its variants. Furthermore, currently, tPA must be administered within a short period (3-5 hours) after symptom onset, presumably because the maturation of the blood clot prevents it from undergoing proteolysis, exposing the C-terminal amino acids (or CTAAs, e.g., CTK), which are not easily "primed" and thus become "rapid" cofactors. Many patients who could have benefited from thrombolytic therapy are excluded from treatment due to this limited time frame. The rFX is an effective fibrinolytic cofactor and can be used to treat subjects with therapeutic thrombolytic agents after symptom onset. While not bound by theory, this disclosure proposes a "supporting cofactor" model of fibrinolysis in which initial plasmin production is increased by modified blood coagulation proteins exhibiting increased intrinsic tPA cofactor activity. CTAA-modified blood coagulation proteins are more susceptible to plasmin-mediated "priming" than fibrin, resulting in a more rapid acquisition of additional CTKs than fibrin, thus accelerating tPA first. For this reason, rFX is useful in the treatment of heart attack, stroke, pulmonary embolism, and deep vein thrombosis.

[0022] This disclosure provides pharmaceutical compositions comprising the rFX and pharmaceutically acceptable excipients described herein. In one embodiment, the pharmaceutical composition further comprises a thrombolytic agent (e.g., tissue plasminogen activator, tissue plasminogen activator variant, urokinase and / or streptokinase). In a further embodiment, the tissue plasminogen variant activator is tenecteplase. In another embodiment, the pharmaceutical composition further comprises an anticoagulant, such as heparin.

[0023] Further methods are provided for dissolving blood clots in subjects where required. Broadly speaking, such methods involve administering a therapeutically effective amount of the rFX or pharmaceutical composition described herein to a subject, such as a mammalian subject (e.g., a human), to dissolve the blood clot.

[0024] Further methods are provided to improve the therapeutic properties of thrombolytic agents. Broadly speaking, these methods involve administering a therapeutically effective amount of the rFX or pharmaceutical composition described herein to a subject together with a thrombolytic agent. In one embodiment, a conventional thrombolytic agent Although administered at very low doses, it is not therapeutic, but at this dose, it reduces the potential bleeding risk of higher therapeutic doses. When combined with rFX or a pharmaceutical composition containing rFX, low doses of conventional thrombolytic agents may have an adjunctive therapeutic effect. In another embodiment, conventional thrombolytic agents are administered at a timing that is considered subtherapeutic when used in the absence of rFX or a pharmaceutical composition. In one embodiment, the conventional thrombolytic agent is, for example, tissue plasminogen activator or tissue plasminogen activator variants, such as tenecteplase.

[0025] In some embodiments, the treatment may include administering rFX or the pharmaceutical composition to the subject in question in a therapeutically effective dose. “Therapeutic dose,” as used herein, refers to an amount (dose) that is effective in mediating a therapeutic benefit (e.g., reducing, dissolving, or preventing blood clots or coagulation). Exemplary doses may range from 0.5 mg / kg to 2 mg / kg. “Pharmacologically effective dose” may be interpreted as an amount that imparts the desired therapeutic effect and may be administered as a single dose or in any dosage or route, alone or in combination with other therapeutic agents, as should also be understood herein. In some embodiments, the agent and other therapeutic agents(s) may be administered simultaneously or within a predetermined time interval (e.g., minutes, hours, days, or weeks). The therapeutic effect includes, but is not limited to, the prevention, treatment, and / or reduction of symptoms of blood coagulation and / or blood clots. [Examples]

[0026] Site-specific mutation generation For initial experiments, the mutation was inserted into a previously generated F10 gene-containing plasmid (Camire, RM, Larson, PJ, Stafford, DW, & High, KA (2000). Enhanced γ-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. Biochemistry, 39(46), 14322-14329). pCMV4-ss-pro-II-FX was modified by substituting the FX signal sequence and propeptide with those of prothrombin, increasing the expression of the functional recombinant protein. Mutation generation was facilitated using the Quikchange® kit according to the manufacturer's protocol. The lysine residue at position 330 was mutated to glutamine to neutralize the negative charge while maintaining the overall size of the side chain. Complementary primers containing the desired mutation(s) were designed using Oligo software developed by Integrated DNA Technologies (Table 2). [Table 2]

[0027] Polymerase chain reaction (PCR) amplification of the pCMV4-ss-pro-II-FX (wtFX) plasmid using mutant primers, followed by restriction enzyme Dpn The parental DNA was digested with I(10U) and converted to XL10-Gold ultracompetent cells in the presence of β-mercaptoethanol, and plated onto Luria-Bertani (LB) agar plates containing ampicillin (10 μg / mL). Six colonies were then selected per mutant and grown in LB medium supplemented with ampicillin (10 μg / mL). DNA was extracted using a miniprep kit (Qiagen), quantified, and fully sequenced to confirm both mutation success and fidelity of the entire F10 gene. Aliquots of cells in LB-ampicillin medium were also stored in 15% glycerol at -80°C for later use.

[0028] For additional experiments, we also purchased the desired sequence F10 and variants, as well as plasmids containing VKOR and PACE / Furin insertions, from Twist Bioscience (San Francisco, USA).

[0029] Stable expression of recombinant factor X The F10-containing plasmid was co-transfected into HEK293 cells with the selectable marker plasmid pcDNA3.1 using Lipofectamine® 2000 according to the manufacturer's protocol. Briefly, both the plasmid and transfection reagent were combined in Opti-MEM medium and incubated at room temperature for 20 minutes. HEK293 cells were then transfected in 6-well plates (at approximately 85% density) using this mixture. 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 the cells were grown overnight at 37°C in 5% carbon dioxide (CO2). The following day, adherent cells were trypsin-treated (0.25% trypsin, 1 mM ethylenediaminetetraacetic acid (EDTA)), and various cell dilutions were cultured in 6-well plates containing the above-mentioned DMEM-F / 12 selective medium (DMEM-F / 12 further supplemented with 6 μg / mL vitamin K and 450 μg / mL geneticin). After 14–21 days, colonies were selected and spread in T150 flasks (Corning CellBIND®) to approximately 90% density before serum depletion and removal of small aliquots of conditioned medium, and assayed for FX production by both Western blotting and coagulation assays.

[0030] To increase the 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, X is Q). Selective medium for these double-transfected cells was supplemented with geneticin and 1.75 μg / mL of puromycin, and a selection reagent for the VKOR-pIRES plasmid. At least 2-3 vials of cells from each clone were frozen in selective medium containing 5% DMSO and stored in liquid nitrogen for large-scale culture after clonal selection.

[0031] Selected clones were thawed and spread in triple flasks (Nunclon®) until a density of 80–90% was reached. The selective medium was then replaced with expression medium (DMEM-F / 12 supplemented with insulin-transferrin-selenium (ITS), 1% L-glutamine, 1% penicillin / streptomycin, 1.75 μg / mL puromycin, 450 μg / mL G418, and 6 μg / mL vitamin K). Conditioned medium 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 medium were also stored at -80°C for use in Western blotting and activity assays.

[0032] Purification of recombinant factor X Conditioned medium from large protein expression (10-20 L) was thawed at 37°C and immediately stored at 4°C or on ice for continued purification (except when coupled to the column at room temperature). The conditioned medium was centrifuged at 15,000 rpm for 30 minutes to remove cell debris and then concentrated in a stirred cell concentrator under nitrogen using a regenerated cellulose ultrafiltration membrane (Millipore) with a molecular weight cutoff limit of 10 kDa. The concentrated medium was then dialyzed overnight against loading buffer (20 mM tris(hydroxymethyl)aminoethane (Tris)-HCl, 150 mM NaCl, 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 before linear gradient elution with NaCl (150-750 mM). The fraction volume ranged from 10 mL (during sample loading) to 1 mL (during elution).

[0033] The collected fractions were assayed for FX activity by a chromogenic assay using S-2765®, a tripeptide substrate designed for FX recognition. Small samples from each fraction were incubated in a 96-well microplate with Russell's viper FX activator (RVV-X, 125 nM) and CaCl2 (2 mM) at room temperature for 20 minutes to generate FXa. S-2765 was diluted to a final concentration of 200 μM in HBS / EDTA (20 mM) (HBS = HEPES-buffered saline, HEPES is (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)). The diluted substrate (150 μL) was added to each reaction well, and FXa activity was kinetically monitored at 405 nm using a Spectramax 190® microplate reader (Molecular Devices). The FX-containing fraction was pooled overnight in loading buffer (8 mM Tris-HCl, 60 mM NaCl, pH 7.4) and then dialyzed. In the case of active-site mutation FX mutants, the fraction was pooled based on Western blot analysis described below.

[0034] The second purification step involves Ca linked to cyanide bromide (CNBr) activated Sepharose 4B, according to the resin manufacturer's protocol (GE Healthcare). 2+A conformation-specific FX antibody (4G3) was used. Partial separation of γ-glutamylcarboxylated FX from fully modified proteins using this antibody has been previously described. Since the ability of the 4G3 antibody to bind to FX is calcium-dependent, the column was equilibrated with a loading buffer containing 2.5 mM CaCl2 immediately before sample application. The dialysate was also mixed with CaCl2 (2.5 mM) and loaded. After binding, the column was washed with the loading buffer and then eluted with EDTA (0–8 mM) on a linear gradient. Fraction volumes were collected as described above. The fractions were assayed for FXa activity as previously described, except that higher concentrations of CaCl2 (up to 25 mM) were required for the EDTA-containing fraction. Several initial fractions collected during sample loading on the 4G3-Sepharose column ("flow-through") were also found to contain FX. These fractions were pooled, the column was re-equilibrated with loading buffer and calcium, loaded onto the column, and eluted again with EDTA. This process was repeated until the flow-through contained a small amount of optical density at A405 nm. All FX-containing fractions were then pooled overnight and dialyzed in loading buffer 3 (1 mM Na2HPO4 / NaH2PO4, pH 6.8).

[0035] The final pooled and dialyzed protein samples from the 4G3-Sepharose column were loaded onto the third and final columns, and onto hydroxyapatite pre-equilibriumized with loading buffer. FX was eluted from the column using a linear gradient of Na2HPO4 / NaH2PO4 (1-400 mM) in 0.5 mL fractions. The FX-containing fraction (asserted by the chromogenic assay described above) was subjected to a 10 kDa molecular weight cutoff. The recombinant FX was pooled and concentrated at 13,000 rpm in a Microcon® centrifuge filter device with a buoyancy control function. Buffer exchange with HBS was also performed in these microtubes. The purified recombinant FX was stored in 50% glycerol at -20°C. Protein concentration was determined by bicinchoninic acid assay (BCA) against a bovine serum albumin (BSA) standard, and confirmed by dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide gel) and Coomassie staining using commercially available human FX derived from plasma as a standard.

[0036] Immunoblot detection of factor X 12% acrylamide SDS-PAGE was transferred to polyvinylidene difluoride and searched for FX. A monoclonal antibody specific to human FX(a) heavy chain was purchased from Green Mountain Antibodies (Vermont, USA). Peroxidase-conjugated goat anti-mouse IgG, used for the detection of FX(a) and its derivatives by Western blotting, was purchased from Jackson ImmunoResearch Laboratories (Pennsylvania, USA) and used in combination with a chemiluminescent ECL-Plus detection system. Purified plasmin used to treat FX in this experiment was purchased from Haemtech (Vermont, USA).

[0037] Plasmin-mediated rFX and mutant cleavage are protected by mutations in Lys330. FX undergoes the same cleavage by plasmin as FXa. Therefore, Lys330 was substituted with Gln (rFX-K330Q) to prevent loss of clot-dissolving function. One of the three key active site amino acids was also modified, with Ser379 being replaced with Ala (rFX-S379A), preventing clot-forming activity that could result from the conversion of FXa in vivo or in plasma. Both single-point mutants and double mutants combining both mutations were produced and purified.

[0038] Figure 1 shows purified plasma-derived or recombinant wild-type (wt) and mutant FX (1 μM) treated with plasmin (0.01 μM) and calcium (5 mM) in the presence of small monolayer vesicles consisting of 75% phosphatidylcholine and 25% phosphatidylserine (50 μM) at room temperature. Cleavage from intact FX to FXβ was observed in all FX morphologies. Further cleavage of FXβ into smaller fragments by plasmin was blocked by the Lys330 to Gln mutation. Purified protein (0.5 μg) was applied to an unreduced, 12% SDS-PAGE, Coomassie blue-stained gel.

[0039] rFX and mutants enhance tPA-mediated plasmin production in vitro. Figure 2 shows tPA (10 nM)-mediated activation of plasminogen (0.5 μM) based on optical density, due to chromogenic substrate cleavage, when generating plasmin in the presence of various forms of recombinant (r)FX (0.1 μM), including wild-type FX (rFX-WT) with the same sequence as normal plasma-derived FX; FX K330Q (rFXc) with mutations at a critical plasmin cleavage site; FX S379A (rFXi) with mutations in the active site that prevents coagulation activity; double mutants of both K330Q and S379A (rFXic); and a negative control in the absence of rFX. Each rFX version enhanced plasminogen activation to plasmin in this purified protein assay (n=3 ± standard deviation).

[0040] The rFX mutant enhances the lysis of plasma clots in vitro. Figure 3 shows the effect of thrombin (10 nM) supplemented with tPA (35 pM) on clot formation initiated in normal plasma and on the dissolution of clots of the indicated form of FX (0.1 μM): rFX-WT; rFXc; plasma-derived (p)FX (pFX); or bi The amount of clot formation and subsequent fibrinolysis was tracked by turbidity analysis (n=3). rFX-K330Q accelerates the dissolution of clots formed in plasma compared to wild-type recombinant FX, plasma-derived FX, or a buffer vehicle.

Claims

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

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

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

4. Recombinant coagulation factor X protein according to any one of claims 1 to 3, further comprising a light chain.

5. The recombinant coagulation factor X protein according to claim 4, wherein the recombinant coagulation factor X protein retains X330 while having at least 90% sequence identity with sequence number 8.

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

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

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

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

10. The recombinant coagulation factor X protein according to claim 5, wherein 10% of the mutations in SEQ ID NO: 8 include S379, and the recombinant coagulation factor X protein retains X330 and A379 while having at least 90% sequence identity with SEQ ID NO:

11.

11. A pharmaceutical composition comprising a recombinant coagulation factor X protein as defined in any one of claims 1 to 10, and a pharmaceutically acceptable excipient.

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

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

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

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

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

17. A method for dissolving a blood clot in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of recombinant coagulation factor X protein as defined in any one of claims 1 to 10 or a pharmaceutical composition as defined in any one of claims 11 to 16.

18. A method for reducing coagulation in a target subject, comprising administering to the target a therapeutically effective amount of recombinant coagulation factor X protein as defined in any one of claims 1 to 10 or a pharmaceutical composition as defined in any one of claims 11 to 16.

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

20. Use of recombinant coagulation factor X protein as defined in any one of claims 1 to 10 or a pharmaceutical composition as defined in any one of claims 11 to 16 for the purpose of reducing coagulation or dissolving blood clots, for the purpose of treating heart attack, stroke, pulmonary embolism or deep vein thrombosis.

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