FUSION PROTEIN OF MUTATED SINGLE-CHAIN HUMAN COAGULATION FACTOR VIII, PREPARATION METHOD THEREFOR, AND USE THEREOF

Abstract

Disclosed is a fusion protein of a mutated recombinant single-chain human coagulation factor VIII (FVIII), a preparation method therefor, and a use thereof. The fusion protein sequentially comprises, from an N-terminus to a C-terminus, a mutated single-chain human FVIII having a partially deleted B-domain, a flexible peptide linker, at least one rigid unit of a carboxyl-terminal peptide of a human chorionic gonadotropin beta subunit, and a half-life prolonging moiety (preferably an IgG Fc variant). The fusion protein has a similar biological activity to a recombinant FVIII, a prolonged active half life in vivo, and better stability in vitro and in vivo, and thus improves the pharmacokinetics and efficacy of the fusion protein.

Claims

1. A fusion protein of human coagulation factor VIII, comprising sequentially from the N-terminus to the C-terminus a mutant single-chain human coagulation factor VIII with a partially deleted B domain, a flexible peptide linker, at least one carboxyl-terminal peptide rigid unit of human chorionic gonadotropin β-subunit, and a half-life extending moiety; wherein the single-chain human coagulation factor VIII has an amino acid sequence as shown in SEQ ID NO: 2, and the half-life extending moiety is selected from an immunoglobulin Fc fragment, albumin, transferrin, or PEG.

2. The fusion protein according to claim 1, wherein the fusion protein is glycosylated by being expressed in Chinese hamster ovary cells.

3. The fusion protein according to claim 1, wherein the mutant single-chain human coagulation factor VIII with a partially deleted B domain comprises an amino acid sequence as shown in SEQ ID NO: 2, or the single-chain human coagulation factor VIII has an amino acid sequence that shares at least 90% identity to the amino acid sequence as shown in SEQ ID NO: 2.

4. The fusion protein according to claim 1, wherein the flexible peptide linker contains two or more amino acids selected from G, S, A, and T residues; wherein, the flexible peptide linker has an amino acid sequence general formula formed by combining cyclic unit (GS)a(GGS)b(GGGS)c(GGGGS)d, wherein a, b, c, and d are integers greater than or equal to 0 and a+b+c+d≥1.

5. The fusion protein according to claim 1, wherein the carboxyl-terminal peptide rigid unit of human chorionic gonadotropin β-subunit comprises an amino acid sequence as shown in SEQ ID NO: 3 or a truncated sequence thereof, wherein the truncated sequence comprises at least two glycosylation sites.

6. The fusion protein according to claim 1, wherein the carboxyl-terminal peptide rigid unit of human chorionic gonadotropin β-subunit shares at least 70%, 80%, 90%, or 95% identity to an amino acid sequence as shown in SEQ ID NO: 3, or to a following amino acid sequence: TABLE-US-00005 (SEQ ID NO: 3) (i) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ; (SEQ ID NO: 12) (ii) SSSSKAPPPSLPSPSRLPGPSDTPILPQ; (SEQ ID NO: 13) (iii) SSSSKAPPPS; or (SEQ ID NO: 14) (iv) SRLPGPSDTPILPQ.

7. The fusion protein according to claim 1, wherein the fusion protein comprises 1, 2, 3, 4, or 5 carboxyl-terminal peptide rigid units of human chorionic gonadotropin β-subunit.

8. The fusion protein according to claim 1, wherein the half-life extending moiety is a human IgG Fc variant having a reduced ADCC effect and/or CDC effect and/or enhanced binding affinity with an FcRn receptor.

9. The fusion protein according to claim 1, wherein the fusion protein has an amino acid sequence as shown in SEQ ID NO: 9.

10. The fusion protein according to claim 1, wherein the fusion protein has activity of greater than 6000 IU/mg.

11. DNA for encoding the fusion protein according to claim 1.

12. (canceled)

13. (canceled)

14. A pharmaceutical composition, comprising a pharmaceutically acceptable carrier, excipient, or diluent and an effective dose of the fusion protein according to claim 1.

15. A method for preparing the fusion protein according to claim 1, comprising: (a) introducing the DNA sequence for encoding the fusion protein into a CHO cell to produce a CHO-derived cell line; (b) screening in step (a) a high-yielding cell strain that expresses more than 1 IU/10.sup.6 (million) cells every 24 hours in a growth medium thereof; (c) culturing the cell strain screened in step (b) to express the fusion protein; (d) harvesting a fermentation broth obtained in step (c) and separating and purifying the fusion protein.

16. The method according to claim 15, wherein a purification process of the fusion protein in step (d) comprises affinity chromatography, hydrophobic chromatography, anion exchange chromatography, and molecular sieve chromatography.

17. A method for prevention or treatment of a hemorrhagic disease in a patient with a congenital or acquired FVIII deficiency, or spontaneous or surgical bleeding in a patient with hemophilia A, comprising administrating the fusion protein of claim 1 to a patient.

18. The fusion protein according to claim 1, wherein the flexible peptide linker is any one selected from the following group consisting of: TABLE-US-00006 (SEQ ID NO: 15) (i) GSGGGSGGGGSGGGGS; (SEQ ID NO: 16) (ii) GSGGGGSGGGGSGGGGSGGGGSGGGGS; (SEQ ID NO: 17) (iii) GGGGSGGGGSGGGGSGGGGS; (SEQ ID NO: 18) (iv) GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS; and (SEQ ID NO: 19) (v) GGGSGGGSGGGSGGGSGGGS.

19. The fusion protein according to claim 1, wherein the carboxyl-terminal peptide rigid unit of human chorionic gonadotropin β-subunit comprises an amino acid sequence selected from the following group consisting of: TABLE-US-00007 (SEQ ID NO: 13) (i) PRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ; (SEQ ID NO: 12) (ii) SSSSKAPPPSLPSPSRLPGPSDTPILPQ; (SEQ ID NO: 13) (iii) SSSSKAPPPS; and (SEQ ID NO: 14) (iv) SRLPGPSDTPILPQ.

20. The fusion protein according to claim 8, wherein the Fc variant is any one selected from the following group consisting of: (i) human IgG1 hinge region, CH2 region, and CH3 region containing Leu234Val, Leu235Ala, and Pro331 Ser mutations; (ii) human IgG2 hinge region, CH2 region, and CH3 region containing Pro331 Ser mutation; (iii) human IgG2 hinge region, CH2 region, and CH3 region containing Thr250Gln and Met428Leu mutations; (iv) human IgG2 hinge region, CH2 region, and CH3 region containing Pro331Ser, Thr250Gln, and Met428Leu mutations; and (v) human IgG4 hinge region, CH2 region, and CH3 region containing Ser228Pro and Leu235Ala mutations.

21. The DNA according to claim 11, which has a sequence as shown in SEQ ID NO: 10.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0080] FIG. 1 is a diagram of SEC-HPLC spectrogram of a purified fusion protein SS-F8.

[0081] FIG. 2 is a diagram of SDS-PAGE electrophoresis of a purified fusion protein SS-F8.

[0082] FIG. 3 shows an effect of a fusion protein SS-F8 on bleeding time of saphenous arteries in SD rats.

[0083] FIG. 4 shows an effect of a fusion protein SS-F8 on APTT of plasma in SD rats.

[0084] FIG. 5 is a statistics diagram of bleeding volume in mice, where *p<0.05, ****p<0.0001.

[0085] FIG. 6 is a statistics diagram of bleeding time in mice, where ns p>0.05, ****p<0.0001.

DETAILED DESCRIPTION

Example 1. Construction of Expression Plasmids for Encoding Mutant Single-Chain FVIII Fusion Proteins

[0086] Gene sequences for encoding an FVIII leader peptide, FVIII protein with a partially deleted B domain, a flexible peptide linker, a CTP rigid unit, and a human IgG vFc variant were artificially optimized codons that CHO cells prefer, and were artificially synthesized. The synthesized full-length DNA fragment of the fusion protein included one restriction endonuclease site, SpeI and EcoRI, at 5′ and 3′ ends, respectively. The full-length DNA fragment was inserted between corresponding restriction sites of pUC57 transfer vector and its sequence was verified through DNA sequencing.

[0087] The full-length gene fragment obtained above of the fusion protein was transferred from the intermediate vector to a self-made expression plasmid PXY1A1M between the corresponding restriction sites to obtain a plasmid that expressed high level of the fusion protein. The PXY1A1M plasmid included, but was not limited to, the following important expression elements: (1) a human cytomegalovirus early promoter, and an enhancer required for exogenous high expression in mammal cells; (2) a double screening marker with kanamycin resistance in bacteria and G418 resistance in mammal cells; (3) murine dihydrofolate reductase (DHFR) gene expression cassette. Methotrexate (MTX) can co-amplify fusion genes and DHFR genes when a host cell is deficient in the DHFR genes (see U.S. Pat. No. 4,399,216). The expression plasmid of the fusion protein was transfected into a mammal host cell line. To achieve stable and high-level expression, a preferred host cell line is a DHFR enzyme deficient CHO-cell (see U.S. Pat. No. 4,818,679). After two days of transfection, the medium was replaced with a screening medium containing 0.6 mg/mL G418, and the cells were seeded in a 96-well plate at a certain concentration (5000-10000 viable cells/well) and cultured for 10-14 days until large discrete cell clones appeared. Transfectants resistant to selected drugs were screened through ELISA analysis method. Wells with high-level fusion proteins were subcloned through the extreme dilution of the 96-well culture plate.

[0088] A series of mutant single-chain FVIII fusion proteins were constructed in the present disclosure, which contained peptide linkers of different lengths, CTP rigid units of different compositions, and several different subtypes of IgG Fc variants (vFc), to verify the effect of linker peptides and Fc variants on the activity of the mutant single-chain FVIII fusion proteins. Details are shown in Table 1. The amino acid sequences of each component are found in SEQUENCE LISTING.

TABLE-US-00003 TABLE 1 Compositions of various single-chain FVIII proteins Compositions of a Series of FVIII Fusion Proteins Code (from the N-terminus to C-terminus) SS-F4 scFVIII-L3-CTP.sup.1-vFcγ.sub.1 SS-F5 scFVIII-L5-CTP.sup.4-vFcγ.sub.4 SS-F6 scFVIII-L1-CTP.sup.3-CTP.sup.3-vFcγ.sub.2-2 SS-F7 scFVIII-L4-CTP.sup.3-vFcγ.sub.2-1 SS-F8 scFVIII-L2-CTP.sup.2-vFcγ.sub.2-3 SS-F9 scFVIII-L2-vFcγ.sub.2-3-CTP.sup.2 55-F10 scFVIII-L1-vFcγ.sub.2-3 SS-F11 scFVIII-L4-vFcγ.sub.2-3

Example 2. Screening of a Stably Transfected Cell Line with High Expression of Fusion Protein

[0089] The above expression plasmid of a fusion protein was transfected into a mammal host cell line to express the mutant single-chain FVIII fusion protein. To maintain stable and high-level expression, a preferred host cell is a DHFR deficient CHO-cell (see U.S. Pat. No. 4,818,679). A preferred transfection method is electroporation. Other methods may be used, such as calcium phosphate co-precipitation, lipofection, and microinjection. The electroporation method used Gene Pulser Electroporator (Bio-Rad Laboratories) with a voltage of 300 V and a capacitance of 1050 pFd, and 50 pg of Pvul linearized expression plasmids was added to 2 to 3×10.sup.7 cells placed in a cuvette, and the cells after electroporation were transferred to a shake flask containing 30 mL of growth medium. After two days of transfection, the medium was replaced with a growth medium containing 0.6 mg/mL G418, and the cells were seeded in a 96-well plate at a certain concentration and cultured for 10-12 days until large discrete cell clones appeared. Transfectants resistant to selected drugs were screened through ELISA analysis method against human IgG Fc. Wells with high-level expression of fusion proteins were subcloned through extreme dilution.

[0090] To achieve the high-level expression of fusion proteins, DHFR gene suppressed by MTX should be used for co-amplification. The transfected fusion protein genes were co-amplified with the DHFR gene in growth media containing MTX with increased concentrations. Positive subclones that expressed DHFR were subjected to limiting dilution, and the pressure was increased gradually to screen out transfectants that could grow in media with MTX of up to 6 μM. The secretion rates of the transfectants were determined and cell lines with high exogenous protein expression were screened. Cell lines with a secretion rate greater than about 1 (preferably about three) IU/10.sup.6 (million) cells/24 h were adaptively suspended using a serum-free medium, and the fusion proteins were then purified using a conditioned medium.

Example 3. Production of Fusion Proteins

[0091] The high-yield cell strains preferably obtained in Example 2 were first subjected to serum-free acclimation and culture in a petri dish, and then transferred to a shake flask for suspension acclimation and culture. After the cells were acclimated to these culture conditions, fed-batch culture was performed in a 300 mL shake flask or perfusion culture was simulated by changing media daily. A CHO-derived cell strain that was screened in Example 2 for producing fusion protein SS-F8 was subjected to fed-batch culture for 14 days in a 300 mL shake flask, where a cumulative yield of expressed recombinant fusion proteins reached 200 mg/L and a maximum viable cell density reached 15×10.sup.6/mL. To obtain more fusion proteins, a 1000 mL shake flask may also be used for culture. In another culture method, the above-mentioned CHO-derived cell strain was cultured in a 100 mL shake flask with media changed daily, where a daily cumulative yield of expressed recombinant fusion proteins was about 20 mg/L and a maximum viable cell density in the shake flask reached 30×10.sup.6/mL. The biological activity of the recombinant fusion proteins produced by the above two methods was determined to be comparable.

Example 4. Purification and Qualitization of Fusion Proteins

[0092] Fusion protein SS-F8 was mainly purified through four-step chromatography in the present disclosure. The four-step chromatography was respectively affinity chromatography, anion exchange chromatography, hydrophobic chromatography, and molecular sieve chromatography. (the protein purifier used in this example was AKTA pure 25 M from GE, U.S; and reagents used in this example were purchased from Sinopharm Chemical Reagent Co., Ltd and had purity at an analytical grade).

[0093] In a first step, affinity chromatography was performed. Sample capture, concentration and the removal of partial pollutants were performed using VIII Select Affinity Chromatography Media from GE. First, the chromatography column was equilibrated with 3-5 column volumes (CVs) of an equilibration buffer containing 10 mM of HEPES, 150 mM of NaCl, 25 mM of CaCl.sub.2), and 0.05% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 50-100 cm/h. The clarified fermentation broth was loaded at a linear flow rate of 50-100 cm/h with a load not higher than 50000 IU/mL. After loading, the chromatography column was equilibrated with 3-5 column volumes (CVs) of an equilibration buffer containing 10 mM of HEPES, 150 mM of NaCl, 5 mM of CaCl.sub.2), and 0.05% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 50-100 cm/h, and unbound components were rinsed. The chromatography column was rinsed with 3-5 column volumes of a decontamination buffer 1 containing 10 mM of HEPES, 1 M of NaCl, 25 mM of CaCl.sub.2), and 0.05% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 50-100 cm/h to remove partial pollutants. The chromatography column was equilibrated with 3-5 column volumes (CVs) of an equilibration buffer containing 10 mM of HEPES, 150 mM of NaCl, 25 mM of CaCl.sub.2), and 0.05% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 50-100 cm/h. Then the target product was eluted at a linear flow rate not higher than 50 cm/h using an elution buffer containing 20 mM of His-HCl, 25 mM of CaCl.sub.2), 0.02% Tween-80, and 45% propylene glycol and with a pH of 6.8-7.2 and target peaks were collected.

[0094] In a second step, anion exchange chromatography was performed. Intermediate purification was conducted by using Q-HP of Bestchrom or other commercially available anion exchange chromatography media (such as Q HP of GE, Toyopearl GigaCap Q-650 of TOSOH, DEAE Beads 6FF of Smart-Lifesciences, Generik MC-Q of Sepax Technologies, Inc, Fractogel EMD TMAE of Merck, and Q Ceramic HyperD F of Pall) to separate structural variants and further remove pollutants such as HCP and DNA. First, the chromatography column was rinsed with 3-5 column volumes (CVs) of an equilibration buffer containing 20 mM of His-HCl, 100 mM of NaCl, 10 mM of CaCl.sub.2, and 0.02% Tween-80 and with a pH of 7.0-7.5 at a linear flow rate of 50-100 cm/h. The target proteins separated through affinity chromatography in the first step were diluted 5-10 times to reduce the concentration of organics. Then samples were loaded with the load being controlled within 5000-10000 IU/mL. After loading, the chromatography column was rinsed with 3-5 column volumes (CVs) of an equilibration buffer containing 20 mM of His-HCl, 100 mM of NaCl, 10 mM of CaCl.sub.2, and 0.02% Tween-80 and with a pH of 7.0-7.5 at a linear flow rate of 50-100 cm/h. Then, the chromatography column was rinsed with 3-5 column volumes (CVs) of a wash buffer containing 20 mM of His-HCl, 500 mM of NaCl, 10 mM of CaCl.sub.2, and 0.02% Tween-80 and with a pH of 7.0-7.5 at a linear flow rate of 50-100 cm/h to remove partial impurity proteins. The chromatography column was then eluted with gradient salt concentrations using an elution buffer containing 20 mM of His-HCl, 1 M of NaCl, 10 mM of CaCl.sub.2, and 0.02% Tween-80 and with a pH of 7.0-7.5, where the condition was elution buffer from 50% and 60%, 3-5 column volumes (CVs), and a linear flow rate not higher than 50 cm/h. Eluted fractions were collected. The samples were sent for detecting protein content, SEC-HPLC, activity, and HCP content separately. A protein concentration and protein activity were determined.

[0095] In a third step, hydrophobic chromatography was performed. Intermediate purification was conducted by using Butyl HP of Bestchrom or other commercially available hydrophobic chromatography media (such as Butyl HP of GE, Toyopearl Butyl-650 of TOSOH, Butyl Beads 4FF of Smart-Lifesciences, Generik MC30-HIC Butyl of Sepax Technologies, Inc, and Fractogel EMD Propyl of Merck) to reduce the content of polymers. The eluate obtained after anion exchange chromatography in the second step still contained a certain proportion of polymers. Because of various reasons for the formation of the polymers such as polymerization with unchanged structures and polymerization with changed structures, the biological activity of the polymers varies greatly and thus causes great interference to the analysis of biological activity. When the target proteins are polymerized, the polymers and monomers differ in property including charge characteristics and hydrophobicity. Polymers and monomers were separated using differences in hydrophobicity. Since the final step of purification would be molecular sieve chromatography, purification was performed using Butyl HP to remove partial polymers to a content less than 10%. First, the chromatography column was equilibrated with 3-5 column volumes (CVs) of an equilibration buffer containing 20 mM of His-HCl, 1.5 M of NaCl, 5 mM of CaCl.sub.2, and 0.02% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 50-100 cm/h. The conductivity of the target proteins separated through anion exchange chromatography in the second step was adjusted using a buffer containing 2 M of (NH.sub.4).sub.2SO.sub.4. Then samples were loaded with a load being controlled lower than 20000 IU/mL. After loading, the chromatography column was rinsed with 3-5 column volumes (CVs) of an equilibration buffer containing 20 mM of His-HCl, 1.5 M of NaCl, 5 mM of CaCl.sub.2), and 0.02% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 50-100 cm/h. Then, the chromatography column was rinsed with 3-5 column volumes (CVs) of a wash buffer containing 20 mM of His-HCl, 1.5 M of NaCl, 5 mM of CaCl.sub.2), and 0.02% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 50-100 cm/h to remove partial polymers. Finally, the target proteins were eluted respectively with 3-5 column volumes (CVs) of 20%, 40%, and 100% of an elution buffer containing 20 mM of His-HCl, 5 mM of CaCl.sub.2), 0.02% Tween-80, and 50% ethylene glycol and with a pH of 6.8-7.2 at a linear flow rate not higher than 60 cm/h. Eluted fractions were collected and sent for detecting SEC-HPLC separately. Target components with the percentage of monomers being greater than 90% were combined for chromatography in the next step.

[0096] In a fourth step, molecular sieve chromatography was performed. Separation was conducted by using superdex 200 of GE or other commercially available molecular sieve media (such as Chromdex 200 prep grade of Bestchrom) to reduce the content of polymers to be lower than 5% and further reduce the content of key pollutans. The chromatography column was rinsed with 2 column volumes (CVs) of an equilibration buffer containing 10 mM of His-HCl, 150 mM of NaCl, 2 mM of CaCl.sub.2), 10 mM of sucrose, and 0.02% Tween-80 and with a pH of 6.8-7.2 at a linear flow rate of 20-40 cm/h. The load was not higher than 3% of the column volume and the samples were rinsed at a linear flow rate of 20 cm/h. Elution components were sequentially collected and combined for SEC detection.

[0097] The SEC-HPLC purity result and SDS-PAGE electrophoresis result of the samples are shown in FIG. 1 and FIG. 2 respectively. The SEC-HPLC result shows that the purity of main peaks of the purified fusion proteins was higher than 98.0%. The SDS-PAGE electrophoretic pattern was as expected. The non-reducing electrophoresis included bands of an unprocessed fusion protein (about 390 kDa), a (LC-L-CTP-Fc).sub.2 dimer fragment (about 210 kDa) formed after shedding of two heavy-chain fragments upon cleavage at another common cleavage site E720, an LC-L-CTP-Fc single-chain fragment (about 100 kDa) and an HC fragment (about 90 kDa). After reduction, clear HC-LC-L-CTP-Fc (about 190 kDa), LC-L-CTP-Fc (about 105 kDa) and HC (about 90 kDa) single-chain bands were obtained. The specific activity of the obtained protein components was 6000-8000 IU/mg.

Example 5. Indirect Determination of the Activity of the Fusion Protein In Vitro by a Chromogenic Substrate Method

[0098] The activity of the mutant single-chain FVIII fusion protein may be determined by the chromogenic substrate method. In this example, the activity was determined using Biophen FVIII:C kit (HYPHEN BioMed, Ref. 221402) based on the following principle: when activated by thrombin, FVIII:C binds to FIXa in the presence of phospholipids and calcium ions to form an enzyme complex which in turn can activate factor X to transform into its active form Xa. The active factor Xa can cause the cleavage of its specific chromogenic substrate (SXa-11) to release chromogenic group pNA. The activity of FXa directly proportional to the amount of pNA may be obtained by determining the amount of produced pNA at 405 nm. In this system, the contents of factor IXa and factor X are certain and excessive, and the activity of FXa is only directly related to the content of FVIIIa. The specific activity of the mutant single-chain FVIII fusion protein determined by this method was about 6000-8000 IU/mg.

Example 6. Studies on the Stability of the Purified Fusion Protein

[0099] To further verify the stability of the mutant single-chain FVIII fusion protein at room temperature of 25° C., the mutant single-chain FVIII fusion protein was stored at room temperature of 25° C. for several days to investigate the effect on the activity of the fusion protein.

[0100] A medicine stability testing chamber (purchased from Shanghai Yiheng Scientific Instrument instruments Co., Ltd.) was used to set a test temperature of 25° C. and a humidity of 75%. Eight copies of SS-F8 and double-chain factor VIII reference drug DS-F8 (a fusion protein retaining the protease cleavage site between Arg1648 and Glu1649 of human wild-type FVIII and having an amino acid sequence as shown in SEQ ID NO: 11) that were diluted to the same concentration, 200 μL for each copy, were stored in the medicine stability testing chamber. One copy of SS-F8 and one copy of DS-F8 were taken and the activity of the fusion proteins was assayed according to the method in Example 5 and recorded as activity values on the first day (d1). Then, the activity of the mutant single-chain FVIII fusion protein was assayed separately on d3, d5, d7 and d14 after the fusion proteins were placed at room temperature (temperature: 25° C., humidity: 75%). The results show that the protein activity of SS-F8 only decreased by about 10% and the protein activity of DS-F8 decreased by more than 25% after 5 days at room temperature; the activity of DS-F8 decreased at a significantly higher rate than that of SS-F8 after 7 days at room temperature; and the protein activity of SS-F8 still remained 80% after 7 days. It can be seen that fusion protein SS-F8 has significantly better stability than DS-F8.

Example 7. Pharmacodynamic Study on the Fusion Protein

[0101] Blood coagulation is in fact enzymatic reactions of a series of coagulation factors. The whole coagulation process is divided into three stages: the first stage is the formation of blood thromboplastin; the second stage is the formation of thrombin; and the third stage is the formation of fibrin, where FVIII and FIX are endogenous coagulation factors and FVII is an exogenous coagulation factor. Bleeding time is the time from natural bleeding to natural hemostasis after skin capillaries are punctured. The procoagulant effect of the fusion protein was detected by observing the effect of the mutant single-chain FVIII fusion protein on the bleeding time of saphenous arteries in SD rats.

[0102] Seven-week-old SD rats (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were selected and randomly divided into two groups. The administration group was administered intravenously with SS-F8 at a single dosage of 200 IU/rat and the control group was administered with the same volume of normal saline. Three hours after administration, rats were induced to be anesthetized. After the surgical position was disinfected, the skin was cut from about 1 cm above the medial malleolus through the knee joint to the artery at leg root; subcutaneous tissues were separated from the supra-vascular protective membrane; and venous vessels, arterial vessels and nerves were exposed in sequence. The saphenous artery and its branches were found at the position of the knee joint. Muscle positions within 5 mm*1 mm near the venous vessels were sharp-dissected with microscopic straight forceps, and the veins, arteries and nerves were lifted from the medial side. The saphenous vein and the saphenous artery were cut with a microscissor and the nerves were not touched or injured. The initial bleeding time was the time when the blood gushed out and recorded as t1. The bleeding site was observed every 30 seconds until no bleeding was seen. The time was recorded as hemostasis time t2. The bleeding time was recorded through a calculation of t2-t1. A statistical analysis was performed on experimental data that was expressed as means±a standard deviation (SD). If the data follows normal distribution, SPSS 18.0 software was used for one-way analysis of variance or student's-test. In the case of non-normal distribution, a non-parametric test such as a Kruskal-wallis test or a Mann-whitney test was used, where P≤0.05 denotes a significant difference and P≤0.01 denotes a very significant difference.

[0103] The experimental results are shown in FIG. 3. After prophylaxis administration, the bleeding time of the SS-F8 group was significantly different from that of the normal saline group in statistics after the bleeding of the saphenous artery caused by a surgery in SD rats. At the dosage of 200 IU/rat, the prophylactic administration of SS-F8 has a significant hemostatic effect on the bleeding model.

Example 8. Direct Determination of the Biological Activity of the Fusion Protein by a Coagulation Method

[0104] The in vitro anticoagulant effect of SS-F8 on SD rat plasma was observed by determining activated partial thromboplastin time (APTT).

[0105] Seven-week-old SD rats (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were selected and randomly divided into 12 groups. After induced to be anesthetized, the rats were cut along the median line of the abdomen with a scalpel under continuous anesthesia, and 10-12 mL of blood was taken from the abdominal aorta. The above blood samples were centrifuged at 20° C. and 1500 rpm for 30 min, and supernatant plasma was separated and added to labeled 1.5 mL centrifuge tubes separately. The whole plasma and test drugs SS-F8 and DS-F8 were prepared at a volume ratio of 6:1 into test samples with concentrations of 25-1000 IU/mL respectively. In a control group, a diluent was added at a volume ratio of 6:1. The APTT values of the above samples were tested with a fully automated coagulator (CS-2000i, Sysmex). The change rate of APTT was calculated according to the following formula: the change rate of APTT=(the APTT value of the administration group − the APTT value of the control group)/the APTT value of the control group. The experimental data was expressed as means±a standard deviation (SD). If the data follows normal distribution, SPSS 18.0 software was used for one-way analysis of variance or student's-test. In the case of non-normal distribution, a non-parametric test such as a Kruskal-wallis test or a Mann-whitney test was used, where P s 0.05 denotes a significant difference and P s 0.01 denotes a very significant difference.

[0106] It can be seen from FIG. 4 that the test drugs SS-F8 and DS-F8 had anticoagulant effect on the plasma of normal rats; the APTT of the test drugs DS-F8 and SS-F8 at a high concentration of 1000 IU/mL decreased by 33.65% and 31.86%, respectively, compared with the vehicle control group; the EC.sub.50 values of DS-F8 and SS-F8 were 139.4 IU/mL and 115.6 IU/mL, respectively; and the EC.sub.50 value of SS-F8 was lower than that of DS-F8, which suggests a lower dosage. With an increase in the concentration of the test drugs, the APTT was significantly reduced and there was a dose-effect relationship.

Example 9. Hemostatic Effect of the Fusion Protein on Acute Hemorrhage in Mice with Hemophilia A

[0107] The acute hemostatic effect of SS-F8 in mice with hemophilia A was evaluated through a tail clip bleeding model of VIII factor gene knockout homozygous hemophilia A (HA). Male HA mice at the age of 6-7 weeks (from Shanghai Model Organisms Center, Inc.) were selected, adaptively fed for one week, and randomly divided into two groups: a HA mouse blank vehicle control group and an SS-F8 administration group. Male C57BL/6 mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd) were selected as a normal control group. Mice were anesthetized through the intraperitoneal injection of 1% sodium pentobarbital (Merck & Co.) at the dosage of 7.5 mL/kg. The C57BL/6 mouse normal control group and the HA mouse blank vehicle control group were administered intravenously via tail veins with a vehicle at the dosage of 10 mL/kg, and the administration group was administered intravenously via tail veins with SS-F8 at the dosage of 112 IU/kg. The tails of the mice were clipped 15 mm from the ends of the tails using a scalpel blade 15 minutes after the administration. The wounds were quickly immersed in 13 mL of normal saline preheated to 37° C. The timing was started, and the blood was collected. The total bleeding time and the bleeding volume within 30 min after the tails were clipped were recorded. Bleeding volume (mL)=(the weight of a centrifuge tube after blood collection (g)−the weight of the centrifuge tube before blood collection (g))/1.0. The experimental groups were compared through T-test testing and analysis software was Graphpad Prism 8.0, where p<0.05 was considered to be statistically significant. Detailed results are shown in Table 2.

[0108] From the analysis of statistical results of the bleeding volume and the sum of bleeding time of each group of animals in FIGS. 5 and 6, the medians of the bleeding volume of the HA mouse blank vehicle control group and the C57BL/6 mouse normal control group were 790.00 μL and 141.15 μL, with an extremely significant difference (P<0.0001), and the medians of the bleeding time were 1800 s and 497 s, with an extremely significant difference (P<0.0001), which indicates that HA mice have an obvious bleeding characteristic compared with normal mice as for the tail-clipping model. After the administration of SS-F8 at the dosage of 112 IU/kg, the medians of the bleeding volume of the administration group and the HA mouse blank vehicle control group were 438.9 μL and 790.00 μL (p<0.0001), and the medians of the bleeding time of the administration group and the HA mouse blank vehicle control group were 739 s and 1800 s (p<0.0001), which indicates a significant coagulant effect and indicates that SS-F8 can be used as an effective coagulant for the acute hemorrhage in hemophilia A, i.e., coagulation factor VIII deficiency.

TABLE-US-00004 TABLE 2 Data statistics of the bleeding volume and bleeding time of each group C57BL/6 Mouse HA Mouse Blank Normal Control Vehicle Control SS-F8 Administration Group Group Group Group Bleeding 141.15 790.00 438.90 volume (μL) Bleeding time 497 1800 739 (s)

[0109] Though the preferred examples of the present disclosure are illustrated and described, it should be understood that those skilled in the art may make various changes in accordance with the teachings herein, and these changes do not violate the scope of the present disclosure.

[0110] All the publications mentioned in the present disclosure are incorporated herein by reference as if each publication is separately incorporated herein by reference. In addition, it should be understood that those skilled in the art, who have read the preceding content of the present disclosure, can make various changes or modifications on the present disclosure, and these equivalent forms fall within the scope of the appended claims.