HIGH-ACTIVITY BLOOD COAGULATION FACTOR XI MUTANT ALA570THR

Abstract

A high-activity blood coagulation factor XI mutant Ala570Thr (A570T), having nucleotide sequences as shown in SEQ ID NOs: 1-4 and an amino acid sequence as shown in SEQ ID NO: 5, is provided. The mutant is resistant to a physiological inhibitor thereof after being activated from a zymogen state to an active enzyme. Therefore, the mutant has a very high blood coagulation activity and a stronger catalytic ability for a non-physiological substrate; and the mutant is applied to the treatment of hemorrhagic diseases, and has good prospects in terms of gene therapy, gene editing and recombinant protein replacement treatments.

Claims

1. A high-activity blood coagulation factor XI mutant Ala570Thr, having: (1) a nucleotide sequence as shown in SEQ ID NO: 1; or (2) a nucleotide sequence as shown in SEQ ID NO: 2; or (3) a nucleotide sequence as shown in SEQ ID NO: 3; or (4) a nucleotide sequence as shown in SEQ ID NO: 4; or a combination of mutations of any other nucleotide at positions 1708, 1709 and 1710.

2. A mutein of the high-activity blood coagulation factor XI mutant Ala570Thr according to claim 1, wherein an amino acid sequence is as shown in SEQ ID NO: 5, and an amino acid, located at position 570 of the mutant is Thr rather than Ala of non-human wild-type FXI; or any other amino acid mutates at the position.

3. A nucleic acid encoding the mutein according to claim 2, or a nucleic acid having the same length as the coding nucleic acid and being completely complementary to the coding nucleic acid.

4. A vector expressing the mutein according to claim 2.

5. A method for preparing a mutein of a high-activity blood coagulation factor XI mutant Ala570Thr, comprising the following steps: (1) ligating a gene encoding the high-activity blood coagulation factor XI mutant Ala570Thr according to claim 1 into a vector to obtain a recombinant vector; (2) transforming the recombinant vector described above into a host cell to obtain clones of mutant cells expressing a recombinant blood coagulation factor XI Ala570Thr; (3) performing continuous perfusion culture on the recombinant cell clones in a serum-free medium to induce expression of the mutein of the recombinant blood coagulation factor XI Ala570Thr; and (4) separating, purifying, filtering, finally filling, and freeze-drying to obtain the expressed high-activity blood coagulation factor XI Ala570Thr mutein.

6. An application of a mutein of a high-activity blood coagulation factor XI mutant Ala570Thr and a coding nucleic acid sequence thereof, wherein the nucleotide sequence according to claim 1 is applied to prepare a gene therapy drug, comprising ligating the nucleotide sequence to a sequence of a promoter and/or a terminator, constructing an expression plasmid, and a viral or non-viral vector for gene therapy, and expressing the mutein of the high-activity blood coagulation factor XI mutant.

7. An application of the mutein of the high-activity blood coagulation factor XI mutant Ala570Thr according to claim 2 and a coding nucleic acid sequence thereof, comprising applying to a viral or non-viral vector or template for gene editing.

8. An application of the mutein of the high-activity blood coagulation factor XI mutant Ala570Thr according to claim 2, comprising applying to preparation of recombinant protein therapeutic drugs for hemophilia or other hemorrhagic diseases.

9. An application of the mutein of the high-activity blood coagulation factor XI mutant Ala570Thr according to claim 2, comprising applying to preparation of a fusion protein of the blood coagulation factor XI Ala570Thr mutant, which is applied to recombinant protein therapeutic drugs for hemophilia or other hemorrhagic diseases.

10. The application according to claim 9, wherein the fusion protein is human albumin, immunoglobulin Fc, transferrin or alpha 1 antitrypsin.

11. An application of a mutein of a high-activity blood coagulation factor XI mutant Ala570Thr and a coding nucleic acid sequence thereof, wherein the nucleotide sequence according to claim 3 is applied to prepare a gene therapy drug, comprising ligating the nucleotide sequence to a sequence of a promoter and/or a terminator, constructing an expression plasmid, and a viral or non-viral vector for gene therapy, and expressing the mutein of the high-activity blood coagulation factor XI mutant.

12. An application of the mutein of the high-activity blood coagulation factor XI mutant Ala570Thr according to claim 3 and a coding nucleic acid sequence thereof, comprising applying to a viral or non-viral vector or template for gene editing.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1-5 are schematic diagrams showing the sequences of the nucleic acid and encoded protein of a high-activity blood coagulation factor XI mutant Ala570Thr (i.e., A570T) of the present invention;

[0029] FIG. 6 is a schematic diagram showing a structure of a vector of the present invention;

[0030] FIG. 7 is a schematic diagram showing the activity of the high-activity blood coagulation factor XI mutant Ala570Thr of the present invention;

[0031] FIG. 8 is a schematic diagram showing correction of thrombin generation in platelet-rich plasma with blood coagulation factor VIII deficiency in vitro by the high-activity blood coagulation factor XI mutant Ala570Thr of the present invention; and

[0032] FIG. 9 is a schematic diagram of thromboelastogram for detecting correction of coagulation defects in acquired hemophilia with antibodies against the coagulation factor VIII by the blood coagulation factor XI mutant Ala570Thr; wherein, a is a blood coagulation factor VIII+buffer control, b is a blood coagulation factor VIII inhibitor+blood coagulation factor XI mutant Ala570Thr of a ⅛ physiological concentration, c is a blood coagulation factor VIII inhibitor+blood coagulation factor XI mutant Ala570Thr of a ¼ physiological concentration, d is a blood coagulation factor VIII inhibitor+blood coagulation factor XI mutant Ala570Thr of a ½ physiological concentration, e is a blood coagulation factor VIII inhibitor+blood coagulation factor XI mutant Ala570Thr of the physiological concentration, f is a blood coagulation factor VIII inhibitor+blood coagulation factor XI mutant Ala570Thr of 2 times physiological concentration, g is a blood coagulation factor VIII deficiency+buffer control, and h is a blood coagulation factor VIII deficiency+blood coagulation factor XI mutant Ala570Thr of the physiological concentration.

DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention will be further described below in connection with specific embodiments. It should be understood that these embodiments are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents of the present invention as taught, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Embodiment 1

[0034] An amino acid sequence of a mutein of a high-activity blood coagulation factor XI mutant Ala570Thr is shown in SEQ ID NO: 5.

[0035] A preparation method of the mutein of the high-activity blood coagulation factor XI mutant Ala570Thr includes the following steps: [0036] (1) ligating a gene encoding a human wild-type coagulation factor XI or the blood coagulation factor XI mutant Ala570Thr into a vector to obtain a recombinant vector; (see FIG. 6) [0037] (2) transforming the recombinant vector described above into a host cell to obtain a recombinant expression cell clone; [0038] (3) culturing the cell clone described above in a serum-free medium to express the mutein of the high-activity blood coagulation factor XI mutant Ala570Thr;

[0039] The serum-free medium is “SAFC Biosciences EX-CELL™ 302” (a commercial reagent). In order to ensure the safety of products and prevent blood-derived preparations from spreading infectious diseases, the serum-free medium was used for mammalian cell culture and protein expression. After the cells reached a steady state after logarithmic phase growth, the cell density was maintained within the target range and the high expression of the blood coagulation factor XI was maintained. [0040] (4) separating, purifying and freeze-drying, so as to obtain the expressed blood coagulation factor XI mutein and related fusion protein.

[0041] After the medium was collected, the medium was clarified and filtered by a deep filter and further separated and purified. The purifying step was divided into two stages: primary purifying and refined purifying, wherein the primary purifying includes the following steps: the filtered and clarified culture solution was concentrated by 10-fold ultrafiltration, and lipid-enveloped viruses, i.e., HIV1/2, HCV, HBV, etc. were inactivated by an organic solvent/detergent method; and the refined purifying includes the following steps: the residual impurities, mainly other proteins secreted by the host cell were further removed from the product by chromatography such as ion exchange (an anion and a cation) and a molecular sieve. The purified protein was subjected to ultrafiltration, liquid exchange, formula adjustment, and then subjected to filtration with a 20 nm nanomembrane for virus removal and freeze-drying. The freeze-drying process includes quick freezing, quenching, freezing, vacuum, main drying and post-drying. The freeze-drying formula is based on inert sugars such as glycine, mannitol, sodium chloride, and calcium chloride, and inorganic salts (composed of glycine, mannitol, sodium chloride, calcium chloride, etc.; and the freeze-drying time is 30 h). [0042] (5) Detection methods for the activity and antigen of the blood coagulation factor XI mutant Ala570Thr. The specific coagulation activity of the blood coagulation factor XI was calculated by comparing the coagulation activity of the blood coagulation factor XI measured by activated partial thromboplastin time (APTT) with the antigen measured by ELISA, as shown in FIG. 7. It can be seen from FIG. 7 that the blood coagulation factor XI mutant Ala570Thr has similar coagulation activity to the wild type.

[0043] Detection methods for the activity and antigen of the blood coagulation factor XI: [0044] {circle around (1)} Detection of the activity of the blood coagulation factor XI by a coagulation method:

[0045] Normal mixed plasma was diluted with OV Buffer at 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320, respectively, and plasma samples to be tested were diluted at 1:10 and 1:20, and a cell supernatant was not treated. 50 μL of the diluted normal mixed plasma, the plasma sample to be tested or a cell supernatant transfected with a blood coagulation factor XI expression vector was taken, 50 μL of blood coagulation factor XI matrix plasma was added, an APTT reagent was added and the mixture was incubated for 3 minutes at 37° C. Then 50 μL of calcium chloride was added, and the coagulation time was recorded on an ST4 semi-automatic hemagglutinator (Stago, France). The activity of the blood coagulation factor XI of the normal mixed plasma diluted at 1:10 was 100%, and a standard curve was established based on the log values of the coagulation time and activity corresponding to different dilutions. If the correlation coefficient R2 is greater than 0.95, the value of the sample to be tested is brought into the calculation to obtain the activity of the blood coagulation factor XI of the sample to be tested. [0046] {circle around (2)} Detection of the antigen of the blood coagulation factor XI by a double-antibody sandwich method:

[0047] An antibody for coating (F9 ELISA kit, Affinity Biologicals, EIA9-0035R1) was diluted at 1:100 with a coating solution (1.59 g/L sodium carbonate and 2.94 g/L sodium bicarbonate, pH 9.6), and the diluted antibody was added at 100 μl/well, and incubated for 2 hours at room temperature. Washing was repeatedly performed for 3 times. Normal mixed plasma was subjected to two-fold dilution at 1:100 with a sample diluent (23.8 g HEPES (free acid)/L, 5.84 g/L NaCl, 3.72 g/L Na.sub.2EDTA, 10 g/L BSA, 0.1% Tween-20, pH 7.2) to 1:3200, respectively. Plasma samples to be tested were diluted at 1:200, 1:400 and 1:800, and cell supernatants were a stock solution, and diluted at 1:10 and 1:100, respectively. 100 μl of the diluted normal mixed plasma or the sample to be tested was added to each well, and left at room temperature for 90 min. Washing was repeatedly performed for 3 times. The detection antibody was diluted with a sample diluent at 1:100, and 100 μl of the diluted detection antibody was added to each well, and left at room temperature for 90 min. Washing was repeatedly performed for 3 times. 100 μl of an OPD substrate was added to each well, and after a stable yellow color appeared (about 5-10 min), 100 μl of a stop solution was added to each well. An absorbance was read at a wavelength of 450 nm with a microplate reader. A standard curve was established, and the value for the antigen of the sample to be tested was calculated. [0048] (6) Correction of the defects of plasma thrombin generation in hemophilia A patients by the blood coagulation factor XI mutant

[0049] A thrombin generation test (TGT) is a comprehensive test used to monitor the thrombin generation ability in plasma. An activator (including a tissue factor and phospholipid) was added into plasma to start a coagulation reaction, and then a thrombin-specific fluorescent substrate was added, and the generated thrombin catalyzed the substrate to release fluorescent groups. The generated fluorescent signal was monitored dynamically by a FLUOROSKAN fluorescence reader, and converted into a thrombin generation curve by using the matching thrombin generation experimental software. The thrombin generation ability was mainly evaluated by several parameters of the curve: (1) lag time, that is, the time required from the start of the reaction to the start of thrombin generation; (2) peak, that is, the maximum amount of generated thrombin; (3) time to peak (ttPeak), that is, the time required from the start of the reaction to the peak of thrombin; and (4) endogenous thrombin potential (ETP), that is, the area under the thrombin generation curve, reflecting the total amount of thrombin generation.

[0050] The thrombin generation test was performed by adding the blood coagulation factor XI mutant Ala570Thr (with the normal physiological concentration of 5 μg/mL) into a platelet-rich plasma (PRP) (deficiency of the coagulation factor VIII) from hemophilia A patients with antibodies (resisting the coagulation factor VIII), as shown in FIG. 8. FIG. 8 shows that the blood coagulation factor XI mutant Ala570Thr of the physiological concentration (5 μg/mL) can correct the thrombin generation disorder caused by blood coagulation factor VIII deficiency.

Embodiment 2

[0051] Detection of Thromboelastogram (See FIG. 9)

[0052] Thromboelastogram (TEG) is a comprehensive test for monitoring the complete coagulation process in whole blood. Thromboelastogram does not require blood sample processing, and uses a small amount of whole blood to monitor the interactions between blood coagulation factors, platelets, fibrinogen, a fibrinolytic system and other cellular components, thus accurately providing a coagulation profile of patients. When testing, an anticoagulant was firstly added to an activation monitoring reagent bottle, and then a certain volume was sucked out and added to a special cylindrical cup (CaCl.sub.2) was added in advance). The cup rotated at a constant speed at an angle of 4° 45′ and a speed of 1 cycle/9 s, and the coagulation state of blood was monitored by a needle immersed in blood and suspended by a spiral wire, and a coagulation speed-intensity curve was drawn by a computer. The coagulation process was mainly evaluated by several parameters of the curve: (1) a R value for the reaction time, that is, the time required from the beginning of detection to the rise of curve amplitude to 2 mm, refers to the time required from the beginning of detection of a specimen to the beginning of fibrin clot formation; (2) a K value for the coagulation time and the clot formation rate α-angle, the K value for the coagulation time is the time required to record from the end of the coagulation time to the curve amplitude of 20 mm, and the clot formation rate α-angle refers to an angle between a tangent line made from the point of clot formation to a maximum arc of the thromboelastogram and a horizontal line, and the K value and the α-angle reflect the result of the joint action of fibrin and platelets at the beginning of blood clot formation, and are mainly influenced by the function of fibrinogen; (3) a MA value refers to a maximum amplitude on the thromboelastogram, that is, the maximum shear force coefficient. The MA value reflects the strongest dynamic characteristics of the interaction between fibrin being formed and platelets and the stability of blood clot formation, where the role of platelets is greater than that of fibrinogen, accounting for about 80%; and (4) a comprehensive coagulation index, i.e., a CI value, is calculated by combining the reaction time, coagulation time, clot formation rate and maximum amplitude of the thromboelastogram curve. The CI value reflects the comprehensive coagulation status of the sample under various conditions, which indicates the presence of low coagulation when the CI value is less than −3, the presence of high coagulation when the CI value is higher than 3, and the presence of normal coagulation when the CI value is −3 to 3.

[0053] It can be seen from FIG. 9 that the blood coagulation factor XI mutant Ala570Thr can correct the coagulation defects caused by coagulation factor VIII deficiency and the presence of antibodies to the coagulation factor VIII.