WO2022052461A1 - 一种高活性凝血因子XI突变体Ala570Thr - Google Patents

一种高活性凝血因子XI突变体Ala570Thr Download PDF

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WO2022052461A1
WO2022052461A1 PCT/CN2021/086785 CN2021086785W WO2022052461A1 WO 2022052461 A1 WO2022052461 A1 WO 2022052461A1 CN 2021086785 W CN2021086785 W CN 2021086785W WO 2022052461 A1 WO2022052461 A1 WO 2022052461A1
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mutant
coagulation factor
ala570thr
protein
recombinant
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PCT/CN2021/086785
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English (en)
French (fr)
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武文漫
王学锋
丁秋兰
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上海交通大学医学院附属瑞金医院
武文漫
王学锋
丁秋兰
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Priority to US18/025,398 priority Critical patent/US20230338479A1/en
Priority to JP2023539205A priority patent/JP2023545320A/ja
Priority to EP21865524.9A priority patent/EP4212623A1/en
Publication of WO2022052461A1 publication Critical patent/WO2022052461A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6443—Coagulation factor XIa (3.4.21.27)
    • A—HUMAN NECESSITIES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21027—Coagulation factor XIa (3.4.21.27)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of hemorrhagic disease treatment, and particularly relates to a highly active coagulation factor XI mutant Ala570Thr.
  • coagulation factor VIII/IX coagulation factor VIII/IX
  • hemophilia type A/type B
  • the coagulation factor VIII/IX activity of severe patients is often lower than 1% of normal, and spontaneous bleeding often occurs leading to muscle hematoma or joint deformity.
  • Infusion of factor VIII/IX preparations currently usually recombinantly expressed coagulation factor VIII/IX proteins in vitro
  • Gene therapy is a treatment method currently in clinical trials.
  • the normal coagulation factor VIII/IX gene is introduced into the patient's body for expression, so as to achieve the purpose of increasing the level of coagulation factor VIII/IX and preventing bleeding.
  • hemophilia A and B can be effectively treated with recombinant or plasma-derived FVIII/FIX, approximately 30% of patients develop antibodies after treatment, rendering the treatment ineffective.
  • Bypass coagulation-active drugs are the best option for treating patients with inhibitor-producing hemophilia.
  • the currently clinically used coagulation factor VIIa (FVIIa) has a short half-life ( ⁇ 2 hours), a large dose (90-100 ⁇ g/kg body weight), and high treatment costs. Therefore, how to obtain better therapeutic effects and Novel drugs in the bypass coagulation pathway with the characteristics of drug metabolism are an urgent problem to be solved in the current treatment of hemophilia.
  • Coagulation factor IX is the physiological substrate of coagulation factor XI (FXI), and the coagulation activity of coagulation factor XI is mainly related to its ability to efficiently cleave and activate coagulation factor IX.
  • factor XI other components of the coagulation reaction may also be catalyzed by factor XI.
  • Recent studies have shown that coagulation factor XI can catalyze the activation of coagulation factor V (FV) and coagulation factor X (FX), thereby directly activating the coagulation common pathway over coagulation factor IX; another study also showed that coagulation factor XI can degrade the tissue factor pathway.
  • TFPI Inhibitor
  • FVIIa exogenous pathway-activated coagulation factor FVII
  • FVIIa exogenous pathway-activated coagulation factor
  • wild-type coagulation factor XI is very inefficient in catalyzing the cleavage of alternative coagulation substrates including FX, FV or TFPI, limiting its ability to promote coagulation through the bypass.
  • the technical problem to be solved by the present invention is to provide a highly active coagulation factor XI mutant, Ala570Thr (A570T), which is resistant to its physiological inhibitor after being activated from the zymogen state to an active enzyme, so it has a high It has a stronger coagulation activity for non-physiological substrates, and is used in the treatment of bleeding diseases, and has a good prospect for gene therapy, gene editing and recombinant protein replacement therapy.
  • A570T highly active coagulation factor XI mutant, Ala570Thr
  • the present invention provides a highly active coagulation factor XI mutant Ala570Thr:
  • nucleotide sequence is shown in SEQ ID NO: 1;
  • nucleotide sequence is as shown in SEQ ID NO:2;
  • nucleotide sequence is as shown in SEQ ID NO:3;
  • nucleotide sequence is as shown in SEQ ID NO: 4.
  • the present invention also provides a mutant protein of highly active coagulation factor XI mutant Ala570Thr, the amino acid sequence of which is shown in SEQ ID NO: 5, and the amino acid of the mutant at 570 is Thr (denoted as Ala570Thr) instead of human wild-type FXI (hFXI) Ala; or any other amino acid change at this position.
  • the present invention also provides a nucleic acid of the mutant protein of the highly active coagulation factor XI mutant Ala570Thr, or a nucleic acid having the same length as the encoding nucleic acid and completely complementary to the encoding nucleic acid.
  • the present invention also provides a carrier for the mutant protein of the highly active coagulation factor XI mutant Ala570Thr.
  • the present invention also provides a method for preparing a mutant protein of a highly active coagulation factor XI mutant Ala570Thr, comprising the following steps:
  • the serum-free medium in the step (3) is "SAFC Biosciences EX-CELL TM 302" (commercialized reagent).
  • the purification in the step (4) includes primary purification and purification.
  • the present invention also provides a plasmid vector expressing the mutant protein Ala570Thr for gene transduction, and its preparation and inspection include the following steps: ligating the cDNA encoding the highly active coagulation factor XI Ala570Thr into a CMV-containing or other eukaryotic cell expression promoter ( gene expression plasmids with or without liver tissue specificity).
  • the mutant protein of the highly active coagulation factor XI mutant Ala570Thr is used in the preparation of gene therapy drugs.
  • the mutant protein of the highly active coagulation factor XI mutant Ala570Thr is used in the preparation of recombinant protein therapeutic drugs for hemophilia or other bleeding diseases.
  • the mutant protein of the highly active coagulation factor XI mutant Ala570Thr is used to prepare a fusion protein of the coagulation factor XI mutant Ala570Thr mutant, and then administer it to a recombinant protein therapeutic drug for hemophilia or other bleeding diseases.
  • the fusion protein is human albumin, immunoglobulin Fc, transferrin or alpha 1 antitrypsin.
  • the pharmaceutical composition or gene therapy vector of the nucleic acid or amino acid sequence of the present invention is used for preventing and/or treating diseases, wherein the diseases mainly include hemorrhagic diseases or bleeding caused by various reasons; among them, the most likely hemorrhagic diseases Is hemophilia A and B, a bleeding disorder due to inherited coagulation factor VIII or IX deficiency, and includes hemophilia A and B in which inhibitory antibody production is present, or acquired acquired due to inhibitor production Factor VIII or IX deficiency; and other bleeding disorders using bypass agents, such as neonatal coagulation disorders; severe liver disease; high-risk surgery; traumatic blood loss; bone marrow transplantation; thrombocytopenia and platelet dysfunction; oral Emergency reversal of anticoagulation; congenital deficiencies in coagulation factors V, VII, X, and XI; Blood loss, cerebral hemorrhage, platelet dysfunction.
  • the diseases mainly include hemorrhagic diseases or bleeding caused by various reasons; among them, the
  • the coagulation factor XI mutant Ala570Thr in the present invention is activated from the zymogen state to have Active enzymes (activated coagulation factor XI, FXIa) are resistant to their physiological inhibitors (such as protease nexin II/KPI, etc.), so they have high coagulation activity and stronger catalytic ability to non-physiological substrates , thereby enhancing the coagulation activity of the alternative pathway, efficiently activating the coagulation response through the alternative pathway through a mechanism independent of coagulation factor IX (FIX)/coagulation factor VIII (FVIII), improving the overall coagulation function of the body, and being applied to bleeding diseases It has good prospects for gene therapy and recombinant protein replacement therapy.
  • Active enzymes activated from the zymogen state to have Active enzymes (activated coagulation factor XI, FXIa) are resistant to their physiological inhibitors (such as protease nexin II/KPI, etc.), so they have high coagulation activity and stronger
  • 1-5 are the sequence diagrams of the nucleic acid and the encoded protein of the highly active coagulation factor XI mutant Ala570Thr (ie A570T) of the present invention
  • Fig. 6 is the schematic diagram of the carrier structure of the present invention.
  • Figure 7 is a schematic diagram of the activity of the highly active coagulation factor XI mutant Ala570Thr of the present invention.
  • Figure 8 is a schematic diagram of the generation of platelet-rich plasma thrombin in the high-active coagulation factor XI mutant Ala570Thr in vitro to correct coagulation factor VIII deficiency;
  • Figure 9 is a schematic diagram of the detection of coagulation factor XI mutant Ala570Thr by thromboelastography to correct the coagulation defect of acquired hemophilia containing anti-coagulation factor VIII antibody; wherein, a is the coagulation factor VIII inhibitor + buffer control, and b is the coagulation factor VIII inhibitor Inhibitor + 1/8 physiological concentration of coagulation factor XI mutant Ala570Thr, c is coagulation factor VIII inhibitor + 1/4 physiological concentration of coagulation factor XI mutant Ala570Thr, d is coagulation factor VIII inhibitor + 1/2 physiological concentration of coagulation factor XI Mutant Ala570Thr, e is coagulation factor VIII inhibitor + physiological concentration factor XI mutant Ala570Thr, f is coagulation factor VIII inhibitor + 2 times physiological concentration coagulation factor XI mutant Ala570Thr, g is coagulation factor VIII deficiency + buffer control , h is factor VIII deficiency + physiological concentration factor XI mutant Ala570Thr.
  • the amino acid sequence of the mutant protein of the hyperactive coagulation factor XI mutant Ala570Thr is shown in SEQ ID NO:5.
  • a method for preparing a mutant protein of a highly active coagulation factor XI mutant Ala570Thr comprising the following steps:
  • the serum-free medium was "SAFC Biosciences EX-CELL TM 302" (commercially available reagent). In order to ensure product safety and prevent blood-derived preparations from spreading infectious diseases, serum-free medium is used for mammalian cell culture and protein expression. After the cells grow to a steady state in log phase, the cell density is maintained within the target range. Maintain high expression of coagulation factor XI.
  • the purification steps are divided into two stages: primary purification and purification.
  • Primary purification the filtered and clarified culture medium is concentrated by 10 times ultrafiltration and then inactivated by the organic solvent/detergent method to inactivate lipid-enveloped viruses, namely HIV1/2, HCV and HBV Etc.;
  • Purification Use ion exchange (anions and cations) and molecular sieves and other chromatographic methods to further remove impurities in the product, mainly other proteins secreted by host cells.
  • the purified protein was subjected to ultrafiltration, liquid exchange, and formula adjustment, followed by virus removal with a 20nm nanomembrane membrane and lyophilization.
  • the freeze-drying process is quick-freezing, quenching, freezing, vacuum, main drying and post-drying.
  • the freeze-dried formula is mainly composed of glycine, mannitol, sodium chloride, calcium chloride and other inert sugars and inorganic salts (the composition is glycine, mannitol, sodium chloride, calcium chloride, etc.; the freeze-drying time is 30 hours).
  • coagulation factor XI mutant Ala570Thr Activity and antigen detection method of coagulation factor XI mutant Ala570Thr.
  • the specific coagulation activity of coagulation factor XI was estimated by comparing the coagulation activity of coagulation factor XI determined by partially activated thromboplastin time (APTT) with the antigen determined by ELISA, see FIG. 7 . It can be seen from Fig. 7 that the coagulation factor XI mutant Ala570Thr has a coagulation activity similar to that of the wild type.
  • Normal mixed plasma was diluted with OV Buffer at 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, and the plasma sample to be tested was diluted at 1:10 and 1:20, and the cell supernatant was Liquid is not processed.
  • Plasma samples to be tested or cell supernatant transfected with coagulation factor XI expression vector add 50 microliters of coagulation factor XI matrix plasma, add APTT reagent, incubate at 37°C for 3 minutes, and then 50 ⁇ l of calcium chloride was added, and the clotting time was recorded on a ST4 semi-automatic coagulometer (Stago, France).
  • the activity of coagulation factor XI diluted 1:10 with normal mixed plasma is 100%, and the standard curve is established by the log value of the clotting time corresponding to different dilutions and the log value of the corresponding activity. If the correlation coefficient R2 is greater than 0.95, the test will be The value of the sample is brought into the calculation to obtain the coagulation factor XI activity of the sample to be tested.
  • the coated antibody (F9ELISA kit, Affinity Biologicals, EIA9-0035R1) was diluted 1:100 with coating solution (1.59g/L sodium carbonate and 2.94g/L sodium bicarbonate, pH 9.6), and 100ul/well of diluted antibody was added. , and incubated at room temperature for 2 hours. Repeat washing 3 times.
  • the normal mixed plasma was diluted 1:100 with sample diluent (23.8g HEPES(free acid)/L, 5.84g/LNaCl, 3.72g/LNa2EDTA, 10g/LBSA, 0.1% Tween-20, Ph 7.2) respectively. to 1:3200.
  • Plasma samples to be tested were diluted 1:200, 1:400 and 1:800, and cell supernatants were diluted to stock solution, 1:10 and 1:100, respectively.
  • the detection antibody was diluted 1:100 with the sample diluent, 100 ul of the diluted detection antibody was added to each well, and it was placed at room temperature for 90 minutes. Repeat washing 3 times.
  • the absorbance was read with a microplate reader at a wavelength of 450 nm. Establish a standard curve and calculate the antigen value of the sample to be tested.
  • Coagulation factor XI mutants correct plasma thrombin generation defects in hemophilia A patients
  • Thrombin generation test is a comprehensive test used to monitor the ability of thrombin generation in plasma.
  • the activator including tissue factor and phospholipid
  • the fluorescent signal was converted into a thrombin generation curve using the matching thrombin generation experimental software.
  • the ability to generate thrombin is 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 thrombin generated.
  • time to peak that is, the time from the start of the reaction to the peak of thrombin
  • EDP endogenous thrombin potential
  • the coagulation factor XI mutant Ala570Thr (at a normal physiological concentration of 5ug/mL) was added to platelet-rich plasma (PRP) (deficient in coagulation factor VIII) in patients with hemophilia A in the presence of antibodies (anti-coagulation factor VIII), A thrombin generation assay was performed, see Figure 8.
  • Figure 8 shows that the factor XI mutant Ala570Thr at physiological concentrations (5 ug/mL) can correct thrombin generation impairment due to factor VIII deficiency.
  • Thromboelastogram is a comprehensive test used to monitor the overall coagulation process in whole blood. It does not require blood sample processing and uses a small amount of whole blood to monitor the interaction between coagulation factors, platelets, fibrinogen, fibrinolytic system and other cellular components, providing an accurate coagulation profile of the patient.
  • TOG Thromboelastogram
  • add anticoagulation to the activation monitoring reagent bottle first, and then suck out a certain volume and add it to a special cylindrical cup (add CaCl 2 in advance).
  • the cup was rotated at a constant speed of 4°45' at a speed of 1 cycle/9s, and the coagulation state of the blood was monitored by a needle immersed in the blood suspended by a helical wire, and the coagulation speed and intensity curves were drawn by a computer.
  • the coagulation process is mainly evaluated by the following curve parameters: (1)
  • the R value of the reaction time that is, the time required from the beginning of the detection to the amplitude of the curve rising to 2 mm, refers to the time from the beginning of the detection of the specimen to the beginning of the formation of the fibrin clot.
  • the K value of the clotting time and the ⁇ angle of the clot formation rate, the agglutination time K value is the time required to record from the end point of the clotting time to the amplitude of the curve reaching 20 mm
  • the ⁇ angle of the clot formation rate refers to the time from the blood clot formation point to the The angle between the tangent line and the horizontal line made by the maximum arc of the tracing graph reflects the result of the joint action of fibrin and platelets at the beginning of blood clot formation, and is mainly affected by the function of fibrinogen
  • MA value refers to the The maximum amplitude, that is, the maximum shear force coefficient.
  • the comprehensive coagulation index is the CI value , is calculated by combining the reaction time, agglutination time, clot formation rate, and maximum amplitude of the thromboelastometry curve. It reflects the comprehensive coagulation status of the sample under various conditions. Below -3 indicates hypocoagulation, higher than 3 indicates hypercoagulation, and between -3 and 3 indicates normal.
  • the coagulation factor XI mutant Ala570Thr can correct the coagulation defect of coagulation factor VIII and the coagulation defect caused by the presence of coagulation factor VIII antibody.

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Abstract

提供了一种高活性凝血因子XI突变体Ala570Thr(A570T)，核苷酸序列如SEQ ID NO:1-4，氨基酸序列如SEQ ID NO:5所示。所述突变体在由酶原状态活化成为具有活性的酶后对其生理抑制物产生抵抗，因此具有很高的凝血活性，对非生理性底物具有更强的催化能力，应用于出血性疾病的治疗，具有很好的基因治疗、基因编辑与重组蛋白替代治疗前景。

Description

一种高活性凝血因子XI突变体Ala570Thr 技术领域
本发明属于出血病治疗领域，特别涉及一种高活性凝血因子XI突变体Ala570Thr。
背景技术
凝血因子缺陷或其它人体凝血功能障碍时会导致出血性疾病的发生，其中由于凝血因子VIII/IX(FVIII/FIX)缺陷所导致的出血性疾病称之为血友病(甲型/乙型)，重型病人的凝血因子VIII/IX活性往往低于正常的1％，经常发生自发的出血导致肌肉血肿或者关节畸形。输注因子VIII/IX制剂(目前通常是体外重组表达的凝血因子VIII/IX蛋白)补充患者体内的凝血因子VIII/IX水平是目前唯一有效的治疗方法，但是需要频繁的给药。基因治疗是目前正在临床试验的治疗方法，将正常的凝血因子VIII/IX基因导入患者体内表达，从而达到提高凝血因子VIII/IX水平，预防出血的目的。虽然应用重组或血浆来源的FVIII/FIX可以有效地治疗血友病A和B，但是约有30％的病人在治疗后产生抗体，使治疗失效。旁路凝血活性药物是治疗有抑制物产生血友病患者的最佳选择。但是目前临床上应用的凝血因子VIIa(FVIIa)，由于半寿期短(～2小时)，所需剂量大(90～100μg/kg体重)，治疗成本高昂，因此如何获得具有更好治疗效果和药物代谢特点的新型旁路凝血途径药物是目前血友病治疗的一个亟待解决的问题。
凝血因子IX是凝血因子XI(FXI)的生理性底物，凝血因子XI的凝血活性主要与其高效裂解活化凝血因子IX的能力有关。但是凝血反应中的其它组分也可能会被凝血因子XI所催化裂解。近年研究表明，凝血因子XI可以催化凝血因子V(FV)和凝血因子X(FX)活化，从而越过凝血因子IX直接激活凝血共同途径；另外一项研究还表明，凝血因子XI可以降解组织因子途径抑制物(TFPI)，从而延长外源途径活化凝血因子FVII(FVIIa)的作用时间，间接地放大外源途径激活的凝血反应。但是野生型凝血因子XI催化裂解包括FX,FV或TFPI在内的旁路凝血反应底物的效率非常低下，限制了它通过旁路促进凝血反应的能力。
发明内容
本发明所要解决的技术问题是提供一种高活性凝血因子XI突变体Ala570Thr(A570T)，该突变体在由酶原状态活化成为具有活性的酶后对其生理抑制物产生抵抗，因此具有很高的凝血活性，对非生理性底物具有更强的催化能力，应用于出血性疾病的治疗，具有很好的基因治疗、基因编辑与重组蛋白替代治疗前景。
本发明提供了一种高活性凝血因子XI突变体Ala570Thr：
(1)核苷酸序列如SEQ ID NO:1所示；
或(2)核苷酸序列如SEQ ID NO:2所示；
或(3)核苷酸序列如SEQ ID NO:3所示；
或(4)核苷酸序列如SEQ ID NO:4所示；
或是任意其他核苷酸在1708位、1709位和1710位突变的组合。
本发明还提供了一种高活性凝血因子XI突变体Ala570Thr的突变蛋白，氨基酸序列如SEQ ID NO:5所示，该突变体位于570的氨基酸为Thr(记为Ala570Thr)而非人野生型FXI(hFXI)的Ala；或是任意其他氨基酸在该位点的改变。
本发明还提供了一种高活性凝血因子XI突变体Ala570Thr的突变蛋白的核酸，或与所述编码核酸长度相同且与所述编码核酸完全互补的核酸。
本发明还提供了一种高活性凝血因子XI突变体Ala570Thr的突变蛋白的载体。
本发明还提供了一种高活性凝血因子XI突变体Ala570Thr的突变蛋白的制备方法，包括如下步骤：
(1)将高活性凝血因子XI突变体Ala570Thr的编码基因连入载体中，得到重组载体；
(2)将上述重组载体转化宿主细胞，得到表达重组凝血因子XI Ala570Thr突变细胞克隆；(3)于无血清培养基中连续灌流培养上述重组细胞克隆，诱导重组凝血因子XI Ala570Thr的突变蛋白的表达；
(4)分离纯化、过滤，最后灌装、冻干，得到所表达的高活性凝血因子XI Ala570Thr突变蛋白。
所述步骤(3)中的无血清培养基为“SAFC Biosciences EX-CELL TM 302”(商品化的试剂)。
所述步骤(4)中的纯化包括初纯和精纯。
本发明还提供了一种表达突变蛋白Ala570Thr的质粒载体进行基因转导，其制备和检验包括如下步骤：将编码高活性凝血因子XI Ala570Thr的cDNA连接入含CMV或其他真核细胞表达启动子(具有或不具有肝组织特异性)的基因表达质粒。
所述高活性凝血因子XI突变体Ala570Thr的突变蛋白应用于制备基因治疗药物。
所述高活性凝血因子XI突变体Ala570Thr的突变蛋白应用于制备血友病或其他出血性疾病的重组蛋白治疗药物。
所述高活性凝血因子XI突变体Ala570Thr的突变蛋白应用于制备凝血因子XI突变体Ala570Thr突变体融合蛋白，并将其施用于血友病或其他出血性疾病的重组蛋白治疗药物。
所述融合蛋白为人白蛋白、免疫球蛋白Fc、转铁蛋白或alpha 1抗胰蛋白酶。
本发明的核酸或氨基酸序列的药物组合物或基因治疗载体，用于预防和/或治疗疾病，其中所述疾病主要包括出血性疾病或各种原因导致的出血；其中，最可能的出血性疾病是血友病A和B，即由于遗传性凝血因子VIII或IX缺乏导致的出血性疾病，并包括其中有抑制性 抗体产生的血友病A和B，或者后天因抑制物产生所导致的获得性凝血因子VIII或IX缺乏；及其它使用旁路制剂的出血性疾病，例如新生儿凝血障碍；严重的肝脏疾病；高风险手术；创伤性失血；骨髓移植；血小板减少症和血小板功能障碍；口服抗凝的紧急逆转；先天性凝血因子V，VII，X和XI的缺陷；血管性血友病，及血管性血友病因子抑制物导致的获得性血管性血友病，与大量损伤有关的失血，大脑出血，血小板功能障碍。
有益效果
与凝血因子XI突变体Gly397Ser由酶原转化成为具有催化活性的酶的速度加快为主的提升凝血因子XI凝血活性机制不同，本发明中的凝血因子XI突变体Ala570Thr在由酶原状态活化成为具有活性的酶(活化的凝血因子XI，FXIa)后对其生理抑制物(如protease nexin II/KPI等)产生抵抗，因此具有很高的凝血活性，对非生理性底物具有更强的催化能力，从而增强了旁路途径凝血活性，通过不依赖于凝血因子IX(FIX)/凝血因子VIII(FVIII)的机制高效的通过旁路途径激活凝血反应，提升机体整体凝血功能，应用于出血性疾病的治疗，具有很好的基因治疗与重组蛋白替代治疗前景。
附图说明
图1-图5为本发明高活性凝血因子XI突变体Ala570Thr(即A570T)的核酸与编码蛋白的序列示意图；
图6为本发明载体结构的示意图；
图7为本发明高活性凝血因子XI突变体Ala570Thr活性示意图；
图8为本发明高活性凝血因子XI突变体Ala570Thr体外纠正凝血因子VIII缺陷富血小板血浆凝血酶生成示意图；
图9为血栓弹力图检测凝血因子XI突变体Ala570Thr纠正含抗凝血因子VIII抗体获得性血友病凝血缺陷示意图；其中，a为凝血因子VIII抑制物+缓冲液对照，b为凝血因子VIII抑制物+1/8生理浓度凝血因子XI突变体Ala570Thr，c为凝血因子VIII抑制物+1/4生理浓度凝血因子XI突变体Ala570Thr，d为凝血因子VIII抑制物+1/2生理浓度凝血因子XI突变体Ala570Thr，e为凝血因子VIII抑制物+生理浓度凝血因子XI突变体Ala570Thr，f为凝血因子VIII抑制物+2倍生理浓度凝血因子XI突变体Ala570Thr，g为凝血因子VIII缺乏+缓冲液对照，h为凝血因子VIII缺乏+生理浓度凝血因子XI突变体Ala570Thr。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
高活性凝血因子XI突变体Ala570Thr的突变蛋白的氨基酸序列如SEQ ID NO:5所示。
高活性凝血因子XI突变体Ala570Thr的突变蛋白的制备方法，包括如下步骤：
(1)将人野生型或凝血因子XI突变体Ala570Thr的人凝血因子XI编码基因连入载体中，得到重组载体；(见图6)
(2)将上述重组载体转化宿主细胞，得到重组表达细胞克隆；
(3)于无血清培养基中培养上述细胞克隆，表达高活性凝血因子XI突变体Ala570Thr的突变蛋白的表达；
无血清培养基为“SAFC Biosciences EX-CELL TM 302”(商品化的试剂)。为保证产品安全，防止血液来源制剂传播感染性疾病，故应用无血清培养基用于哺乳动物细胞培养、蛋白表达，细胞经对数期生长达到稳态后，将细胞密度维持在目标区间内，维持凝血因子XI高表达。
(4)分离纯化与冻干，从而得到所表达的凝血因子XI突变蛋白及相关融合蛋白。
在培养基收集后，经深层滤器澄清过滤并进一步分离纯化。纯化步骤分为初纯和精纯两个阶段，初纯：将过滤澄清的培养液在10倍超滤浓缩后通过有机溶剂/洗涤剂法灭活脂包膜病毒即HIV1/2、HCV以及HBV等；精纯：以离子交换(阴离子和阳离子)和分子筛等色谱层析方法进一步去除产品中残留以宿主细胞分泌的其他蛋白为主的杂质。纯化蛋白经过超滤换液、调节配方后再进行即20nm纳米膜除病毒过滤并冻干。冻干过程为速冻、淬火、冷冻、真空、主干燥、后干燥。冻干配方以甘氨酸、甘露醇、氯化钠、氯化钙等惰性糖类以及无机盐为主(组成为甘氨酸、甘露醇、氯化钠、氯化钙等；冻干时间为30小时)。
(5)凝血因子XI突变体Ala570Thr活性和抗原检测方法。凝血因子XI的特异性凝血活性由部分活化的凝血活酶时间(APTT)所测定的凝血因子XI凝血活性与ELISA测定的抗原比较推算而得，见图7。由图7可知，凝血因子XI突变体Ala570Thr具有与野生型类似的凝血活性。
凝血因子XI活性和抗原检测方法：
①凝固法检测凝血因子XI活性：
正常混合血浆用OV Buffer分别进行1:10,1:20,1:40,1:80,1:160,1:320稀释，血浆待测样本进行1:10和1:20稀释，细胞上清液不处理。取50微升稀释的正常混合血浆、待测血浆样本或转染凝血因子XI表达载体的细胞上清液，加入50微升凝血因子XI基质血浆，加入APTT试剂，37℃温育3分钟，再加入50微升氯化钙，ST4半自动血凝仪(Stago公司，法国)上记录凝固时间。以正常混合血浆1:10稀释的凝血因子XI活性为100％，以不同稀释度对应的凝固时间的log值和对应的活性得log值建立标准曲线，若相关系数R2大于0.95，则将待测 样本的值带入计算，得到待测样本的凝血因子XI活性。
②双抗夹心法检测凝血因子XI的抗原：
用包被液(1.59g/L碳酸钠和2.94g/L碳酸氢钠，pH 9.6)将包被抗体(F9ELISA试剂盒,Affinity Biologicals,EIA9-0035R1)1:100稀释，加入稀释抗体100ul/孔，室温孵育2小时。重复洗涤3次。将正常混合血浆用样品稀释液(23.8g HEPES(free acid)/L,5.84g/LNaCl,3.72g/LNa2EDTA,10g/LBSA,0.1％吐温-20，Ph 7.2)分别1:100对倍稀释至1:3200。待测血浆样本以1:200,1:400和1:800稀释，细胞上清液分别为原液、1:10和1:100稀释。每孔加入100ul稀释好的正混血浆或待测样品，室温放置90min。重复洗涤3次。将检测抗体用样品稀释液1:100稀释，每孔加入100ul的稀释完的检测抗体，室温放置90min。重复洗涤3次。每孔加入100ul的OPD底物，待出现稳定的黄色之后(约5-10min)，每孔加入100ul的终止液。用酶标仪在450nm的波长下读取吸光度。建立标准曲线，并计算待测样本的抗原值。
(6)凝血因子XI突变体纠正血友病A患者血浆凝血酶生成缺陷
凝血酶生成试验(thrombin generation test,TGT)：是用于监测血浆中凝血酶生成能力的综合性实验。将激活剂(含组织因子及磷脂)加入血浆中启动凝血反应，再加入凝血酶特异性的荧光底物，生成的凝血酶催化底物释放出荧光基团，使用FLUOROSKAN荧光读数仪动态监测生成的荧光信号，使用配套的凝血酶生成实验软件将信号转换成一条凝血酶生成曲线。主要通过曲线的几个参数评价凝血酶生成能力：(1)延迟时间(lag time)，即从反应开始到凝血酶开始生成所需时间；(2)峰值(peak)，即生成的凝血酶最大量；(3)达峰时间(time to peak,ttpeak)，即从反应开始到凝血酶达峰值所需时间；(4)凝血酶生成潜力(endogenous thrombinpotential,ETP)，即凝血酶生成曲线下面积，反应凝血酶生成总量。
在存在抗体(抗凝血因子VIII)的血友病A患者的富含血小板血浆(PRP)中(乏凝血因子VIII)中加入凝血因子XI突变体Ala570Thr(浓度为正常生理浓度5ug/mL)，进行凝血酶生成检测，见图8。图8显示生理浓度(5ug/mL)凝血因子XI突变体Ala570Thr可以纠正由于凝血因子VIII缺乏造成的凝血酶生成障碍。
实施例2
检测血栓弹力图(见图9)
血栓弹力图(thromboelastogram，TEG)：是用于监测全血中全部凝血过程的综合性试验。它不需要血标本处理，用少量全血监测凝血因子、血小板、纤维蛋白原、纤溶系统和其他细胞成分之间的相互作用，准确地提供患者的凝血概况。检测时先将抗凝血加至活化监测试剂瓶中，再吸出一定体积加至特制圆柱形杯中(提前加入CaCl 2)。杯子以4°45'的角度和1周/9s的速度匀速转动，通过一根浸泡在血液中由螺旋丝悬挂的针监测血液的凝固状态，并 由计算机绘制凝血速度和强度曲线。主要通过以下几个曲线参数评估凝血过程：(1)反应时间R值，即检测起始至曲线振幅上升至2mm所需时间，是指从标本开始检测至纤维蛋白凝块开始形成所需的时间；(2)凝集时间K值和凝集块形成速率α角，凝集时间K值是记录从凝血时间终点至曲线振幅达20mm所需时间，凝集块形成速率α角是指从血凝块形成点至描记图最大弧度所作切线与水平线的夹角，它们反映纤维蛋白和血小板在血凝块开始形成时的共同作用的结果，主要受纤维蛋白原功能影响；(3)MA值是指描记图上的最大振幅，即最大切变力系数。反映正在形成的纤维蛋白与血小板相互联结的最强动力学特性及血凝块形成的稳定性，其中血小板的作用要比纤维蛋白原大，约占80％；(4)凝血综合指数即CI值，是结合了血栓弹力图曲线的反应时间、凝集时间、凝集块形成速率、最大振幅结合推算得出。反映样本在各种条件下的凝血综合状态，低于-3提示存在低凝，高于3提示存在高凝，介于-3和3提示为正常。
由图9可知，凝血因子XI突变体Ala570Thr可以纠正凝血因子VIII缺陷及凝血因子VIII抗体存在时所导致的凝血缺陷。

Claims (10)

  1. 一种高活性凝血因子XI突变体Ala570Thr，其特征在于：
    (1)核苷酸序列如SEQ ID NO:1所示；
    或(2)核苷酸序列如SEQ ID NO:2所示；
    或(3)核苷酸序列如SEQ ID NO:3所示；
    或(4)核苷酸序列如SEQ ID NO:4所示；
    或是任意其他核苷酸在1708位、1709位和1710位突变的组合。
  2. 一种如权利要求1所述的高活性凝血因子XI突变体Ala570Thr的突变蛋白，其特征在于：氨基酸序列如SEQ ID NO:5所示，该突变体位于570的氨基酸为Thr而非人野生型FXI的Ala；或是任意其他氨基酸在该位点的改变。
  3. 编码如权利要求2中所述的突变蛋白的核酸，或与所述编码核酸长度相同且与所述编码核酸完全互补的核酸。
  4. 一种表达如权利要求2中所述的突变蛋白的载体。
  5. 一种高活性凝血因子XI突变体Ala570Thr的突变蛋白的制备方法，包括如下步骤：
    (1)将如权利要求1所述的高活性凝血因子XI突变体Ala570Thr的编码基因连入载体中，得到重组载体；
    (2)将上述重组载体转化宿主细胞，得到表达重组凝血因子XI Ala570Thr突变细胞克隆；
    (3)于无血清培养基中连续灌流培养上述重组细胞克隆，诱导重组凝血因子XI Ala570Thr的突变蛋白的表达；
    (4)分离纯化、过滤，最后灌装、冻干，得到所表达的高活性凝血因子XI Ala570Thr突变蛋白。
  6. 一种高活性凝血因子XI突变体Ala570Thr的突变蛋白及其编码核酸序列的应用，其特征在于：应用权利要求1或3所述的核苷酸序列于制备基因治疗药物，包括将其与启动子和/或终止序列连接，构建表达质粒，基因治疗病毒或非病毒载体，表达权利要求2中的高活性凝血因子XI蛋白。
  7. 一种如权利要求2或3所述的高活性凝血因子XI突变体Ala570Thr的突变蛋白及其编码核酸序列的应用，其特征在于：用于基因编辑的病毒或非病毒载体或模板。
  8. 一种如权利要求2所述的高活性凝血因子XI突变体Ala570Thr的突变蛋白的应用，其特征在于：应用于制备血友病或其他出血性疾病的重组蛋白治疗药物。
  9. 一种如权利要求2所述的高活性凝血因子XI突变体Ala570Thr的突变蛋白的应用，其特征在于：应用于制备凝血因子XI Ala570Thr突变体融合蛋白，并将其施用于血友病或其他出血性疾病的重组蛋白治疗药物。
  10. 根据权利要求9所述的应用，其特征在于：所述融合蛋白为人白蛋白、免疫球蛋白Fc、转铁蛋白或alpha 1抗胰蛋白酶。
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