WO2004022598A1 - Proteine hybride assurant les fonctions d'hemolyse et d'anticoagulation - Google Patents

Proteine hybride assurant les fonctions d'hemolyse et d'anticoagulation Download PDF

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Publication number
WO2004022598A1
WO2004022598A1 PCT/CN2003/000743 CN0300743W WO2004022598A1 WO 2004022598 A1 WO2004022598 A1 WO 2004022598A1 CN 0300743 W CN0300743 W CN 0300743W WO 2004022598 A1 WO2004022598 A1 WO 2004022598A1
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WO
WIPO (PCT)
Prior art keywords
fusion protein
protein
hirudin
anticoagulant
thrombolytic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2003/000743
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English (en)
French (fr)
Chinese (zh)
Inventor
Bingxing Shi
Zuze Wu
Aiping Yu
Chunna Dong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Radiation Medicine of CAMMS
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Institute of Radiation Medicine of CAMMS
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Publication date
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Priority to US10/526,682 priority Critical patent/US8212003B2/en
Priority to JP2004533177A priority patent/JP2006516113A/ja
Priority to DE60316967T priority patent/DE60316967T2/de
Priority to AU2003261606A priority patent/AU2003261606A1/en
Priority to EP03793572A priority patent/EP1541589B1/en
Priority to CNB038207729A priority patent/CN1301268C/zh
Publication of WO2004022598A1 publication Critical patent/WO2004022598A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • 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)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • 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)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to a fusion protein of thrombolytic and anticoagulant proteins and a connecting peptide thereof, and more particularly, the present invention relates to an amino acid sequence of an anticoagulant protein and a protein molecule having plasminogen activation through an amino acid sequence that can be recognized and cleaved by a coagulation factor.
  • a fusion protein linked by a linker peptide the medical application of the fusion protein is the use of a linker peptide capable of being recognized by a coagulation factor sequence for linking thrombolytic and anticoagulant proteins.
  • Cardiovascular disease is the number one killer facing human beings in recent years.
  • the main thrombolytic drugs currently used in clinical thrombotherapy are urokinase and tissue-type plasminogen activator, and the main anticoagulants are heparin and hirudin.
  • thrombolytic therapy has played an important role in reducing the mortality of patients with thrombosis, small clots produced during thrombolytic therapy enter the blood circulation and easily cause re-embolization. Therefore, in clinical practice, heparin or hirudin is usually used as an anticoagulant drug and a thrombolytic drug in combination.
  • thrombolytic and anticoagulant drugs often cause hemorrhage and anticoagulation in the whole body due to their poor selectivity in the clinic, thus causing systemic bleeding.
  • tissue-type plasminogen activator t-PA
  • streptokinase S :
  • urokinase UK
  • urokinase-like plasminogen activator u-PA
  • SAK Staphylokinase
  • Hirudin is a new generation of natural anticoagulant small molecule protein drugs, which has high affinity and selective inhibition of thrombin.
  • hirudin is prone to cause systemic bleeding clinically, and hirudin does not have a corresponding antagonist.
  • An object of the present invention is to develop and provide a fusion protein capable of having both thrombolytic and anticoagulant functions.
  • the fusion protein has the following characteristics: the fusion protein remains soluble Thromboprotein activates plasminogen activity and retains thrombolytic function; when the N-terminus of anticoagulant protein such as hirudin is connected to the C-terminus of thrombolytic protein, the intact fusion protein does not have anticoagulation in vitro and in non-thrombotic sites of the blood system Activity, thereby avoiding or reducing the side effects of systemic bleeding caused by anticoagulant proteins such as hirudin; using the strong affinity of the C-terminus of anticoagulant proteins such as hirudin and thrombin to confer fusion protein targeting, thereby improving the drug The local concentration of the protein in the thrombus site reduces the clinical dosage.
  • the thrombus site After the fusion protein reaches the thrombus site with the blood circulation, the thrombus site The unique coagulation factors involved in thrombosis can quickly degrade the fusion protein at the designed recognition site, and release the free thrombolytic and anticoagulant proteins to obtain complete anticoagulant and thrombolytic activities.
  • the discovery of the present invention based on the above characteristics has now been completed.
  • the first aspect of the present invention relates to a fusion protein containing a thrombolytic protein, an anticoagulant protein, and a linker peptide.
  • Yet another aspect of the present invention relates to a method for preparing a fusion protein containing lysin and anticoagulant protein, which comprises linking a lysogenic thrombin protein and an anticoagulant gene with a corresponding base containing an IEGR or GPR sequence into a fusion protein gene, and The fusion protein gene is expressed in the system of E. coli, yeast or animal cells to obtain a fusion protein.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable carrier or excipient.
  • a further aspect of the present invention relates to the use of a linker peptide containing a coagulation factor recognition sequence for the preparation of a fusion protein containing lysed thrombus and anticoagulant protein.
  • a further aspect of the present invention relates to a linker peptide capable of being recognized by a coagulation factor and cleaving a sequence as a linker that dissolves thrombus and anticoagulant proteins.
  • Yet another aspect of the invention relates to a method of treating diseases and symptoms associated with thrombosis, which comprises administering a therapeutically effective amount of a fusion protein to a patient with a thrombus.
  • Figure 1 Schematic representation of the fusion protein.
  • FIG. 1 Electrophoresis of fusion protein gene, staphylokinase (SAK) gene, hirudin (HV2) gene.
  • SAK staphylokinase
  • HV2 hirudin
  • thrombolytic protein refers to a protein that can promote thrombolysis, such as staphylokinase (SAK), tissue-type plasminogen activator (t-PA), streptokinase (SK), urokinase (UK ), Urokinase-like plasminogen activator (u-PA), snake venom and other proteins that have the ability to activate other hemolytic factors or have a direct thrombolytic effect, or their mutants. Staphylokinase (SAK) or a mutant thereof is preferred.
  • anticoagulant protein or "anticoagulant protein” refers to a protein capable of anticoagulation, such as hirudin, antithrombin III, snake venom, etc., or a mutant thereof. Hirudin or a mutant thereof is preferred.
  • linker peptide of the coagulation factor recognition sequence refers to the amino acid tetrapeptide sequence IEGR (IleGluGlyArg) or a peptide containing IEGR, or the amino acid tripeptide sequence GPR (GlyProAr) or a peptide containing GPR.
  • the term "disease and symptoms related to thrombosis” refers to any disease or symptom caused by a thrombus, such as cerebral thrombosis, arterial thrombosis, stroke, atherosclerosis, and the like.
  • the term "patient” refers to mammals, especially humans.
  • the fusion protein of the present invention is preferably a fusion protein SFH (SAK-GSIEGR-HV2), a plasminogen activator (t-PA) and hirudin, which is a fusion protein formed by linking staphylokinase and hirudin through a linker peptide GSIEGR.
  • Fusion protein tPA-PRIEGR-HV2 linked by a linker peptide PRIEGR or fusion protein (SAK-GSGPR-HV2) linked by staphylokinase and hirudin through a linker peptide GSGPR.
  • the fusion protein of the present invention can be expressed in E. coli, Pichia, Saccharomyces cerevisiae or animal cells, preferably in E. coli or yeast.
  • Example 1 Preparation of SAK-GSIEGR-HV2 fusion protein (SFH) and its thrombolytic and anticoagulant activities
  • the synthetic primer was 5, CG GGA TCC ATC GAA GGT CGT ATT ACT TAC ACT GAT TGT ACA GAA TCG-3 ', hirudin downstream primers were synthesized with Pst I restriction sites; BamH I and Pst I were used to cut hirudin genes with FXa recognition sequence, and BamH I and Pst I double-digested the above pBVSAK vector, and the digested and recovered hirudin gene fragment was ligated to the pBVSAK vector to obtain the pBVSFH plasmid.
  • a splicing overlap PCR method may be used to connect the two gene fragments.
  • the pBVSFH plasmid was transformed into E.
  • the fusion protein (SFH) with a purity of 96% or more was obtained by ion exchange and gel filtration.
  • the fusion protein contained three functional regions: SAK amino acid sequence, FXa recognition sequence GSIEGR, and hirudin amino acid sequence.
  • the amino acid sequence of the fusion protein (SFH) is
  • fusion protein was assayed by chromogenic substrate (S-2251). The fusion protein was lysed by a mouse tail. SFH has significant anticoagulant activity compared to SAK.
  • Xho I and Avr II digestion sites were added upstream and downstream of the tPA gene, respectively, to construct a tPA gene without a stop codon to the pPIC9 vector.
  • Avr II digestion site and FXa recognition were added upstream of the hirudin gene by PCR.
  • Amino acid coding bases, Avr II digestion sites and FXa recognition amino acid sequence coding bases were added using a primer synthesis method upstream of hirudin gene 5 and before the hirudin downstream primer synthesis with Not I enzyme Cleavage site; Avr II and Not I double digestion with FXa recognition sequence
  • the hirudin gene was connected to the tPA downstream of the pPIC9 vector with the tPA gene constructed above to form the fusion gene PAFH.
  • the plasmid pPAFEL was double digested with BamH I and Sal I to pPAFH and pPIC9K plasmids.
  • the PAFH gene was constructed into pPIC9K to obtain ⁇ -K gene.
  • the pPAFH-K plasmid was linearized, electrotransformed and recombined into the yeast genome, and expression was induced by methanol.
  • the fusion target protein contains tPA amino acid sequence, FXa recognition sequence and hirudin amino acid sequence.
  • STH SAK-GSLGPR-HV2 fusion protein
  • the primer synthesis method is used to synthesize to the hirudin gene Upstream 5, before the end, hirudin downstream primer synthesis with a Pst I digestion site; BamH I and Pst I double digestion of a hirudin gene with a thrombin (FXIIa) recognition sequence, and BamH I and Pst I dual enzymes
  • the pBVSAK vector was cut, and the hirudin gene fragment recovered by digestion was ligated to the pBVSAK vector to obtain the pBVSTH plasmid, which was identified by enzyme digestion.
  • a splicing overlap PCR method can be used to connect the two gene fragments.
  • the pBVSTH plasmid was transformed into E. coli, and expression was induced at 42 ° C.
  • the target fusion protein (STH) with a purity of 96% or more was obtained through ion exchange and gel filtration.
  • the fusion protein contained three functional regions: the SAK amino acid sequence, the thrombin (FXIIa) recognition sequence, and the hirudin amino acid sequence.

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PCT/CN2003/000743 2002-09-03 2003-09-03 Proteine hybride assurant les fonctions d'hemolyse et d'anticoagulation Ceased WO2004022598A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
US10/526,682 US8212003B2 (en) 2002-09-03 2003-09-03 Bifunctional fusion protein with thrombolytic and anticoagulant activities and uses thereof
JP2004533177A JP2006516113A (ja) 2002-09-03 2003-09-03 血栓溶解および抗凝固両方の機能を有する融合タンパク質、ならびにその使用
DE60316967T DE60316967T2 (de) 2002-09-03 2003-09-03 Kondensiertes protein, das sowohl thrombolytisch als auch gerinnungshemmend wirkt, und dessen verwendung
AU2003261606A AU2003261606A1 (en) 2002-09-03 2003-09-03 Fused protein with the function of both hemolysis and anticoagulation and the use of it
EP03793572A EP1541589B1 (en) 2002-09-03 2003-09-03 Fused protein with the function of both thrombolysis and anticoagulation and the use of it
CNB038207729A CN1301268C (zh) 2002-09-03 2003-09-03 溶栓和抗凝双功能融合蛋白及应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNA021290865A CN1480466A (zh) 2002-09-03 2002-09-03 一类溶栓抗凝双功能融合蛋白及应用
CN02129086.5 2002-09-03

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WO2004022598A1 true WO2004022598A1 (fr) 2004-03-18

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PCT/CN2003/000743 Ceased WO2004022598A1 (fr) 2002-09-03 2003-09-03 Proteine hybride assurant les fonctions d'hemolyse et d'anticoagulation

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US (1) US8212003B2 (enExample)
EP (1) EP1541589B1 (enExample)
JP (2) JP2006516113A (enExample)
CN (2) CN1480466A (enExample)
AT (1) ATE376001T1 (enExample)
AU (1) AU2003261606A1 (enExample)
DE (1) DE60316967T2 (enExample)
WO (1) WO2004022598A1 (enExample)

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Publication number Priority date Publication date Assignee Title
CN1896108B (zh) * 2005-06-01 2012-01-04 中国人民解放军军事医学科学院放射与辐射医学研究所 特异性抗凝血物质的制备及其应用
CN102443065A (zh) * 2005-06-01 2012-05-09 中国人民解放军军事医学科学院放射与辐射医学研究所 特异性抗凝血物质的制备及其应用

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EP1816201A1 (en) * 2006-02-06 2007-08-08 CSL Behring GmbH Modified coagulation factor VIIa with extended half-life
US8968728B2 (en) * 2006-05-12 2015-03-03 Bharat Biotech International Limited Chimeric fusion proteins
EP2103630B1 (en) * 2006-12-15 2011-11-16 Institute Of Radiation Medicine Academy Of Military Medical Sciences, PLA Preparation of low bleeding anticoagulant fusion protein and its use
CN100519585C (zh) * 2007-02-06 2009-07-29 中国人民解放军军事医学科学院基础医学研究所 P11与sak的融合蛋白及其制备方法和用途
KR101629702B1 (ko) * 2007-02-12 2016-06-13 체에스엘 베링 게엠베하 카잘-형 세린 프로테아제 억제제의 치료학적 적용
DK2915564T3 (da) * 2007-09-28 2021-02-08 Alexion Pharma Inc Antidoter mod faktor XA-inhibitorer og fremgangsmåder til anvendelse deraf
DE102009010611A1 (de) * 2009-02-25 2010-08-26 Siemens Aktiengesellschaft Vorrichtung und Verfahren zur Steuerung einer mit mehreren Brennern ausgestatteten Turbine für flüssige oder gasförmige Brennstoffe
KR20130099027A (ko) * 2010-08-05 2013-09-05 카운슬 오브 사이언티픽 앤드 인더스트리얼 리서치 혈전용해 및 응고억제 특성을 갖는 단백질 융합 구조물
CN102180973B (zh) * 2011-03-18 2012-08-29 重庆大学 靶向多功能防栓融合蛋白及其制备方法和应用
BR112015004734A2 (pt) * 2012-09-07 2017-11-21 Sanofi Sa proteínas de fusão para tratar uma síndrome metabólica
CN106366200B (zh) * 2015-07-23 2020-04-10 武汉光谷人福生物医药有限公司 制备重组葡激酶-水蛭素融合蛋白的方法
JP7028904B2 (ja) * 2019-03-21 2022-03-02 アカデミア シニカ 合成ペプチド、それを備える薬学的組成物及び血栓塞栓症関連疾患の治療におけるその使用
CN113150168B (zh) * 2021-01-29 2022-11-11 武汉真福医药股份有限公司 一种qk纤溶酶基因-水蛭素融合蛋白的制备方法及应用
KR102789819B1 (ko) * 2021-10-21 2025-03-31 중앙대학교 산학협력단 트롬빈 검출용 신규한 펩타이드 및 이의 용도

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1896108B (zh) * 2005-06-01 2012-01-04 中国人民解放军军事医学科学院放射与辐射医学研究所 特异性抗凝血物质的制备及其应用
CN102443065A (zh) * 2005-06-01 2012-05-09 中国人民解放军军事医学科学院放射与辐射医学研究所 特异性抗凝血物质的制备及其应用

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AU2003261606A1 (en) 2004-03-29
DE60316967T2 (de) 2008-05-08
JP2006516113A (ja) 2006-06-22
EP1541589A1 (en) 2005-06-15
ATE376001T1 (de) 2007-11-15
US8212003B2 (en) 2012-07-03
DE60316967D1 (de) 2007-11-29
US20060127389A1 (en) 2006-06-15
EP1541589A4 (en) 2005-11-16
EP1541589B1 (en) 2007-10-17
CN1678636A (zh) 2005-10-05
CN1301268C (zh) 2007-02-21
CN1480466A (zh) 2004-03-10

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