[go: up one dir, main page]

WO2006066960A1 - Variante de tfpi possedant une activite antithrombotique accrue - Google Patents

Variante de tfpi possedant une activite antithrombotique accrue Download PDF

Info

Publication number
WO2006066960A1
WO2006066960A1 PCT/EP2005/013962 EP2005013962W WO2006066960A1 WO 2006066960 A1 WO2006066960 A1 WO 2006066960A1 EP 2005013962 W EP2005013962 W EP 2005013962W WO 2006066960 A1 WO2006066960 A1 WO 2006066960A1
Authority
WO
WIPO (PCT)
Prior art keywords
tfpi
seq
polypeptide
factor
amino acid
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/EP2005/013962
Other languages
English (en)
Inventor
Bernd Engelmann
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.)
Ludwig Maximilians Universitaet Muenchen LMU
Original Assignee
Ludwig Maximilians Universitaet Muenchen LMU
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Ludwig Maximilians Universitaet Muenchen LMU filed Critical Ludwig Maximilians Universitaet Muenchen LMU
Publication of WO2006066960A1 publication Critical patent/WO2006066960A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8114—Kunitz type inhibitors

Definitions

  • the present invention relates to variant Tissue Factor Pathway Inhibitor - 1 alpha (TFPI - I alpha) characterized by increased resistance to inactivation and enhanced anticoagulant activity as compared to a native TFPI - I alpha protein. More specifically, the invention relates to variant TFPI- I alpha characterized by increased resistance to protease cleavage and increased inhibitory effect on formation of the initiation complex of coagulation .
  • TF tissue factor
  • TF activates factor X by forming a complex with factor Vila , thereby initiating the coagulation cascade .
  • the ternary initiation complex of coagulation thus formed is rapidly shut off by TFPI , which interrupts the coagulation cascade at its earliest stage (Mousa et al . , 2004 , Methods MoI . Med. 93 : 133 -155 ; Kato 2002 , Arterioscler . Thromb .
  • TFPI is expressed and stored in endothelial cells , platelets and other cells .
  • endogenous inhibitors including antithrombin III , protein C, and others .
  • TFPI native TFPI might contribute to prevent pathological thrombus formation.
  • overexpression of TFPI can prevent arterial thrombosis (Kopp et al , 2004 , Blood, 103 : 1653 - 1661 ) .
  • TFPI has been proposed as a therapeutic agent in humans (WO03/055442 ) .
  • This document discloses a TFPI analog that contains an additional N-terminal Alanine for improved expression in E . coli . Delivery of TFPI by recombinant adeno- associated virus has been proposed (WOOl/09360 ) .
  • chimeric proteins are disclosed that comprise portions of TFPI - I and TFPI -2.
  • Other structural derivatives of TFPI namely isolated Kunitz domains 1 to 3 are disclosed in WO93/14120 , WO93 /14121 and WO93 /14122.
  • TFPI participates in the pathogenesis of sepsis , in particular in the pathological induction of coagulation during sepsis known as disseminated intravascular coagulation (Levi et al , 2003 , Cardiovasc . Res . 60 : 26 -39 ; Osterud & Bj orklid 2001 , Semin . Thromb . Hemost . 27 : 605-617 ) .
  • disseminated intravascular coagulation Levi et al , 2003 , Cardiovasc . Res . 60 : 26 -39 ; Osterud & Bj orklid 2001 , Semin . Thromb . Hemost . 27 : 605-617 .
  • the participation of TFPI in a variety of further diseases has been documented, including different types of inflammatory conditions (Golino P, Cimmino G . Semin Vase Med .
  • an inhibitor of blood coagulation suitable as a therapeutic agent , in particular an inhibitor suitable for the prevention and treatment of a disease involving TFPI and the tissue factor/factor Vila/factor Xa-complex, such as thrombosis .
  • the present invention solved this aim by providing TFPI variants having increased resistance to fragmentation by pathophysiological ⁇ relevant proteases , in particular serine proteases , and increased anti-coagulant activity.
  • the present invention relates to : 1.
  • a variant of the polypeptide of SEQ ID No : 1 having increased protease resistance at one or more protease cleavage sites , and an increased anti -coagulant activity as compared to the amino acid sequence according to SEQ ID No : 1.
  • polypeptide according to item 1 wherein said one or more protease cleavage sites are one or more of cathG, HNE , MMP , thrombin and plasmin cleavage sites .
  • polypeptide according to any one of item 1 to 4 comprising one or more variations affecting the cleavage site between Threonin 87 and Threonin 88 of SEQ ID No : 1.
  • polypeptide according to any one of item 1 to 5 comprising one or more variations affecting the cleavage site between Leucine 89 and Glutamine 90 and / or Tyrosine 159 and Glycine 160 of SEQ ID No : 1.
  • polypeptide according to item 10 comprising the substitutions T87F and L89A.
  • a vector comprising the polynucleotide of item 12.
  • a host cell comprising the vector of item 13.
  • a polypeptide expressed by the host cell of item 14 wherein said polypeptide is encoded by the vector of item 13.
  • a pharmaceutical composition comprising the polypeptide of any one of item 1 to 11 or 15 ⁇
  • a pharmaceutical composition comprising the polynucleotide of item 12.
  • a pharmaceutical composition comprising the vector of item 13.
  • polypeptide according to any one of item 1 to 11 or 15 for the manufacture of a medicament for the treatment and/or prevention of a disease involving TFPI and the tissue factor/factor Vila/factor Xa-complex 20.
  • polynucleotide according to item 12 for the manufacture of a medicament for the treatment and/or prevention of a disease involving TFPI and the tissue factor/factor Vila/factor Xa-complex .
  • the present invention relates to variants of TFPI -I alpha protein .
  • TFPI - I is a glycosylated molecule comprising 276 amino acid residues (SEQ ID No : 1) .
  • the terms TFPI - I alpha and TFPI - I are used interchangeably.
  • "Native " TFPI-I alpha protein relates to a TFPI -I polypeptide with an amino acid sequence that can be isolated from a human being .
  • native TFPI - I comprises the natural diversity with respect to the amino acid sequence as can be found in humans .
  • the native TFPI polypeptide has an amino acid sequence as set forth in SEQ ID No : 1.
  • SEQ ID No : 1 comprises , starting from the N-terminus in sequence : An N-terminal amino acid sequence followed by the Kunitz- 1 domain, followed by an amino acid sequence between the Kunitz- 1 and Kunitz-2 domains , the Kunitz-2 domain, an amino acid sequence between the Kunitz-2 and Kunitz-3 domain, the Kunitz-3 domain and a C-terminal amino acid sequence .
  • Kunitz domains are distinct domains of TFPI - I characterised by three disulfide bridges each .
  • Kunitz domains I , II and III are commonly defined as spanning amino acid residues 26 to 76 , 97 to 127 and 189 to 239 respectively, of SEQ ID No : 1.
  • variant polypeptide as used herein relates to the polypeptide according to SEQ ID No : 1 , which contains a variation of the amino acid sequence , provided said variant polypeptide has increased protease resistance at one or more protease cleavage sites , and an increased anti -coagulant activity as compared to the protein with the amino acid sequence according to SEQ ID No : 1.
  • Preferable variations comprise additions , substitutions or deletions of one or more amino acid residues .
  • the variant of the present invention has at least 75% homology to SEQ ID No : 1 , more preferably at least 85% homology, more preferably at least 95% homology .
  • homology means % identity of amino acid residues when comparing the variant amino acid sequence with the native sequence .
  • a variant consists of 100 amino acid residues of which 90 are identical to corresponding parts of the native sequence , that variant has 90% homology .
  • Corresponding parts of the native sequence can be determined e . g . by sequence alignment by methods commonly known in the art .
  • variants are obtained by introducing amino acid variations by methods well known to the person skilled in the art .
  • molecular biology methods are used to introduce changes to a polynucleotide sequence encoding SEQ
  • the variant polypeptide may preferably be a fragment which lacks an amino acid sequence at the N-terminal end and/or the C-terminal end, or comprise deletions within the amino acid sequence .
  • the fragment may lack part of the C-terminal sequence following the Kunitz-3 domain, and optionally in addition lack the
  • Kunitz- 3 domain and optionally in addition lack all or part of the amino acid sequence spanning Kunitz-2 and Kunitz-3 domains in SEQ ID No : 1.
  • a fragment may also lack all or part of the N-terminal amino acid sequence preceding the Kunitz-1 domain .
  • the fragment lacking the amino acid sequence at the N-terminal end and/or the C-terminal end can be obtained by a standard method known to the person skilled in the art .
  • the variants carrying a deletion can in addition carry one or more variations affecting one or more protease cleavage sites .
  • affecting a cleavage site means that the resistance to cleavage by a given protease at this particular cleavage site is increased in comparison with the native sequence , in particular in comparison to SEQ ID NO:
  • Increased protease resistance means that upon exposure to the protease , the variant polypeptide is cleaved to a lesser degree as compared to the native polypeptide .
  • the variant TFPI can be incubated with a given protease , and fragments analysed by SDS-PAGE . Increased protease resistance will result in a decrease in fragment formation as compared to TFPI not carrying the variation .
  • Fragment formation can be quantified by various means , e . g . by quantification of the bands on the SDS gel by densitometry, or other methods .
  • this method can optionally be combined with N- terminal sequencing of fragments e . g . isolated from the gel . Preferred methods are specified in the examples .
  • Resistance to protease cleavage is considered to be increased when it is preferably at least 20% higher as compared to the native amino acid sequence , more preferably at least 40% higher, more preferably at least 60% higher, more preferably at least 80% higher, and most preferably at least 100% higher, wherein preferably the native amino acid sequence is SEQ ID No : 1.
  • the variant of the present invention is characterized by increased anticoagulant activity .
  • the skilled person readily knows methods to determine anticoagulant activity of the variants of the invention . Any known method to determine blood clotting can be employed, preferably a method wherein clotting is induced by recombinant tissue factor (TF) . Preferred methods are provided in the examples .
  • anti-coagulant activity is measured in whole blood, or, alternatively, in an assay utilizing platelets and neutrophils .
  • Anti -coagulant activity is considered to be increased, when the readout of the coagulation test is preferably at least 20% higher as compared to the native amino acid sequence , more preferably at least 40% higher, more preferably at least 60% higher, and most preferably at least 80% higher, wherein preferably the native amino acid sequence is SEQ ID No : 1.
  • the anti -coagulant activity is enhanced 1-2 fold, more preferably 2 -4 fold, most preferably 2.4 -3.6- fold .
  • Preferred readouts of coagulation tests comprise the quantification of generation of the initiation complex of coagulation, more preferably the quantification of generation of factor Xa .
  • Another preferred readout of a coagulation test is the determination of coagulation time by thromboelastography, preferably in whole blood .
  • the variants of the present invention comprise variations at one or more amino acid residues directly adj acent to one or more protease cleavage sites .
  • the skilled person can readily determine the cleavage sites of TFPI -I for any protease of interest .
  • a standard method comprises exposing the substrate , i . e . TFPI to the protease , separating fragments , e . g . by SDS-polyacrylamide gel electrophoresis (PAGE) , and N-terminal sequencing of the fragments .
  • the N- terminal sequence can be aligned with the known amino acid sequence of the substrate , and thus the position of the cleavage site identified .
  • Other methods than SDS-PAGE can be employed to separate cleavage fragments .
  • HNE human neutrophil elastase
  • cathepsin G cathepsin G
  • MMP matrix metalloproteases
  • protease cleavage sites identified in TFPI are mostly non-canonical .
  • no canonical cleavage sites for CatG, HNE and MMP-9 are known in TFPI . It is therefore not surprising that even when exposed to proteases in vitro, no rapid cleavage (as would be expected for natural protease substrates) is observed . Examples of such natural substrates are the members of the coagulation cascade , where several serine proteases cleave their substrates within seconds or less .
  • in vitro cleavage of TFPI has been reported to follow a substantially slower time-course , e . g . in the range of minutes to hours (Cunningham et al , Biochem. J . 2002 , 367 , 451-458 ) .
  • Variations can be limited to one side of the cleavage site , e . g . to amino acid 87 when the cleavage site lies between amino acids 87 and 88 , or affect both sides of the cleavage site .
  • the present invention also encompasses variations affecting amino acid residues not directly adj acent to the cleavage site , provided they show the said effect on protease resistance and anti -coagulation activity .
  • the present invention relates to variants with increased resistance at the cleavage sites of one or more of cathepsin G (cathG) , human neutrophil elastase (HNE) , matrix metalloproteinase (MMP) , thrombin and plasmin, more preferably one or more of cathG, HNE , and MMP and most preferably cathG and / or HNE .
  • cathG cathepsin G
  • HNE human neutrophil elastase
  • MMP matrix metalloproteinase
  • thrombin plasmin
  • Preferred matrix metalloproteinases comprise MMP- I , MMP- 7 , MMP- 8 , MMP- 9 and MMP- 12.
  • the variant comprises a variation affecting preferably one or more protease cleavage sites in SEQ ID No : 1 of a protease that does not cleave Kunitz domains , more preferably one or more cleavage sites of proteases cleaving within at least one of the amino acid sequences linking Kunitz domains , and most preferably the cleavage sites of one or more proteases that cleave within the amino acid sequence between the Kunitz- 1 and Kunitz-2 domains .
  • Proteases that cleave within the amino acid sequence between the Kunitz- 1 and Kunitz-2 domains comprise MMP- 12 , Cath G, Thrombin, Plasmin and HNE .
  • variants comprise one or more variations affecting the cleavage sites between Threonin 87 and Threonin 88 of SEQ ID No : 1 , and / or Leucine 89 and Glutamine 90 , and / or Tyrosine 159 and Glycine 160 of SEQ ID No : 1. More preferably, the variants according to the present invention comprise a variation affecting the cleavage site between Threonin 87 and Threonin 88 of SEQ ID No : 1 , and a variation affecting the cleavage site between Leucine 89 and Glutamine 90.
  • the amino acid at position 87 of SEQ ID No : 1 is substituted by Phenylalanine (T87F)
  • / or the amino acid at position 89 of SEQ ID No : 1 is substituted by Alanine (L89A)
  • / or the amino acid at position 159 of SEQ ID No : 1 is substituted by Alanine (Y159/A) .
  • any other amino acid can be substituted at these positions , which has an effect on resistance to protease cleavage , which can be experimentally determined as described above .
  • an exchange of amino acids based on theoretical considerations only is not possible .
  • the variant comprises the substitutions T87F and L89A. It is likely that apart from providing resistance towards proteolytic degradation, the variations introduced into the variants of the present invention elicit additional functional changes in the protein, such as , e . g . , providing an increased affinity for the TFPI substrates factor Vi la and Xa . Also , the variants may alternatively or in addition be characterized by an increased plasma half life . Thus , the variants generated retain the canonical anticoagulant function of TFPI , but in addition increase its competence as inhibitor of the start process of fibrin formation .
  • the polypeptide of the invention can be produced by standard methods well known in the art .
  • the polypeptide can be produced by recombinant expression ( see below) .
  • the effects of different hosts for recombinant expression on glycosylation are well known in the art .
  • the skilled person can select the appropriate host system for the desired pattern of glycosylation .
  • polypeptide of the present invention can be isolated and purified by the following means :
  • glycosylated TFPI using mammalian cell hosts including mouse C127 cells , baby hamster kidney cells , Chinese hamster ovary cells and human SK hepatoma cells has been reported (Day et al , 1990 , Blood 76 : 1538 - 1545 ; Pedersen et al , 1990 , J . Biol . Chem. 265 : 16786- 16793 ;
  • the polypeptides can be chemically synthesized by methods such as direct peptide synthesis using solid-phase techniques .
  • fragments of the polypeptide of the invention can be separately synthesized and combined using chemical methods to produce a full -length molecule .
  • variants of SEQ ID No : 1 also include variants in which one or more of the amino acids therein has an altered side chain .
  • derivatized polypeptides include, for example , those comprising amino acids in which free amino groups form amine hydrochlorides , p-toluene sulfonyl groups , carobenzoxy groups ; the free carboxy groups form salts , methyl and ethyl esters ; free hydroxl groups that form 0-acyl or O-alkyl derivatives as well as naturally occurring amino acid derivatives , for example , 4 -hydroxyproline , for proline , 5-hydroxylysine for lysine , homoserine for serine , ornithine for lysine etc .
  • amino acid derivatives that can alter covalent bonding, for example , the disulfide linkage that forms between two cysteine residues that produces a cyclized polypeptide .
  • the variants of the invention also comprise polypeptides cyclizised by other means than a disulfide bridge .
  • the polypeptides of the present invention may also be modified by a modifying agent .
  • modifying agent include polyethylene glycol (PEG) , dextran, poly (N-vinyl -pyrrolidone) , polypropylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol , polyvinyl alcohol , and the like .
  • the modification of the protein with PEG can be performed by, for example , a method in which activated ester derivatives of PEG is reacted with the protein, a method in which aldehyde derivatives at the terminal portion of PEG is reacted with the protein in the presence of a reducing agent , and the like .
  • polynucleotide of the invention Another embodiment of the invention relates to a polynucleotide encoding a variant polypeptide of SEQ ID No : 1 having increased protease resistance at one or more protease cleavage sites and an increased anti -coagulant activity as compared to the amino acid sequence according to SEQ ID No : 1.
  • polynucleotide is to be construed in the broadest sense and comprises any polymer of nucleotides .
  • polynucleotide " and “nucleic acid” are used interchangeably .
  • the polynucleotide of the present invention can be in the form of DNA (deoxyribonucleic acid) which contains the bases adenine , thymine , guanine and cytosine or RNA (ribonucleic acid) which contains the bases adenine , uracil , guanine and cytosine or mixtures of the two .
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the polynucleotides of the present invention also include modifications of the polynucleotide wherein one or more of the bases of the nucleic acids can be replaced by modified bases .
  • modified bases include 2 -methyladenine , 1 - methylguanine , 5-methylcytosine , and 5-hydroxymethylcytosine .
  • a polynucleotide according to the invention may be inserted into a vector .
  • vector refers to a plasmid, virus or other vehicle known in the art that can be manipulated by insertion or incorporation of a polynucleotide .
  • Such vectors can be used for genetic manipulation (i . e . , " cloning vectors " ) or can be used to transcribe or translate the inserted polynucleotide ( "expression vectors " ) .
  • a vector generally contains at least an origin of replication for propagation in a cell and a promoter .
  • Control elements including expression control elements as set forth herein, present within an expression vector are included to facilitate proper transcription and translation (e . g .
  • control element is intended to include , at a minimum, one or more components whose presence can influence expression, and can also include additional components , for example , leader sequences and fusion partner sequences .
  • an expression control element refers to one or more nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked .
  • An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate , translation of the nucleic acid sequence .
  • an expression control element can include , as appropriate , promoters , enhancers, transcription terminators , a start codon (e . g . , ATG) in front of a protein-encoding gene .
  • "Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner .
  • promoter is meant a minimal sequence sufficient to direct transcription .
  • constitutive and inducible promoters are included in the invention (see e . g . , Bitter et al . , Methods in Enzymology 153 : 516 - 544 , 1987) .
  • Inducible promoters are activated by external signals or agents .
  • those promoter elements which are sufficient to render promoter-dependent gene expression controllable for specific cell -types , tissues or physiological conditions ; such elements may be located in the 5 ' , 3 ' or intronic regions of the gene . Promoters useful in the invention also include conditional promoters .
  • conditional promoter is a promoter which is active only under certain conditions .
  • the promoter may be inactive or repressed when a particular agent , such as a chemical compound, is present . When the agent is no longer present , transcription is activated or derepressed .
  • constitutive promoters such as T7 and the like , as well as inducible promoters such as pi , of bacteriophage X, plac , ptrp, ptac (ptrp-lac hybrid promoter) may be used .
  • constitutive promoters such as SV40 , RSV, CMV including CMV- IE , and the like or inducible promoters derived from the genome of mammalian cells (e . g . , metallothionein promoter) or from mammalian viruses (e . g . , the mouse mammary tumor virus long terminal repeat ; the adenovirus late promoter) may be used .
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention .
  • Mammalian expression systems which utilize recombinant viruses or viral elements to direct expression may be engineered .
  • nucleic acid of interest may be ligated to an adenovirus transcription/translation control complex, e . g . , the late promoter and tripartite leader sequence .
  • the vaccinia virus 7.5K promoter may be used .
  • vectors based on bovine papilloma virus which have the ability to replicate , as extrachromosomal elements . Shortly after entry of an extrachromosomal vector into mouse cells , the vector replicates to about 100 to 200 copies per cell . Because transcription of the inserted cDNA does not require integration of the plasmid into the host ' s chromosome , a high level of expression occurs .
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene , for example .
  • the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the nucleic acid of interest in host cells . High level expression may also be achieved using inducible promoters , including, but not limited to, the metallothionein RA promoter and heat shock promoters .
  • yeast a number of vectors containing constitutive or inducible promoters may be used .
  • a constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used .
  • vectors that facilitate integration of foreign nucleic acid sequences into a yeast chromosome , via homologous recombination for example are known in the art and can be used .
  • a nucleic acid of interest according to the present invention may be inserted into an expression vector for expression in vitro (e . g .
  • a cell into which a vector can be propagated and its nucleic acid transcribed, or encoded polypeptide expressed is referred to herein as a "host cell " .
  • the term also includes any progeny of the subj ect host cell .
  • a nucleic acid of interest according to the present invention may be inserted into an expression vector for expression in vivo for somatic gene therapy for example .
  • these vectors for example , retroviral vectors , lentivirus vectors , Adenovirus vectors , Adeno-associated virus vectors , plasmid expression vectors , the nucleic acids of the invention are expressed upon infection/introduction of the vector into a suitable tissue/cell of the subj ect to be treated .
  • Host cells include but are not limited to microorganisms such as bacteria, yeast , insect and mammalian organisms .
  • bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors containing a nucleic acid of interest e. g . , bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors containing a nucleic acid of interest ; yeast transformed with recombinant yeast expression vectors containing a nucleic acid of interest ; plant cell systems infected with recombinant virus expression vectors (e . g . , cauliflower mosaic virus , CaMV; tobacco mosaic virus , TMV) or transformed with recombinant plasmid expression vectors (e . g .
  • Ti plasmid containing a nucleic acid of interest ; insect cell systems infected with recombinant virus expression vectors (e . g . , baculovirus) containing a nucleic acid of interest ; or animal cell systems infected with recombinant virus expression vectors (e . g . , retroviruses , lentivirus , adenovirus , vaccinia virus ) containing a nucleic acid of interest , or transformed animal cell systems engineered for stable expression .
  • recombinant virus expression vectors e . g . , baculovirus
  • animal cell systems infected with recombinant virus expression vectors e . g . , retroviruses , lentivirus , adenovirus , vaccinia virus
  • expression vectors which contain viral origins of replication for example, cells can be transformed with a nucleic acid of interest controlled by appropriate control elements (e . g . , promoter/enhancer sequences , transcription terminators , polyadenylation sites , etc . ) .
  • the expression vector also can contain a nucleic acid encoding a selectable or identifiable marker conferring resistance to a selective pressure thereby allowing cells having the vector to be identified, grown and expanded .
  • the selectable marker can be on a second vector that is cotransfected into a host cell with a first vector containing an invention polynucleotide .
  • a number of selection systems may be used, including, but not limited to the herpes simplex virus thymidine kinase gene , hypoxanthine-guanine phosphoribosyltransferase gene , and the adenine phosphoribosyltransferase genes can be employed in tk- , hgprt or aprt cells respectively .
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate ; the gpt gene , which confers resistance to mycophenolic acid; the neomycin gene , which confers resistance to the aminoglycoside G-418 ; and the hygromycin gene , which confers resistance to hygromycin .
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • transformation means a genetic change in a cell following incorporation of DNA exogenous to the cell ' .
  • a "transformed cell” is a cell into which (or a progeny of which) a DNA molecule has been introduced by means of recombinant DNA techniques .
  • Transformation of a host cell with DNA may be carried out by- conventional techniques known to those skilled in the art .
  • methods of DNA transformation include , for example , calcium phosphate co-precipitates , conventional mechanical procedures such as microinj ection, electroporation, insertion of a plasmid encased in liposomes , and viral vectors .
  • Eukaryotic cells also can be cotransformed with DNA sequences encoding a nucleic acid of interest , and a second foreign DNA molecule encoding a selectable phenotype , such as the those described herein .
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40 ) or bovine papilloma virus , to transiently infect or transform eukaryotic cells and express the protein .
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • said RNA molecule can be delivered to a subj ect by transforming the cells of said subj ect with an expression vector that comprises a nucleotide sequence encoding said RNA molecule under control of sequence that allows for the transcription of the nucleotide sequence .
  • the protein molecule can be delivered to the cells of a subj ect by transforming said cell with an expression vector that comprises a nucleotide sequence encoding said protein under control of sequence that allows for the transcription of the nucleotide sequence .
  • the present invention relates to a vector, preferably an expression vector comprising a nucleic acid sequence encoding a polypeptide of the invention.
  • the present invention relates to a host cell comprising the vector of the invention, preferably a host cell transformed with the above mentioned expression vector .
  • the present invention also relates to polypeptides expressed by the host cell of the invention .
  • the polypeptides can be obtained using recombinant expression methods as disclosed herein .
  • polynucleotide encoding the protein can be produced, inserted into a vector and transformed into host cells using well known techniques described herein and further known in the art (Sambrook et al . , Molecular Cloning : A Laboratory Manual , Cold Spring Harbor Laboratory, N.Y . , 1989) .
  • protein may be isolated and purified in accordance with conventional methods . For example , lysate prepared from an expression host (e .g .
  • bacteria can be purified using HPLC,_ size-exclusion chromatography, gel electrophoresis , affinity chromatography, or other purification technique .
  • Other protein purification methods known in the art additionally can be used (see e . g . , Deutscher et al . , Guide to Protein Purification : Methods in Enzymology, Vol . 182 , Academic Press , 1990 ) .
  • compositions of the invention are provided.
  • the present invention also relates to pharmaceutical compositions comprising the polypeptide or polynucleotide of the invention .
  • the pharmaceutical composition of the invention comprises the vector of the invention .
  • the pharmaceutical composition according to the invention can also comprise further therapeutic compounds .
  • Such further compounds comprise other substances suitable to reduce coagulant activity .
  • additional compounds do not affect factor Xa, such as salicylic acid or clopidogrel .
  • factors like heparin or coumarin may also be used.
  • the composition of the present invention may also comprise substances administered to patients suffering from cardiovascular diseases , such as ⁇ -blockers , angiotensin converting enzyme (ACE) inhibitors , or substances affecting blood lipids , such as statins .
  • ACE angiotensin converting enzyme
  • compositions with an amount of one or more compounds (polypeptide / polynucleotide) according to the invention and/or their use in the application according to the invention occurs in the customary manner by means of common pharmaceutical technology methods .
  • the compounds according to the invention are processed together with suitable , pharmaceutically acceptable excipients , carriers etc . to medicinal forms suitable for the various indications and types of application .
  • the medicaments can be produced in such a manner that the respective desired release rate is obtained, for example a quick flooding and/or a sustained or depot effect .
  • a pharmaceutical composition comprising one or more polypeptides of the invention can be produced by mixing medically acceptable diluent , stabilizer, carrier, and/or other additives with the polypeptides of the present invention .
  • the polypeptides of the present invention may be modified by a modifying agent , such as PEG, as described above .
  • a pharmaceutical composition comprising the polynucleotides of the invention can comprise agents that increase cell membrane permability and/or cellular uptake of the nucleic acids .
  • these agents are polyamines as described for example by Antony, T . et al . ( 1999) Biochemistry 38 : 10775- 10784 ; branched polyamines as described for example by Escriou, V . et al ( 1998) Biochem. Biophys . Acta 1368 (2 ) : 276 - 288 ; polyaminolipids as described for example by Guy-Caffey, J . K. et al . ( 1995 ) J . Biol . Chem.
  • DOTMA desribed by Feigner , P . L . et al . ( 1987 ) PNAS USA 84 (21 ) : 7413 -7417 and cationic porphyrins as described for example by Benimetskaya , L . et al . (1998 ) NAR 26 (23 ) : 5310 -5317.
  • a usual method used for transfer of a gene into animal cells is employed for the transfer of the gene for the therapy into the target tissue or cell of the subj ect to be treated .
  • Examples include a method using a vector for animal cells derived from virus utilized for a gene therapy such as retrovirus vectors such as Moloney mouse leukemia virus , adenovirus vectors , adeno-associated virus (AAV) vectors , herpes simplex virus vectors , and HIV vectors (with respect to a vector for gene therapy, see Verma, I . M. , Nature , 389 : 239 , 1997) ; calcium phosphate transfection, DEAE-dextran transfection, electroporation, the liposome method, the lipofection method, the microinj ection method, and the like .
  • retrovirus vectors such as Moloney mouse leukemia virus , adenovirus vectors , adeno-associated virus (AAV) vectors , herpes simplex virus vectors , and HIV vectors
  • retrovirus vectors such as Moloney mouse leukemia virus , adenovirus vectors , adeno-associated virus (
  • the pharmaceutical composition of the present invention is a preparation for systemic application, preferably for parenteral use , more preferably inj ections and infusions .
  • inj ections are prepared either in the form of vials or also as so-called ready-to-use inj ection preparations , for example as ready-to-use syringes or single use syringes in addition to perforation bottles for multiple withdrawals .
  • Administration of the inj ection preparations can occur in the form of subcutaneous (s . c ) , intramuscular (i . m. ) , intravenous (i . v . ) , internodal (i . n . ) or intracutaneous (i . e . ) application, preferably i . v ..
  • the respective suitable inj ection forms can especially be produced as solutions , crystal suspensions , nanoparticular or colloid-disperse systems , such as for example , hydrosols .
  • the inj ectable formulations can also be produced as concentrates which can be adjusted with aqueous isotonic dilution agents to the desired dosage of the compounds of the invention . Furthermore , they can also be produced as powders , such as for example lyophilisates , which are then preferably dissolved or dispersed immediately before application with suitable diluents .
  • the infusions can also be formulated in the form of isotonic solutions , fat emulsions , liposome formulations , microemulsions and liquids based on mixed micells , for example , based on phospholipids . As with inj ection preparations , infusion formulations can also be prepared in the form of concentrates to dilute .
  • the inj ectable formulations can also be applied in the form of continuous infusions as in stationary as well as in out patient therapy, for example in the form of mini -pumps .
  • Albumin, plasma expanders , surface active compounds , organic solvents , pH influencing compounds , complex forming compounds or polymeric compounds can be added to the parenteral medicinal forms with the aim of decreasing the adsorption of the compounds of the present invention to materials such as inj ection instruments or packaging materials , for example plastic or glass .
  • the compounds according to the invention can be bound to nanoparticles in the preparations for parenteral use , for example on finely dispersed particles based on poly (meth) acrylates , polyacetates , polyglycolates , polyamino acids or polyether urethanes .
  • the parenteral formulations can also be constructively modified as depot preparations , for example on the multiple unit principle , where the compounds of the present invention are incorporated in a most finely distributed and/or dispersed, suspended form or as crystal suspensions , or on the single unit principle , where the compounds according to the invention are enclosed in a medicinal form, for example , a tablet or a seed which is subsequently implanted .
  • these implantation or depot medicaments in single unit and multiple unit medicinal forms consist of so-called biodegradable polymers , such as for example , polyether urethanes of lactic and glycolic acid, polyether urethanes , polyamino acids , poly (meth) acrylates or polysaccharides .
  • Sterilized water pH value influencing substances , such as for example organic and inorganic acids or bases as well as their salts , buffer substances for setting the pH value , agents for isotonicity, such as for example sodium chloride , monosodium carbonate , glucose and fructose , tensides and/or surface active substances and emulsifiers , such as for example , partial fatty acid esters of polyoxyethylene sorbitan (Tween ® ) or for example fatty acid esters of polyoxethylene (Cremophor ® ) , fatty oils such as for example .peanut oil , soybean oil and castor oil , synthetic fatty acid esters , such as for example ethyl oleate , isopropyl myristate and neutral oil (Miglyol ® ) as well as polymer adjuvents such as for example gelatin, dextran, polyvinylpyrrolidone , organic solvent additives which increase solubility
  • pressed implants are suitable which are preferably formulated on the basis of so-called biodegradable polymers .
  • so-called electronically controlled release systems can also be formulated by which release of the compounds of the present invention can be selectively adjusted to individual needs .
  • the pharmaceutical composition of the invention can also be applied locally at a site that is particularly prone to thrombus formation, e . g . by means of an intraluminal catheter.
  • the pharmaceutical composition can be applied at the site of vascular or heart surgery, in particular surgery performed using indwelling catheters or devices , or can be applied at a site of thrombolytic intervention .
  • compositions of the present invention can be applied directly, e . g . in form of an inj ection, or can be applied indirectly .
  • the pharmaceutical compositions can be comprised in a coating on a medical device .
  • the coating may be a fixed layer, or may liberate the composition of the invention to the surrounding fluid or tissue .
  • Such coating may optionally comprise further substances that result in a prolonged release of the composition of the present invention .
  • Such substances are well known to the skilled person.
  • Examples of preferred medical devices are intraluminal catheters , artificial cardiac valves , hemodialysis equipment or any other devices that extend into the lumen of blood vessels , or are otherwise in contact with blood .
  • a preferred embodiment of a medical device is a stent or a vascular implant with a coating comprising the polypeptide or the polynucleotide of the invention . Stents for treatment of vascular stenosis are particularly preferred .
  • the present invention also relates to the use of the polypeptide or polynucleotide of the invention for the manufacture of a medicament for the treatment and / or prevention of a disease involving TFPI and the tissue factor/factor Vila/factor Xa-complex .
  • Preferred examples of such diseases comprise thrombosis or conditions associated with an increased risk of thrombosis .
  • Further preferred examples comprise ischemia/reperfusion damage , cancer, and inflammatory conditions .
  • Thrombosis comprises arterial and venous thrombosis that may be the primary cause of a disease or syndrome or be associated with a disease or syndrome as a secondary event .
  • Thrombosis is e . g . responsible for the mortality resulting from myocardial infarction, stroke , or lung emboli .
  • arterial thrombosis represents a maj or mediator of the lethal complications associated with sepsis .
  • the polypeptide and / or polynucleotide are used for the manufacture of a medicament for the prevention of thrombosis in patients with an increased risk of thrombosis .
  • the risk of thrombosis is enhanced e . g . in subj ects suffering from arteriosclerosis , patients having undergone surgery, patients suffering from inflammatory disorders , in particular disorders affecting blood vessels , bed ridden patients , patients suffering from a malignant disease as well as patients with ischemia / reperfusion damage .
  • the risk of thrombosis can be enhanced due to alterations of blood cells , plasma, blood flow and / or vessel walls .
  • Such alterations can be determined by a range of clinical parameters , such as e . g . increased aggregation of thrombocytes , erythrocytes and increased blood viscosity, elevation of pro-coagulant factors (e . g . fibrinogen) or reduction of anti-coagulant (e . g . antithrombin III , protein C) and abnormalities of fibrinolysis .
  • risk factors for arteriosclerotic diseases such as smoking, hypercholesterinaemia , hypertension as well as established vessel abnormalities such as coronary heart disease or arterial stenosis , lead to an increased risk of thrombosis .
  • Patients who are presently treated with anti -coagulants such as heparin or with substances inhibiting thrombocyte aggregation, such as salicylic acid can be regarded as patients with an increased risk of thrombosis .
  • polypeptides and / or polynucleotides of the invention can also be used for the manufacture of a medical device , in particular a device coated or filled with said compounds .
  • Figure Ia The isolated T87F mutant and the native TFPI (SEQ ID No : 1 ) were incubated for 10 min at 37 0 C with HNE ( 10 nM) . While the native TFPI was nearly completely degraded, T87F remained intact .
  • Figure Ib After incubation of native TFPI (SEQ ID No : 1) with MMP- 9 (200 nM) , the native TFPI was substantially cleaved, whereas the T87F/L21A variant remained intact .
  • Figure Ic Cathepsin G (50 nM) elicited near complete degradation of the native TFPI (SEQ ID No : 1 ) . Under the same conditions , the T87F/L89A mutant was not cleaved .
  • FIG. 2 Factor Xa formation by a mixture of human platelets and human neutrophils (P/N) in response to collagen was reduced by addition of native TFPI (5 nM, nTFPI , SEQ ID No : 1) by 35% .
  • native TFPI 5 nM, nTFPI , SEQ ID No : 1
  • the same concentration of the variant T87F lead to a reduction by 64% .
  • a similar level of reduction was achieved by the variant T87F/L21A, whereas the variant T87F/L89A resulted in 85% reduction .
  • Figure 3 Thromboelastography tracings of whole blood ' ( I ) , whole blood mixed with collagen ( II ) , whole blood mixed with collagen in the presence of native TFPI (SEQ ID No : 1) (III) or the variant T87F/L89A ( IV) .
  • the variant resulted in a 160% prolongation of coagulation time as compared to II .
  • Figure 4 Polynucleotide and amino acid sequence of a variant polypeptide of the invention comprising the variation L21A, T87F, L89A and Y159A (SEQ ID No : 6 and 7) .
  • the sequences of fig . 4 also depict the N-terminal signal sequence (the first 9 amino acids) , and a C-terminal strep-tag for purification (the last 23 amino acids) , as is also apparent from comparing SEQ ID No : 1 with SEQ ID Nos . 6 and 7.
  • Figure 5 Schematic representation of TFPI ( SEQ ID No : 1 ) excluding N-terminal signal peptide and C-terminal strep-tag indicating various protease cleavage sites
  • Example 1 production of native and variant TFPI polypeptides
  • E . coli was grown in LB and SOC medium.
  • the full- length human TFPI -I gene (alpha isoform) (excluding the N-terminal signal peptide) was amplified from arterial vessel wall cDNA using outer and inner primers , the forward outer primer 5 1 GTACATGCACTTTGGGCTTC-3 ' (SEQ ID No : 2 ) and the reverse outer primer S ' -TCACATATCTTTAACAAGAATTC -3 ' (SEQ ID No : 3 ) .
  • the inner primers (forward : 5 ' - CGGGATCCGCTGATTCTGAGGAAG-3 ' (SEQ ID No : 4 ) ; reverse : 5 ' - GGAATTCGCTGATTCTGAGGAAG- S ' (SEQ ID No : 5 ) ) contained restriction sites for EcoRI and BamHI .
  • Said full length TFPI was cloned in the expression vector pASK- IBA3 containing the Strep-Tag coding sequence ( IBA Germany) .
  • Site directed mutagenesis was performed with GeneTailorTM Site-Directed Mutagenesis System (Invitrogen, Germany) ' . All the constructs were sequenced in their entirety to verify for the correctness of the mutated sites (Seqlab, Goettingen, Germany) .
  • the following mutants of human TFPI were designed : For • modification of the HNE cleavage site between Threonin 87 and Threonin 88 , the amino acid in position 87 was substituted by Phenylalanin (T87F) . In order to additionally modify the cath G specific degradation sites between Leucin 89 and Glutamine 90 , and between Tyrosine 159 and Glycine 160 , the amino acids in positions 89 and 159 were both replaced by Alanine (T87F/L89A; T87F/Y159A) . To alter the MMP cleavage site between residues 20 and 21 , Leucine 21 was substituted by Alanine (T87F/L21A) . The modifications were introduced as given below . Culture conditions and expression of TFPI protein
  • Luria-Bertani (LB) broth medium was used to produce TFPI native and the mutant forms of the protein .
  • 500 ml of LB amp ( lOO ⁇ g/ml amp . ) medium was inoculated with 10 ml- of BL21 E . coli cells , transfected with the expression vectors for the variant TFPI polypeptides , from an overnight culture .
  • the mixture was shaken in an incubator at 37 0 C and 190 rpm.
  • 50 ⁇ l anhydrotetracyline IBA, Germany
  • the cell culture was incubated for an additional 2 h at 25 0 C .
  • TFPI TFPI
  • the culture suspensions were centrifuged at 5000 rpm for 20 min at 4 0 C to pellet the cells .
  • the pellets were resuspended in 10 ml of pre-cooled buffer W ( 100 ⁇ iM Tris/HCI , pH 8.0 , 150 mM NaCl , 1. O mM EDTA) , and sonicated under ice-cooling conditions until the lysis was complete .
  • the suspension was then centrifuged at 6 , 000 rpm for 15 min at 4 0 C to remove the cell debris , the supernatant was additionally centrifuged at 35 , 000 g for 30 min at 4 0 C to remove the residual proteins .
  • elution buffer 100 mM Tris/HCI , pH 8.0 , 1. O mM EDTA, 0.02% w/v NaN3 , and 2.5 mM desthiobiotin
  • the eluent was collected ( 0.5 ml/tube) .
  • Thirty microliters of each fraction was analyzed using a 1.5 mm 12% SDS- polyacrylamide gel .
  • the amount of protein was also quantified using UV spectrophotometry .
  • Equal amounts of the recombinant proteins were dissolved in 20 ⁇ l of Tris-HCl , 50 mM, pH 6.8 , containing 2 - mercaptoethanol ( 1%) , sodium dodecyl sulfate (SDS) (2%) , glycerol (20% ) , bromphenol blue ( 0.04%) .
  • the samples were heated at 100 0 C for 5 min . and then were subj ected to SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto nitrocellulose membranes (Hibond,
  • Example 2 in vitro resistance to protease cleavage of native and variant TFPI polypeptides
  • TFPI were incubated for 10 min at 37 °C with HNE ( 10 nM (Calbiochem 50 ⁇ g Package) ) and analyzed by Western blot as described above . While the native TFPI was nearly completely degraded, T87F remained entirely intact (Fig . Ia) . After incubation of native TFPI with MMP- 9 (200 nM (Calbiochem ( 5 ⁇ g Package) , the native TFPI was substantially- cleaved as analyzed by Western blot as described above . In contrast , the T87F/L21A variant was not degraded (Fig . Ib) .
  • Cathepsin G (200 nM (Sigma 0.25 U Package) elicited near complete degradation of the native TFPI as analyzed by Western blot as described above . Under the same conditions , the T87F/L89A mutant was not cleaved (Fig . Ic) .
  • the variant TFPI molecules showed enhanced resistance to protease cleavage in vitro as compared to native TFPI .
  • Example 3 Effect of the TFPI variants on the activity of the initiator complex of coagulation
  • TFPI variants In order to assess the anticoagulant efficiency of the TFPI variants , their effects on the formation of factor Xa were evaluated in mixtures of activated human platelets and human neutrophils . Platelets were isolated from venous blood of healthy volunteers . The blood was drawn into sodium citrate ( 0.38%final concentration) and subsequently centrifuged at 19Og for 15 minutes . The upper two thirds of the supernatant were supplemented with apyrase ( 0.2 U/ml) and iloprost ( 10 ng/ml ) .
  • the suspension was centrifuged at 33Og for 10 min, and the platelet pellet was recovered and resuspended in a buffer composed of 145 mM NaCl , 10 mM HEPES (N-2 - hydroxyethylpiperazine-N 1 -2 -ethanesulfonic acid) , 5 mM KCl , 1 mM MgC12 , and 5 mM glucose (pH 7.4 ; resuspension buffer) .
  • Human neutrophils were prepared by incubation of freshly obtained buffy coats with microbeads coupled to anti-CD15 antibodies (Miltenyi Biotec) for 15 min at 8 °C .
  • the suspensions were thereafter applied onto the positive selection column, and neutrophils were eluted with the antibody buffer (phosphate-buffered saline [PBS] supplemented with 0.13 % EDTA [ethylenediaminetetraacetic acid] and 0.15% bovine serum albumin) .
  • the isolated neutrophils and platelets were incubated in resuspension buffer for 5 min with collagen (type I , 12 ⁇ g/ml ) at 37 0 C .
  • the medium contained 0.88 U/ml factor VII of a coagulation factor concentrate ( factors II , VII , IX, X (Beriplex P/N 500 ; Aventis-Behring) ) , together with 8 mM
  • variant TFPI T87F diminished the factor Xa generation by 64% .
  • Additional mutation at position 21 did not augment the inhibitory influence of T87F (55% reduction) , indicating that the mutation in position 21 did not enhance the anticoagulant influence of TFPI at the particular selected in vitro conditions .
  • T87F/L89A elicited the strongest inhibition of the factor Xa formation, lowered its generation by 85% (Fig . 2 ) .
  • the variant TFPI polypeptides exhibited an enhanced capability of reducing the initiator complex of coagulation .
  • the effect of the TFPI variants was also tested under ex vivo conditions in whole blood .
  • the coagulation time ( fibrin formation) was determined mechanically by thrombelastography (TEG) (roTEG, Instrumentation Laboratories) . 0.3 ml of the
  • the fibrin formation time r (the distance between the start of the recalcification and the point where the amplitude of the tracing reaches 2 mm) was markedly accelerated by addition of collagen (Fig . 3 ; II vs . I ) .
  • the time r was prolonged by 44% in the presence of the native TFPI ( III ) .
  • addition of T87F/L89A prolonged the coagulation time by 160% ( IV) .
  • the T87F/L89A mutant enhances the anticoagulant efficiency by 2.4 -3.6 -fold .

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une variante de l'inhibiteur du trajet du facteur tissulaire-1 alpha (TFPI-1 alpha) caractérisée par une résistance accrue à l'inactivation, en particulier, une résistance accrue au clivage de protéase, ainsi que par une activité anticoagulante augmentée par rapport à une protéine native de TFPI-1 alpha. Cette variante de TFPI-1 alpha permet d'obtenir un anticoagulant approprié à une application clinique dans des maladies dans lesquelles jouent un rôle TFPI et le complexe facteur tissulaire/facteurVIIa/facteur Xa. Il est possible, en particulier, de traiter au moyen de cette variante de TFPI-1 alpha des maladies associées à la thrombose ou provoquées par cette dernière.
PCT/EP2005/013962 2004-12-23 2005-12-23 Variante de tfpi possedant une activite antithrombotique accrue Ceased WO2006066960A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP04030687 2004-12-23
EP04030687.0 2004-12-23

Publications (1)

Publication Number Publication Date
WO2006066960A1 true WO2006066960A1 (fr) 2006-06-29

Family

ID=36089892

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/013962 Ceased WO2006066960A1 (fr) 2004-12-23 2005-12-23 Variante de tfpi possedant une activite antithrombotique accrue

Country Status (1)

Country Link
WO (1) WO2006066960A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991002753A1 (fr) * 1989-08-18 1991-03-07 Novo Nordisk A/S Proteine anticoagulante
WO1996004378A2 (fr) * 1994-08-05 1996-02-15 Chiron Corporation Proteines chimeriques et muteines des inhibiteurs des voies du facteur tissulaire de la coagulation tfpi et tfpi-2
WO2000011034A1 (fr) * 1998-08-25 2000-03-02 Knut Kleesiek Mutant de l'inhibiteur de la voie de la thromboplastine tissulaire, sa sequence d'adn et son utilisation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991002753A1 (fr) * 1989-08-18 1991-03-07 Novo Nordisk A/S Proteine anticoagulante
WO1996004378A2 (fr) * 1994-08-05 1996-02-15 Chiron Corporation Proteines chimeriques et muteines des inhibiteurs des voies du facteur tissulaire de la coagulation tfpi et tfpi-2
WO2000011034A1 (fr) * 1998-08-25 2000-03-02 Knut Kleesiek Mutant de l'inhibiteur de la voie de la thromboplastine tissulaire, sa sequence d'adn et son utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BAJAJ MADHU S ET AL: "Tissue factor pathway inhibitor: Potential therapeutic applications", THROMBOSIS AND HAEMOSTASIS, vol. 78, no. 1, 1997, pages 471 - 477, XP009064749, ISSN: 0340-6245 *
BROZE JR G J ET AL: "REGULATION OF COAGULATION BY A MULTIVALENT KUNITZ-TYPE INHIBITOR", BIOCHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, PA, US, vol. 29, no. 33, 21 August 1990 (1990-08-21), pages 7539 - 7546, XP000268583, ISSN: 0006-2960 *
GIRARD T J ET AL: "FUNCTIONAL SIGNIFICANCE OF THE KUNITZ-TYPE INHIBITORY DOMAINS OF LIPOPROTEIN-ASSOCIATED COAGULATION INHIBITOR", NATURE, NATURE PUBLISHING GROUP, LONDON, GB, vol. 338, April 1989 (1989-04-01), pages 518 - 520, XP000857349, ISSN: 0028-0836 *
MOATTI D ET AL: "POLYMORPHISMS OF THE TISSUE FACTOR PATHWAY INHIBITOR (TFPI) GENE IN PATIENTS WITH ACUTE CORONARY SYNDROMES AND IN HEALTHY SUBJECTS. IMPACT OF THE V264M SUBSTITUTION ON PLASMA LEVELS OF TFPI", ARTERIOSCLEROSIS, THROMBOSIS, AND VASCULAR BIOLOGY, vol. 19, no. 4, April 1999 (1999-04-01), pages 862 - 869, XP000856947, ISSN: 1079-5642 *

Similar Documents

Publication Publication Date Title
EP0487591B1 (fr) Proteine anticoagulante
US6087487A (en) Nematode-extracted serine protease inhibitors and anticoagulant proteins
JP3051995B2 (ja) 酸化抵抗性トロンボモジュリン類縁体
JP3350549B2 (ja) ヒトＫｕｎｉｔｚ型プロテアーゼ阻害剤変異体
US7407475B2 (en) Modified annexin proteins, and methods and compositions for using them
US7090986B2 (en) Protein for blocking platelet adhesion
JP5699393B2 (ja) トロンビンインヒビター
US20010020003A1 (en) Protease inhibitor peptides
PL188387B1 (pl) Białko o aktywności hamującej proteazę serynową, kompozycja farmaceutyczna, wyodrębniona sekwencja,samopowtarzający się wektor, zastosowanie białka o aktywności proteazy serynowej, sposób hamowania aktywności proteazy serynowej ex vivo i sposób wytwarzania białka
JPH09509838A (ja) クニッツドメイン拮抗剤蛋白質
CA2220497A1 (fr) Peptides inhibiteurs de proteases de type kunitz
US11897924B2 (en) Anticoagulant fusion proteins and uses thereof
CZ402097A3 (cs) Deriváty prothrombinu
KR20010005529A (ko) 덴드로아스핀 스캐폴드에 기초한 이 또는 다기능성 분자
JP2004527452A (ja) 医薬用途のためのトロンボモジュリン
RU2183214C2 (ru) Природный и рекомбинантный ингибиторы тромбина, их получение и применение
WO2006066960A1 (fr) Variante de tfpi possedant une activite antithrombotique accrue
JP5236952B2 (ja) 改善された特性を有するｆｘｉｉｉバリアント
JP2000128803A (ja) ティッシュ・ファクター・パスウェイ・インヒビター−２抗体
JP2002509757A (ja) 体外血液循環を受ける患者において血栓症を阻害するための方法
WO1999060126A9 (fr) Inhibiteur de protease dependant de la proteine k
WO2008059917A1 (fr) Mutant de la thrombine
WO1994020537A1 (fr) Analogues de tfpi non glycosylates
TW200846356A (en) Preparation and use of variants of the kunitz domain 2 of the human placental bikunin gene
JPH10113195A (ja) 凍結乾燥製剤の製造法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05820649

Country of ref document: EP

Kind code of ref document: A1