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WO1994020537A1 - Analogues de tfpi non glycosylates - Google Patents

Analogues de tfpi non glycosylates Download PDF

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Publication number
WO1994020537A1
WO1994020537A1 PCT/DK1994/000085 DK9400085W WO9420537A1 WO 1994020537 A1 WO1994020537 A1 WO 1994020537A1 DK 9400085 W DK9400085 W DK 9400085W WO 9420537 A1 WO9420537 A1 WO 9420537A1
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Prior art keywords
tfpi
amino acid
glu
asn
analogue
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PCT/DK1994/000085
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English (en)
Inventor
Ole Nordfang
Jens G. Litske Petersen
Søren Erik BJØRN
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Novo Nordisk A/S
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Priority to AU62022/94A priority Critical patent/AU6202294A/en
Publication of WO1994020537A1 publication Critical patent/WO1994020537A1/fr

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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
    • C07K14/81—Protease inhibitors
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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 present invention relates to non-glycosylated tissue factor pathway inhibitor (TFPI) analogues.
  • Blood coagulation is a complex process involving many acti ⁇ vating and inactivating coagulation factors.
  • Anticoagulant proteins are known to be important for regulation of the coa ⁇ gulation process and anticoagulants are thus important in the treatment of a variety of diseases, e.g. thrombosis, myocardial infarction, disseminated intravascular coagulation etc.
  • heparin is used clinically to increase the activity of antithrombin III and heparin cofactor II.
  • Antithrombin III is used for the inhibition of factor Xa and thrombin.
  • Hirudin is used for the inhibition of thrombin and protein C may be used for the inhibition of factor V and factor VIII.
  • Anticoagulant proteins may also be used in the treatment of cancer.
  • Tissue factor is a protein cofactor for factor Vll/VIIa and binding of tissue factor en ⁇ hances the enzymatic activity of factor Vila (FVIIa) towards its substrates factor IX and factor X.
  • tissue factor pathway inhibitor TFPI
  • TFPI has been shown to be a potent inhibitor of TF/FVIIa-induced coagulation (R.A. Gra zinski et al., Blood 21 (1989) 983-989) .
  • TFPI binds and inhibits factor Xa (FXa) and the complex between TFPI and FXa inhibits TF/FVIIa (Rapaport, Blood 21 (1989) 359-365) .
  • TFPI is especially interesting as an anticoagulant/antimetastatic agent as many tumor cells express TF activity (T. Sakai et al., J. Biol. Chem. 264 (1989), 9980- 59988) and because TFPI shows anti-Xa activity like antistatin which has antimetastatic properties.
  • TFPI has been recovered by Broze et al. (supra) from HepG2 he- patoma cells (Broze EP A 300988) and the gene for the protein has been cloned (Broze EP A 318451) .
  • a schematic diagram over 0 the secondary structure of TFPI is shown in copending patent application No. 07/828,920 (WO 91/01253).
  • the amino acid sequence of TFPI with its natural 28 amino acid signal peptide (Sequence ID Number 1 and 2) is shown in Fig. 1 where the N- terminal amino acid Asp is given the number 1.
  • the protein 5 consists of 276 amino acid residues and has, in addition to three inhibitor domains of the Kunitz type, three potential glycosylation sites at position Asnll7, Asnl67 and Asn228. The molecular weight indicates that some of these sites are gly ⁇ cosylated. Furthermore, it has been shown that the second 0 Kunitz domain binds FXa while the first Kunitz domain binds FVIIa/TF (T.J. Girard et al., Nature 338 (1989) 518-520). TFPI has also been isolated from HeLa cells (WO 90/08158) and it was shown that HeLa TFPI binds heparin.
  • TFPI 5 analogues are described as retaining this TFPI activity as well as anti Xa activity although parts of the molecule have been deleted. Furthermore, these analogues show a much lower af ⁇ finity for heparin than full-length TFPI, making them more useful as therapeutic agents than the native molecule.
  • the TFPI analogues furthermore have a longer half life as compared with native TFPI which will further reduce the amount of active ingredients for the medical treatment.
  • These TFPI analogues are thus characterized in having TFPI activity but with no or low heparin binding capacity unde physiological conditions (pH, ionic strength) .
  • low heparin binding capacity is meant to indicate binding capacity of about 50%, more preferably of about 25% an most preferably less than about 10% of that of native TFPI a physiological pH and ionic strength.
  • the heparin binding capacity i substantially lost when the sequence from amino acid residu number 162 to amino acid residue number 276 is deleted from th TFPI molecule. It was therefore concluded that the hepari binding domain is located in this part of the TFPI molecule an it was assumed that the heparin binding domain comprises a least a region from Arg246 to Lys265 near the C-terminal end o the TFPI molecule which is rich in positively charged amin acid residues.
  • TFPI analogues lacking C-terminal parts of th molecule surprisingly are expressed in good yields in yeast.
  • These TFPI analogues contain at least the first and secon Kunitz domain and lack part of the C-terminal end of the nativ TFPI molecule, more specifically the third Kunitz domain fro amino acid Cysl89 to amino acid Cys239 and a substantial par of the amino acid sequence from Lys240 to Met276.
  • substantial part is meant from about 70% to 100%.
  • the present invention relates to a non-glycosylated TFPI analogue containing at least the first and second Kunitz domai and lacking the third Kunitz domain and a substantial part o the amino acid sequence from amino acid Lys240 to Met276 o native TFPI, said analogue being modified at either or both o the two N-glycosylation triads Asnll7-Glnll8-Thrll9 and Asnl67- Asnl68-Serl69 to avoid N-glycosylation.
  • the present invention is based on the surprising finding that non-glycosylated TFPI,,.. 161 has the same activity as the glycosylated TFPI_,. 161 and on the finding that a glycosylation mutant of TFPI.,.., ⁇ has pharmacokinetics which makes it very suitable for use as an anticoagulant for infusion.
  • the half- life is significantly increased compared with the glycosylated variant produced in yeast. Therefore the amount of active ingredient in the pharmaceutical preparation can be reduced.
  • the half life is still sufficiently short to obtain a reasonably fast clearance of the protein in case of bleeding complications seen in some patients suffering from thrombosis.
  • glycosylation modifications may change the pharmacokinetics of proteins (see e.g. P. Stanley, Glycobiology 2 . (1992) 99-107) .
  • the biological activity of the protein may be changed and the biological half life may either be increased or decreased by such modifications.
  • the TFPI analogues may also contain a Ser residue as the N- terminal residue for efficient cleavage of a signal peptide by a signal peptidase.
  • the N-terminal in the TFPI molecule may be replaced by a Ser or an additional Ser may be inserted adjacent to the original N-terminal residue.
  • the TFPI analogues may furthermore lack part of the N-terminal sequence of native TFPI such as the sequence from amino acid residue 1 to 24.
  • the present invention also relates to a non-glycosylated TFPI analogue containing at least the amino acid sequence from Phe25 to Glu 148 of the native TFPI molecule and lacking the third Kunitz domain from amino acid Cysl89 to amino acid Cys239 and a substantial part of the amino acid sequence from Lys240 to Met276 of the native TFPI molecule, said TFPI analogue being modified at either or both of the two N-glycosylation triads Asnll7-Glnll8-Thrll9 and Asnl67-Asnl68-Serl69 to avoid N- glycosylation.
  • the present invention relates to a non- glycosylated TFPI analogue containing at least the amino acid sequence from Aspl to Glul48 of the native TFPI molecule and lacking the third Kunitz domain from Cysl89 to Cys239 and a substantial part of the amino acid sequence from Lys240 to Met276 of the native TFPI molecule, said TFPI analogue being modified at either or both of the two N-glycosylation triads Asnll7-Glnll8-Thrll9 and Asnl67-Asnl68-Serl69 to avoid N- glycosylation.
  • the present invention relates to a non-glycosylated TFPI analogue lacking the amino acid sequence from Glnl62 to Met276 of the native TFPI molecule in yeast, said analogue being modified at the N-glycosylation triad Asnll7-Glnll8-Thrll9 to avoid N-glycosylation.
  • the modification of the N-glycosylation triad may be in the form of a deletion and/or substitution of one or more of the three amino acid residues of the triad. Asn in position 117 may thus be replaced by any other naturally occurring amino acid residue; Gin in position 118 may be replaced by Pro or Asp; Thr in position 119 may be replaced by any other naturally occurring amino acid residue except Ser or may be deleted and Asnll7, Glnll ⁇ and Thr 119 may all be deleted.
  • the modification according to the present invention is intended to cover any combination of such modifications.
  • the present invention is related to a DNA sequence encoding the novel, non-glycosylated TFPI analogues.
  • the present invention furthermore relates to recombinant expression vectors comprising DNA sequences pemitting gene expression, including a promoter and a terminator, functionally fused to a DNA sequence encoding the TFPI analogue and capable of expressing the TFPI analogue according to the invention in a transformed or transfected eukaryotic host cell.
  • the present invention relates to eucaryotic cells containing a recombinant expression vector as defined above and to a method of making the novel, non- glycosylated TFPI analogues which process comprises culturing a eukaryotic cell line as defined above in a suitable nutrient medium under conditions permitting the expression of the TFPI analogues and recovering the resulting TFPI analogues from the culture.
  • TFPI analogues The cDNA for the native TFPI has been cloned and sequenced (T.- C. Wun et al., J. Biol. Chem. 263 (1988) 6001-6004).
  • DNA sequences encoding the TFPI analogues according to the present invention may be constructed by altering TFPI cDNA by site- directed mutagenesis using synthetic oligonucleotides in accordance with well-known procedures (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor, NY) .
  • the DNA sequence encoding the TFPI analogue of the invention may also be prepared synthetically by established standard methods.
  • oligonucleotides may be synthesized by phosphoamidite chemistry in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
  • the expression vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and which is capable of expressing the TFPI analogues in the selected eukaryotic cell.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence encoding the TFPI analogue will be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the DNA encoding the TFPI analogues of the invention in mammalian cells are the SV 40 promoter (Subramani et al., Mol.Cell Biol. 1 (1981) 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al..
  • yeast promoters include promoters from yeast glycolytic genes
  • TPI1 promoter T. Alber and G. Kawasaki, op.cit and US patent 4,599,311
  • ILV5 J.G.L. Petersen and S. Holmberg, Nucl. Acids Res. JL4 . (1986) 9631-9651 promoter.
  • the DNA sequence encoding the TFPI analogues may also be operably connected to a suitable terminator sequence which show transcription termination activity in a host cell.
  • Suitable terminators may be the human growth hormone terminator (Palmiter et al., op.cit.) .
  • the terminator sequences may be derived from the 3 ' untranslated regions of yeast genes such as TPI1 (T. Alber and G. Kawasaki, op. cit.) and ILV5 (J.G.L. Petersen and S. Holmberg, op. cit.) .
  • the vector may further comprise elements such as polyadenylation signals, transcriptional enhancer sequences and translational enhancer sequences.
  • TFPI analogues in host cells that can secrete the analogues into the culture media.
  • a secretory signal sequence is operably linked to the TFPI analogue DNA sequence.
  • the secretory signal should preferably be cleaved in vivo, e.g. by a signal peptidase or in yeast by the yeast KEX2 protease (D. Julius et al., Cell 2 (1984) 1075- 1089) during export of the fusion protein to allow for secretion of a TFPI analogue having the correct N-terminal amino acid.
  • a suitable signal sequence for mammalian cells is the t-PA signal sequence (Friezner et al., J. Biol. Chem. 261 (1986) 6972-6985) .
  • Suitable secretory signals for yeast include the ⁇ -factor prepropeptide (J. Kurjan and I. Herskowitz, Cell 10 (1982) 933-943; U.S. Patent No. 4,546,082 and EP 116,201), the PH05 signal peptide (WO 86/00637) , secretory signal sequences derived from the BAR1 gene (U.S. Patent No. 4,613,572 and WO 87/002670), the SUC2 signal peptide (M. Carlson et al., Mol. Cell. Biol.
  • a secretory signal sequence may be synthesized according to the rules established, for example, by G. von Heijne (Nucl. Acids Res. .14. (1986) 4683- 4690) . Examples of synthetic secretory signal sequences are described in WO 89/02463 and WO 92/13065.
  • Suitable yeast vectors include YRp7 (K. Struhl et al., Proc. Natl. Acad Sci.
  • Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
  • Preferred selectable markers are those that complement host cell 0 auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include for yeast the genes LEU2 (Broach et al., op.cit.), URA3 (D. Botstein et al.. Gene 8 (1979) 17-24), HIS3 (K. Struhl et al., op.cit.) or POT1 (US Patent No. 4,931,373).
  • suitable 5 selectable markers are the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hygromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the host cell may be any eukaryotic cell which is capable of producing the TFPI analogues and is preferably a mammalian cell 0 or a yeast cell.
  • suitable mammalian cell lines are the COS (ATCC CRL 1650 and 1651), BHK (ATCC CRL 1632, ATCC CCL 10) or CHO (ATCC CCL 61) cell lines.
  • the yeast host cell may be any yeast species which is capable of producing the TFPI analogue.
  • suitable yeast host 5 cells include strains of Saccharomyces spp., Schizosaccharo- mvces spp. Kluweromyces spp. , Pichia spp. and Hansenula spp. , in particular strains of Saccharomyces cerevisiae.
  • the host strain carry a mutation, such as the yeast pep4 mutation (E.W. Jones, Genetics 5 . (1977) 23-33) , which results in reduced proteolytic activity.
  • the recombinant expression vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the yeast 2-micron sequence and the SV40 origin (for mammalian cells) .
  • the transformed or transfected host cells are grown according to standard methods in a growth medium containing nutrients required for growth of the particular host cells.
  • suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
  • the growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.
  • Suitable growth conditions for yeast cells for example, include culturing in a medium comprising a nitrogen sourc (e.g.
  • yeast extract or nitrogen-containing salts inorgani salts, vitamins and essential amino acid supplements a necessary at a temperature between 4°C and 37°C, with 30° being particularly preferred.
  • the pH of the medium i preferably maintained at a pH greater than 2 and less than 8, more preferably pH 5-6.
  • the medium used to culture mammalian cells may be an conventional medium suitable for growing mammalian cells, suc as a serum-containing or serum-free medium containin appropriate supplements. Suitable media are available fro commercial suppliers or may be prepared according to publishe recipes (e.g. in catalogues of the American Type Cultur Collection) .
  • the TFPI analogues will preferably be secreted to the growt medium and may be recovered from the medium by conventiona procedures including separating the host cells from the mediu by centrifugation or filtration, precipitating th proteinaceous components of the supernatant or filtrate b means of a salt, e.g ammonium sulphate, followed b purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or th like.
  • a salt e.g ammonium sulphate
  • the present invention also relates to a pharmaceutica composition
  • a pharmaceutica composition comprising a TFPI analogue of the inventio together with a pharmaceutically acceptable carrier or diluent.
  • the TFPI analogue may b formulated by any of the established methods of formulatin pharmaceutical compositions, e.g. as described in Remington' Pharmaceutical Sciences. 1985.
  • the composition may typically b in a form suited for systemic injection of infusion and may, a such, be formulated with sterile water or an isotonic saline o glucose solution.
  • the compositions may be sterilized b conventional lyophilized preparation being combined with the sterile aqueous solution prior to administration.
  • the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • concentration of the TFPI analogue of the invention may vary widely, i.e. from less than about 0.5%, such as from 1%, to as much as 15-20% by weight.
  • a unit dosage of the composition may typically contain from about 0.1 to about 100 mg of the TFPI analogue of the invention.
  • the pharmaceutical preparations may be in a buffered aqueous solution with appropriate stabilizers and preservatives.
  • the solution may be heat treated and may be contained in ampoules or in carpoules for injection pens.
  • the stabilized solution may be freeze dried and contained in ampoules or in two chamber injection systems with freeze dried substance in one chamber and solvent in the other chamber.
  • the TFPI analogue of the invention is contemplated to be advantageous to use for the therapeutic applications suggested for full-length TFPI. These include, but are not limited to treatment of patients with coagulation disorders or cancer as described in European patent application No. 0 487 591.
  • a specific coagulation disorder which may be treated with the TFPI analogue is disseminated intravascular coagulation (DIC) which is a common and serious complication occurring in patients with sepsis, trauma, burns, haemolytic anemia, metastatic cancer, etc.
  • DIC is characterized by fibrin deposition in various organs, e.g.
  • coagulation factors fibrinogen, FVII etc.
  • coagulation inhibitors e.g. antithrombin III
  • platelets e.g. platelets
  • TF tissue factor
  • monocytes and endothelial cells leading to formation of complexes between TF and activated factor VII (FVIIa) resulting in activation of the coagulation system by the extrinsic pathway.
  • FVIIa activated factor VII
  • Fig. 1 shows a synthetic gene and the corresponding amino acid sequence for human TFPI including the signal peptide.
  • Fig. 2 shows DNA sequences and corresponding amino acid sequences for the prepropeptide of human serum albumin pp HSA (Sequence ID Number 3 and 4) and the synthetic secretion signal 212spx3 (Sequence ID
  • Fig. 3 shows the synthetic gene for TFPI 1 . 161 -ll7Gln fused to the synthetic secretion signal 212spx3 (Sequence ID
  • Fig. 4 shows restriction site maps of plasmid pY-ppTFPI161 and plasmid pP-212TFPI161-117Q (the map of the third expression plasmid described in this study, pP- 212TFPI161, is similar to that of pP-212TFPI161-
  • Fig. 5 shows a Western analysis of secreted TFPI 1 . 161 and its
  • Fig. 6 illustrates the construction of the URA3-2_ yeas expression plasmid pYES-GykTFPI161-117Q encoding a fusion protein consisting of the synthetic secretion peptide yk and TFPI 1 . 161 -117Q.
  • the sizes of the plasmids are given in base pairs. Only relevant restriction endonuclease sites are shown.
  • Fig. 7 illustrates the construction of five URA3-2u yeast plasmids derived from pYES-GykTFPI161-117Q for expression of secreted unglycosylated two-domain TFPI analogues with different polypeptide lengths.
  • the four TFPI expression plasmids not depicted by drawings are very similar to pYES-GykTFPI161-117Q and pYES-GykT21-161-Q differing only in the coding region for the TFPI precursors.
  • the sizes of the plasmids are given in base pairs. Only relevant restriction endonuclease sites are shown.
  • Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs. Modified T7 DNA polymerase (Sequenase) was obtained from United States Biochemicals. Restriction endonucleases and other enzymes were used in accordance with the manufacturers recommendations.
  • pBS-i- (Stratagene) was used as cloning vector for construction of the synthetic TFPI gene by cloning of synthetic DNA fragments.
  • Strains of Saccharomyces cerevisiae used as hosts for expression of TFPI analogues were the two diploids E18 (MATa/MAT ⁇ tpi: :LEU2/ ⁇ tpi: :LEU2 Ieu2/leu2 +/his4 pep4-3/pep4- 3) (US Patent No. 4,931,373) and YNG452 (MAT ⁇ /MAT ⁇ ura3- 52/ura3-52 Ieu2- ⁇ 2/Ieu2- ⁇ 2 +/his4 pep4- ⁇ l/pep4- ⁇ l) .
  • the latter was derived from strain JC482 (J.F. Cannon and K. Tatchell, Mol. Cell. Biol. 2 (1987) 2653-2663).
  • Yeast expression vectors used for expression of TFPI analogues in yeast were of the POT-type (US Patent No. 4,931,373) or the URA3-LEU2d-2 ⁇ plasmid pAB24 (P.J. Barr et al., in Proc. Alko Symp. on Industrial Yeast Genetics (Korkola and Nevalainen, eds.) Found. Biotech. Industr. Ferment. Res. 5_ (1987) 139-148).
  • DNA sequences were determined by the dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. 7_4 (1977) 5463- 5467) using double stranded plasmid DNA as template and 3 P- or 35 S labelled primers and Sequenase.
  • SDS polyacrylamide gel elecrrophoresis under reducing conditions was performed according to U.K. Laemmli (Nature 227 (1979) 680-685) using 12.5% separating gels. Protein was stained with Coomassie Brillant Blue R-250 (Sigma) .
  • N-terminal sequence analysis was carried out by automated Edman degradation using an Applied Biosystems 470A gas-phase sequencer. Analysis by on-line reverse-phase HPLC was performed for the detection and quantification of the liberated PTH amino acids from each sequence cycle.
  • TFPI activity was measured in a chromogenic microplate assay, modified according to the method of Sandset et al., (Thromb. Res. 4_7 (1987) , 389-400) .
  • Heat treated plasma pool was used as a standard. This standard is defined as containing 1 U/ml of TFPI activity.
  • Standards and samples were diluted in buffer A (0.05 M Tris-HCl, 0.1 M NaCl, 0.1 M Na-citrate, 0.02% NaN 3 , pH 8.0) containing 2 g/ml polybrene and 0.2% bovine serum albumin.
  • FVIIa/TF/FX/CaCl 2 combination reagent was prepared in buffer A and contained 1.6 ng/ml FVIIa (Novo Nordisk A/S), human tissue factor diluted 60 fold, 50 ng/ml FX (Sigma) and 18 mM CaCl 2 .
  • the assay was performed in microplate strips at 37°C. 50 ⁇ l of samples and standards were pipetted into the strips and 100 ⁇ l combination reagent was added to each well. After 10 minutes incubation, 25 ⁇ l of FX (3.2 ⁇ .g/ml) was added to each well and after another 10 minutes 25 ⁇ l of chromogenic substrate for FXa (S2222) was added 10 minutes after the ad ⁇ dition of substrate. The reaction was stopped by addition of 50 ⁇ l 1.0 M citric acid pH 3.0. The microplate was read at 405 nm.
  • the gene had 26 silent nucleotide substitutions in degenerate codons as 0 compared to the cDNA resulting in fourteen unique restriction endonuclease sites in order to facilitate the introduction of mutations in TFPI as well as the in-frame insertion of new secretion signals at the N-terminal of mature TFPI.
  • the DNA sequence of the 922 bp Sail fragment and the corresponding 5 amino acid sequence of human TFPI (pre-form) is shown in Fig. 1.
  • TFPI analogues were constructed from the synthetic TFPI gene by replacing portions 0 of the TFPI gene with appropriate synthetic DNA fragment.
  • the DNA fragments were annealed oligodeoxynucleotides synthesized by phosphoramidite chemistry. Resulting plasmids were propagated in E. coli and the nucleotide sequences verified by DNA sequencing.
  • TFPI.,.. 161 was expressed as a fusion protein with an N-terminal addition of 24 amino acids corresponding to the prepropeptide of human serum albumin (Fig. 2) , or with the synthetic secretion sequence 212spx3 (Fig.
  • TFPI 1 . 161 has one consensus-site, Asnll7, for the addition of N- linked carbohydrate characteristic of many eukaryotic cells like mammals and fungi, including yeast (M.A. Kukuruzinska et al., Ann. Rev. Biochem. 55 (1987) 915-944).
  • Asnll7 N- linked carbohydrate characteristic of many eukaryotic cells like mammals and fungi, including yeast
  • TFPI genes in S. cerevisiae In order to express the TFPI genes in S. cerevisiae. two yeast expression plasmids for TFPI,. ⁇ ,, pY-ppTFPI161 and pP- 212TFPI161, were constructed, while one plasmid, pP-212TFPI161- 117Q, was constructed for TFPI 1 . 161 -ll7Gln. The restriction site maps for two of these plasmids are shown in Fig. 4. High-level expression was achieved by placing the genes behind the strong constitutive promoters of the TPIl or ILV5 genes of S. cerevisiae. Transcription termination sequences were derived from the same genes. Plasmid pY-ppTFPI161 (Fig.
  • plasmids pP-212TFPI161 and pP-212TFPI161-117Q were based on a vector of the POT-type (G. Kawasaki and L. Bell, US patent 4,931,373). All expression plasmids carried, in addition to selective markers for transformation of the plasmids into suitable host strains of S. cerevisiae. DNA sequences of the 2-micron plasmid of yeast for high plasmid- copy numbers in yeast.
  • Plasmid pY-ppTFPI161 was transformed into strain YNG452 derived from strain JC482 (J.F. Cannon and K. Tatchell, Mol. Cell. Biol. 2 (1987) 2653-2663)) under selection for uracil independence (H. Ito et al., J. Bacteriol. 153 (1983) 163-168). Plasmids pP-212TFPI161 and pP-212TFPI161-117Q were transformed into strain E18 selecting for ability to grown on media with glucose as the carbon source by complementation of the disrupted triose phosphate isomerase gene with the POT-marker (P.R. Russell, Gene 40 (1985) 125-130.
  • TFPI 1-161 was expressed in strain YNG452 as a fusion to the prepropeptide of HSA. However, more than a 10-fold increase in secreted activity was seen when the same analogue was expressed in strain E18 as a fusion to the 212spx3 prepropeptide, and this increase in activity was unaffected by substitution of Asnll7 to Gin.
  • Example 2 R Reellaattiivvee ⁇ anticoagulant activities of TFPI 1 161 and TFPI., 161 -ll7Gln in a PT-clotting assay
  • transformants YNG452[pY-ppTFPI161] and E18[pP-212TFPI161-117Q] were grown in pilot- or laboratory-scale fermentors in fed-batch processes with glucose, and the analogues purified from the supernatant medium by a combination of ion exchange chromatography and gel- filtration. Subsequent analysis of similar activity amounts (P.M. Sandset et al., op.cit) by SDS-polyacrylamide gel electrophoresis and Western blotting using an antiserum raised against an N-terminal peptide of TFPI (A.H.
  • the N-terminal amino acid sequences were determined on purified preparations of the analogues or on proteolytic fragments thereof. In both cases the expected N-terminal sequence for mature TFPI was obtained.
  • TFPI 1 . 161 and TFPI 1 . 161 -ll7Gln were prepared as described in the preceding examples and dissolved in 10 mM glycylglycine, 100 mM NaCl, 30 g/1 mannitol, pH 7.0 to a concentration of 1 mg/ l.
  • Six female rabbits (New Zealand) with a mean weight of 2.56 kg were anaesthetized with pentobarbital sodium. Test compounds were administered via a catheter placed in vena jugularis, and blood samples were obtained from a catheter placed in a. carotis on the opposite side. The first 5 ml of blood were discarded. Two groups of rabbits were treated with either TFPI,,. 161 or TFPI 1 .
  • TFPI activity was measured in the chromogenic activity assay.
  • Alpha- and beta half-lives, clearance and mean residence time were calculated by non-liniar regression by using a two- compartmental model.
  • the fittings were performed by use of the SIMPLEX procedure written in a program adopted from K. Yamaoka et al., (A pharmacokinetic analysis program ( ulti) for microcomputer. J. Pharm. Dyn. 4. (1981) 879) .
  • the following pharmacokinetic parameters were obtained:
  • the ⁇ half life of the unglycosylated variant was increased 3-4 fold compared with the glycosylated form and the clearance rate was reduced by half.
  • the amount of TFPI 1-161 needed to keep a steady state plasma level will be reduced two fold by using the unglycosylated variant (117Gln) .
  • the clearance rate is sufficient to obtain clearance in case of bleeding complications.
  • the six TFPI analogues were expressed as fusion proteins with 10 an N-terminal addition of a 54 amino acid, Kex2-cleavable synthetic secretion peptide, denoted yk.
  • the secretion sequence consisted of the putative 21 amino acid signal peptide of the aspartyl protease of S. cerevisiae encoded by the YAP3 gene (M.
  • yeast plasmids encoding the analoques, a yeast plasmid encoding TFPI 1 . 161 -ll7Gln fused to
  • Plasmid pP-212TFPI161-117Q (Fig.4 and Fig.6) was digested with restriction endonucleases Sphl and Xbal in a double digestion, and the 1.1 kb Sphl-Xbal fragment consisting of the TPIl promoter and the coding region for the 212spx3
  • the plasmid was cleaved with EcoRI and PflMI in order to remove the coding region for the modified ⁇ -amylase signal peptide of the 212spx3 secretion sequence. Subsequent insertion of a synthetic double-stranded EcoRI-PflMI oligonucleotide with codons for the YAP3 signal peptide created the DNA sequence encoding the yk secretion sequence fused to the N-terminus of TFPI 1 . 161 -ll7Gln. The gene fusion was assured by DNA sequencing around the EcoRI and PflMI sites.
  • TPIl promoter fragment was replaced by a DNA fragment containing a 0.44 kb fragment of the promoter for the glyceraldehyde-3-phosphate dehydrogenase gene GPP (G3PDA) of S. cerevisiae (J.P. Holland and M.J. Holland, J. Biol. Chem. 254 (1979) 9839-9845; G.A. Bitter and K.M. Egan, Gene 12. (1984) 263-274) with an Sphl site inserted immediately upstream of position -452 and an EcoRI site immediately downstream of position -12, as this promoter was expected to be somewhat stronger than the TPIl promoter.
  • G3PDA glyceraldehyde-3-phosphate dehydrogenase gene GPP
  • the resulting 6.4 kb yeast plasmid pYES-GykTFPI161-117Q with the gene for the yk/TFPI ⁇ , ⁇ - 117 Gin fusion protein under the control of the GPP promoter is shown in Fig. 6.
  • yeast plasmids encoding C-terminal truncations of the TFPI.,_ 161 -117 Gin precursor pYES-GykTFPI161-117Q was digested with Xhol and Xbal (Fig. 7) . This digestion removed the coding sequence for amino acids 142 to 161 in TFPI 1 . 161 (see Fig.3; an Xbal site is located 6 nucleotides downstream of the translational stop codon) . Insertion of a synthetic double- stranded Xhol-Xbal oligonucleotide restored the TFPI coding sequence with a stop codon after amino acid 160Gly resulting in a plasmid encoding ykTFPI 1 .
  • yeast plasmids with N-terminal truncations of TFPI 1 . 161 -ll7Gln
  • the coding sequence for the ykTFPI 1 . 161 -H7Gln fusion was isolated as a 0.68 kb EcoRI-Xbal fragment from pYES- GykTFPI161-117Q and inserted into the polylinker region of plasmid pUC19 (C. Yanisch-Perron, J. Viera and J. Messing, Gene 33 (1985) 103-119) (Fig.7).
  • Yeast plasmids with C-terminal truncations of the precursor ykTFPI 21 . 161 -H7Gln were constructed in a manner similar to the plasmids for C-terminal truncations of the ykTFPI l . 161 -H7Q described above.
  • pYES-GykT21-161-Q was digested with Xhol and Xbal and the two different synthetic double-stranded oligonucleotides inserted in order to introduce stop codons after amino acids 160Gly or 149Asp.
  • the pYES plasmids encoding the six TFPI analogues fused to the yk secretion sequence were transformed into the haploid S. cerevisiae strain YNG318 (genotype MAT ⁇ ura3-52 Ieu2- ⁇ 2 his4 pep4- ⁇ l; an isogenic derivative of strain JC482 (J.F. Cannon and K. Tatchell, Mol. Cell. Biol. 2 (1987) 2653-2663)).
  • the plasmids were introduced by the alkali cation transformation procedure (H. Ito, Y. Fukuda, K. Murata and A. Kimura, J. Bacteriol.
  • Plasmid TFPI-117 Gin Secreted TFPI analogue activity (U/A 600 -ml )
  • the tested TFPI-117 Gin analogues are effectively secreted by yeast transformants, and they are produced in active form.
  • the 2-3 fold difference in activity levels observed for the analogues may be due to differences in e.g. gene expression levels, the amounts of TFPI polypeptides secreted, different specific activities, or it may reflect experimental variance.
  • the analogues can be purified from the culture supernatants and characterized further.
  • Cys Lys Ala lie Met Lys Arg Phe Phe Phe Asn lie Phe Thr Arg Gin

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Abstract

De nouveaux analogues de TFPI (inhibiteurs de voie de facteur tissulaire) non glycosylatés contiennent le premier et le deuxième domaines de Kunitz mais pas le troisième domaine de Kunitz, et ils offrent une demi-vie prolongée par rapport aux analogues de TFPI glycosylatés correspondants.
PCT/DK1994/000085 1993-03-02 1994-03-02 Analogues de tfpi non glycosylates WO1994020537A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2738016A1 (fr) * 1995-08-21 1997-02-28 Inst Oenologie Procede d'integration stable de genes dans la levure
US5772629A (en) * 1995-10-23 1998-06-30 Localmed, Inc. Localized intravascular delivery of TFPI for inhibition of restenosis in recanalized blood vessels
WO1998042850A1 (fr) * 1997-03-26 1998-10-01 Rpms Technology Limited Proteines hybrides anticoagulantes ancrees dans des membranes cellulaires

Citations (2)

* 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
WO1991019514A1 (fr) * 1990-06-19 1991-12-26 Novo Nordisk A/S Preparation anticoagulante

Patent Citations (2)

* 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
WO1991019514A1 (fr) * 1990-06-19 1991-12-26 Novo Nordisk A/S Preparation anticoagulante

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2738016A1 (fr) * 1995-08-21 1997-02-28 Inst Oenologie Procede d'integration stable de genes dans la levure
US5772629A (en) * 1995-10-23 1998-06-30 Localmed, Inc. Localized intravascular delivery of TFPI for inhibition of restenosis in recanalized blood vessels
WO1998042850A1 (fr) * 1997-03-26 1998-10-01 Rpms Technology Limited Proteines hybrides anticoagulantes ancrees dans des membranes cellulaires
EP1676920A3 (fr) * 1997-03-26 2007-07-25 Imperial Innovations Limited Proteine-fusion anticoagulante ancrée à une membrane cellulaire
US9376684B2 (en) 1997-03-26 2016-06-28 Imperial Innovations Limited Anticoagulant fusion protein anchored to cell membrane

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