WO1990010081A1 - Analogues solubles de la thrombomoduline - Google Patents

Analogues solubles de la thrombomoduline Download PDF

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Publication number
WO1990010081A1
WO1990010081A1 PCT/US1990/000955 US9000955W WO9010081A1 WO 1990010081 A1 WO1990010081 A1 WO 1990010081A1 US 9000955 W US9000955 W US 9000955W WO 9010081 A1 WO9010081 A1 WO 9010081A1
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amino acid
ala
sequence
delta
analog
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PCT/US1990/000955
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English (en)
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Michael John Morser
Charles Glaser
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Codon
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Priority to KR1019910700941A priority Critical patent/KR920701459A/ko
Publication of WO1990010081A1 publication Critical patent/WO1990010081A1/fr
Priority to US08/463,605 priority patent/US5827824A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3153—Streptokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/7455—Thrombomodulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation

Definitions

  • the present invention relates to the production and use of recombinant DNA technology to manufacture soluble thrombomodulin analogs useful, e.g., in antithrombotic therapy.
  • Novel proteins, nucleic acid sequences, vectors, pharmaceuticals and methods of inhibiting thrombotic activity are disclosed herein.
  • antithrombotic compounds capable of preventing clot formation, as well as decreasing the chance of a reocclusion event after fibrinolytic therapy.
  • Several disease states require treatment with an effective, safe anticoagulant.
  • anticoagulants are used for treatment of pulmonary embolism, to prevent deep venous thrombosis in patients undergoing prolonged bed rest or following certain surgical procedures, and for treatment of acute myocardial infarction with or without thrombolytic therapy.
  • Heparin in particular has several other physiological effects that impinge on hemodynamics and hemostasis. Also, these treatments necessitate very stringent laboratory monitoring, as patients are at severe risk for bleeding episodes. In these therapies, it is difficult to determine the proper dose that will provide
  • Warfarin therapy often takes several days to become effective, and the effects require even longer to subside once the therapy is discontinued.
  • Thrombomodulin a 105 kDa cell membrane protein, has been shown to have anticoagulant
  • thrombomodulin is widely distributed on the endothelium of blood vessels and lymphatics of all organs except the central nervous system and helps modulate the balance between the formation and the dissolution of blood clots.
  • thrombomodulin controls the activity of thrombin, a central enzyme in the coagulation of blood. Thrombin's activities include conversion of fibrinogen to fibrin and stimulation of platelet activation and aggregation, ultimately resulting in blood clot
  • the complex does not promote clot formation but instead acts as an anticoagulant by inhibiting the direct procoagulant actions of thrombin, and by
  • Activated Protein C is a potent anticoagulant enzyme which disrupts the coagulation cascade.
  • thrombomodulin The DNA sequence encoding thrombomodulin has been isolated and sequenced both in its genomic form and as a cDNA molecule (R. Jackman ejt al. (1986), Proc. Natl. Acad. Sci. USA., 83:8834-38 and (1987), 84:6425-29, both of which are incorporated herein by reference) and domains of thrombomodulin have been suggested (D. Wen et al. (1987), Biochemistry. 26:4350-57).
  • EGF epidermal growth factor
  • Native thrombomodulin is membrane bound due to its inherent amino acid sequence. It is insoluble without detergent treatment, generally rendering it unsuitable for systemic therapies such as for
  • Soluble thrombomodulin-like molecules have been reported in human plasma and urine.
  • thrombomodulin H. Ishii and Majerus, P., (1985), J. Clin. Invest., 76:2178-81). These forms of thrombomodulin are present in such low amounts
  • thrombomodulin have been described and partially sequenced (see Ishii, (1985), supra; Kurosawa et al.,
  • thrombomodulin protein Analogs of thrombomodulin have been described in WO 88/05053 which discloses analogs with varying numbers of EGF domains.
  • This invention provides sequences of nucleic acid encoding a peptide selected from the group
  • ala.val.val.pro.arg.ser. - Q where Y is selected from the sequence of amino acids provided in Table 2 ranging from amino acid 227 to amino acid 462, and where Q is selected from the sequence of amino acids provided in Table 2 ranging from amino acid 227 to amino acid 462, amino acid 350 to amino acid 462 or amino acid 227 to amino acid 497.
  • the sequences represented by Y and Q denote the epidermal growth factor-like domains of the native TM, the fourth, fifth and sixth EGF-like domains and the six EGF-like domains plus the O-linked glycosylation domain.
  • sequence is selected from the group consisting of:
  • X represents the nucleic acid bases numbered 879 to 1586
  • Z represents the nucleic acid bases numbered 879 to 1586, 1251 to 1586 or 879 to 1690 all numbers refer to bases depicted in Table 2 and vectors comprising such nucleotide sequences.
  • compositions may be those wherein the peptide is a species of the peptide which exhibits about the expected molecular weight for said peptide under non-reducing chromatography.
  • chromatographic gel within about 10% of the predicted molecular weight of the peptide based on its structure (exclusive of any glycosylation that might be present) . They may also be in a dry and salt-free form, a
  • compositions may comprise one of the described peptides chemically linked to a fibrinolytic enzyme, preferably where the chemical linkage arises from a covalent bond between the enzyme and an amino acid residue of the peptide.
  • the fibrinolytic enzyme in such a complex is preferably a streptokinase molecule bound to a plasminogen
  • compositions having antithrombotic activity comprising a sterile
  • compositions preparation of a unit dose of thrombomodulin-like protein and having an amino acid sequence described above are disclosed, as well as methods for controlling thrombotic activity in a mammal by administering an effective amount of such compositions.
  • compositions which comprise a biocompatible polymer having a surface which has peptides bound thereto selected from the group of peptides described above and methods for inhibiting blood clotting induced by polymers implanted into a living mammal wherein a thrombomodulin-like protein is bonded to the polymer and the polymer is implanted into the mammal.
  • This invention additionally dislcoses an isolated DNA fragment containing a DNA sequence coding for multi-functional soluble human thrombomodulin (TM) analog, said analog consisting essentially of amino acid residues 350 to 462 or 390 to 462 or 227 to 462 of native TM and a targeting component.
  • the targeting component is a sequence of amino acids capable of binding to fibrin and preferably also imparting
  • the analogs are preferably operably linked to a DNA sequence coding for a fusion protein comprising: a first amino acid sequence comprising a thrombomodulin fragment without a stop transfer domain; and a second amino acid sequence comprising a targeting component; wherein the bifunctional TM analog is secreted from the host.
  • Preferred sequences of TM used in these analogs are as follows: delta 1-389, delta 463-557 TM;
  • the bifunctional analog may have fibrinolytic activity resulting from the fusion with t-PA as
  • the invention further provides for methods of suppressing coagulation in a patient comprising
  • TM analog effective to suppress further coagulation, wherein the analog comprises TM amino acids as
  • the invention also provides for treating a patient suffering form acute myocardial infarction caused by a thrombus, said method comprising administering to the patient an amount of bifunctional soluble TM analog effective to suppress further
  • bifunctional TM analog comprises TM amino acids as described above wherein said TM amino acid sequence is fused at the C-terminus or the N-terminus to amino acids 4-530 of human t-PA.
  • the invention also provides for a
  • bifunctional TM analog capable of binding thrombin with substantially the same affinity for thrombin as native thrombomodulin, said analog comprising native
  • bifunctional analog is further characterized by the ability to bind fibrin and cleave plasminogen to plasmin.
  • analogs which consist essentially of the amino acid sequence set forth in Table 9.
  • TM analogs wherein said analogs consist essentially of the signal sequence of t-PA covalently attached to the amino terminus of the 6 EGF-like domain of thrombomodulin and the 6 EGF-like domain of
  • This invention further relates to soluble thrombomodulin analogs and methods for making such analogs. More specifically these processes for making soluble thrombomodulin analogs comprising the steps of: culturing a host cell transformed with a DNA sequence encoding the analog; and collecting analogs secreted by the host cell; wherein the analogs lack a stop transfer sequence functional in the host cell.
  • the host cells are preferably eukaryote cells such as yeast or
  • the analog be derived from an isolated DNA fragment containing a DNA sequence coding for a soluble human thrombomodulin analog, capable of being secreted by a host cell transfected with said DNA fragment, wherein said DNA sequence encodes a
  • polypeptide comprising amino acid resides 227-462 of native TM as depicted in Table 2.
  • the preferred analogs have been describe above for the bifunctional fusion analogs and the analogs. Recombinant cells producing such analogs, the protein analogs themselves, pharmaceutical preparations and methods of using the analogs are also disclosed herein.
  • the analogs preferably retain substantially the same or increased affinity to thrombin as native thrombomodulin.
  • Solubility is preferably achieved by deleting the stop transfer domain entirely or in part.
  • Drawing 1 presents a schematic description of the role of thrombomodulin as an anti-coagulant
  • Drawing 2A and 2B schematically illustrate analogs disclosed herein.
  • Drawing 3 illustrates two of the synthetic oligonucleotides used in Example 1 and the plasmid pUC19pcrTM7.
  • Drawing 4 illustrates eukaryotic expression plasmid pTM108 as described in Example 2.
  • Drawing 5 illustrates the construction of transient mammalian expression vector pTHR13 from pTHR5 as described in Example 3.
  • Drawing 6 illustrates the construction of baculovirus transfer vector pTMHYlOl as described in Example 4.
  • Drawing 7 illustrates vectors used to produce fusion proteins that have both antithrombotic and fibrinolytic activity as described in Example 9.
  • Drawing 8 schematically illustrates a
  • multifunctional molecule comprising a fibrinolytic exzyme (such as lys-plasminogen streptokinase complex or t-PA) covalently conjugated to a TM analog.
  • a fibrinolytic exzyme such as lys-plasminogen streptokinase complex or t-PA
  • Drawing 9 illustrates an elution and activity profile produced during the separation of TM analog 6h- 227/462 varient forms using anion exchange
  • Drawing 10A shows a graph comparing the
  • Drawing 10B shows a dose response curve for TM analog 6h/227-462 in the Protein C activation assay.
  • Drawing 11A illustrates the activity of TM 6h/227-462 in the APTT.
  • Drawing 11B shows the activity of TM analog 6h/227-462 compared to antithrombin III and heparin in the activated partial thromboplastin time assay.
  • Drawing 12 shows a graph depicting the prolongation of clotting time by TM analog 6h/227-462 measured in three assays; APTT, TCT and PT.
  • Drawing 13 shows the direct inhibition of thrombin mediated conversion of fibrinogen to fibrin by TM analog 6h/227-462.
  • thrombomodulin except that they are made in a soluble form that is secreted from the producer cell. Also provided are methods for the production of these compositions. These soluble thrombomodulin analogs can be produced economically and are easily purified and administered. A variety of therapeutic uses are anticipated, particularly with respect to anticoagulant and/or antithrombotic therapies. In order to fully appreciate the invention, the following detailed description is set forth.
  • Thrombomodulin or “TM”, as used herein, refers to a protein with the biological activities of mammalian thrombomodulin typically found on endothelial cell surfaces and capable of acting as a receptor for thrombin (N. Esmon, (1987) Seminars in Thrombosis and Hemostasis. 13(4):454-463 and European Patent
  • a DNA sequence encoding the full-length native human thrombomodulin protein has been isolated (European Patent Application No. 88870079.6, which is incorporated herein by reference).
  • the cDNA sequence encodes a 60.3 kDa protein of 575 amino acids, which includes a signal sequence of about 18 amino acids.
  • Thrombomodulin within this definition includes natural allelic variations that may exist between individuals.
  • thrombomodulin exhibit a high degree of homology with one another.
  • domain refers to a discrete amino acid sequence that can be associated with a particular function or characteristic.
  • the full length thrombomodulin gene encodes a precursor peptide containing the following domains:
  • the TM analogs of the present invention have been modified to embrace selected specific regions of the native protein, that is (i) EGF-like domains 4, 5, and 6, (ii) all six of the six EGF-like domains EGF-like domains or (iii) the six EGF-like domains plus the O-linked glycosylation domain.
  • Table 1 and Drawings 2A and 2B provide the specific analogs to which this invention is drawn.
  • the two peptides 11/6 and 11/2 refer to peptides which are similar to the elastase cleaved TM.
  • Elastase is an endopeptidase which cleaves at small neutral amino acids.
  • the 11/6 refers to the number of amino acid residues present at the amino and carboxy termini respectively, which are outside the 227-462 region of the 6 EGF-like domains.
  • This peptide is analogous to the rabbit TM elastase fragment in the number of residues which are outside this region.
  • the second TM analog is the 11/2 peptide.
  • the 11 refers to the number of residues on the amino terminus which are beyond the 227 residue and the 2 refers to the 2 amino acids residing beyond the 462 residue.
  • the 11/2 analogue is believed to be the elastase fragment of human TM.
  • TM analogs 6h/227-462, 6h/227:462:227-462, 6h/350-462, and 6h/227-497 all have six additional amino acids from the hypodermin A signal sequence.
  • These TM analogs are comprised of one or more of the EGF regions and/or the O-linked glycosylation domain. The specific regions embraced by these species are listed in the table below. Additional species are shown in Table 1 and Drawings 2A and 2B.
  • X is the number of amino acids derived from the signal sequence.
  • TM multifunctional proteins
  • heterologs are composed of a first functionality that is associated with native thrombomodulin, thrombin binding for example, and a second functionality that is heterologous, ie. is a functionality from a source other than native thrombomodulin.
  • the second functionality may effect localization of the TM
  • heterologous peptide sequence may provide an additional biological activity, such as a proteolytic activity.
  • proteolytic activity is the enzymatic cleavage of plasminogen to plasmin.
  • the targeting components of this invention are designated as targeting to fibrin. It is preferred that the target also have fibrolytic activity.
  • the multifunctional TM heterologs disclosed herein may be fusion proteins, wherein they have been provided with heterologous protein domains by N- and/or C-terminal extensions of the original TM sequences. Additionally, the multifunctional TM heterolog may be a chemical conjugate to an heterologous protein through a spacer molecule. Ruger et al, (1987), Proc. Natl.
  • Fibrinolysis 2:189-195 have described chemical linkages between t-PA and other molecules. These molecules have an altered affinity for cell surfaces, or enhanced affinity for fibrin.
  • Preferred heterologous protein domains may be encoded by nucleotide sequences isolated from tissue plasminogen activator or pro-urokinase.
  • Suitable t-PA sequences may include, in addition to the entire protein:
  • a particularly preferred embodiment of a heterologous domain of the present invention includes amino acids 4-530 of human tissue plasminogen activator (t-PA).
  • the TM analogs described are secreted from the eukaryotic cells in which they are produced.
  • a "soluble TM analog” is a TM analog which is soluble in an aqueous solution and can be secreted by a cell.
  • the soluble TM analog may optionally be combined with phospholipid vesicles, detergents or other similar compounds well known to those skilled in the art of pharmacological
  • TM analogs of the present invention are soluble in the blood stream, making the analogs useful in various anticoagulant and other therapies.
  • the TM analogs of the present invention are active in one or more functional assays that measure aspects of clotting. These can include the activation of Protein C, by acting as a co-factor of thrombin, the inhibition of the conversion of fibrinogen to fibrin by thrombin (thereby substantially inhibiting blood coagulating activity) or the
  • the latter two assays can be run on an automatic coagulation timer according to the
  • Multifunctional proteins will also be active in assays that measure the activities not normally associated with TM.
  • the fibrinolytic activity of fusion proteins combining TM analog sequences with, for example, t-PA can be measured in any of several assays, such as the fibrin plate assay described by Haverkatet and Brakman, (1975) Prog. in Chem. Fibrin. Thromb. 1:151-159.
  • Nucleic acid sequences encoding these proteins may be used to transform cells.
  • sequences most typically DNA, may or may not contain an associated signal sequence.
  • transformed or transfected cells can be cultured to readily produce these proteins in large quantities.
  • the nucleic acid sequences described are operable and useful in a number of host cells which are adapted to tissue culture.
  • the cells are eukaryotic cells, preferably human, that can grow rapidly in standard media preparations.
  • Prokaryotic or yeast cells may also be suitable for the use of the described invention.
  • This invention embraces molecular genetic manipulations that can be achieved in a variety of known ways.
  • the recombinant cells, plasmids, and DNA sequences of the present invention provide a means to produce pharmaceutically useful compounds wherein the compound, secreted from recombinant cells, is a soluble derivative of thrombomodulin.
  • Thrombomodulin Analogs as an Anticoagulant/ Antithrombotic.
  • the underlying pathology of thrombotic disorders is that a clot forms in response to a
  • This stimulus such as, for example, a damaged vessel wall.
  • This stimulus triggers the coagulation cascade generating thrombin, which has the ability to convert fibrinogen to fibrin, the matrix of the clot.
  • Soluble thrombomodulin analogs administered systemically will protect against thrombus formation because they will inhibit the generation of thrombin, via the activated Protein C system, and/or inhibit the action of thrombin on fibrinogen without disturbing other coagulation parameters.
  • the use of soluble thrombomodulin analogs will be both safe and effective at preventing unwanted thrombus formation.
  • the effect of thrombomodulin can be overcome by the large amounts of thrombin generated by a serious injury to vessels allowing a hemostatic plug to form.
  • thrombomodulin analogs alone or in combination with thrombolytics are useful for treatment, either to cure the disease or to prevent its progression to a more severe state. Soluble thrombomodulin analogs also provide a safe and effective anticoagulant, for
  • bioprostheses such as heart valves or patients requiring extracorporeal circulation.
  • These compounds may replace heparin and warfarin in the treatment of, for example, pulmonary embolism or acute myocardial infarction.
  • PE pulmonary embolism
  • antithrombotic which is effective at preventing DVT without predisposing the patient to bleeding or other complications could make a significant impact on patient recovery and well-being.
  • Angioplasty is a procedure frequently used for restoring patency in occluded arteries. Although patency may be restored, this procedure often damages the endothelial lining of the artery, and blood clots begin to form as a result. Soluble thrombomodulin analogs administered in conjunction with angioplasty will prevent this deleterious side effect.
  • urokinase Use of these agents can lead to serious bleeding complications. Patients who have had a thrombus removed by fibrinolytic therapy and in whom the blood flow has been restored frequently reocclude the affected vessel, i.e., a clot reforms. Attempts have been made to prevent reocclusion by increasing the dose or duration of treatment with a thrombolytic agent, but the incidence of bleeding then increases. Thus the therapeutic index for these drugs is narrow.
  • soluble thrombomodulin analogs provides protection against reocclusion, and its specific action is local rather than systemic, i.e., where thrombin is being generated or being released from a clot. Therefore, when used in combination with a thrombolytic agent, whose dose can then be decreased, the risk of bleeding can be substantially reduced.
  • multifunctional proteins direct the soluble TM heterolog to the site of the fibrin clot.
  • the fibrinolytic activity conferred upon the compound by the heterologous domain provides a superior thrombolytic agent.
  • the TM domain(s) are inherently located precisely where needed to bind thrombin and inhibit any further growth of the clot matrix.
  • This thrombin may be either newly generated by the coagulation pathway or released from the dissolving clot.
  • the therapeutically effective dose of the multifunctional protein will be less than the doses of each molecule administered individually, reducing any concerns about the broader systemic action of either the TM analog or the t-PA and any consequential undesirable side effects.
  • soluble thrombomodulin analogs would be by a bolus intravenous injection, by a constant intravenous infusion or by a combination of both routes. Also, soluble thrombomodulin mixed with appropriate excipients may be taken into the
  • a therapeutically effective dose is defined as that level of TM analog required to prevent formation of pathological clots.
  • Systemic treatment with thrombomodulin analogs can be monitored by determining the activated partial thromboplastin time (APTT) on serial samples of blood taken from the patient.
  • APTT activated partial thromboplastin time
  • the coagulation time observed in this assay is prolonged when a sufficient level of TM analog is achieved in the circulation.
  • analogs of this invention should offer an improved pharmaceutical. It is anticipated that these analogs will offer superior characteristics from a
  • Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by S.L. Beaucage and M.H. Caruthers, (1981)
  • oligonucleotides Purification of oligonucleotides was by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in J.D. Pearson and F.E. Regnier, (1983) J. Chrom., 255:137-149.
  • the sequence of the cloned genes and synthetic oligonucleotides can be verified using the chemical degradation method of A.M. Maxam et al. (1980) Methods in Enzymology. 65:499-560. The sequence can be confirmed after the assembly of the oligonucleotide fragments into the double-stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double-stranded templates of R.B. Wallace et al. (1981) Gene. 16:21-26. Southern Blot hybridization techniques were carried out according to Southern et al. (1975) J. Mol. Biol.,
  • This invention relates to cloning and use of genes for expression vectors in eukaryotic or
  • prokaryotic cells Intermediate vectors are cloned for amplification in prokaryotes such as E. coli, Bacillus or Streptomyces. Most preferred is E . coli because that organism is easy to culture and more fully
  • E. coli E. coli
  • Strain MH-1 is preferred unless otherwise stated. All E. coli strains are grown on Luria broth (LB) with glucose, or M9 medium supplemented with glucose and acid-hydrolyzed casein amino acids. Strains with resistance to antibiotics were maintained at the drug concentrations described in Maniatis. Transformations were performed according to the method described by D.A. Morrison, (1977) J. Bact. , 132:349-351 or by J.E. Clark-Curtiss and R. Curtiss, (1983) Methods in
  • vector refers to viral expression systems, autonomous self-replicating circular DNA
  • Plasmids and includes both the expression and nonexpression plasmids. Where a recombinant microorganism or cell culture is described as hosting an "expression vector,” this includes both
  • the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or is incorporated within the host's genome.
  • promoter is a region of DNA involved in binding the RNA polymerase to initiate transcription.
  • operably linked refers to a juxtaposition wherein the components are configured so as to perform their usual function.
  • control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • control sequence refers to a DNA sequence or sequences which are capable, when properly ligated to a desired coding sequence, of affecting its expression in hosts compatible with such sequences. Such control sequences include at least promoters in both prokaryotic and eukaryotic hosts, and optionally, transcription termination signals. Additional factors necessary or helpful in effecting expression may also be identified. As used herein, "control sequences” simply refers to whatever DNA sequence may be useful to result in expression in the particular host used.
  • polysaccharide composition from that naturally produced by the human body.
  • compositions of the peptides described herein are those which migrate largely as distinct bands on reducing gels in standard electrophoresis techniques never mind. It is
  • compositions having less than about 10% contamination and most preferably less than 3% contamination with peptides other than that desired.
  • the analogs of the present invention are soluble derivatives which lack a stop transfer
  • the full length gene for thrombomodulin can be prepared by several methods. Human genomic
  • Oligonucleotide probes specific to the thrombomodulin gene, can be synthesized using the published gene sequence. Methods for screening genomic libraries with oligonucleotide probes are known. The publication of the gene sequence for thrombomodulin demonstrates that there are no introns within the coding region. Thus a genomic clone provides the necessary starting material to construct an expression plasmid for thrombomodulin using known methods.
  • RNA prepared from endothelial cells provides suitable starting material for the preparation of cDNA.
  • Synthetic oligonucleotides can be used to construct TM genes. This is done using a series of over-lapping oligonucleotides usually 40-120 bp in length, representing both the sense and non-sense strands of the gene. These DNA fragments can be annealed, ligated and cloned.
  • An alternative and preferred method combines the use of synthetic oligonucleotide primers with polymerase extension on a mRNA or DNA template.
  • This polymerase chain reaction (PCR) method amplifies the desired nucleotide sequence. Restriction endonuclease sites can be incorporated into the primers.
  • Patents 4,683,195 and 4,683,202 describe this method. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • Genes encoding soluble thrombomodulin analogs may be constructed using the gene encoding full length thrombomodulin as a starting material. Alternatively, analogs can be constructed from synthetic
  • oligonucleotides or by use of the polymerase chain reaction on either a DNA or RNA template to produce a pure fragment of the desired gene which can then be cloned. Combinations of these methods can be used in order to obtain the DNA sequences that are desired.
  • DNA codons may be selected to minimize secondary structure that would otherwise interfere with
  • restriction sites find use in construction of a vector capable of expressing a gene encoding a soluble thrombomodulin analog. It may also be useful to design particular codon usage for efficiency in expression by a particular host cell.
  • soluble thrombomodulin analogs may have more than one copy of a native domain.
  • a soluble thrombomodulin analogs may have more than one copy of a native domain.
  • thrombomodulin analog of the present invention may have more than one copy of the 6 EGF-like domains. Such a molecule may have enhanced utility in a pharmaceutical product since this region binds thrombin.
  • soluble TM analogs described herein are secreted when expressed in eukaryotic cell culture.
  • Secretion may be obtained by the use of the native signal sequence of the thrombomodulin gene.
  • thrombomodulin analogs of the present invention may be ligated in proper reading frame to a signal sequence other than that corresponding to the native
  • thrombomodulin gene For example, the signal sequence of t-PA, (see commonly assigned co-pending USSN 074,083 filed July 16, 1987 incorporated herein by reference) or of hypodermin A or B (see commonly assigned copending U.S. Serial No. 148,749, filed January 27, 1989 incorporated hereby by reference) can be linked to the polypeptide.
  • the signal sequence of t-PA which contains the second intron of the human t-PA gene. The inclusion of the intron enhances the productivity of the adjacent structural gene (see commonly assigned co-pending USSN #003,611 filed
  • a stop codon so that translation will be terminated at the desired position.
  • a stop codon can be provided by the desired expression plasmid.
  • polyadenylation sequence is necessary to ensure proper processing of the mRNA in eukaryotic cells encoding the soluble thrombomodulin analog. Also, it may be
  • Such sequences may be provided from the native gene or by the expression plasmid.
  • TM analogs described may be encoded by genes wherein at least one N-terminal or C-terminal domain is deleted from the naturally occurring human DNA sequence encoding TM, and replaced with a DNA sequence encoding a fibrinolytic enzyme.
  • T-PA and prourokinase are fibrinolytic enzymes useful for such an analog. It may be desirable to replace both an N-terminal and a C-terminal domain of the native
  • the gene encoding full length TM is modified so that the signal sequence and N-terminal domains, comprising amino acids -18 through 226 are deleted and replaced with DNA encoding the signal sequence of t-PA (amino acids -32 to -1 according to the numbering system in Table 6).
  • the TM analog described above may be further modified by the use of DNA encoding amino acids 4 to 530 of t-PA to replace the O-linked glycosylation, stop transfer, and
  • cytoplasmic domains of native thrombomodulin amino acids 463-557.
  • Such a recombinant DNA molecule can be transfected into a host cell, thus providing a
  • multifunctional protein capable of binding fibrin, activating Protein C, and converting plasminogen to plasmin.
  • a preferred source of the t-PA gene can be obtained by isolating the t-PA gene from an E. coli culture (strain MH-1) on deposit with American Type
  • TM analogs may be desirable for other TM analogs described herein, and the use of the gene encoding pro-urokinase would have analogous utility in creating a multifunctional protein.
  • thrombomodulin analogs of this invention are described by their amino acid sequences and by their DNA sequence, it being understood that the analogs include their biological equivalents such that this invention includes minor substitutions and
  • Cloning vectors suitable for replication and integration in prokaryotes or eukaryotes and containing transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of soluble thrombomodulin analogs are described herein.
  • the vectors are comprised of
  • a cloned gene such as a DNA sequence encoding a soluble thrombomodulin analog in a
  • prokaryotic system it is essential to construct expression vectors which contain, at the minimum, a strong promoter to direct mRNA transcription
  • regulatory regions suitable for this purpose are the promoter and operator region of the E. coli ⁇ -galactosidase gene, the E. coli
  • tryptophan biosynthetic pathway or the leftward promoter from the phage lambda.
  • selection markers include the genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
  • pUC19 is used as a vector for the subcloning and amplification of desired gene sequences.
  • the DNA sequence encoding a soluble TM analog can be ligated to various expression vectors for use in transforming host cell cultures.
  • the vectors typically contain marker genes and gene sequences to initiate transcription and translation of the soluble
  • the vectors preferably contain a marker gene to provide a phenotypic trait for selection of
  • transformed host cells such as dihydrofolate reductase, metallothionein, hygromycin, or neomycin
  • the nuclear polyhedral viral protein from Autographa californica is useful to screen transfected insect cell lines from Spodoptera
  • hygromycin is included as a eukaryotic selection marker in CHL-1 cells.
  • eukaryotic cell systems useful for the expression of soluble TM analogs, there are numerous cell systems to select from.
  • Illustrative examples of mammalian cell lines include RPMI 7932, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7, C127 or MDCK cell lines.
  • insect cell lines include
  • the expression vector ex. plasmid, which is used to transform the host cell, preferably contains gene sequences to initiate the transcription and sequences to control the translation of the soluble TM analog protein gene sequence. These sequences are referred to as expression control
  • illustrative expression control sequences include but are not limited to the following: the retroviral long terminal repeat promoters ((1982) Nature. 297:479-483), SV40 promoter ((1983) Science. 222:524-527. thymidine kinase promoter (J. Banerji et al. (1982) Cell, 27:299-308), or the beta-globin promoter (P.A. Luciw et al. (1983) Cell, 33:705-716).
  • the recipient vector nucleic acid containing the expression control sequences is cleaved using the retroviral long terminal repeat promoters ((1982) Nature. 297:479-483), SV40 promoter ((1983) Science. 222:524-527. thymidine kinase promoter (J. Banerji et al. (1982) Cell, 27:299-308), or the beta-globin promoter (P.A. Luciw et al. (1983) Cell, 33
  • This segment is ligated to a DNA
  • heterologous proteins For expression of heterologous proteins in yeast, the following promoters are useful for expression of heterologous proteins in yeast.
  • GAL1,10 ((1984) Mol. and Cell. Biol.,
  • polyadenylation or transcription termination sequences need to be incorporated into the vector.
  • An example of a polyadenylation sequence is the polyadenylation sequence from SV40, which may also function as a transcription terminator.
  • sequences incorporated into the appropriate vectors can be used to direct synthesis of proteins in either transient expression systems or in stable clones. In the former case yields are low, but the experiments are quick. In the latter case it takes more time to isolate high producing clones. Different vectors may be used for the two different types of experiments.
  • sequences may be included within the plasmid that allow the plasmid to replicate to a high copy number within the cell. These sequences may be derived from virus such as SV40 (e.g. C. Doyle et al. (1985) J. Cell Biol., 100:704-714) or from chromosomal replicating sequences such as murine autonomous
  • expression should also contain a strong promoter such as the SV40 early promoter (e.g., A. van Zonnenfeld et al. (1987) Proc. Natl. Acad. Sci. USA., 83:4670- 4674) to control transcription of the gene of
  • transient expression provides a rapid method for assay of gene products
  • the plasmid DNA is not incorporated into the host cell chromosome.
  • use of transient expression vectors does not provide stable transfected cell lines.
  • a description of a plasmid suitable for transient expression is provided by A. Aruffo & B. Seed, (1987) Proc. Natl. Acad. Sci. USA., 84:8573-8577.
  • CHL-1 cells These are derived from RPMI 7932 melanoma cells, a readily available human cell line.
  • the CHL-1 cell line has been deposited with the ATCC according to the conditions of the Budapest Treaty and has been assigned #CRL 9446, deposited June 18, 1987.
  • the soluble TM analogs may be expressed in yeast cells.
  • the expression of heterologous proteins in yeast is well known. F. Sherman et al., Methods in Yeast Genetics. Cold Spring Harbor Laboratory, (1982) is a well recognized work describing the various methods which have utility for producing soluble thrombomodulin analogs in yeast.
  • Soluble TM analogs may alternatively be produced in the insect cell lines described above using the baculovirus system. This system has been described by V.A. Luckow and M.D. Summers (1988) Bio/Technology. 6:47-55. Generally, this expression system provides for a level of expression higher than that provided by most mammalian systems.
  • the baculovirus infects the host insect cells, replicates its genome through numerous cycles, and then produces large amounts of polyhedron crystals.
  • the polyhedron gene can be replaced with a TM analog gene.
  • the promoter will then make large amounts of analog protein following infection of the culture host cell and replication of the baculovirus genome.
  • the nonsecreted gene product is harvested from the host 3-7 days post infection.
  • the soluble TM protein may be secreted from the cells if appropriate signal sequences are present on the protein.
  • the host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate
  • liposomes containing the DNA liposomes containing the DNA, electroporation and microinjection of the DNA directly into the cells.
  • the host cell is capable of rapid cell culture and able to appropriately
  • transfected cells are grown up by means well known in the art. For examples, see Biochemical Methods in Cell Culture and Virology, Kuchler, R. J., Dowden, Hutchinson and Ross, Inc. (1977). The
  • expression products are harvested from the cell medium in those systems where the protein is secreted from the host cell or from the cell suspension after disruption of the host cell system by, e.g., mechanical or
  • the present invention provides soluble TM analogs which are secreted by cultured recombinant eukaryotic cells.
  • the analogs are produced in serumfree or serum supplemented media and are secreted intact. If prokaryotic cells are used, the TM analogs may be deposited intracellularly. The analogs may be glycosylated or non-glycosylated.
  • this "conditioned media" is harvested. The conditioned media is then clarified by centrifugation or filtration to remove cells and cell debris.
  • the proteins contained in the clarified media are concentrated by adsorption to any suitable resin such as, for example, Q Sepharose or metal chelators, or by use of ammonium sulfate
  • Recombinant TM analogs may be produced in multiple conformational forms which are detectable under nonreducing chromatographic conditions. Removal of the higher molecular weight species and those species having a low specific activity is desirable and is achieved by a variety of chromatographic techniques including anion exchange or size exclusion
  • Recombinant TM analogs may be concentrated by pressure dialysis and buffer exchanged directly into volatile buffers (e.g., N-ethylmorpholine (NEM), ammonium bicarbonate, ammonium acetate, and pyridine acetate).
  • volatile buffers e.g., N-ethylmorpholine (NEM), ammonium bicarbonate, ammonium acetate, and pyridine acetate.
  • samples can be directly freezedried from such volatile buffers resulting in a stable protein powder devoid of salt and detergents.
  • freeze-dried samples of recombinant analogs can be efficiently resolubilized before use in buffers compatible with infusion (e.g., phosphate buffered saline).
  • suitable buffers might include
  • streptokinase also called APSAC or EminaseTM (Beecham Research Labs., Ltd., Middlesex, U.K.)
  • APSAC APSAC
  • EminaseTM Beecham Research Labs., Ltd., Middlesex, U.K.
  • the acylation of the active site of the lys-plasminogen streptokinase complex leaves the fibrin binding site free, thus the complex retains the ability to bind to an unwanted thrombus.
  • the active fibrinolytic complex is produced in situ after slow deacylation by hydrolysis.
  • the same technique can be used to chemically link other molecules, such as a soluble TM analog, to the active site of a fibrinolytic enzyme through free amine groups on the analog.
  • the peptides disclosed herein can be chemically linked to a fibrinolytic enzyme such as lys-plasminogen
  • streptokinase streptokinase, t-PA or the like through a covalent bond between the enzyme, and an amino acid residue within the peptide.
  • lys-plasminogen streptokinase complex can be linked specifically and reversibly to a TM analog by using a heterobifunctional inverse substrate of plasmin (see, for example, Drawing 8).
  • the fibrinolytic enzyme linked to the TM analog can be tissue plasminogen activator (t-PA).
  • t-PA tissue plasminogen activator
  • acyl-enzyme is reacted with thiolated TM analog.
  • the acylation reaction is highly specific for the active center serine residue in either the plasminogen or the t-PA molecules.
  • Thiolated TM is preferably produced using analogs having only one free amine group, 11/2, 4t-227/462 or 6h-227/462, which is at the N-terminus of the molecule.
  • Thiolated TM analog is produced by reacting the analog with 2-iminothiolane as described in Smith (1988), supra.
  • TM>SK:Plg or TM>t-PA acylated fibrinolytic enzyme
  • the activity of the TM analog or the fibrinolytic enzyme can be individually assayed by methods described herein. Hydrolysis of the acylenzyme conjugate can be monitored on SDS gels and by release of fibrinolytic activity.
  • acylenzyme pro-drugs Chemical linkages are used to produce acylenzyme pro-drugs.
  • This approach provides a reversible TM>fibrinolytic conjugate with the combined properties of both molecules, thus providing a route to thrombus specificity, via the lys-plasminogen streptokinase complex or t-PA, with a kinetic approach to the control of bleeding.
  • the TM>fibrinolytic acyl-enzyme pro-drug acts as a relatively slowly cleared reservoir of fibrinolytic activity.
  • the half-life of the fibrinolytic and the clot specificity of the TM analog is increased and the clearance rates and systemic effects of both are decreased.
  • Thrombomodulin activity can be detected in a variety of assays that depend on alterations in the action of thrombin. The ability of thrombomodulin or its soluble analogs to accelerate the thrombin
  • this assay consists of two steps. The first step is the incubation of the test
  • thrombomodulin or TM analog with thrombin and Protein C under defined conditions.
  • the thrombin is inactivated with hirudin or antithrombin III and heparin, and the activity of the activated Protein C is determined by use of a chromogenic
  • This assay is carried out with purified reagents.
  • TM analog can be measured using plasma in clotting time assays such as the activated partial thromboplastin time (APTT), thrombin clotting time (TCT) and/or prothrombin time (PT).
  • APTT activated partial thromboplastin time
  • TCT thrombin clotting time
  • PT prothrombin time
  • Soluble TM analogs which have utility as therapeutic agents are able to activate Protein C in physiological concentrations of calcium ion.
  • Formulation and Use of Thrombomodulin Analogs are able to activate Protein C in physiological concentrations of calcium ion.
  • Soluble thrombomodulin analogs described herein may be prepared in a lyophilized or liquid formulation.
  • the material is to be provided in a concentration suitable for pharmaceutical use as either an injectable or intravenous preparation.
  • These compounds can be administered alone or as mixtures with other physiologically acceptable active materials, such as one-chain t-PA, or inactive materials, or with suitable carriers such as, for example, water or normal saline. These compounds can be administered parenterally, for example, by
  • injection can be subcutaneous, intravenous or intramuscular. These compounds are administered in pharmaceutically effective amounts and often as
  • Such salts can include, e.g.,
  • hydrochloride hydrobromide, phosphate, sulphate, acetate, benzoate, malate, citrate, glycine, glutamate, and aspartate, among others.
  • the analogs described herein may display enhanced in vivo activity by
  • An antithrombotic agent can be prepared using the soluble TM analogs described herein and can consist of a completely purified thrombomodulin analog alone or in combination with a thrombolytic agent as described above.
  • Compounds of the present invention which are shown to have the above recited physiological effects can find use in numerous therapeutic applications such as, e.g., the inhibition of blood clot formation.
  • the compounds disclosed can be useful for treatment of systemic coagulation disorders such as DIC, which is often associated with septicemia, certain cancers and toxemia of pregnancy.
  • these compounds can be administered to mammals for veterinary use, such as with domestic animals, and for clinical use in humans in a manner similar to other therapeutic agents, that is, in a physiologically acceptable carrier.
  • the administration dosage will range from about 0.0001 to 100 mg/kg, and more usually 0.001 to 0.1 mg/kg of the host body weight.
  • These dosages can be administered by constant infusion over an extended period of time, until a desired circulating level has been attained, or preferably as a bolus injection.
  • thrombogenesis in association with prosthetic endovascular or cardiovascular devices includes the following sequence:
  • thrombin dissolution which requires plasmin generation and fibrinolysis.
  • the surface will rapidly acquire a layer of absorbed plasma proteins which will mediate subsequent thrombotic events. This series of events also follows when blood is circulated through an extracorporeal device, such as a heart/lung machine.
  • Thrombomodulin represents a new class of molecule suitable for creating a thromboresistant surface. It is especially suitable as such a surface since it has no known inhibitors and will be available to function in this capacity for extended periods of time.
  • the TM analogs described herein are particularly advantageous for this purpose as they are closely related to the protein fragment which is derived when full length TM is digested with porcine pancreatic elastase.
  • the long-term stability of immobilized proteins is of paramount importance.
  • the smaller, proteolytically resistant TM analog is more advantageous than the full length molecule which can be proteolysed by enzymes in the blood, resulting in the potential loss of active component from the biomaterial surface.
  • the TM analogs will be
  • the TM analogs may be used to coat polymers used in a wide variety of biological applications including, but not limited to, arteriovenous shunts, intravascular shunts (eg., umbilical, angiographic), vascular grafts, heart valves, artificial joints, pacemakers, left ventricle assist devices, and the like.
  • Biocompatible polymers may be any suitable polymeric biomaterial or combination thereof known and used in the art for biological application such as polyurethanes, silicone elastomers, hydrogels (e.g., poly(hydroxyethyl methacrylate), polyesters,
  • polyethers polyvinyl alcohol, and the like.
  • the TM analog may be bonded to coat the polymer material following activation of the
  • Activation methods are known in the art and may utilize amino, carboxyl, hydroxyl or sulfhydryl functions on the compound to be coated. Activation may be achieved through a variety of known mono- and/or bifunctional reagents, including, but not limited to, glutaraldehyde, carbodiimide activated COOH,
  • isocyanate isocyanate, cyanuric acid, or hydrosuccinimide esters.
  • Spacer arms known in the art, may optionally be used.
  • the single amine function at the N-terminus of some of the TM analogs disclosed herein, such as 11/2, 4t/227-462 and 6h/227-462 provide a highly selective means to covalently couple this protein in a highly oriented array to activated biomaterial
  • biocompatible polymer Once the biocompatible polymer has been coated, it may be implanted in a mammal as necessary according to the teaching in the art for the procedure at hand or used in any device that contacts blood where the blood must remain anticoagulated.
  • Human DNA was used to isolate a gene encoding the 6 EGF-like domains of thrombomodulin corresponding to amino acids 227-462. This DNA was isolated from fetal liver according to the method of Blin, N and D. W. Stafford, (1976) Nucleic Acids Res. 3:2303. The DNA was then used as a template in a polymerase chain reaction with synthetically derived primers selected to embrace the desired regions (see Drawings 2 and 3, and Tables 1 and 3) .
  • the sequence of the #1033 and #1034 primers correspond to the 5' and 3' ends of the desired domain; but they have been modified so that they contain a BamHI site.
  • a termination codon (TGA) was introduced following base 1586.
  • the polymerase chain reaction was run under the conditions described by Saiki, et al., (1988) Science, 320:1350-1354 except that the initial temperature of annealing was 37oC. After 10 cycles, the annealing temperature was raised to 45oC for the remaining 30 cycles. An aliquot of the reaction products was separated on a 5% polyacrylamide gel and visualized by ethidium bromide staining. A band of the predicted size (700 bp) could clearly be seen. To confirm the identity of the insert, one can optionally sequence this band or hybridize it to an internal probe. b. Isolation of genes encoding other regions of TM.
  • reaction mixture described in part (a.) above was restricted with BamHI, separated on a 5% polyacrylamide gel, and the 700 bp band was excised and eluted. It was ligated to pUC19 that had been restricted with BamHI and the new plasmid was transformed into E. coli strain DH5 ⁇ .
  • Plasmid pTM309 contains amino acids 350-462 of native TM (EGF-like domains 4,5,& 6) and pTM323 contains amino acids 227-497 (EGF-like domains 1-6 + O-linked glycosylation domain).
  • Additional plasmids were constructed that contain other TM analog gene sequences. (See Drawing 2A and Table 1).
  • Mammalian Cells a. Mammalian expression vectors for TM analogs This example provides a mammalian expression vector comprising the analog genes of Example 1.
  • Drawing 4 provides an overview of the construction of one of these vectors.
  • the genes are operably linked to the signal sequence of human tissue plasminogen
  • the expression plasmid, pPA124 contains a promoter contained within the three copies of the long terminal repeats derived from Harvey Sarcoma virus for the expression of cloned genes. This plasmid was derived from pPA119, and pSC672, both described in detail in co-pending USSN #074,083, filed July 16, 1987, incorporated herein by reference. A BglII - BclI fragment containing the SV40 polyadenylation region was isolated from pSC672. This fragment was cloned into pPA119 which had been digested with BglII and BclI. In the resulting plasmid, pPA124, both the BglII and BclI sites remained intact. Plasmid pPA124 contains the t-PA signal sequence adjacent to an appropriate
  • restriction site and this signal sequence also contains the second intron of the human t-PA gene.
  • the gene encoding the soluble TM analog was removed from pUC19pcrTM7 by treatment with BamHI and ligated to pPA124 that had been treated with BglII.
  • Transformants were screened for the presence of the insert in the correct orientation, that is in which the t-PA signal sequence was linked to the 5' end of the thrombomodulin insert encoding an open reading frame.
  • This plasmid, pTM101 was then digested with Clal and ligated to a ClaI fragment containing the dhfr gene under the control of the SV40 promoter. The Clal fragment is described in WO88/02411 at page 26.
  • Transformants were screened for the presence of this dhfr cassette and then the orientation relative to the plasmid was determined by restriction mapping (pTM103).
  • Plasmid pTM103 containing the dhfr sequence in the divergent direction to the thrombomodulin sequence, was treated with Bell and a DNA fragment encoding a gene providing hygromycin resistance on a BamHI fragment was ligated into the plasmid.
  • Clones were selected, after transformation into E. coli strain DH5 ⁇ , by their ability to grow on plates containing both ampicillin and hygromycin B.
  • the orientation of the hygromycin B gene relative to the plasmid was determined by restriction mapping.
  • One plasmid, pTM108 in which the hygromycin B gene lies in the opposite orientation to the TM gene, was grown up in culture.
  • This plasmid has the sequences encoding the TM analog under the control of the triple LTR promoter, with both a gene that confers hygromycin B resistance and one that encodes dhfr present on the plasmid.
  • the thrombomodulin sequence was linked to the tissue plasminogen activator signal sequence, ensuring its secretion. (See Table 6) b. Transfection, selection and amplification of stable mammalian clones.
  • CHL-1 cells transfected with the bacterial hygromycin B gene can survive growth in 0.3 mg/ml hygromycin B.
  • the transfection or selection frequency was 2/10 3 and was determined as the number of colonies arising after selection, divided by the total number of cells plated.
  • the culture supernatant was shown to contain 1.5 U/ml TM activity after 24 hours in contact with the cells.
  • a population of cells resistant to the first selection conditions were then subjected to a second round of selective pressure. Either 100nM or 500nM methotrexate (MTX) was added to the growth medium to select for transfectants that expressed the dhfr gene. Only clones which had amplified the dhfr gene would be able to grow in this high level of MTX. In the process of gene amplification, other plasmid sequences will be co-amplified with the dhfr gene and thus lead to increased gene expression of the non-selectable gene as well. Resistant clones were apparent after 5 to 6 weeks. Individual clones resistant to these levels of MTX were isolated and assayed. A culture after
  • Plasmid pPA133 is a modification of plasmid pL1 (Okayma, H. and Berg P., (1983) Mol. Cell Biol. 3:280-289) purchased from Pharmacia LKB Biochemical (Piscataway, NJ).
  • This plasmid, PL1 carries the SV40 origin of replication the early region transcription promoter and mRNA splicing sequences. It was used to construct a plasmid containing a polylinker sequence that adds a unique series of restriction sites, the t-PA signal sequence under the control of the SV40 promoter and the SV40 origin or replication. The mRNA splicing sites were removed.
  • pPA129 Construction of this plasmid, pPA129, is described in detail in co-pending, commonly assigned patent application USSN 345,372, incorporated herein by reference.
  • pPA133 pPA129 was digested with BglII and another polylinker sequence was inserted immediately downstream from the t-PA signal sequence. This polylinker creates a plasmid with a unique series of restriction sites (BglII, NotI, SnaBI and SplI). ii. pTHR5
  • Plasmid pTHR5 is a modification of plasmid pCDM8 purchased from Invitrogen (San Diego, CA) and carries the cytomegalovirus immediate early promoter. Plasmid pPA133 was digested with XhoI and Notl to release the t-PA signal sequence which was isolated and cloned into pCDM8 that had been digested with XhoI and Notl so that the t-PA signal sequence is under the control of the cytomegalovirus promoter. (See Drawing 5). b. Transient Expression Plasmids Containing TM Analog Genes.
  • the cloning plasmids containing TM analog gene sequences described in Example l were digested with BamHI and/or BglII and/or NotI.
  • the TM gene sequences were isolated and ligated into either pPA133 or pTHR5 immediately adjacent to the t-PA signal sequence to insure that the transiently expressed peptides would be secreted into the cell culture medium.
  • a model plasmid, pTHR13, is shown in Drawing 5.
  • Table 2 lists other TM analog gene containing plasmids and their parent plasmids.
  • Plasmid pTHR13 was made by digesting cloning plasmid pTM301 with BamHI and BglII and ligating the TM analog gene fragment into the BglII site of pTHR5.
  • pTHR13 contains the gene sequence coding for the 6 EGF-like domains of thrombomodulin operably linked to the t-PA signal sequence.
  • COS-1 cells were either transfected with the parental plasmids or mock transfected with phosphate buffered saline (PBS). Protein C activation assays were done using conditioned cell culture medium to determine the presence of TM analog. Table 7 depicts a list of transient expression plasmids.
  • Example 4 Production of human thrombomodulin analogs in insect cells infected with a baculovirus expression vector.
  • Autographa California nuclear polyhedrosis virus was used as an expression vector for human thrombomodulin (TM).
  • TM human thrombomodulin
  • the hybrid hypodermin A-TM analog genes were inserted downstream of the polyhedrin transcription signal, and the TM-polyhedrin hybrid genes were transferred by homologous recombination to an infectious AcNPV expression vector.
  • the wild type AcNPV polyhedrin gene has been replaced with the TM analog gene in the
  • COD#1198 and COD#1199 were synthesized, see Table 6. These oligomers contain the Hypodermin A signal sequence, a translation initiation codon, a BglII cloning site, a BamHI 5' overhand and a Kpnl 3' overhang. COD#1198 and COD#1199 were annealed and cloned into pSC654, a pUC19 derivative, creating pHY1. See Drawing 6.
  • Plasmid pHY1 was restricted with BamHI and EcoRI, releasing the hypodermin A signal sequence.
  • Plasmid pSC714 is a derivative of pVL1393, obtained from Summers, et al. The only
  • the BamHI fragment from pUC19pcrTM7 was cloned into the BglII site of pHY1 and the orientation was chosen such that the hypodermin A signal sequence was adjacent to amino acid 227. This plasmid is
  • Plasmid pHY101 was treated with BamHI/EcoRI which releases the Hypodermin A signal sequence linked to the TM analog coding sequence.
  • Shuttle vector pVL1393 contains a partially deleted AcNPV polyhedrin gene and unique BamHI and EcoRI cloning sites. The BamHI/EcoRI fragment from pHY101 was inserted
  • Transfer plasmids containing other TM analog gene sequences were constructed using a strategy similar to that outlined above. Fragments from the cloning plasmids described in Example 1 above were cloned into pSC716 in frame so that the TM analog gene sequence was fused to the hypodermin A signal sequence. See Table 8 for a list of transfer vectors and the gene sequences contained therein. b. Construction of AcNPV Transfer Vectors
  • a new signal sequence is synthesized that contains a BalI cloning site, a BamHI 5' overhang and a KpnI 3' overhang.
  • the synthetic fragments are annealed and cloned into pSC654 as described above creating pHY2.
  • New genes consisting of human analog 11/6 or human analog 11/2 are isolated by using human DNA in the polymerase chain reaction as described in Example 1.
  • the primers introduce a BalI site at the 5' end and a termination codon and an EcoRI site at the 3' end (see Table 3).
  • the genes are cloned into pHY2, which has also been treated with Ball and EcoRI.
  • the EcoRI site is a unique site in the original pSC654 vector used to make pHY2.
  • the new plasmids pHY102 and pHY103 contain the signal sequence of hypodermin A fused to genes that encode analog 11/6 and analog 11/2, respectively.
  • Plasmids pHY102 and pHY103 are treated with
  • sf9 cells cotransfected into sf9 cells basically as follows: a T25 flask was seeded with 2 ⁇ 10 6 cells. The cells were allowed to attach for one hour at room temperature. Then one microgram of transfer vector and 1 ⁇ g AcNPV DNA coprecipitated in calcium phosphate were incubated with SF9 cells for 4 hrs. TMN-FH media supplemented with 10% FBS was then replenished. A stock virus was created. The genes contained on plasmids pTMHY102 and pTMHY103 are transferred into the AcNPV genome in the same manner as described above. d. Detection and Purification of Recombinant
  • Plaque assays were performed to detect recombinant viruses.
  • the transfection stocks were diluted to 10 -4 , 10 -5 , 10 -6 on day 7 post transfection.
  • a control vector in which ⁇ -galactosidase is under the control of the polyhedrin promoter was also included in the experiment. The results showed that the control vector assayed with X-gal gave an average of 2%
  • Occlusion negative plaques from the cells transfected with the TM transfer vectors were picked and replated at 10 -1 , 10 -2 and 10 -3 dilution of plaque assay. On day 7, the plates showed 100% pure occlusion negative recombinant plaques.
  • 2 ⁇ 10 6 sf9 cells were infected with 1 ml of the transfection stock as described above. On day 3 the cells were pelleted and resuspended in serum free
  • T25 flasks were seeded at a density of
  • infected insect cells which secrete other TM analogs including 6h/(227-462)2, 6h/350-462, 6h/227-497, etc.
  • the t-PA gene is used for the assembly of genes encoding chimaeric fusions of a fibrinolytic enzyme with thrombomodulin polypeptide sequences.
  • the t-PA gene used for this purpose was isolated from plasmid pPA003, which is on deposit with the ATCC, accession No. 67293. Plasmid pPA003 is described in detail in co-pending, commonly assigned, patent
  • Tables 7 and 8 depict a list of mammalian expression plasmids and baculovirus transfer plasmids containing thrombomodulin analog genes fused with the t-PA gene. a. TM Heterologs Synthesized in Mammalian cells
  • TM Analogs fused to the N-terminus of t-PA The 3' ends of the TM analog genes described in the previous examples all have termination codons which must be removed in order to create chimaeric proteins containing thrombomodulin gene sequences fused to the N-terminus of t-PA. Primers for use in a PCR were synthesized so that when incorporated into the DNA sequences of the TM analogs the termination codons at the 3' ends were deleted and a BglII site was
  • the plasmids that result contain a gene that encodes a fusion protein consisting of a signal sequence from t-PA followed by the TM analog which is fused to mature t-PA under control of either the SV40 promoter or the cytomegalovirus promoter. See Drawing 7.
  • TM Analogs fused to the C-terminus of t-PA The termination codon at the 3' end of the t- PA gene was deleted using the same method described above. Two primers were synthesized for the PCR that introduced a new BglII site immediately 3' to the proline at amino acid 530, which also deletes the termination codon, and creates a BamHI site at amino acid 4 in place of BglII site found in the native t-PA gene sequence.
  • the t-PA gene used as a template for the PCR was isolated from plasmid pPA509.
  • This plasmid is on deposit with the ATCC, accession #67443, and is described in detail in co-pending, commonly assigned, patent application USSN 074,083, incorporated herein by reference.
  • the new t-PA gene fragment was inserted into the BglII site of pTHR5 so that the t-PA gene is fused to the t-PA signal sequence, creating plasmid pTHR16. See Drawing 7.
  • Thrombomodulin analog gene sequences digested with BglII and/or Bam HI were inserted into pTHR16 at the BglII site creating plasmids that encode soluble secreted fusion proteins whose N-terminus is the natural N-terminus of t-PA fused at the natural C-terminus of t-PA to the N-terminus of the TM analog gene.
  • Schematic examples of these fusion peptides are shown in Drawing 2B.
  • the DNA and amino acid sequence of t-PA fused with the 6 EGF-like domains of TM is shown in Table 9.
  • TM heterolog gene sequences for expression in insect cells were constructed by methods similar to those described above. For example, a plasmid for producing one TM heterolog in which the 6 EGFs were operably linked to the N-terminus of t-PA was
  • pTHR6 is a transient expression vector containing the 6 EGFs fused to t-PA for expression in mammalian cells.
  • pTHR10 is a baculovirus transfer vector containing gene sequences for the 6 EGF-like domains.
  • the resulting plasmid, pTHR25 contains gene sequences for the 6 EGF-like domains and t-PA in a baculovirus transfer vector.
  • TM analog 6h/227-462 was purified from conditioned media from cells infected with AcNPV-6h/227-462 by removal of cell debris, followed by five chromatography steps: 1) Q Sepharose, 2) thrombin affinity, 3) gel filtration, 4) anion exchange, and 5) a second gel filtration step. Substantially pure
  • chromatographic resins were purchased from commerical sources.
  • Q Sepharose and Sephadex G25 were purchased from Sigma (St. Louis, MO), and Mono Q 5/5TM from Pharmacia LKB (Piscataway, NJ).
  • Thrombin activity was measured using the Kabi S-2238 substrate and indicated that >86% of the
  • thrombin was removed from the solution, and presumably coupled to the resin, giving a final concentration of about 6 mg of thrombin per ml of resin.
  • the enzymatic activity of the DFP treated resin was ⁇ 1% of the starting activity.
  • Conditioned media was harvested and clarified by cross-flow filtration on a Microgon 0.3 ft 2 hollow fiber filter unit at about pH 6.0, then diluted by 50% with ultra-pure water. The pH was adjusted from about 6.0 to about pH 5.2 with glacial acetic acid. The adjusted media was then loaded onto a column of Q
  • Sepharose resin at a flow rate of about 110-150 ml/min.
  • the column had previously been equilibrated with about 4 column volumes of wash buffer 1 (117 mM Na acetate, 0.02% NaN 3 pH 5.0). After loading, the column was washed with wash buffer 1 followed by wash buffer 2 (25mM Na acetate, 0.1MNaCl, pH5.0) then the TM analog was eluted with wash buffer 2 containing 0.3 M NaCl, pH 5.0. This column matrix can be reused by stripping with wash buffer 2 + 2.0 M NaCl.
  • Nonreduced samples were diluted in Laemmli sample solubilization buffer (50 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, and .01% bromphenol blue) and loaded directly onto the gel. Reduced samples were incubated for 7 minutes at 100oC in sample
  • solubilization buffer containing 10 mM dithiothreitol. Following incubation, iodoacetamide was added to a final concentration of 50 mM, and the sample incubated an additional 5 minutes at room temperature, before loading on the gel.
  • Pharmacia LMW Calibration Kit protein standards were used for MW markers, and the gels were silver stained.
  • TM activity all material visualized on the non-reduced gel had TM activity.
  • the lower MW bands seen in non-reduced gels were the predominant form or species of the isolated protein.
  • the preferred species of peptide is one having a higher TM activity being free of other less active thrombomodulin-like peptides. This preferred species corresponds to the slower migrating of the two lower MW bands.
  • Such peptide species were purified below to provide under nonreducing chromatography a single species having the expected apparent molecular weight predicted for the particular peptide.
  • the TM analog 6h/227-462 gene has two
  • affinity purified product was shown to be glycosylated by treatment with N-glycanase which resulted in an apparent decrease in MW of about 2000 daltons.
  • Amino Acid Composition and Sequencing were shown to be glycosylated by treatment with N-glycanase which resulted in an apparent decrease in MW of about 2000 daltons.
  • the resultant solution was reduced with a 43 fold molar excess of DTT over disulfide (2.67 ⁇ moles, 0.89 ⁇ M final concentration) for 3 hr at 37oC, then alkylated for 60 minutes at 37oC by the addition of a 2.6 fold molar excess of iodoacetic acid over DTT (6.94 ⁇ moles, 2.3 ⁇ M final concentration).
  • the sample was purified using a Waters Delta PakC-4 reverse phase HPLC with a linear gradient of water + 0.1% TFA to 75% CH.CN + 0.1% TFA. Only a single protein peak was recovered following the reduction and alkylation protocol. Aliquots were dried down in a Savant evaporator for sequencing and amino acid analysis.
  • amino acid composition was determined as described above in the determination of specific activity starting at the hydrolysis step.
  • the amino acid composition found for the protein agrees well with what was expected (see Table 4).
  • the exceptions are tryptophane and cysteine, which are not accurately measured by the methods used here,
  • the substantially pure, active TM analog 6h/227-462 was applied to a Sephadex G25 column and recovered in 0.2% N-ethylmorpholine acetate (NEM) pH 7.0. This step removes GuHCl and NaCl. ii. Anion Exchange Chromatography
  • NEM N-ethylmorpholine
  • Protein C activation assay The specific activities are listed in the table below. No inactive peptides were detectable in any of the fractions.
  • Test Material Specific Activity (U/mg) Mono Q Load 166 , 000 ⁇ 12 , 000 fxns 32 - 35 416,000 ⁇ 19,000 fxns 40 - 44 262,000 ⁇ 4,000 fxns 70 - 71 67,600 ⁇ 5,000
  • Conditioned cell culture media was collected and the ionic strength adjusted to be equivalent to about the ionic strength of 0.3 M NaCl using 1 M NaCl.
  • the adjusted media was loaded directly onto a thrombin affinity column that had previously been washed with 0.3 M NaCl, 20 mM Tris HCl, 0.5 mM CaCl 2 and 0.02% NaN3, pH7.5 (wash buffer). After loading, the column was washed with wash buffer, then the TM analog was removed from the column matrix with 1.5 M GuHCl, 2.0 M NaCl, 20 mM Tris, 1 mM Na EDTA and 0.02% NaN 3 .
  • a peak containing TM activity was collected and applied to a Sephadex G25 column and washed through with 0.2% NEM to effect a buffer exchange (removal of NaCl and GuHCl).
  • the active fractions were pooled and applied to a column of Mono Q 5/5 (Pharmacia, LKB) in 0.2% NEM, pH 7.0.
  • the TM analog was selectively eluted with a gradient of 0 to 0.6 M NaCl in 0.2% NEM pH7.0.
  • Fractions containing TM activity were pooled and samples run on SDS-PAGE gels under both reducing and non-reducing conditions. The TM analog appeared to be about 90-95% pure.
  • the remaining high molecular weight contaminating material was removed by molecular exclusion chromatography.
  • the activity containing fractions from the Mono Q column were lyophilized to dryness and resuspended in (380 ul) of 1.0% NEM, pH 7.0 then applied to a Superose 12 TM (Pharmacia, Piscataway, NJ) column at 0.5 ml/min.
  • the active material collected from this chromatography step was estimated to be 99% pure TM analog by silver stained SDS-PAGE gel analysis.
  • Rabbit thrombomodulin, hirudin and human Protein C were supplied by American Diagnostica. Human thrombin is available from a variety of noncommercial and commercial sources. Bovine thrombin was purchased from Miles Labs, Dallas, Texas. D-valyl-L-leucyl-L- arginine-p-nitroanilide (S-2266) and D-Phe-Pip-Arg-p-nitroanilide (S-2238) were purchased from Kabi
  • Bovine serum albumin fraction V
  • citrated human plasma citrated human plasma
  • APTT reagent APTT reagent
  • Microtiter plates were supplied by Corning (#25861-96). All other reagents were of the highest grade available. b. Methods and Results.
  • thrombomodulin sample unknown or standard
  • thrombin 3 nM
  • Protein C 1.5 ⁇ M
  • the assay diluent for each protein was 20 mM Tris-HCl, 0.1 M NaCl, 2.5 mM CaCl 2 , 5 mg/ml BSA, pH 7.4.
  • the wells were incubated for 2 hours at 37'C, after which Protein C activation was terminated by the addition of 20 ⁇ l of hirudin
  • the amount of activated Protein C formed was detected by adding 100 ⁇ l of 1.0 mM S-2266 (in assay diluent), and continuing to incubate the plate at 37oC.
  • the absorbance at 405nm in each well was read every 10 seconds for 30 minutes, using a Molecular Devices plate reader.
  • the absorbance data was stored, and the change in absorbance per second (slope) in each well was calculated.
  • the change in absorbance per second is proportional to pmole/ml of activated Protein C.
  • a unit of activity is defined as 1 pmole of activated Protein C generated per ml/min under the reagent conditions defined above.
  • the response of both purified rabbit TM and conditioned medium containing analog 6h/227-462 in this assay is shown in Drawing 10A.
  • Drawing 10B the enhancement of protein C activation by purified TM analog is depicted.
  • the activity values reported were calculated using rabbit thrombomodulin as a standard.
  • TM analogs showed Protein C activation enhancing activity including 4t/227-462, 4t/227-462:227-462, PA:227-462 (mammalian cells), 6h/227-462:PA, 6h/227-462:227-462, PA:227-462 (insect cells), 6h/350-462, and 6h/227-497. ii. Determination of Specific Activity of TM Analog 6h/227-462.
  • TM analog samples were dialyzed exhaustively against 0.2% ethylmorpholine acetate, pH 7.5, and assayed for activity and amino acid composition (no activity was lost due to the dialysis).
  • Samples were prepared for analysis by dialysing samples into 0.2% N-ethylmorpholine-acetate, 0.02% NP-40, pM 7.5 using a 6000 Spectropor membrane and dried down in hydrolysis tubes. Hydrolysis was performed in evacuated tubes in a vapor phase at 110oC for 22 hours using 6 N constant boiling HCl containing 1% phenol. The amino acid analysis was performed after samples were dried down and derivatized with phenylisothiocyanate (PITC).
  • PITC phenylisothiocyanate
  • APTT reagent brain cephalin in ellagic acid
  • calcium ion The time required for the clot to form is reproducible and increases proportionally with the addition of
  • thrombomodulin Reagents for the APTT are incubated at 37oC. before mixing, except for the citrated plasma, which is kept at 4oC.
  • the reaction was carried out as follows: 100 ⁇ l of Sigma Citrated Plasma was added to a plastic cuvette (Sarstedt #67.742), incubated at 37oC for 1 min; 100 ⁇ l of Sigma APTT reagent was added and the mixture incubated for 2 min at 37oC; 100 ⁇ l of test sample (or control buffer) and 100 ⁇ l 25 mM CaCl 2 were added and the cuvette was immediately placed in a
  • Hewlett-Packard 8451A spectrophotometer equipped with a circulating water bath to keep the cuvette at 37oC during reading.
  • the absorbance due to light scattering at 320 nm was measured every 0.5 seconds, from 15 to 120 seconds, timed from the addition of CaCl 2 .
  • a plot of absorbance vs. time yields a sigmoidal curve, with the clotting time defined as the time at which the slope is the steepest, corresponding to the inflection point of the curve.
  • TM analog 6h/227-462 was compared to heparin and antithrombin III in the APTT assay. As seen in Drawing 11B, TM analog 6h/227-462 showed a greater ability to prolong the APTT at lower amounts than either heparin or AT III. The quantities of heparin and AT II used in this experiment are not effective doses in vivo suggesting that a TM analog will be more effective than either. iv. Inhibition of thrombin clotting time (TCT) and prothrombin reaction (PT).
  • TCT thrombin clotting time
  • PT prothrombin reaction
  • Both the PT and TCT are determined using the Hewlett-Packard 8452 A diode-array spectrophotometer used for the APTT.
  • 90 ul of either TM analog 6h/227-462 or PBS was added to 20 ul thromboplastin and 90 ul 25 mM CaCl 2 in a cuvette. The mixture was incubated for 1 minute at 37 C, then 100 ul of citrated plasma was added. After loading the cuvette into the spectrophotometer,the absorbance due to light scattering at 320 nm was measured every 0.5 seconds, from 15 to 120 seconds, timed from the
  • a plot of absorbance vs. time yields a sigmoidal curve, with the clotting time defined as the time at which the slope is the steepest, corresponding to the inflection point of the curve.
  • the TCT is evaluated in the same manner.
  • the initial reaction mixture contains 100 ul citrated plasma, 25 ul of 100 mM CaCl 2 and 162.5 ul of either PBS or TM analog. After 1 minute, 12.5 ul of thombin is added. The clotting time is measured as described above.
  • Drawing 12 shows that the TM analog 6h/227-462 prolongs both the PT and the TCT.
  • v Direct anticoagulant activity - Inhibition of thrombin catalyzed conversion of fibrinogen to fibrin.
  • Thrombin and varying amounts of TM analog 6h/227-462 were incubated for 2 minutes at 37 C in microtitre plate wells.
  • the total initial reaction volume was 50 ul PBS +7.5 mM CaCl 2 and 90 nM thrombin.
  • 100 ul of 3.75 mg/ml human fibrinogen was added per well, and the thrombin induced formation of fibrin was followed by measuring the change in absorbance at 405 nm in a Molecular Devices Vmax spectrophotometer (Molecular Devices, Menlo Park, CA).
  • the end-point of the assay was the time at which 50% of the final absorbance was reached. Residual thrombin activity was determined by reference to a thrombin standard curve, which linearly relates the reciprocal of the thrombin concentration to the
  • TM analog 6h/227-462 inhibited the ability of thrombin to convert fibrinogen to fibrin in a dose dependant manner. vi. Inhibition of platelet activation and aggregation.
  • TM analog 6h/227-462 inhibited the thrombin mediated activation and aggregation of platelets.
  • TM heterologs The fibrinolytic activity of TM heterologs, fusions of TM analogs and t-PA, was evaluated using zonal clearing on plasminogen-enriched fibrin plates as described by Haverkatet and Brakman, (1975) Prog. in Chem. Fibrin. Thromb. 1:151-159.
  • TM heterologs PA: 227- 462 (mammalian cells), PA: 350-462 (mammalian cells), 6h/227-462:PA, and PA:227-462 (insect cells) all
  • TM analog 6h/227-462 inhibited the activation of Factor XIII by thrombin as measured by the method of Polgar, et al., (1987) Thromb. Haemostas. 58:140. ix. Additional measures of TM antithrombotic activity.
  • TM analog's inhibition of the inactivation of protein S by thrombin is measured by the method described by Thompson & Salem, J. Clin. Invest..
  • mice Three groups of six mice each were anaesthetised and injected with 37.5 units of thrombin via the tail vein. In mice receiving no other treatment this caused pulmonary embolism rapidly leading to death. Two groups were pretreated by injection with TM analog 6h/227-462 (3 min. prior) at 1x or 2x the concentration of thrombin.
  • mice in the control group were comatose for 30 minutes whereas mice in the pretreatment groups
  • the TM analog can be concentrated by drying and resolubilization as
  • Dialysis was performed using 12,000-14,000 molecular weight cutoff dialysis tubing at 4oC.
  • TM analog (1,921 U/ml) in 2 M NaCl, 1.5 M guanidine-HCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.5, was extensively dialyzed against 0.2% N-ethylmorpholine (NEM) (pH 8.8). No loss in activity was detected although the protein was diluted by a factor of 1.2 (final concentration was 1,661 U/ml).
  • NEM N-ethylmorpholine
  • the Protein C activity assay was carried out on all samples. The results are provided in Table 5.
  • TM analog 6h/227-462 was buffer exchanged on Sephades G25 into PBS then diluted 5-fold with pure water.
  • TM analog can be efficiently dialysed from the thrombin eluent buffer into the volatile buffer N- ethylmorpholine at pH 8.8 in the absence of detergent
  • TM analog samples in the NEM buffer can be freezedried and stored in the absence of salts, detergents or other stabilizers
  • samples of the TM analog can be resolubilized in either NEM or PBS with or without NP- 40 resulting in little or no loss of biological activity.
  • Other salts that are of use include
  • hydrobromide, sulfate, acetate, citrate, malate, borate, lactate, glycine, glutamate and aspartate, among others, and d) samples can be effectively
  • a prokaryotic expression vector was contructed for the expression of a TM analog in E.
  • the parent plasmid was pRIT5, purchased from Amersham, which contains a gene coding for Protein A under the control of the Protein A promoter and having the Protein A signal sequence.
  • This plasmid was digested with BamHI and a BamHI fragment from plasmid pUC19pcrTM7 was inserted.
  • the BamHI fragment carries the gene coding for the 6 EGF-like domains of native thrombomodulin.
  • the resulting plasmid, pTHR8 was transformed into E coli.
  • a culture was grown up and a partial purification of the TM analog protein was done as described by Amersham using an IgG Sepharose column. This protein was active in the Protein C activation assay.
  • Oligomer primers for t-PA Oligomer primers for t-PA
  • COD #1482 GATCATGCTCAAGTTTGTTATTTTATTGTGCAGTATTGCCTATGTT
  • +/- indicates presence or absence of a stop codon at the 3' end of the TM gene
  • +/- indicates presence or absence of a stop codon at the 3' end of the TM gene

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Abstract

L'invention concerne la production et l'utilisation de techniques de recombinaison d'ADN afin de produire des analogues solubles de la thrombomoduline utiles par exemple en thérapie anti-thrombotique. L'invention concerne également de nouvelles protéines, de nouvelles séquences d'acide nucléique, de nouveaux vecteurs et de nouveaux procédés et substances pharmaceutiques d'inhibition de l'activité thrombotique.
PCT/US1990/000955 1989-02-17 1990-02-16 Analogues solubles de la thrombomoduline WO1990010081A1 (fr)

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WO1991009118A2 (fr) * 1989-12-07 1991-06-27 British Bio-Technology Limited Proteines fibrinolytiques et antithrombotiques activables
EP0544826A1 (fr) * 1990-08-15 1993-06-09 Berlex Laboratories, Inc. Analogues ameliores de thrombomoduline d'usage pharmaceutique
WO1993015755A1 (fr) * 1992-02-05 1993-08-19 Schering Aktiengesellschaft Analogues de thrombomoduline resistant a la protease
WO1993025675A1 (fr) * 1992-06-10 1993-12-23 Schering Aktiengesellschaft Mutants des domaines du facteur de croissance epidermique de la thrombomoduline humaine
US5585095A (en) * 1993-09-14 1996-12-17 Regents Of The University Of Minnesota Method to enhance thrombomodulin APC generation using cationic proteins
US5762921A (en) * 1994-06-16 1998-06-09 Genentech, Inc. Composition and methods for the treatment of tumors
US5827824A (en) * 1989-04-28 1998-10-27 Schering Aktiengesellschaft Protease-resistant thrombomodulin analogs
US7250168B2 (en) * 2002-05-01 2007-07-31 Bayer Schering Pharma Ag Tissue factor targeted thrombomodulin fusion proteins as anticoagulants
US7579000B2 (en) 2002-05-01 2009-08-25 Bayer Schering Pharma Ag Tissue factor targeted antibodies as anticoagulants
US20130136731A1 (en) * 2010-08-05 2013-05-30 Council Of Scientific & Industrial Research Protein Fusion Constructs Possessing Thrombolytic and Anticoagulant Properties

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CN101189334B (zh) 2005-06-03 2013-11-06 持田制药株式会社 抗cd14抗体融合蛋白质

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US6063763A (en) * 1989-04-28 2000-05-16 Schering Aktiengesellschaft Protease-resistant thrombomodulin analogs
US5863760A (en) * 1989-04-28 1999-01-26 Schering Altiengesellschaft Protease-resistant thrombomodulin analogs
US5827824A (en) * 1989-04-28 1998-10-27 Schering Aktiengesellschaft Protease-resistant thrombomodulin analogs
WO1991009118A2 (fr) * 1989-12-07 1991-06-27 British Bio-Technology Limited Proteines fibrinolytiques et antithrombotiques activables
WO1991009125A1 (fr) * 1989-12-07 1991-06-27 British Bio-Technology Limited Proteines et acides nucleiques
WO1991009118A3 (fr) * 1989-12-07 1991-10-31 British Bio Technology Proteines fibrinolytiques et antithrombotiques activables
EP0544826A4 (fr) * 1990-08-15 1994-04-27 Berlex Laboratories, Inc.
EP0544826A1 (fr) * 1990-08-15 1993-06-09 Berlex Laboratories, Inc. Analogues ameliores de thrombomoduline d'usage pharmaceutique
AU675422B2 (en) * 1992-02-05 1997-02-06 David Richard Light Protease-resistant thrombomodulin analogs
WO1993015755A1 (fr) * 1992-02-05 1993-08-19 Schering Aktiengesellschaft Analogues de thrombomoduline resistant a la protease
WO1993025675A1 (fr) * 1992-06-10 1993-12-23 Schering Aktiengesellschaft Mutants des domaines du facteur de croissance epidermique de la thrombomoduline humaine
US5585095A (en) * 1993-09-14 1996-12-17 Regents Of The University Of Minnesota Method to enhance thrombomodulin APC generation using cationic proteins
US5674489A (en) * 1993-09-14 1997-10-07 Regents Of The University Of Minnesota Method to enhance thrombomodulin APC generation using cationic proteins
US5762921A (en) * 1994-06-16 1998-06-09 Genentech, Inc. Composition and methods for the treatment of tumors
US7579000B2 (en) 2002-05-01 2009-08-25 Bayer Schering Pharma Ag Tissue factor targeted antibodies as anticoagulants
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KR920701459A (ko) 1992-08-11
JPH04505554A (ja) 1992-10-01
AU646633B2 (en) 1994-03-03
EP0458903A1 (fr) 1991-12-04
CA2046906A1 (fr) 1990-08-18
AU5191790A (en) 1990-09-26
EP0458903A4 (en) 1992-06-17

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