Classifications

• • 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
  • C07K14/435—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)
• • 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
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
• • 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

Definitions

• the present invention relates to authentic and modified human thrombomodulin produced by recombinant DNA techniques, DNA sequences coding for these molecules, pharmaceutical preparations containing such molecules and their use in human therapy and profylaxis for the treatment and prevention of thrombic episodes.
• the coagulation of blood is the classical example of chemical amplification.
• a miniscule stimulus of key substances such as platelets, factor XII and/or factor VII, can trigger off complete gelation of the blood.
• the amplification is mediated by a series of proteolytic activations, involving coagulations factors XII, IX, VIII, X, V and prothrombin, which leads to precipitation of fibrin in plasma and entrapment of the cellular elements.
• blood coagulation must be controlled by other interactions that counterbalance procoagulant impulses, and tune the inherently explosive cascade reaction to local physiological needs. Without these influences, initiated coagulation would run away and disseminated intravascular coagulation ensue.
• the endothelial cells that form the interior lining of blood vessels are one source of potent antithrombotic materials. Heparan sulfate on the endothelial surface greatly potentiates antithrombin III capable of inhibiting the clotting process at several levels .
• SUBSTITUT __ * l Tissue plasminogen activator another blood fluidity regulatory principle, is released from endothelial cells after certain stimuli and initiates reactions to redissolve already formed fibrin fibers.
• Thrombomodulin is a glycoprotein on the surface of endotheliu and certain other cells which contributes to the control of coagulation (W. Owen and C. Esmon, J.Biol.Chem. 256:5532-5535; N. Esmon et al., J.Biol.Chem. 257:859-864, 1982; see also L. Clouse and P. Comp, N. Engl.J.Med. 314:1298-1304, 1986)). It binds stoichiometrically to thrombin, the penultimate step in the coagulation cascade, and alters its proteolytic specificity. Normally, thrombin converts fibrinogen into insoluble fibrin.
• the thrombin-thrombo odulin complex activates Protein C.
• the activated Protein C (PCa) in combination with protein S, degrades the activated cofactors Va and Villa, and thus turns coagulation off (R. arlar et al., Blood 59:1067- 1072, 1982).
• Activated Protein C also degrades the inhibitor of plasminogen activator (PAI) thereby facilitating fibrinolysis (V. van Hinsburgh et al., Blood 65:444-451, 1985).
• PAI plasminogen activator
• Thrombomodulin-initiated feedback thus appears to play an important regulatory role in the body's hemostatic mechanism.
• solubilized thrombomodulin has anticoagulant properties i vitro.
• a concentration of approximately 4 nM, kept in solution by detergent, has been reported to double the partial thromboplastin time of human plasma (S. Kurosawa and N. Aoki, Thromb.Res. 37:353-364, 1985).
• Human thrombomodulin has been purified from placenta (H. Salem et al. , J.Biol.Chem. 259:12246-12251; S. Kurosawa and N. Aoki, _ibid. ) . Briefly, the procedures consisted of preparation of membranes, solubilization in a non-ionic detergent, and twice affinity chromatography on solid phase thrombin, whose protease activity had been
• SUBSTITUTE SHEET destroyed by diisopropylphosphofluridate. Both purifications included an additional step, either ion exchange chromatography or size exclusion chromatography. Thrombomodulin is an extraordinarily stable glycoprotein. Activity can be quantitatively recovered from samples that have been treated with 1% SDS, 8 M Urea, pH 2, pH 10, or even boiled for 10 minutes. However, treatment with mercaptoethanol or pepsin completely destroys activity. The molecule is very poorly soluble in the absence of detergents.
• Purified preparations of thrombomodulin show one dominating band in gelelectrophoresis. Biological activity can be recovered from the gels and coincide with the band. The electrophoretic mobility corresponds roughly to a molecular weight of 88-105,000 dalton, but estimates appears rather dependent on the gel system. The isoelectric point is low, approximately 4. An estimated aminoacid composition has been published (S. Kurosawa and N. Aoki, ibid. ) , but detailed characterization has been seriously hampered by paucity of material.
• a partial cDNA sequence of bovine thrombomodulin comprising the C-terminal half indicates that this portion has a certain resemblance to the LDL receptor (R. Jackman et al., Proc.Natl.Acad.Sci, USA 8_3:8834-8838, 1986).
• An approximately 240 amino acid region contains cysteine residues whose spacing would be consistent with the formation of six modules resembling epidermal growth factors (EGF) .
• EGF epidermal growth factors
• SUBSTITUTE SHEET analogy to the LDL receptor could carry O-linked sugar chains. Then follows a stretch of 24 hydrophobic amino acids probably representing a transmembrane region, and finally a 56 amino acid segment starting with multiple positively charged residues, which probably represents a short cytoplasmic domain. As described below, our results indicate that human and bovine thrombomodulin are at least partly homologous.
• Rabbit thrombomodulin was recently separated by ion-exchange into an acidic and a non-acidic fraction which differed in their anticoagulant properties (M.C. Bourin et al. , Proc.Natl.Acad.Sci . USA 83:5924-5928, 1986). Both fractions contained thrombin-cofactor activity, required for Protein C activation. The acidic fraction, in addition, prevented fibrinogen clotting by thrombin and accelerated thrombin neutralization by antithrombin in a heparin-like fashion. The two latter properties were abolished by the polycation Polybrene and by heparinase. These results would indicate that direct and indirect thrombin-neutralizing activities depend on secondary glycosylation of the peptide chain, while Protein C coactivator activity does not.
• Complementary DNA or cDNA A DNA molecule or sequence which have been enzymatically synthesized from sequences present in a mRNA template.
• DNA Construct A DNA molecule, or a clone of such a molecule, either single- or double-stranded, which may be isolated in partial form -from a naturally occurring gene or which has been modified to contain segments of DNA which are combined and juxtaposed in a manner which would not otherwise exist in nature.
• SUB S TITUTE SHEET Plasmid or Vector A DNA construct containing genetic information which may provide for its replication when inserted into a host cell.
• a plasmid generally contains at least one gene sequence to be expressed in the host cell, as well as sequences encoding functions which facilitate such gene expression, including promoters and transcription initiation sites. It may be a linear or closed circular molecule.
• Joined DNA sequences are said to be joined when the 5' and 3 ' ends of one sequence are attached by phosphodiester bonds to the 3' and 5' ends, respectively, of an adjacent sequence. Joining may be achieved by such methods as ligation of blunt or cohesive termini, by synthesis of joined sequences through cDNA cloning, or by removal or intervening sequences through a process of directed mutagenesis.
• Pre-pro region An amino acid sequence which generally occurs at the amino termini of the precursors of certain proteins, and which is generally cleaved from the protein, at least in part, during secretion.
• the pre-pro region comprises, in part, sequences directing the protein into the secretory pathway of the cell.
• Domain or Module A three-dimensional, self-assembling array of amino acids of a protein molecule, which contains structural elements necessary for a specific biological activity of that protein.
• Thrombomodulin may have three separate activities. First, it binds to thrombin with high affinity. Secondly, it specifically endows the thrombin- thrombomodulin complex with the ability to activate
• this binding may modify the reactivity of the complexed thrombin to a number of different components, including fibrinogen, platelets, and antithrombin.
• thrombomodulin activity equivalent to the first and second of these activities, and disregard the third type of activity. Consequently, thrombomodulin derivatives or generally thrombomodulins will be considered active if they possess protein C coactivator activity, regardless whether or not they modify other reactivities of the bound thrombin.
• Protein C is a two-chain glycoprotein with a molecular weight of approximately 57,000 dalton.
• One chain designated the light chain consists of an amino-terminal gammacarboxylated region, followed by two domains with homology to epidermal growth factor (EGF) .
• the other chain designated the heavy chain contains -a serine protease domain.
• Activation of protein C consists of cleavage of a single peptide bond, Arg-12 to Leu-13 in the heavy chain. It is this reaction whose catalysis by the thrombin- thrombomodulin complex can accelerate dramatically, typically between 100 and 20000 fold. Indeed, the
• this invention relates to a group of compounds which in this specification has been designated thrombomodulins, comprising human thrombomodulin and derivatives thereof.
• the thrombomodulins of the invention all have certain features in common which have been alluded to above and will be specified in further detail below. It is foreseen that said thrombomodulins will be of use in the therapeutical control of coagulation. By increasing the amount of thrombomodulins in a patient it will be possible to increase the activation of Protein C, thereby potentiating the patient's anticoagulant capacity. It is anticipated that the thrombomodulin- derived anticoagulants of the invention will be superior to well known anticoagulants, such as heparin and Vitamin K antagonists.
• Vitamin K antagonists indavertently diminish the physiological anticoagulatory response of Protein C by blocking the formation of gammacarboxylated proteins indiscriminately, and thrombic episodes which typically manifest themselves as skin necrosis have occurred early in anti-Vitamin K treatment (A. Broekmans et al.., Thromb Haemost 49 :251, 1983) .
• Thrombomodulins in contrast, will reinforce natural anticoagulant and fibrinolytic mechanisms by limiting survival of PAI and activated factors Va and Villa, but leave the reservoir of coagulation factors in plasma largely intact. Spatial specificity will be high as the activity is dependent on local formation of thrombin, and systemic activity should thus be negligible.
• the anticoagulant activity of the thrombomodulins of the invention should not interfere directly with formation of the hemostatic plug, neither does it change the structure of the fibrin. Therefore fewer complications in the form of bleeding episodes are anticipated compared to the use of the traditional anticoagulant substances.
• Thrombomodulins will be particularly useful as anticoagulants in patients with increased bleeding risk or in patients where such a risk cannot be accepted.
• Such patients include those who have recently suffered from a stroke caused by a thrombus in a cerebral vessel, patients who recently have undergone surgery, or such that have a potential source of bleeding (tumor, ulcer etc).
• Fig. 1 shows the sequence of a 60-mer DNA probe used for identifying human thrombomodulin cDNA clones
• SUBSTITUTE SHEET Fig. 2 shows an alignment of DNA sequences with deduced amino acid sequences from bovine thrombomodulin (B) cDNA (nucleotides 850 to 1035) (Jackman- et al. , PNAS, USA 8_3: 8834-8838, 1986, Fig. 3) and from the corresponding region in human thrombomodulin (H) cDNA clone p2.1 (this invention).
• B bovine thrombomodulin
• H human thrombomodulin
• Fig. 3 shows an- alignment of DNA sequences with deduced amino acid sequences from bovine thrombomodulin (B) cDNA (Jackman et al. , PNAS, USA 83_: 8834-8838, 1986, Fig. 3) and from the corresponding regions in the human thrombomodulin (H) cDNA clone p2.1 (this invention). Two regions (bovine nucleotide number 1 to 66 and 145 to 186) are compared, and identities between the two molecules are boxed,
• Fig. 4 shows a human thrombomodulin cDNA sequence from clone p2.1 (this invention). The sequence shows 365 bp corresponding to nucleotides 1754 to 2123 (369 bp) in the 3' untranslated part of the bovine cDNA sequence (Jackman et al., PNAS, USA 8J3: 8834-8838, 1986, Fig. 3),
• Fig. 5 shows a RNA blot analysis of human cell line A549 mRNA. Five micrograms of mRNA from two different preparations were separated on a 1% agarose gel, blotted to nitrocellulose, and hybridized with a nick-translated restriction fragment from plasmid p2.1. The nucleotide length of known DNAs is indicated, Fig. 6 shows the amino acid sequence of human tissue plasminogen activator with selected domains marked A, B, and C,
• SUBSTITUTE SHEET Fig. 7 shows the DNA sequence of the 3640 bp insert in p2.1.
• the amino acid sequence of human thrombomodulin, with its signal peptide at the amino terminus, is shown with all the amino acid residues given by their one-letter abbreviations,
• Fig. 8 shows the sequence of the BamHI insert of plasmid pBoel743-2-9-8. Regions constructed from synthetic oligonucleotides are underlined with horizontal arrows. The amino acid sequence of the encoded human tPA signal peptide and the human thrombomodulin mutant is given by the one-letter code for all amino acid residues. The position of some restriction endonuclease recognition sites are indicated,
• Fig. 9 shows the construction of the mammalian expression vector pBoel-TMl.
• Fig. 10 shows the construction of the mammalian expression vector pBoel-TM2.
• this invention relates to a group of compounds having thrombomodulin activity as defined by a high affinity binding to thrombin, and the capacity of endowing a complex between such a compound and thrombin with the ability to activate Protein C, and comprising from the C-terminal part of the molecule two or more of the following structural elements:
• SUBSTITUTE SHEET b) a transmembrane region of approximately 24 amino acids, c) a region rich in serine, threonine and proline residues, d) a domain comprising at least two EGF domains, and e) an N-terminal where one or more of the elements a), b), and/or c) may be omitted or replaced by affinity imparting entities or entities enhancing the solubility in physiological solutions, or physiologically compatible derivatives thereof.
• the present invention describes molecules with thrombomodulin activity, which have been imparted improved solubility characteristics, relative to authentic human thrombomodulin in physiological solutions.
• Other molecules of the invention have been imparted affinity for specific tissue structures occurring in vivo, or lend themselves to conjugation to surfaces or other molecules or entities.
• modified thrombomodulins contain part of the amino acid sequences of human thrombomodulin, but the C-terminal portion, including part or all of the hydrophobic membrane spanning region has been deleted or replaced by affinity imparting entities.
• One preferred site to terminate the human thrombomodulin sequence is at the start of the hydrophobic, membrane-spanning region, corresponding to His-12 to Gly-14 (bases 883-891) in figure 2 (corresponding to His-514 to Gly-516 (bases 1670-1678) in fig. 7).
• a second preferred site to terminate the human thrombomodulin sequence is at the C-terminal of the sequence of growth factor domains.
• SUBSTITUTE SHEET Further preferred embodiments comprise molecules containing only regions e) and d) where the number of EGF domains is equal to or greater than four.
• the human thrombomodulin sequence is terminated at the N-terminal of any of the two first growth factor domains, and C-terminally at the start of the hydrophobic, membrane spanning region, or the C-terminal of any of growth factor domains 4, 5, or 6.
• the modified thrombomodulins may at the same time be fusion proteins, in that they have been provided with new desirable heterologous protein domains as N- or C-terminal extensions, said domains having desirable affinity to specific physiological structures, i.e. fibrin clots, membrane surfaces, receptor molecules, or extracellular matrix components.
• One preferred site to fuse the human thrombomodulin sequence to a suitable heterologous domain is at the start of the membrane spanning region, corresponding to His-12 to Gly-14 in figure 2.
• a second preferred site to fuse the human thrombomodulin to a suitable heterologous domain is at the C-terminal of the growth factor module sequence.
• Further preferred sites to fuse the human thrombomodulin to a suitable heterologous domain are at the N-terminal of any of the growth factor domains 1, 2, or 3, or at the C-terminal of any of the growth factor domains 4, 5, or 6.
• Domains with specific affinity for physiological structures which domains, are suitable as fusion partners encompass growth factor modules, kringles, finger-modules, vitamin K-dependent calcium-binding gammacarboxylated regions, and antibody-derived, antigen-recognizing structures.
• heterologous domains with affinity for physiological structures encompass the finger modules of tissue plasminogen activator and fibronectin, the growth factor modules of urokinase, tissue plasminogen activator, and protein C, the kringle module of tissue plasminogen activator and the first, fourth and fifth kringle module of plasminogen.
• the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin, up to Ser-13 of figure 2, C-terminally extended to include the finger domain of tissue plasminogen activator (region A in fig. 6, encompassing amino acids 4 to 50). This would endow the molecule with affinity to fibrin.
• the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin up to Ser-13 of figure 2, C-terminally extended to include the second kringle domain (region B in fig. 6, encompassing amino acids 176 to 263) of tissue plasminogen activator. This would also endow the molecule with affinity to fibrin.
• the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin up to Ser-13 of figure 2, C-terminally extended to include the growth factor module of tissue plasminogen activator (region C in fig. 6, encompassing amino acid 50 to 87).
• the modified thrombomodulin consists of the N- terminal sequences of human thrombomodulin up to but not including the first cystein residue of its last growth factor module, C-terminally extended to include the growth factor module of tissue plasminogen activator starting at the first cystein residue in this module (amino acid 51 in figure 6) .
• the modified thrombomodulin consists of the four carboxyterminal epidermal growth factor domains together with the extracellular O-glycosylation rich domain N- terminally joined to the signal peptide from human tissue type plasminogen activator (tPA) and a short adaptor sequence (fig. 8).
• a sixth specific embodiment corresponds to the fifth embodiment above, but excluding the extracellular O-glycosylation rich domain.
• the molecules may contain a C- teri inal extension including a free cysteine residue, or one or more lysine residues to facilitate covalent conjugation in vitro or simply a highly charged region, rich in lysine and arginine, or glutamate and aspartate, so as to mediate noncovalent adhesion to surfaces.
• the modified thrombomodulins may.consist of the N-terminal sequences of human thrombomodulin up to the cut off sites mentioned in the preceding paragraphs which N-terminal sequences through a spacer molecule are conjugated to affinity providing entities such as antibodies or fragments thereof.
• affinity providing entities such as antibodies or fragments thereof.
• the present invention provides for means to produce authentic as well as the aforementioned modified human thrombomodulins by expression of a cloned nucleic acid construct encoding all or parts of the complete human thrombomodulin molecule in a suitable cell.
• S thereof may be expressed alone or as fusions with other domains so as to facilitate production, or provide desired extra domains as expounded above.
• the authentic or modified thrombomodulins are encoded in a cloned nucleic acid construct in a form including the natural regions of the human thrombomodulin gene which regions are responsible for the expression of such amino acids that are actively involved in the export of the natural protein from the cell, and which amino acids eventually completely or at least in part are cleaved from the protein.
• the authentic or modified thrombomodulins are encoded in a cloned nucleic acid construct in a pre-pro form including the pre-pro region of tissue plasminogen activator.
• the present invention describes pharmaceutical compositions containing authentic or modified thrombomodulins, useful in the treatment of patients with a view to prevent or revert a thrombic condition.
• SUBSTITUTE SHEET The construction of DNA sequences encoding preferred terminated human thrombomodulins and of DNA sequences encoding fusions between parts of human thrombomodulins and preferred functional domains from other proteins are best carried out after the introduction of specific restriction enzyme recognition sites at specific points in the human thrombomodulin cDNA and in the cDNAs of the preferred domain-donor molecules. Introduction of restriction enzyme recognition sites at specific points in a DNA molecule can advantageously be obtained with the use of one of several well known, highly efficiency procedures for oligodeoxyribonucleotide directed site-specific mutagenesis (e.g. Y. Morinaga et al., BIO/TECHNOLOGY 2: 636-639, 1984).
• C- terminally deleted modified human thrombomodulins can advantageously be obtained through the use of oligonucleotide directed site-specific mutagenesis.
• One such preferred modified human thrombomodulin terminating at the start of the hydrophobic, membrane-spanning region can be constructed by the specific introduction of a stop-codon at the position of the existing Gly-codon GGC (at base numbers 889 to 891 in fig. 2).
• an oligonucleotide could be used that also introduced a convenient restriction enzyme recognition site just 3' to the introduced stop-codon hereby facilitating further construction work with this shortened version of human thrombomodulin cDNA.
• the different parts of the human thrombomodulin cDNA and the different parts of other cDNAs from which specific domains are to be recruited can most conveniently be subcloned in small DNA vectors (such as pUCl9 , pBR322 and pGEM3) before mutagenesis. After appropriate mutagenesis to introduce new restriction sites at joining-positions, the cDNAs should be sequenced to confirm the mutated genotype. The mutated fragments can then be joined directly together by small DNA vectors (such as pUCl9 , pBR322 and pGEM3) before mutagenesis. After appropriate mutagenesis to introduce new restriction sites at joining-positions, the cDNAs should be sequenced to confirm the mutated genotype. The mutated fragments can then be joined directly together by
• Various host cells may be used to produce the proteins including mammalian cells, yeast, fungi and bacteria. However, cultured mammalian cells are preferred.
• One particularly preferred cell line is the BHK cell line tk-tsl3 (Waechter and Baserga, Proc .Natl.Acad.Sci. USA 79: 1106-1110, 1982). Methods for expressing cloned genes in each of these types of host are known in the art.
• expression vectors containing cloned thrombomodulin sequences are introduced into the cells by appropriate transfection techniques, such as calcium phosphate-mediated transfection (Graham and Van der Eb, Virology 52: 456-467, 1973; as modified by Wigler et al., Proc.Natl.Acad.Sci. , USA 77: 3567-3570, 1980).
• a DNA-calcium phosphate precipitate is formed, and this precipitate is applied to the cells.
• a portion of the cells take up the DNA and maintain it inside the cell for several days.
• a small fraction of the cells integrate the DNA into the genome of the host cell.
• integrants are identified by cotransfection with a gene that confers a selectable phenotype (a selectable marker).
• a preferred selectable marker is the mouse dihydrofolate reductase (DHFR) gene, which imparts cellular resistance to the drug methotrexate (MTX) .
• Thrombomodulin active compounds produced by the transfected cells may be purified from the cell culture media by adsorption to an ion-exchange column or affinity chromatography.
• One preferred affinity column contains inactivated, immobilized thrombin (H. Salem et al. , J.Biol.Chem. 259: 12246-12251, 1984).
• Another technique which will be of advantage is by using monoclonal antibodies or fragments thereof directed against specific antigenic determinants on the desired thrombomodulin. These can be used as affinity reagents conjugated to a solid phase to provide efficient column purification.
• the raising of monoclonal antibodies, their conjugation to column matrices and the use of antibody affinity columns for isolation and purification are well known in the art.
• the human cell line A549 (American Type Culture Collection CCL 185) which was isolated from lung carcinomatous tissue (Giard et al. (1972) J. Natl. Cancer Inst. 51.:1417-1423 ) has been shown to express about 10,000 molecules of thrombomodulin per cell (ref) .
• A549 was used as a source for mRNA preparation.
• A549 was grown to a total cell number of 9.4 x 10 in RPMI 1640 containing 10% fetal calf serum and antibiotics.
• RNA was isolated by the quanidinium thiocyanate method (Chirgwin et al. (1979) Biochemistry 113:5293-5299) and purified by CsCl gradient centrifugation. A total of 950 ⁇ g RNA was obtained, and
• S UBSTITUTE SHE ⁇ T mRNA was isolated by use of an oligo(dT)-cellulose column (Aviv & Leder (1972) PNAS 6_9:1408-1412 ) . Sixty-one micrograms of mRNA were obtained from 750 ⁇ g total RNA (one cycle) . After ethanol precipitaton, this preparation of mRNA was resuspended in 10 mM Tris HCL pH 7.5, 0.1 mM
• the mRNA prepared as described was used for construction of a cDNA library and mRNA blot analysis.
• a cDNA library was constructed by the method described by Okayama & Berg (Mol. Cell. Biol. 2_ :161-170 (1982); Mol. Cell. Biol. :28 °- 289 (1983)).
• E. coli K12 (MC1061) (Casadaban & Cohen C.J. Mol. Biol. 138:179-207) was used for transformation.
• the hybridization solution contained 6xSSC,
• Plasmid preparations were prepared, subjected to Hind III digestion, electrophoresed in 1% agarose, blotted onto nitrocellulse filters, and hybridized with the above 60-mer.
• One of the plasmids designated p2.1 gave a strong hybridization signal and was selected for further characterization.
• p2_l had a cDNA insert of approximately 3.8 kb. , and within this insert.
• partial DNA sequences Maxam and Gilbert, Methods Enzymol. 65:499-560, 1980. Sanger et al., PNAS, USA 74:5463-5467, 1977) were obtained to further characterize and identify the clone.
• Fig. 2 and 3 show sequence homology between selected regions of the bovine (Jackman et al., PNAS, USA 83:8834-8838, 1986, Fig. 3) and the human cDNA as obtained from p2.1. Regions with amino acid identities are boxed-. From Fig. 2 it can be noted that within the 24 amino acid residues long putative transmembrane region in the bovine sequence (from Gly-14 (889) to Leu-37(960), only 2 amino acid substitutions are identified.
• SUBSTITUTE SHEET Fig. 4 shows a sequence from a 3' untranslated region of p2.1. In this part of the molecule well- conserved regions between the bovine and the human sequences can also be identified. In order to estimate the completeness of this clone the following experiment was performed.
• the gel was rinsed 2 x 10 min in 1 x SSC (1 x SSC is 0,150 M NaCl and 0.015 m sodium citrate (pH 7)) before blotting of the mRNA to a Gene Screen (TM) ' hybridization transfer membrane overnight in 10 x SSC.
• TM Gene Screen
• the blotted mRNA was baked to the membrane for 2 h at 80°C.
• Prehybridization (5h) and hybridization (20h) were done in 50% formamide; 0.1% each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone; 5 x SSC, 1% SDS, and 0,5 mg/ml of heat denatured salmon sperm DNA at 42°C.
• a nick-translated p2.1 cDNA restriction fragment was used as a hybridization probe. After hybridization, the mRNA blot was washed successively in 2 x SSC for 2 x 5 min at room temperature, 2 x SSC, 0,5% SDS for 2 x 30 min at 65°C, and in 0,1 x SSC for 2 x 30 min at room temperature. In fig. 5 the autoradiography from this experiment is shown. Lanes 1 and 2 show the mRNAs from two different preparations, and lane 3 the DNA molecular weight markers. By this experiment an approximately 3,8 kb long human thrombomodulin mRNA was identified.
• the complete sequence of the cDNA in the plasmid p2.1 was determined (Tabor, S. and Richardson, C.C. (1987) Proc. Natl. Acad. Sci USA, 84: 4767 - 4771.), and is presented in Fig- 7.
• the plasmid contained stretches of G:C ho opolymer tails at the 5 1 end in addition to a poly(A) tail in the 3 1 end of the cDNA insert.
• Desirable human thrombomodulin (hTM) mutants should be expressed as soluble proteins without the membrane spanning region. To obtain such mutants, the ollowing steps were performed. p2.1 was digested with PstI, and a 870 bp fragment (nucleotide 952 to 1821 in Fig 7) was isolated and subcloned in pGEM3 (Promega Biotec) . A subclone with the Kpnl site in the hTM cDNA oriented towards the BamHI site of the multilinker in pGEM3 was designated pBoel743-2. The insert in this plasmid was mutagenized (Morinaga, Y. , et al. (1984) BIO/TECHNOLOGY, 2 z 636 - 639) with an oligodeoxyribonucleotide, NOR 570:
• cleotide introduced an ApaLI (GTGCAC) site just 5' to the new translation stop signal, an Ayrll (CCTAGG) site spanning the stop signal and a BamHI (GGATCC) site on its 3' side.
• a correct mutant pBoel743-2-9 was identified through colony hybridization and through mapping with appropriate restriction enzymes.
• the expressed protein contained only the four carboxyterminal epidermal growth factor homologous domains together with the extracellular O-glycosylation rich domain.
• This mutant was generated as follows.
• pBoel743-2-9 was digested with BamHI and Hindi, and a 0.53 kb Hindi/- BamHI fragment was isolated. This fragment was joined to synthetic DNA, which encoded the signal peptide from the human tissue type plasminogen activator (tPA) (Pennica et al., (1983) Nature, 301: 214 - 221) and a short adaptor sequence, in BamHI digested pUC13.
• tPA human tissue type plasminogen activator
• a correct recombinant pBoel743-2-9-8 was identified with restriction enzyme digestions, and the synthetic region was sequenced.
• the 711 bp sequence of the tPA encoding region, the adaptor and the HindI/BamHI fragment of pBoel743-2-9-8 is shown in Fig 8.
• SUBSTITUTE SHEET plasmid also carries a mouse DHFR (dihydrofolate reduc- tase) cDNA under control of SV40 regulatory elements to provide a selectable marker in transfected mammalian cells.
• a correct recombinant pBoel-TMl was isolated and characterized with restriction enzyme digestions.
• pBoel- TM1 was propagated in E. coli in large scale, and purified on CsCl/Ethidium Bromide gradients by ultracentrif gation.
• Another desirable hTM mutant should be expressed as a soluble protein without the region rich in O-glycosylation and without the membrane spanning region. To obtain such a mutant, the following steps were performed.
• pBoel743-2 was mutagenized (Fig 10) with an oligonucleotide, NOR 571:
• This mutation resulted in the introduction of a TAG translation stop codon at the position of the Glycine-487 codon GGT.
• the oligonucleotide introduced a BamHI (GGATCC) site just 3* to the transla ⁇ tion stop signal.
• a correct mutant pBoel743-2-10 was identified through colony hybridization, through mapping with restriction enzymes, and through DNA sequencing.
• pBoel743-2-10 was digested with Hindi and BamHI, and a 0.44 kb fragment was purified on a polyacrylamide gel. This DNA fragment was joined to a 0.18 kb BamHI/- Hindi fragment from pBoel743-2-9-8 in BamHI digested and alkaline phosphatase treated Zem219b to generate pBoel- TM2.
• pBoel-TM2 encodes a mutant hTM precursor in which the four carboxyterminal epidermal growth factor homologous domains can be secreted from a mammalian cell under control of the human tPA signal peptide.
• pBoel-TM2 was grown in large scale in E.coli to prepare enough pure plasmid for transfection of mammalian cells.
• mutant hTM in cultured BHK cells (Syrian Hamster, thymidine kinase mutant line tk tsl3 ,
• each petri dish was washed with serum free Dulbeccos Modified Eagle Medium (containing 25 mM N-2-hydroxyethyl-piperazine N'-2- ethanesulfonic acid (HEPES) , pH 7.4, 10 mg/1 insulin, 0.2 % Bovine Serum Albumin) , and incubated in the same medium for 24 hrs.
• the used medium was harvested and assayed for TM activity in an assay described below.
• cells Forty-eight hours after transfection, cells were trypsinized and diluted into medium containing 400 nM methotrexate (MTX) . After 10 to 12 days, individual colonies were cloned out and expanded separately. The expanded cultures were propagated for 24 hours in serum free medium as described above, and producer clones were identified using an assay for protein C coactivator activity.
• MTX methotrexate
• Protein C activation was measured by a method modified from Bourin et.al. (Proc. Natl. Acad. Sci. US 8_3,5924 (1986)). Two hundred ⁇ l of serum free supernatant from transfected cultures were mixed with 40 ⁇ l Protein C and 10 ⁇ l thrombin ( final concentrations 1 ⁇ M and 20 nM, respectively) , and calcium chloride was added to a final concentration of 2 mM. The mixtures were then incubated at 37"C for 30 minutes and the reaction was stopped with 200 pmole Antithrombin III and 5 U heparin.
• the volume was adjusted to 650 ⁇ l with 50 mM Tris.HCl/0.1 M NaCl, pH 8.3 and 100 ⁇ l 1 mM S-2266 (D-Val-Leu-Arg-p_-nitroanilide, KabiVitrum) in the same buffer was added.
• modified thrombomodulins of this invention have a Protein C co- activating activity.
• Coagulation was measured in seconds.

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