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WO1989010401A1 - Activateurs de plasminogenes a selectivite accrue pour la fibrine - Google Patents

Activateurs de plasminogenes a selectivite accrue pour la fibrine Download PDF

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Publication number
WO1989010401A1
WO1989010401A1 PCT/US1989/001255 US8901255W WO8910401A1 WO 1989010401 A1 WO1989010401 A1 WO 1989010401A1 US 8901255 W US8901255 W US 8901255W WO 8910401 A1 WO8910401 A1 WO 8910401A1
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WIPO (PCT)
Prior art keywords
kringle
scu
plasminogen activator
plasminogen
amino acid
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PCT/US1989/001255
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English (en)
Inventor
Jen-I Mao
David Miller Abercrombie
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Collaborative Research, Inc.
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Publication of WO1989010401A1 publication Critical patent/WO1989010401A1/fr

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    • 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
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • 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)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

  • plasminogen activators have replaced the original agents, urokinase and streptokinase, because these new agents offer relatively more fibrin selective plasminogen activation.
  • these agents activate plasminogen in the vicinity of the fibrin clot but only minimally elsewhere in the general circulation.
  • they spare other important circulating proteins such as alpha-2-antiplasmin and fibrinogen from degradation by the rather non-specific protease plasmin.
  • both of these naturally-occurring plasminogen activators exhibit half-lives in the circulation of only two to five minutes.
  • fibrin selective plasminogen activators have been extracted from normal and tumor tissues and are produced by certain cells in culture.
  • plasminogen activators with high fibrin selectivity - - a single-chain urinary plasminogen activator (scu-PA) also known as prourokinase, and tissue-type plasminogen activator (t-PA) - - which are easily distinguished immunologically.
  • the single-chain urinary plasminogen activator is a trypsin-like serine protease 411 amino acid residues in length.
  • the molecule contains two other domains: a cysteine-rich amino-terminal region of 45 amino acid residues which is homologous with epidermal growth factor (EGF) and thus is termed the EGF domain, and immediately adjacent to that domain is an 87 amino acid residue so-called "kringle” region which is highly homologous to the multiply disulfide bonded "kringle” domains of plasminogen and prothrombin.
  • EGF epidermal growth factor
  • kringle 87 amino acid residue so-called "kringle” region which is highly homologous to the multiply disulfide bonded "kringle” domains of plasminogen and prothrombin.
  • Hydrolysis of the lysine 158-isoleucine 159 bond by plasmin converts scu-PA into urokinase, a two-chain u-PA (tcu-PA) in which the chains are linked to one another by at least one disulfide bridge.
  • Urokinase (plasmin-produced tcu-PA) is known to exist in either of two forms, a high molecular weight form (HMW urokinase) consisting of amino acid residues 1-411, and a low molecular weight form (LMW urokinase) consisting of amino acid residues 136-411 or 137-411. Cleavage by plasmin may play an important physiological role and has been demonstrated to produce a molecule with quite different properties as measured both in vitro and in vivo. For example, plasmin-produced tcu-PA is more active than scu-PA on small molecule substrates such as S2444 (N-pyro-Glu-Gly-p-nitroanilide; Kabi Vitrum, Sweden).
  • scu-PA is fibrin-selective whether it is full length, or truncated having an amino terminus at leucine 144 and lacking the EGF-like and kringle domains (Stump et al., 1987, supra; Stump, D.C., Lijnen, H.R., and Collen, D., 1986, J. Biol. Chem. 261: 17120-17126).
  • the kringle domain of scu-PA appears to play no role in f ibrin selectivity; instead, the connecting region between the kringle and the serine protease domains seems important for this property.
  • plasmin-produced tcu-PA ie., urokinase
  • tcu-PA ie., urokinase
  • Tissue-type plasminogen activator is also a trypsin-like serine protease and is composed of a single polypeptide chain of 527 amino acids.
  • the primary structure of t-PA shares a high degree of homology with that of scu-PA.
  • t-PA is converted by plasmin cleavage of the arginine 275-isoleucine 276 bond to a two-chain form also linked by at least one disulfide bond.
  • current theory holds that there are no substantial differences between the activities of two-chain t-PA and single-chain t-PA.
  • T-PA is also composed of several domains, including not only the EGF-like, the kringle, and serine protease domains analogous to those of u-PA, but also a second kringle and a so-called "finger" domain resembling the fibrin-binding region of fibronectin.
  • the second kringle domain and the "finger” domain are both considered to contribute fibrin affinity to t-PA.
  • the finger domain is principally responsible for the fibrin affinity of t-PA, and the second kringle domain alone may be responsible for the fibrin-selectivity of t-PA action (Larsen et al., 1988., J. Biol. Chem. 263: 1023-1029).
  • Plasminogen the substrate of both scu-PA and t-PA, is another multi-domain trypsin-like serine protease involved in thrombolysis. It is composed of two types of domains - - namely, kringle domains, of which there are five, and a serine protease domain. Recently, human plasminogen cDNA was cloned and sequenced (Forsgren, M., Raden, B., Israelsson, M., Larsson, K., and Lars-Olof, H., 1987, FEBS Lett. 213: 254-260).
  • the scu-PA kringle and the first kringle of t-PA exhibit no measurable affinity for fibrin, while the second kringle of t-PA and the first and fourth kringles of plasminogen exhibit high affinity for lysine-Sepharose and fibrin.
  • both t-PA and scu-PA exhibit fibrin selective plasminogen activation in vivo, but they appear to do so by different mechanisms.
  • binding studies demonstrate an affinity of t-PA for fibrin, and kinetic studies indicate that interaction with soluble fibrin fragments dramatically decreases the K m of t-PA for plasminogen, thus rendering it a more efficient plasminogen activator.
  • scu-PA exhibits little or no affinity for fibrin and soluble fibrin fragments have only modest affects on its kinetic parameters of plasminogen activation, and yet it activates plasminogen with a fibrin selectivity virtually indistinguishable from that of t-PA, sparing alpha-2-antiplasmin and fibrinogen in vivo.
  • plasminogen is the mediator for scu-PA fibrin selectivity and it is scu-PA's resistance to interaction with circulating plasminogen-activator inhibitors which permits it to reach the fibrin clot.
  • plasminogen or plasminogen bound to fibrin is both a substrate and an allosteric effector which stabilizes a more active form of scu-PA (Ellis, V., Scully, M.F., and Kakkar, V.V., 1987, J. Biol. Chem. 262: 14998-15003).
  • scu-PA is less active on small molecule substrates than tcu-PA, but it appears to be sufficiently active on the large substrate plasminogen to initiate plasminogen activation.
  • plasminogen activator which exhibits better discrimination between plasminogen bound to, or in the vicinity of, the fibrin clot versus plasminogen in the general circulation. If such an agent were available, then larger doses could be administered without fear of breakdown of the patient ' s thrombogenic system, thereby permitting more rapid dissolution of the thrombus.
  • Fibrin-selectivity in plasminogen activation is understood to mean that plasminogen activation occurs primarily at the site of the fibrin clot so that little active plasmin is distributed in the general circulation where it can damage other thrombogenic proteins.
  • fibrin-selectivity could arise by either or both of at least two mechanisms: (a) a plasminogen activator may activate plasminogen more efficiently at the surface of a fibrin clot, or (b) a plasminogen activator may activate plasminogen less efficiently in the general circulation.
  • the second mechanism would be expected to play a role in a plasminogen activator which bound to fibrin and thus spent less time in the general circulation.
  • the t-PA molecule appears to have features required for both mechanisms to operate, while the scu-PA molecule appears to have only a feature for the first mechanism.
  • Recently, several groups have attempted to provide u-PA with kringle domains from other proteins. For example, both Nakayama et al. (1986, Thrombosis & Haemostasis 56: 364-370) and Robbins and Tanaka (1986, Biochemistry 25: 3603-3611) have built hybrids of the heavy chain of human plasminogen (containing all five kringle domains) with the serine protease domain of LMW urokinase by fusing the two chains by means of disulfide bonds between existing cysteine residues.
  • the kringle-fusions to u-PA described in the present invention were made at the cDNA level and represent fusions to the truncated single-chain form of u-PA, the form which exhibits excellent fibrin selectivity in its plasminogen activation.
  • the plasminogen kringle-scu-PA hybrids of this invention provide increased fibrin selectivity beyond that demonstrated for the molecules made by Nakayama et al. (1986, supra) and Robbins and Tanaka (1986, supra).
  • the result is a modified scu-PA with better fibrin selectivity than plasmin-produced tcu-PA but poorer fibrin selectivity than scu-PA itself or t-PA.
  • a modified plasminogen activator having greater fibrin selectivity than the unmodified plasminogen activator from which it is derived is provided.
  • the modified plasminogen activator includes at least one of the following domains: a kringle that is more homologous with a kringle of plasminogen than is any kringle of the unmodified plasminogen activator; a first hybrid t-PA kringle, the first hybrid kringle being a fusion of a portion of t-PA kringle 1 and a portion of t-PA kringle 2; or a second hybrid t-PA kringle, the second hybrid kringle being a fusion of a portion of t-PA kringle 2 and a portion of a scu-PA kringle.
  • scu-PA is modified to provide increased fibrin selectivity in its activation of plasminogen as compared to unmodified scu-PA.
  • fusion of plasminogen kringles 1 and/or 4 to a truncated scu-PA lacking its own EGF-like and kringle domains, or replacement of most of the scu-PA kringle with most of kringles 1 or 4 of plasminogen or with kringle 2 of t-PA results in a plasminogen activator with increased fibrin selectivity.
  • replacement of the scu-PA EGF-like and kringle domains with the t-PA EGF-like and finger domains together with a t-PA kringle 1-kringle 2 fusion domain also provides a more fibrin-specific plasminogen activator. While the precise sites of fusion are not absolutely critical, preferred embodiments of this invention is shown in TABLE 1.
  • leucine-80 to histidine, glutamine-81 to arginine, glutamine-82 to proline, threonine-83 to arginine, arginine-108 to aspartate, leucine-122 to arginine, and valine-123 to tryptophan (see TABLE 1).
  • Other changes may be made but these suffice to demonstrate improved fibrin selectivity of the plasminogen activator and the feasibility of further improvements.
  • the location of these amino acid residues in the folded protein has not been determined at this time, because the x-ray crystallographic structure is only known for the kringle of prothrombin.
  • the molecules of the present invention are produced by using recombinant DNA- methodology to provide new cDNA genes encoding new plasminogen activators.
  • the first and/or fourth kringles from plasminogen or a hybrid of t-PA kringle l and kringle 2 together with the t-PA finger and EGF-like domains are added to a truncated scu-PA lacking its own kringle.
  • Truncated scu-PA is a relatively fibrin selective plasminogen activator (Stump et al., 1986, supra), but the addition of a plasminogen kringle domain provides increased fibrin selectivity.
  • plasminogen kringle 1 or 4 or t-PA kringle 2 replaces most of the scu-PA kringle to provide a scu-PA with increased fibrin selectivity in its activity.
  • the scu-PA kringle itself is modified at between four and seven amino acid residues resulting in a scu-PA molecule with improved fibrin selectivity in its activation of plasminogen.
  • Another object of the invention is to provide novel DNA sequences encoding the foregoing plasminogen activators.
  • Another object of the invention is to provide cloned vectors in transformed host cells capable of expressing the foregoing plasminogen activators.
  • Another object of this invention is to provide a modified urinary plasminogen activator in accordance with the preceding objects having altered amino acid residues in the kringle domain.
  • Still another object of this invention is to provide a modified plasminogen activator in accordance with the first and second objects with most of the first or fourth kringle from human plasminogen.
  • a further object of this invention is to provide a modified scu-PA in accordance with the first and second objects with a new kringle domain from human plasminogen or from human t-PA replacing the kringle and EGF domains of scu-PA.
  • a further object of the present invention is to provide means and methods of producing the new plasminogen activators of the preceding objects.
  • Yet another object of the invention is to provide improved methods and products for treating certain medical conditions.
  • FIG. 1 is a schematic drawing of plasmid pCGM16
  • FIG. 2 is a schematic drawing of plasmid pCGM38;
  • FIGS. 3A and 3B show the nucleotide sequences of synthetic oligodeoxy-nucleotides encoding plasminogen kringles 1 and 4;
  • FIG. 4 is a schematic drawing of plasmids pCGE242 and pCGE241 which contain the plasminogen kringle DNA of FIGS. 3A and 3B respectively cloned between the HindIII and Bglll sites of pSV2-gpt;
  • FIG. 5 is a schematic drawing of plasmids pCGM61 and pCGM74 which contain the plasminogen kringle fusions to truncated scu-PA;
  • FIG. 6 is a schematic drawing of plasmid pCGM34 which contains a portion of t-PA encoding DNA fused to DNA coding for scu-PA amino acid residues 132-411;
  • FIG. 7 shows the nucleotide sequences of a synthetic oligodeoxy-nucleotide encoding a modified scu-PA kringle, with lines denoting the individual oligodeoxynucleotides synthesized, annealed, and ligated for the construction of plasmid pCGM99.
  • sc-uPA was obtained from human kidney cells (Kohno, T., Hopper, P., Lillquist, J.S., Suddith, R.L., Greenlee, R. and Moir, D.T., 1984, Biotechnology 2: 628-634).
  • Sc-uPA is defined as that plasminogen activator having a single polypeptide chain and exhibiting plasminogen activator activity expressed in terms of CTA (Committee on Thrombolytic Agents) units determined by means of a fibrin plate assay (Brakman, P., 1967, “Fibrinolysis: a standardized fibrin plate method and a fibrinolytic assay of plasminogen", Scheltema and Holkema, Amsterdam, pp. 1-124) and having an amino acid and encoding nucleotide sequence substantially as shown in Fig. 1 of Holmes, W.E., et al. (1985; supra).
  • the cells were grown to confluency in Dulbecco's Modified Eagles medium supplemented with 5% heat-inactivated NuSerum (Collaborative Research, Inc., Bedford, Massachusetts). Confluent cells were harvested by centrifugation after treatment with 0.25 percent trypsin for 15 minutes at 37°C and were frozen in liquid nitrogen.
  • the poly (A) RNA was isolated according to the method of Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982, "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 191-198). Briefly, 5 grams of frozen cells were lysed with NP-40 and the nuclei were removed by ultra-centrifugation. The cytoplasmic fraction was treated with proteinase K, and the protein was removed by repeated phenol: chloroform:isoamyl alcohol extractions. The total cytoplasmic RNA was recovered by precipitation with ethanol with a yield of about 16 milligrams.
  • the poly (A) RNA was isolated by oligo (dT) cellulose chromatography with a yield of about 200 micrograms. The intactness of the isolated poly (A) RNA was verified by in vitro translation in a rabbit reticulocyte lysate system. Conversion of poly (A) mRNA to double stranded cDNA was by standard procedures (see Wickens, M. P., Buell, G. N. and Schimke, R. T., 1978, J. Biol. Chem.
  • oligo (dT) as the primer of the first strand synthesis by reverse transcriptase, DNA polymerase I to synthesize the second strand, nuclease SI treatment to insure that the ends are flush and chromatography over Biogel A150M (Bio-Rad, Richmond, California) for size selection.
  • the size-selected double-stranded cDNA was ligated first to Xbal synthetic oligonucleotide linkers (Collaborative Research, Inc., Bedford, MA) and then into a single-stranded f1 phage vector (Zinder, N.D. and Boeke, J.D., 1982, Gene 19: 1-10; Bowden, D.W., Mao, J., Gill, T., Hsiao, K., Lillquist, J.S., Testa, D. and Vovis, G.F., 1984, Gene 27: 87-99).
  • the cDNA library of recombinant fl phage was transfected into E. coli by standard methods to yield fl phage plaques suitable for preparing nitrocellulose filter lifts and probing (see below).
  • oligodeoxynucleotide probes were synthesized by the automated phosphoramidite procedure (automated DNA synthesizer, Applied
  • the probes were labeled with 32P by means of polynucleotide kinase and gamma- 32P-ATP and isolated from the residual label by means of a polyacrylamide gel.
  • the resultant labeled probes were mixed and used to probe nitrocellulose plaque lifts from the library essentially according to the method of Benton, W. D. and Davis, R. W. (1977, Science 196: 180-182).
  • the use of multiple probes insured that unexpected occasional polymorphisms within the gene or poor hybridization by certain probes due to unpredictable structure or sequence context problems would not prevent identification of the clones containing prourokinase. In this manner, approximately 50,000 clones were screened for prourokinase sequences.
  • FIG. 1 depicts the introduction of this full-length cDNA encoding scu-PA into the eukaryotic expression vector pSV2-gpt (Mulligen, R. and Berg, P., 1981, Proc. Nat'l. Acad. Sci. USA 78: 2072-2076).
  • scu-PA cDNA was isolated from plasmid pCGE194 after restriction with Xbal followed by filling-out the ends with E. coli DNA polymerase I (Klenow fragment) and all four deoxynucleotide triphosphates.
  • DNA of plasmid pSV2-gpt was cut with restriction endonucleases HindIII and Bglll, and the ends were filled out with E. coli DNA polymerase (Klenow fragment) and all four deoxynucleotide triphosphates.
  • the approximately 2.3 kb scu-PA encoding fragment from pCGEl94 was ligated to the approximately 5.25 kb vector fragment from pSV2-gpt, and the ligation mixture was used to transform competent E. coli cells to ampicillin resistance.
  • Cells carrying the desired plasmid pCGM16 were identified by restriction endonuclease digestion analysis of plasmid DNA isolated from them.
  • Plasmid pCGM16 carrys the full length scu-PA encoding cDNA inserted in pSV2-gpt with the Xbal site but not the HindIII site regenerated at the SV40 promoter end of pSV2-gpt and the Xbal and Bglll sites lost at the other end of the insert.
  • Restriction endonuclease sites also are shown throughout the figures. Relative positions of restriction endonuclease cleavage sites are depicted by straight lines and are labeled in italics. Sites which were present in the parental vectors but lost upon ligation are shown in parentheses.
  • this vector After transformation of mammalian cells and integration into the chromosomes, this vector is capable of replication in mammalian cells and directing the expression of the added scu-PA cDNA from the SV40 early promoter.
  • the secretion of sc-uPA from Chinese hamster ovary (CHO)cells which had been transformed with pCGM16 was verified by analysis of conditioned medium in the fibrin plate assay (Brakman, 1967, supra) and by assaying it for amidolytic activity (Kohno et al., 1984, supra).
  • plasmid In order to facilitate construction of expression/secretion vectors for genes encoding hybrid plasminogen activators, a plasmid was constructed having a unique Bglll restriction endonuclease access site at the end of the scu-PA secretion signal coding region.
  • pCGM38 FOG 2
  • new DNA fragments encoding useful portions of proteins with fibrin affinity may be inserted between DNA sequences coding for the scu-PA secretion signal and portions of the scu-PA structural gene.
  • plasmid pCGM16 was cleaved with BssHII, which cuts within the alanine codon at position -15 in the secretion signal region (see FIG.
  • the two synthetic deoxyoligonucleotides each 44 nucleotides in length, were synthesized as described previously. They replace the portion of the signal sequence coding region which was deleted above and provide a new Bglll site at the end of the signal sequence codons.
  • the sequences of the synthetic nucleotides are as follows:
  • the first or fourth kringle from human plasminogen was fused to human scu-PA lacking its own EGF and kringle domains. This was accomplished in these examples at the DNA level by building the fusion gene for expression in living cells.
  • FIG. 3A Lines denote the individual oligodeoxynucleotides synthesized, annealed, and ligated for the construction of plasmids pCGE242 (FIG. 3A) and pCGE241 (FIG. 3B).
  • FIG. 4 Schematic diagrams of the entire plasmids pCGE242 and pCGE241 are shown in FIG. 4.
  • DNA sequences encoding the amino acid sequences of the first (Plg-K1) and fourth (Plg-K4) kringles of human plasminogen were derived by synthesis of overlapping oligodeoxynucleotide fragments corresponding to the published amino acid sequence of the respective regions of plasminogen (Forsgren, M., Raden, B., Israelsson, M., Larsson, K, and Lars-Olof, H., 1987, FEBS Lett. 213: 254-260). Using standard methods known in the art, sixteen such synthetic oligodeoxynucleotides (FIG.
  • FIG. 3B sixteen synthetic oligodeoxynucleotides (FIG. 3B) were ligated together between the HindIII and Bglll sites of pSV2-gpt to yield a DNA sequence encoding the fourth plasminogen kringle (amino acid residues 354-435; numbering from sequence of Forsgren, et al., as described above) cloned in a suitable vector.
  • An E. coli strain carrying the desired plasmid pCGE241 (FIG. 4) was isolated. Shown in Fig.
  • oligodeoxynucleotides used to make pCGE242 and pCGE241; however, many other sequences would suffice provided that they encode the same amino acid sequence, that they are cohesive in the proper order, and that they provide the necessary restriction endonuclease sites for the remainder of the constructions described below.
  • the Plg-K1-scu-PA and Plg-K4-scu-PA fusions were constructed by fusing the plasminogen kringle nucleotide sequences built as described above to the scu-PA nucleotide sequence in vector pCGM38 (FIG. 2). The fusions were performed at the common Fspl and Bglll sites. Plasmid DNA from pCGE242 (FIG. 4) and from pCGE241 (FIG.
  • Plasmid pCGM38 (FIG. 2) was cut with restriction endonucleases Bglll which cuts uniquely in the scu-PA cDNA, and with Kpnl which cuts in the vector sequences. The resulting approximately 5.17 kb fragment carrying most of the pSV2-gpt vector DNA, the SV40 early promoter and the scu-PA secretion signal coding DNA was isolated. A second aliquot of pCGM38 (FIG.
  • DNA was cut with restriction endonucleases Fspl, which cuts between codons 131 and 132 in the scu-PA cDNA, and with Kpnl, which cuts in the pSV2-gpt vector DNA.
  • Fspl restriction endonucleases
  • Kpnl restriction endonucleases
  • the resulting approximately 2 kb DNA fragment carrying scu-PA cDNA (codons 88-411) fused to a portion of pSV2-gpt vector DNA was isolated.
  • the DNA encoding each of the plasminogen kringles (K1 and K4) were ligated to the two fragments derived from pCGM38. Both ligation mixtures were used to transform competent E. coli cells, and transformant strains carrying the desired plasmids were identified.
  • plasmids carry either Plg-K1 (pCGE242) or Plg-K4 (pCGE241) encoding DNA fused to truncated scu-PA(132-411) cDNA in the mammalian expression vector pSV2-gpt, but the kringles are out of reading frame with the scu-PA DNA.
  • the kringle-scu-PA junctions were placed in translational-reading-frame by restriction with BglII, blunting the ends by digestion with S1 nuclease, restriction with SnaBI, and religation.
  • the resulting plasmids pCGM74 (FIG. 5) and pCGM61 (FIG. 5) carry functional transcriptional units encoding Plg-K1-scu-PA and Plg-K4-scu-PA fusions, respectively, both transcribed from the mammalian SV40 promoter and containing the scu-PA secretion signal for efficient secretion of the resulting protein (see Summary in Table 1).
  • Example 3 In addition, as another example of this invention, both Plg-K1 and Plg-K4 encoding sequences were fused to the scu-PA encoding cDNA at the codon for amino acid ala-132 to yield a scu-PA containing both plasminogen kringles known to bind to fibrin. DNA of plasmid pCGM74 (FIG.
  • Plasmid pCGM75 carrys the SV40 promoted gene for the scu-PA secretion signal fusion to Plg-K1(101-162)-Plg-K4(375-438)-scu-PA(132-411), all contained in vector pSV2-gpt (Mulligan and Berg, 1981, supra; see summary Table I below)
  • Example 4 the major part of plasminogen kringle 1 (Plg-K1; amino acid residues 102-162) replaces most of the kringle of scu-PA (amino acid residues 68-131) to yield a hybrid PA with improved fibrin selectivity.
  • DNA of plasmid pCGM16 (FIHut to completion with restriction endonuclease Xbal at the SV40 promoter/scu-PA junction and partially with Ncol (desired site is at scu-PA amino acid codon 66), and an approximately 320 bp fragment containing scu-PA codons 1-66 was isolated.
  • Plasmid pCGM74 (FIG.
  • the resulting ligation mixture was transformed into competent E. coli cells, and a transformant was identified carrying the desired plasmid pCGM107, containing the scu-PA(SS)-scu-PA(1-67)-Plg-K1 (102-162)-scu-PA(132-411) coding sequence.
  • Example 5 In a related additional example of this invention, the major part of plasminogen kringle 4 (Plg-K4; amino acid residues 376-435) replaces most of the kringle of scu-PA (amino acid residues 68-131) to yield another hybrid PA with improved fibrin selectivity.
  • Plg-K4 amino acid residues 376-435
  • scu-PA amino acid residues 68-131
  • a strategy identical to that described above in Example 4 was used, except that the second DNA fragment encoded most of Plg-K4 fused to scu-PA(132-411) and derived from plasmid pCGM61 (FIG. 5) instead of pCGM74.
  • the synthetic oligodeoxy-nucleotides are of a different sequence, and that is shown below.
  • t-PA-K2 amino acid residues 198-261
  • scu-PA kringle amino acid residues 68-131
  • Plasmid pCGM16 (FIG. 1) was cut with restriction endonucleases Xbal at the promoter/scu-PA junction and Kpnl in the vector sequences, and a 5.1 kb fragment containing most of the vector sequences and the SV40 promoter region was isolated. Another aliquot of pCGM16 was cut completely with restriction endonuclease Kpnl and partially with Fspl (desired site is between codons 131 and 132), and a 1.99 kb fragment carrying cDNA encoding scu-PA from codons 132 to 411 and a portion of vector sequences was isolated. These two isolated fragments were ligated together in the presence of two synthetic oligodeoxynucleotides having a sequence encoding t-PA from codons 255 through 261 as shown below:
  • Competent E. coli cells were transformed with the ligated DNA, and a clone carrying plasmid pKHlll containing t-PA (codons 255-261) fused to scu-PA (codons 132-411) inserted adjacent to the SV40 promoter in pSV2-gpt was obtained.
  • Plasmid pCGM33 was used as the source for the remainder of the t-PA coding sequence. This plasmid carrys cDNA encoding full-length t-PA flanked by Xbal DNA linkers and located adjacent to the SV40 promoter in pSV2-gpt.
  • the t-PA cDNA was obtained from RNA isolated from Bowes melanoma cells by standard methods of cDNA cloning.
  • the t-PA sequence contained in pCGM33 is essentially the same as that described by others (Pennica, D., Holmes, W.E., Kohr, W. J., et al., 1983, Nature 301: 214-221).
  • Plasmid pCGM33 was cut with restriction endonucleases Xbal at the promoter/t-PA junction and with Seal at codon 255 in the t-PA coding sequence, and an approximately 0.88 kb DNA fragment encoding amino acid residues 1-255 of t-PA was isolated. Plasmid pKHlll was cut with restriction endonuclease Xbal at the SV40-t-PA junction, blunted with S1 nuclease, and cut further with endonuclease Kpnl in the vector sequences.
  • a gene encoding a hybrid scu-PA having most of its kringle replaced with kringle 2 of t-PA was constructed from plasmids pCGM34 and pCGM16 as follows. Plasmid pCGM16 was cut to completion with restriction endonuclease Kpnl and partially with Ncol (desired site is at codon 66), and an approximately 5.43 kb DNA fragment carrying most of the vector sequences, the SV40 promoter, and scu-PA cDNA (codons 1-66) was isolated.
  • Plasmid pCGM34 was cut completely with Kpnl and partially with EcoRI (desired site is at t-PA codon 205), and a 2.16 kb DNA fragment carrying t-PA cDNA (codons 205-261) fused to scu-PA cDNA (codons 132-411) together with a small portion of vector sequence was isolated. These two isolated fragments were ligated to each other and to two synthetic oligodeoxy-nucleotides carrying codon 67 of scu-PA fused to codons 198-205 of t-PA as shown below.
  • the ligation mixture was used to transform competent E. coli cells, and a clone containing the desired plasmid pCGM105 carrying SV40-promoted scu-PA(codons 1-67)-t-PA(codons 198-261)-scu-PA(codons 132-411) was identified (see Summary in Table 1). Plasmid DNA was prepared and used to transform mammalian cells so that they produce the recombinant protein.
  • a hybrid kringle consisting of a portion of t-PA-K1 and a portion of t-PA-K2 was fused to truncated scu-PA(132-411), thereby replacing the scu-PA kringle.
  • plasmid pCGM34 was cut with restriction endonuclease Narl at t-PA codon 110 and at Kpnl in the vector.
  • An approximately 5.29 kb DNA fragment containing most of the pSV2-gpt vector, the promoter, and t-PA codons 1-110 was isolated.
  • a second aliquot of pCGM34 was cut partially with EcoRI (desired site is at t-PA codon 205) and completely with Kpnl in the vector, and an approximately 2.2 kb DNA fragment containing t-PA codons 205-261 fused to scu-PA codons 132-411 was isolated.
  • Plasmid pCGM103 encodes t-PA(SS)-t-PA-K1(1-111)-t-PA-K2-(200-261)-scu-PA(132-411) transcribed from the SV40 early promoter and carried on vector pSV2-gpt (see Summary in Table 1).
  • Examples 8 & 9 In two additional examples of this invention, specific amino acid residues of the scu-PA kringle were altered to yield a modified scu-PA with increased fibrin selectivity.
  • threonine-83, arginine-108, leucine-122, and valine-123 were changed to arginine, aspartate, arginine, and tyrosine, respectively. This was accomplished by replacing the scu-PA codons for amino acid residues 67-131 in plasmid pCGM16 with synthetic oligodeoxy-nucleotides incorporating new codons for the altered residues described above. Briefly, plasmid pCGM16 (FIG.
  • a second modified scu-PA cDNA containing three additional altered amino acid codons was produced by modification of plasmid pCGM99 as follows. DNA from plasmid pCGM99 was digested with restriction endonucleases AlwNI and Bglll (see FIG. 7), and the large fragment was isolated. It contains the entire plasmid except for the small sequence encoding the twelve amino acids between the AlwNI and Bglll sites. Two synthetic oligodeoxynucleotides of sequence shown below provide a modified set of codons for the twelve amino acids such that leucine-80 is changed to histidine, glutamine-81 is changed to arginine, and glutamine-82 is changed to proline .
  • oligodeoxynucleotides were annealed and ligated together between the AlwNI and Bglll sites of the isolated large fragment from pCGM99.
  • the resulting ligation mixture was used to transform competent E. coli HB101 cells to ampicillin resistance, and a clone carrying the desired plasmid pCGMlOl (see Summary in Table 1) was isolated.
  • Example 10 In another example of this invention, plasminogen kringles 1 and 4 are fused to t-PA residues 262 through 529 to create a new plasminogen activator molecule with greater fibrin specificity and longer in vivo half-life than naturallyoccurring t-PA or scu-PA.
  • a gene for the new hybrid plasminogen activator is constructed as follows for expression in mammalian cells. First, plasmid .
  • pCGM75 (see Example 3, supra) is cut completely with restriction endonuclease Kpnl in the pSV2-gpt vector sequence and partially with Fspl (desired site is at the Plg-K4-scu-PA junction), and an approximately 5.7 kb DNA fragment carrying most of the vector sequence together with all of Plg-Kl and Plg-K4 is isolated.
  • DNA encoding t-PA (codons 280-529) is isolated by cutting plasmid pCGM33 (see Example 6, supra) with restriction endonucleases Kpnl in the pSV2-gpt sequences and with Banll in the codon for amino acid residue 280. An approximately 1.69 kb DNA fragment is isolated. Finally, both of these isolated fragments are ligated together in the presence of two annealed synthetic oligodeoxy-nucleotides of the following sequence.
  • oligodeoxynucleotides span the gap between the Fspl and Kpnl sites of the isolated DNA fragments from plasmids pCGM75 and pCGM33, and they provide the coding sequence for amino acid residues 262 through 280 of t-PA.
  • the ligation mixture is used to transform competent E. coli cells to ampicillin resistance, and a transformed clone is identified carrying the desired plasmid pCGMlll.
  • Plasmid pCGM111 is pSV2-gpt carrying a fusion of scu-PA secretion signal-Plg-K1(70-162)-Plg-K4(354-438)-t-PA(262-529) to the SV40 early promoter (see summary in Table 1).
  • Plasmid pCGM74 is used in conjunction with a second plasmid supplying a selectable marker to co-transform Chinese hamster ovary cell line DG44 (Urlaub, G., Kas, E., Carothers, A.M., and Chasin, L.A., 1983, Cell 33: 405-412) which completely lacks the diploid DHFR locus.
  • the co-transformed selectable plasmid is either pSV2-DHFR (Subramani, S., Mulligan, R., and Berg, P., 1981, Mol. Cell. Biol.
  • plasminogen activator To increase the plasminogen activator expression level, resulting co-transformants are carried through a step-wise gene amplification procedure which involves challenging the cells with increasing concentrations of methotrexate (MTX) (Kaufman, R. and Sharp, P., 1982, J. Mol. Biol. 159: 601-621).
  • MTX methotrexate
  • Suitable high level plasminogen activator producing clones are identified by amidolytic assay (Kohno et al, 1984, supra) of cultures of cells which are resistant to high levels of MTX. Clones are grown in medium consisting of 1:1 DME:F12 which lacks thymidine and hypoxanthine (Gibco, Grand Island, NY) but is supplemented with 10% fetal bovine serum. Following growth in T-flasks and roller bottles, conditioned medium containing between 2 and 20 ug/ml of plasminogen activator is harvested.
  • the mutant plasminogen activators are purified by means of the following procedure.
  • Conditioned medium is filtered through a 0.2 micron pleated capsule filter (Gelman, Ann Arbor, MI) to remove particulate material, titrated to pH7, if necessary, and applied to a column of anti-scu-PA-Sepharose equilibrated with a buffer consisting of 10 mM sodium phosphate (pH7.4), 0.14 M sodium chloride, 10 KlU/ml Aprotinin (Sigma, St. Louis, MO).
  • Anti-scu-PA-Sepharose was made by coupling scu-PA-specific monoclonal antibody to CNBr-activated Sepharose (purchased from Pharmacia, Piscataway, NJ).
  • the monoclonal antibody was prepared by fusion of scu-PA immunized mouse spleen cells with antibody-secreting myeloma cells according to published procedures (Oi, V. & Touchberg, L., 1980 in Selected Methods in Cellular Immunology, pp. 351-372. W.H. Freeman & Co., San Francisco). Analysis of the antibody showed that it is about 30 times more specific for scu-PA than tcu-PA.
  • the coupling procedure was done essentially according to standard methods (see Pharmacia publication - Affinity Chromatography, Principles and Methods; also see Axen, R., Porath, J., & Ernback, S., 1967 Nature 214:1302-1304, and March, S.C., Parikh, I., & Cuatrecasas, P., 1974, Analytical Biochemistry 60:149-152) except that 20 mg of purified monoclonal antibody was coupled per ml of gel. Briefly, the procedure for coupling was as follows. A specified mass of dried cyanogen bromide-activated Sepharose was measured and swollen in 1 mM hydrochloric acid solution (200ml/g).
  • the swollen gel was washed with coupling buffer (0.1 M sodium bicarbonate, pH 8.3, 0.5 M NaCl, 0.8 mM CaCl 2 , 0.5 mM MgCl,) to equilibrate it before adding protein.
  • the monoclonal antibody was dissolved in the same coupling buffer so that approximately 20 mg would couple per ml of gel.
  • the protein solution was added to the cyanogen bromide-activated Sepharose suspension and then mixed end-over-end at room temperature for 3 hrs. The amount of monoclonal antibody added at the beginning of the reaction was estimated by absorbancy at 280 nm.
  • the coupled Sepharose was filtered to remove the coupling buffer containing unreacted protein (estimated again by absorbancy at 280 nm). Remaining active groups on the cyanogen bromide-activated Sepharose were blocked next by adding a solution of 1M ethanolamine (in coupling buffer) and mixing the suspension at room temperature for 2 hours. The coupled Sepharose was filtered again and washed with 0.1 M acetate, pH 4, 1 M NaCl followed by 10 mM Na phosphate, pH 7.2, 1 M NaCl for a total of three times.
  • the coupled resin was washed again with 3 M sodium thiocyanate dissolved in 10 mM Na phosphate, pH 7.2, 0.14 M NaCl, and then washed finally with 10 mM Na phosphate, pH 7.2, 0.14 M NaCl.
  • the resin was then used as an affinity matrix for purification of scu-PA related proteins. After washing the column with the equilibration buffer, it is developed with 50 mM glycine (pH2) to elute the protein which had been bound to the antibody-Sepharose.
  • the eluate is diluted with one-fourth volume of 100 mM sodium acetate (pH5.3), 1 M sodium chloride, adjusted to a pH of 5.3, and applied to a column of p-aminobenzamidine-Sepharose (Collaborative Research, Inc., Bedford, MA) equilibrated with a buffer consisting of 20 mM sodium acetate (pH5.3), 0.1 M sodium chloride. Protein flowing through the column is monitored by absorbancy at 280 nm, collected and concentrated to about 1 mg/ml for further analyses described below.
  • the purified mutant plasminogen activator is examined in the in vitro fibrin clot lysis model of Gurewich, V., Pannell, R., Louie, S., Kelley, P., Suddith, R.L., and Greenlee, R. (1983, J. Clin. Invest. 73: 1731-1739). Briefly, aliquots of citrated human plasma containing 1.5 uCi IBRIN
  • the mutant plasminogen activator exhibits potency equal to that of wild-type scu-PA, but shows increased fibrin specificity over that of wild-type scu-PA.
  • Example 12 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 12 is identical to Example 11 except plasmid pCGM61 is used in place of plasmid pCGM74.
  • Example 13 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 13 is identical to Example 11 except plasmid pCGM75 is used in place of plasmid pCGM74.
  • Example 14 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 14 is identical to Example 11 except plasmid pCGM107 is used in place of plasmid pCGM74.
  • Example 15 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 15 is identical to Example 11 except plasmid pCGM109 is used in place of plasmid pCGM74.
  • Example 16 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 16 is identical to Example 11 except plasmid pCGM105 is used in place of plasmid pCGM74.
  • Example 17 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 17 is identical to Example 11 except plasmid pCGM103 is used in place of plasmid pCGM74.
  • Example 18 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 18 is identical to Example 11 except plasmid pCGM99 is used in place of plasmid pCGM74.
  • Example 19 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 19 is identical to Example 11 except plasmid pCGM101 is used in place of plasmid pCGM74.
  • Example 20 Demonstration of fibrin selectivity during clot lysis in vitro initiated by a mutant plasminogen activator.
  • Example 20 is identical to Example 11 except plasmid pCGM111 is used in place of plasmid pCGM74.
  • Example 21 Demonstration of fibrin specificity during clot lysis in vivo.
  • the mutant plasminogen activator of Example 11 is also examined for potency and specificity during clot lysis in the rabbit jugular venous thrombosis model according to the protocol of Collen, D., Stassn, J.M., and Verstraete, M. (1983, J. Clin. Invest. 71: 368-376). Potency is judged by the time and dose dependence of clot lysis; fibrin specificity is determined by measurement of residual plasma fibrinogen and alpha-2-antiplasmin levels by means of standard assays (Clauss, A., 1957, Acta. Hematol.
  • mutant plasminogen activators exhibit potency approximately equal to that of wild-type scu-PA, but show increased fibrin specificity over that of wild-type scu-PA.
  • Example 22 Demonstration of fibrin specif icity during clot lysis in vivo.
  • Example 22 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM61 and is derived from Example 12.
  • Example 23 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 23 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM75 and is derived from Example 13.
  • Example 24 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 24 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM107 and is derived from Example 14.
  • Example 25 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 25 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM109 and is derived from Example 15.
  • Example 26 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 26 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM105 and is derived from Example 16.
  • Example 27 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 27 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM103 and is derived from Example 17.
  • Example 28 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 28 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM99 and is derived from Example 18.
  • Example 29 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 29 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM101 and is derived from Example 19.
  • Example 30 Demonstration of fibrin specificity during clot lysis in vivo.
  • Example 30 is identical to Example 21 except that the purified plasminogen activator is encoded by plasmid pCGM111 and is derived from Example 20.
  • Example 31 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • the purified mutant plasminogen activator of Example 11 is also analyzed for rate of disappearance from the circulation of a rabbit.
  • aliquots of rabbit serum are withdrawn, processed to obtain platelet-poor plasma, and analyzed for the level of plasminogen activator according to the ELISA described by Stump, D.C., Kieckens, L. DeCook, F., and Collen, D.(1987, J.Pharmacol. Exp. Ther. 242: 245-250). Samples are analyzed immediately prior to infusion, at 1, 2, 3, and 4 hrs of infusion, and at 1, 3, 5, 7, 9, 15, 30 and 60 minutes after infusion.
  • the rate of disappearance of the mutant plasminogen activator following bolus injection into the circulation of a rabbit is also determined as follows.
  • the purified mutant plasminogen activator of Example 11 is injected as a single bolus into the circulation of a rabbit to achieve an initial concentration of about 1 microgram per ml.
  • samples are taken, platelet-poor plasma prepared, and the amount of plasminogen activator antigen present in the plasma quantitated by ELISA described by Stump et al. (1987, supra).
  • Plasma plasminogen activator levels are plotted as a function of time and the harmacokinetic parameters are calculated by the method of Welling (1986, in "Pharmacokinetic Processes and Mathematics," ACS Monograph 185, Washington, D .C.).
  • the mutant plasminogen activator encoded by plasmid pCGM74 and purified as in Example 11 is found to exhibit a longer circulating half-life than natural scu-PA.
  • Example 32 Demonstration of a longer half-life for a. mutant plasminogen activator in the circulation of a rabbit.
  • Example 32 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM61 and is derived from Example 12.
  • Example 33 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • Example 33 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM75 and is derived from Example 13.
  • Example 34 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • Example 34 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM107 and is derived from Example 14.
  • Example 35 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • Example 35 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM109 and is derived from Example 15.
  • Example 36 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • Example 36 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM105 and is derived from Example 16.
  • Example 37 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • Example 37 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM103 and is derived from Example 17.
  • Example 38 Demonstration of a longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • Example 38 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM99 and is derived from Example 18.
  • Example 39 Demonstration of a_ longer half-life for a mutant plasminogen activator in the circulation of a rabbit.
  • Example 39 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGM101 and is derived from Example 19.
  • Example 40 Demonstration of a longer half-life for a_ mutant plasminogen activator in the circulation of a rabbit.
  • Example 40 is identical to Example 31 except that the purified plasminogen activator is encoded by plasmid pCGMlll and is derived from Example 20.
  • SS refers to the DNA encoding the secretion signal sequences of either scu-PA (nucleotides -20 through -1 of sequence of Holmes et al. (supra) or t-PA (nucleotides -35 through -1 of sequence of Pennica et al. supra).
  • Abbreviations for amino acid substitutions in Examples 8 and 9 are by the single letter code; the first letter designates the original amino acid at the numbered position, and the letter following the number designates the new amino acid at that position.

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Abstract

Un activateur de plasminogènes modifié présente une sélectivité pour la fibrine supérieure à l'activateur de plasminogènes non modifié dont il est dérivé. L'activateur de plasminogènes modifié comporte au moins un des domaines suivants: un ''kringle'' qui est plus homologue d'un ''kringle'' de plasminogène que ne l'est n'importe quel ''kringle'' de l'activateur de plasminogènes non modifié; un premier ''kringle'' hybride t-PA, le premier ''kringle'' hybride étant une fusion d'une portion du ''kringle'' t-PA 1 et d'une portion du ''kringle'' t-PA 2; ou bien un second ''kringle'' hybride t-PA, le second ''kringle'' hybride étant une fusion d'une portion du ''kringle'' t-PA 2 et d'une portion d'un ''kringle'' scu-PA.
PCT/US1989/001255 1988-04-22 1989-03-23 Activateurs de plasminogenes a selectivite accrue pour la fibrine WO1989010401A1 (fr)

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Cited By (4)

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WO1992004450A1 (fr) * 1990-09-01 1992-03-19 Beecham Group Plc Activateurs de plasminogene hybrides
US5595736A (en) * 1991-04-22 1997-01-21 Eli Lilly And Company Compounds and methods for treatment of thromboembolic disorders
WO2020140101A1 (fr) 2018-12-28 2020-07-02 Catalyst Biosciences, Inc. Polypeptides activateurs de plasminogène de type urokinase modifiés et leurs procédés d'utilisation
US11613744B2 (en) 2018-12-28 2023-03-28 Vertex Pharmaceuticals Incorporated Modified urokinase-type plasminogen activator polypeptides and methods of use

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GB8901422D0 (en) * 1989-01-23 1989-03-15 Fujisawa Pharmaceutical Co New tissue plasminogen activator

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BIOCHEMISTRY, Volume 27, No. 7, issued April 1988, (DE VRIES et al.), "Artificial Exon Shuffling between Tissue-type Plasminogen Activator (t-PA) and Urokinase (u-PA): A comparative study on the Fibrinolytic Properties of t-PA/u-PA Hybrid Protease", see pages 2565-2572, see particularly pages 2565-2568 and 2570-2572. *
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JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 262, No. 22, issued August 1987, (NELLES et al.), "Characterization of a Fusion Protein Consisting of Amino Acids 1 to 263 of Tissue-type Plasminogen Activator and Amino Acids 144 of Urokinase-type Plasminogen Activator", see pages 10855-10862, see particularly pages 10855 and 10861. *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004450A1 (fr) * 1990-09-01 1992-03-19 Beecham Group Plc Activateurs de plasminogene hybrides
US5595736A (en) * 1991-04-22 1997-01-21 Eli Lilly And Company Compounds and methods for treatment of thromboembolic disorders
US5658788A (en) * 1991-04-22 1997-08-19 Eli Lilly And Company Compounds and methods for treatment of thromboembolic disorders
WO2020140101A1 (fr) 2018-12-28 2020-07-02 Catalyst Biosciences, Inc. Polypeptides activateurs de plasminogène de type urokinase modifiés et leurs procédés d'utilisation
US11613744B2 (en) 2018-12-28 2023-03-28 Vertex Pharmaceuticals Incorporated Modified urokinase-type plasminogen activator polypeptides and methods of use
US12331334B2 (en) 2018-12-28 2025-06-17 Vertex Pharmaceuticals, Incorporated Modified urokinase-type plasminogen activator polypeptides and methods of use

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