EP0366661A1 - PROTEINES HUMAINES A MUTANTS DE SUBSTITUTION t-PA(u-PA), ADN RECOMBINANT PREVU A CET EFFET, CELLULES HOTES TRANSFECTEES PAR TRANSMISSION, PREPARATION DES PROTEINES A MUTANTS ET COMPOSITIONS PHARMACEUTIQUES - Google Patents

PROTEINES HUMAINES A MUTANTS DE SUBSTITUTION t-PA(u-PA), ADN RECOMBINANT PREVU A CET EFFET, CELLULES HOTES TRANSFECTEES PAR TRANSMISSION, PREPARATION DES PROTEINES A MUTANTS ET COMPOSITIONS PHARMACEUTIQUES

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Publication number
EP0366661A1
EP0366661A1 EP88903965A EP88903965A EP0366661A1 EP 0366661 A1 EP0366661 A1 EP 0366661A1 EP 88903965 A EP88903965 A EP 88903965A EP 88903965 A EP88903965 A EP 88903965A EP 0366661 A1 EP0366661 A1 EP 0366661A1
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human
chain
substitution
mutant proteins
mutant
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Hans Pannekoek
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Stichting Centraal Laboratorium Van de Bloedtransfusiedienst Van
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Stichting Centraal Laboratorium Van de Bloedtransfusiedienst Van
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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
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • 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

Definitions

  • This invention relates to the construction of a recombinant DNA molecule and the expression of the recombinant DNA molecule in host cells.
  • the invention also relates to a process for preparing human substitution- mutant proteins built up from a segment of the glycoprotein tissue type plasminogen activator (t-PA) and from a segment of the glycoprotein urokinase (u-PA).
  • t-PA glycoprotein tissue type plasminogen activator
  • u-PA glycoprotein urokinase
  • the invention further relates to pharmaceutical compositions having an anti-thrombotic activity.
  • t-PA human glycoprotein tissue type plasminogen activator
  • t-PA The human glycoprotein tissue type plasminogen activator (t-PA) is a serine protease, which is synthesized in vascular endothelial cells and also in various cell lines, such as the Bowes melanoma cell line ( if in et al., 1974; Rijken et al., 1980).
  • t-PA performs an essential role in fibrinolysis, the process which is responsible for solubilizing and hence removing a blood clot.
  • t-PA catalyses the conversion of the zymogen plasminogen into active serine protease plasmin- Plasmin is the enzyme considered to be primarily respons ⁇ ible for the lysing of the most important protein component in blood clots, namely fibrine.
  • fibrin In the absence of fibrin, t-PA only has a very low plasminogen activator activity. while in the presence of fibrin the activity of t- PA is accelerated by a factor of 100 to more than 1000. Owing to this property of t-PA, plasmin is generated virtually exclusively on the fibrin surface of a clot, and a systemic formation of plasmin in the circulation is avoided.
  • t-PA As a useful anti-thrombolytic.
  • the isolation on a large scale of t-PA from its natural environment (blood plasma) would meet with substantial practical and logistic problems owing to its low concentration (about 1 ng per ml).
  • Recombinant DNA techniques can offer a solution here to produce relatively large quantitie It has indeed been shown that t-PA synthesized by means of these techniques represents a useful preparation for the treatment of patients suffering from myocardial infarction (Verstraete et al. , 1985; Williams et al., 1986) .
  • t-PA intravenous administration of an effective dose of t-PA in these studies requires relatively large quantities and, in a number of cases, may lead to the decomposition of components other than fibrin and to undesirable hemorrhagic tendencies.
  • the need of administering large amounts of t-PA in an anti-thrombotic- therapy is specifically related to the rapid clearance of t-PA in vivo (its half-life is a few minutes). This phenomenon is attributed to interaction with an hepatic component through which t-PA is removed from circulation (Emeis et al. , 1985) and to inactivation through complexing with serine protease inhibitors, specifically with the endothelial plasminogen activator inhibitor PAI-1 (Sprenger and K Kunststoff, 1987).
  • t-PA molecula variants of t-PA be developed, which exhibit a more effective anti-thrombolytic activity than does t-PA, for example, by virtue of such variants being less effi ⁇ ciently inhibited by PAI-1.
  • t-PA is synthesized as a molecule consisting of one chain, and can be converted by plasmin into a molecule consisting of two chains by cutting a single arginine-isoleucine peptide bond (Wallen et al., 1980).
  • Two-chain t-PA consists of an amino-terminal "heavy" chain (H; 38 kD) and a carboxy-terminal "light” chain (L; 34 kD) , which are kept together by a single disulfide bond.
  • H amino-terminal "heavy” chain
  • L carboxy-terminal "light” chain
  • the S variant consists of 527 amino acids, while the L variant is composed of 530 residues. Concluding, an amino-terminal segment of either 32 or 35 amino acids is split off from the precursor protein to generate two types of t-PA. There are no indications that other properties are to be attributed to the S and the L variants.
  • the signal peptide followed by the pro-sequence, segments similar to the pre-pro portion of, e.g., serum albumin (Patterson and Geller, 1977; Lawn et al., 1981).
  • F finger
  • F fibronectin
  • c The "epidermal growth factor-like” (EGF or E) domain, which exhibits structural similarities to both human and mouse EGF (Savage et al., 1972; Gregory and Preston, 1977) .
  • EGF or E epidermal growth factor-like domain
  • the carboxy-terminal L chain of t-PA exhibits homology with the "family of trypsin-like" serine proteases It has recently been shown in our laboratory that this part of the t-PA molecule contains the catalytic centre for the plasminogen activator activity and also is the principal interaction site for the physiological inhibitor of t-PA, the endothelial plasminogen activator inhibitor PAI-1 (MacDonald et al., 1985; Van Zonneveld et al., " 1986a) .
  • PAI-1 endothelial plasminogen activator inhibitor
  • This invention relates to the construction of cDNA's coding for substitution-mutants of t-PA, in which a constant portion of the t-PA cDNA constructed in our laboratory is fused with a different portion of a (partial) cDNA coding for the so-called B chain of the human urokinas (u-PA) (Verde et al., 1984).
  • the object is to construct a recombinant DNA molecule coding for a fusion protein in which the properties of both t-PA and u-PA are repre ⁇ sented.
  • urokinase is a plasminogen activator and is isolated from urine or from conditioned medium of various cultured cell lines.
  • HMW u-PA high-molecular
  • LMW u-PA low-molecular
  • HMW u-PA high-molecular
  • LMW u-PA low-molecular
  • the amino acid sequence of both HMW u-PA and LMW u-PA, and also the position of asparaginine-coupled glycosylation sites have been completely explained (G ⁇ nzler et al., 1982a; G ⁇ nzler et al., 1982b; Steffens et al., 1982).
  • HMW u-PA consists of two chains linked by a single disulfide bond.
  • the amino-terminal chain (A chain) contains 157 residues (1 - 157) and the carboxy-terminal chain (B chain) contains 253 amino acids (159 - 411).
  • LMW u-PA also consists of two chains linked by a disulfide bond in the same way and at the same position as HMW u-PA.
  • the amino- terminal chain (Al) consists of 22 residues (136n - 157) and the carboxy-terminal B chain is identical to the B chain of HMW u-PA (-159 - 411) .
  • u-PA cDNA a recombinant DNA molecule carrying the complete genetic information- for human u-PA (so-called u-PA cDNA) and the expression of biologically active u-PA synthesized in Escherichia coli bacteria, transformed with a plasmid containing u-PA cDNA has been described (Heyneker et al., 1983).
  • the nucleotide sequence of full-length u-PA cDNA was determined in these studies, so that the amino acid sequence could be derived from it.
  • u-PA is synthesized as a precursor molecule consisting of 431 amino acids. The amino-terminal segment of 20 amino acids, which probably has a signal peptide function, is removed.
  • u-PA cDNA was a messenger RNA (mRNA) preparation containing mRNA still including introns.
  • mRNA messenger RNA
  • the presence of these two introns in the u-PA cDNA makes it necessary in expression studies to use eukaryotics or tissue culture cells of eukaryotics as host cells, as prokaryotics such as Escherichia coli will not be able to eliminate these introns from the corresponding mRNA.
  • mRNA messenger RNA
  • the u-PA protein can be regarded as a molecule built up from a number of discrete domains each performing an autonomous function.
  • Urokinase has been used on a large scale as an anti-thrombolytic. However, u-PA has a property which seriously hinders such uses. Unlike t-PA, u-PA has a substantial plasminogen activator activity in the absence of fibrin- The result of this property is that when administered _i ⁇ vivo both circulating plas ⁇ minogen and fibrin-bonded plasminogen are converted into plasmin without any distinction. The systemic formation of plasmin leads to the proteolytic degradation of fibrinogen and of the blood clotting factors Factor V and Factor VIII, possibly resulting in heavy bleeding complications.
  • the inventors have now succeeded in constructing two different human molecules (substitution-mutant cDNA's) and expressing these in host cells.
  • These recombinant DNA molecules contain in addition to a vector portion a DNA sequence coding for the most important part of the H chain of t-PA and a DNA sequence coding at least for the complete B chain of u-PA.
  • the object of this is to add the property that the plasminogen activator activity of t-PA i_> greatly accelerated by the interaction of the kringle 2 (K2) domain and the finger (F) domain of t-PA with fibrin , to the plasminogen activator activity of u-PA, which is located on the B chain of u-PA.
  • the invention is further embodied in a process for preparing human t-PA(u-PA) substitution-mutant proteins, which comprises culturing, in a known per se manner, host cells that have been transfected using such a recom ⁇ binant DNA, and recovering the human t-PA(u-PA) substitution mutants produced by the host cells.
  • t-PA(u-PA) substitution- mutant proteins are more active plasminogen activators than t-PA, and in addition are less sensitive to the physiological inhibitor of t-PA, i.e., the endothelial plasminogen activator inhibitor (PAI-1) .
  • the invention is also embodied in a pharmaceutical composition having an effect on blood clotting and/or on fibrinolysis, comprising human t-PA(u-PA) substitution mutant proteins produced using such a process, and in host cells which, as a result of transfection by means of a recombinant DNA molecule as defined above, are capable of producing human t-PA(u-PA) substitution- mutant proteins.
  • Plasmid pSV2/t-PA carries the complete genetic information (cDNA) for human t-PA, and the nucleotide sequence of this plasmid has been fully determined (Mulligan and Berg, 1981; Van Zonneveld et al., 1986b).
  • plasmid pHUK-1 which contains a partial cDNA coding for at least the B chain of human u-PA (Verde et al., 1984).
  • the nucleotide sequence of plasmid pHUK-1 has also been fully determined (Okayama and Berg, 1983; Verde et al., 1984).
  • the numberin of restriction sites in t-PA cDNA has been taken from Pennica et al. (1983), while the numbering of restriction sites in u-PA DNA and cDNA has been indicated by Verde et al (1984) .
  • the DNA of the vector being the replicative double-stranded form of the bacteriophage M13mp8 (Vieira and Messing, 1982) was fully digested with the restriction endonuclease EcoRI.
  • the now linear vector was mixed with the Fragments A and B and, by means of the enzyme DNA ligase, a recombinant DNA construct was prepared consisting of the above three components, in which the PstI end of t-PA (position 1312) is fused with the PstI end of u-PA (position 386) and the two EcoRI ends (position 801 of t-PA and position 727 of u-PA) are fused with the two EcoRI ends of M13mp8.
  • This construct was called Starting from the single-stranded form of the
  • the 5' terminalhalves of the two primers are identical to the base pairs 958 to 975 of t-PA cDNA coding for the amino acids asparagic acid (D), valine (V), proline (P) , serine (S), cysteine (C) and serine (S).
  • the 3' terminal half of primer I correspon to the base pairs 677 to 698 of u-PA DNA coding for the amino acids glutamine (Q), cysteine (C) , glycine (G)V glutamine (Q) , lysine (K) and threonine (T) .
  • the 3' terminal half of primer II corresponds to the base pairs 638 to 655 of u-PA and code for the amino acids glycine (G) , lysine (K) , lysine (K) , proline (P), serine (S) and serine (S).
  • the Fusion Fragments at the 5' side are f sed with the adjacent t-PA cDNA from the full-length t-PA cDNA, and at the 3* side the Fusion Fragments are fused with the adjacent u-PA (c)DNA from the plasmid pHUK-1.
  • the DNA of the plasmid pSV2/t-PA (Van Zonneveld et al., 1986b) was fully digested with the restriction enzymes Hindlll and EcoRI, as a result " of which we were able to isolate a restriction fragment extending from the Hindi!I restriction site on the vector portion of pSV2/t-PA and the EcoRI site at position 801 in the t-PA cDNA portion.
  • pHUK-1 DNA (Verde et al., 1984) was fully digested with the restriction endonuclease PstI and partially with the restriction endonuclease EcoRI, avoiding "cleavage" of the EcoRI restriction site at position 1364. In this way we were able to isolate an EcoRI PstI fragment corresponding to the positions
  • the vector used for the following construction is the plasmid pUCl9, which was digested with the restriction endonucleases HindiII and PstI (Yanisch-Peron et al., 1985).
  • Fusion Fragment A (222 base pairs; EcoRI ends) was mixed with the Hindlll-EcoRI fragment from pSV2/t-PA, the EcoRI-PstI fragment from pHUK-1 and the digested vector pUC19.
  • the Fusion Fragment B (261 base pairs) was, separately, mixed with the same three fragments.
  • the Hindlll-BamHI fragmen contain the fusions between t-PA cDNA, coding for the most important portion of the H chain of t-PA, and the B chain of u-PA (with the difference between the two fragments being expressed in that when primer II is used there are 13 amino acids more than when primer I is used).
  • the two Hindlll-BamHI fragments are separately used to replace the full-length t-PA cDNA portion on the plasmid pSV2/t-PA.
  • the DNA of plasmid pSV2/t-PA was fully digested with the restriction endonucleases Hindlll and Bglll and the fragment containing the vector portion was isolated.
  • the restriction endonucleases BamHI and Bglll generate the same DNA ends, we were subsequently able to ligate the above Hindlll-BamHI fragments with the digested vector.
  • DH1 bacteria the desired two types of recombinant DNA plasmids were isolated and purified (Maniatis et al., 1982).
  • the nucleotide sequence of the relevant sequences of these final plasmids called pSV2/tPA: :u-PA-I and pSV2/t-PA: :u-PA-I.I, was fully determined by DNA sequencing (Sanger et al., 1977) and the relevant part of the fusion between t-PA cDNA and u-PA (c)DNA, and also the correspond ⁇ ing amino acid sequence, are shown in the accompanying Figure 1.
  • the mutant proteins to be discussed in the following paragraphs were called t-PA::u-PA-I and t-
  • PA::u-PA-II and are encoded by, respectively, pSV2/t- PA::u-PA-I DNA and pSV2/tPA: :u-PA-II DNA. It is finally noted that, as a result of the constructions described, the plasmids last mentioned contain only one intron in the u-PA portion r originating from the plasmid pHUK- 1.
  • An E. coli K12 strain DHl pSV2/t-PA: :u-PA-II was deposited at the Centraal Bureau voor Schimmelcultures (CBS) at Baarn, The Netherlands, on April 28 1987, and was accorded number CBS 293.87. From the plasmid thus rendered accessible, the shorter plasmid pSV2/tPA::u- PA-I can be constructed with facility, for example, via the outlooping technique.
  • mouse fibroblast cell line (mouse Ltk-) was cultured in so-called "Iscove's minimal medium", to which were added penicillin, streptomycin and 10% (v/v) foetal calf serum. Transfection of these cells was carried out as described (Lopata et al., 1984; Van Zonneveld et al., 1986b). After transfection the cells were incubated in the above medium, but without foetal calf serum. Five days after transfection, conditioned media were' harvested, whereafter Tween-80 and sodium azide were added to a final concentration of, respectively, 0.01%
  • the concentration of the secreted mutant proteins t-PA: :u-PA-I, t-PA: :u-PA-II, and also of control trans- fection experiments with pSV2/t-PA DNA coding for recom ⁇ binant t-PA (rt-PA) was determined by means of an immuno- radiometric method (IRMA).
  • IRMA immuno- radiometric method
  • the IRMA is based on the presence of kringle 1 (Kl) of t-PA in all proteins being investigated and the availability of two different mono ⁇ clonal anti-t-PA antibodies directed against Kl (Van Zonneveld et al., 1986c). These two antibody preparations have been called CLB-t-PA 72 and CLB-t-PA 16 (Van Zonnevel et al.
  • the monoclonal anti-t-PA antibody CLB-t-PA.72 was coupled to cyanogen bromide activated Sepharose and incubated in 10.5 mM sodium phosphate (pH 7.4), 150 mM sodium chloride (PBS), to which had been added 1% (w/v) bovine serum albumin, 0.1% (v/v) Tween-20 and various dilutions of the conditioned media of the transfec tissue culture cells, previously concentrated by means of Amicon centricon 30 filters by a factor of about 50.
  • PBS sodium chloride
  • a characteristic concentration assay gave the following results: rt-PA 16 ng/ml; t-PA::u-PA-I 0.6 ng/ml and t-PA: :u-PA-II 0.4 ng/ml, being the concentrations in the unconcentrated conditioned media for transfected cells treated under comparable conditions.
  • the lower expression level of the mutant plasminoge activator proteins, as compared with rt-PA, is possibly to be attributed to the presence of an intron in the u-PA portion of the t-PA substitution mutant. If splicing of this intron is a limitative step, this could result in a lower expression level for the mutant proteins as compared with the expression of rt-PA.
  • the total reaction volume was 250 /ul and the reactions were carried out in 96- well microtiter plates.
  • a Titertek Multiscan spectrophoto- meter equipped with a thermostat was used to monitor the development of the optical density at 405 nm for 5 to 6 hours with intervals of 10 minutes.
  • Initial reactio velocities were obtained by graphically plotting the relationship between the increase in optical density at 405 nm per minute against the incubation time.
  • the Glu-plasminogen concentration was varied from 1.7 to 27.5 nM for 0.08- 0.2 ng/ml of each of the mutant proteins, 0.32-0.64 ng/ml rt-PA and 0.1-0.2 ng/ml HMW u-PA.
  • the experiments were duplexed and repeated four times.
  • so-called "Lineweaver-Burk plots” were plotted, using the "weighed” linear regression analysis, whereafter we were able to determine the so-called Km and v(max) values.
  • k(cat) value could be calculated by using the molecular weights of the various plasminogen activators, namely, t-PA::u-PA-I 72 kD, t-PA: :u-PA- II 74 kD, rt-PA 70 kD and HMW uPA 54 kD.
  • t-PA t-PA::u-PA-I 72 kD
  • t-PA :u-PA- II kD
  • rt-PA 70 kD HMW uPA 54 kD.
  • the affinity for the substrate Glu-plasminogen markedly increases for all plasminogen activators.
  • the affinity increases by a factor of 122 to 220 (Km * from 0.61-0.88 ⁇ M to 0.005-0.004 ⁇ M)
  • the affinity increases by a factor of at least 3125 (Km from > 50 ⁇ M to 0.016 ⁇ M)
  • for HMW u-PA by a factor of 30 (Km from 2.2 ⁇ M) to 0 -073 ⁇ M) ' .
  • PAI-1 complex formation of the mutant plasminogen activators with the endothelial plasminogen activator inhibitor (PAI-1) and the inhibition of the mutant plasminogen activators by PAI-1
  • the endothelial plasminogen activator inhibitor (PAI-1) is regarded as the physiological inhibitor of the fibrinolytical process and therefore as an important regulator in vivo of the "nett fibrinolytic activity" (survey article: Sprengers and Kluft, 1987) .
  • PAI-1 is synthesized, among other method, by cultured vascular endothelial cells. PAI-1 complexes very fast both with u-PA and with t-PA (second-order reaction constant in excess of 10 7 M ⁇ l sec -1 (Sprengers and K Kunststoff, 1987).
  • the complex of the plasminogen activators with PAI-1 is resistent to sodium dodecyl sulphate (SDS), so that the formation of a complex can be analyzed by means of SDS-polyacrylamide gel electrophoresis techniques.
  • SDS sodium dodecyl sulphate
  • the activity of the plasminogen activators in complex with PAI-1 can only be demonstrated after treat ent with Triton X-100 (Loskutoff et al., 1983), possibly as a result of intrinsic plasminogen activator activity of the complex or as a result of dissociation of plasminogen activator from the complex.
  • PAI-1 which is synthesized by and secreted by cultured endothelial cells, is found as a mixture of inactive, latent and active inhibitor. The latent form can be activated by treatment with certain chemicals, such as SDS.
  • PAI-1 cDNA which harbours the complete genetic information for the human PAI-1 glycoprotein (Ny et al., 1986; Pannekoek et al., 1986; Ginsburg et al., 1986; Andreasen et al., 1986).
  • the amino acid sequence of PAI-1 could be derived af er the elucidation of the complete nucleotide sequence.
  • the amino acid sequence of PAI-1 shows significant homology with proteins belonging to the "family" of serine protease inhibitors (so-called serpins).
  • a buffer consisting of 2% (w/v) sucrose, 5/ ⁇ g/ml bromo- phenol blue and 2.5% (w/v) SDS, and the samples were analyzed by means of SDS-polyacryl amide gel electrophoresi After electrophoresis, a fibrin-agarose indicator gel was prepared which also contained plasminogen (Granelli- Piperno and Reich, 1978) and used as an overlay for the SDS-polyacryl amide gel treated with Triton X-100.
  • plasminogen Gramnelli- Piperno and Reich, 1978
  • PAI-1 forms a complex with both t-PA: :u-PA-II and the control plasminogen activators t-PA and HMW u-PA. This conclusion can be drawn from the observation that the molecular weight of the plasminoge activator activity increases after pre-incubation with PAI-1.
  • the latent fraction of the PAI-1 preparation (total about 130 ng) was previousl activated with SDS (final concentration 0.1% (w/v)).
  • SDS final concentration 0.1% (w/v)
  • Various dilutions of the activated starting preparation were treated with an equal volume (55 ⁇ l) 0.2 M TRIS-HC1 (pH 7.4), 0.2% (v/v) Tween-80, 6% (v/v) Triton X-100 at room temperature for 15 minutes.
  • one of the plasminogen activators was added in a volume of 30 ⁇ l, which corresponded to 0.024 ng t-PA: :u-PA-II, 0.024 ng rt-PA or 0.024 ng HMW u-PA.
  • the mixtures were incubated at room temperature for 30 minutes.
  • the remaining plasminogen activator activity of the various activators was finally determined as follows.
  • t-PA :u-PA-II 4.1 x 10 _12 M rt-PA 0.6 x 10 "12 M HMW u-PA 0.24 x 10 ⁇ 12 M
  • Determination of plasminogen activator activity in this determination, use is made of the chromo- genic substrate S-2251, which is specific for plasmin.
  • the activation of Gl ⁇ -plasminogen by rt-PA, ' Bowes melanoma t-PA and other t-PA derivatives is greatly accelerated by the addition of fission products of fibrinogen treated with cyanogen bromidei. This last component is called
  • the PAI-1 preparation used is either conditioned (serum-free) medium of cultured human vascular endothelial cells (concentration PAI-1: 2.4 ⁇ g/ml) or a purified preparation PAI-1 (100 ⁇ g/,l; Lambers et al., 1987). The two preparations gave corresponding results.
  • HMW u-PA two-chained was a gift of Dr. G.Cassani
  • the Km values are given in iM.
  • the k(cat) values are given in sec ⁇ l, so that the quotient k(cat)/Km has the dimension ⁇ M ⁇ l.sec ⁇ l.
  • Fig. 1 shows the nucleotide sequence of the cDNA area coding for the fusion portion of, respectively, t-PA::u-PA-I (top) and t-PA: :u-PA-II (bottom). Also shown are the corresponding amino acids of the t-PA portions and the u-PA portions.
  • the numbering above the nucleotide sequences corresponds to that of the t-PA nucleotide sequence (955 etc.) and the u-PA nucleotide sequence (679 etc., 640 etc.).
  • the numbering under the amino acid sequences corresponds to that of the t-PA amino acid sequence (256 etc.) and the u-PA amino acid sequence 147 etc., 134 etc.).
  • the underligned nucleotide sequence indicates the synthetic primers used for the construction of the two mutants.

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  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Protéines recombinantes formés à partir d'une partie d'activation (t-PA) de plasminogène humain de type tissulaire et à partir d'une partie d'urokinase humaine (u-PA). La partie de t-PA comprend la chaîne H ou une chaîne H modifiée de t-PA, et la partie u-PA comprend la chaîne B de u-PA. La chaîne L de t-PA et la chaîne A de u-PA sont virtuellement absentes. L'invention se rapporte également à des informations génétiques recombinantes codant pour ces protéines recombinantes, à un procédé de préparation des protéines recombinantes, et à des compositions pharmaceutiques une protéine recombinante d'après l'invention.
EP88903965A 1987-04-29 1988-04-28 PROTEINES HUMAINES A MUTANTS DE SUBSTITUTION t-PA(u-PA), ADN RECOMBINANT PREVU A CET EFFET, CELLULES HOTES TRANSFECTEES PAR TRANSMISSION, PREPARATION DES PROTEINES A MUTANTS ET COMPOSITIONS PHARMACEUTIQUES Withdrawn EP0366661A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL8701021A NL8701021A (nl) 1987-04-29 1987-04-29 Humane t-pa(u-pa) substitutie-mutant eiwitten, daarvoor coderend recombinant dna, getransfecteerde gastheercellen, bereiding van de mutant eiwitten, en farmaceutische preparaten.
NL8701021 1987-04-29

Publications (1)

Publication Number Publication Date
EP0366661A1 true EP0366661A1 (fr) 1990-05-09

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EP88903965A Withdrawn EP0366661A1 (fr) 1987-04-29 1988-04-28 PROTEINES HUMAINES A MUTANTS DE SUBSTITUTION t-PA(u-PA), ADN RECOMBINANT PREVU A CET EFFET, CELLULES HOTES TRANSFECTEES PAR TRANSMISSION, PREPARATION DES PROTEINES A MUTANTS ET COMPOSITIONS PHARMACEUTIQUES

Country Status (4)

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EP (1) EP0366661A1 (fr)
JP (1) JPH02504462A (fr)
NL (1) NL8701021A (fr)
WO (1) WO1988008451A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE462829B (sv) * 1989-01-10 1990-09-10 Kabigen Ab Trombolytiskt aktiva kompositioner innehaallande en modifierad plasminogenaktivator av vaevnadstyp samt en normal human t-pa, streptokinas eller humant urokinas
US5910481A (en) * 1995-11-13 1999-06-08 Immuno Ag Hybrid proteins with modified activity
JP7604376B2 (ja) 2018-12-28 2024-12-23 バーテックス ファーマシューティカルズ インコーポレイテッド 改変ウロキナーゼ型プラスミノゲンアクティベータポリペプチドおよび使用方法

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Publication number Priority date Publication date Assignee Title
GB8334498D0 (en) * 1983-12-24 1984-02-01 Beecham Group Plc Compounds
DK149786A (da) * 1985-04-04 1986-10-04 Beecham Group Plc Proteolytisk enzym
DE3781420T2 (de) * 1986-01-31 1993-02-25 Central Glass Co Ltd Hybrides plasminogenaktivatoraehnliches polypeptid.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8808451A1 *

Also Published As

Publication number Publication date
NL8701021A (nl) 1988-11-16
WO1988008451A1 (fr) 1988-11-03
JPH02504462A (ja) 1990-12-20

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