WO1990003436A1 - POLYPEPTIDES ANALOGUES AU tPA, LEUR FABRICATION ET LEUR UTILISATION - Google Patents

POLYPEPTIDES ANALOGUES AU tPA, LEUR FABRICATION ET LEUR UTILISATION Download PDF

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Publication number
WO1990003436A1
WO1990003436A1 PCT/EP1989/001040 EP8901040W WO9003436A1 WO 1990003436 A1 WO1990003436 A1 WO 1990003436A1 EP 8901040 W EP8901040 W EP 8901040W WO 9003436 A1 WO9003436 A1 WO 9003436A1
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Prior art keywords
tpa
polypeptides
dna
sequences
cells
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PCT/EP1989/001040
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German (de)
English (en)
Inventor
Alfred Bach
Martin Schmidt
Karl-Hermann Strube
Verena Baldinger
Margarete Schwarz
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Basf Aktiengesellschaft
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Publication of WO1990003436A1 publication Critical patent/WO1990003436A1/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
    • C12N9/6459—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
    • 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)

Definitions

  • the present invention relates to new tPA-like polypeptides with plasma activator activity, processes for their preparation and their use in combating diseases.
  • the human, mature tissue plasmin activator is a polypeptide of 527 amino acids and a molecular weight of about 68 kD (Pennica et al. 1983, Nature 301, 214-221 and Ny et al. 1984, Proc. Natl. Acad. Sei USA 81, 5355-5359) (see Fig. La-lc).
  • the molecule contains several distinct regions, so-called domains, to which various functions are assigned.
  • the N-terminus is formed by a finger region that is homologous to the fibronectin. This is followed by a region that is similar to several growth factors. This is followed by two Kringel domains.
  • the protease domain follows a cleavage site at which the single-chain molecule can be cleaved into two chains; this contains the active center and has homology to other serine proteases.
  • tissue plasminogen activator has a strong affinity for fibrin, the plasminogen activation is dependent on fibrin; as a protease, tPA cleaves plasminogen to plasmin, which in turn breaks down fibrin into cleavage products. tPA can be inhibited by plasminogen activator inhibitors. Furthermore, tPA has a relatively short half-life; the molecule breaks down rapidly in the liver.
  • tPA specifically activates plasminogen to plasmin in blood clots and causes the vascular current to be restored by breaking down the fibrin.
  • tPA can therefore be used in fibrinolytic therapy, e.g. B. after a heart attack.
  • tPA-like polypeptides which have the amino acid sequence of tPA or sequences derived therefrom, but in which 1-6 tripeptides are replaced by the tripeptide RGD, have improved properties.
  • sequences derived from the tPA are to be understood as naturally occurring allele variants and synthetic variants which, in their primary structure, agree more than 90% with the natural tP ⁇ and have a plasminogen-activating effect.
  • the new polypeptides preferably contain one or more RGD
  • polypeptides which contain the tripeptide RGD in position 130-132 are particularly preferred. Also preferred are polypeptides which contain one to two additional RGD tripeptides in addition to the RGD sequence in position 130-132.
  • the invention further relates to DNA sequences for the mutated
  • Encode polypeptides as well as vectors containing these DNA sequences.
  • the new polypeptides can be produced genetically using known methods.
  • the starting point for the constructions described here is a cDNA clone, hereinafter called pUCtpa, which contains the coding sequence of tPA and 5'- and 3'- untranslated regions *. After a "leader” and pro sequence, the mature protein begins with Ser (1) and ends after Pro (527).
  • PUCtPA is isolated as follows: RNA is isolated from human uterine tissue and transcribed into double-stranded cDNA. After inserting this cDNA into the commercially available cloning vector pUC9, a cDNA library is created. The methods used can be found, for example, in Maniatis et al., Molecular Cloning, CSH-press. 5 “Screening” of such gene banks with radioactively labeled oligonucleotide probes has also become a widely used and described method. A cDNA clone containing the coding region and adjacent regions can be isolated by this method.
  • the genes or gene fragments can be provided with suitable chemically synthesized control regions or isolated from bacteria, phages, eukaryotic cells or their viruses, which control the of the proteins.
  • the transformation or transfection of the hybrid plasma obtained in this way into suitable host organisms is also known and has been described in detail.
  • the hybrid plasmids can also be provided with corresponding signal sequences which allow the polypeptides to be secreted into the medium.
  • vectors can be used which place the gene to be expressed, in this case the mutated tPA cDNA, under the control of the mouse metallothionein or the SV40 viral promoter.
  • Clones are then isolated which have copies of these vectors as episomes or integrated into the genome.
  • the integration and expression of the foreign gene based on the bovine papillo virus are particularly advantageous.
  • the construction of so-called “shuttle” vectors is possible.
  • the plasmid is first constructed and propagated in bacterial cells; the conversion into the eukaryotic cells then takes place, for example into the mouse fibroblast cell line C127.
  • yeast, insect cells and other mammalian cells such as CHO, L and 293 cells, can also be used for the expression.
  • the tissue plasma activator receives glycoside side chains at amino acids 117, 184 and 448 (Asn) when expressed in eukaryotic cells.
  • Bacteria are unable to synthesize glycoside side chains.
  • bacteria are often unable to split off the initiator amino acid methionine from the finished protein. These difficulties can be avoided by using secretion systems.
  • the new polypeptides are purified by separating them from the culture medium by affinity and ion exchange chromatography using known methods.
  • the polypeptides obtained according to the invention have improved properties in at least one of the points mentioned below: clot specificity, half-life, inhibitor binding, proteolytic activity and can therefore be used for thrombolysis. They show improved properties compared to human tissue plasminogen activator.
  • the invention therefore also relates to medicaments which contain at least one of the new polypeptides, optionally in a pharmaceutically acceptable carrier or binder.
  • the drugs can also combinations of the new proteins with other fibrinolytics, such as prourokinase, urokinase or streptokinase or their derivatives, as well as with other pharmaceutical proteins, e.g. B. Superoxide disase included. Further embodiments of the invention are described in more detail in the following examples.
  • RNA was sedimented by centrifugation through a 5.7 M CsCl cushion overnight at 45,000 rpm.
  • the polyA + -containing RNA fraction was then separated by affinity chromatography on oligo (dT) cellulose.
  • the polyA + RNA was rewritten into single-stranded cDNA.
  • the second strand was synthesized using E. coli DNA polymerase I.
  • Sall linkers were set up on the double-stranded cDNA with the aid of the enzyme T4-DNA ligase.
  • the commercially available plasmid pUC9 was linearized with the restriction enzyme Sall. Both DNAs were ligated together and transformed with the hybrid CaCl2-treated, competent cells of the E. coli strain HB101 thus obtained. The cells were plated on LB plates with 100 g / ml A picillin - and incubated overnight at 37 ° C.
  • oligonucleotide probes each comprising 17 bases of the tPA-DNA. They consist of the following sequences:
  • probes were labeled with j - 32p-ATP at the 5 'end. They were then incubated with the prehybridized filters in a solution containing 6 x SET, 0.1% SDS, 5 x Denhardt's and 10% dextran sulfate overnight at 42 ° C with gentle shaking. The filters are then washed several times in 6 x SET / 0.1% SDS at 42 ° C., dried and exposed to an X-ray film. Clones that gave a radioactive response during screening were isolated and grown up. A clone, hereinafter called pUCtPA, contained an approximately 2.1 kb insert which contained the coding region and 5 'and 3' non-coding regions (FIG. 2). Plasmid DNA from pUCtPA was prepared by lysozyme digestion and SDS-alkali treatment of the bacterial culture and subsequent CsCl gradient centrifugation.
  • the starting point was the plasmid pUCtPA described in Example 1. It was cut preparatively with the restriction enzyme Sall. The fragments obtained were separated by gel electrophoresis. The Sall fragment, which contained the DNA sequence encoding tPA, was eluted electrophoretically from the gel. 30 ng of this fragment were ligated at 4 ° C. with 100 ng of the Sall cut, commercially available cloning vector mpl9. The volume of the ligation mixture was 10 ⁇ l. The ligation was terminated by heating at 80 ° C. for ⁇ minutes.
  • the mPl9 plasmid which contained the cDNA of tPA in the orientation that the + strand is the tPA cDNA component of the phage genome when single-stranded M13 phages are formed, was designated as mptPA. 0
  • bacteria of the Sta ⁇ mies JM 101 or cells derived therefrom are transformed with the plasmid mptPA, single-stranded bacteriophages which contain the + strand of the tPA cDNA are released into the medium when these cells multiply.
  • the single-stranded DNA can then be isolated using 15 conventional and widely described methods.
  • oligonucleotide 21-27mer
  • the oligonucleotide was previously phosphorylated using the T polynucleotide kinase
  • the filters were washed at 6 ⁇ SET / 0.1% SDS at a temperature of 55-65 ° C. depending on the base composition and length of the oligonucleotide hybridization sample used. Hybridization was carried out in each case with the oligonucleotide which was used in the corresponding mutagenization batch. Positive clones were made autoradiographically visible. These substitutions had the effect that the tPA cDNA contained in mpl8 now codes for a polypeptide which contains the amino acid motif RGD and three further amino acid substitutions at one or more locations, the total number of amino acids of these polypeptides in each case being 527.
  • the mptPADNA described in Example 2 was generated using the oligonucleotides
  • This mutated mptPADNA was the starting point for the following examples 3.1 to 3.13.
  • the washing temperature was 60 ° C in 6xSET / 0.1% SDS.
  • the washing temperature was 62 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGD2. 10
  • the washing temperature was 60 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGD3. 20th
  • the washing temperature was 60 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGD4. 30
  • the washing temperature was 60 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGD6.
  • the washing temperature was 65 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGD7.
  • the washing temperature was 61 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGD10.
  • the washing temperature was 65 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGDll. 10
  • the washing temperature was 55 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGDl2. 10
  • the washing temperature was 60 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGDl3. 20th
  • the washing temperature was 55 ° C in 6xSET / 0.1% SDS.
  • the resulting positive clones were named mptPA-RGD14. 30
  • DNA of the monkey virus SV40 was cut with the restriction enzymes Ba HI and Bell and the 0.24 kb fragment was prepared by gel electrophoresis (Fig. 5). The ends were filled with the Klenow fragment of the DNA polymerasel in the presence of the four deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP. Xhol linkers were then ligated.
  • the commercially available vector pUCl ⁇ was linearized with the enzyme Smal 1. Then Xhol linkers were also used. DNA of this vector (“pUCl ⁇ Xho") was inerted with Xhol 1, treated with alkaline phosphatase and ligated with the 0.24 kb XhoI-SV40 fragment (see above). PSVpA was created. pSVpA DNA was preparatively cleaved with Xhol and incubated as * above with Klenow polymerase in the presence of the four dNTPs. The 0.24 kb fragment was gel isolated.
  • the eukaryotic expression vector CL28XhoBPV created by ligation of CL28X and pB2-2 (according to Reddy et al. 1987, DNA 6, 461-72), was partially cut with the restriction enzyme Xbal, ie it was incubated for a limited time so that molecules arose that were split at only one of the two Xbal recognition sequences, i.e. linearized (Fig. 6). The approach was then implemented with Klenow polymerase and dNTPs as described. The linear molecules were then isolated by gel electrophoresis.
  • the linear pCL28XhoBPV fragments were then ligated with the pretreated 0.24 kb fragment from SV40. After transformation and screening of minilysates, a clone was isolated which carried the SV40 fragment in the former Xbal site approximately 0.15 kB 3 'to the Xhol site; this DNA (“pCL28XhoBPV-SVpolyA") carried the SV40 transcription stop signals of the "early" genes.
  • Plasmid DNA from pCL28XhoBPV-SVpolyA was linearized with the restriction enzyme Xhol and treated with alkaline phosphatase.
  • mptPARGDl-15 was cleaved with the restriction enzyme Sall and the 2.1 kb fragment was isolated. Both fragments were linked together using T4 ligase. After transformation and minilysates, a clone was isolated which contained the mutated tPA DNA simply and in the correct orientation: pCL28BPV-tPA-RGDl-15.
  • 25 BPV genomes contain, to recognize as collections transformed cells, so-called foci. .
  • Foci expressing the tPA muteins described above were identified by a casein agar overlay (see below). After 2-3 h incubation at 37 ° C; 7% C0 2 were in the cloudy casein agar lysis yards on the
  • the cell lines were kept in serum-free DMEM after reaching confluence.
  • the tPA mutein from the serum-free cell culture supernatant obtained in this way can now be characterized and purified.
  • the cells are washed twice in 60 mm petri dishes with serum-free medium. Then 2 ml of "overlay agarose" are carefully pipetted into the petri dishes. The petri dishes are left to cool and solidify the agarose at room temperature. It is then incubated for 2-3 hours in an incubator at 37 ° C. with the addition of 7% CO 2 and the size and number of the lysis yards are determined.
  • the tPA mutein was isolated from the serum-free cell culture supernatant obtained according to Example 5 by affinity chromatography on Erythrina trypsin inhibitor Sepharose (ETI-Sepharose, 1 cm x 3 cm). To produce the affinity matrix, 5 mg of ETI per 1 ml of CNBr-activated Sepharose 4B were coupled. The gel material was equilibrated with 20 mM Na phosphate, 0.15 M NaCl, 0.01% ⁇ Tween 80, pH 7.0 before applying the cell supernatant. After application, the gel material was treated with the same buffer in order to remove non-specifically bound material.
  • ETI-Sepharose Erythrina trypsin inhibitor Sepharose
  • the specifically bound mutein was then desorbed by elution with 0.1 M glycine, 0.1 M arginine / HCl, 0.01% ⁇ Tween 80, pH 3.0.
  • the eluate was combined and adjusted to pH 5.0 with 0.1 M sodium hydroxide solution.
  • the following polypeptides were obtained, which differ as follows from the tPA shown in Fig. La-d:
  • the numerals denote the amino acid positions in which the polypeptide differs from the tPA shown in Fig. La-d, while the letter stands for the 20 amino acid, which is in the mutein at the position indicated by the digit.
  • the oligosaccharide residues contain ⁇ -linked galactose residues which, according to 40 Fig. 8, are linked to ⁇ -linked subterminal galactose residues.
  • the ⁇ -galactose residues are preferably localized on the galactose ( T_ (numbering of the sugar residues see Fig. 8).
  • the other subterminal galactose residues are either substituted by sialic acid or other ⁇ -linked galactose.

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Abstract

On décrit des polypeptides analogues au tPA, ainsi que leur fabrication. Les nouveaux polypeptides sont appropriés pour combattre des maladies.
PCT/EP1989/001040 1988-09-17 1989-09-07 POLYPEPTIDES ANALOGUES AU tPA, LEUR FABRICATION ET LEUR UTILISATION WO1990003436A1 (fr)

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DE3831714A DE3831714A1 (de) 1988-09-17 1988-09-17 Tpa-aehnliche polypeptide, ihre herstellung und verwendung
DEP3831714.1 1988-09-17

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WO1990003436A1 true WO1990003436A1 (fr) 1990-04-05

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JP (1) JPH04500306A (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007874A1 (fr) * 1990-10-24 1992-05-14 British Bio-Technology Limited Proteines pharmaceutiquement actives et comprenant une proteine active et une sequence d'affinite a l'integrine

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5612311A (en) 1990-04-06 1997-03-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
CN104479025B (zh) * 2014-08-08 2018-04-20 重庆医科大学 一种组织型纤溶酶原激活剂的构建、表达纯化及功能鉴定

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0093619A1 (fr) * 1982-05-05 1983-11-09 Genentech, Inc. Plasminogène-activateur de tissu humain, compositions pharmaceutiques le contenant, procédés pour sa préparation ainsi que ADN et intermédiaires cellulaires transformés pour la mise en oeuvre de ces procédés
WO1987004722A1 (fr) * 1986-01-31 1987-08-13 Genetics Institute, Inc. Nouvelles proteines thrombolytiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0093619A1 (fr) * 1982-05-05 1983-11-09 Genentech, Inc. Plasminogène-activateur de tissu humain, compositions pharmaceutiques le contenant, procédés pour sa préparation ainsi que ADN et intermédiaires cellulaires transformés pour la mise en oeuvre de ces procédés
WO1987004722A1 (fr) * 1986-01-31 1987-08-13 Genetics Institute, Inc. Nouvelles proteines thrombolytiques

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007874A1 (fr) * 1990-10-24 1992-05-14 British Bio-Technology Limited Proteines pharmaceutiquement actives et comprenant une proteine active et une sequence d'affinite a l'integrine

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DE3831714A1 (de) 1990-03-22
JPH04500306A (ja) 1992-01-23
EP0403598A1 (fr) 1990-12-27

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