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EP0273774A2
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EP
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Plasminogen activators, DNA encoding the same and their preparation and use
Assignee: Novartis Vaccines and Diagnostics Inc
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Filing: 1987-12-30
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Granted: —
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Expires: 2007-12-30
Abstract
Abstract Novel plasminogen activators are disclosed. Of the novel plasminogen activators, one is a modified urokinase that is altered to make it less susceptible to forming two chain-urokinase and to increase the fibrin specificity of urokinase. Also among the novel plasminogen activators are fibrinolytic proteins comprised of at least one domain capable of interacting with fibrin and the protease domain of urokinase. The domain capable of interacting with fibrin may have an amino acid sequences substantially the same as those of kringle-2 of t-PA, kringle-1 and/or -4 of plasminogen and the finger domain of t-PA. The protease domain may have an amino acid sequence substantially the same as that of 30 Kd urokinase. DNA sequences encoding the novel plasminogen activators, expression vectors and expression systems therefor are also disclosed and claimed.
Inventors
- 👤 James Joseph Devlin
- 👤 Patricia Egan Devlin
- 👤 David Fu-Chi Mark
- 👤 Robin Clark
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-27 21:06
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6424; C12N9/6456
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
62 words
A protein comprising urokinase modified at at least one amino acid wherein said modified urokinase is less susceptible than unmodified urokinase to cleavage in the region corresponding to lysine 158 in urokinase and a second modification of at least one amino acid wherein said second modification produces a modified urokinase with increased activity compared to a molecule having only said first modification.
Claim 2
Dependent Claim
62 words
A protein comprising urokinase modified at at least one amino acid wherein said modified urokinase is less susceptible than unmodified urokinase to cleavage in the region corresponding to lysine 158 in urokinase and a second modification of at least one amino acid wherein said second modification produces a modified urokinase with increased activity compared to a molecule having only said first modification.
Claim 3
Dependent Claim
28 words
The protein of claim 1 wherein at least one neutral or negatively charged amino acid is substituted for the amino acid residue corresponding to lysine 158 in urokinase.
Claim 4
Dependent Claim
32 words
The protein of claim 1 or claim 2 wherein at least one neutral or positively charged amino acid is substituted for the amino acid residue corresponding to aspartic acid 355 in urokinase.
Claim 5
Dependent Claim
37 words
A recombinant plasminogen activator protein comprising a domain capable of interacting with fibrin and a protease domain wherein said protease domain comprises an amino acid sequence that is substantially the same as the protease domain of urokinase.
Claim 6
Dependent Claim
37 words
A recombinant plasminogen activator protein comprising a domain capable of interacting with fibrin and a protease domain wherein said protease domain comprises an amino acid sequence that is substantially the same as the protease domain of urokinase.
Claim 7
Dependent Claim
107 words
A recombinant plasminogen activator protein comprising at least one domain capable of interacting with fibrin and a protease domain wherein said protease domain comprises an amino acid sequence that is substantially the same as the protease domain of the modified urokinase of any one of claims 1 to 3, for example wherein the lysine corresponding to lysine 158 of urokinase is altered whereby said protease domain is resistant to cleavage at said lysyl residue and optionally wherein the aspartic acid corresponding to aspartic acid 355 of urokinase is altered whereby the altered protein has an increased activity compared to a protein having only said lysine 158 modification.
Claim 8
Dependent Claim
30 words
The protein of claim 4 wherein the domain capable of interacting with fibrin comprises an amino acid sequence selected from the group consisting of lysine-binding kringle domains and finger domains.
Claim 9
Dependent Claim
21 words
The protein of claim 6 wherein said finger domain is substantially the same as the finger region of tissue plasminogen activator.
Claim 10
Dependent Claim
30 words
The protein of claim 6 wherein said lysine-binding kringle domain is substantially the same as one selected from the group consisting of kringle-2 of t-PA, kringle-1 and kringle-4 of plasminogen.
Claim 11
Dependent Claim
29 words
The protein of claim 8 which further comprises a finger domain comprising an amino acid sequence that is substantially the same as the finger region of tissue plasminogen activator.
Claim 12
Dependent Claim
18 words
A DNA sequence encoding an amino acid sequence comprising the protein of any of claims 1 to 9.
Claim 13
Dependent Claim
30 words
A DNA sequence encoding a protein of any one of claims 4 to 9 in operable linkage with control sequences for the expression of said protein in a host cell.
Claim 14
Dependent Claim
26 words
A DNA sequence according to claim 11 wherein said control sequences are selected from those operable in prokaryotic cells, plant cells, mammalian cells, and insect cells.
Claim 15
Dependent Claim
23 words
An expression system comprising a prokaryotic cell or a mammalian cell or an insect cell transformed with the DNA sequence of claim 11.
Claim 16
Dependent Claim
33 words
A DNA sequence encoding a heterologous secretory signal peptide in proper translational reading frame with the DNA sequence of claim 11, wherein said heterologous secretory signal peptide is encoded by the DNA sequence.
Claim 17
Independent Claim
80 words
A method for producing a polypeptide with plasminogen activator activity comprising cotransfecting a susceptible host insect cell with a recombinant baculovirus transfer vector and baculovirus DNA wherein said transfer vector comprises at least one DNA sequence encoding the protein of any one of claims 1 to 9 under the transcriptional control of a vaculovirus promoter, isolating a recombinant baculovirus expression vector, infecting insect cells under suitable conditions with said isolated expression vector, and recovering said polypeptide from the culture medium.
Claim 18
Independent Claim
81 words
A method of facilitating the secretion of a protein, which method consists of designing a secretory signal peptide comprising amino acid residues within the region encompassing positions -13 through +2 of said protein, wherein one or more of the residues are selected from among the four most frequently used residues at each selected postion as determined from a database of known secretory peptides, optionally the selection of said amino acid residues being fruther adjusted for the expected frequency of each residue.
Claim 19
Independent Claim
81 words
A method of facilitating the secretion of a protein, which method consists of designing a secretory signal peptide comprising amino acid residues within the region encompassing positions -13 through +2 of said protein, wherein one or more of the residues are selected from among the four most frequently used residues at each selected postion as determined from a database of known secretory peptides, optionally the selection of said amino acid residues being fruther adjusted for the expected frequency of each residue.
Claim 20
Dependent Claim
19 words
The use of a protein of any one of claims 1 to 9 in the manufacture of a medicament.
References Cited (Backward Citations)
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