Human uterine tissue plasminogen activator produced by recombinant DNA

• US 5,344,773 A
• Filed: 10/01/1985
• Issued: 09/06/1994
• Est. Priority Date: 10/01/1984
• Status: Expired due to Term

• Current Assignee: Genzyme Corporation (Sanofi )
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Dackowski, William, Wei, Cha-Mer, Reddy, Vermuri B., Lau, David Tai-Yui, Douglas, Richard, Purcell, Richard D. Jr., Cole, Edward S., Lemontt, Jeffrey F., Hsiung, Nancy
• Primary Examiner(s): Hill, Jr., Robert J.
• Assistant Examiner(s): Allen, Marianne Porta
• Application Number:
  US06/782,686
• Time in Patent Office: 3,262 Days
• Field of Search: 435/172.3, 435/320, 435/240.2, 435/253, 435/255, 435/212, 435/172.1, 435/226, 435/320.1, 435/, 935/27, 935/28, 935/29, 935/32, 935/14, 935/69, 935/70, 935/72, 935/50, 536/27, 536/23.5, 536/23.2
• US Class Current: 435/226
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2795/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/702 yeast
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 9/6459 t-plasminogen activator (3.4.21.68), i.e. tPA
• CPC Class Codes: C12Y 304/21069 Protein C activated (3.4.21.69)

Abstract

A replicable expression vector capable, in a transformed host mammallian cell, of expressing a DNA sequence encoding active human TPA which, when expressed in the host cell, yields active TPA which lacks one or more of the TPA-bound N-linked carbohydrate moieties of naturally occurring human TPA.

Claims

1. A recombinant DNA molecule which encodes human tissue plasminogen activator (TPA) and which differs from a naturally occurring DNA molecule encoding naturally occurring human TPA in that, in place of at least one of the Asn codons in the three DNA sequences which encode an Asn-X-(Ser or Thr) signal for N-linked glycosylation in said naturally occurring human TPA corresponding to amino acids 114-116, 181-184, and 445-447 of FIG. 1A-C hereof, there is substituted a Gln codon.

2. A mammalian cell transformed with the recombinant DNA molecule of claim 1.

3. The recombinant DNA molecule of claim 1 wherein one said substituted Gln codon replaces a naturally occurring Asn codon encoding an amino acid in the glycosylation signal corresponding to amino acids 114-116 of FIG. 1A-C hereof.

4. The recombinant DNA molecule of claim 1 or claim 3 wherein one said substituted Gln codon replaces a naturally occurring Asn codon encoding an amino acid in the glycosylation signal corresponding to amino acids 181-184 of FIG. 1A-C hereof.

5. The recombinant DNA molecule of claim 4 wherein one said substituted Gln codon replaces a naturally occurring Asn codon encoding an amino acid in the glycosylation signal corresponding to amino acids 445-447 of FIG. 1A-C hereof.

6. The recombinant DNA molecule of claim 1 or claim 3 wherein one said substituted Gln codon replaces a naturally occurring Asn codon encoding an amino acid in the glycosylation signal corresponding to amino acids 445-447 of FIG. 1A-C hereof.

7. The recombinant DNA molecule of claim 1 wherein there is substituted a Gln codon for the naturally occurring Asn codon in two of said three DNA sequences.

8. A method of making active human TPA which lacks one or more of the N-linked carbohydrate moieties of naturally occurring human TPA, said method comprising transforming a mammalian cell with the recombinant DNA molecule of claim 1,

culturing said mammalian cell in culture medium under conditions in which said recombinant DNA molecule is expressed to yield said TPA, and

isolating said TPA from said cell or said culture medium.

9. The recombinant DNA molecule of claim 1 wherein there is substituted a Gln codon for the naturally occurring Asn codon in one or two of said three DNA sequences.

10. The recombinant DNA molecule of claim 1 wherein there is substituted a Gln codon for the naturally occurring Asn codon in one of said three DNA sequences.

11. The recombinant DNA molecule of claim 1 wherein there is substituted a Gln codon for the naturally occurring Asn codon in three of said three DNA sequences.

12. Modified tissue plasminogen activators wherein the modification consists of amino acid substitutions for at least one residue in one, or more than one, of the three Asn-X-(Ser/Thr) glycosylation signals corresponding to amino acids 114-116 181-184, and 445-447 of FIG. 1A-C hereof.

13. Modified human tissue plasminogen activator (TPA) which differs from naturally occurring human TPA in that, in at least one of the three Asn-X-(Ser or Thr) signals for N-linked glycosylation in said naturally occurring human TPA corresponding to amino acids 114-116, 181-184, and 445-447 of FIG. 1A-C hereof, there is substituted a Gln for the Asn.

14. The modified human TPA of claim 13 wherein there is substituted a Gln for the Asn in one or two of said three glycosylation signals.

15. The modified human TPA of claim 13 wherein one said substituted Gln replaces a naturally occurring Asn in the glycosylation signal corresponding to amino acids 114-116 of FIG. 1A-C herein.

16. The modified human TPA of claim 13 or claim 15 wherein one said substituted Gln replaces a naturally occurring Asn in the glycosylation signal corresponding to amino acids 181-184 of FIG. 1A-C hereof.

17. The modified human TPA of claim 16 wherein one said substituted Gln replaces a naturally occurring Asn in the glycosylation signal corresponding to amino acids 445-447 of FIG. 1A-C hereof.

18. The modified human TPA of claim 13 or claim 15 wherein one said substituted Gln replaces a naturally occurring Asn in the glycosylation signal corresponding to amino acids 445-447 of FIG. 1A-C hereof.

19. The modified human TPA of claim 13 wherein there is substituted a Gln for the Asn in three of said three glycosylation signals.

20. The modified human TPA of claim 13 wherein there is substituted a Gln for the Asn in one of said three glycosylation signals.

21. The modified human TPA of claim 13, wherein there is substituted a Gln for the Asn in two of said three glycosylation signals.