# US 5,344,773 A · Human uterine tissue plasminogen activator produced by recombinant DNA

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- Number: US 5,344,773 A
- Document ID: US5344773A
- Status: Granted
- Kind: A
- Application number: 06/782,686
- Filed: 1985-10-01
- Granted: 1994-09-06
- Earliest claimed: 1985-10-01
- Assignees: Genzyme Corporation
- Inventors: Cha-Mer Wei; Nancy Hsiung; Vermuri B. Reddy; Jeffrey F. Lemontt; William Dackowski; Richard Douglas; Edward S. Cole; Richard D. Purcell, Jr.; David Tai-Yui Lau
- Web page: https://patentgrep.com/patent/US5344773A

## CPC

- C12N 9/6459: {t-plasminogen activator (3.4.21.68), i.e. tPA}
- C12N 15/81: for yeasts
- C12N 15/85: for animal cells
- C12Y 304/21069: Protein C activated (3.4.21.69)
- 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)
- C12N 2795/14143: viral genome or elements thereof as genetic vector
- C12N 2830/00: Vector systems having a special element relevant for transcription
- C12N 2830/002: inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- C12N 2830/702: yeast
- C12N 2830/85: mammalian
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1, 3)

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.

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 1, 3)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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.

### Claim 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.

### Claim 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.

### Claim 14 (depends on 13)

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.

### Claim 15 (depends on 13)

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.

### Claim 16 (depends on 13, 15)

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.

### Claim 17 (depends on 16)

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.

### Claim 18 (depends on 13, 15)

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.

### Claim 19 (depends on 13)

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

### Claim 20 (depends on 13)

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

### Claim 21 (depends on 13)

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