# US 5,700,677 A · Protein analogues of tissue plasminogen activator

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- Number: US 5,700,677 A
- Document ID: US5700677A
- Status: Granted
- Kind: A
- Application number: 07/799,769
- Filed: 1991-11-27
- Granted: 1997-12-23
- Earliest claimed: 1986-03-28
- Assignees: Creative BioMolecules, Inc.; A. Menarini S.A.S.
- Inventors: Roberto Crea; Roy Hoi Loi Pang; Hermann Oppermann; Peter C. Keck; Gabriel Alvarado-Urbina; Gay-May Wu; Charles M. Cohen
- Web page: https://patentgrep.com/patent/US5700677A

## CPC

- C07K 14/31: from Staphylococcus (G)
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- C12N 9/6459: {t-plasminogen activator (3.4.21.68), i.e. tPA}
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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

Protein analogues of tissue plasminogen activator (tPA) are described. The analogues exhibit the proteolytic function of natural tPA and optionally fibrin binding activity, but are molecules, generally of lower molecular weight than natural tPA, designed for efficient expression in prokaryotic host cell systems. The analogues can comprise a catalytic fragment of tPA or a catalytic fragment of tPA linked to a polypeptide which stabilizes the catalytic fragment, provides for efficient expression of the fragment or confers a fibrin binding capability. Fibrin binding polypeptides can be a polypeptide fragments derived from tPA which embody the fibrin binding domain(s) of natural tPA or they can be an exogenous (non-tPA) polypeptides of eukaryotic or prokaryotic origin which exhibit fibrin binding affinity such as the antigen binding fragment of an antifibrin immunoglobulin or the B domain of protein A. Genetic constructs for expression of the analogues are also provided.

## Claims

### Claim 1

An analogue of tissue plasminogen activator (tPA) of the formula:
H.sub.2 N--X--L--CF.sub.tPA --COOH
wherein CF.sub.tPA represents a catalytic fragment of tPA;
X represents a fibrin binding domain of protein A present in single or multiple units; and
L represents a peptide bond linkage between X and CF.sub.tPA or an oligopeptide linking X and CF.sub.tPA, wherein said analogue possesses fibrinolytic and fibrin-binding activities.

### Claim 2 (depends on 1)

A tPA analogue of claim 1, wherein X is the B domain of protein A.

### Claim 3

An analogue of tissue plasminogen activator (tPA) of the formula:
H.sub.2 N--(F.sub.BProA).sub.n --L--CF.sub.tPA --COOH
Wherein CF.sub.tPA represents a catalytic fragment of tPA;
F.sub.B ProA represents the B domain of protein A;
L represents a peptide bond or an oligopeptide linker linking F.sub.BProA and CF.sub.tPA ; and
n is an integer from 1 to 5, wherein said analogue possesses fibrinolytic and fibrin-binding activities.

### Claim 4 (depends on 3)

An analogue of claim 3, wherein CF.sub.tPA is the catalytic fragment spanning amino acid residues 262-527 of tPA as shown in FIG. 1 or a fragment having substantially equivalent thrombolytic activity.

### Claim 5 (depends on 3)

A tPA analogue of claim 3, having the formula:
H.sub.2 N --F.sub.B ProA --CF.sub.tpa --COOH.

### Claim 6 (depends on 3)

A tPA analogue of claim 3, having the formula:
H.sub.2 N--F.sub.B ProA --F.sub.B ProA CF.sub.tPA --COOH.

### Claim 7

An analogue of tissue plasminogen activator (tPA) comprising a domain of protein A that binds fibrin linked through its carboxy terminus to the amino terminus of a catalytic fragment of tPA, wherein said analogue possesses fibrinolytic and fibrin-binding activities.

### Claim 8 (depends on 7)

An analogue of tissue plasminogen activator of claim 7, wherein the domain of protein A that binds fibrin is the B domain of protein A or multiple units thereof.

### Claim 9

An analogue of tissue plasminogen activator (tPA) of the formula:
H.sub.2 N--X.sub.n --L--CF.sub.tpA --COOH
wherein CF.sub.tPA is the catalytic fragment spanning amino acid residues 262-527 of tPA as depicted in FIG. 1
X represents a fibrin binding domain of protein A;
L represents a peptide bond or an oligopeptide linker separating X and CF.sub.tPA ; and
n is an integer from 1 to 5.

### Claim 10

An analogue of tissue plasminogen activator (tPA), comprising a catalytic fragment of human tPA linked at its amino terminus directly to the B domain of protein A or multiple units thereof.

### Claim 11 (depends on 10)

An analogue of claim 10, wherein the catalytic fragment of human tPA comprises amino acid residues 262-527 of tPA as shown in FIG. 1.

### Claim 12

An analogue of tissue plasminogen activator (tPA), comprising a catalytic fragment of human tPA spanning amino acid residues 262-527 of tPA as shown in FIG. 1 linked at its amino terminus directly to a B domain of protein A.