# US 9,518,255 B2 · Mutants of staphylokinase carrying amino and carboxy-terminal extensions for polyethylene glycol conjugation

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- Number: US 9,518,255 B2
- Document ID: US9518255B2
- Status: Granted
- Kind: B2
- Application number: 14/206,984
- Filed: 2014-03-12
- Published: 2015-04-30
- Granted: 2016-12-13
- Earliest claimed: 2008-12-05
- Assignees: Council of Scientific and Industrial Research
- Applicants: Council of Scientific & Industrial Research
- Inventors: Satish Singh; Kanak Lata Dikshit
- Web page: https://patentgrep.com/patent/US9518255B2

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- A61K 38/4886: {Metalloendopeptidases (3.4.24), e.g. collagenase}
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61P 9/04: Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C12N 9/48: acting on peptide bonds (3.4)
- 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)
- C12Y 304/24029: Aureolysin (3.4.24.29)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES

## Abstract

The present invention relates to the development of new derivatives of a bacterial plasminogen activator, Staphylokinase (SAK), having one or more amino acid residues with single or multiple cysteines at the amino and/or carboxy terminal ends and their conjugation with PEG (Polyethylene Glycol), resulting in new Staphylokinase derivatives that display altered oligomeric states, enhanced thermal and protease stability and extended plasma half-life. Also included is the cloning and expression in a suitable bacterial host; purification of Staphylokinase derivatives to homogeneity and their chemical modification by integrating a PEG molecule to create new biologically active Staphylokinases having higher protein stability and improved in vivo plasma half life, that may enhance the clinical potential of Staphylokinase in thrombolytic therapy for the treatment of cardiovascular diseases.

## Claims

### Claim 1

A Staphylokinase variant comprising the amino acid sequence of SEQ ID NO: 5, wherein the C-terminal cysteine residue of SEQ ID NO: 5 is conjugated to a polyethylene glycol (PEG) molecule.

### Claim 2 (depends on 1)

The Staphylokinase variant of claim 1, wherein the conjugated PEG molecule is a branched PEG molecule.

### Claim 3 (depends on 2)

The Staphylokinase variant of claim 2, wherein the molecular size of the PEG molecule ranges from 5-20 Kilodaltons.

### Claim 4 (depends on 1)

The Staphylokinase variant of claim 1, wherein the variant has proteolytic stability which is greater than the proteolytic stability of the polypeptide identified as SEQ ID NO:1, wherein proteolytic stability of the Staphylokinase variant and of the polypeptide identified as SEQ ID NO:1 is measured in vitro at a pH of about 8 and at a temperature of about 37° C. in the presence of trypsin.

### Claim 5 (depends on 1)

The Staphylokinase variant of claim 1, wherein the variant has in vivo immunogenicity which is less that the in vivo immunogenicity of the polypeptide identified as SEQ ID NO:1.

### Claim 6 (depends on 1)

The Staphylokinase variant of claim 1, wherein the variant has an in vivo half life which is greater than the in vivo half life of the polypeptide identified as SEQ ID NO:1.

### Claim 7 (depends on 1)

The Staphylokinase variant of claim 1, wherein the variant has temperature stability which is greater than the temperature stability of the polypeptide identified as SEQ ID NO:1, wherein the temperature stability is determined at a temperature ranging from about 20° C. to about 80° C.

### Claim 8 (depends on 1)

The Staphylokinase variant of claim 1, wherein the conjugated PEG molecule is a linear PEG molecule.