Mutants of Staphylokinase Carrying Amino and Carboxy-Terminal Extensions for Polyethylene Glycol Conjugation

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.

1 . A Staphylokinase variant, wherein at least one cysteine residue is added to the amino-terminal region of SEQ ID NO:1, to the carboxy-terminal region of SEQ ID NO:1 or to the amino-terminal region and the carboxy-terminal region of SEQ ID NO:1. 
     
     
         2 . The Staphylokinase variant of  claim 1 , wherein the at the least one cysteine residue is added to the amino-terminal residue of SEQ ID NO:1. 
     
     
         3 . The Staphylokinase variant of  claim 1 , wherein the at least one cysteine residue is added to the carboxy-terminal residue of SEQ ID NO:1. 
     
     
         4 . A Staphylokinase variant of  claim 1 , comprising the sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. 
     
     
         5 . The Staphylokinase variant of  claim 1 , wherein the at least one cysteine residue is modified with a cysteine-reactive moiety. 
     
     
         6 . The Staphylokinase variant of  claim 1 , wherein the at least one cysteine residue is conjugated to a polyethylene glycol (PEG) molecule. 
     
     
         7 . The variant of  claim 6 , wherein the PEG molecule is linear or branched. 
     
     
         8 . The variant of  claim 7 , wherein the molecular size of the PEG molecule ranges from 5-20 Kilodaltons. 
     
     
         9 . The variant of  claim 6 , wherein the variant has proteolytic stability which is greater than the proteolytic stability of the polypeptide identified as SEQ ID NO:1. 
     
     
         10 . The variant of  claim 6 , wherein the variant has in vivo immunogenicity which is less that the in vivo immunogenicity of the polypeptide identified as SEQ ID NO:1. 
     
     
         11 . The variant of  claim 6 , 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. 
     
     
         12 . The 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. 
     
     
         13 . A recombinant  E. coli  host cell comprising a vector, wherein the vector comprises a DNA sequence encoding a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6. 
     
     
         14 . The host cell of  claim 13 , wherein the host cell is deposited in the International Depository Authority (IDA), and wherein the host cell has the deposit number MTCC 5437, MTCC 5438, MTCC 5439, or MTCC 5440. 
     
     
         15 . A pharmaceutical composition comprising the Staphylokinase variant of  claim 6 , further comprising a pharmaceutically acceptable carrier. 
     
     
         16 . A method for treating a cardiovascular disorder selected from the group consisting of myocardial infarction, vascular thromboses, pulmonary embolism, stroke a vascular event, disease or disorder selected from a group consisting of myocardial infarction, angina, pulmonary embolism, transient ischemic attack, deep vein thrombosis, thrombotic re-occlusion subsequent to a coronary intervention procedure, heart surgery or vascular surgery, peripheral vascular thrombosis, Syndrome X, heart failure, and a disorder in which a narrowing of at least one coronary artery occurs, comprising administering the pharmaceutical composition of  claim 15 .