Modified plasminogen activator inhibitor type-1 and methods based thereon

Abstract

The present invention is based upon the discovery that modified plasminogen activator inhibitor type-I (PAI-1) in which two or more amino acid residues that do not contain a sulfhydryl group have been replaced with amino acid residues that contain a sulfhydryl group and, therefore, forms intramolecular disulfide bonds, have increased in vivo half-life. Also disclosed are the modified PAI-1 proteins, derivatives and analogs thereof, specific antibodies, nucleic acid molecules and host cells. Methods for producing modified PAI-1, derivatives and analogs are also provided. The invention further relates to Therapeutics, pharmaceutical compositions and method of using the composition for treatment. The invention may be used to inhibit angiogenesis in a subject, thereby treating diseases or conditions associated with undesired angiogenesis and cell proliferation. Such conditions include psoriasis, chronic inflammation, tumor invasion and metastasis and conditions in which angiogenesis is pathogenic. The modified PAI-1 molecules of the present invention are useful for the treatment, prophylaxis, management and amelioration of cardiovascular diseases such as, but not limited to those that are related to hyperfibrinolysis, hemophilia, and vessel leakage syndrome.

1 - 23 . (canceled) 
     
     
         24 . A modified plasminogen activator inhibitor type-1 (PAI-1) molecule comprising the amino acid sequence that has at least 80% overall similarity to the amino acid sequence of SEQ ID NO: 2, in which one or more amino acid residues are each substituted by an amino acid residue that contains a sulfhydryl group, such that one or more disulfide bridges are formed at a position selected from the group consisting of positions 10-40, 70-120, 150-220 or 300-400 of SEQ ID NO:2, wherein said modified PAI-1 molecule has a half-life that is longer than the half-life of a corresponding wild-type PAI-1 molecule, and wherein said modified PAI-1 molecule inhibits urokinase plasminogen activator. 
     
     
         25 . The modified PAI-1 molecule of  claim 24  wherein said residue that contains a sulfhydryl group is cysteine or methionine. 
     
     
         26 . The modified PAI-1 molecule of  claim 24  that further comprises one or more amino acid substitutions that are not substitutions with a sulfhydryl-containing residue. 
     
     
         27 . A method of treating a disease or disorder in a subject, said method comprising administering the modified PAI-1 molecule of  claim 24  to the subject. 
     
     
         28 . The method of  claim 27  wherein said disease or disorder is a cardiovascular disease, hyperfibrinolysis, hemophilia, or vessel leakage syndrome. 
     
     
         29 . A method of treating a disease or disorder that is mediated by uPA in a subject, said method comprising administering the modified PAI-1 molecule of  claim 24  to the subject. 
     
     
         30 . A method of treating a disease or disorder that is mediated by tPA in a subject, said method comprising administering the modified PAI-1 molecule of  claim 24  to the subject. 
     
     
         31 . The modified PAI-1 molecule of  claim 24 , comprising an A3 strand and an A5 strand, each comprising a top and a bottom part, and said one or more disulfide bridges link said top part of A3 strand, said top part of A5 strand, said bottom part of A3 strand, said bottom part of A5 strand, and/or said helix D region. 
     
     
         32 . The modified PAI-1 molecule of  claim 24  wherein said molecule comprises an amino acid sequence which has at least 95% overall similarity to SEQ ID NO: 2. 
     
     
         33 . The modified PAI-1 molecule of  claim 24  wherein said group consists of positions 31, 97, 192, 197, 347 and 355. 
     
     
         34 . The modified PAI-1 molecule of  claim 24  wherein said group consists of positions 29-32, 92-107, 180-197, 246-249, 341-353, 353-374 and 381-391.