Platelet glycoprotein ib alpha variant fusion polypeptides and methods of use thereof

Abstract

The present invention provides compositions and methods for treating or preventing vascular-associated disorders.

1 . A protein comprising an amino acid sequence with one to 20 amino acid substitutions, deletions or insertions relative to amino acids 1-290 of a human GPlbα protein sequence (SEQ ID NO:1), wherein said polypeptide has at least one activity selected from the group consisting of:
 lower affinity binding to alpha thrombin relative to binding to alpha thrombin of a polypeptide comprising the amino acid sequence of SEQ ID NO:2; 
 lower aggregation relative to aggregation of a polypeptide comprising the amino acid sequence of SEQ ID NO:2; and 
 increased resistance to proteolysis than a polypeptide comprising the amino acid sequence of SEQ ID NO:2. 
 
     
     
         2 . The polypeptide of  claim 1 , wherein said first polypeptide has no more than 15 substitutions, insertions, or deletions relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         3 . The polypeptide of  claim 1 , wherein said first polypeptide has no more than 12 substitutions, insertions, or deletions relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         4 . The polypeptide of  claim 1 , wherein said polypeptide has no more than 10 substitutions, insertions, or deletions relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         5 . The polypeptide of  claim 1 , wherein said polypeptide has no more than 5 substitutions, insertions, or deletions relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         6 . The polypeptide of  claim 1 , wherein said polypeptide binds with lower affinity to alpha thrombin relative to binding to alpha thrombin of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         7 . The polypeptide of  claim 6 , wherein said polypeptide comprises at least one of the amino acid substations Y276F, Y278F, Y279V, or a conservative variant thereof, relative to the amino acid sequence of SEQ ID NO:2. 
     
     
         8 . The polypeptide of  claim 6 , wherein said polypeptide comprises at least two of the amino acid substations Y276F, Y278F, Y279V, or a conservative variant thereof, relative to the amino acid sequence of SEQ ID NO:2. 
     
     
         9 . The polypeptide of  claim 6 , wherein said polypeptide comprises the amino acid substitutions Y276F, Y278F, Y279V, or a conservative variant thereof, relative to the amino acid sequence of SEQ ID NO:2. 
     
     
         10 . The polypeptide of  claim 1 , wherein aggregation of said polypeptide is lowered relative to aggregation of a polypeptide comprising the amino acid sequence of SEQ ID NO:2. 
     
     
         11 . The polypeptide of  claim 10 , wherein said polypeptide comprises the amino acid substitution C65S, or a conservative variant thereof, relative to the amino acid sequence of SEQ ID NO:2. 
     
     
         12 . The polypeptide of  claim 1 , wherein said polypeptide is more resistant to proteolysis than a polypeptide comprising the amino acid sequence of SEQ ID NO:2. 
     
     
         13 . The polypeptide of  claim 12 , wherein said polypeptide comprises the amino acid substitution K237V, or a conservative variant thereof, relative to the amino acid sequence of SEQ ID NO:2. 
     
     
         14 . A polypeptide comprising an amino acid sequence with 1 to 10 amino acid substitutions, insertions, or deletions relative to amino acids 1-290 of SEQ ID NO:2, provided that said amino acid sequence includes 233V, 239V, and comprises the amino acid substitution Y276F. 
     
     
         15 . The polypeptide of  claim 14 , wherein said amino acid sequence comprises the amino acid substitution K237V. 
     
     
         16 . The polypeptide of  claim 14 , wherein said amino acid sequence further comprises the amino acid substitution C65S. 
     
     
         17 . The polypeptide of  claim 14 , wherein said amino acid sequence further comprises the amino acid substitution Y278F or Y279F. 
     
     
         18 . The polypeptide of  claim 1 , wherein said amino acid sequence further comprises the amino acid substations Y278F and Y279F. 
     
     
         19 . The polypeptide of  claim 18 , further comprising the amino acid substitution K237V. 
     
     
         20 . The polypeptide of  claim 19 , further comprising the amino acid substitution C65S. 
     
     
         21 . The polypeptide of  claim 14 , wherein said polypeptide binds with higher affinity to von Willebrand factor than a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         22 . A polypeptide comprising one or more substitutions in the amino acid sequence SEQ ID NO:2, wherein said one or more substitutions is
 Y276F;   Y276F K237V;   Y276F C65S;   Y276F Y278F Y279F;   Y276F Y278F Y279F K237V; or   Y276F Y278F Y279F K237V C65S.   
     
     
         23 . A fusion polypeptide comprising the polypeptide of  claim 1  linked to a second polypeptide. 
     
     
         24 . The fusion polypeptide of  claim 23 , wherein said second polypeptide comprises a region of a heavy chain immunoglobulin polypeptide. 
     
     
         25 . The fusion polypeptide of  claim 24 , wherein said second polypeptide comprises an Fc region of an immunoglobulin heavy chain. 
     
     
         26 . The fusion polypeptide of  claim 25 , wherein said second polypeptide has less effector function than the effector function of a Fc region of a wild-type immunoglobulin heavy chain. 
     
     
         27 . The fusion polypeptide of  claim 24 , wherein said second polypeptide binds with low or no affinity to a Fc receptor. 
     
     
         28 . The fusion polypeptide of  claim 24 , wherein said second polypeptide binds with low or no affinity to complement protein Clq. 
     
     
         29 . The fusion polypeptide of  claim 24 , wherein said second polypeptide comprises a region of a heavy chain immunoglobulin polypeptide. 
     
     
         30 . The fusion polypeptide of  claim 24 , wherein said second polypeptide comprises an Fc region of an immunoglobulin heavy chain. 
     
     
         31 . The fusion polypeptide of  claim 24 , wherein said second polypeptide has less effector function than the effector function of a Fc region of a wild-type immunoglobulin heavy chain. 
     
     
         32 . The fusion polypeptide of  claim 24 , wherein the second polypeptide comprises the sequence of SEQ ID NO:3. 
     
     
         33 . The fusion polypeptide of  claim 24 , further comprising a signal sequence. 
     
     
         34 . The fusion polypeptide of  claim 33 , wherein said signal sequence comprises the sequence MPLLLLLLLLPSPLHP (SEQ ID NO:8) or MPLQLLLLLI LLGPGNSLQL WDTWADEAEK ALGPLLARDR R (SEQ ID NO:9). 
     
     
         35 . A multimeric polypeptide comprising the fusion polypeptide of  claim 24 . 
     
     
         36 . The multimeric polypeptide of  claim 35 , wherein said multimeric polypeptide is a dimer. 
     
     
         37 . A DNA molecule encoding the polypeptide of  claim 1 . 
     
     
         38 . A vector comprising the DNA of  claim 37 . 
     
     
         39 . A cell comprising the vector of  claim 38 . 
     
     
         40 . A method for expressing glycoprotein Ibα polypeptide-immunoglobulin fusion polypeptide, the method comprising culturing the cell of  claim 39  under conditions that result in expression of said glycoprotein Ibα polypeptide-immunoglobulin fusion polypeptide. 
     
     
         41 . A pharmaceutical composition comprising the polypeptide of  claim 1 . 
     
     
         42 . A pharmaceutical composition comprising the fusion protein of  claim 23 . 
     
     
         43 . A method of inhibiting adherence of a blood cell to a biological tissue in a biological system, the method comprising adding to said biological system the fusion polypeptide of  claim 23  in an amount sufficient to inhibit adherence of said blood cell to said biological tissue. 
     
     
         44 . The method of  claim 43 , wherein said biological system is an in vitro system. 
     
     
         45 . The method of  claim 43 , wherein said biological system is an ex vivo system. 
     
     
         46 . The method of  claim 43 , wherein said biological system is an in vivo system. 
     
     
         47 . The method of  claim 43 , wherein said blood cell is a platelet. 
     
     
         48 . The method of  claim 47 , wherein said platelet express glycoprotein Ib α, P-selectin or thrombin. 
     
     
         49 . The method of  claim 43 , wherein said blood cell is a leukocyte. 
     
     
         50 . The method of  claim 49 , wherein said leukocyte express Mac-1 or a selectin ligand. 
     
     
         51 . The method of  claim 49 , wherein said biological tissue is complexed with von Willebrand Factor, thrombin, glycoprotein Ib α, or P-selectin. 
     
     
         52 . A method of inhibiting adherence of a protein to a biological tissue in a biological system, the method comprising adding to said biological system the fusion polypeptide of  claim 1  in an amount sufficient to inhibit adherence of said protein to said biological tissue. 
     
     
         53 . The method of  claim 52 , wherein said biological system is an in vitro system. 
     
     
         54 . The method of  claim 52 , wherein said biological system is an ex vivo system. 
     
     
         55 . The method of  claim 52 , wherein said biological system is an in vivo system. 
     
     
         56 . The method of  claim 52 , wherein said protein is membrane associated. 
     
     
         57 . The method of  claim 52 , wherein said protein is glycoprotein Ibα, P-selectin, von Willebrand Factor or thrombin. 
     
     
         58 . The method of  claim 52 , wherein said protein is in solution. 
     
     
         59 . The method of  claim 58 , wherein said protein is von Willebrand Factor or thrombin. 
     
     
         60 . The method of  claim 52 , wherein said biological tissue is complexed with a protein selected from the group consisting of glycoprotein Ibα, Mac-1, P-selectin, von Willebrand Factor and thrombin. 
     
     
         61 . A method of treating a disorder associated with platelet activation in a subject, the method comprising administering to a subject in need thereof the fusion polypeptide of  claim 1 . 
     
     
         62 . The method of  claim 61 , wherein said disorder is associated with thrombotic disease. 
     
     
         63 . The method of  claim 61 , wherein said disorder is ischemic heart disease, angina, acute myocardial infarction, stroke, venous thrombosis, atherosclerosis, or arterial thrombosis. 
     
     
         64 . The method of  claim 61 , wherein said disorder is angina. 
     
     
         65 . The method of  claim 64 , wherein said angina is unstable angina. 
     
     
         66 . The method of  claim 61 , wherein said subject is a human. 
     
     
         67 . The method of  claim 61 , further comprising administering to said subject a compound selected from the group consisting of acetylsalicylic acid, heparin, a glycoprotein IIb/IIIa antagonist, clopidogrel, a P-selectin antagonist, a thrombin inhibitor and a thrombolytic enzyme. 
     
     
         68 . A protein comprising an amino acid sequence with one to 20 amino acid substitutions, deletions or insertions relative to amino acids 1-290 of a human GPIb2V protein sequence (SEQ ID NO:2), wherein said first polypeptide binds with higher affinity to von Willebrand factor than does a polypeptide comprising SEQ ID NO:2. 
     
     
         69 . The protein of  claim 68 , wherein said protein is a fusion protein. 
     
     
         70 . The protein of  claim 1 , wherein said protein is a fusion protein. 
     
     
         71 . The fusion protein of  claim 70  comprising a region of an immunoglobulin.