Expression and export of angiogenesis inhibitors as immunofusins

Abstract

Disclosed are nucleotide sequences, for example, DNA or RNA sequences, which encode an immunoglobulin Fc-angiogenesis inhibitor fusion protein. The angiogenesis inhibitors can be angiostatin, endostatin, a plasminogen fragment having angiostatin activity, or a collagen XVIII fragment having endostatin activity. The nucleotide sequences can be inserted into a suitable expression vector and expressed in mammalian cells. Also disclosed is a family of immunoglobulin Fc-angiogenesis inhibitor fusion proteins that can be produced by expression of such nucleotide sequences. Also disclosed are methods using such nucleotide sequences and fusion proteins for treating conditions mediated by angiogenesis.

What is claimed is:  
     
         1 . A DNA molecule encoding a fusion protein comprising: 
 (a) a signal sequence;    (b) an immunoglobulin Fe region; and    (c) a target protein sequence selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, a collagen XVIII fragment having endostatin activity, and combinations thereof.    
     
     
         2 . The DNA of  claim 1  wherein said signal sequence, said immunoglobulin Fc region and said target protein sequence are encoded serially in a 5′ to 3′ direction.  
     
     
         3 . The DNA of  claim 1 , wherein said signal sequence, said target sequence, and said immunoglobulin Fc region are encoded serially in a 5′ to 3′ direction.  
     
     
         4 . The DNA of  claim 1  wherein said immunoglobulin Fc region comprises an immunoglobulin hinge region.  
     
     
         5 . The DNA of  claim 1  wherein said immunoglobulin Fc region comprises an immunoglobulin hinge region and an immunoglobulin constant heavy chain domain.  
     
     
         6 . The DNA of  claim 1  wherein said immunoglobulin Fc region comprises a hinge region and an CH 3  domain.  
     
     
         7 . The DNA of  claim 1  wherein said immunoglobulin Fc region lacks at least the CH 1  domain.  
     
     
         8 . The DNA of  claim 1  wherein said immunoglobulin Fc region encodes at least a portion of immunoglobulin gamma.  
     
     
         9 . A replicable expression vector for transfecting a mammalian cell, said vector comprising the DNA of  claim 1 .  
     
     
         10 . A mammalian cell harboring the DNA of  claim 1 .  
     
     
         11 . A fusion protein comprising an immunoglobulin Fc region, and a target protein selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, a collagen XVIII fragment having endostatin activity, and combinations thereof.  
     
     
         12 . The fusion protein of  claim 11  wherein said plasminogen fragment has molecular weight of approximately 40 kD and comprises an amino acid sequence set forth in SEQ ID No: 3.  
     
     
         13 . The fusion protein of  claim 11  wherein said target protein comprises amino acid sequence set forth in SEQ ID No: 3.  
     
     
         14 . The fusion protein of  claim 11  wherein of said collagen XVIII fragment comprises the amino acid sequence set forth in SEQ ID No: 1.  
     
     
         15 . The fusion protein of  claim 11  wherein said target protein comprises at least two molecules selected from the group consisting of angiostatin, endostatin, a plasminogen fragment, and a collagen XVIII fragment, wherein said two molecules are linked by a polypeptide linker.  
     
     
         16 . The fusion protein of  claim 11  wherein said target protein is linked to an N-terminal end of said immunoglobulin Fc region.  
     
     
         17 . The fusion protein of  claim 11  wherein said target protein is linked to a C-terminal end of said immunoglobulin Fc region.  
     
     
         18 . A multimeric protein comprising at least two fusion proteins of  claim 11  linked via a disulfide bond.  
     
     
         19 . The multimeric protein of  claim 18  wherein the target protein of at least one said fusion protein is angiostatin and the target protein of at least one said fusion protein is endostatin.  
     
     
         20 . The multimeric protein of  claim 18  wherein the target protein of both of said fusion proteins is angiostatin.  
     
     
         21 . The multimeric protein of  claim 18  wherein the target protein of both of said fusion proteins is endostatin.  
     
     
         22 . The fusion protein of  claim 11  further comprising a second target protein selected from the group consisting of angiostatin, endostatin, a plasminogen fragment having angiostatin activity, and a collagen XVIII fragment having endostatin activity.  
     
     
         23 . The fusion protein of  claim 22  wherein said second target protein is linked by a polypeptide linker to said first target protein.  
     
     
         24 . The fusion protein of  claim 22  wherein said first target protein is connected to an N-terminal end of said immunoglobulin Fc region and said second target protein is connected to a C-terminal end of said immunoglobulin Fc region.  
     
     
         25 . A multimeric fusion protein comprising at least two fusion proteins of  claim 11 , wherein said fusion proteins are linked by a polypeptide bond.  
     
     
         26 . A method of producing a fusion protein, the method comprising the steps of: 
 a) providing the mammalian cell of  claim 10;  and    b) culturing the mammalian cell to produce said fusion protein.    
     
     
         27 . The method of  claim 26  comprising the additional step of collecting said fusion protein.  
     
     
         28 . The method of  claim 26  comprising the additional step of cleaving said immunoglobulin Fc region from said target protein.  
     
     
         29 . A method of treating a condition mediated by angiogenesis comprising the step of administering the DNA of  claim 1  to a mammal in need of an angiogenesis inhibitor.  
     
     
         30 . A method of treating a condition mediated by angiogenesis comprising the step of administering the vector of  claim 9  to a mammal in need of an angiogenesis inhibitor.  
     
     
         31 . A method of treating a condition alleviated by the administration of angiostatin or endostatin comprising the step of administering an effective amount of the fusion protein of  claim 11  to a mammal having said condition.