Variants of vascular endothelial cell growth factors having antagonistic properties, nucleic acids encoding the same and host cells comprising those nucleic acids

Abstract

The present invention involves the preparation of vascular endothelial growth factor (VEGF) antagonist molecules comprising variant VEGF polypeptides which are capable of binding to and occupying cell surface VEGF receptors without inducing a VEGF response, thereby antagonizing the biological activity of the native VEGF protein. Specifically, the variant VEGF polypeptides of the present invention comprise modifications of at least one cysteine residue in the native VEGF sequence, thereby inhibiting the ability of the variant polypeptide to dimerize through the formation of disulfide bonds. The present invention is further directed to methods for preparing such variant VEGF antagonists and to methods, compositions and assays utilizing such variants for producing pharmaceutically active materials having therapeutic and pharmacologic properties that differ from the native VEGF protein.

1 . A VEGF antagonist molecule comprising a variant vascular endothelial growth factor polypeptide, said variant polypeptide comprising an amino acid modification of at least one cysteine residue, wherein said amino acid modification inhibits the ability of said variant polypeptide to properly dimerize with another vascular endothelial growth factor polypeptide monomer, wherein said antagonist molecule is capable of binding to vascular endothelial growth factor receptors without significantly inducing a vascular endothelial growth factor response, and functional derivatives of said antagonist molecule.  
     
     
         2 . The antagonist molecule according to  claim 1  wherein said amino acid modification is a substitution of said at least one cysteine residue with a different amino acid which is incapable of participating in a disulfide bond.  
     
     
         3 . The antagonist molecule according to  claim 2  wherein said substitution is of the cysteine residue at amino acid position 51 and/or 60 of the native VEGF amino acid sequence.  
     
     
         4 . The antagonist molecule according to  claim 3  wherein aspartic acid is substituted for cysteine.  
     
     
         5 . The antagonist molecule according to  claim 4  comprising the substitution C51 D.  
     
     
         6 . The antagonist molecule according to  claim 4  comprising the substitution C60D.  
     
     
         7 . The antagonist molecule according to  claim 1  wherein said amino acid modification is a chemical modification of said at least one cysteine residue which renders said cysteine residue incapable of participating in a disulfide bond.  
     
     
         8 . The antagonist molecule according to  claim 7  wherein said chemical modification is of the cysteine residue at amino acid position 51 and/or 60 of the native VEGF amino acid sequence.  
     
     
         9 . The antagonist molecule according to  claim 1  containing further amino acid modifications that do not otherwise affect the essential biological characteristics.  
     
     
         10 . An isolated nucleic acid sequence comprising a sequence that encodes the VEGF antagonist molecule of  claim 1 .  
     
     
         11 . A replicable expression vector capable in a transformant host cell of expressing the nucleic acid of  claim 10 .  
     
     
         12 . Host cells transformed with the vector according to  claim 11 .  
     
     
         13 . Host cells according to  claim 12  which are Chinese hamster ovary cells.  
     
     
         14 . A composition of matter comprising the VEGF antagonist molecule according to  claim 1  compounded with a pharmaceutically acceptable carrier.  
     
     
         15 . A method of treatment which comprises administering a composition according to  claim 14.