Non-natural ribonuclease conjugates as cytotoxic agents

Abstract

The present invention is directed toward the delivery of a toxic protein to pathogenic cells, particularly cancer cells. In preferred embodiments, the toxic protein is a ribonuclease that has been modified to make it toxic to target cells and that can be conjugated to a target cell-specific delivery vector, such as an antibody, for delivery to pathogenic cells.

1 . A composition comprising a non-natural human ribonuclease one (human RNase I) conjugated to a cell- or disease-specific targeting moiety, wherein said ribonuclease is configured to kill said cell or degrade pathogenic RNA. 
     
     
         2 . The composition of  claim 1 , wherein said non-natural human ribonuclease one has a variant sequence that disrupts binding to the ribonuclease inhibitor. 
     
     
         3 . The composition of  claim 1 , wherein said non-natural human ribonuclease one has a variant sequence compared to a natural ribonuclease one selected from the group consisting of: N88C/L86E, N88R, G89D, R91D/R4C, L86E, N88R, G89D, R91D, V118C/L86E, N88C, R91D/R4C, L86E, N88C, R91D, V118C/R4C, N88C, V118C/K7A, L86E, N88C, R91D/K7A, L86E, N88R, G89D, R91D/R4C, K7A, L86E, N88C, R91D, V118C/R4C, G38R, R39D, L86E, N88R, G89D, R91D, V118C/ and R4C, K7A, L86E, N88R, G89D, R91D, V118C. 
     
     
         4 . The composition of  claim 1 , wherein said cell is a cancer cell, virally infected cell, or immune system cell. 
     
     
         5 . The composition of  claim 1 , wherein said pathogenic RNA is of viral origin. 
     
     
         6 . The composition of  claim 1 , wherein said ribonuclease is conjugated to said cell- or disease-specific targeting moiety by a linker. 
     
     
         7 . The composition of  claim 6 , wherein said linker is attached to a non-native cysteine of said ribonuclease. 
     
     
         8 . The composition of  claim 1 , wherein said targeting moiety comprises an antigen binding protein or immunoglobulin. 
     
     
         9 . The composition of  claim 8 , wherein said immunoglobulin comprises a human or humanized antibody. 
     
     
         10 . The composition of  claim 8 , wherein said immunoglobulin comprises an antibody fragment. 
     
     
         11 . The composition of  claim 1 , wherein said targeting moiety comprises a receptor ligand. 
     
     
         12 . The composition of  claim 1 , wherein said targeting moiety comprises a small molecule. 
     
     
         13 . The composition of  claim 12 , wherein said small molecule comprises a lipid or carbohydrate. 
     
     
         14 . The composition of  claim 1 , wherein said targeting moiety comprises an engineered non-natural protein. 
     
     
         15 . The composition of  claim 1 , wherein said targeting moiety comprises a polymer. 
     
     
         16 . The composition of  claim 1 , wherein said targeting moiety is conjugated to said ribonuclease within a loop region of said ribonuclease corresponding to amino acids 84-95 of bovine ribonuclease A. 
     
     
         17 . The composition of  claim 1 , wherein said ribonuclease and said targeting moiety comprise a fusion protein. 
     
     
         18 . A composition comprising a nucleic acid molecule that encodes the composition of  claim 1 . 
     
     
         19 . A method for killing a cell comprising the step of exposing a cell to the composition of  claim 1 . 
     
     
         20 . The method of  claim 19 , wherein said cell is a cancer cell virally infected cell, or immune system cell. 
     
     
         21 . The composition of  claim 10 , wherein said antibody fragment is an Fc fragment.