High efficiency tissue specific compound delivery system using streptavidin-protein a fusion protein

Abstract

The present invention relates to methods and compositions that can be employed to introduce toxins and nucleic acids into the cytoplasm or nucleus of a eukaryotic cell, particularly a cell of a higher vertebrate. The invention particularly concerns the use of a fusion protein of streptavidin and protein A sequences to form a non-covalent complex of a toxin or nucleic acid and an antibody.

We claim:  
     
         1 . A complex for transferring a compound into a cell, which comprises: 
 (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;    (b) an antibody, bound to the antibody binding site, which antibody is specific for a cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody; and    (c) a biotinylated compound, bound to the biotin binding site.    
     
     
         2 . The complex of  claim 1  in which the biotinylated compound is selected from the group consisting of biotinylated multidrug resistance (MDR) gene product, biotinylated single stranded nucleic acid, double stranded DNA that forms triplex structure with a biotinylated single stranded nucleic acid having a homopurine or homopyrimidine portion, and biotinylated protein of a pathological bacteria or virus.  
     
     
         3 . The complex of  claim 1  in which there are four antibody binding sites and four biotin binding sites.  
     
     
         4 . The complex of  claim 1  in which the streptavidin component of said streptavidin-protein A fusion protein has a modified RYD sequence.  
     
     
         5 . The complex of  claim 1  which further comprises an antibody that binds transferrin receptor.  
     
     
         6 . The complex of  claim 1 , wherein the antibody recognizes a surface antigen selected from the group consisting of: 
 (a) human lymphocyte antigen (HLA-DR);    (b) cluster of differentiation (CD33);    (c) cluster of differentiation (CD34); and    (d) epidermal growth factor (EGF) receptor.    
     
     
         7 . The complex of  claim 1  in which the antibody is an IgG antibody.  
     
     
         8 . A pharmaceutical composition, comprising: 
 (a) the complex of  claim 1 , and    (b) a pharmaceutically acceptable carrier, which composition is substantially free of biotinylated compound not bound to the streptavidin-protein A fusion protein.    
     
     
         9 . A complex for transferring a prodrug into a cell, which comprises: 
 (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;    (b) an antibody, bound to the antibody binding site, which antibody is specific for a cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody; and    (c) a biotinylated prodrug, bound to the biotin binding site.    
     
     
         10 . A complex for transferring a multidrug resistance (MDR) gene product into a cell, which comprises: 
 (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;    (b) an antibody, bound to the antibody binding site, which antibody is specific for a cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody; and    (c) a biotinylated MDR gene product, bound to the biotin binding site.    
     
     
         11 . A complex for transferring a single stranded nucleic acid into a cell, which comprises: 
 (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;    (b) an antibody, bound to the antibody binding site, which antibody is specific for a cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody; and    (c) a biotinylated single stranded nucleic acid, bound to the biotin binding site.    
     
     
         12 . A complex for transferring a duplex nucleic acid into a cell, which comprises: 
 (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;    (b) an antibody, bound to the antibody binding site, which antibody is specific for a cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody;    (c) a biotinylated single stranded nucleic acid, having a homopurine or homopyrimidine portion, bound to the biotin binding site; and    (d) a double stranded DNA that forms triplex structure with the biotinylated single stranded nucleic acid.    
     
     
         13 . A complex for transferring protein of a pathological bacteria or virus into a cell, which comprises: 
 (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;    (b) an antibody, bound to the antibody binding site, which antibody is specific for a cell antigen presenting cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody; and    (c) a biotinylated protein of a pathological bacteria or virus, bound to the biotin binding site.    
     
     
         14 . A method for transferring a compound into a cell, which comprises the steps of: 
 (a) forming a complex comprising 
 i) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;  
 ii) an antibody, bound to the antibody binding site, which antibody is specific for a cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody; and  
 iii) a biotinylated compound, bound to the biotin binding site;  
   (b) isolating the complex from the biotinylated compound that is not bound to the biotin binding site; and    (c) exposing the isolated complex to a cell, so that the compound enters the cell.    
     
     
         15 . The method of  claim 14 , wherein the compound is selected from the group consisting of toxin, multidrug resistance (MDR) gene product, single stranded nucleic acid, and protein of a pathological bacteria or virus.  
     
     
         16 . The method of  claim 14 , wherein the complex has four antibody binding sites and four biotin binding sites.  
     
     
         17 . The method of  claim 14 , wherein streptavidin component of said streptavidin-protein A fusion protein has a modified RYD sequence.  
     
     
         18 . The method of  claim 14 , wherein the complex further comprises an antibody that binds transferrin receptor.  
     
     
         19 . The method of  claim 14 , wherein the antibody recognizes a surface antigen selected from the group consisting of: 
 (a) human lymphocyte antigen (HLA-DR);    (b) cluster of differentiation (CD33);    (c) cluster of differentiation (CD34); and    (d) epidermal growth factor (EGF) receptor.    
     
     
         20 . The method of  claim 14 , wherein the antibody is an IgG antibody.  
     
     
         21 . A method for transferring double stranded DNA into a cell, which comprises the steps of: 
 (a) forming a complex comprising: 
 i) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site;  
 ii) an antibody, bound to the antibody binding site, which antibody is specific for a cell surface protein, and which cell surface protein undergoes endocytosis after binding with the antibody;  
 iii) a biotinylated single stranded nucleic acid, bound to the biotin binding site; and  
 iv) a double stranded DNA that forms triplex structure with the biotinylated single stranded nucleic acid; and  
   (b) exposing the complex to a cell, so that the double stranded DNA enters the cell.    
     
     
         22 . The method of  claim 21 , wherein the complex has four antibody binding sites and four biotin binding sites.  
     
     
         23 . The method of  claim 21 , wherein streptavidin component of said streptavidin-protein A fusion protein has a modified RYD sequence.  
     
     
         24 . The method of  claim 21 , wherein the complex further comprises an antibody that binds transferrin receptor.  
     
     
         25 . The method of  claim 21 , wherein the antibody recognizes a surface antigen selected from the group consisting of: 
 (a) human lymphocyte antigen (HLA-DR);    (b) cluster of differentiation (CD33);    (c) cluster of differentiation (CD34); and    (d) epidermal growth factor (EGF) receptor.    
     
     
         26 . The method of  claim 21 , wherein the antibody is an IgG antibody.