High efficiency tissue-specific compound delivery system using streptavadin-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.

1 . A complex for transferring a compound to a cell produced by a method which comprises the steps of:
 Incubating a. a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site, wherein said streptavidin-protein A fusion protein forms tetramers;   b. an antibody, bound to the antibody binding site, in which the antibody is specific for a cell surface protein, and in which the cell surface protein undergoes endocytosis after binding with the antibody; and   c. a biotinylated compound to be transferred to said cell, bound to the biotin binding site, thereby forming a complex   wherein said complex is capable of transferring said compound to said cell when contacted with said cell after forming said complex, resulting in the transfer of said compound into said cell.   
     
     
         2 . The complex of  claim 1  in which the biotinylated compound is selected from the group consisting of 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, biotinylated enzyme 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 , 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. 
     
     
         6 . The complex of claim I in which the antibody is an IgG antibody. 
     
     
         7 . 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. 
     
     
         8 . A complex for transferring an enzyme into a cell produced by a method which comprises the steps of:
 Incubating (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site, wherein said streptavidin-protein A fusion protein forms tetramers; (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 enzyme bound to the biotin binding site, thereby forming a complex   wherein said complex is capable of transferring said enzyme to said cell when contacted with the cell after forming said complex resulting in expression of said enzyme activity inside said cell.   
     
     
         9 . A complex for transferring a protein of a pathological bacteria or virus into a cell, produced by a method which comprises the steps of:
 Incubating (a) a streptavidin-protein A fusion protein having an antibody binding site and a biotin binding site, wherein said streptavidin-protein A fusion protein forms tetramers; (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, thereby forming a complex   wherein said complex is capable of transferring said complex into said cell when contacted with said cell after forming said complex, resulting in the transfer of said protein into said cell.