Chimera comprising bacterial cytotoxin and methods of using the same

Abstract

This invention provides, a recombinant polypeptide encoding a chimera. The chimera includes a DNase I fragment or a homologue thereof and a Cdt fragment or a homologue thereof. Further, the invention provides methods, utilizing the recombinant polypeptide encoding the chimera, such as a method for inhibiting the proliferation of a neoplastic cell, a method for treating a neoplastic disease in a human subject, a method for inhibiting or suppressing a neoplastic disease in a human subject, and a method for reducing the symptoms associated with a neoplastic disease in a human subject.

1 . A recombinant polypeptide comprising a chimera, wherein said chimera comprises a DNase I fragment or a homologue thereof and a Cdt fragment or a homologue thereof. 
     
     
         2 . The recombinant polypeptide of  claim 1 , wherein said Cdt fragment is a CdtB fragment. 
     
     
         3 . The recombinant polypeptide of  claim 2 , wherein said CdtB fragment is as set forth in SEQ ID NO: 14 or a fragment thereof. 
     
     
         4 . The recombinant polypeptide of  claim 2 , wherein said CdtB fragment comprises a mutation. 
     
     
         5 . The recombinant polypeptide of  claim 4 , wherein said mutation is G55R, L62E, S99Y, R100E, V134R, Y174P or their combination. 
     
     
         6 . The recombinant polypeptide of  claim 4 , wherein said DNase I fragment comprises a substitution of SEQ ID NO: 58 for SEQ ID NO: 59. 
     
     
         7 . The recombinant polypeptide of  claim 1 , wherein said DNase I fragment is encoded by a sequence as set forth in SEQ ID NO: 1-7 or a fragment thereof. 
     
     
         8 . The recombinant polypeptide of  claim 1 , wherein said chimera is His 6 -tagged. 
     
     
         9 . The recombinant polypeptide of  claim 1 , wherein said chimera comprises (a) said CdtB fragment in the amino terminus of said chimera and said DNase I fragment in the carboxy terminus of said chimera or (b) a homologue of a DNase I fragment in the carboxy terminus of said chimera and a Cdt fragment in the amino terminus of said chimera or (c) a DNase I in the carboxy terminus of said chimera and a homologue of a Cdt fragment in the amino terminus of said chimera or (d) a homologue of a DNase I fragment in the carboxy terminus of said chimera and a homologue of a Cdt fragment in the amino terminus of said chimera. 
     
     
         10 . The recombinant polypeptide of  claim 1 , wherein the amino acid sequence of said chimera is as set forth in SEQ ID NO: 24-27. 
     
     
         11 . The recombinant polypeptide of  claim 1 , wherein said recombinant polypeptide binds both CdtA and CdtC. 
     
     
         12 . The recombinant polypeptide of  claim 1 , wherein said recombinant polypeptide inhibits proliferation of a neoplastic cell. 
     
     
         13 . The recombinant polypeptide of  claim 1 , wherein said recombinant polypeptide inhibits proliferation of a neoplastic cell and binds both CdtA and CdtC. 
     
     
         14 . The recombinant polypeptide of  claim 1 , wherein the amino acid sequence of said recombinant polypeptide is as set forth in SEQ ID NO: 29. 
     
     
         15 . A recombinant polynucleotide encoding the chimera of  claim 1 . 
     
     
         16 . The recombinant polynucleotide of  claim 15 , wherein the nucleic acid sequence encoding said chimera is as set forth in SEQ ID NO: 22-23. 
     
     
         17 . The recombinant polynucleotide of  claim 15 , wherein the nucleic acid sequence of said chimera is as set forth in SEQ ID NO: 28. 
     
     
         18 . A DNA vector comprising the recombinant polynucleotide of  claim 15 . 
     
     
         19 . A plasmid comprising the recombinant polynucleotide of  claim 15 . 
     
     
         20 . The DNA vector of  claim 17 , wherein said vector further comprises a promoter. 
     
     
         21 . The DNA vector of  claim 19 , wherein said promoter is a constitutively active promoter. 
     
     
         22 . A method for inhibiting the proliferation of a neoplastic cell comprising the step of contacting said cell with a recombinant polypeptide according to  claim 1  or a nucleic acid molecule encoding the same. 
     
     
         23 .- 39 . (canceled) 
     
     
         40 . A method for treating a neoplastic disease in a subject comprising the step of administering to said subject a recombinant polypeptide according to  claim 1  or a nucleic acid encoding the same. 
     
     
         41 .- 57 . (canceled) 
     
     
         58 . A method for inhibiting or suppressing a neoplastic disease in a subject comprising the step of administering to said subject a recombinant polypeptide according to  claim 1  or a nucleic acid encoding the same. 
     
     
         59 .- 75 . (canceled) 
     
     
         76 . A method for reducing the symptoms associated with a neoplastic disease in a subject comprising the step of administering to said subject a recombinant polypeptide according to  claim 1  or a nucleic acid encoding the same. 
     
     
         77 .- 93 . (canceled) 
     
     
         94 . A composition comprising: a recombinant CdtA polypeptide comprising at least one of the mutations C149A and C178A, said polypeptide operably linked to a ligand that binds specifically to an antigen expressed on the surface of a cancerous epithelial cell type. 
     
     
         95 . (canceled) 
     
     
         96 . (canceled) 
     
     
         97 . The composition of  claim 94 , wherein said cell type is a progenitor stem cell. 
     
     
         98 . The composition of  claim 94 , wherein said cancer is a squamous cell carcinoma. 
     
     
         99 . The composition of  claim 94 , wherein said mutant CdtA polypeptide encodes a genotoxin. 
     
     
         100 . The composition of  claim 94 , wherein said mutant CdtA polypeptide is set forth in SEQ ID NO: 9. 
     
     
         101 . The composition of  claim 94 , wherein said antigen is prominin-1 or CD133. 
     
     
         102 . The composition of  claim 94 , wherein said ligand is a protein that binds specifically to said antigen. 
     
     
         103 . The composition of  claim 94 , wherein said ligand is an antibody that binds specifically to said antigen. 
     
     
         104 . The composition of  claim 103 , wherein said antibody is a monoclonal antibody. 
     
     
         105 . The composition of  claim 104 , wherein said antibody is anti-prominin-1 or anti-CD133 antibody. 
     
     
         106 . A recombinant CdtA polypeptide operably linked to a ligand that binds specifically to an antigen expressed on the surface of a cancerous epithelial cell type, wherein said recombinant CdtA polypeptide comprises at least one of the mutations C149A and C178A. 
     
     
         107 . A chimeric CdtA polypeptide comprising at least one of the mutations C149A and C178A. 
     
     
         108 . An isolated nucleic acid sequence encoding (i) a CdtA polypeptide comprising at least one of the mutations C149A and C178A or (ii) a nucleic acid sequence that is at least 85% identical to the sequence of (i). 
     
     
         109 . A method for inhibiting the proliferation of a cancerous epithelial cell type comprising: administering a recombinant CdtA polypeptide according to  claim 106 . 
     
     
         110 .- 119 . (canceled) 
     
     
         120 . A method for inhibiting the proliferation of a cancerous epithelial cell comprising: contacting said cell with a recombinant CdtA polypeptide according to  claim 106 . 
     
     
         121 . A method for treating cancer comprising: administering a recombinant CdtA polypeptide according to  claim 106 . 
     
     
         122 . (canceled) 
     
     
         123 . (canceled)