Receptor-mediated uptake of an extracellular bcl-xl fusion protein inhibits apoptosis

Abstract

Apoptosis-modifying fusion polypeptides, and the corresponding nucleic acid molecules, are disclosed. Pharmaceutical compositions comprising these polypeptides, and the use of these polypeptides to modify apoptosis are also provided.

1 . A functional pro-apoptosis-modifying fusion protein capable of binding a target cell, comprising: 
 (a) a first domain capable of enhancing apoptosis in the target cell; and    (b) a second domain capable of specifically targeting the fusion protein to the target cell, wherein the fusion protein integrates into or otherwise crosses a cellular membrane of the target cell upon binding.    
     
     
         2 . The fusion protein of  claim 1 , wherein the first domain is capable of inducing or enhancing apoptosis.  
     
     
         3 . The functional purified apoptosis-modifying fusion protein of  claim 1 , comprising an amino acid sequence as set forth as the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence that differs from SEQ ID NO: 4 by one or more conservative amino acid substitutions, but which retains targeting and pro-apoptosis-modifying abilities.  
     
     
         4 . The functional pro-apoptosis-modifying fusion protein of  claim 1 , further comprising: 
 (c) a linker connecting the first domain to the second domain.    
     
     
         5 . The protein of  claim 1 , wherein the first domain is a Bcl-2 family protein, or a variant or fragment thereof that retains an apoptosis-enhancing property.  
     
     
         6 . The protein of  claim 5 , wherein the first domain is Bad, or a variant or fragment thereof that enhances apoptosis in the target cell to which the protein is exposed.  
     
     
         7 . The protein of  claim 6 , wherein the first domain is a variant of Bad having an amino acid other than serine at amino acid position 112 and/or position 136.  
     
     
         8 . The protein of  claim 6 , wherein the first domain consists essentially of Bad.  
     
     
         9 . The protein of  claim 7 , wherein the target cell is a tumor cell, a cancer cell, a neoplasm cell, a hyper-proliferative cell, or an adipocyte.  
     
     
         10 . The protein of  claim 1 , wherein the second domain comprises a receptor-binding domain derived from a bacterial toxin, a monoclonal antibody, a growth factor, or a cytokine.  
     
     
         11 . The protein of  claim 10 , wherein the second binding domain comprises a receptor-binding domain derived from diphtheria toxin or anthrax toxin.  
     
     
         12 . The protein of  claim 10 , wherein the second binding domain comprises a receptor-binding domain derived from epidermal growth factor.  
     
     
         13 . The protein of  claim 10 , wherein the receptor-binding domain comprises diphtheria toxin receptor binding domain, or a variant or fragment thereof that targets the fusion protein to the target cell to which the protein is exposed.  
     
     
         14 . The protein of  claim 10 , wherein the second domain further comprises a translocation domain of diphtheria toxin.  
     
     
         15 . The protein of  claim 4 , wherein the linker is 5-100 amino acid residues in length.  
     
     
         16 . The protein of  claim 4 , wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 6.  
     
     
         17 . The protein of  claim 16 , wherein the linker consists of the amino acid sequence shown in SEQ ID NO: 6.  
     
     
         18 . The functional apoptosis-modifying fusion protein of  claim 1 , comprising: 
 (a) Bad;    (b) a diphtheria toxin translocation domain; and    (c) a bacterial toxin receptor binding domain,    wherein (a), (b), and (c) are functionally linked.    
     
     
         19 . The fusion protein of  claim 18 , wherein (c) is a diphtheria toxin or anthrax toxin receptor binding domain.  
     
     
         20 . A composition comprising the protein according to  claim 1 .  
     
     
         21 . A pharmaceutical composition comprising the composition according to  claim 20 , and a pharmaceutically acceptable carrier.  
     
     
         22 . A combined pharmaceutical composition comprising a fusion protein according to  claim 19 , and a sufficient amount of PA to enable measurable transport of the fusion protein into a target cell.  
     
     
         23 . The protein of  claim 1  for use in enhancing apoptosis in a target cell.