Modified caspase polypeptides and uses thereof

Abstract

The technology relates in part to compositions comprising modified Caspase-9 polypeptides, compositions comprising nucleic acids coding for modified Caspase-9 polypeptides, chimeric modified Caspase-9 polypeptides, and methods of use thereof, including methods for cell therapy. Methods for cell therapy include modifying transfused cells to express an inducible modified Caspase-9 protein, with reduced basal activity in the absence of the inducer.

1 . A composition comprising a nucleic acid comprising a nucleotide sequence that encodes a chimeric protein comprising a multimeric ligand binding region and a modified Caspase-9 polypeptide, wherein the modified Caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and comprises at least one amino acid substitution selected from the group consisting of T317S D330A, F404Y, F406L, F406T, F404W, T317A, S144A, S144D, S196A, S183A, S195A, F404T, F404W, N405F, F406T, D315A, A316G, F319W, 5307A, Y153A, and Y153F. 
     
     
         2 . The composition of  claim 1 , wherein the chimeric protein comprising the modified Caspase-9 polypeptide has decreased basal activity compared to a chimeric protein comprising a Caspase-9 polypeptide comprising the amino acid sequence of SEQ ID NO:9. 
     
     
         3 . The composition of  claim 2 , wherein the basal activity is measured using a secreted alkaline phosphatase (SEAP) reporter-based surrogate killing assay. 
     
     
         4 . The composition of  claim 1 , wherein the least one amino acid substitution is selected from the group consisting of T317S, D330A, F404Y, F406T, F404W, T317A, S195A, S144A, S144D, S196A, and S183A. 
     
     
         5 . The composition of  claim 1 , wherein the multimeric ligand binding region is selected from the group consisting of FKBP ligand binding region, cyclophilin receptor ligand binding region, steroid receptor ligand binding region, cyclophilin receptor ligand binding region, and tetracycline receptor ligand binding region. 
     
     
         6 . The composition of  claim 1 , wherein the multimeric ligand binding region comprises an F v′ F vls  amino acid sequence. 
     
     
         7 . The composition of  claim 1 , wherein Caspase-9 activity is induced upon binding of a ligand to the multimeric ligand binding region of the chimeric protein. 
     
     
         8 . The composition of  claim 7 , wherein the ligand is dimeric. 
     
     
         9 . The composition of  claim 8 , wherein the ligand is dimeric FK506, or a dimeric FK506-like analog. 
     
     
         10 . The composition of  claim 8 , wherein the ligand is AP1903 or AP20187. 
     
     
         11 . An isolated cell transformed or transfected with the composition of  claim 1 . 
     
     
         12 . A method of administering cells to a human subject, comprising
 a) transfecting or transducing cells with a nucleic acid comprising a nucleotide sequence that encodes a chimeric protein comprising a multimeric ligand binding region and a modified Caspase-9 polypeptide, wherein the modified Caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and comprises at least one amino acid substitution selected from the group consisting of N405Q, T3175, D330A, F404Y, F406L, F406T, F404W, T317A, S144A, S144D, S196A, S183A, S195A, F404T, F404W, N405F, F406T, D315A, A316G, F319W, 5307A, Y153A, and Y153F; and   b) administering the transfected or transduced cells to a human subject.   
     
     
         13 . The method of  claim 12 , wherein the cells are selected from the group consisting of inducible progenitor cells (iPS), embryonic stem (ES) cells, mesenchymal stem cells, plasma (B) cells, myocytes and T cells. 
     
     
         14 . The method of  claim 12 , further comprising administering to the subject a ligand that binds to the multimeric ligand binding region. 
     
     
         15 . The method of  claim 14 , wherein the ligand is AP1903. 
     
     
         16 . The method of  claim 14 , wherein after administration of the ligand, the number of transfected or transduced cells is reduced. 
     
     
         17 . A method of controlling the survival of transplanted therapeutic cells in a subject, comprising
 a) preparing or obtaining therapeutic cells;   b) transfecting or transducing the therapeutic cells with a nucleic acid comprising a nucleotide sequence that encodes a chimeric protein comprising a multimeric ligand binding region and a modified Caspase-9 polypeptide, wherein the modified Caspase-9 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 9, and comprises at least one amino acid substitution selected from the group consisting of N405Q, T3175, D330A, F404Y, F406L, F406T, F404W, T317A, S144A, S144D, S196A, S183A, S195A, F404T, F404W, N405F, F406T, D315A, A316G, F319W, 5307A, Y153A, and Y153F;   c) transplanting the transfected or transduced therapeutic cells into a subject; and   d) after (c), administering to the subject a ligand that binds to the multimeric ligand binding region, wherein transplanted therapeutic cells that express the modified Caspase-9 polypeptide are killed following administration of the ligand.   
     
     
         18 . The method of  claim 17 , wherein the therapeutic cells are selected from the group consisting of inducible progenitor cells (iPS), embryonic stem (ES) cells, mesenchymal stem cells, plasma (B) cells, myocytes and T cells. 
     
     
         19 . The method of  claim 17 , wherein the ligand is AP1903. 
     
     
         20 . The method of  claim 17 , wherein the subject has been diagnosed with a hyperproliferative disease or an immune disease.