Lentiviral vectors for regulated expression of a chimeric antigen receptor molecule

Abstract

The invention relates to the regulated expression of a chimeric antigen receptor (CAR) within a lentiviral vector. The CAR comprises a hook-binding domain that interacts with a hook, preferably encoded by the same lentiviral vector, which prevents proper processing and release of the CAR to the cell membrane. The invention encompasses vectors, methods of making the vectors, and methods of using them, including medicinal uses. The vectors can be used for administration to humans to induce immune responses and to treat cancers and tumors.

1 - 18 . (canceled) 
     
     
         19 . An isolated cell comprising a nucleic acid molecule, a nucleic acid vector or a lentiviral vector encoding a chimeric antigen receptor, the said chimeric antigen receptor comprising:
 a binding domain;   a transmembrane domain;   a hook-binding domain comprising a streptavidin-binding peptide; and   an activation domain comprising a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34.   
     
     
         20 . The isolated cell according to  claim 19 , wherein the isolated cell is a cell of the immune system. 
     
     
         21 . The isolated cell according to  claim 19 , wherein the isolated cell is
 (i) a T cell, or   (ii) a NK cell.   
     
     
         22 . The isolated cell according to  claim 19 , wherein the isolated cell is a mammalian cell, particularly a human cell. 
     
     
         23 . The isolated cell according to  claim 19 , wherein the binding domain comprises a single-chain Fv antibody or a nanobody. 
     
     
         24 . The isolated cell according to  claim 19 , wherein the hook comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:32. 
     
     
         25 . The isolated cell according to  claim 19 , wherein the nucleic acid vector or the lentiviral vector comprises a β2-micro g lobulin, ubiquitin, MHCI or MHCII promoter. 
     
     
         26 . The isolated cell according to  claim 19 , wherein the chimeric antigen receptor comprises the amino acid sequence of any of SEQ ID NO:46, SEQ ID NO:48, or SEQ ID NO:50. 
     
     
         27 . An isolated cell comprising a vector or a lentiviral vector particle expressing a chimeric antigen receptor, the said chimeric antigen receptor comprising:
 a binding domain;   a transmembrane domain;   a hook-binding domain comprising a streptavidin-binding peptide; and   an activation domain comprising a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34.   
     
     
         28 . The isolated cell according to  claim 27 , wherein the isolated cell is a cell of the immune system. 
     
     
         29 . The isolated cell according to  claim 27 , wherein the isolated cell is
 (i) a T cell, or   (ii) a NK cell.   
     
     
         30 . The isolated cell according to  claim 27 , wherein the isolated cell is a human cell. 
     
     
         31 . The isolated cell according to  claim 27 , wherein the binding domain comprises a single-chain Fv antibody or a nanobody. 
     
     
         32 . The isolated cell according to  claim 27 , wherein the hook comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:32. 
     
     
         33 . The isolated cell according to any one of  claims 27 , wherein the vector or the lentiviral vector particle comprises a β2-microglobulin, ubiquitin, MHCI, or MHCII promoter. 
     
     
         34 . The isolated cell according to claim  9 , wherein the chimeric antigen receptor comprises the amino acid sequence of any of SEQ ID NO:46, SEQ ID NO:48, or SEQ ID NO:50. 
     
     
         35 . A method for the induction of an immune response in a human comprising the administration to said human of an isolated cell according to  claim 19 . 
     
     
         36 . The method according to  claim 35 , wherein the isolated cell is intramuscularly, intravenously, intra-articularly or intra-tumoraly administered to the human. 
     
     
         37 . The method according to  claim 35 , wherein biotin is administered to the human subsequently to the administration of the cell. 
     
     
         38 . A method for the treatment of cancers and tumors in a human comprising the administration to said human of an isolated cell according to  claim 19 . 
     
     
         39 . The method according to  claim 38 , wherein the isolated cell is intramuscularly, intravenously, intra-articularly or intra-tumoraly administered to the human. 
     
     
         40 . The method according to  claim 38 , wherein biotin is administered to the human subsequently to the administration of the cell. 
     
     
         41 . A method for the treatment of auto-immune diseases in a human comprising the administration to said human of an isolated cell according to  claim 19 . 
     
     
         42 . The method according to  claim 41 , wherein the isolated cell is intramuscularly, intravenously, intra-articularly or intra-tumoraly administered to the human. 
     
     
         43 . The method according to  claim 41 , wherein biotin is administered to the human subsequently to the administration of the cell. 
     
     
         44 . A method for expressing a chimeric antigen receptor in a cell comprising:
 expressing a nucleic acid molecule, a nucleic acid vector or a lentiviral vector encoding the chimeric antigen receptor, or a vector expressing the chimeric antigen receptor in the cell, or   transducing the cell with a lentiviral vector particle expressing the chimeric antigen receptor,   under conditions that allow the expression of the chimeric antigen receptor;   said chimeric antigen receptor comprising:
 a binding domain; 
 a transmembrane domain; 
 a hook-binding domain comprising a streptavidin-binding peptide; and 
 an activation domain comprising a T cell activating fragment of at least 100 amino acids of SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, SEQ ID NO:33, or SEQ ID NO:34. 
   
     
     
         45 . The method according to  claim 44 , wherein it further comprises harvesting or isolating the chimeric antigen receptor. 
     
     
         46 . The method according to  claim 44 , wherein the cells are treated with biotin. 
     
     
         47 . The method according to  claim 46 , wherein the cells are treated with biotin at an initial concentration of at least 0.2, 0.4, 0.8, 1.6, 2.5, 5, 10, 20, 40 or 80 μM.