Engineered Dendritic Cells and Uses for the Treatment of Cancer

Abstract

This invention provides the field of therapeutics. Most specifically present invention provides methods of generating in vitro engineered dendritic cells conditionally expressing interleukin-12 (IL-12) under the control of a gene expression modulation system in the presence of activating ligand and uses for therapeutic purposes in animals including human.

1 . A vector for conditionally expressing a protein having the function of interleukin-12 (IL-12), comprising a polynucleotide encoding a gene switch, said gene switch comprising (1) at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, operably linked to a promoter, and (2) a polynucleotide encoding a protein having the function of IL-12 linked to a promoter which is activated by said ligand-dependent transcription factor. 
     
     
         2 . The vector of  claim 1 , wherein said polynucleotide encoding a gene switch comprises (1) at least one transcription factor sequence, operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding a protein having the function of IL-12 linked to a promoter which is activated by said ligand-dependent transcription factor. 
     
     
         3 . The vector of  claim 2 , which is an adenoviral vector. 
     
     
         4 . The vector of  claim 3 , which is rAD.RheoIL12. 
     
     
         5 . The vector of  claim 2 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch. 
     
     
         6 . The vector of  claim 5 , wherein said polynucleotide encoding a gene switch comprises a first transcription factor sequence and a second transcription factor sequence under the control of a promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. 
     
     
         7 . The vector of  claim 6 , wherein said first transcription factor and said second transcription factor are connected by an internal ribosomal entry site. 
     
     
         8 . The vector of  claim 7 , wherein said internal ribosomal entry site is an EMCV IRES. 
     
     
         9 . The vector of  claim 2 , wherein said polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. 
     
     
         10 . The vector of  claim 9 , wherein the first transcription factor sequence comprises a nucleic acid encoding for a VP-16 transactivation domain and a retinoic acid-X-receptor (RXR) protein. 
     
     
         11 . The vector of  claim 10 , wherein the second transcription factor sequence comprises a nucleic acid encoding for a GAL-4 DNA binding domain and Ecdysone receptor (EcR) protein. 
     
     
         12 . The vector of  claim 11 , wherein said vector further comprises a polynucleotide encoding a protein having the function of IFN-alpha linked to a promoter which is activated by said ligand-dependent transcription factor. 
     
     
         13 . The vector of  claim 11 , wherein said polynucleotide encoding a protein having the function of IL-12 encodes human IL-12. 
     
     
         14 . The vector of  claim 13 , wherein said polynucleotide encoding a protein having the function of IL-12 encodes a polypeptide at least 85% identical to wild type human IL-12. 
     
     
         15 . A method of producing a population of dendritic cells conditionally expressing a protein having the function of IL-12, comprising modifying at least a portion of dendritic cells by introducing into said dendritic cells the vector of  claim 1 . 
     
     
         16 . The method of  claim 15 , wherein said dendritic cells are human dendritic cells. 
     
     
         17 . The method of  claim 16 , wherein said dendritic cells are bone marrow dendritic cells. 
     
     
         18 . An in vitro engineered dendritic cell comprising the vector of  claim 1 . 
     
     
         19 . The in vitro engineered dendritic cell of  claim 18  further comprising a vector conditionally expressing a protein having the function of IFN-alpha, said vector comprises a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding a protein having the function of IFN-alpha linked to a promoter which is activated by said ligand-dependent transcription factor. 
     
     
         20 . A pharmaceutical composition comprising a population of the in vitro engineered dendritic cells of  claim 18 . 
     
     
         21 . The pharmaceutical composition of  claim 20 , wherein the pharmaceutical composition is suitable for intratumoral, intraperitoneal or subcutaneous administration. 
     
     
         22 . The pharmaceutical composition of  claim 20 , wherein said population of in vitro engineered dendritic cells contains at least 10 4  cells. 
     
     
         23 . The pharmaceutical composition of  claim 20 , wherein said population of in vitro engineered dendritic cell contains at least 10 7  cells. 
     
     
         24 . A method of treating a tumor in a mammal comprising administering to a mammal in need thereof a population of the in vitro engineered dendritic cells of  claim 18 . 
     
     
         25 . The method of  claim 24 , wherein said tumor is a benign tumor. 
     
     
         26 . The method of  claim 24 , wherein said tumor is a malignant tumor. 
     
     
         27 . The method of  claim 24 , wherein said tumor is a melanoma. 
     
     
         28 . The method of  claim 27 , wherein said tumor is a malignant melanoma skin cancer. 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 28 , wherein an effective amount of a ligand which activates the ligand-dependent transcription factor is further administered to said mammal. 
     
     
         31 . The method of  claim 30 , wherein said ligand is selected from the group consisting of RG-115819, RG-115932, and RG-115830. 
     
     
         32 . The method of  claim 30 , wherein said ligand is an amidoketone or oxadiazoline. 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 30 , wherein said ligand is administered less than one hour before or after said in vitro engineered dendritic cell. 
     
     
         35 . The method of  claim 30 , wherein said ligand is administered less than 24 hours after said in vitro engineered dendritic cell. 
     
     
         36 . The method of  claim 30 , wherein said ligand is administered less than 48 hours after said in vitro engineered dendritic cell. 
     
     
         37 .- 50 . (canceled) 
     
     
         51 . A kit comprising said in vitro engineered dendritic cells of  claim 18 . 
     
     
         52 . (canceled) 
     
     
         53 . A method of inducing conditional expression of a protein having the function of interleukin-12 (IL-12) in the dendritic cells comprising administering to a mammal in need thereof a population of the in vitro engineered dendritic cells of  claim 18 . 
     
     
         54 .- 73 . (canceled)