Chimeric transcription factor variants with augmented sensitivity to drug ligand induction of transgene expression in mammalian cells

Abstract

Provided herein is a system for inducible expression of a chimeric antigen receptor in cells, such as mammalian cells. The system comprises: a) a first nucleic acid comprising a first promoter inducible by a drug, wherein the first nucleic acid is operably linked to a first polynucleotide that encodes a chimeric antigen receptor, which comprises a ligand binding domain that is specific for a ligand selected from the group consisting of a tumor specific molecule, a viral specific molecule, and any other selected molecule expressed on a target cell population, wherein the ligand elicits recognition, modulation, inhibition, and/or elimination by a lymphocyte, a second polynucleotide, which encodes a spacer or an optimized polypeptide spacer, a third polynucleotide, which encodes a transmembrane domain and a fourth polynucleotide, which encodes an intracellular signaling domain and b) a second nucleic acid comprising a second promoter that is operably linked to a nucleic acid encoding a transcriptional activator for the first promoter inducible by drug, wherein the system is inducible by an amount of the drug that is less than a comparable system utilizing a wild type HEA3 chimeric transcription factor, or the system has an enhanced transcriptional expression at a given concentration of the drug compared to a wild type HEA3. Methods of making such cells and methods of treatment using these cells are also provided.

1 . (canceled) 
     
     
         2 . A method for inducing expression of a polynucleotide in a cell, comprising:
 (a) obtaining a cell, wherein the cell comprises:
 (i) a first nucleic acid comprising a first promoter inducible by an agent, wherein the first nucleic acid is operably linked to the polynucleotide, and 
 (ii) a second nucleic acid comprising a second promoter operably linked to a nucleic acid encoding a transcriptional activator for the first promoter, wherein the transcriptional activator comprises an amino acid sequence set forth in any one of SEQ ID NOs:01-11 or 121; and 
   (b) contacting the cell with the agent and a stimulator, wherein the stimulator is selected from phorbol 12-myristate 13-acetate (PMA), ionomycin, or a combination thereof.   
     
     
         3 . The method of  claim 2 , wherein the agent is selected from tamoxifen, a metabolite of tamoxifen, an analog of tamoxifen, a salt of tamoxifen, a hydrate of tamoxifen, 4-hydroxytamoxifen, fulvestrant, an estrogen analog, or CMP8. 
     
     
         4 . The method of  claim 2 , wherein the drug induces expression of the polynucleotide at a concentration of less than 10 nM. 
     
     
         5 . The method of  claim 2 , wherein the transcriptional activator comprises the amino acid sequence set forth in any one of SEQ ID NOs:01-03, 08-11 or 121. 
     
     
         6 . The method of  claim 5 , wherein the transcriptional activator comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2, 3, 8-11 or 121. 
     
     
         7 . The method of  claim 6 , wherein the transcriptional activator comprises the amino acid sequence set forth in SEQ ID NO: 121. 
     
     
         8 . The method of  claim 2 , wherein the transcriptional activator comprises the amino acid sequence set forth in any one of SEQ ID NOs: 4-7. 
     
     
         9 . The method of  claim 2 , wherein the first promoter comprises the nucleotide sequence set forth in SEQ ID NO:23. 
     
     
         10 . The method of  claim 2 , wherein the second promoter is a constitutive promoter. 
     
     
         11 . The method of  claim 2 , wherein the polynucleotide encodes a payload is selected from a chimeric antigen receptor, a cytokine, a chemokine receptor, a chimeric cytokine receptor, or a microRNA. 
     
     
         12 . The method of  claim 2 , further comprising (d) contacting the cell with an activating stimulator selected from an anti-CD3 antibody or antigen binding fragment thereof, or an anti-CD28 antibody or antigen binding fragment thereof. 
     
     
         13 . The method of  claim 2 , wherein the cell is selected from a T cell, a precursor T cell, or a hematopoietic stem cell. 
     
     
         14 . The method of  claim 13 , wherein the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of a naïve CD8+ T cell, a central memory CD8+ T cell, an effector memory CD8+ T cell, and a bulk CD8+ T cell; or a CD4+ T helper lymphocyte cell selected from the group consisting of a naïve CD4+ T cell, a central memory CD4+ T cell, an effector memory CD4+ T cell, and a bulk CD4+ T cell. 
     
     
         15 . The method of  claim 2 , wherein the cell is ex vivo. 
     
     
         16 . The method of  claim 2 , wherein the cell is in vivo. 
     
     
         17 . A method for inducible expression of a polynucleotide in a subject, comprising:
 (a) introducing a cell into the subject, wherein the cell comprises:
 (i) a first nucleic acid comprising a first promoter inducible by an agent, wherein the first nucleic acid is operably linked to the polynucleotide, and 
 (ii) a second nucleic acid comprising a second promoter operably linked to a nucleic acid encoding a transcriptional activator for the first promoter, wherein the transcriptional activator comprises an amino acid sequence set forth in any one of SEQ ID NOs:01-11 or 121; and 
   (b) administering to the subject the agent in combination with a stimulator, wherein the stimulator is selected from phorbol 12-myristate 13-acetate (PMA), ionomycin, or a combination thereof.   
     
     
         18 . The method of  claim 17 , wherein the agent is selected from tamoxifen, a metabolite of tamoxifen, an analog of tamoxifen, a salt of tamoxifen, a hydrate of tamoxifen, 4-hydroxytamoxifen, fulvestrant, an estrogen analog, or CMP8. 
     
     
         19 . The method of  claim 17 , wherein the polynucleotide encodes a cytokine, chimeric antigen receptor, a chemokine receptor, a chimeric chemokine receptor, a polypeptide capable of regulating apoptosis, a polypeptide capable of modulating an extracellular environment, a polypeptide capable of modulating checkpoint signaling, or a microRNA. 
     
     
         20 . The method of  claim 17 , wherein the cell is selected from a T cell; a precursor T cell; a hematopoietic stem cell; a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of a naïve CD8+ T cell, a central memory CD8+ T cell, an effector memory CD8+ T cell, and a bulk CD8+ T cell; or a CD4+ T helper lymphocyte cell selected from the group consisting of a naïve CD4+ T cell, a central memory CD4+ T cell, an effector memory CD4+ T cell, or a bulk CD4+ T cell. 
     
     
         21 . The method of  claim 17 , wherein the cell is autologous to the subject.