Gold Optimized CAR T-cells

Abstract

Control Devices are disclosed including RNA destabilizing elements (RDE), RNA control devices, and destabilizing elements (DE) combined with Chimeric Antigen Receptors (CARs) or other transgenes in eukaryotic cells. Multicistronic vectors are also disclosed for use in engineering host eukaryotic cells with the CARs and transgenes under the control of the control devices. These control devices can be used to optimize expression of CARs in the eukaryotic cells so that, for example, effector function is optimized. CARs and transgene payloads can also be engineered into eukaryotic cells so that the transgene payload is expressed and delivered after stimulation of the CAR on the eukaryotic cell.

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         21 . A method of controlling a transgene, comprising the steps of: obtaining a primary T-cell comprising a receptor, and a heterologous nucleic acid comprising a polynucleotide encoding the transgene that is operably linked to a polynucleotide encoding a RNA degradation element (RDE), wherein the RDE is an AU rich element, wherein the heterologous nucleic acid is transcribed to make a transcript encoding the transgene operably linked to the RDE; exposing the primary T-cell to a ligand for the receptor wherein binding of the ligand by the receptor activates the primary T-cell and thereby changes a metabolic state of the primary T-cell; expressing the transgene wherein the amount of polypeptide made from the transgene is increased after the change in metabolic state of the primary T-cell; and exposing the primary T-cell to a glycolysis inhibitor, wherein the exposure to the glycolysis inhibitor occurs after the exposure to the ligand, wherein the amount of the polypeptide made from the transgene is decreased after the exposure to the glycolysis inhibitor. 
     
     
         22 . The method of  claim 21 , wherein the glycolysis inhibitor is a rapamycin, a 2-deoxyglucose, a 3-bromophyruvic acid, an iodoacetate, fluoride, an oxamate, a ploglitazone, or a dichloroacetic acid. 
     
     
         23 . The method of  claim 22 , wherein the glycolysis inhibitor is a rapamycin. 
     
     
         24 . The method of  claim 21 , wherein the transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, or a checkpoint inhibitor. 
     
     
         25 . The method of  claim 21 , wherein the transgene encodes an IL-2, an IL-10, an IL-12, an IL-15, an IL-18, an interferon gamma, a TNFα, or a TGF-β. 
     
     
         26 . The method of  claim 25 , wherein the transgene encodes an IL-12. 
     
     
         27 . The method of  claim 21 , wherein the receptor is a chimeric antigen receptor or a T-cell receptor. 
     
     
         28 . The method of  claim 27 , wherein the chimeric antigen receptor or the T-cell receptor is activated by a ligand found on a target cell. 
     
     
         29 . The method of  claim 28 , wherein the target cell is a cancer cell. 
     
     
         30 . The method of  claim 29 , wherein the cancer cell is a solid tumor cell. 
     
     
         31 . The method of  claim 30 , wherein the transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, a microRNA, or a checkpoint inhibitor. 
     
     
         32 . The method of  claim 30 , wherein the wherein the transgene encodes an IL-2, an IL-10, an IL-12, an IL-15, an IL-18, an interferon gamma, a TNFα, or a TGF-β. 
     
     
         33 . The method of  claim 32 , wherein the transgene encodes an IL-12. 
     
     
         34 . The method of  claim 21 , wherein the ligand is a PSMA, a MUC1, a FAP, a folate receptor 1, a NY-ESO-1, or a CD33. 
     
     
         35 . The method of  claim 34 , wherein the transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, a microRNA, or a checkpoint inhibitor. 
     
     
         36 . The method of  claim 35 , wherein the wherein the transgene encodes an IL-2, an IL-10, an IL-12, an IL-15, an IL-18, an interferon gamma, a TNFα, or a TGF-β. 
     
     
         37 . The method of  claim 36 , wherein the ligand is a MUC1 found on a target cell. 
     
     
         38 . The method of  claim 21 , wherein the ligand is a CD123, a CD19, a CD22, a SLAMF7, a CLL-1 or a FLT-3. 
     
     
         39 . The method of  claim 38 , wherein the transgene encodes a cytokine, a FasL, an antibody, a growth factor, a chemokine, an enzyme that cleaves a polypeptide or a polysaccharide, a granzyme, a perforin, a microRNA, or a checkpoint inhibitor. 
     
     
         40 . The method of  claim 39 , wherein the transgene encodes an IL-2, an IL-10, an IL-12, an IL-15, an IL-18, an interferon gamma, a TNFα, or a TGF-β.