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 natural killer cell comprising a chimeric antigen receptor, and a heterologous nucleic acid comprising a polynucleotide encoding the transgene that is operably linked to a polynucleotide encoding a RNA degradation element, wherein the RNA degradation element is an AU rich element, wherein the heterologous nucleic acid is transcribed to make a transcript encoding the transgene operably linked to the RNA degradation element, wherein the transgene encodes a payload; exposing the natural killer cell to a ligand for the chimeric antigen receptor, wherein the ligand is an antigen found on a target cell, wherein binding of the ligand by the chimeric antigen receptor activates the natural killer cell and thereby changes a metabolic state of the natural killer cell; and expressing the transgene wherein the amount of polypeptide made from the transgene is increased after the change in metabolic state of the natural killer cell. 
     
     
         22 . The method of  claim 21  wherein the payload is 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. 
     
     
         23 . The method of  claim 21 , wherein the payload is an IL-2, an IL-12, an IL-15, a membrane bound IL-15, an IL-18 or a TNFα. 
     
     
         24 . The method of  claim 21 , wherein the target cell is a cancer cell. 
     
     
         25 . The method of  claim 24 , wherein the cancer cell is in a solid tumor cell. 
     
     
         26 . The method of  claim 21 , wherein the natural killer cell further comprises a membrane bound IL-15. 
     
     
         27 . The method of  claim 24 , wherein the ligand is a MUC1, a CD19, or a CD123. 
     
     
         28 . The method of  claim 24 , wherein the cancer cell is from a leukemia, a lymphoma, or an ovarian cancer. 
     
     
         29 . The method of  claim 27 , wherein the cancer cell is a lymphoma cell. 
     
     
         30 . The method of  claim 27 , wherein the payload is an IL-2, an IL-12, an IL-15, a membrane bound IL-15, an IL-18 or a TNFα. 
     
     
         31 . The method of  claim 28 , wherein the payload is an 11-2, an IL-12, an IL-15, a membrane bound IL-15, an IL-18 or a TNFα. 
     
     
         32 . The method of  claim 27 , wherein the payload is an IL-2. 
     
     
         33 . The method of  claim 27 , wherein the payload is an IL-12. 
     
     
         34 . The method of  claim 27 , wherein the payload is an IL-15. 
     
     
         35 . The method of  claim 27 , wherein the payload is an IL-18. 
     
     
         36 . The method of  claim 27 , wherein the payload is a TNFα. 
     
     
         37 . The method of  claim 27 , further comprising the step of killing the target cell. 
     
     
         38 . The method of  claim 25 , wherein the payload is an IL-2, an IL-12, an IL-15, a membrane bound IL-15, an IL-18 or a TNFα. 
     
     
         39 . The method of  claim 27 , wherein the RDE is from a 3′-UTR of INFg or a 3′-UTR of IL6. 
     
     
         40 . The method of  claim 27 , wherein the RDE is a SEQ ID NO: 30.