Gold optimized CAR T-cells
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.
1. A method of controlling a transgene, comprising the steps of: obtaining a primary T-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 (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 chimeric antigen receptor wherein binding of the ligand by the chimeric antigen receptor activates the primary T-cell and thereby changes a metabolic state of the primary T-cell; and 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.
2. The method of claim 1 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.
3. The method of claim 1 , wherein the transgene encodes an IL-2, an IL-12, an IL-15, an IL-18 or a TNF-α.
4. The method of claim 3 , wherein the transgene encodes an IL-12.
5. The method of claim 1 , wherein the transgene encodes a reporter.
6. The method of claim 5 , wherein the reporter can be imaged and further comprising the step of imaging a target site that expresses the ligand.
7. The method of claim 1 , wherein the ligand is an antigen found on a target cell.
8. The method of claim 7 , wherein the target cell is a cancer cell or a bacterium.
9. The method of claim 8 , wherein the target cell is a cancer cell.
10. The method of claim 9 wherein the ligand is a cancer associated antigen.
11. The method of claim 9 , wherein the cancer cell is a tumor cell.
12. The method of claim 11 , wherein the ligand is a tumor associated antigen.
13. The method of claim 8 , further comprising the step of killing the target cell.
14. The method of claim 1 , wherein the RDE in the transcript is bound by a glycolytic enzyme selected from the group consisting of a glyceraldehyde phosphate dehydrogenase, an enolase, a phosphoglycerate kinase, a triose phosphate isomerase, an aldolase A or a phosphoglycerate mutase.
15. The method of claim 14 , wherein the glycolytic enzyme is a glyceraldehyde phosphate dehydrogenase.
16. The method of claim 15 , wherein the activation of the primary T-cell induces a glycolysis wherein the activation of the glycolysis results in a reduction of binding of the RDE in the transcript by the glycolytic enzyme.
17. The method of claim 16 , further comprising the step of binding a second RDE binding protein to the RDE in the transcript.
18. The method of claim 17 , wherein binding of the second RDE binding protein increases the half-life of the transcript with the RDE.
19. The method of claim 1 , wherein the RDE is from a 3′-UTR of INFg or a 3′-UTR of IL6.
20. The method of claim 19 , wherein the RDE is a SEQ ID NO: 30.