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 for expressing a transgene, comprising the steps of:
(a) 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 destabilizing 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, wherein an RDE binding protein binds to the RDE and regulates expression of the transgene;
(b) binding the primary T-cell to a target ligand for the chimeric antigen receptor at a target site in a subject wherein binding of the target ligand by the chimeric antigen receptor activates the primary T-cell, wherein activation of the primary T-cell changes a metabolic state of the primary T-cell and this changes binding of the RDE by the RDE binding protein; and
(c) expressing the transgene wherein the amount of polypeptide made from the transgene is increased after activation of the cell by the chimeric antigen receptor.
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, a checkpoint inhibitor, a chimeric antigen receptor or a membrane bound receptor.
3. The method of claim 2 , wherein the transgene encodes a cytokine.
4. The method of claim 3 , wherein the cytokine is an IL-12.
5. The method of claim 1 , wherein the primary T-cell displays an amount of 10 to 100,000 chimeric antigen receptors per primary T-cell.
6. The method of claim 5 , wherein the primary T-cell displays 100 to 5,000 chimeric antigen receptors.
7. The method of claim 1 , wherein the target cell is an AML, cell.
8. The method of claim 7 , wherein the target ligand is a CD43.
9. The method of claim 8 , further comprising the step of killing the AML cell.
10. 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.
11. The method of claim 10 , wherein the glycolytic enzyme is a glyceraldehyde phosphate dehydrogenase.
12. The method of claim 11 , wherein the activation of the primary T-cell increases a glycolysis wherein the activation of the glycolysis results in a reduction of binding of the RDE in the transcript by the glyceraldehyde phosphate dehydrogenase.
13. The method of claim 12 , further comprising the step of binding a second RDE binding protein to the RDE in the transcript.
14. The method of claim 13 , wherein binding of the second RDE binding protein increases a half-life of the transcript, or a translation rate of the transcript.