While chimeric antigen receptor (CAR)-T immunotherapies have shown remarkable efficacy against leukemias and lymphomas, improved CAR-T efficacy and persistence are needed to overcome solid tumors, without compromising safety. Here, we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to their cognate ligands. Cell activation is controlled by the homodimerizer rimiducid that triggers signaling cascades downstream of MyD88 and CD40 via an engineered chimeric protein termed iMC. A rapamycin-controlled pro-apoptotic switch, iRC9, is co-expressed, which induces dimerization of the caspase-9 domain to mitigate possible toxicity from excessive CAR-T function. When combined with a first generation CAR, these molecular switches allow for specific and efficient regulation of engineered T cells.
Methods & Results:
T cells were activated and co-transduced with the HER2 GoCAR (SFG-iMC.2A-CAR.ζ) and RapaCIDe (SFG-iRC9.2A-ΔCD19) vectors to generate “Dual-switch GoCAR-T” cells. Combined transduction of RapaCIDe and HER2 GoCAR vectors into T cells did not adversely affect the antitumor efficacy of the GoCAR-T cells, which eliminated OE19 esophageal tumor cells in a 7-day coculture assay at a 1:20 effector to target ratio (3.9±4.3% OE19-eGFPFluc cells remained in GoCAR-modified cultures vs. 1.1±0.1% for the dual-switch GoCAR), and promoted T cell expansion (53.4±9.4% CAR+ for GoCAR vs. 44.6±13.2% for the dual-switch). When challenged in an OE19 tumor-bearing mouse model, rimiducid stimulation of the dual-switch GoCAR-T cells enhanced tumor killing and T cell expansion.
Deployment of the off-switch induced fast (½ Vmax ~ 8 hours) and efficient elimination of T cells (Dual-switch GoCAR-T = 94.1% AnnV+/PI+ vs. GoCAR-T = 5.1%) in a caspase-3 activation assay with real-time (IncuCyte) monitoring as well as AnnV/PI detection by flow cytometry. In vivo assessment of the RapaCIDe switch was performed via eGFPluciferase (eGFPfluc)-labeled RapaCIDe-T cells in NSG mice. Rapamycin, but not rimiducid, treatment efficiently eliminated RapaCIDe-T cells within 24 hours, which is similar to the rate observed by the clinically validated rimiducid-regulated CaspaCIDe switch.
Dual-switch GoCAR-T, a novel platform comprising a first-generation CAR combined with regulated costimulation and apoptotic signaling elements, effectively controlled tumor growth and T cell expansion and elimination in vitro and in vivo. This dual-switch technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells.
Citation Format: MyLinh T. Duong, Matthew R. Collinson-Pautz, Eva Morschl, Mary E. Brandt, Ming Zhang, Kevin W. Slawin, Aaron E. Foster, J. Henri Bayle, David M. Spencer. Dual-switchGoCAR-T cells: small molecule-regulated “GO” and “STOP” switches to target solid cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-184. doi:10.1158/1538-7445.AM2017-LB-184