Abstract LB-141: A unified dual-switch CAR vector to target solid cancer with controllable "on" and "off" states
Authors: MyLinh Duong, Eva Morschl, Aruna Mahendravada, Matthew Collinson-Pautz, Mary Brandt, Ming Zhang, Aaron Foster, J. Henri Bayle, David Spencer
Journal: Cancer Research (2018)

Abstract Background Despite impressive efficacy in liquid tumors, improved CAR-T efficacy and persistence appear necessary to control solid tumors, but this increased potency will likely increase the risk of toxicity. Here, we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to cognate ligands. Cell activation is controlled by rimiducid (Rim), which triggers signaling cascades downstream of MyD88 and CD40 via an engineered chimeric protein termed iMC. A rapamycin (Rap)-controlled apoptotic switch, iRC9, is co-expressed, which induces dimerization of caspase-9 active domains to mitigate possible toxicity from excessive CAR-T function. When combined with a first generation CAR in a single vector, these molecular switches allow for specific and efficient regulation of engineered T cells to eliminate HER2+ and PSCA+ cancer cells. Results Transduction of activated T cells using γ-retroviruses encoding the unified dual-switch (DS) HER2- or PSCA-CAR components (SFG-iRC9.2A-CAR.ζ.2A-iMC) yielded efficient transgene expression despite the large insert size (60.5±8.7% CAR+). Greater transduction efficiency was achieved with single-cell producer clones encoding the PSCA DS CAR vector (79.3±2.9% CAR+). When exposed to target antigen in a coculture assay with tumor cells, robust IL-2 and IFN- γ production by DS CAR-T cells was Rim-dependent. Tumor cell killing and T cell expansion were also enhanced by Rim stimulation. In an OE19 tumor-bearing mouse model, Rim stimulation of HER2 DS CAR-T cells significantly enhanced tumor killing (205.8±58.3 mm2 vehicle (veh)-treated vs 55.9±10.9 mm2 Rim-treated, p&amp;lt;0.05) and T cell expansion (4.6E5 average radiance veh-treated vs 1.9E6 Rim-treated). When targeting PSCA+ HPAC cancer cells, PSCA DS CAR-T cells proliferated in a Rim dose-dependent manner and tumor control was maintained even after cessation of Rim treatment. In a second pancreatic tumor model, SU8686, Rim treatment induced long-term tumor control (&amp;gt;80 days). When necessary, deployment of the off-switch (iRC9) rapidly (½ Vmax ~ 8 hours) and efficiently eliminated DS CAR-T cells in a caspase-3 activation assay with real-time (IncuCyte) monitoring, as well as AnnV/PI detection by flow cytometry (3.2±1.0% AnnV+/PI+ veh-treated vs 80.0±5.2% 10nM Rap-treated, p&amp;lt;0.005). In vivo assessment of the iRC9 switch was performed via eGFPluciferase (eGFPfluc)-labeled PSCA dual-switch CAR-T cells in NSG mice. Temsirolimus (Tem; a Rap prodrug) treatment efficiently eliminated PSCA DS CAR-T cells within 6 hours (6.1E4 average radiance veh-treated vs 2.1E4 0.4mg/kg Tem-treated, p&amp;lt;0.0001). Summary Dual-switch CAR-T, a novel platform comprising a CAR combined with regulated costimulation and apoptotic signaling elements, effectively control solid tumor growth and T cell expansion and elimination. This technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells. Citation Format: MyLinh Duong, Eva Morschl, Aruna Mahendravada, Matthew Collinson-Pautz, Mary Brandt, Ming Zhang, Aaron Foster, J. Henri Bayle, David Spencer. A unified dual-switch CAR vector to target solid cancer with controllable "on" and "off" states [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-141.