Abstract 2294: Reversible regulation of chimeric antigen receptor surface expression
Authors: Richman Sarah A. , Wang Liang-Chuan , Moon Edmund K. , Albelda Steven M. , Milone Michael C. 
Published: July 2016

The purpose of this study is to develop and characterize a chimeric antigen receptor (CAR) T cell platform that can be rapidly and reversibly down-regulated. The rationale for this approach is to mitigate the risk of tissue toxicity as CAR T cell therapies are expanded from hematologic malignancies to solid tumors. Whereas off-tumor/on-target activity can be tolerable in the case of the B cell hypoplasia that occurs with CART therapy for ALL, targeting of other normal tissues predicted to occur in the solid tumor setting likely will be more clinically challenging. Currently implemented design approaches to arresting CAR-mediated toxicity have involved suicide domains. While effective in triggering CAR T cell apoptosis, these approaches are not reversible, and additional therapy would require repeated infusions, which may not be feasible under certain circumstances. A system of temporary down-regulation could overcome this obstacle and provide a useful alternative to current strategies. To this end, we have fused a mouse anti-human GD2 neuroblastoma antigen-specific CAR to a LID (ligand-induced degradation) domain. The LID domain, developed at Stanford University, targets the polypeptide for degradation upon the addition of rapamycin or a similar ligand, “shield.” In the study presented here, bulk human T cells were transduced with lentivirus encoding either the control anti-GD2 CAR or the anti-GD2-CAR-LID fusion construct. Transduced T cells, after a standard procedure of stimulation and expansion, were then incubated in either standard media alone or media with shield ligand. CAR expression was measured at a range of shield ligand concentrations and at various time points up to 24 hours by flow cytometry using goat-anti mouse antibody. We observed a shield dose-response reduction in CAR expression and found that this shield-induced reduction in expression started at 8 hours and approached ∼ 20% of baseline by 24 hours. As early as 24 hours following the removal of the shield-containing media and washing, CAR surface expression had returned to ∼65% of baseline. Next, we assessed in vitro T cell effector functions including proliferation as measured by flow cytometric cell counting, IFN-gamma release as measured by ELISA, and cytotoxicity as measured by chromium release. We observed that in the presence of shield ligand, T cell effector functions were reduced to near background levels. The shield compound appeared to have no effect on the control anti-GD2 CAR that did not contain the LID domain. Our conclusion from these in vitro experiments is that the CAR-LID construct has the potential to allow for temporary and tunable down-regulation of CAR T cell activity in a clinical setting and warrants further characterization. In vivo experiments designed to test the CAR-LID T cells in a mouse model of xenografted disseminated neuroblastoma are ongoing, and we plan to expand this approach to other tumor systems.

Citation Format: Sarah A. Richman, Liang-Chuan Wang, Edmund K. Moon, Steven M. Albelda, Michael C. Milone. Reversible regulation of chimeric antigen receptor surface expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2294.