An oxygen sensitive self-decision making engineered CAR T-cellA key to the success of chimeric antigen receptor (CAR) T-cell based therapies greatly rely on the capacity to identify and target antigens with expression restrained to tumor cells. Here we present a strategy to generate CAR T-cells that are only effective locally (tumor tissue), potentially also increasing the choice of targetable antigens. By fusing an oxygen sensitive subdomain of HIF1α to a CAR scaffold, we generated CAR T-cells that are responsive to a hypoxic environment, a hallmark of certain tumors. Along with the development of oxygen-sensitive CAR T-cells, this work also provides a basic framework to use a multi-chain CAR as a platform to create the next generation of smarter self-decision making CAR T-cells.
651. Integration of Dual Signal Inputs Strategies in Novel Chimeric Antigen Receptors to Control the CAR T-Cell FunctionsAdoptive immunotherapy using engineered T-cells has emerged as a powerful approach to treat cancer. The potential of this approach relies on the ability to redirect the specificity of T cells through genetic engineering. Novel specificities in T cells have been typically implemented through the genetic transfer of the so-called chimeric antigen receptors (CARs). CARs are synthetic receptors composed of an extracellular targeting moiety and one or more intracytoplasmic signaling domain derived from lymphocyte activation receptors. Present CAR architectures are designed to combine all relevant domains within a single polypeptide, thereby; they combine advantages of MHC unrestricted target recognition to the potent native effector mechanisms of the T cell. Although adoptive transfer of CAR T cells is proven to be an effective cancer therapy, potential adverse effects such as cytokine release syndrome (CRS) and/or the risk of on-target off-tumor targeting are still a major concern. Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. Our ability to engineer synthetic systems in primary T-cells that function as Boolean logic gates responding to multiple inputs would benefit adoptive immunotherapy using engineered T-cells. Exogenous or endogenous environmental signal integration by a modular AND gate may represent an important advancement in improving our control of the safety of the CAR T-cell technology. Here, we describe the development of novel CAR designs that integrate new components directly within the CAR architecture to improve our capacity to spatiotemporally control and switch the CAR T-cells functions between on and off states. In particular, we showed that such a system can be engineered to control the CAR through addition of an exogenous small molecule (Rapamycin or synthetic rapalogs) ultimately inducing the cytolytic properties of the engineered T-cell. Alternatively, properties of the tumor microenvironment can also be used as additional endogenous input to the target antigen recognition. We showed that low oxygen levels can be used to trigger the CAR surface presentation, creating a so called “self-decision making” CAR T-cell.