Abstract
BackgroundEngineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells. Therefore, the development of strategies to control CAR expression at the surface of T-cells and their functions is under intense investigations.ResultsHere, we report the development and evaluation of an off-switch directly embedded within a CAR construct (SWIFF-CAR). The incorporation of a self-cleaving degradation moiety controlled by a protease/protease inhibitor pair allowed the ex vivo tight and reversible control of the CAR surface presentation and the subsequent CAR-induced signaling and cytolytic functions of the engineered T-cells using the cell permeable Asunaprevir (ASN) small molecule.ConclusionsThe strategy described in this study could, in principle, be broadly adapted to CAR T-cells development to circumvent some of the possible hurdle of CAR T-cell manufacturing. This system essentially creates a CAR T-cell with an integrated functional rheostat.
Highlights
Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity
We focused on a strategy that would allow us to control the stability and degradation properties of the Chimeric antigen receptor (CAR) at the protein level using a small molecule
We fused the degradation moiety, composed of a protease target site, the HCV NS3 protease, and the degron, to the C-terminal end of the CAR, such that upon cleavage at the protease target site, a short 8-amino-acid sequence would remain at the C-terminus of the CD3z domain that contains the Immunoreceptor tyrosine-based activation motif (ITAM)
Summary
Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. In the past few years, the adoptive transfer of engineered T-cells has emerged as a key player in the development of new treatments against cancer [1, 2] The success of such therapies relies, in part, on the ability to engineer chimeric antigen receptor (CAR) to target tumor cells that present a predefined antigen. Adoptive T-cell therapy with CARexpressing T-cells targeting the B cell antigen CD19 have induced durable, sustained antitumor responses in patients with leukemias and lymphomas Inspired by this success, the scientific community has been quickly extended the number and identity of targeted tumor antigen far beyond CD19, raising new challenges in the antigen selection and for the manufacturing of these engineered cells. There is still a need for systems that precisely control CAR T-cell functions ex vivo in order to circumvent some of the difficulties encountered during manufacturing of these engineered cells and to overall expand and improve the possibilities for producing CAR T-cells targeting novel tumor antigens
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