Reversible Transgene Expression Reduces Fratricide and Permits 4-1BB Costimulation of CAR T Cells Directed to T-cell Malignancies

Abstract

T cells expressing second-generation chimeric antigen receptors (CARs) specific for CD5, a T-cell surface marker present on normal and malignant T cells, can selectively kill tumor cells. We aimed to improve this killing by substituting the CD28 costimulatory endodomain (28.z) with 4-1BB (BB.z), as 28.z CD5 CAR T cells rapidly differentiated into short-lived effector cells. In contrast, 4-1BB costimulation is known to promote development of the central memory subpopulation. Here, we found BB.z CD5 CAR T cells had impaired growth compared with 28.z CD5.CAR T cells, due to increased T-cell-T-cell fratricide. We demonstrate that TRAF signaling from the 4-1BB endodomain upregulated the intercellular adhesion molecule 1, which stabilized the fratricidal immunologic synapse between CD5 CAR T cells. As the surviving BB.z CD5 CAR T cells retained the desired central memory phenotype, we aimed to circumvent the 4-1BB-mediated toxicity using a regulated expression system that reversibly inhibits CAR expression. This system minimized CAR signaling and T-cell fratricide during <i>in vitro</i> expansion in the presence of a small-molecule inhibitor, and restored CAR expression and antitumor function of transduced T cells <i>in vivo</i> These studies reveal a mechanism by which 4-1BB costimulation impairs expansion of CD5 CAR T cells and offer a solution to mitigate this toxicity. <i>Cancer Immunol Res; 6(1); 47-58. ©2017 AACR</i>.