Key Points: Genomic disruption of CD7 prior to CAR transduction allows generation of CD7 CAR T cells without extensive self-antigen-driven fratricide. CD7 CAR T cells have robust activity against T-cell malignancies in vitro and in vivo.

Antigen-independent tonic signaling by chimeric antigen receptors (CARs) can increase differentiation and exhaustion of T cells, limiting their potency. Incorporating 4-1BB costimulation in CARs may enable T cells to resist this functional exhaustion; however, the potential ramifications of tonic 4-1BB signaling in CAR T cells remain unclear. Here, we found that tonic CAR-derived 4-1BB signaling can produce toxicity in T cells via continuous TRAF2-dependent activation of the nuclear factor κB (NF-κB) pathway and augmented FAS-dependent cell death. This mechanism was amplified in a non-self-inactivating gammaretroviral vector through positive feedback on the long terminal repeat (LTR) promoter, further enhancing CAR expression and tonic signaling. Attenuating CAR expression by substitution with a self-inactivating lentiviral vector minimized tonic signaling and improved T cell expansion and anti-tumor function. These studies illuminate the interaction between tonic CAR signaling and the chosen expression platform and identify inhibitory properties of the 4-1BB costimulatory domain that have direct implications for rational CAR design.

Chimeric antigen receptor (CAR) T cell therapy for the treatment of acute myeloid leukemia (AML) has the risk of toxicity to normal myeloid cells. CD7 is expressed by the leukemic blasts and malignant progenitor cells of approximately 30% of AML patients but is absent on normal myeloid and erythroid cells. Since CD7 expression by malignant blasts is also linked with chemoresistance and poor outcomes, targeting this antigen may be beneficial for this subset of AML patients. Here, we show that expression of a CD7-directed CAR in CD7 gene-edited (CD7KO) T cells effectively eliminates CD7+ AML cell lines, primary CD7+ AML, and colony-forming cells but spares myeloid and erythroid progenitor cells and their progeny. In a xenograft model, CD7 CAR T cells protect mice against systemic leukemia, prolonging survival. Our results support the feasibility of using CD7KO CD7 CAR T cells for the non-myeloablative treatment of CD7+ AML.

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 in vitro expansion in the presence of a small-molecule inhibitor, and restored CAR expression and antitumor function of transduced T cells in vivo. 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. Cancer Immunol Res; 6(1); 47–58. ©2017 AACR.