The outcome of acute myeloid leukemia (AML) remains poor, and immunotherapy has the potential to improve this. T cells expressing chimeric antigen receptors (CARs) or bispecific T cell engagers targeting CD123 are actively being explored in preclinical and/or early phase clinical studies. We have shown that T cells expressing CD123-specific bispecific T cell engagers (CD123.ENG T cells) have anti-AML activity. However, like CAR T cells, their effector function diminishes rapidly once they are repeatedly exposed to antigenpositive target cells. Here we sought to improve the effector function of CD123.ENG T cells by expressing inducible costimulatory molecules consisting of MyD88 and CD40 (iMC), MyD88 (iM), or CD40 (iC), which are activated by a chemical inducer of dimerization (CID). CD123.ENG T cells expressing iMC, iM, or iC maintained their antigen specificity in the presence of CID as judged by cytokine production (IFNc, IL-2) and their cytolytic activity. In repeat stimulation assays, activating iMC and iM, in contrast to iC, enabled CD123.ENG T cells to secrete cytokines, expand, and kill CD123-positive target cells repeatedly. Activating iMC in CD123.ENG T cells consistently improved anti-tumor activity in an AML xenograft model. This translated into a significant survival advantage in comparison to mice that received CD123.ENG or CD123.ENG.iC T cells. In contrast, activation of only iM in CD123.ENG T cells resulted in donor-dependent antitumor activity. Our work highlights the need for both toll-like receptor (TLR) pathway activation via MyD88 and provision of costimulation via CD40 to consistently enhance the antitumor activity of CD123.ENG T cells.
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