A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ–induced CD38 expression

• A single-chain CD38 T-cell engager targets CD38-negative LSCs activated by IFN-γ and induces CD38 expression on AML blasts.

• CD38 T-cell engager activates T cells against autologous leukemia cells and in AML mouse models.

Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38^pos), LSCs are frequently enriched in the CD34^posCD38^neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38^pos and CD38^neg LSC-enriched blasts. IFN-γ–induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38^pos AML cells and both CD8^pos and CD4^pos T cells. We demonstrate that BN-CD38 engages autologous CD4^pos and CD8^pos T cells and CD38^pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34^posCD38^neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34^pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.