TP53 -mutant acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS) are characterized by chemotherapy resistance and represent an unmet clinical need. Chimeric antigen receptor (CAR) T-cells might be a promising therapeutic option for TP53 -mutant AML/MDS. However, the impact of TP53 deficiency in AML cells on the efficacy of CAR T-cells is unknown. We here show that CAR T-cells engaging TP53 -deficient leukemia cells exhibit a prolonged interaction time, upregulate exhaustion markers, and are inefficient to control AML cell outgrowth in vitro and in vivo compared to TP53 wild-type cells. Transcriptional profiling revealed that the mevalonate pathway is upregulated in TP53 -deficient AML cells under CAR T-cell attack, while CAR T-cells engaging TP53 -deficient AML cells downregulate the Wnt pathway. In vitro rational targeting of either of these pathways rescues AML cell sensitivity to CAR T-cell-mediated killing. We thus demonstrate that TP53 deficiency confers resistance to CAR T-cell therapy and identify the mevalonate pathway as a therapeutic vulnerability of TP53 -deficient AML cells engaged by CAR T-cells, and the Wnt pathway as a promising CAR T-cell therapy-enhancing approach for TP53 -deficient AML/MDS.

CAR T-cell products targeting lineage-specific cell-of-origin antigens, thereby eliminating both tumor and healthy counterpart cells, are currently clinically approved therapeutics in B- and plasma-cell malignancies. While they represent a major clinical improvement, they are still limited in terms of efficacy by e.g. single, sometimes low-expressed antigen targeting, and in terms of safety by e.g., lack of on-off activity. Successful cell-of-origin non-discriminative targeting of heterogeneous hematopoietic stem and progenitor cell malignancies, such as acute myeloid leukemia (AML), will require antigen-versatile targeting and off-switching of effectors in order to then allow rescue by hematopoietic stem cell transplantation (HSCT), preventing permanent myeloablation. To address this, we developed adaptor-CAR (AdFITC-CAR) T-cells targeting fluoresceinated AML antigen-binding diabody adaptors. This platform enables the use of adaptors matching the AML-antigen-expression profile and conditional activity modulation. Combining adaptors significantly improved lysis of AML cells in vitro. In therapeutic xenogeneic mouse models, AdFITC-CAR T-cells co-administered with single diabody adaptors were as efficient as direct CAR T-cells, and combinatorial use of adaptors further enhanced therapeutic efficacy against both, cell lines and primary AML. Collectively, this study provides proof-of-concept that AdFITC-CAR T-cells and combinations of adaptors can efficiently enhance immune-targeting of AML.

Background: Chimeric antigen receptor (CAR) T cells targeting CD19 induce durable responses in B-cell acute lymphoblastic leukemia (B-ALL). However, the contribution of the tumor microenvironment to the therapeutic response after CAR T-cell treatment remains incompletely understood. Methods: We performed single-cell RNA sequencing and spectral flow cytometry-based analyses of bone marrow-resident immune cells from B-ALL patients before and after CAR T-cell treatment. Results: We observed profound changes in the microenvironment in response to CAR T-cell-mediated inflammation, including an increase in myeloid cells. Significant induction of the interferon response, hypoxia, and TGF-β signaling was accompanied by expansion of myeloid-derived suppressor cells (MDSCs) and endogenous exhausted CD8+ T cells. PD-1 expression in endogenous T cells post-treatment was associated with a lack of durable response in the cohort of patients analyzed. Further, we revealed that HIF-1α, VEGF, and TGFBR2 are key players in the intercellular communication between CAR T cells and the immune niche, potentially driving widespread T-cell dysfunction. Infusion of anti-CD19 CAR T cells led to increased accumulation of human MDSCs, exacerbation of a hypoxic environment and T-cell exhaustion in hematopoietic stem/progenitor cell-humanized mice bearing a human tumor. Conclusions: In conclusion, CAR T-cell-mediated myeloid activation is associated with pathways of immune dysregulation that may antagonize the effects of therapy.