Adapter CAR-T cells to counteract T cell exhaustion and enable flexible targeting in AML

Although the landscape for treating acute myeloid leukemia (AML) patients has changed substantially in recent years, the majority of patients will eventually relapse and succumb to their disease. Allogeneic stem cell transplantation provides the best anti-leukemia treatment strategy, but is only suitable in a minority of patients. In contrast to B-cell neoplasias, chimeric antigen receptor (CAR) T-cell therapy in AML has encountered challenges in target antigen heterogeneity, safety, and T-cell dysfunction. We developed a Fab-based adapter CAR (AdCAR) T-cell platform with flexibility of targeting and control of AdCAR T-cell activation. Utilizing AML cell lines and a long-term culture assay for primary AML cells, we were able to demonstrate AML-specific cytotoxicity using anti-CD33, anti-CD123, and anti-CLL1 adapter molecules in vitro and in vivo. Notably, we show for the first time the feasibility of sequential application of adapter molecules of different specificity in primary AML co-cultures. Importantly, utilizing the AML platform, we were able to demonstrate that continuous adapter molecule exposure led to AdCAR T-cell exhaustion through chronic AdCAR stimulation, which was counteracted through treatment-free intervals. As CAR T-cell exhaustion is a well-known cause of resistance, the AdCAR platform might ameliorate CAR T-cell dysfunction. Adapter CAR T-cells of three healthy donors stimulated continuously with anti-CD33 adapter molecule or with a treatment-free interval (TFI) from day 7-14 were sorted and analyzed. Samples: Continuously stimulated = day 0, 14, 21; TFI-stimulated = day 14, 21.