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-AML 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 established 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, using the AML platform, we were able to demonstrate that chronic T-cell stimulation and exhaustion can be counteracted through introduction of treatment-free intervals. As T-cell exhaustion and target antigen heterogeneity are well-known causes of resistance, the AdCAR platform might offer effective strategies to ameliorate these limitations.

Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

Currently, only a few chimeric antigen receptor (CAR) T cell therapies have been approved by the Food and Drug Administration and European Medicines Agency for the treatment of B-cell malignancies. To enable broader application of the CAR T cell technology in other indications, improved control and flexible targeting of multiple tumor antigens are required. Here, we developed a novel adapter CAR (AdCAR) T cell platform for flexible targeting of multiple tumor antigens. This platform is based on a short peptide tag derived from an interdomain region of fibroblast growth factor receptor 2 (FGFR2), commonly mutated in cancer. To select AdCARs specific for mutated FGFR2-derived peptide tags, a multistep pooled screening approach in primary T cells was employed, incorporating MACS separation and next-generation sequencing. The resulting AdCAR was highly specific for the FGFR2-derived peptide tag. Using different in vitro and in vivo model systems, the activity of AdCAR T cells was shown to be strictly dependent on the presence of the adapter and corresponding target antigen. Moreover, AdCAR T cells could be redirected to different target antigens by the addition of respective adapter molecules (AM). Finally, in situ expression of functional AM in primary T cells under control of a drug-inducible promoter system was demonstrated, highlighting the potential for controlling the activity of AdCAR T cells by cellular micropharmacies.