Acute myeloid leukemia (AML) remains a significant challenge in oncology, with high relapse rates despite advancements in treatment. Allogeneic stem cell transplantation offers the best chance of remission but is only applicable to a limited number of patients. Chimeric antigen receptor (CAR) T-cell therapy, highly effective in B-cell neoplasms, has proven less successful in AML. This is primarily due to three key factors: the heterogeneous expression of target antigens on AML cells, safety concerns related to severe side effects, and the rapid onset of T-cell dysfunction or exhaustion. This research addresses these issues by developing a novel CAR T-cell platform that offers greater flexibility in targeting and activation, enhances safety, and reduces T-cell exhaustion. The study aims to establish the efficacy and safety of this new platform in pre-clinical models, paving the way for clinical trials.

The existing literature highlights the limitations of current CAR T-cell therapies in AML. Studies have shown limited success with CAR T-cells targeting CD123 or combinations of CD33/CLL1, with only a minority of patients experiencing favorable outcomes. The phenomenon of T-cell exhaustion, characterized by decreased effector function and increased expression of inhibitory markers like PD-1 and TIM-3, is a significant obstacle to long-term efficacy. Previous research has suggested that intermittent T-cell stimulation, through treatment-free intervals (TFIs) or molecular switches, can mitigate exhaustion and enhance CAR T-cell persistence. This study builds upon this knowledge, proposing a novel approach to overcome these limitations.

The researchers developed a Fab-based adapter CAR (AdCAR) platform. In vitro cytotoxicity assays were performed using AML cell lines (MV-41, HL-60, OCAML-3) and primary AML (pAML) cells in long-term co-cultures. Different adapter molecules (AMs) targeting CD33, CD123, and CLL-1 were employed, assessing specific lysis using flow cytometry. Internalization assays, utilizing pHrodo Red Avidin labeling, were conducted to study receptor-mediated endocytosis of AMs. Long-term AdCAR T-cell stimulation assays with treatment-free intervals (TFIs) were implemented to evaluate T-cell exhaustion. In vivo studies were performed in NOD.scid.gc-IL2Rγnull/Il2rg-/- mice engrafted with OC-AML2 cells, assessing leukemia growth by bioluminescence imaging (BLI). RNA sequencing was used to analyze transcriptional changes in AdCAR T-cells under continuous versus intermittent stimulation.

The AdCAR T-cell platform demonstrated specific and efficient lysis of AML cell lines and pAML cells in vitro and in vivo. Sequential targeting using different AMs against CD33, CD123, and CLL-1 proved successful in eliminating pAML cells in ex vivo long-term co-cultures. Receptor-mediated endocytosis of AMs was observed, potentially impacting AM half-life and cytotoxicity. Prolonged continuous stimulation of AdCAR T-cells led to T-cell exhaustion, characterized by reduced cytotoxicity and increased expression of inhibitory markers. The introduction of treatment-free intervals (TFIs) effectively counteracted T-cell exhaustion, leading to improved AdCAR T-cell functionality and persistence. RNA sequencing revealed transcriptional reprogramming in AdCAR T-cells under TFI conditions, with downregulation of cell cycle and metabolic pathways associated with exhaustion and upregulation of genes associated with effector function. In vivo studies using a highly aggressive AML model showed equivalent leukemia growth elimination by AdCAR T-cells compared to conventional CD33 CAR T-cells.

This study demonstrates the potential of the AdCAR T-cell platform as a novel approach for AML immunotherapy. The platform's flexibility in targeting multiple antigens addresses the heterogeneity of AML, while the use of TFIs effectively mitigates T-cell exhaustion. The shorter half-life of Fab-based AMs compared to antibody-based formats contributes to a potentially safer therapeutic profile by allowing for better controlled activation and reducing the risk of cytokine release syndrome. The findings highlight the importance of considering receptor-mediated endocytosis in AM design and dosing regimens. Future research should focus on optimizing AMs to further enhance their efficacy and persistence, as well as exploring the clinical translation of this platform.

The AdCAR T-cell platform offers a promising approach for AML immunotherapy, addressing key limitations of current CAR T-cell therapies. Its flexibility, safety profile, and ability to counteract T-cell exhaustion warrant further investigation in clinical trials. Future studies should focus on refining AM design, optimizing treatment schedules, and exploring combinations with other therapeutic modalities.

The study primarily utilized pre-clinical models. While the findings are encouraging, further research is needed to confirm the efficacy and safety of the AdCAR T-cell platform in clinical settings. The study focused on a limited number of AML-associated antigens; further investigation is needed to determine the platform's effectiveness against other AML targets and subtypes.