A novel CAR-T cell approach for T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is a precursor T-cell neoplasm that has attracted attention within the scientific community. Conventional therapeutic approaches, such as chemotherapy and allogeneic transplantation, have improved patient outcomes, achieving five-year event-free survival rates ranging from 70 to 83% and an overall survival rate of approximately 80% in pediatric cases. Nevertheless, these treatments are often associated with significant toxicity and high failure rates. Moreover, refractory or relapsed (r/r) disease continues to represent a critical clinical issue, accounting for nearly 20% of patient mortality. These limitations highlight the need for novel, more effective, and targeted therapeutic strategies, among which chimeric antigen receptor (CAR)-T cell therapy is emerging as an attractive candidate. Since CAR-T cell therapy has demonstrated remarkable clinical success in B-cell malignancies, research efforts have increasingly focused on exploring its applicability to T-cell hematological disorders as well. However, in this field, the use of CAR-T treatment is particularly challenging due to both biological and technical limitations, primarily the lack of tumor-specific antigens. In T-ALL, malignant cells often share surface markers with healthy T-cells, increasing the risk of fratricide among engineered and wild-type T lymphocytes. Therefore, when designing the therapy, it is fundamental to identify targets that are differentially expressed by T-ALL cells. This strategy allows the cytotoxic activity of CAR-T lymphocytes to be directed mainly against cancer cells, minimizing damage to healthy tissue. In the present work, a surface target was identified as a potentially effective target, being reported in the literature as expressed in more than 70% of T-ALL cases, including over 85% of r/r cases. Importantly, the risk of potential on-target/off-tumor toxicity is considered very low, as this receptor has been detected only in a small fraction of normal lymphocytes (<5%). Consequently, a novel CAR construct was designed to target this surface antigen, and its functional activity was assessed. T lymphocytes used in the experiments were isolated, activated, and expanded from healthy donors, and subsequently transduced with a retroviral vector encoding a second-generation CAR construct incorporating an alternative extracellular domain for target recognition. Co-cultures of engineered CAR-T cells with T-ALL cell lines, MOLT-4 and HSB-2 (target-positive and target-negative cell line, respectively), were established. The in vitro results highlighted the potential of this novel CAR design, demonstrating specificity toward the target and a sustained anti-tumor activity over time. Furthermore, the risk of fratricide was carefully evaluated, and no such activity was observed, thus providing an additional safety feature to the product. Collectively, these findings identify this innovative CAR construct as an ideal candidate for clinical translation.