Resumen de Pre-tcr specific car-t cells as a novel immunotherapy for t-cell acute lymphoblastic leukaemia
T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy that primarily affects children and young adults. Despite therapeutic advances, the disease remains challenging to manage, particularly in cases that are refractory to standard therapies or that relapse. Relapsed/refractory (r/r) T-ALL has limited therapeutic options and poor prognosis, especially in adults, with no standard targeted immunotherapies available so far. Therefore, developing novel, effective and safe targeted therapies for T-ALL patients is an unmet clinical need. T-cells engineered to express chimeric antigen receptors (CARs) against tumours have emerged as a powerful tool against r/r haematopoietic malignancies such as B-ALL. However, the application of CAR-T therapy to T-ALL is challenging due to the antigen sharing by malignant and healthy T-cells, which can lead to CAR-T cell fratricide and T-cell aplasia, resulting in impaired anti-tumour efficacy and life-threatening immunodeficiency. Hence, identification of antigens exclusively expressed in T-ALL blasts is critical for the development of CAR-T cells against T-ALL. The pre-TCR is a surface complex expressed in distinct T-ALL subgroups. While conflicting results have been produced on the requirement of pre-TCR in T-ALL pathogenesis, recent results from our group showed that pre-TCR is critically involved in T-ALL progression, uncovering its novel role as a biomarker of leukaemia initiating cells, which are the cells responsible for relapse. Given that pre-TCR expression in normal tissues is restricted to a particular temporal window during intrathymic T-cell development, our hypothesis is that pre-TCR should be an optimal target for TALL immunotherapy. Therefore, the general aim of this study was to develop and validate a pre- TCR specific CAR-T strategy, as a novel, safe and efficacious immunotherapy specific for T-ALL. Using a proprietary monoclonal antibody developed against the extracellular domain of the pTα chain of the human pre-TCR, we produced two 2nd-generation CAR constructs, containing different hinge, transmembrane and either CD28 or 4-1BB-derived co-stimulatory domains (CARpTα.28 and CARpTα.41BB, respectively), that were expressed on T cells. Functional assays showed that CARpTα.41BB triggered a specific and effective T-cell activation upon pre-TCR recognition, inducing robust and specific cytotoxic activity, while high constitutive cell activation derived from tonic signalling was induced by CARpTα.28. The efficacy of CARpTα.41BB T cells was further validated in vivo in xenograft models of pre-TCR+ T-ALL, in which they displayed a potent anti-leukaemic impact, hampering tumour progression of both T-ALL cell lines and patientderived T-ALLs that correlated with increased mouse survival. Notably, CARpTα.41BB T cells were fratricide resistant, persisting long-term in vivo mostly as effector memory CD8+ cells, with no symptoms of exhaustion. Collectively, our results provide proof-of-concept of a novel, specific and efficacious anti-pre-TCR CAR-T immunotherapy for the treatment of a significant number of r/r T-ALL patients
