BackgroundChimeric antigen receptor (CAR)-T cells have emerged as a breakthrough treatment for relapse/refractory hematological tumors, showing impressive complete remission rates. However, around 50% of the patients relapse before 1-year post-treatment. T-cell ‘fitness’ is critical to prolong CAR-T persistence and activity. Allogeneic T cells from healthy donors are less dysfunctional or exhausted than autologous patient-derived T cells; in this context, Delta One T cells (DOTs), a recently described cellular product based on MHC/HLA-independent Vδ1+γδ T cells, represent a promising allogeneic platform.MethodsHere we generated and preclinically validated, for the first time, 4-1BB-based CAR-DOTs directed against the interleukin-3α chain receptor (CD123), a target antigen widely expressed on acute myeloid leukemia (AML) blasts.ResultsCD123CAR-DOTs showed vigorous, superior to control DOTs, cytotoxicity against AML cell lines and primary samples both in vitro and in vivo, even on tumor rechallenge.ConclusionsOur results provide the proof-of-concept for a DOT-based next-generation allogeneic CAR-T therapy for AML.

BackgroundThe dismal clinical outcome of relapsed/refractory (R/R) T cell acute lymphoblastic leukemia (T-ALL) highlights the need for innovative targeted therapies. Although chimeric antigen receptor (CAR)-engineered T cells have revolutionized the treatment of B cell malignancies, their clinical implementation in T-ALL is in its infancy. CD1a represents a safe target for cortical T-ALL (coT-ALL) patients, and fratricide-resistant CD1a-directed CAR T cells have been preclinically validated as an immunotherapeutic strategy for R/R coT-ALL. Nonetheless, T-ALL relapses are commonly very aggressive and hyperleukocytic, posing a challenge to recover sufficient non-leukemic effector T cells from leukapheresis in R/R T-ALL patients.MethodsWe carried out a comprehensive study using robustin vitroandin vivoassays comparing the efficacy of engineered T cells either expressing a second-generation CD1a-CAR or secreting CD1a x CD3 T cell-engaging Antibodies (CD1a-STAb).ResultsWe show that CD1a-T cell engagers bind to cell surface expressed CD1a and CD3 and induce specific T cell activation. Recruitment of bystander T cells endows CD1a-STAbs with an enhancedin vitrocytotoxicity than CD1a-CAR T cells at lower effector:target ratios. CD1a-STAb T cells are as effective as CD1a-CAR T cells in cutting-edgein vivoT-ALL patient-derived xenograft models.ConclusionsOur data suggest that CD1a-STAb T cells could be an alternative to CD1a-CAR T cells in coT-ALL patients with aggressive and hyperleukocytic relapses with limited numbers of non-leukemic effector T cells.

Chimeric Antigen Receptor (CAR)-T cells have emerged as a breakthrough treatment for relapse/refractory (r/r) hematological tumors, showing impressive complete remission rates in B-cell malignancies. However, around 50% of the patients relapse before 1-year post-treatment. T-cell fitness is critical to prolong the persistence and activity of the adoptively transferred product. Allogeneic T cells from healthy donors are less dysfunctional or exhausted than autologous patient-derived T cells, enabling a very attractive and cost-effective off-the-shelf therapy option. In this context, Delta One T cells (DOTs), a recently described cellular product based on MHC/HLA-independent Vd1+ gd T cells generated from the peripheral blood of healthy donors, represent a robust platform of allogeneic effector T cells. Here we generated and pre-clinically validated 4-1BB-based CAR-DOTs directed against the IL-3a; chain receptor (CD123), a target antigen widely expressed on acute myeloid leukemia (AML) blasts. CD123CAR-DOTs showed vigorous, superior to control DOTs, cytotoxicity against AML cell lines and primary samples both in vitro and in vivo. Continuous administration of IL-15 supported the long-term persistence of a single-dose CD123CAR-DOTs in patient-derived xenograft models, sustaining their anti-leukemic efficacy as demonstrated in a re-challenge assay in vivo. Our results provide proof-of-concept for an allogeneic next-generation therapy based on CD123CAR-DOTs for r/r AML patients.