Patient derived CAR T cell expressing the Suicide gene iC9 represent a safe approach in case of CAR+ leukemia relapse

Use of chimeric antigen receptor T cells (CAR T) for treatment of relapsing/refractory acute lymphoblastic leukemia (ALL) has been accompanied by exciting clinical results. However, lentiviral vector-based clinical approaches revealed a potential risk of relapse associated with CD19-/CAR+ leukemic clones. To verify if also use of a γ-retroviral platform may lead to emergence of CD19-/CAR+ leukemia cells, either peripheral blood or bone marrow-derived mononuclear cells of patients with >40% of blasts at diagnosis (CD45dim+/CD34+/CD19+/CD22+/CD10+), were transduced with a second-generation CAR.CD19 in frame with a suicide gene (namely, inducible caspase 9, iC9). Patient-derived CAR-T cells showed a phenotype not significantly different from that seen in healthy-donors. In particular, both flow-cytometry analysis and Real-Time-quantitative PCR failed to identify leukemic cells in the final CAR-T cell product generated from B-cells precursor ALL (Bcp-ALL) patients. In vitro studies also showed that CD19+ leukemic cell lines transduced with the iC9.CAR.CD19 vector, mimicking the model in which leukemic cells could be genetically modified with CAR, were efficiently eliminated by activation of iC9. Moreover, by applying a long-term time-frame in vivo mouse experiment, and we provided evidences that use of the γ-retroviral CAR platform is not associated with the expansion of leukemia cells characterized by CAR positivity. Taken together these data suggest that the use of a γ-retroviral platform with the addition of the iC9 suicide gene allows to obtain a highly functional and safe CAR.T product, even when the production starts from biological material characterized by heavy contamination with leukemia blasts.