LAUSR

Background Anti-CD19 chimeric antigen receptor (CAR)-expressing T cells have shown remarkable remission rates in acute B cell leukemias. It has been reported that anti-CD19 CAR-T cells sustain complete remission of leukemia even when their numbers in circulation are below the limit of detection by flow cytometry (FC) and detectable only by quantitative PCR (qPCR). Thus, developing a monitoring strategy that combines high sensitivity and multiparametric immunophenotypic evaluation is crucial to predict and improve the clinical response to CAR-T cell therapies. Methods To facilitate CAR-T cell tracking by FC in preclinical models, a new lentiviral vector was made by cloning the enhanced green fluorescent protein (EGFP) gene downstream the anti-CD19 CAR gene. The specificity of this new vector was assessed in vitro and in vivo. We also established a high sensitive protocol for qPCR-based detection of CAR-T cell to be used in clinical and preclinical models. Results After transduction of T cells with the new vector, we found that all CAR+ cells (44.8%) were also EGFP+. We next assessed the in vitro cytotoxicity of CAR19GFP cells by coculturing them with luciferase (luc)-expressing tumor cell lines either CD19+ (RAJI) or CD19− (K562). Within 24h of coculture, bioluminescence data showed 75% of eradication of CD19+ tumoral cells and sparing of CD19− tumor cells, demonstrating functionality and selectivity. Lastly, NSG mice previously grafted with RAJI-luc cells were treated with a single intravenous injection of CAR19GFP cells. This treatment promoted a 1000-fold reduction in tumor burden and prolonged survival. Flow cytometry analysis of peripheral blood showed a steady persistence of Conclusion Combined, this data demonstrates the generation of a fully functional in vitro and in vivo CAR/EGFP reporter vector and the establishment of a protocol for molecular detection of CAR-T cells. This CAR-T cell-monitoring platform will provide invaluable data in preclinical and clinical studies on CAR-T cell persistence, a parameter intrinsically linked to therapeutic efficacy. Support: FAPESP grants 2013/08135-2,2019/18672-1 and 2019/18702-8.