ANTI-CD19 CAR T CELL TRACKING PLATFORM FOR PRECLINICAL AND CLINICAL STUDIES SHOWS GREAT TUMOR BURDEN REDUCTION LINKED TO STEADY PERSISTENCE

Abstract

Introduction: The emerging of chimeric antigen receptor (CAR) T cell therapy has shown promising results leading to the FDA approval of five CAR T cell products by now. Anti CD19 CAR T cells have shown >80% complete remission rates in acute B cell leukemias, however due to their limited persistence and exhaustion post transplantation the therapeutic efficacy is low or absent for other hematological malignancies and solid tumors. It is known that anti-CD19 CAR T cells support complete remission of leukemia and normal B cell aplasia even if their numbers in circulation are below the limit of detection by flow cytometry (FC) and only detectable by quantitative PCR (qPCR). Hence, developing a tracking strategy that benefits fromhigh sensitivity and multiparametric immunophenotypic evaluation is crucial to predict and improve the clinical response to CAR-T cell therapies. Objective: Develop a platform for tracking anti-CD19 CAR-T cells in vivo using multiparametric FC and qPCR. Methods: For easy CAR-T cell monitoring by FC in preclinical models, we generated a new lentiviral vector by cloning the enhanced green fluorescent protein (EGFP) gene downstream the anti-CD19 CAR gene. The specificity of this new vector was tested in vitro and in vivo. We also established a highly sensitive protocol for qPCR-based detection of CAR-T cell to be used in clinical and preclinical models. Results and discussion: After transduction of T cells with the new vector, all CAR+ cells (44.8%) were also EGFP+. Particularly, the median fluorescence intensity of EGFP was 3.8-fold higher than that of CAR stained with an Alexa 647-conjagated antibody. Thus, through EGFP fluorescence, this new construct allows direct CAR-T cell tracking by FC with a higher sensitivity compared to antibody staining. We next assessed the in vitro cytotoxicity of CAR-T cells by coculturing them with luciferase (luc)-expressing tumor cell lines. Within 24h of coculture, bioluminescence data showed 75% of eradication of CD19+ tumor cells (RAJI) and sparing of CD19- tumor cells (K562), demonstrating functionality and selectivity. Lastly, NSG mice previously grafted with RAJI-luc cells were treated with a single intravenous injection of CAR-T cells. This treatment promoted a 1,000-fold reduction in tumor burden and prolonged survival even with flow cytometry analysis of peripheral blood showing a stable persistence of <1% of CAR-T cells throughout the experiment. To complement our detection toolkit, we designed pair of primers and probe spanning the 4-1BB and CD3ζ domains of our anti-CD19 CAR to be amplified by qPCR. With this method, we were able to detect as little as 10 copies of the anti-CD19 CAR with a high linearity (R2 = 0.99) and an amplification efficiency of 100%. Conclusion: Combined, this data represents 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 crucial data in preclinical and clinical studies on CAR-T cell persistence, a parameter intrinsically linked to therapeutic efficacy. Funding: FAPESP grants 2013/08135-2, 2019/18672-1 and 2019/18702-8.