A real-time quantitative PCR targeting the viral vector for the monitoring of patients treated with axicabtagene ciloleucel.

Baras, Agathe; Bocket, Laurence; Beauvais, David; Hallaert, Christophe; Varlet, Pauline; Agha, Ibrahim Yakoub; Hober, Didier; Alidjinou, Enagnon Kazali

Axicabtagene ciloleucel or axi-cel (CD19-CAR T-cells) has been recently approved for refractory/ relapsed diffuse large B cell lymphoma and primary mediastinal B-cell lymphoma. Proliferation of CAR T-cells after infusion and their persistence have been reported as important factors. Laboratory tools are needed for the monitoring of patients. We developed a vector-based, simple and accurate real-time qPCR to measure axi-cel vector copy number (VCN) in peripheral blood samples. Primers and probe targeting the 5'LTR region of the gammaretroviral vector (mouse stem cell virus or MSCV) were designed for amplification. To generate standard curves MSCV plasmid was subcultured and quantified using droplet digital PCR (ddPCR). The method was applied to quantify VCN in blood samples from patients treated with axi-cel. The limit of quantification of the qPCR assay was established at 2.2 copies/μL in DNA eluate. The qPCR method was well correlated with flow cytometry (FC) findings; however, the assay appeared to be more sensitive than FC. The kinetics observed in blood samples from treated patients was in agreement with previously reported findings. In conclusion, we developed a sensitive and accurate qPCR assay for the quantification of transgenic CAR T-cells, which can be a useful additional tool for the monitoring of patients treated with of axi-cel.