Comparison of single copy gene-based duplex quantitative PCR and digital droplet PCR for monitoring of expansion of CD19-directed CAR T cells in treated patients

Chimeric antigen receptor (CAR) T cell therapy with axicabtagene ciloleucel, tisagenlecleucel and brexucabtagen ciloleucel has been adopted as the standard of care for patients with refractory and/or relapsed CD19-positive lymphoid malignancies. Monitoring of kinetics of CAR T cells after administration is crucial for patient follow-up and important to guide clinical decisions for patients subjected to CAR T cell therapy. Information of transgene copies within a CAR T cell product prior to administration, i.e. vector copy numbers, is of high importance to warrant patient safety. However, experimental assays for quantitative CAR T cell monitoring in the open domain are currently lacking. Several institutions have established in-house assays to monitor CAR T cell frequencies. In the present study, the quantitative (q)PCR assay established at the Heidelberg University Hospital (Heidelberg, Germany), i.e. single copy gene-based duplex qPCR, was compared with the digital droplet PCR assay established at the University Medical Center Hamburg-Eppendorf (Hamburg, Germany). Both methods that were independently developed enable accurate and comparable CAR T cell frequency assessment and are useful in the clinical setting.