A study on optimized lentiviral transduction conditions in CAR-T cells targeting CD30

Background and purpose: Chimeric antigen receptor T (CAR-T) cell technology has been widely used in the field of blood tumor treatment. Lentivirus transduction is a key link in the preparation of CAR-T cells, which is closely related to the quality of CAR-T cells. Therefore, the parameters involved in the lentivirus transduction process still need to be further optimized. This study aimed to investigate the effects of multiplicity of infection (MOI), incubation density, activation time before transduction and transduction system height on anti-CD30 CAR-T cells in lentiviral transduction T cells carrying CD30 antibody sequences, optimize the transduction conditions of CAR-T cells, and improve transduction efficiency and CAR-T cells function. Methods: Different MOI, incubation density, activation time before transduction and transduction system height were used to optimize the transduction of human peripheral blood-derived T cells, then proliferation capacity, transduction efficiency, cell viability and killing efficiency in vitro of anti-CD30 CAR-T cells were detected. Results: When the MOI was 1.00, 1.50 and 3.00, the transduction efficiency and effective cell number were significantly higher than those in the 0.00, 0.25 and 0.50 groups. When the incubation density was greater than 0.5×107 cells/mL, the incubation density had no significant effect on the transduction efficiency of T cells. The cell viability rate of the 72 h group was lower than 80%, which was lower compared with other groups significantly, the transduction efficiency of 24 and 48 h was significantly higher compared with the 0 h, 8 h and 16 h groups, and the expansion rate of CAR-T cells in the 48 h group was significantly higher compared with the 24 h group. The transduction efficiency and proliferation capacity of 0.16 mm in height of transduction system were significantly higher compared with the 0.53 mm group, however, had no significant effect on killing efficiency of CAR-T cells in vitro. Conclusion: Through the comprehensive evaluation of CAR-T cells function, the optimal transduction conditions for lentivirus include MOI of 1.00, incubation density of 1.0×107 cells/mL, activation before transduction for 48 h and transduction system height of 0.16 mm.