Comparative analysis and optimization of protocols for producing recombinant lentivirus carrying the anti-Her2 chimeric antigen receptor gene.

BACKGROUND: The production of anti-Her2 chimeric antigen receptor (CAR) T cells needs to be optimized to make it a reliable therapy.

METHODS: Three types of lentiviral vectors expressing anti-Her2 CAR together with packaging plasmids were co-transfected into 293 T-17 cells. The vector with the best packaging efficiency was selected, and the packaging cell culture system and packaging plasmid system were optimized. Centrifugation speed was optimized for the concentration of lentivirus stock. The various purification methods used included membrane filtration, centrifugation with a sucrose cushion and the novelly-designed instantaneous high-speed centrifugation. The recombinant lentiviruses were transduced into human peripheral T cells with an optimized multiplicity of infection (MOI). CAR expression levels by three vectors and the efficacy of CAR-T cells were compared.

RESULTS: When co-transfected, packaging cells in suspension were better than the commonly used adherent culture condition, with the packaging system psPAX2/pMD2.G being better than pCMV-dR8.91/pVSV-G. The optimal centrifugation speed for concentration was 20 000 g, rather than the generally used ultra-speed. Importantly, adding instantaneous centrifugation for purification significantly increased human peripheral T cell viability (from 13.25% to 62.80%), which is a technical breakthrough for CAR-T cell preparation. The best MOI value for transducing human peripheral T cells was 40. pLVX-EF1a-CAR-IRES-ZsGreen1 expressed the highest level of CAR in human peripheral T cells and the cytotoxicity of CAR-T cells reached 63.56%.

CONCLUSIONS: We optimized the preparation of recombinant lentivirus that can express third-generation anti-Her2 CAR in T cells, which should lay the foundation for improving the efficacy of CAR-T cells with respect to killing target cells.