2.1. Plasmid construction and lentivirus preparation
The target TR1419CAR consisted of TR1419 scFv, CD28 or 4-1BB costimulatory domains, and CD3-ζ signaling domain [24,25]. They were obtained by overlap PCR amplification, PCR products were clone into lentivirus vector pWPXL and verified by enzymatic digestion and sequencing. To produce the lentivirus supernatant, 8×105 HEK-293T cells were plated in 6-well. After 24h, 293T cells were transfected with pMD.2G encoding VSV-G envelope, pSPAX2 lentivirus plasmid, and the CAR-pWPXL plasmids by using xfect Transfection Reagent (Takara), according to the manufacturer’s instructions. The lentivirus supernatants were collected and filtered with 0.45 μM filter at 48h after transfection.