Lentivirus vector construction and production
DNA sequences encoding MAGE-B2-TCR alpha and beta chains separated by a self-cleaving T2A peptide were cloned into the pALD-Lenti expression vector (Aldevron) by isothermal assembly, with an EF1a promoter driving TCR gene expression. A total of five MAGE-B2 TCRs were tested. DNA sequences encoding CARs or GFP driven by a MSCV promoter were also cloned into a lentiviral expression vector for evaluation. Lentivirus was generated by transient transfection of HEK293 cells with third generation LVV packaging plasmids (p-ALD-Lenti, Aldevron) and the expression construct. Lentivirus was harvested from the supernatant 3 days post-transfection,concentrated using high-speed centrifugation, formulated in a sucrose solution and stored at -80 degC(Gandara, Affleck, & Stoll, 2018). Infectious titers were determined by using a flow cytometry cell-based assay on HEK293 and Jurkat cells(Kutner, Zhang, & Reiser, 2009).