CAR construction and generation of CAR Lentivirus

A codon-optimized single-chain fragment variant (ScFv) comprising the V_H and V_L segments of the humanized, and affinity-matured antibody of 7G3, CSL362, was synthesized and developed¹³. The ScFv was fused to a CAR backbone comprising a short IgG4-Fc hinge spacer, a CD28 transmembrane, CD28 followed by an OX40 co-stimulatory moiety, and the CD3ζ signaling domain. The anti-CD123 CAR lentivirus was produced in HEK-293T cells. Briefly, 293T cells were plated one day prior to transfection and cultured in hi-glucose Dulbecco’s minimum essential medium (DMEM) supplemented with 10% FCS and 1% penicillin/streptomycin to achieve 60-80% confluence on the day of transfection. On the day of transfection, 5.5 μg each of the anti-CD123 lentiviral DNA, pMDL-pRRE, pRSV-REV, and PMD2.G packaging plasmid DNA were added to serum-free media (Opti-MEM; Sigma Aldrich, Germany) and Lipofectamine 3000. The mixture was added to the HEK-293T cells following 30 min incubation at room temperature (RT). Cell culture media was replaced after 16 h and cultured for a further 48 h. The viral supernatant was harvested, centrifuged, and filtered through a 0.45 µm membrane filter. The viral supernatant was concentrated by ultracentrifugation, resuspended in 1/150th of the original culture volume with cold sterile 1× PBS, and stored in single-use aliquots at −80 °C until use.