Engineered CAR T-cell Constructs

The ErbB2-specific scFv 4D5 (25 (link)) sequence derives from the humanized mAb that was used to produce Herceptin. The sequence for the anti-VEGFR2-specific scFv 2C6 (26 (link)) was derived from a human Ab. Both CARs were designed and synthesized by PCR using a web-based DNA codon optimization algorithm (27 (link)). Supplemental Fig. 1 shows the sequences of the 4D5 and 2C6 scFvs (including the linker sequence) and the primers used to synthesis these genes.⁴ The synthesized DNA fragments were sequence confirmed and subcloned in frame into MSGV-1-based vector (28 (link)) containing CD28 and CD3ζ signaling moieties (12 (link)) to generate MSGV-4D5–28Z or MSGV-2C6–28Z. Variant signaling domains were constructed by mega-primer overlap PCR of specific signaling domains and assembled in the order described in figure legends (details available upon request). An MSGV-1-based vector encoding TCRα and TCRβ specific for NY-ESO-1 (MSGV-1G4-AIB) was described previously (29 (link)). FMC63–28Z is a CAR vector in which scFv against human CD19 derived from mAb FMC63 as described (30 (link)) was constructed and subcloned into MSGV-1-based retroviral vector containing CD28-CD3ζ cassette. A trinitrophenyl-specific CAR, SP6–28Z CAR (22 (link)), and a CAR containing the extracellular domain of LNGFR were used as controls.