FIGURE 1.

Recognition of ErbB2-expressing tumor cell lines by Herceptin 4D5 CAR-transduced PBLs. A, ErbB2 expression of tumor cell lines. An Affibody reagent specific for ErbB2 was used to determine expression on tumor cell lines (solid line), and an isotype Ab was used as negative control (dotted line). B, A schematic representation of the retroviral vector MSGV-4D5–28Z (4D5–28Z) encoding the Herceptin (4D5)-based CAR against ErbB2. SP, signal peptide; VL, variable L chain, VH, variable H chain. Supplemental Fig. 1 shows the complete sequence of the CAR and primers used for synthesizing the scFv. C, Cytokine production of 4D5 CAR-transduced T cells. IFN-γ secretion of 4D5–28Z-transduced T cells following coculture with the indicated tumor lines. Nontransduced T cells (NV) were used as a negative control. D, Specific tumor cell lysis by 4D5 CAR-transduced T cells. 4D5 CAR-transduced PBLs (4D5–28Z) were tested in a ⁵¹Cr release assay, where nontransduced PBLs (NV) served as a control. PBLs were cocultured with ⁵¹Cr-labeled control tumor line MDA468 (ErbB2^–), and ErbB2⁺ tumor lines SK-OV3, SK-BR3, and 624.38mel at the indicated E:T ratio for 4 h, after which the percentage lysis of target cells was calculated. E, Proliferation of 4D5 CAR-transduced PBLs. 4D5–28Z or NY-ESO-1 TCR (1G4-AIB)-transduced PBLs were cocultured with tumor lines MDA468 (ErbB2^–/NY-ESO-1^–), MDA231 (ErbB2⁺/NY-ESO-1^–), and 624.38mel (ErbB2⁺/NY-ESO-1⁺) for 3 days. [³H]thymidine ([³H]TdR) was added for the final 17 h of culture and incorporation was measured. Data are representative of three experiments.