Fig. 2.

Human T cells can be specifically redirected toward melanoma cells. (A) CD3⁺ T cells obtained from a healthy volunteer (allogeneic) and from melanoma patient 1 (autologous) were engineered by retroviral gene transfer to express the HMW-MAA, the melanotransferrin (mtf), and the CD20-specific CAR, respectively. Anti-CD19 CAR and mock-transduced T cells were used as controls. CAR expression on the cell surface was monitored by flow cytometry using a PE-conjugated antihuman IgG1 mAb, which binds to the CAR extracellular spacer region, and a FITC-conjugated anti-CD3 mAb. (B) To record antigen specificity of CAR-mediated T-cell activation, engineered CD3⁺ T cells with the HMW-MAA, CD19, and the CD20-specific CAR, respectively, and unmodified T cells without CAR (w/o) were coincubated for 48 h in serial dilutions with MeWo cells and Daudi cells (each 2.5 × 10⁴ cells per well), respectively. IFN-γ secreted into the culture supernatant, was recorded by ELISA. (C) Primary melanoma cells (5 × 10⁴ cells per well) from a melanoma lesion of patient 1 were coincubated with 2.5 × 10⁴ allogeneic CAR-expressing T cells per well or 0.625 × 10⁴ autologous CAR-expressing T cells per well, both without (w/o) or with CARs specific for mtf, HMW-MAA, and CD20, respectively. The ratios of engineered T cells: tumor cells were 1:2 in the allogeneic and 1:8 in the autologous settings. Specific cytotoxicity was recorded after 48 h. Data represent the mean of triplicates ± SEM. (D) Engineered T cells are cytolytic against HMW-MAA⁺ tumor cells in an in vivo coincubation assay. Allogeneic or autologous T cells engineered to express the HMW-MAA– or the mtf-specific CAR (1 × 10⁶ CAR-expressing T cells) or mock-modified T cells without CAR (w/o) were s.c. inoculated together with 1 × 10⁶ melanoma cells in NIH-III mice. Growth of the individual tumors was recorded; the bold line represents the mean tumor growth. The overall survival of treated mice is shown in a Kaplan–Meier plot. The median survival time and the P values of significance compared with mice treated with T cells without CAR (w/o) were calculated using the log-rank test and the XLSTAT2010 software.