Figure 1.

Characterization and in vitro activity of ErbB2CAR-T cells: (A) The ErbB2CAR construct is comprises the ScFv specifically directed against ErbB2 followed by the co-stimulation domain of CD28 and the activation domain from FcγR. (B,C): Retroviral supernatant collected from ErbB2-expressing packaging cells (PG13) was used to infect PBLs from a healthy donor. Infected and non-infected cell cultures were grown for an additional 3 days following transduction. On the day they were harvested, the cells were analyzed by flow cytometry and the CAR expression level was calculated. (B) A representative flow cytometry analysis of lymphocytes transduced with ErbB2CAR-GFP. (C) Average transduction expression of the ErbB2CAR-GFP. The dots indicate four independent experiments, and the lines show mean ± STDEV. (D) Expression of CD4/CD8 gated from CD3 positive cells out of ErbB2CAR-GFP transduced T cells. Transductions of four donors are shown; a line indicates the average. (E) Expression of ErbB2 antigen on the human OC cell lines NAR, OVCAR8, and SKOV3. Cells were stained with anti-human ErbB2-APC or mouse IgG1k-APC isotype control and analyzed by flow cytometry. (F) Killing assay using effector (E) and target cells (T). Cells were incubated for 16 h with ErbB2CAR or UNT T cells (as control) at different E:T ratios as indicated. The percentage of killing was measured by methylene blue colorimetric assay. An average of five ErbB2CAR versus UNT experiments are presented with ± SEM. * p < 0.05; ** p < 0.01. (G) T cells transduced with ErbB2CAR (black bars) or UNT cells (gray bars) were co-cultured with the indicated ovarian cell lines at an E:T ratio of 2:1. After 16 h, IFNγ levels in culture supernatants were measured by ELISA. The results shown are the average of three experiments ±STDEV. ErbB2CAR versus UNT * p < 0.05; ** p < 0.01. F/G statistical analysis was performed using Student’s t-test between ErbB2CAR versus UNT treatment.