Figure 3.

TILs can be successfully transduced with anti-Her2 CAR.

TILs were thawed and activated using CD3/28 beads with IL-2 (600 IU/ml) and transduced with retroviral vector in the presence of retronectin and selected with G418 for expression of anti-He2-CAR. Non-transduced TILs underwent the transduction protocol in the presence of an empty viral vector. Cells were examined for CAR expression using flow cytometry. TIL-CAR were stained with c-Myc tag (red), with an isotype (IgG2A; blue). Cells are gated on morphology, viability, CD3⁺ and c-Myc tag staining. CAR expression was calculated based on <1% staining of the non-transduced CAR stained with c-Myc tag. (a) Maximal staining of transduction of TIL#10 and (b) TIL#11 transduced with anti-Her2 CAR. (c) CAR expression using flow cytometry shown for multiple transductions. Results are expressed with mean and SEM for each patient. (d) CAR density on transduced T cells using a proprietary bead standard kit (Simply Cellular Beads, BANGSLABS laboratories). CAR density on TILs was pooled and data shown ± SEM performed in triplicate wells.

CAR, chimeric antigen receptor; PBMC, peripheral blood mononuclear cell; TIL, tumor-infiltrating lymphocyte