Figure 4.

Transduced TIL#10 retain CAR expression following REP.

TILS were thawed and activated using CD3/28 beads with IL-2 (600 × IU/ml). Five days following activation they were 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 to control for any changes induced by the process of transduction. Following confirmation of successful transduction and CAR expression using flow cytometry, to increase cell numbers for experiments, TILs underwent a second activation using REP. REP is a restimulation process utilizing soluble OKT3 and irradiated donor PBMCs. Cells were examined for CAR expression using flow cytometry. Data are shown for c-Myc tag (αHer2 CAR; red) with isotype control (blue). (a) TIL#10 expressed the CAR (92%) following transduction after CD3/28 bead activation and G418 selection. (b) Following further expansion, by REP, cells maintain CAR expression (73%). Control cells transduced with an empty viral vector maintain negative expression for CAR pre-REP (c) and post-REP (d). Cells are gated on cell morphology, viability, CD3⁺ and stained for c-Myc tag expression (CAR) or isotype (IgG2A; control).

CAR, chimeric antigen receptor; OKT3, anti-CD3; PBMC, peripheral blood mononuclear cell; REP, rapid expansion protocol; TIL, tumor-infiltrating lymphocyte.