Improved in vivo migration and antitumor activity of T cells transduced with a bicistronic vector encoding both CCR4 and CAR-CD30. For in vivo experiments, T cells transduced with CAR-CD30 or the bicistronic vector encoding CCR4 and CAR-CD30 were further transduced to express eGFP-FFLuc to monitor their migration in vivo in SCID mice using the IVIS system. (A) The expression of eGFP-FFLuc on CAR-CD30⁺ and CCR4⁺CAR-CD30⁺ cells in one representative donor. (B) The bioluminescence signal from T cells in 3 representative SCID mice engrafted with TARC⁻ tumor (Karpas/wt) on the left side and the TARC⁺ tumor (Karpas/TACR) on the right side. Whereas no significant expansion of the bioluminescent signal was observed at either tumor site in mice receiving CAR-CD30⁺ T cells (top panels), a significant increase of bioluminescence was observed in mice receiving CCR4⁺CAR-CD30⁺ T cells (bottom panels) at the site of tumor-producing TARC. (C) The fold change of bioluminescence signal between K/wt and K/TARC site for CAR-CD30⁺ (□) and CCR4⁺CAR-CD30⁺ (■) T cells. Data are mean ± SD of 8 mice. Data confirm that CCR4 expressed by T cells using a bicistronic vector remain functional in vivo. To evaluate in vivo antitumor activity of T cells transduced with the bicistronic vector, the HL-derived cell line HDLM-2 was further transduced with FFLuc and inplanted subcutaneously into NOG-SCID mice (7 mice/group). Tumor growth was monitored by measuring bioluminescence signals with the IVIS system. Mice then received intravenous T cells transduced with retroviral vectors encoding either CCR4 or CAR-CD30, or the bicistronic vector CCR4(I)CAR-CD30. (D) The bioluminescence signal of tumor cells in 4 representative mice/group. In mice receiving CCR4⁺ or CAR-CD30⁺ T cells, the bioluminescence signal, and thus tumor, increased over time. In contrast, in mice that received CCR4⁺CAR-CD30⁺ T cells, the signal remained stable, indicating tumor control. (E) The data from all 7 mice/group.