Improved migration of activated T lymphocytes genetically modified to overexpress CCR4 and a CAR targeting the CD30 antigen expressed by HL. A bicistronic vector encoding CCR4 and CAR-CD30 was constructed and used to transduce T cells from 8 healthy donors. (A) The expression of CCR4 and CAR-CD30 by T cells transduced with a retroviral vector encoding CAR-CD30 (□) or with a bicistronic retroviral vector encoding CCR4 and CAR-CD30 (■). Surface expression of the CCR4 and CAR was evaluated by FACS analysis. (B) The migration of CAR-CD30⁺ (□) and CCR4⁺CAR-CD30⁺ (■) T cells toward TARC gradients, using a transwell migration assay. T-cell migration was evaluated using culture supernatants collected from HDLM-2 and L428, which physiologically produce high amounts of TARC, and against Karpas genetically modified to produce TARC (K/TARC). Karpas wild type (K/wt) was used as a control. The panel indicates that migration toward TARC is significantly improved for T cells genetically modified to overexpress CCR4 using the bicistronic vector CCR4(I)CAR-CD30. This improved migration was TARC mediated as it was inhibited by addition of anti-TARC antibodies but not by addition of an isotype control. (C) Killing of CD30⁺ (HDLM-2, ; Karpas, ■) and CD30⁻ (□) tumor cells by CAR-CD30⁺ and CCR4⁺CD30-CAR⁺ T cells. (D) The measurement of IL-2 cytokine released in the supernatant of T cells cocultured with or without HDLM-2 and assessed using a specific ELISA assay. T cells were transduced with either CAR-CD30 or CCR4(I)CAR-CD30, where CAR molecules also incorporate the CD28 endodomain. As a control, T cells were also transduced with the same CAR targeting CD30 but lacking the CD28 endodomain (CD30CARζ). As anticipated, we observed enhanced production of IL-2 by T cells transduced with the CAR containing the CD28 endodomain (CAR-CD30), regardless of coexpression of CCR4, but not by T cells transduced with CD30CARζ lacking CD28. The figure indicates that T cells transduced with CCR4(I)CAR-CD30 vector produce IL-2 in amounts comparable with that of T cells transduced with the vector encoding CAR-CD30 alone, confirming that the CD28 pathway is not impaired by the coexpression of CCR4. Data are mean ± SD of 10 donors.