Improved Homing of Antigen-Specific T Cells to Hodgkin’s Disease (HD) Tumor Cells by Forced Expression of CCR4 Receptor.
Authors: Di Stasi A. , Dotti G. , Foster A. , Zhang L. , Heslop Helen H. , Rooney C. , Brenner Malcolm K. , Savoldo B. 
Published: April 2020

Abstract
One crucial requirement for the success of any adoptive T cell transfer is that the effector T cells should migrate efficiently to the tumor site. Such an effect has been documented following adoptive transfer of Epstein-Barr virus specific cytotoxic T cells (EBV-CTLs). Antigenic stimulus from (normal and malignant) cells persistently infected with EBV led to expansion and sustained survival, and was also associated with activity against the EBV+ HD tumors. Since most HD patients have tumors that are EBV- but CD30+, we attempted to extend this approach by incorporating a CD30 chimeric receptor (CD30CAR) into the EBV-CTLs. Pre-clinical animal studies showed that CD30CAR+ EBV-CTLs readily migrated to EBV+/CD30+ tumors, but had limited capacity to localize to EBV−/CD30+ tumor cells. The likeliest explanation for this observation is that EBV- HD tumors produce the chemokine TARC, which attracts Th2 and regulatory T cells, but has little effect on EBV-CTLs, since these express low levels of the TARC receptor, CCR4. We hypothesized that forced expression of CCR4 by redirected EBV-CTLs would improve their homing to the EBV−/CD30+ HD cells. The full length of CCR4 receptor was cloned into the SFG retroviral vector and used to transduce both activated T cells and EBV-CTLs obtained from 6 and 4 healthy donors, respectively. Expression of CCR4 was 12±8% on activated T cells (mainly on CD4+ cells, 12±6%) and 4±5% on EBV-CTLs. After transduction with a CCR4, but not a control vector, expression of CCR4 increased to 53±24% (CD4+ 27±15% and CD8+ 17±9%) on activated T cells and 30±19% on EBV-CTLs. We then evaluated the capacity of control and transgenic T cells and EBV-CTLs to migrate in response to TARC, using a trans-well migration assay. Migration was tested against different CD30+ tumor lines producing TARC at low (Karpas wild type, &lt;32 pg/ml) or high levels (Karpas engineered to produce TARC, HDLM-2 and L428, all TARC &gt;2000pg/mL), measured by ELISA. The percent of cells migrating in the trans-well assay was significantly increased for CCR4 transgenic CD8+ selected T cells (54±11% with Karpas/TARC vs. 8±2% with media vs. 8±4% with Karpas-wt). Migration of control OKT3/28 blasts was less than 15% in all the conditions. Migration was significantly inhibited by the addition of antibody blocking TARC (&lt;10%). An improved migration of CCR4+ CD8+ cells was observed also towards HDLM-2 (48±7%) and L428 (45±16%) as compared to control T cells (21±7% and 23±7%, respectively). Similarly, CCR4+ EBV-CTL showed improved migration towards Karpas/TARC (26±14%) as compared to Karpas/wt (12±8%) or media (10±7%). This genetic modification did not modify either the phenotype or the antigen specificity of EBV-CTLs, which retained the capacity to kill autologous EBV+ lymphoblastoid cells. We then determined if forced expression of CCR4 receptor was functional in vivo. To track homing of transgenic T cells, we transduced them with the FireFlyluciferase (FFLuc) vector and followed signal with a bioluminescent system (IVIS, Xenogen). Sublethally irradiated SCID mice were implanted s.c. with Karpas/wt on the left side and Karpas/TARC on the right side. When tumors were measurable, FFLuc+CCR4+ T cells were injected iv. By 24 hours post injection, signal was detectable only at the side of TARC secreting tumor cells, and signal persisted at 10 to 16 fold higher levels on the TARC+ side for &gt;7 days. These data suggest that the migration of CARCD30 EBV-CTL to EBV−/CD30+ HD can be augmented by co-expressing the CCR4 receptor.