Abstract 3150: GD2-specific chimeric antigen receptor T cells targeting retinoblastoma
Authors: Jatuporn Sujjitjoon, La-ong Sri Atchaneeyasakul, Shih-Ting Tsao, Elias Sayour, Pa-thai Yenchitsomanus, Lung-Ji Chang
Journal: Cancer Research (2015)

Abstract Introduction: Retinoblastoma (RB) is an aggressive childhood cancer of high incidence (1/20,000). The conventional treatment is enucleation followed by chemotherapy. Patients with poor prognosis often face resistant relapses after chemo- and radiation therapies. Immunotherapy based on chimeric antigen receptor (CAR)-engineered T cells targeting cancer-specific antigens has shown great potential in treating hematological malignancies. This study aims to identify RB-specific surface antigens and investigate the RB tumor targeting function of a 4th generation CAR-T cells with specificity for disialoganglioside 2 (GD2) protein. Methods: We examined RB surgical specimens from eight patients for GD2 expression by immunohistochemistry, and a RB cell line (Y79RB) by flow cytometry. Activated T-cells were engineered to express a 4th generation CAR specific to GD2 protein by using lentiviral transduction. Y79RB cells were labeled with a green fluorescence protein and used as target cells in a CAR-T/RB co-culture experiment. Tumor cell lysis was monitored under a fluorescent microscope, and apoptotic cell death was analyzed by AnnexinV/PI staining and flow cytometry. Results: All eight RB tumor specimens were strongly positive for GD2 based on immunohistochemistry. In addition, GD2 expression was detected on the cell surface of Y79RB at a very high level by flow cytometry analysis as compared with known GD2 -positive tumor cells such as neuroblastoma and Ewing's sarcoma. In vitro co-culture experiments showed that Y79RB cells were targeted and killed by GD2 CAR-Ts but not control CD19 CAR-Ts. At a ratio of 1:1, the RB cells were completely eradicated after 3 days of co-culture with GD2 CAR-Ts. When we increased the tumor cell ratio in the coculture, we observed tumor resistance to GD2 CAR-Ts. Further analysis of the escaped Y79RB cells indicated that their surface GD2 expression was down-regulated. In addition, we found that PD-L1 was up-regulated in Y79RB cells when cocultured with GD2 CAR-Ts but not control CD19 CAR-Ts. We further demonstrated that the increased PD-L1 expression in Y79RB interacted with PD-1 on activated CAR-Ts and suppressed the CAR-T effector functions. Conclusion: This is the first report that GD2-specific CAR-Ts can effective target and kill RB. Decreased CAR-T killing efficiency may occur due to the up-regulation of PD-L1 and down-regulation of GD2 expressions on the tumor cells. The combination of GD2-specific CAR-Ts and anti-PD-L1 antibody may improve the CAR-T immunotherapy efficacy. Citation Format: Jatuporn Sujjitjoon, La-ong Sri Atchaneeyasakul, Shih-Ting Tsao, Elias Sayour, Pa-thai Yenchitsomanus, Lung-Ji Chang. GD2-specific chimeric antigen receptor T cells targeting retinoblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2015-3150