Abstract 4754: Targeting of Ewing sarcoma by CAR-reengineered GD2-specific T cells

Abstract

Abstract Primary metastatic Ewing sarcoma involving the bone and/or bone marrow has remained largely incurable. Relapses after intensive multimodal treatment regimens are attributed to a rare subpopulation of residual cells capable of reinitiating tumor growth. Cellular immunotherapy with chimeric antigen receptor (CAR) transduced T cells is a promising strategy to eradicate minimal residual disease and prevent relapse. Based on the observation that Ewing sarcoma cells express the ganglioside antigen GD2, we have explored the capacity of T cells transduced with GD2-specific CARs to functionally interact with Ewing sarcoma cells. Surface GD2 expression was detected on 10 of 11 Ewing sarcoma cell lines by flow cytometry analysis, and on 11 of 12 primary Ewing tumor samples by immunofluorescence staining. To obtain GD2-specific effector T cells, CD3/CD28 stimulated T cells from five healthy donors were retrovirally transduced with the second generation GD2-specific CAR 14.G2aCD28ζ, resulting in CAR surface expression in 83.8±2.7% (81.1-87.8%) of the cells. Ewing sarcoma cell lines expressing various levels of GD2 were used as target cell lines for 14.G2aCD28ζ-mediated cytolysis in a 51Cr release assay. Both GD2hi VH-64 and GD2intermediate Cado-ES-1 cells were efficiently lysed by gene-modified T cells (36.6±7.7% and 25.3%±8.2 lysis at a 40:1 effector-to-target cell ratio) in the absence of background cytolysis of GD2-negative tumor cells and by non-transduced T cells. Moreover, efficient and antigen-specific granzyme B secretion was found after coincubation of various Ewing sarcoma cell lines and a primary Ewing sarcoma cell culture with CAR gene-modified T cells. 14.G2aCD28ζ T cells further eradicated Ewing sarcoma cells in a serum-free spheroid model for anchorage-independent micrometastatic tumor growth. Functional activation of CAR-expressing T cells in response to GD2 positive Ewing sarcoma cells was further demonstrated by significant secretion of IFN-γ and TNFα and significant proliferative responses to various cell lines expressing low, intermediate or high levels of GD2. Thus, GD2 is a promising target antigen for the redirected antitumor activity of T cells not only in neuroblastoma and melanoma, but also in Ewing sarcoma. To address the preclinical in vivo efficacy of 14.G2aCD28ζ T cells, we have now established an in vivo model of disseminated disease by intravenous transfer of Ewing sarcoma cells to sublethally irradiated NOD/scid mice. Magnetic resonance imaging allows to visualize metastatic tumor manifestations in xenotransplanted mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4754. doi:10.1158/1538-7445.AM2011-4754