9531 Background: Genetic manipulation of T cells may provide promising new therapies for pediatric solid tumors. T cells can be modified to express chimeric antigen receptors (CAR), which link the antigen-recognition region of an antibody to a CD3-zeta signaling domain and various costimulatory domains. CARs allow for non-MHC-restricted tumor recognition, followed by T cell activation, expansion and cytolytic activity. We constructed a series of CARs specific for the disialoganglioside GD2. GD2 is a well-established target for neuroblastoma, with anti-GD2 moAb therapy improving survival in high risk neuroblastoma and GD2-CAR therapy showing clinical activity in this disease. GD2-CARs also have preclinical activity against melanoma and GD2 expression has been reported in some rhabdomyosarcomas, osteosarcomas and Ewing sarcomas. Methods: We observed that GD2 is overexpressed in the following established laboratory and recent patient-derived cell lines: Rh18 (rhabdomyosarcoma: 94.8% expression), 143B and MG63 (osteosarcoma: 78.5% and 98.2%, respectively) and Tc71 (Ewing sarcoma: 96.1%). These cell lines were used to evaluate our GD2-CARs in vitro. We compared activity between second generation, with one costimulatory molecule (CD28), and third generation, with two costimulatory molecules (CD28 & CD137), GD2-CAR constructs, with or without an extracellular CH2-CH3 hinge domain from human IgG1. Results: Second and third generation GD2-CAR modified T cells show similar high level lysis of GD2+ rhabdomyosarcoma, osteosarcoma and Ewing sarcoma cell lines as seen with neuroblastoma lines. Addition of the CH2-CH3 hinge region consistently improved cytolytic activity by 5-10%, measured by chromium-release assay, suggesting that this extracellular structural alteration may improve CAR function. Studies evaluating the expression of GD2 in tumor tissues and the efficacy of GD2-CAR modified T cells in osteosarcoma and rhabdomyosarcoma xenograft models are underway. Conclusions: GD2-CAR based therapy may prove effective for patients with GD2+ rhabdomyosarcoma, osteosarcoma and Ewing sarcoma in addition to patients with neuroblastoma and melanoma.