A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing

// Malvina Prapa 1 , Sara Caldrer 2 , Carlotta Spano 1 , Marco Bestagno 3 , Giulia Golinelli 1 , Giulia Grisendi 1 , Tiziana Petrachi 1 , Pierfranco Conte 4 , Edwin M. Horwitz 5 , Dario Campana 6 , Paolo Paolucci 1, * , Massimo Dominici 1, * 1 Department of Medical and Surgical Sciences for Children & Adults, Division of Oncology, University-Hospital of Modena and Reggio Emilia, Modena, Italy 2 Department of Pathology and Diagnostics, University of Verona, Verona, Italy 3 International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 4 Istituto Oncologico Veneto, Padova, Italy 5 Departments of Pediatrics and Medicine, Division of Hematology/Oncology/BMT, Nationwide Children's Hospital, The Ohio State University College of Medicine, Columbus, Ohio, USA 6 Department of Pediatrics, National University of Singapore, Singapore * These authors have contributed equally to this work Correspondence to: Massimo Dominici, e-mail: massimo.dominici@unimore.it Keywords: GD2, chimeric antigen receptor, anti-GD2 IgM-derived, neuroblastoma, T lymphocytes Received: May 11, 2015 Accepted: July 08, 2015 Published: July 20, 2015 ABSTRACT Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.