Immunotherapy of acute leukemia by chimeric antigen receptor-modified lymphocytes using an improved Sleeping Beauty transposon platform

// Chiara F. Magnani 1 , Nice Turazzi 1 , Fabrizio Benedicenti 2 , Andrea Calabria 2 , Erika Tenderini 2 , Sarah Tettamanti 1 , Greta M.P. Giordano Attianese 1, 3 , Laurence J.N. Cooper 3 , Alessandro Aiuti 2 , Eugenio Montini 2 , Andrea Biondi 1 , Ettore Biagi 1 1 Tettamanti Research Center, Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital/Fondazione MBBM, Monza, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 3 University of Texas MD Anderson Cancer Center, Houston, Texas, USA Correspondence to: Andrea Biondi, email: abiondi.unimib@gmail.com Ettore Biagi, email: e.biagi@hsgerardo.org Keywords: chimeric antigen receptor, Sleeping Beauty transposon, cytokine-induced killer cells, acute lymphoblastic leukemia, acute myelogenous leukemia Received: February 11, 2016     Accepted: May 20, 2016     Published: June 13, 2016 ABSTRACT Chimeric antigen receptor (CAR)-modified T-cell adoptive immunotherapy is a remarkable therapeutic option proven effective in the treatment of hematological malignancies. In order to optimize cell manufacturing, we sought to develop a novel clinical-grade protocol to obtain CAR-modified cytokine-induced killer cells (CIKs) using the Sleeping Beauty (SB) transposon system. Administration of irradiated PBMCs overcame cell death of stimulating cells induced by non-viral transfection, enabling robust gene transfer together with efficient T-cell expansion. Upon single stimulation, we reached an average of 60% expression of CD123- and CD19- specific 3rd generation CARs (CD28/OX40/TCRzeta). Furthermore, modified cells displayed persistence of cell subsets with memory phenotype, specific and effective lytic activity against leukemic cell lines and primary blasts, cytokine secretion, and proliferation. Adoptive transfer of CD123.CAR or CD19.CAR lymphocytes led to a significant anti-tumor response against acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) disseminated diseases in NSG mice. Notably, we found no evidence of integration enrichment near cancer genes and transposase expression at the end of the differentiation. Taken all together, our findings describe a novel donor-derived non-viral CAR approach that may widen the repertoire of available methods for T cell-based immunotherapy.