CAR T cells generated using Sleeping Beauty transposon vectors and expanded with an EBV-transformed lymphoblastoid cell line (LCL) display antitumor activity in vitro and in vivo.

CAR T cell immunotherapy for the treatment of cancer is now an approved treatment for B-cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR-T cell therapies. Non-viral integrative vectors such as Sleeping Beauty (SB) transposons provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBζ CAR T cells using a lymphoblastoid cell line (LCL), and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to Mock condition. CAR+ lymphocytes showed increased proliferation compared to Mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared to day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NSG mice grafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBζ CAR T cells showed improved overall survival when compared to Mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the LCL provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.