These authors contributed equally to this work.Chimeric antigen receptor (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. Nonviral 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 19BBf CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell 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 with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBf 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 lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.
Recently approved by the FDA and European Medicines Agency, CART cell therapy is a new treatment option for B-cell malignancies. Currently, CART cells are manufactured in centralized facilities and face bottlenecks like complex scaling up, high costs, and logistic operations. These difficulties are mainly related to the use of viral vectors and the requirement to expand CART cells to reach the therapeutic dose. In this paper, by using Sleeping Beauty-mediated genetic modification delivered by electroporation, we show that CART cells can be generated and used without the need for ex vivo activation and expansion, consistent with a point-of-care (POC) approach. Our results show that minimally manipulated CART cells are effective in vivo against RS4;11 leukemia cells engrafted in NSG mice even when inoculated after only 4 h of gene transfer. In an effort to better characterize the infused CART cells, we show that 19BBz T lymphocytes infused after 24 h of electroporation (where CAR expression is already detectable) can improve the overall survival and reduce tumor burden in organs of mice engrafted with RS4;11 or Nalm-6 B cell leukemia. A side-by-side comparison of POC approach with a conventional 8-day expansion protocol using Transact beads demonstrated that both approaches have equivalent antitumor activity in vivo. Our data suggest that POC approach is a viable alternative for the generation and use of CART cells, overcoming the limitations of current manufacturing protocols. Its use has the potential to expand CAR immunotherapy to a higher number of patients, especially in the context of low-income countries.
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