Abstract

Abstract Introduction: CD19-targeted CAR-T cell immunotherapy is now an approved treatment for B cell leukemias and lymphomas. Current protocols for producing CAR-T cells use viral vectors for genetic modification and take 12-15 days to expand sufficient numbers of T cells, constituting an important bottleneck for the widespread use of this therapy. The development of non-viral, plasmid-based systems like Sleeping Beauty (SB) transposons, coupled with the emergence of large scale, closed electroporation devices, present the opportunity of generating high numbers of CAR-T cells in a short period of time and transforming this in a point-of-care (POC) therapy. Methods and Results: In this work, mononuclear cells were isolated using Ficoll and electroporated using Nucleofector IIb electroporator combined with plasmids encoding 19BBz CAR (in the pT3 SB transposon backbone) and SB100x transposase. Cells were rested for 24h and used for in vitro and in vivo experiments. The phenotype was assessed by flow cytometry. The in vitro cytotoxicity assay was performed using Calcein-AM dye on target cells incubated with different ratios of effector cells. 8-12-week-old-female-NSG were injected iv. 5 × 106 RS4;11 GFP or 105 Nalm-6 GFP and after 3 days were treated with different doses of recently electroporated CAR-T cells. CAR expression on day 1 following electroporation ranged between 5-15% and in vitro cytotoxic activity against RS4;11 or Nalm-6 CD19+ B cell precursor leukemia lines was low or absent. NSG mice engrafted with RS4;11 and treated with 1 × 105 19BBz+ cells showed improved survival when compared to mice treated with mock electroporated cells. POC approach was also efficient against Nalm-6, where 19BBz+CAR-T cells (7 × 105 per mice) improved the survival of mice. In both models, decreased tumor burden in blood and spleen was observed in mice treated with 19BBz. Head to head comparison of 19BBz cells used in POC approach or expanded for 8-12 days in vitro showed similar antitumor activity in vivo against RS4;11 cells, leading to equivalent improvements in mice survival. Conclusion: In summary, we show that the POC strategy is a viable approach for the use of CAR-T cells, obviating the need for T cell expansion while having the potential to greatly decrease the total cost and time of CAR-T cell therapy manufacturing. Citation Format: Luiza Abdo, Luciana Rodrigues Barros, Mariana Saldanha Viegas, Luisa Vieira Marques, Priscila de Sousa Ferreira, Leonardo Chicaybam, Martín Hernán Bonamino. Development of CAR-T cell therapy for B-ALL using a point-of-care approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3247.