CAR-T cell manufacturing in Brazil: a partnership for accessible immunotherapy in hematologic malignancies

Introduction: Chimeric Antigen Receptor (CAR) T-cell immunotherapy, which involves genetically modified autologous T lymphocytes, has shown exceptional success in treating of hematologic malignancies.While commercially approved therapies are effective, high production costs limit their widespread use, especially in public healthcare systems.We have set a partnership between the Brazilian National Cancer Institute (INCA) and the Children's Hospital of Philadelphia (CHOP-USA) for validating in Brazil a similar approach to the one adopted by CHOP.Objectives: Validate the production of anti-CD19 CAR-T cell product using a closed automated platform.Methodology: For manufacturing validation, CAR-T cells were produced from B cell precursor acute lymphoblastic leukemia pediatric patient leukapheresis material on the platform CliniMACS Prodigy platform (Miltenyi Biotec).A total of 2x10 8 purified T cells were expanded following lentiviral transduction with the CAR-CD19-BBz transgene.On the 6th day of expansion, microbiological and flow cytometry analyses were performed.Once the target CAR-T cell count was reached, the cells were harvested and cryopreserved.Samples were sent to CHOP for quality control testing of final product release, using pre-approved validation protocols.An in vivo study was conducted using NSG mice inoculated with the NALM-6 leukemia cell line and treated with 1.4x10 6 CAR-T cells.Results: On the 6th day post-transduction, 1.8x10 9 cells were obtained, with 37% CAR+ expression.Production process was successfully validated, passing all quality control tests.After thawing (prior to infusion in animals), cells retained 97% viability and 34% CAR+ expression.Survival curve analysis revealed a significant difference between treated and control groups (p = 0.018).Moreover, the average bioluminescence intensity-proportional to tumor burden-was reduced by 90% in the treated group by the 16th day post-treatment. Conclusion:These results validate our first CAR-T cell manufacturing process, ensuring a high-quality final product.The protocol successfully achieved transduction and cell expansion at 6 days of expansion, with high viability and receptor expression post-freezing, as well therapeutic efficacy.These findings highlight the viability of this automated-based protocol for CAR-T production in our institution, enhancing accessibility and benefiting patients from the public health system with hematological neoplasms.