hematol transfus cell ther. 2 0 2 0; 4 2(2) :150–158 www.htct.com.br Hematology, Transfusion and Cell Therapy Original article Establishment of a simple and efficient platform for car-t cell generation and expansion: from lentiviral production to in vivo studies Virgínia Picanc ¸o-Castro a,∗ , Pablo Diego Moc ¸o a , Amanda Mizukami a , Leticia Delfini Vaz a , Marcelo de Souza Fernandes Pereira a , Renata Nacasaki Silvestre a , Júlia Teixeira Cottas de Azevedo a , Aline de Sousa Bomfim a , Mario Soares de Abreu Neto a , Kelen Cristina Ribeiro Malmegrim a,b , Kamilla Swiech a,b , Dimas Tadeu Covas a,c a Universidade de São Paulo (USP), Hemocentro, Centro de Terapia Celular CTC, Ribeirão Preto, SP, Brazil b Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Ribeirão Preto (FCFRP), SP, Brazil c Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto da Ribeirão Preto (FMRP-USP), SP, Brazil a r t i c l e i n f o Article history: Received 28 March 2019 Accepted 10 June 2019 Available online 9 October 2019 Keywords: Lentiviral vector production CAR-T cells CD19 Cell expansion In vitro Cytotoxicity In vivo xenograft model Immunotherapy a b s t r a c t Introduction: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, pro- liferation, and cytokine production. Objectives: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Methods: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3- endodomain. Conclusions: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progres- sion in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice. © 2019 Associac ¸˜ ao Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ∗ Corresponding author at: Hemocentro, Universidade de São Paulo (USP), Ribeirão Preto, SP, Brazil Rua Tenente Catão Roxo 2501, Ribeirão Preto, SP, Brazil. E-mail address: virginia.picanco@hemocentro.fmrp.usp.br (V. Picanc ¸ o-Castro). https://doi.org/10.1016/j.htct.2019.06.007 2531-1379/© 2019 Associac ¸˜ ao Brasileira de Hematologia, Hemoterapia e Terapia Celular. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).