GMP-compliant manufacturing of NKG2D CAR Memory T cells using CliniMACS prodigy
EntityUAM. Departamento de Pediatría; Instituto de Investigación Sanitaria Hospital Universitario de La Paz (IdiPAZ)
10.3389/fimmu.2019.02361Frontiers in Immunology 10.October (2019): 2361
Funded byThis study was funded in part by the National Health Service of Spain, Instituto de Salud Carlos III (ISCIII), FONDOS FEDER grant (FIS) PI18/01301, by the Unoentrecienmil Foundation and by CRIS Cancer Foundation to beat Cancer (http://criscancer. org). LF, AF, IM, and AE are granted by CRIS Cancer Foundation to beat cancer
ProjectGobierno de España. PI18/01301
SubjectsNKG2D CAR; Memory T cells; Automated production; Large-scale; Clinical-grade; CliniMACS prodigy; Medicina
Rights© 2019 Fernández, Fernández, Mirones, Escudero, Cardoso, Vela, Lanzarot, de Paz, Leivas, Gallardo, Marcos, Romero, Martínez-López and Pérez- Martínez.
Esta obra está bajo una Licencia Creative Commons Atribución 4.0 Internacional.
Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3z signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients’ own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA− memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA+ fraction was depleted from healthy donors’ non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 108 CD45RA− cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3z lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency ofMedicines andMedical Devices (AEMPS) for themanufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies
Google Scholar:Fernández, Lucía - Fernández, Adrián - Mirones, Isabel - Escudero, Adela - Cardoso, Leila - Vela, María - Lanzarot, Diego - Paz, Raquel de - Leivas, Alejandra - Gallardo, Miguel - Marcos, Antonio - Romero, Ana Belén - Martínez-López, Joaquín - Pérez Martínez, Antonio
This item appears in the following Collection(s)
Showing items related by title, author, creator and subject.
Fernández, Adrián; Escudero, Adela; Matamala, Nerea; Mirones, Isabel; Pernas, Alicia; Cobo, Marta; Casado, Gema; Lanzarot, Diego; Vela, María; Ferreras, Cristina; Mestre, Carmen; Viejo, Aurora; Leivas, Alejandra; Martínez, Joaquín; Fernández, Lucía; Navarro‐Zapata, Alfonso; Ruz‐Caracuel, Beatriz; Rodríguez‐Antolín, Carlos; Pérez‐Martínez, Antonio
Fernández, Adrián; Navarro‐zapata, Alfonso; Escudero, Adela; Matamala, Nerea; Ruz‐caracuel, Beatriz; Mirones, Isabel; Pernas, Alicia; Cobo, Marta; Casado, Gema; Lanzarot, Diego; Rodríguez‐antolín, Carlos; Vela, María; Ferreras, Cristina; Mestre, Carmen; Viejo, Aurora; Leivas, Alejandra; Martínez, Joaquín; Fernández, Lucía; Pérez Martínez, Antonio
Leivas, Alejandra; Valeri, Antonio; Córdoba, Laura; García-Ortiz, Almudena; Ortiz, Alejandra; Sánchez-Vega, Laura; Graña-Castro, Osvaldo; Fernández, Lucía; Carreño-Tarragona, Gonzalo; Pérez, Manuel; Megías, Diego; Paciello, María Liz; Sánchez-Pina, Jose; Pérez Martínez, Antonio; Lee, Dean A.; Powell, Daniel J.; Río, Paula; Martínez-López, Joaquín