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dc.contributor.authorFernández, Lucía
dc.contributor.authorFernández, Adrián
dc.contributor.authorMirones, Isabel
dc.contributor.authorEscudero, Adela
dc.contributor.authorCardoso, Leila
dc.contributor.authorVela, María
dc.contributor.authorLanzarot, Diego
dc.contributor.authorde Paz, Raquel
dc.contributor.authorLeivas, Alejandra
dc.contributor.authorGallardo, Miguel
dc.contributor.authorMarcos, Antonio
dc.contributor.authorRomero, Ana Belén
dc.contributor.authorMartinez-Lopez, Joaquin 
dc.contributor.authorPérez-Martínez, Antonio
dc.identifier.citationCancers (Basel). 2019 ;12(1).es_ES
dc.description.abstractNatural 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 CD3ζ 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-CD3ζ 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 of Medicines and Medical Devices (AEMPS) for the manufacture 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.es_ES
dc.description.sponsorshipThis 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 (, AF, IM, and AE are granted by CRIS Cancer Foundation to beat cancer.es_ES
dc.relation.isversionofPublisher's versiones_ES
dc.subjectCliniMACS prodigyes_ES
dc.subjectNKG2D CARes_ES
dc.subjectautomated productiones_ES
dc.subjectmemory T cellses_ES
dc.titleGMP-Compliant Manufacturing of NKG2D CAR Memory T Cells Using CliniMACS Prodigyes_ES
dc.rights.licenseAtribución-NoComercial-CompartirIgual 4.0 Internacional*
dc.contributor.funderUnoentrecienmil Foundationes_ES
dc.contributor.funderInstituto de Salud Carlos III - ISCIII (España)es_ES
dc.contributor.funderCRIS against Cancer foundationes_ES
dc.identifier.journalFrontiers in immunologyes_ES
dc.repisalud.orgCNIOCNIO::Unidades técnicas::Unidad de Investigación Clínica de Tumores Hematológicos H12O-CNIOes_ES

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Atribución-NoComercial-CompartirIgual 4.0 Internacional
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