GMP-compliant manufacturing of NKG2D CAR Memory T cells using CliniMACS prodigy

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dc.contributor.author Fernández, Lucía
dc.contributor.author Fernández, Adrián
dc.contributor.author Mirones, Isabel
dc.contributor.author Escudero, Adela
dc.contributor.author Cardoso, Leila
dc.contributor.author Vela, María
dc.contributor.author Lanzarot, Diego
dc.contributor.author Paz, Raquel de
dc.contributor.author Leivas, Alejandra
dc.contributor.author Gallardo, Miguel
dc.contributor.author Marcos, Antonio
dc.contributor.author Romero, Ana Belén
dc.contributor.author Martínez-López, Joaquín
dc.contributor.author Pérez-Martínez, Antonio
dc.contributor.other UAM. Departamento de Pediatría es_ES
dc.contributor.other Instituto de Investigación Sanitaria Hospital Universitario de La Paz (IdiPAZ) es_ES
dc.date.accessioned 2020-09-11T10:11:38Z
dc.date.available 2020-09-11T10:11:38Z
dc.date.issued 2019-10-10
dc.identifier.citation Frontiers in Immunology 10.October (2019): 2361 en_US
dc.identifier.issn 1664-3224 es_ES
dc.identifier.uri http://hdl.handle.net/10486/691848
dc.description.abstract 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 en_US
dc.description.sponsorship This 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 en_US
dc.format.extent 12 pag. es_ES
dc.format.mimetype application/pdf en
dc.language.iso eng en
dc.publisher Frontiers Media en_US
dc.relation.ispartof Frontiers in Immunology en_US
dc.rights © 2019 Fernández, Fernández, Mirones, Escudero, Cardoso, Vela, Lanzarot, de Paz, Leivas, Gallardo, Marcos, Romero, Martínez-López and Pérez- Martínez. es_ES
dc.subject.other NKG2D CAR es_ES
dc.subject.other Memory T cells en_US
dc.subject.other Automated production en_US
dc.subject.other Large-scale en_US
dc.subject.other Clinical-grade en_US
dc.subject.other CliniMACS prodigy en_US
dc.title GMP-compliant manufacturing of NKG2D CAR Memory T cells using CliniMACS prodigy en_US
dc.type article en
dc.subject.eciencia Medicina es_ES
dc.relation.publisherversion https://doi.org/10.3389/fimmu.2019.02361 es_ES
dc.identifier.doi 10.3389/fimmu.2019.02361 es_ES
dc.identifier.publicationfirstpage 2361-1 es_ES
dc.identifier.publicationissue October es_ES
dc.identifier.publicationlastpage 2361-12 es_ES
dc.identifier.publicationvolume 10 es_ES
dc.relation.projectID Gobierno de España. PI18/01301 es_ES
dc.type.version info:eu-repo/semantics/publishedVersion en
dc.rights.cc Reconocimiento es_ES
dc.rights.accessRights openAccess en
dc.authorUAM Pérez Martínez, Antonio (262086)


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