GMP-compliant manufacturing of NKG2D CAR Memory T cells using CliniMACS prodigy
Entity
UAM. Departamento de Pediatría; Instituto de Investigación Sanitaria Hospital Universitario de La Paz (IdiPAZ)Publisher
Frontiers MediaDate
2019-10-10Citation
10.3389/fimmu.2019.02361
Frontiers in Immunology 10.October (2019): 2361
ISSN
1664-3224DOI
10.3389/fimmu.2019.02361Funded by
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 cancerProject
Gobierno de España. PI18/01301Editor's Version
https://doi.org/10.3389/fimmu.2019.02361Subjects
NKG2D CAR; Memory T cells; Automated production; Large-scale; Clinical-grade; CliniMACS prodigy; MedicinaRights
© 2019 Fernández, Fernández, Mirones, Escudero, Cardoso, Vela, Lanzarot, de Paz, Leivas, Gallardo, Marcos, Romero, Martínez-López and Pérez- Martínez.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
Files in this item
Google Scholar:Fernández, Lucía
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Fernández, Adrián
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Mirones, Isabel
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Escudero, Adela
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Cardoso, Leila
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Vela, María
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Lanzarot, Diego
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Paz, Raquel de
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Leivas, Alejandra
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Gallardo, Miguel
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Marcos, Antonio
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Romero, Ana Belén
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Martínez-López, Joaquín
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Pérez Martínez, Antonio
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