Good manufacturing practice-grade generation of CD19 and CD123-specific CAR-T cells using piggyBac transposon and allogeneic feeder cells in patients diagnosed with B-cell non-Hodgkin lymphoma and acute myeloid leukemia
Jazyk angličtina Země Švýcarsko Médium electronic-ecollection
Typ dokumentu časopisecké články
PubMed
39192973
PubMed Central
PMC11347927
DOI
10.3389/fimmu.2024.1415328
Knihovny.cz E-zdroje
- Klíčová slova
- CAR-T cells, PiggyBac PB transposon, electroporation, leukemia, lymphoma,
- MeSH
- akutní myeloidní leukemie * terapie imunologie genetika MeSH
- allogeneické buňky imunologie MeSH
- antigeny CD19 * imunologie genetika MeSH
- B-buněčný lymfom terapie imunologie genetika MeSH
- chimerické antigenní receptory * genetika imunologie MeSH
- elektroporace MeSH
- imunoterapie adoptivní * metody MeSH
- lidé MeSH
- podkladové buňky MeSH
- T-lymfocyty imunologie metabolismus MeSH
- transpozibilní elementy DNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny CD19 * MeSH
- chimerické antigenní receptory * MeSH
- transpozibilní elementy DNA * MeSH
BACKGROUND: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. METHODS: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. RESULTS: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. CONCLUSIONS: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.
Faculty of Science Charles University Prague Czechia
Institute of Hematology and Blood Transfusion Prague Czechia
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