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GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD(2) and Releasing Inducible IL-18

Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4(th) advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T...

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Detalles Bibliográficos
Autores principales: Glienke, Wolfgang, Dragon, Anna Christina, Zimmermann, Katharina, Martyniszyn-Eiben, Alexandra, Mertens, Mira, Abken, Hinrich, Rossig, Claudia, Altvater, Bianca, Aleksandrova, Krasimira, Arseniev, Lubomir, Kloth, Christina, Stamopoulou, Andriana, Moritz, Thomas, Lode, Holger N., Siebert, Nikolai, Blasczyk, Rainer, Goudeva, Lilia, Schambach, Axel, Köhl, Ulrike, Eiz-Vesper, Britta, Esser, Ruth
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8988144/
https://www.ncbi.nlm.nih.gov/pubmed/35401506
http://dx.doi.org/10.3389/fimmu.2022.839783
Descripción
Sumario:Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors. Thus, approaches using 4(th) advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T cells redirected for universal cytokine-mediated killing”), are currently under investigation. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD(2) using the CliniMACS Prodigy(®) system using a recently described “all-in-one” lentiviral vector combining constitutive anti-GD(2) CAR expression and inducible IL-18. Starting with 0.84 x 10(8) and 0.91 x 10(8) T cells after enrichment of CD4(+) and CD8(+) we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 10(9) and 3.6 x 10(9) engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD(2)-CAR mediated activation after co-cultivation with GD(2)-expressing target cells. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD(2)-expressing target cells indicated by an increased expression of activation markers (e.g. CD25, CD69) on both CD4(+) and CD8(+) T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-2, granzyme B, IFN-γ, perforin, TNF-α). Manufactured TRUCKs showed a specific cytotoxicity towards GD(2)-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD(2)-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.