GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 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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Published in | Frontiers in immunology Vol. 13; p. 839783 |
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Main Authors | , , , , , , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Frontiers Media S.A
24.03.2022
|
Subjects | |
Online Access | Get full text |
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Summary: | 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors share senior authorship Reviewed by: Paul G. Schlegel, University Children’s Hospital Würzburg, Germany; Manish Malviya, Memorial Sloan Kettering Cancer Center, United States These authors have contributed equally to this work and share first authorship Edited by: Francesca Del Bufalo, Bambino Gesù Children’s Hospital (IRCCS), Italy This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology |
ISSN: | 1664-3224 1664-3224 |
DOI: | 10.3389/fimmu.2022.839783 |