GMP-Compliant Manufacturing of TRUCKs: CAR T Cells targeting GD2 and Releasing Inducible IL-18

Abstract

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 4th 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 GD2 using the CliniMACS Prodigy® system using a recently described “all-in-one” lentiviral vector combining constitutive anti-GD2 CAR expression and inducible IL-18. Starting with 0.84 x 108 and 0.91 x 108 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 109 and 3.6 x 109 engineered T cells from the two donors, respectively, within 12 days. Preclinical characterization demonstrated antigen-specific GD2-CAR mediated activation after co-cultivation with GD2-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 GD2-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 GD2-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 GD2-expressing target cells. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products.

Enhancing CAR T cell therapy using Fab-Based Constitutively Heterodimeric Cytokine Receptors

“…The IL18 receptor signals via MYD88 (45), which plays a critical role in innate immunity (46). MYD88 have used in adoptive cell therapy as an alternative CAR co-stimulatory domain, showing improved CAR T-cell effector function in the setting of chronic antigen stimulation (47)(48)(49). Furthermore, several approaches have been characterized by delivering an engineered IL18 cytokine or an IL18 receptor signal within the tumor microenvironment, showing increased chemokine and Th1 cytokine secretion (26,50,51).…”

Current and future concepts for the generation and application of genetically engineered CAR-T and TCR-T cells

“…Because the TRUCK concept is dependent on CD3ζ-mediated NFAT translocation, it is applicable not only for CAR-T but also for TCR-T cells ( 13 ). The most common transgenic proteins for this approach are IL-12 and IL-18 ( 14 – 17 ) but many other cytokines and enzymes are currently explored ( 12 ). The TRUCK concept is of particular interest for the treatment of solid tumors by combining T cell-mediated killing with immune modulation of the tumor microenvironment (TME) through the secretion of cytokines.…”

Small-Molecule Compounds Boost CAR-T Cell Therapy in Hematological Malignancies

“…These cytokines can promote T cell growth, survival, and expansion while providing resistance to immune suppression. So far, IL-2, IL-7 [127], IL-9 [128], IL-12 [129], IL-13 [130], IL-15 [131], IL-18 [132], IL-21 [133], IL-23 [134], IL-33 [135], and other cytokines have been used in pre-clinical studies in conjugations with CAR-T cells, while some cytokines, such as IL-1 [136,137], IL-6 [138,139], IL-10 [140], TNF-α [136,141], IFN-γ [138], and GM-CSF [142], have some negative effects on CAR-T cell therapy participating in the development of CRS during CAR-T cell therapy. Due to further research on the mechanisms of CRS and ICANS, as well as the continuous exploitation and validation of related cytokine inhibitors, concrete results have been achieved in the treatment of adverse reactions during CAR-T cell therapy.…”