Design and Characterization of an "All-in-One" Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines.

Katharina Zimmermann,Britta Eiz-Vesper,Bianca Altvater,Melanie Galla,Loreen Sophie Rudek,Axel Schambach,Christina Kloth,Belal Neyazi,Johann Meyer,Thomas Moritz,Michael Alexander Morgan,I Erol Sandalcioglu,Anna Christina Dragon,Claudia Rossig,Johannes Kuehle,Hinrich Abken

doi:10.3390/cancers12020375

Abstract

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T cells redirected for universal cytokine-mediated killing”), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular “all-in-one” vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the GD2-specific CAR via GD2+ target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the “all-in-one” vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2+ tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy.

Highlights

Adoptive cell therapy using chimeric antigen receptor (CAR)-modified T cells is one of the greatest breakthroughs in tumor therapy in recent years [1,2,3]
We demonstrated the reliability of the established “all-in-one” vector construct regarding tight and effective induction of reporter genes and anti-cancer cytokines in primary human T cells that led to a potent anti-tumor effect in vitro, with the potential to reshape the tumor microenvironment (TME) in the case this system would be applied in vivo to treat solid tumors
The GD2 CAR contains a single-chain variable fragment derived from the monoclonal antibody 14.G2a that has been approved for therapeutic use in children with neuroblastoma and has been used in previous clinical trials [33], an IgG1 (HCH2 CH3 ) hinge domain followed by the CD28 transmembrane (TM) domain, and the intracellular 4-1BB and CD3ζ signaling domains [25] (Figure 1A)

Summary

Introduction

Adoptive cell therapy using chimeric antigen receptor (CAR)-modified T cells is one of the greatest breakthroughs in tumor therapy in recent years [1,2,3]. For example, interleukin (IL), provided some anti-tumor activity, as demonstrated by tumor regression and prolonged survival in murine studies [13,14], but was linked with severe systemic toxicities [15,16]. In this direction, the TRUCK (“T cell redirected for universal cytokine-mediated killing”) concept was established, which equips CAR-redirected T cells with an additional NFAT (nuclear factor of activated T cells) inducible cytokine response that results in the restricted locoregional release of a transgenic cytokine upon CAR engagement of cognate target cells.

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Results

Discussion

Conclusion