Engineering safer theranostic CAR-T cells with a novel cytokine sensor-dependent therapeutic gene switch

• Applicant: Dr. Ayse Nur Menevse
• Subject Area: Hematology, Oncology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525007351

Engineering T cells to express artificial chimeric antigen receptors (CARs) revolutionized cancer immunotherapy. Despite the clinical success of CAR-T cells against B cell malignancies, major problems remain to be solved such as poor tumor infiltration, low persistence, restricted efficacy in solid tumors and severe toxicities such as cytokine release syndrome (CRS). Furthermore, CAR-T cells so far have been engineered to target surface antigens, where soluble antigens could not activate CAR signaling. To improve CAR-T cell persistence and potency, TRUCKs have been engineered to release any therapeutic transgenic protein in an inducible manner. However, CAR tonic signaling can lead to non-coordinated expression of the transgene and thereby exacerbating toxicities. Therefore, a novel versatile synthetic biology tool is essentially needed to reprogram theranostic CAR-T cells with the capacity to diagnose the dynamic changes in the tumor microenvironment by sensing soluble factors such as i) toxicity-associated inflammatory cytokines MCP-1 and IL-6 or ii) immunosuppressive cytokines IL-10 and TGF-β and to react according to therapeutical needs in a tightly controlled manner. Generalized extracellular molecule sensor (GEMS) platform, established by Prof. Fussenegger`s group, enables the engineering of chimeric sensors in a versatile manner by interchanging any target-binding domain with various signal transduction domains. GEMS devices can be engineered to sense any disease-related soluble factors and respond by expressing any therapeutic transgene. To this end, the GEMS platform is a valuable tool to equip CAR-T cells with additional novel functions. In this project, I aim to exploit the potential of the GEMS platform, to improve cellular immunotherapy. For the first time, CAR-T cells will be programmed to express GEMS devices that are able to monitor CRS-associated MCP-1 and IL-6 levels with AND-Gate logic. Serially linked receptor-based synthetic signaling circuits will use the CAR-T cell activation as an input to induce the expression of the first sensor (GEMSMCP1). Only in the presence of MCP-1, the engineered CAR-T cells will express the second sensor (GEMSIL6), which in turn will trigger the expression of a suicide gene and induce apoptosis in the CAR-T cells. To overcome the limitations of the current clinically used suicide genes such as immunogenicity and dependency on the administration of an ectopic small molecule, I aim to generate a novel suicide gene which will be an engineered form of DNASE1L3 (eDNASE1L3). Additionally, I will test other transgenes such as a shRNA/miRNA or neutralizing scFVs targeting TNFα and IFNγ to potentially reduce CRS-associated toxicities without eliminating CAR-T cells. This project will be a proof-of-concept for programming theranostic CAR-T cells using GEMS devices as cytokine sensors and drivers of therapeutic transgene expression.

• DFG Programme: WBP Fellowship
• International Connection: Switzerland
• Host: Professor Dr. Martin Fussenegger