Endogenous CAR T cell activation signal-based dual CAR construct

Authors

Asmita Khaniya, Medical Microbiology Interdisciplinary Program, Graduate School, Chulalongkorn UniversityFollow
Koramit Suppipat M.D., Cellular Immunotherapy Research Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, ThailandFollow
Supannikar Tawinwung Ph.D., Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, ThailandFollow
Nattiya Hirankarn M.D., Ph.D., Center of Excellence in Immunology and Immune-mediated Diseases, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, BangkokFollow

Abstract

Background: Despite the notable success of chimeric receptor (CAR) T-cell therapy in treating hematological malignancies, many challenges limit its therapeutic effectiveness. To overcome antigen loss mediated disease relapses, strategies like dual antigen-targeted CAR constructs have been used. However, this strategyrequires robust promoters, to ensure sufficient CAR expression on the cell surface. Utilization of multiple promoters cause the constitutive and simultaneous expression of CARs which lead to issues with CAR T cell tonic signaling and exhaustion.

Objective: This study aimed to utilize the endogenous CAR T cell activation signals to explore the dual CAR T cell-based platform.

Methods: Using scFv of CD19 and CD20 as a proof of concept, in this study we generated CD19/CD20 dual CAR T cells, and aimed to conditionally express the second CAR (CD20 CAR) upon activation of the first CAR (CD19 CAR) after binding to its cognate antigen. The study utilized a sleeping beauty (SB) transposon-based CAR construct and tested different transcription factor binding motifs (8xNFAT, 8xNR4A, 4xNFAT+4NR4A) for conditional CD20 CAR expression.

Results: Our study demonstrated the robust activity of the EF-1α promoter in effectively driving CAR expression within a complex construct. However, the high background expression of the second CAR before encountering the antigen was observed in this study, most likely due to endogenous TCR stimuli. Despite a high background, the 8xNR4A promoter showed the highest inducibility after 72 hours.

Conclusions: This study indicated the feasibility of utilizing endogenous CAR T cell-based activation-induced motifs to drive the expression of CAR T cells in dual antigen-targeted CAR T cell platform. Further exploration of effector-to-target ratios is deemed essential for understanding dynamic regulation in inducible CAR T cells.

Res

DOI

10.56808/2673-060X.5406

Recommended Citation

Khaniya, Asmita; Suppipat, Koramit M.D.; Tawinwung, Supannikar Ph.D.; and Hirankarn, Nattiya M.D., Ph.D. (2024) "Endogenous CAR T cell activation signal-based dual CAR construct," Chulalongkorn Medical Journal: Vol. 68: Iss. 3, Article 4.
DOI: https://doi.org/10.56808/2673-060X.5406
Available at: https://digital.car.chula.ac.th/clmjournal/vol68/iss3/4