Introduction
Chimeric antigen receptor T (CAR-T) cells have performed poorly in patients with advanced solid cancers. A critical hurdle for CAR-T cell efficacy is the immunosuppressive tumor microenvironment (TME). CAR-T cell function is profoundly inhibited by transforming growth factor-beta (TGF-β) enriched in the TME. Current strategies to address this issue focus on the abrogation of TGF-β signaling. However, these strategies can have toxic side effects due to an imbalance in T cell homeostasis induced by complete blockade of TGF-β signaling.
Method
To address these challenges, we designed a novel chimeric switch receptor comprising a TGF-β-binding domain and a T-cell co-stimulatory domain to initiate CAR-T-cell activation upon TGF-β binding. We firstly engineered the switch receptor to enhance signal conversion while minimizing its effects on endogenous TGF-β signaling. Flow cytometry-based and confocal microscopy-based Förster resonance energy transfer (FRET) assays were utilized to investigate the protein-protein interaction between the switch receptors and endogenous TGF-b receptors. RNA sequencing, flow cytometry and phosphorylation mass spectrometry were used to explore the downstream signaling of the switch receptor. In vitro chromium release assay, AlphaLISA cytokine assay and CTV proliferation assays were used to characterize the response of switch CAR-T cells to tumor cells in the presence of TGF-β. A TGF-βhigh DU-145 prostate cancer model was then introduced to assess the in vivo efficacy of switch CAR-T cells.
Result
Using FRET assays, we found only the switch receptor with a short intracellular sequence (SIS) augmented ligand-dependent homodimers without disturbing the endogenous TGF-β receptor complex. A switch receptor incorporating a 4-1BB signaling domain showed significantly enhanced cytotoxicity and higher levels of cytokine secretion in the presence of TGF-β. Switch CAR-T cells also demonstrated higher proliferation and increased mitochondrial biogenesis in response to TGF-β. This superior CAR-T-cell function was induced by altered cell metabolism and MAPK signaling. Furthermore, both switch and conventional CAR-T cells had equivalent levels of SMAD2 phosphorylation in response to TGF-β, indicating that switch CAR-T cells retained endogenous TGF-β homeostatic signaling. Finally, tumor-bearing mice treated with switch CAR-T cells showed significantly better tumor control, which was associated with decreased TGF-β and increased IFN-γ levels within the tumor.
Conclusion
In conclusion, the novel switch receptor activated CAR-T cells in response to TGF-β, leading to improved CAR-T-cell function in a tumor-specific manner. Switch CAR-T cells also preserved endogenous TGF-β signaling and balanced T cell homeostasis, indicating these cells will be safe to use in the clinic.