Introduction
Chimeric antigen receptor T (CAR-T) cells have limited efficacy in advanced solid cancers. A critical hurdle to success is the immunosuppressive tumor microenvironment (TME) wherein CAR-T cell function is profoundly inhibited by transforming growth factor-beta (TGF-β). Current strategies to address this issue include systemic blockade of TGF-β signaling but toxic side effects can arise from imbalanced T-cell homeostasis.
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. The conformation of the switch receptor was evaluated by Förster resonance energy transfer (FRET) assays, and signal transduction assessed by RNAseq and mass spectrometry. In vitro chronic stimulation assay, mitochondria stress test, chromium release assay, AlphaLISA cytokine assay, and CTV proliferation assays were used to characterize the response of switch CAR-T cells. Their in vivo efficacy was assessed in TGF-βhigh prostate and breast cancer models. Further characterization of switch CAR-T cells in the peripheral blood, spleen and tumor was performed in the MDA-MB468 orthotopic breast cancer model.
Result
Using FRET assays, we found only the switch receptor with a short intracellular sequence 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 TNF-α secretion in the presence of TGF-β. Switch CAR-T cells also demonstrated higher proliferative capacity in short-term and under chronic antigen stimulation with increased mitochondrial biogenesis in response to TGF-β. This superior function was induced by enhanced oxidative metabolism and MAPK signaling. 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 signaling. Next, tumor-bearing mice treated with switch CAR-T cells showed significantly better tumor control, this was associated with decreased TGF-β and increased IFN-γ levels within the tumor. In a TGF-βhigh breast cancer xenograft model, tumor-infiltrating switch CAR-T cells showed increased activation (CD69 and CD137 levels) and lower expression of immune inhibitor receptors. Importantly, switch CAR-T cells showed no evidence of activation at site outside the tumor (peripheral blood and spleen), indicating their potential as a safe therapeutic.
Conclusion
In conclusion, the novel switch receptor activated CAR-T cells in response to TGF-β, leading to improved CAR-T-cell fitness in a tumor-specific manner. Switch CAR-T cells also preserved endogenous TGF-β signaling and balanced T-cell homeostasis, indicating they will be safe to use in the clinic.