Article Text
PDF
Abstract
Background Immunosuppression in the tumor microenvironment (TME) poses a critical hurdle for immunotherapy in many advanced solid tumors, wherein clinical activity has been limited for chimeric antigen receptor T (CAR-T) cells. Notably, transforming growth factor-beta (TGF-β) hinders anti-tumor immunity within the TME. Treatment regimens to inhibit TGF-β have been developed. However, considering their role in maintaining T cell homeostasis, immune-related toxicity with systemic suppression of TGF-β signaling limits their clinical use and the design of new TGF-β-targeted immunotherapies.
Methods We designed a novel chimeric switch receptor (TGFBRII.BB) comprising a truncated type II TGF-β receptor (TGFBRII) linked to a 4-1BB intracellular costimulatory domain. Förster resonance energy transfer (FRET) assays were performed to evaluate and optimize the receptor structure. The signaling network and in vitro functional assays were assessed upon TGF-β stimulation. In vivo studies were investigated in TGF-βhi prostate and breast tumor models. Further mechanisms were explored by flow cytometry, single cell RNA-sequencing and ATAC-sequencing coupled with bulk RNA-sequencing.
Results Through FRET assays, the switch receptor was optimized to augment ligand-dependent homodimerization without disturbing the endogenous TGF-β receptor complex. Then the switch receptor was co-expressed in Lewis-Y or HER2-specific CAR-T cells (switch CAR-T). Equivalent levels of SMAD2-phosphorylation were observed in conventional and switch CAR-T cells upon TGF-β ligation, suggesting the preserved TGF-β homeostatic signal in switch CAR-T cells. Upon TGF-β and CAR-stimulation, switch CAR-T cells counteracted the TGF-β-mediated immunosuppression with enhanced effector function and increased TNF-α secretion. This improved anti-tumor activity was primarily mediated by MAPK-driven immune activation. Furthermore, switch CAR-T cells showed increased oxidative phosphorylation and mitochondrial fitness in response to TGF-β, suggesting a favorable metabolic profile driven by 4-1BB activation. This also aligned with preserved proliferative capacity of switch CAR-T cells after chronic antigen exposure in the presence of TGF-β. Moreover, switch CAR-T cells resulted in superior efficacy in TGF-βhi prostate and breast cancer xenograft models and significantly improved survival (figure 1). Tumor-infiltrating switch CAR-T cells showed increased activation and reduced expression of immune inhibitory molecules. In parallel, switch CAR-T cells upregulated genes encoding cytotoxic granules and exhibited low expression of immune exhaustion-related genes. Importantly, the switch CAR-T cells preserved a naïve/memory-like phenotype and remained inactive in the periphery (figure 2).
Conclusions The switch receptor rewires the TGF-β signal into tumor-specific immune activation in the TME, leading to enhanced efficacy in immunosuppressive tumor models. Switch CAR-T cells preserved endogenous TGF-β signaling for T cell homeostasis, providing a safe strategy for cancer patients.
Ethics Approval Peter MacCallum Cancer Centre Animal Ethics E696.
Switch TGFBRII.BB CAR-T cells lead to enhanced anti-tumor efficacy in TGF-βhi pros- tate and breast cancer xenograft models. NSG mice were injected either orthotopically with 2 x 106 MDA-MB-468 cells in the mammary fat pad or subcutaneously with 1x 106 DU145 tumor cells. A. Kaplan-Meier survival curves for untreated mice and mice treated with empty vector-T cells, conventional CAR-T cells and TGFBRII.BB CAR-T cells in an orthotopic breast cancer xenograft model. B. Kaplan-Meier survival curves in a prostate cancer xenograft model. Log rank Mantel-Cox test was performed for significance analysis
Mechanism of switch CAR-T cells overcoming TGF-β-induced immunosuppression in the TME. Switch CAR-T cells convert the TGF-β suppressive signal into immune activation mainly via MAPK pathway and promote mitochondrial fitness via 4-1BB signaling. Homeostatic SMAD-dependent TGF-β signal was also preserved. Switch CAR-T cells maintain naïve/memory phenotype in the periphery only in the presence of TGF-β without the CAR antigen while they exhibit enhanced activation and cytotoxic capacity at the tumor site
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.