Oral Presentation MIN Spring Retreat 2023

Engineering a TGF-β switch receptor enhances CAR-T cell function in a suppressive TGF-β-enriched tumor microenvironment (#26)

Vicky Qin 1 2 , Niko Thio 1 , Xiao Jing Ong 1 2 , Arthur Xuan Wang 1 2 , Paul J Neeson 1 2 , Criselle D’Souza 1 2 , Joe Jiang Zhu 1 2
  1. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
  2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia

Introduction
Chimeric antigen receptor T (CAR-T) cells have limited efficacy in patients with advanced solid cancers. A critical hurdle 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 focus on abrogating TGF-β signaling. However, complete blockade of TGF-β signaling can have toxic side effects due to imbalanced T cell homeostasis.

Method
We designed a novel chimeric switch receptor comprising a TGF-β-binding domain and a T-cell costimulatory domain to initiate CAR-T-cell activation upon TGF-β binding. The structure of switch receptor was optimized to minimize its effects on endogenous TGF-β signaling and examined by Förster resonance energy transfer (FRET) assays. RNA sequencing and mass spectrometry revealed the switch receptor signaling network. The anti-tumor effect of switch CAR-T cells was characterized by in vitro functional assays. A TGF-βhigh prostate cancer model was introduced to assess their in vivo efficacy.

Result
Using FRET assays, we found only the switch receptor with a short intracellular sequence augmented ligand-dependent homodimers without disturbing endogenous TGF-β receptors. A switch receptor incorporating a 4-1BB signaling domain significantly enhanced cytotoxicity, proliferation and TNF-α secretion in CAR-T cells in response to TGF-β. This superior function was facilitated by altered cell metabolism and MAPK signaling. Furthermore, switch and conventional CAR-T cells had equivalent levels of SMAD2 phosphorylation induced by TGF-β, indicating that switch CAR-T cells retained TGF-β homeostatic signaling. Finally, mice treated with switch CAR-T cells showed significant tumor control, which was associated with decreased TGF-β and increased IFN-γ levels within the tumor.

Conclusion
The novel switch receptor activated CAR-T cells via 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.