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385 Engineering a TGF-β switch receptor enhances CAR-T cell function in a suppressive TGF-β-enriched tumor microenvironment
  1. Joe Jiang Zhu,
  2. Criselle DSouza,
  3. Vicky Qin,
  4. Niko Thio,
  5. Michael Kershaw,
  6. Joe Trapani,
  7. Phillip Darcy and
  8. Paul Neeson
  1. Peter MaCallum Cancer Centre, Melbourne, Australia, Melbourne, Australia


Background 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.

Methods We engineered a novel chimeric switch receptor comprising a TGF-β receptor domain and a T cell costimulatory domain to initiate T cell costimulation upon TGF-β binding. To maintain T cell homeostasis balance, we optimized the intracellular sequence of the switch receptor to reduce its effect on endogenous TGF-β signaling and decrease the potential side effects. The switch receptor was co-expressed in LeY-specific CAR-T cells (switch CAR-T). The in vitro function of switch CAR-T cells were investigated in the presence of TGF-β. We also performed RNAseq to explore genes involved in the switch receptor activation. Finally, we demonstrated the anti-tumor efficacy of switch CAR-T cells in vivo.

Results In the presence of TGF-β, switch CAR-T cells showed significantly enhanced cytotoxicity and higher levels of TNF secretion compared with conventional CAR-T cells (figure 1). In the presence of both CAR stimulation and TGF-β, but not TGF-β alone, switch CAR-T cells also had significantly higher proliferation and increased mitochondrial biogenesis, indicating an antigen-specific response. 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 signaling (figure 2). RNAseq analysis showed that switch CAR-T cells have a unique gene expression profile in response to CAR stimulation and TGF-β. Finally, tumor-bearing mice treated with switch CAR-T cells showed significantly better tumor control compared with conventional CAR-T cells (figure 3). This finding was associated with decreased TGF-β and increased IFN-γ levels within the tumor.

Conclusions The novel switch receptor activated CAR-T cells in response to immunosuppressive TGF-β leading to improved CAR-T cell function. This effect also required CAR-T cell activation through CAR-stimulation, suggesting a tumor-specific response. Furthermore, these switch CAR-T cells provided improved in vivo anti-tumor control. By fine-tuning the intracellular sequence of the switch receptor, we also successfully retained the endogenous TGF-β signaling. Switch CAR-T cells can preserve this important homeostatic mechanism and will be safe to use in the clinic.

Abstract 385 Figure 1

Switch CAR-T cells exhibited superior cytotoxicity and cytokine production. (A) Cytokine production of TNF-α after 16 hours of co-culture with LeY+ DU-145 cells and switch CAR-T cells or conventional CAR-T cells with or without TGF-β, measured by AlphaLISA assay (mean ± SEM of triplicate cultures). (B) Relative lysis of LeY+ DU-145 after 16 hours co-culture, measured by 51Cr release assay (mean ± SEM of triplicate cultures).

Abstract 385 Figure 2

The switch receptor enhanced the proliferation of switch CAR-T cells in the presence of TGF-β and maintained endogenous TGF-β signaling. (A) Contour plots showing proliferation of switch CAR-T cells and conventional CAR-T cells labelled with cell trace violet when cultured in the presence of TGF-β. (B) Histogram overlay of phosphorylated SMAD2 gated on CAR+ cells when cultured with TGF-β.

Abstract 385 Figure 3

Switch CAR-T cells showed significantly enhanced tumor control in vivo. (A) NSG mice were inoculated subcutaneously with DU-145 cells and treated with CAR-T cells when tumors were established. The growth kinetics was plotted (n=6). *** p<0.001. (B) Survival analysis was assessed, and the significance of Kaplan-Meier survival analysis was determined by log-rank Mantel-Cox test.

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