Tumor microenvironment (TME) immune suppression is a key hurdle for CAR-T cell efficacy in patients with prostate cancer. In particular, TGF-β is produced by prostate cancer associated fibroblasts and macrophages that suppresses CAR-T cell function. To address this issue we engineered a novel switch receptor (TGFB-41BB) to bind TGFβ and transduce a co-stimulation signal via a 4-1BB signalling domain. When this novel molecule was co-expressed with a CAR in T cells, this created switch CAR-T cells.
We first showed that the TGFβ-41BB switch receptor assembled as a homodimer in the T cell membrane and importantly did not disrupt the endogenous TGFβ receptor. In the presence of TGF-β, the switch CAR-T cells demonstrated significantly improved in vitro function including switch CAR-T cell proliferation, cytotoxicity and TNF secretion compared to conventional CAR-T cells. TGF-β also increased switch CAR-T cell mitochondrial mass and increased oxidative consumption rate. Despite this, the switch CAR-T cells still retained endogenous TGF-β signaling for homeostatic control. To better understand downstream signaling from the switch receptor, we used phosphoproteome mass spectrometry. In the context of CAR activation and TGF-β the switch CAR-T cells activated the MAPK related and cell cycle signaling networks and showed increased expression of genes associated with T cell activation, MAPK signalling and immune metabolism pathways.
When tested in TGF-β+ tumour bearing mouse models, the switch CAR-T cells had significantly improved tumour control and mouse survival compared to both dominant negative dn.TGFβRII or conventional CAR-T cells. These findings were consistent for switch CAR-T cells derived from healthy donor or patient T cells. At a single cell level, the switch CAR-T cells in the tumour (not the periphery) had a gene expression profile of activated highly cytotoxic effector cells, but also had reduced levels of exhaustion. Finally compared to dn.TGFβRII CAR-T cells, the switch CAR-T cells had distinct epigenetic regulation of effector protein genes following chronic stimulation.
In conclusion, our novel switch receptor CAR-T cells showed a unique profile with increased activation and cytotoxic differentiation specifically at the tumour site despite high levels of TGF-β, leading to improved tumour control.