BACKGROUND: Immune excluded solid tumors are resistant to T cell based therapies. To address this issue, we created a switch receptor (sr,TGFB-41BB) to co-stimulate CAR-T cells specifically in the TGFβ+ tumor microenvironment (TME). We evaluated the mechanisms for srCAR-T cell anti-tumor efficacy in pre-clinical prostate and breast cancer models.
RESULTS: The sr molecule assembled as homodimers in the cell membrane and did not disrupt the endogenous TGFβ receptor. In the presence of TGF-β, the srCAR-T cells significantly improved function in vitro [including proliferation, cytotoxicity and TNF secretion] compared to conventional CAR-T cells. srCAR-T cells also showed increased gene accessibility for cytotoxicity (PRF,GZMB) and memory (LEF1, SELL) and reduced accessibility for PDCD1. In the context of CAR activation and TGFβ, the srCAR-T cells activated the MAPK related signaling network, increased mitochondrial mass and increased oxidative consumption rate. Mouse srHER2-CAR-T cells showed significantly increased control of established e0771Her2 breast tumors compared to dn.TGFBRII-CAR-T cells. In the TME, srCAR-T increased mitochondrial mass, activation (CD137, CD25, PD-1,TCF-1, Tbet), cytokines (IFN-g, TNF, IL2), and CD107a, gzmB. In tumor draining lymph nodes (tdLN) of treated mice, the srCAR-T cells were TCF-1+ and decreased Tox, T-bet and Ki-67. ‘Tumor free’ mice were resistant to re-challenge at day 60 with eO771-HER2 or eO771 parental tumors. To explore the mechanisms for this epitope spreading effect, eO771-HER2/OVA tumor bearing mice were treated with either srHER2-CAR-T cells or dn.TGFβRII-HER2-CAR-T cells. We showed increased migratory cDC1 bearing OVA antigen in both the TME and the tdLN in srHER2-CAR-T treated but not dn.TGFβRII-HER2-CAR-T treated mice, and co-existing increased OVA tetramer+ CD8+ T cells in both the tdLN and TME, suggesting cross-priming of tumor-specific T cells in the tDLN. When tested in TGF-b+ prostate and breast cancer models in NSG mice, human srCAR-T cells had significantly improved tumour control and mouse survival compared to both dominant negative dn.TGFβRII or conventional CAR-T cells using both healthy donor or patient T cells. At a single cell level, the switch CAR-T cells in the tumour (not the periphery) were activated highly cytotoxic effector cells with reduced levels of exhaustion.
CONCLUSIONS: srCAR-T cells showed increased activation and cytotoxicity specifically at the TGF-b+ tumor site, leading to improved tumor control. Importantly, srCAR-T cells treatment led to engagement of the endogenous immune system and control of heterogenous antigen expressing tumors. This work highlights the strong translational potential of this approach in patients.