We developed a systematic approach to identify genetic perturbations that could render T cells resistant to a range of inhibitory signals encountered in the TME. The suppressive TME and T cell intrinsic checkpoints can impinge on the efficacy of engineered T cells targeting solid tumours 14. Finally, we show that RASA2-ablation in antigen-specific T cells can enhance tumour control and extend survival in multiple preclinical models of liquid and solid tumours.

We find that ablation of RASA2 enhances sensitivity to antigen and improves both effector function and persistence of CAR T and TCR T cells. Here we describe unbiased genetic screens performed under several immunosuppressive conditions commonly encountered in the tumour microenvironment (TME) that uncovered ablation of the RASA2 gene as a strategy for T cells to overcome multiple inhibitory cues. We previously developed a discovery platform in primary human T cells and applied it to identify novel genetic regulators of T cell proliferation 13. Large-scale CRISPR screens can accelerate the discovery of genetic perturbations that can boost the efficacy of engineered T cells 3, 8, 9, 10. However, the optimal gene targets in human T cells have not been explored systematically. Targeted manipulation of select genes is being tested as a strategy to boost the efficacy of adoptive T cell therapies 5, 6, 7. In addition, persistent exposure to antigen can lead to T cell dysfunction, highlighting the need to balance effector function and long-term persistence in engineered T cells 3, 12. Within the tumour mass, the immunosuppressive microenvironment poses a substantial barrier to the efficacy of anti-tumour immunity 2, 11. However, many cancers, especially solid tumours, do not respond to current T cell therapies or rapidly progress after the initial response. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.ĬAR T cells have been transformative in a subset of aggressive haematological malignancies, and T cell receptor (TCR)-transgenic T cells (TCR T cells) have shown promising results in early-phase clinical studies for solid tumours 1. Ablation of RASA2 in multiple preclinical models of T cell receptor and CAR T cell therapies prolonged survival in mice xenografted with either liquid or solid tumours. RASA2-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. Repeated tumour antigen stimulations in vitro revealed that RASA2-deficient T cells show increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. These screens converged on RASA2, a RAS GTPase-activating protein (RasGAP) that we identify as a signalling checkpoint in human T cells, which is downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. Here we performed multiple genome-wide CRISPR knock-out screens under different immunosuppressive conditions to identify genes that can be targeted to prevent T cell dysfunction. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function 3, 4, 5, 6, 7, 8, 9, 10. The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints 1, 2. Nature volume 609, pages 174–182 ( 2022) Cite this article

RASA2 ablation in T cells boosts antigen sensitivity and long-term function

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