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

Abstract

The efficacy of adoptive T cell therapies for cancer treatment can be limited by suppressive signals from both extrinsic factors and intrinsic inhibitory checkpoints1,2. Targeted gene editing has the potential to overcome these limitations and enhance T cell therapeutic function3–10. 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. 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. RASA2 ablation enhanced MAPK signalling and chimeric antigen receptor (CAR) T cell cytolytic activity in response to target antigen. 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-knockout CAR T cells had a competitive fitness advantage over control cells in the bone marrow in a mouse model of leukaemia. 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. Together, our findings highlight RASA2 as a promising target to enhance both persistence and effector function in T cell therapies for cancer treatment.

Modular Pooled Discovery of Synthetic Knockin Sequences to Program Durable Cell Therapies

“…For control TCR RNA-seq experiments (Figure S5C-E), a dataset from a manuscript recently accepted for publication was analyzed ( (Carnevale et al, 2022), GSE204862). For GD2 CAR validation experiments, edited cells were sorted for CAR+/TCR-expression on day 7 (single inserts) or on day 6 and day 14 (combo inserts) after electroporation.…”

“…CRISPR/Cas9 mediated gene ablation of inhibitory receptors induced in dysfunctional T cellsstarting with PD1 -has recently been attempted in clinical trials (Stadtmauer et al, 2020). Lossof-function screens continue to nominate perturbations that can increase T cell fitness such as knockout (KO) of Regnase-1 (Wei et al, 2019), Roquin (Zhao et al, 2021a), Ptpn2 (LaFleur et al, 2019), SOCS1 (Sutra Del Galy et al, 2021) or RASA2 (Carnevale et al, 2022;Shifrut et al, 2018), in murine and/or human T cells. Third, large-scale gain-of-function screens using either CRISPR activation (CRISPRa) (Schmidt et al, 2022) or a lentiviral library of open reading frames (ORFs) have revealed promising perturbations such as overexpression of lymphotoxin B receptor (LTBR) (Legut et al, 2022).…”

Strategies to enhance CAR-T persistence

“…Finally, p38 inhibition improved the persistence and antitumor efficacy of CAR-based adoptive immunotherapies [ 191 ]. Similar screening studies have also been related to T cell proliferation, cytokine regulation, and tumor cell resistance, but these studies differed in biosample types, library sources, sgRNA numbers, and screening protocols (Table S1) [ 192 – 194 ].…”

CAR-T cell therapy for hematological malignancies: Limitations and optimization strategies

“…Other than PD-1 and TIM-3, Carnevale et al ( 50 ), identified a novel immune checkpoint, RAS P21 Protein Activator 2 (RASA2), in T cells. RASA2 is a RAS GTPase-activating protein and is upregulated upon chronic stimulation.…”

“…RASA2 is a RAS GTPase-activating protein and is upregulated upon chronic stimulation. As demonstrated in multiple murine models including leukemia, CD19-CAR-T cells with RASA2-ablation experience increased RAS/MAPK signaling and activation, leading to enhanced and persistent anti-tumor activity ( 50 ).…”