🧬 CAR T cells demonstrate the power of engineered cells as therapeutics. But they fail for most patients. Can CRISPR help here? Our new paper in Nature (www.nature.com/articles/s41...) presents a screening platform to optimize immunotherapies & discover boosters of CAR T cell function. (1/13)

⚙️ We developed CELLFIE (“cell engineering for immunotherapy enhancement”), a CRISPR platform to make & test gene-edited CAR T cells at scale. CELLFIE supports in vitro & in vivo screens with various clinically relevant readouts, plus combinatorial & base-editing screens. (2/13)

🩸 Using CELLFIE, we conducted 58 genome-wide CRISPR screens, with readouts for CAR T cell proliferation, target cell recognition, activation, apoptosis & fratricide, and exhaustion. The screens identified known genes (PD-1, CTLA4, TIM3, TIGIT etc.) and promising new hits. (3/13)

💡 Conceptually, our CRISPR screens create a form of “artificial evolution” by which we optimize CAR T cells for their tasks as cancer therapeutics. This is important because CAR T cells are products of cell engineering and lack task-specific evolutionary optimization. (4/13)

🐭 But not everything that makes CAR T cells proliferate or kill better in vitro translates into more effective therapies. For scalable validation in mice, we conducted pooled in vivo CRISPR screening and observed strong positive effects of RHOG, PRDM1, and FAS knockouts. (5/13)

🐁 We performed extensive in vivo validations and found that RHOG knockout CAR T cells achieve strong reductions in cancer cell numbers and prolonged survival in an aggressive mouse model of human leukemia, with consistent results across different CARs and T cell donors. (6/13)

🔍 RHOG is a small GTPase involved in cell signaling. How does it influence CAR T cells ? We found that RHOG knockout increases the proliferative capacity of CAR T cells and helps them retain a highly functional state with reduced exhaustion and enhanced memory phenotype. (7/13)

💪 We also observed prolonged survival for FAS knockout CAR T cells, likely because these cells are less effective at killing each other (“fratricide”). Combining RHOG & FAS knockout, we obtained more & better CAR T cells, which further improved survival in leukemic mice. (8/13)

🔬 From a technical perspective, we are excited how our new in vivo CROP-seq method improves gRNA detection (reading from an mRNA transcript as in nature.com/articles/nme...) and reduces experimental noise (by using UMIs), which enables larger screens with fewer mice. (9/13)

🔥 What’s next? Our discovery of strong combined effects for RHOG & FAS knockout underlines the potential of synergistic gene edits for boosting CAR T cell function. We thus integrated combinatorial screening into CELLFIE, using the Blainey lab’s CROPseq-multi method. (10/13)

⚕️ Our CELLFIE platform supports clinical translation of CRISPR-boosted CAR T cells. For example, to avoid the DNA double-strand breaks introduced by CRISPR knockout, we performed a tiling base-editing screen across RHOG and identified promising gRNA for clinical testing. (11/13)

🤝 This was a large project & great teamwork by: P. Datlinger*, E.V. Pankevich*, C.D. Arnold*, N. Pranckevicius, J. Lin, D. Romanovskaia, M. Schäfer, F. Piras, A.-C. Orts, A. Nemc, P. Biesaga, M. Chan, T. Neuwirth, A. Artemov, W. Li, S. Ladstätter, T. Krausgruber, C. Bock (12/13)