Abstract 7034: Pooled CRISPR screening coupled with single-cell sequencing identifies modifiers of CAR T cell state in the context of chronic antigen stimulation
Authors: Joshi Sahil , Wozniak Glenn , Gagnon John , Vucci Kristina , Merrell Allyson , Simon Mandi , Oh Catherine , Cardozo Andrew , DeTomaso David , Chow Julie , Zheng Grace , Boroughs Angela , Joho Keith , Thakore Pratiksha , Oh Soyoung , Freimer Jake , Cass Ashley , Sail Vibhavari , Tocchini Carla , Sandoval Marian , Liu Andrea , Cui Eric , Drever Matt , Galvin Brendan , Milush Jeff , Gray-Rupp Levi , Wheeler Emily , Chen Bob , Levine Jacob , Eidenschenk Celine , Schartner Jill , Geiger-Schuller Katie , Huetter Jan-Christian , Rutz Sascha , Rozenblatt-Rosen Orit , Mellman Ira , Haining W. Nicholas 
Published: March 2024

T cell exhaustion resulting from chronic antigen stimulation and an immunosuppressive tumor microenvironment limits the efficacy of T cell therapies in the solid tumor setting. The onset of T cell exhaustion is associated with distinct epigenetic and transcriptional changes. We hypothesized that genetic perturbations which shift T cells away from exhaustion associated states could increase the potency of immunotherapies. To this end, we utilized pooled, in vitro CRISPR/Cas9-based screening paired with deep sequencing readouts to characterize perturbation dependent T cell states in the context of chronic antigen stimulation. In order to achieve this, we developed a lentivirus-based workflow to perform CRISPRko, CRISPRi and CRISPRa pooled screens in human CAR T-cells that allowed for assessment of T cell phenotypes mediated by knockout, knockdown or overexpression of a large pool of target genes with single cell transcriptome readout. Subjecting these engineered CAR T-cells to an antigen-specific, cell-based repetitive stimulation assay (RSA) led to the progressive loss of T cell proliferation and effector function enabling in vitro modeling of T cell exhaustion. Moreover, characterization of the CAR T-cells by single cell sequencing recapitulated key hallmarks of the transcriptional and epigenetic landscape of T cell exhaustion. We also discovered T cell intrinsic gene perturbations that govern T cell states in the context of chronic antigen stimulation. These results demonstrate the power of pooled CRISPR screening with single cell readouts to identify novel target genes to enhance CAR T-cell therapies.

Citation Format: Sahil Joshi, Glenn Wozniak, John Gagnon, Kristina Vucci, Allyson Merrell, Mandi Simon, Catherine Oh, Andrew Cardozo, David DeTomaso, Julie Chow, Grace Zheng, Angela Boroughs, Keith Joho, Pratiksha Thakore, Soyoung Oh, Jake Freimer, Ashley Cass, Vibhavari Sail, Carla Tocchini, Marian Sandoval, Andrea Liu, Eric Cui, Matt Drever, Brendan Galvin, Jeff Milush, Levi Gray-Rupp, Emily Wheeler, Bob Chen, Jacob Levine, Celine Eidenschenk, Jill Schartner, Katie Geiger-Schuller, Jan-Christian Huetter, Sascha Rutz, Orit Rozenblatt-Rosen, Ira Mellman, W. Nicholas Haining. Pooled CRISPR screening coupled with single-cell sequencing identifies modifiers of CAR T cell state in the context of chronic antigen stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7034.