Genome-wide CRISPR Knockout Screen of Healthy Donor CART19-28? Cells in the Manuscript IL-4 Drives Exhaustion of CD8+ CART Cells

Durable response to chimeric antigen receptor T (CART) cell therapy remains limited in part due to CART cell exhaustion. Here, we investigate the regulation of CART cell exhaustion with three independent approaches including: a genome-wide CRISPR knockout screen using an in vitro model for exhaustion, RNA and ATAC sequencing on baseline and exhausted CART cells, and RNA and ATAC sequencing on pre-infusion CART cell products from responders and non-responders in the ZUMA-1 clinical trial. Each of these approaches identify interleukin (IL)-4 as a regulator of CART cell dysfunction. Further, IL-4-treated CD8+ CART cells develop signs of exhaustion independently of the presence of CD4+ CART cells. Conversely, IL-4 pathway editing or the combination of CART cells with an IL-4 monoclonal antibody improves antitumor efficacy and reduces signs of CART cell exhaustion in mantle cell lymphoma xenograft mouse models. Therefore, we identify both a role for IL-4 in inducing CART exhaustion and translatable approaches to improve CART cell therapy. Overall design: In an effort to investigate the epigenetic regulation of exhaustion, we first developed an in vitro model for exhaustion. In this model, CART19-28? cells are chronically stimulated with the CD19-expressing JeKo-1 tumor cell line by adding fresh JeKo-1 cells every other day for two weeks. Then, we used this in vitro model for exhaustion to complete a genome-wide CRISPR knockout screen in healthy donor CART19-28? cells.