Abstract 4094: Enhanced CRISPR-Cas9 edited CAR-T cells through simultaneous inactivation of multiple T cell exhaustion pathways
Authors: Sergeeva Oksana , Fearnley Amber , Chin Shu Shien , An Xingyue , Pels Nana Adjoa , Metzler Kristen , Joinville Kyla , Calderon Lizet Aquino , Abulizi Abudukadier , Karp Hayden , Rodriguez Julio , Matis Louis , Fine Jay , Myers Jeremy 
Published: April 2023

Abstract
T cell exhaustion is a hallmark of tumor-infiltrating T cells and a major therapeutic challenge for immunotherapy which is not sufficiently understood. To identify T cell exhaustion promoting genes which may be targeted by new therapies, we combined highly efficient CRISPR/Cas9 gene knockouts in primary human T cells with functional assessment of effector function under exhaustion-inducing chronic T cell activation conditions. We conducted an initial CRISPR/Cas9 T cell exhaustion screen focused on previously identified genes reported as enhancers of anti-tumor T cell response in mouse tumor models, published exhaustion genes, and genes associated with CD8 T cell activation. Based on these results, we constructed gene networks from these screen hits and performed a secondary CRISPR-Cas9-based screen. We found that eight genes, when efficiently knocked out, limited T cell exhaustion, as measured by increased proliferation under chronic activation, reduced expression of T cell exhaustion markers, and improved tumor cell killing in CD3-bispecific antibody-mediated tumor/T cell co-culture assays. Prioritized T cell exhaustion gene hits were also evaluated in assays assessing anti-CD19 CAR-T cell function. The CRISPR-mediated ablation of five T cell exhaustion gene hits resulted in significantly more effective CD19-positive human tumor cell killing, compared to the non-targeting control CAR-T cells. These five genes included a transcriptional regulator, a member of a CD28-independent costimulatory pathway, and a series of ligand/receptor pairs that have been previously associated with T cell exhaustion and dysfunction. Interestingly, knockdown of combinations of the top three T cell exhaustion gene hits resulted in CAR-T cells that were more effective in tumor killing than any of the single gene knockouts, suggesting a non-redundant role for each gene, most likely comprising independent pathways. Furthermore, enhanced tumor cell killing was observed when each T cell exhaustion gene hit was ablated in combination with PDCD1 (PD-1) in double knockout CAR-T cells, suggesting that the T cell exhaustion pathways we identified were complementary to the PD1-PDL1 immune checkpoint pathway in regulating effector T cell function. By using a multidimensional, functional CRISPR-Cas9-based T cell exhaustion screen, we have uncovered genes that are involved in T cell exhaustion. These gene knockouts can be universally applied individually or in combination to create CAR-T cells with more durable effector activity, with the potential to achieve superior patient outcomes in a variety of tumor indications.
Citation Format: Oksana Sergeeva, Amber Fearnley, Shu Shien Chin, Xingyue An, Nana Adjoa Pels, Kristen Metzler, Kyla Joinville, Lizet Aquino Calderon, Abudukadier Abulizi, Hayden Karp, Julio Rodriguez, Louis Matis, Jay Fine, Jeremy Myers. Enhanced CRISPR-Cas9 edited CAR-T cells through simultaneous inactivation of multiple T cell exhaustion pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4094.