Abstract 5329: High throughput screening strategies in the development of logic gated cell therapies
Authors: Wang Li , Panagiotopoulou Sofia Kyriazopoulou , Harari-Steinfeld Rona , De Silva Dasmanthie , Tan Michelle , Lim Laura , Boroughs Angela , Sue Cate , Chen Jon , Thomas Jamie , Chua Mary , Yashin Ed , Shieh Christine , Fong Ryan , Xu Sophie , Zheng Grace , Galvin Brendan , Cooper Aaron , Mikkelsen Tarjei , Haining Nicholas 
Published: April 2023

Abstract
Chimeric antigen receptor T cell (CAR T) therapy has demonstrated unprecedented therapeutic activity in hematologic malignancies. However, generating potent clinical responses against solid tumors remains a challenge for CAR T therapy. As the field strives to improve the therapeutic efficacy of CAR T cells with novel target antigens and enhanced potency, the risks of on-target toxicity pose a major barrier to progress. To address these challenges, we have developed engineered CAR T cells to target solid tumors through AND logic gates, where CAR expression is conditionally induced by a transcription factor released from a priming receptor (PrimeRTM) upon binding to the PrimeR antigen. The AND gate limits off-tumor toxicity as it requires both CAR and PrimeR antigen expression in the tumor microenvironment.
To ensure PrimeR expression and signal transduction upon antigen binding, while minimizing residual ‘‘leaky’’ CAR induction in the absence of PrimeR antigen, we screened hundreds of PrimeR binders using both arrayed and pooled strategies. In an arrayed strategy, we engineered T cells from four donors in multiwell plates using CRISPR-mediated, non-viral, site-specific integration of logic gates bearing a variable PrimeR binder and a fixed MSLN CAR. In addition, we employed a pooled screening strategy, where we engineered T cells from two independent donors with a pool containing a subset of &gt;300 of the same logic gates.
Engineered T cells from both strategies were co-cultured with cell lines bearing either both CAR and PrimeR antigens or a single antigen, in order to evaluate fidelity and on-target functionality. In the arrayed setting, on-target functionality was quantified based on the levels of CAR induction, cytokine secretion, T cell activation, and target cell killing in the presence of both antigens, while fidelity was assessed based on the absence of these activity signals in the presence of a single antigen. In the pooled setting, sorting based on functional markers was performed and sequencing was used to quantify the relative abundance of cells with each logic gate in different sorted populations. On-target activity and circuit fidelity were then quantified based on enrichments in different sorted populations.
Results from the pooled and arrayed screens were highly concordant. We combined the screen readouts to nominate a small set of PrimeR binders that exhibited both high fidelity and on-target functionality. We confirmed the desired characteristics of these binders with targeted arrayed screens in additional conditions as well as in in-vivo models. We have applied both screen strategies to select a small set of leads from hundreds of candidate PrimeR binders in the context of a logic-gated MSLN CAR. As pooled and arrayed screens come with different sets of limitations and advantages, both serve as important tools for the effective selection of receptors in the development of novel cell therapies.
Citation Format: Li Wang, Sofia Kyriazopoulou Panagiotopoulou, Rona Harari-Steinfeld, Dasmanthie De Silva, Michelle Tan, Laura Lim, Angela Boroughs, Cate Sue, Jon Chen, Jamie Thomas, Mary Chua, Ed Yashin, Christine Shieh, Ryan Fong, Sophie Xu, Grace Zheng, Brendan Galvin, Aaron Cooper, Tarjei Mikkelsen, Nicholas Haining. High throughput screening strategies in the development of logic gated cell therapies. [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 5329.