Abstract LB028: Development of logic-gated CAR-NK cells to reduce target-mediated healthy tissue toxicities
Authors: Gonzalez Alba , Roguev Assen , Frankel Nicholas W. , Garrison Brian S. , Lee Derrick , Gainer Marcus , Mullenix Alyssa , Gordley Russell M. , Loving Kathryn A. , Chien Jenny , Lee Gary 
Published: July 2021

Abstract
CAR-based cell therapies have revolutionized cancer treatment, however, applications beyond targeting lineage antigens are challenging due to expression of targeted antigens in healthy cells posing a risk for on-target off-tissue toxicities. This presents an opportunity to leverage synthetic biological logic gated gene circuits, such as NOT gate, to expand cancer targets for CAR-based cell therapies. We have constructed a first-in-class NOT gate in CAR-NK cells to protect healthy cells from CAR-mediated cytotoxicity. An inhibitory CAR (iCAR) recognizes a safety antigen expressed on healthy cells and suppresses activating CAR (aCAR) functions, significantly reducing NK cell activity. Multiple iCARs with intracellular co-inhibitory domains containing immunoreceptor tyrosine-based inhibitory motifs have shown to suppress over 50% of aCAR-mediated killing (p&lt;0.05) and significantly reduce TNFa secretion (p&lt;0.0005) in an antigen-specific manner. Here we describe a robust cancer and safety antigen pairing discovery method for the development of NOT gated CAR-NK therapies. A bioinformatics pipeline uses transcriptomics data to discover and prioritize tumor and healthy tissue antigens. We have identified genes differentially expressed in healthy vs tumor tissue and selected leads based on antigens' co-expression in healthy tissue, subcellular localization, antigen topology (presence of extracellular domain(s)), and antibody availability. Such antigen pairs have been validated in primary tissue samples. In AML, targeting the critical leukemic stem cell (LSC) population via antigens such as FLT3 leads to hematopoietic toxicity due to expression in healthy hematopoietic stem cells (HSCs). Comparative bioinformatic analysis between AML and healthy human bone marrow mononuclear cell (BMMC) samples identified 10 surface antigens differentially expressed between HSCs and AML cells that could be used as NOT gate targets to protect HSCs from CAR-mediated toxicity. We further validated one of the top candidate targets, EMCN, by flow cytometry and confirmed significant differential protein expression between HSCs and LSCs. Similarly, in CEA+ tumors, significant on-target off-tissue toxicities occur in healthy epithelium resulting in colitis and lung damage. We prioritized 3 healthy tissue antigens preferentially expressed in intestinal and lung epithelial cells compared to cancer cells: VSIG2, CPM and SLC26A2. IHC analysis confirmed that these targets are expressed at higher levels in the healthy tissues compared to CEA+ tumor, making them attractive candidates to use in a NOT gate circuit to improve the therapeutic window of CEA CAR-NK cells. Using a bioinformatics discovery and validation pipeline coupled with NOT logic gated CAR-NK cells, we can selectively target tumor antigens, while protecting healthy tissues, to create cell therapies with greater efficacy, precision and control.
Citation Format: Alba Gonzalez, Assen Roguev, Nicholas W. Frankel, Brian S. Garrison, Derrick Lee, Marcus Gainer, Alyssa Mullenix, Russell M. Gordley, Kathryn A. Loving, Jenny Chien, Gary Lee. Development of logic-gated CAR-NK cells to reduce target-mediated healthy tissue toxicities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB028.