Abstract 2848: Incorporation of inhibitory signaling domains into chimeric antigen receptors (iCAR) designed for self-regulation of canonical CAR-T to treat solid tumors

Abstract

Abstract Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking cancer treatment that has produced remarkable clinical efficacy for hematopoietic malignancies, yet “on-target off-tumor” toxicity due to lack of target specificity limits the therapeutic potential of CAR-T cells in solid tumors. We are developing dual CAR-T cells composed of a canonical activator CAR (aCAR), intended to elicit efficacy in solid tumors, and an inhibitory CAR (iCAR) designed to efficiently inhibit the aCAR activity in normal tissue and vital organs. The iCAR and aCAR scFvs of this system bind distinct cell-surface antigens that are widely co-expressed on the normal epithelial from which solid tumors originate. Upon antigen binding, signal propagation through iCAR cytoplasmic domain (iDomain) counteracts aCAR induction of CAR T-cell activation and killing; thus protecting normal tissue. Dual CAR-T cells are activated and kill when exposed to tumor cells that have lost iCAR target expression due to chromosomal loss-of-heterozygosity (LOH) which is common in all cancers. iCAR-targeted LOH generates absolute and irreversible tumor specificity therefore tumor-specific overexpression of the iCAR or aCAR targets is not required. We have carried out a comprehensive screen using human T-cells to identify the most effective inhibitory iDomains derived from cell-surface receptors that naturally inhibit or moderate immune cell activation. The cell-based assay suite developed for this screen consisted of cell lines that co-express iCAR and aCAR target at clinically relevant levels and isogenic partners that mimic LOH through CRISPR editing of the iCAR target. The expression of dual CARs in T cells as biscistronic constructs introduced with lentiviral vectors was successful in a limited number of cases, and it was necessary to develop alternative methods of iCAR aCAR co-expression to complete the screen. Combining aCAR transduction with iCAR mRNA Electroporation (T-REP) proved to be a useful method to disentangle iDomain potency and expression level. The screen identified new iDomains that may expand the potential of iCAR technology in developing strategies to treat solid tumors without compromising efficacy for safety. Citation Format: David Bassan, Jason Yi, Neta Chaim, Nir Bujanover, Sarit Tabak, Tanya Kim, Yael Lopesco, Leehee Weinberger, Kristina Vucci, Michael Weist, Caitlin Schnair, Gregor B. Adams, Orit Foord, Frank J. Calzone, Rick Kendall, Adi Sharbi-Yunger. Incorporation of inhibitory signaling domains into chimeric antigen receptors (iCAR) designed for self-regulation of canonical CAR-T to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2848.