Abstract 4078: Optimized chimeric antigen receptors (CARs) for CAR-NK cell therapies
Authors: Yang Cuiqing , Wang Yifang , Liu Tingting , Jiang Fuwei , Wang Qingyang , Wang Qin , Ye Gang , Tang Renhong , Cao Zhouxiao 
Published: April 2023

Abstract
Introduction: CAR-T-cell therapy has shown great success in treating hematopoietic malignancies. Allogenic NK cell-based therapies have also been shown to mount potent responses against hematopoietic malignancies. Unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as serious cytokine release syndrome, neurotoxicity, or graft-vs-host-disease. Several groups have demonstrated the clinical efficacy of allogeneic CAR-expressing NK cells that utilize CARs developed for T-cells. However, activating receptor signaling in NK cells is different from T cells. NK cell-mediated cytotoxicity is regulated by a repertoire of activating and inhibitory receptors. Activating receptors include natural cytotoxic receptors (NCRs) such as NKG2D, CD16, LFA-1, CD244 (2B4), and CD137 (4-1BB) and so on. These activating cell surface receptors have the capacity to trigger cytolytic programs as well as cytokine and chemokine secretion via intra-cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs) such as in 2B4 and 4-1BB and/or via other transmembrane signaling adaptors. Recent studies using NK cell-based activating receptor signaling modules such as NKG2D and 2B4 as CAR co-stimulatory domains proved increased anti-tumor activity compared to T-cell CARs.
Methods: The goal of this study is to develop next-generation NK cell-optimized CARs to improve targeting and potency of NK cell treatment. To achieve this, we constructed a library of CARs containing signaling modules from diverse T cell signaling receptors and NK cell specific signaling receptors, and then screened for CAR activity using short-term and long-term NK cell cytotoxicity assays. In comparison with T cell signaling receptors, CAR-NK cells with NK cell signaling receptors showed more potent cytotoxicity. Using a repeated rounds of cancer cell clearance assay, serial re-stimulation by fresh cancer target cells was tested. CAR-NK cells with NK cell signaling receptors showed remarkable persistence and antigen-mediated expansion of CAR-NK cells compared with T cell signaling receptors.
Results: We identified a NK cell-CAR containing two NK cell co-stimulatory domain and CD3z signaling domain,showing better persistence than T-cell CAR (CD28-CD28-zeta) and 2 previously described NK cell-CARs (NKG2D-2B4-zeta and 2B4).
Conclusions: We have successfully identified an optimized CAR suitable for NK cells that enable increased potency of NK cells compared to other CAR designs. The most potent CARs identified in our screen are being engineered into a highly effective dual targeting BCMA-GPRC5D CAR-NK cell product as a next-generation “off-shelf” cell therapy.
Citation Format: Cuiqing Yang, Yifang Wang, Tingting Liu, Fuwei Jiang, Qingyang Wang, Qin Wang, Gang Ye, Renhong Tang, Zhouxiao Cao. Optimized chimeric antigen receptors (CARs) for CAR-NK 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 4078.