Abstract 554: Development of an iPSC-derived NK cell screening platform for discovery of NK cell optimized Chimeric Antigen Receptors (CARs) for next-generation CAR-NK cell immunotherapies
Authors: Swanson Lee , Allen Gabrielle , Lyon Kenyon , Tadros Wael , Shabla Max , Kaufman Dan S. , Zhu Huang 
Published: June 2022

Abstract
Chimeric Antigen Receptor (CAR) immunotherapy has transformed cancer therapeutics and has shown great success in treating hematopoietic malignancies. To date, autologous CAR-T-cell therapy has been the main clinical approach. However, autologous CAR-T cells have several major limitations including high-cost and complexity of production, donor-to-donor variability, and potentially severe toxicities. Allogenic NK cell-based therapies have also been shown to mount potent responses against hematopoietic malignancies (i.e., AML). However, unlike T-cell therapies, allogeneic NK cells do not cause toxicities such as cytokine release syndrome, neurotoxicity, or graft-vs-host-disease. Therefore, this approach provides an important platform for allogeneic CAR-enabled therapies. Several groups have demonstrated the clinical efficacy of allogeneic CAR-expressing NK cells that utilize CARs developed for T-cells. However, 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. The goal of this study was to develop next-generation NK cell-optimized CARs to improve targeting and potency of our iPSC-derived NK cell therapies. To achieve this, we developed a platform to comprehensively screen immune cell signaling modules in iPSC-derived NK cells. First, we constructed a library of 44 novel CAR constructs containing signaling modules from diverse NK cell specific signaling receptors. Next, we developed a viral-based CAR expression protocol in mature iPSC-derived NK cells that yields high CAR expression (&gt;75% CAR+) while maintaining high viability (&gt;90%). We then screened for CAR activity using two co-culture target cell killing assays (eSight impedance assay and caspase 3/7 killing assay) and two NK cell-resistant CD19+ target cell lines (Raji and SupB15). Our screen identified 7 novel CAR signaling modules that performed better than both a second-generation T-cell CAR (CD28-CD28-zeta) and 3 previously described NK cell-CARs (NKG2D-2B4-zeta, CD28-OX40-zeta, and CD28-OX40L-zeta). These results were consistent across both functional assays and both target cell lines tested adding support to our findings. Overall, we have successfully developed an efficient CAR screening platform in iPSC-derived NK cells and have identified NK optimized CARs that enable increased potency of NK cells compared to current CAR solutions. The most potent CARs identified in our screen are being engineered into CISH-knockout iPSC-derived NK cells which were previously developed by us that demonstrate improved in vivo anti-tumor activity, in vivo persistence, metabolic fitness, polyfunctionality and resistance to cell exhaustion to create next-generation CAR-NK cell-based immunotherapies.
Citation Format: Lee Swanson, Gabrielle Allen, Kenyon Lyon, Wael Tadros, Max Shabla, Dan S. Kaufman, Huang Zhu. Development of an iPSC-derived NK cell screening platform for discovery of NK cell optimized Chimeric Antigen Receptors (CARs) for next-generation CAR-NK cell immunotherapies [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 554.