Abstract 5512: CBLB, CISH and CD70 multiplexed gene knockout with CRISPR/Cas9 enhances cytotoxicity of CD70-CAR NK cells and provides greater resistance to TGF-β for cancer immunotherapy
Authors: Guo Chao , Fan Yanying , Aronov Alexander , Zhang Qi , Sahu Sombeet , Dequéant Mary-Lee , Guo Changan , Karnik Sushant , Leary Glenn D. , Massilamany Chandirasegaran , Chan Ivan H. , Trager James B. 
Published: June 2022

Abstract
Natural killer (NK) cells provide an attractive platform for development of effective cancer immunotherapies. NK cells are known for their ability to kill tumor cells and do not elicit graft-versus-host disease, making them a potential source of ‘off-the-shelf’ allogeneic cell therapy. NK cells are also amenable to CRISPR genomic engineering to enhance the antitumor activity of NK cells by increasing their cytotoxicity, overcoming suppression within the tumor microenvironment, or promoting their persistence and homing to tumor sites. Cytokine inducible SH2-containing protein (CISH) is a NK cell checkpoint for IL-15 mediated NK survival, proliferation, cytotoxicity, and anti-tumor immunity. The E3 ubiquitin ligase CBLB is also a negative regulator of NK cell function and has been shown to mediate TGF-β sensitivity by downregulating inhibitory SMAD7 in primary T cells. We hypothesized that knockout of both CISH and CBLB would not only improve NK cell effector function and but also render NK cells resistant to TGF-β mediated suppression. In this study, we utilized CRISPR-Cas9 ribonucleoproteins (RNPs) to disrupt CISH and CBLB genes in isolated peripheral blood NK cells from healthy donors. Given that CD70 expression is present on activated NK cells, to target CD70 on renal cell carcinoma (RCC) with CAR NK cells, CD70 was knocked out in NK cells to avoid fratricide. Western blotting, FACS and TIDE/Amplicon NGS Sequencing data confirmed all three genes were successfully disrupted. Then we expanded these edited NK cells by using IL-2 and stimulation using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. Furthermore, we were able to transduce CRISPR/Cas9 edited NK cells to express a CD70-CAR construct and membrane bound IL-15. CAR expression was assessed by flow cytometry. In vitro cytotoxicity was measured using the IncuCyte S3 live cell analysis system. CD70/CISH/CBLB triple knockout CD70-CAR NK cells could be produced efficiently and exhibited similar persistence as CD70/CISH or CD70/CBLB double knockout CD70-CAR NK cells in culture. Cytotoxicity assays demonstrated that CD70/CISH/CBLB triple knockout CD70-CAR NK cells had greater tumor growth control after multiple rechallenges. In the presence of exogenous TGF-β, CD70/CISH/CBLB triple knockout CD70-CAR NK cells showed greater resistance to TGF-β inhibition of cytotoxicity. In summary, we show CD70/CISH/CBLB triple knockout CD70-CAR NK cells demonstrate enhanced anti-tumor activity against relevant solid tumor cell lines and provide greater resistance to tumor microenvironment inhibition. These data support the further exploration of CD70/CISH/CBLB triple gene knockout CD70 CAR NK cells for clinical application.
Citation Format: Chao Guo, Yanying Fan, Alexander Aronov, Qi Zhang, Sombeet Sahu, Mary-Lee Dequéant, Changan Guo, Sushant Karnik, Glenn D. Leary, Chandirasegaran Massilamany, Ivan H. Chan, James B. Trager. CBLB, CISH and CD70 multiplexed gene knockout with CRISPR/Cas9 enhances cytotoxicity of CD70-CAR NK cells and provides greater resistance to TGF-β for cancer immunotherapy [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 5512.