Abstract 891: CRISPR-Cas9-gRNA RNP mediated gene knockout of TGFBR2 and CISH enhances CD19-CAR NK cell function and provides resistance to TGFβ

Abstract

Abstract Natural killer (NK) cells, known for their ability to kill tumor cells, represent a promising ‘off the shelf' therapeutic for cell-based cancer immunotherapy. One of the major challenges with NK cell-based therapies includes their lack of persistence and expansion after adoptive transfer. In addition, the presence of inhibitory signals within the tumor microenvironment and mediated by tumor cells also contributes to the poor infiltration and activation of NK cells at the tumor site. To improve NK cell function, we developed an optimized CRISPR-Cas9 system utilizing electroporation of CRISPR-gRNA ribonucleoproteins (RNPs) to disrupt important regulatory genes to enhance and/or protect primary human NK cell function. This method allowed efficient knockout of the TGFBR2 and CISH genes in NK cells, genes that have a functional impact on NK cells. FACS and Amplicon NGS Sequencing data confirmed both genes were successfully disrupted. Furthermore, we were able to transduce and expand CRISPR/Cas9 edited NK cells to express a CD19-CAR construct and membrane bound IL-15. Cytotoxicity assays demonstrated that TGFBR2 knockout CD19-CAR NK cells were resistant to TGFβ inhibition of cytotoxicity. We also showed that CISH gene-edited CD19-CAR NK cells have significantly improved proliferation, survival, cytokine production, and cancer cell cytotoxicity. In summary, this data demonstrates that this platform enables effective primary human NK cell gene knockout to enhance NK function and provide resistance to tumor microenvironment inhibition. Citation Format: Chao Guo, Ivan H. Chan, Luxuan Buren, Yanying Fan, Alexander Aronov, James B. Trager. CRISPR-Cas9-gRNA RNP mediated gene knockout of TGFBR2 and CISH enhances CD19-CAR NK cell function and provides resistance to TGFβ [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 891.