Celularity is currently developing a proprietary allogeneic NK cells (PNK) derived from human placental CD34+ progenitors for treatment of a broad spectrum of blood disorders and malignant diseases. To augment the anti-tumor function of CD34-derived PNK-cells, a study was conducted to simultaneous knock-out (KO) CBLB (negative regulator of NK cytotoxicity) and TGFβ receptor II TGFBR2 (tumor microenvironment) genes in PNK-cells using CRISPR/Cas9. In DKCT (Double Knockout CBLB TGFBR2) cells, high efficiency of editing (>80%) was achieved for both genes and did not affect expansion and differentiation of CD34 cells into DKCT PNK-cells. The results showed that knocking-out CBLB (CBLB-KO) in PNK-cells led to an increase (2–4 fold) of cytotoxicity and cytokine secretion against a range of hematological and solid cancer cell lines as well as primary tumor cells. However, these enhanced functions from CBLB-KO PNK-cells were still found to be sensitive to TGFβ-mediated suppression. TGFBR2-KO rendered PNK-cells the ability to maintain high-level expression of NK activating receptors and resist inhibition of cytotoxicity after TGFβ exposure. The improved cytolytic function of DKCT cells in vitro was confirmed using xCELLigence platform. Furthermore, DKCT PNK-cells were insensitive to exogenous TGFβ and exhibited improved (2–4 fold) cytotoxicity. Subsequently, we compared the anti-tumor activity of DKCT to controls in an in vivo model of AML, where conditioned NSG mice were engrafted with HL60-GFP-luc cells 3 days prior to PNK-cell infusion. All four PNK-cell groups exhibited increased survival compared to vehicle. In CBLB-KO and DKCT groups, the tumor burden was similar but significantly reduced compared to Cas9 control or TGFBR2-KO. Future studies will address the benefit of DKCT PNK-cells in TGFβ secreting tumor-bearing animal models to pave the way for genetically modified NK cell therapy.

Citation Format: James Li, Xuan Guo, Hemlata Rana, Andrea DiFiglia, Joseph Gleason, Uri Herzberg, Robert Hariri, Xiaokui Zhang. Genetic modification potentiates the antitumor activity of human placental CD34+ cells-derived NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 935.