Abstract 4853: Knockout of hypoxia inducible factor 1 subunit alpha (HIF1a) augments the antitumor activity of EGFR CAR-NK cells

Abstract Chimeric antigen receptor T-cell (CAR-T) therapies have gained remarkable success with durable remissions in hematologic malignancies. However, the potential adverse effects, including cytokine release syndrome (CRS), graft-versus-host disease (GVHD), neurotoxicity, and off-tumor toxicity, and high production cost have limited the clinical application of CAR-T cells in oncological treatment. Recently, natural killer (NK) cells has emerged as a promising alternative immunotherapy with high biosafety and universal application. Nevertheless, the persistence and efficacy of CAR-NK cells remains to be solved. Knockout of immune checkpoint molecules (ICPs) has been proved to be an effective way to enhance the potencies of CAR-NK cells, such as cytokine inducible SH2-containing protein (CISH) and Cbl proto-oncogene B (CBLB). In solid tumors, hypoxic microenvironment can drive tumor-infiltrating NK cells dysfunction. Cellular response to hypoxia is mediated by hypoxia-inducible factors. Hypoxia inducible factor 1 (HIF1) is a master regulator and inhibits activation markers and effector molecules of tumor-infiltrating NK cells in hypoxic conditions. In this study, we disrupted HIF1a, alpha subunit of HIF1, in NK cells derived from peripheral blood mononuclear cells (PBMCs) to evaluated the anti-tumor effects. Then EGFR CAR molecules were transduced into the NK cells with retroviral vector. Successful knockout was performed by CRISPR/Cas9 and confirmed by TIDE, ICE and qPCR analysis. Our results showed that the NK cell viability was slightly affected by electroporation and gradually returned, leading to a slight reduction in knockout NK cell expansion. However, the freeze-thaw viabilities viability of knockout NK cells was comparable to that of control cells. Flow cytometry assay demonstrated that several stimulatory receptors were up-regulated in HIF1a-/- NK cells, such as CD57, CD62L, CD69, and NKp44, and some inhibitory and activating receptors were down-regulated, including NKG2A, CD38, NKp30 and NKp46. Using luciferase-based serial killing analysis, the knockout group exhibited greater in vitro cytotoxicity against HCT116 and SHP77 tumor cells compared to that of unedited CAR-NK. Moreover, HIF1a knockout significantly enhanced the anti-tumor activity of CAR-NK cells in SHP77 subcutaneously inoculated xenograft mouse model. Taken together, our data suggested that HIF1a deficiency could enhance the cytotoxicity and persistence of CAR-NK cells, and supported the further exploration for clinical application. Citation Format: Fanxiang Gao, Tingting Liu, Qin Wang, Yanxue Gong, Chao Wang, Feng Zhou, Zhuoxiao Cao. Knockout of hypoxia inducible factor 1 subunit alpha (HIF1a) augments the antitumor activity of EGFR CAR-NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4853.