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Natural killer (NK) cells are a critical component of the innate immune system and do not elicit graft-versus-host disease, making them an attractive source of ‘off-the-shelf’ allogeneic cell therapy. NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Another important anti-tumor role of NK cells is via antibody-dependent cell mediated cytotoxicity (ADCC). The process is mediated by CD16a of NK cells, which binds the Fc portion of antibodies. After NK cell activation, the CD16a ectodomain is rapidly cleaved from the NK cell surface by a disintegrin, Metalloprotease-17 (ADAM17), that is produced by both tumor cells and activated NK cells. This dampens NK cell activity by preventing NK cell attachment to antibody-coated target cells and diminishes CD16a signaling. Inhibiting ADAM17-mediated CD16a cleavage leads to augmented ADCC activity and increased cytokine production by human NK cells. Other NK cell activating receptors are known to be regulated by a similar mechanism1. In this study, we have utilized CRISPR-Cas9 ribonucleoproteins (RNPs) to disrupt the ADAM17 gene in isolated peripheral blood NK cells from healthy donors. FACS and Amplicon NGS Sequencing data confirmed successful disruption of the gene. Edited NK cells were expanded using IL-2 and stimulation with NKSTIM, a modified K562 stimulatory cell line expressing a 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 transduced ADAM17 edited NK cells to express a CD70 CAR2 or CD19 CAR3 construct and mbIL-15. CAR expression was assessed by FACS. In vitro cytotoxicity was measured by IncuCyte. Using an artificial activation system (phorbol 12-myristate 13-acetate [PMA]), we showed that ADAM17 KO NK cells maintain dramatically higher surface expression of CD16a and CD62L than control NK cells. In IncuCyte cytotoxicity assays against Raji tumor cells in the presence of rituximab (anti-CD20), ADAM17 KO NK cells and ADAM17 KO CD19 CAR NK cells demonstrated higher cytotoxicity compared to control NK cells. Similarly, ADAM17 KO NK cells and ADAM17 KO CD70 CAR NK cells also showed enhanced cytotoxicity against 786-O tumor cells in the presence of anti-EGFR antibody, cetuximab. Furthermore, ADAM17/CISH/CBLB triple KO CD70 CAR NK cells exhibited enhanced antitumor efficacy in vivo in a 786-O RCC tumor and a HL60 AML xenograft model. Taken together, we show ADAM17 KO NK or ADAM17 KO CAR NK cells demonstrate improved ADCC and ADAM17 KO can enhance in vivo anti-tumor activity of CISH/CBLB KO CD70 CAR NK cells against relevant tumor models. These data support the further exploration of ADAM17 gene knockout NK cells for clinical application and to improve efficacy of therapeutic antibodies in combination with adoptive transfer of engineered NK cells. Citation Format: Chao Guo, Yanying Fan, Alexander Aronov, Nikki Kimura, Damie Juat, Jessica Sood, Ivan H. Chan, James B. Trager. ADAM17 knockout NK or CAR NK cells augment antibody dependent cellular cytotoxicity (ADCC) and anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 890. |
