Abstract A009: Enhanced antitumor efficacy of IL-15/IL-15 receptor α/IL-21-armed ROR1 CAR-NK cells against solid tumors

Abstract Primary natural killer (NK) cells derived from CD3-depleted peripheral blood mononuclear cells (PBMCs) were cultured in the presence of IL-15 and IL-21 and electroporated with mRNA encoding chimeric antigen receptor (CAR) constructs. The CAR constructs contained an anti-ROR1 single-chain variable fragment (scFv) as the ectodomain and 4-1BB, DAP10, and CD3ζ as the endodomains (ROR1 CAR-NK). Cytotoxicity assays revealed that the optimal endodomain configuration was 4-1BB–DAP10–CD3ζ (4-1BB–DAP10–CD3ζ > 4-1BB–CD3ζ–DAP10>4-1BB–CD3ζ). To enhance cytokine support, secretory IL-15/IL-15 receptor α (IL15LI ROR1 CAR-NK) or IL-21/IL-15/IL-15 receptor α (IL21-IL15LI ROR1 CAR-NK) modules were incorporated into the 4-1BB–DAP10–CD3ζ design. IL15LI ROR1 CAR-NK cells secreted ∼5 ng/ml IL-15, whereas IL21-IL15LI ROR1 CAR-NK cells secreted ∼5 ng/ml IL-15 and ∼10 ng/ml IL-21 simultaneously. Expression of anti-ROR1 scFv and ROR1 antigen-binding efficiency were both >90%. Notably, IL21-IL15LI ROR1 CAR-NK cells displayed upregulated activating receptors, including NKp46 and CD25, and downregulated inhibitory receptors, such as TIGIT and PD-1. In addition, they demonstrated enhanced proliferation compared with control groups. Functionally, these cells displayed significantly improved cytotoxicity against ROR1high solid tumor cells, whereas their activity against ROR1low tumor cells showed no substantial difference from that of conventional ROR1 CAR-NK cells. These results were further supported by real-time cytotoxicity assays. In vivo xenograft models further demonstrated that cytokine-armed ROR1 CAR-NK cells suppressed tumor growth in a dose-dependent manner. Collectively, these in vitro and in vivo results indicate that cytokine-armed ROR1 CAR-NK cells exhibit strong antitumor activity against ROR1-positive solid tumors by modulating NK cell receptor expression and NK cell growth.This work was supported by the Industrial Technology Innovation Program [P0028496] of the Ministry of Trade, Industry and Energy, Republic of Korea. Citation Format: Inpyo Choi, Soobin Jung, Heeju Jeon, Soo-Yeon Park, Gwangsig Ryu, Jinok Koh. Enhanced antitumor efficacy of IL-15/IL-15 receptor α/IL-21-armed ROR1 CAR-NK cells against solid tumors [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr A009.