Signaling intact membrane-bound IL-15 enables potent anti-tumor activity and safety of CAR-NK cells

Xiaodi Xu¹Peiyu Cao¹Meng Wang¹Yan Wan¹Shuwen Sun¹Yuxin Chen¹Yilin Liu¹Tong Su¹Ge Gao¹Xinze Liu²Weixiang Zhong¹Xi Chen¹Xiaoyuan Lu³Buze Chen¹Junnian Zheng^4*Gang Wang^1*Huizhong Li^1*

• ¹Cancer Institute, Xuzhou Medical University, Xuzhou, China
• ²The Second Clinical Medical School, Nanjing Medical University, Nanjing, China
• ³Department of Obstetrics and Gynecology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China
• ⁴Center of Clinical Oncology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, China

Background: Chimeric antigen receptor (CAR)-NK cells are a promising and safe alternative to CAR-T cells. However, the limited persistence in vivo restricts their clinical application and sustained therapeutic responses. IL-15 has been extensively used to improve CAR-NK cell persistence and effectiveness. Nevertheless, accumulation of IL-15 might induce uncontrolled proliferation of CAR-NK cells and thus lead to fatal side-effects. Therefore, it is essential to develop a safe and effective alternative strategy to improve the persistence and anti-tumor activity of CAR-NK cells. Methods: A signaling intact membrane-bound IL-15 (mbIL-15) was designed by fusing IL-15 and full-length IL-15Rα and was systematically compared with secretory IL-15 (sIL-15) in a B7H3-targeting CAR-NK cell system regarding their functionality and safety through various in vitro and in vivo experiments. Results: Both expression of sIL-15 or mbIL-15 significantly enhanced the proliferation by activating STAT5 and improved anti-tumor activity of CAR-NK cells in vitro and in vivo. Although CAR-NK cells with sIL-15 quickly eliminated intraperitoneal ovarian cancer, the mice experienced severe consequences, including dysregulated CAR-NK cell expansion, intense inflammatory responses, and irreversible organ damages. In contrast, CAR-NK cells carrying mbIL-15 showed moderate cell proliferation and potent tumor killing activity without observable adverse effects in both local treatment and systemic administration models. Conclusion: Head-to-head comparative studies demonstrated that signaling intact mbIL-15 significantly improved therapeutic efficacy and safety of CAR-NK cells compared to sIL-15, which provided preclinical evidence for future clinical development.