Abstract A031: Optimizing immunotherapeutic efficacy of off-the-shelf liver natural killer cells for hepatocellular carcinoma: role of IL-37 in overcoming cryopreservation challenges
Authors: Imaoka Yuki , Ohira Masahiro , Nakayama Toshihiro , Sasaki Kazunari , Ohdan Hideki 
Published: February 2025

Background: Hepatocellular carcinoma (HCC) is characterized by high mortality and recurrence rates worldwide. Liver transplantation (LT) is considered the best treatment option, as it removes both the tumor and carcinogenic liver tissue, reducing recurrence risk. Despite this, effective adjuvant therapies to prevent metastasis after LT or hepatectomy remain lacking. Fresh liver natural killer (NK) cell therapy has shown promise, and phase I clinical trials have been completed. Previous research demonstrated that the PI3K/AKT/FOXO1 pathway is crucial for NK cell function and that interleukin-33 (IL-33) suppresses NK cell antitumor activity. This study focused on interleukin-37 (IL-37), an inhibitor of the PI3K/AKT/FOXO1 pathway, aiming to develop “off-the-shelf” liver NK cell therapy that maintains consistent antitumor activity independent of donor variability. Methods: Lymphocytes were isolated from liver transplant donors and cultured for three days with the combination of IL-2 and IL-37, followed by anti-CD3 antibody treatment to deplete T cells. NK cell activation markers and cytotoxicity were assessed using flow cytometry. Serum IL-37 levels were measured in 120 patients who underwent initial liver resection, and their association with clinical outcomes was analyzed. Results: Liver NK cells were highly sensitive to cryopreservation stress, leading to reduced viability and cytotoxic function after thawing. IL-37 significantly increased the proportion of TRAIL-positive and NKp44-positive NK cells in fresh samples (N=6), enhancing cytotoxicity against the K562 cell line. IL-33 levels in the culture supernatant showed an inverse correlation with the proportion of TRAIL-positive and CD69-positive NK cells (N=20). IL-2 and IL-37 treatment also partially restored TRAIL expression in liver NK cells post-cryopreservation. Patient characteristics were similar between high and low IL-37 groups. However, higher serum IL-37 levels were significantly associated with better recurrence-free survival (RFS) and a lower rate of early recurrence within two years (Fig. 2). IL-37 was identified as an independent prognostic factor for 2-year (HR 0.99, p=0.047) and 5-year RFS (HR 0.99, p&lt;0.01). Conclusions: The findings of this study suggest that liver NK cell therapy using IL-37 has the potential to retain its immunotherapeutic efficacy even after cryopreservation and thawing. The development of "off-the-shelf" NK cells capable of maintaining their functional properties post-thawing represents an ideal adjuvant therapy that can overcome temporal and geographical constraints associated with current treatments. Furthermore, this approach could facilitate multiple administrations and personalized immunotherapy for high-risk HCC patients, enhancing treatment outcomes and potentially reducing recurrence rates.

Citation Format: Yuki Imaoka, Masahiro Ohira, Toshihiro Nakayama, Kazunari Sasaki, Hideki Ohdan. Optimizing immunotherapeutic efficacy of off-the-shelf liver natural killer cells for hepatocellular carcinoma: role of IL-37 in overcoming cryopreservation challenges [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A031.