Abstract 4741: Targeting sorafenib-resistant HCCs by protein X-armed T cell immunotherapy

Abstract Hepatocellular carcinoma (HCC) is the most common type of liver cancer globally, ranking fourth according to the World Health Organization's GLOBOCAN database. Sorafenib and lenvatinib are single-drug therapies used for advanced HCC, but only around 30% of patients benefit from them, and drug resistance typically develops within a few months. Our previous study has demonstrated the specific IL-6-IGF-1R-YAP signaling in regulating the stemness expressions, tumor metastasis, and drug-resistance of the sorafenib resistant HCC (2015 Clinical Cancer Res, 2019 J Exp Clinical Cancer Research, 2021 Cancers, 2023 Cancers). In advance our previous study, this study aims to develop a novel immune therapeutic strategy for sorafenib-resistant HCC treatment. We found, when compared to peri-tumor tissues, the immunohistochemical staining showed a high co-expression of both protein X and PD-L1 in liver tumor tissues (6. 25%, n=32 versus 28. 125%, n = 64, respectively). To further confirm this observation, we used in vitro cell line models with sorafenib-naïve HCCs (HepG2215N, Hep3BN, Huh7N, and PLC5N) and sorafenib-resistant HCCs (HepG2215R, Hep3BR, Huh7R, and PLC5R) in this study. As PD-L1 on tumor cells can dampen T cell cytotoxicity, we generated CD3/protein X bispecific antibodies, which can bind to both CD3 and protein X, and coupled them to T cells (Arm-T cells) and normal T cells (N-T cells). We tested their cytotoxic effects on sorafenib-resistant HCCs using the sorafenib-resistant HepG2215R cells, co-cultured with Arm-T cells and N-T cells for 24 hours. Our results showed that Arm-T cells exhibited a significantly stronger cytotoxic effect on sorafenib-resistant HepG2215R cells, with several notable findings: (1) The binding time of Arm-T cells to HepG2215R cells was significantly longer than that of N-T cells (248. 9 ± 146. 6 minutes, n=23 versus 28. 3 ± 13. 49 minutes, n=24) ; (2) The cytotoxicity of Arm-T cells was significantly higher than that of N-T cells (29. 7 ± 2. 3% versus 13. 6 ± 3. 3%) ; and (3) The secreted levels of INFγ and TNFα by Arm-T cells were significantly higher than those of N-T cells (138. 1 ± 7. 2 ng/ml and 34. 6 ± 0. 7 ng/ml versus 31. 4 ± 2. 5 ng/ml and 3. 6 ± 1. 1 ng/ml, respectively). These findings were also consistent when using Hep3B-R cells (data not shown). To summarize, our study shows that Arm-T cells possess strong cytotoxic abilities against sorafenib-resistant HCC. These findings have implications for developing effective immuno-therapeutic strategies for patients with sorafenib-resistant HCCs. Citation Format: Mai-Huong Thi Ngo, Michael Chen, Pei-Chi Lan, Kuo-Hsiang Chuang, Yen-Hua Huang. Targeting sorafenib-resistant HCCs by protein X-armed T cell immunotherapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4741.