A conceptual exploration on the synergistic anti-tumor effects of high-order combination of OHSV2-DSTE FAP5/CD3 , CAR-T cells, and immunotoxins in hepatocellular carcinoma

Shuang Dong¹Xin Chen²Xiaoyu Li¹Yang Wang³Qing Huang¹Yuanxiang Li¹Jing Jin⁴Xianmin Zhu¹Yi Zhong¹Qian Cai¹Xue Chang¹Fang Guo⁵Le Huang²Mingqian Feng²Binlei Liu³Sheng Hu^6*

• ¹Department of Medical Oncology, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hebei Province, China
• ²College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei Province, China
• ³Hubei Key Laboratory of Industrial Microbiology, Hubei University of Technology, Hubei, Hebei Province, China
• ⁴Wuhan Binhui Biopharmaceutical Co., Ltd, Wuhan, China
• ⁵Department of Pathology, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and Technology., Wuhan, Hebei Province, China
• ⁶Department of medical oncology, Huazhong University of Science and Technology, Tongji Medical College, Hubei Cancer Hospital, Hubei Cancer Hospital, Wuhan, China

BACKGROUND: Although the treatment landscape for advanced hepatocellular carcinoma (HCC) has seen significant advancements in the past decade with the introduction of immune checkpoint inhibitors and antiangiogenic drugs, progress has fallen short of expectations. Recently, a novel engineered oncolytic virus (OHSV2) that secretes dual-specific T cell engagers (DSTEs) targeting the fibroblast activation protein (FAP) was developed and combined with GPC3-targeting CAR-T cells and immunotoxins to exert a synergistic antitumor effect. METHODS: OHSV2-DSTEFAP5/CD3 was initially generated by transducing the DSTEs engaging FAP5 on fibroblasts into the backbone of our oncolytic virus OHSV2. An innovative high-order combination was devised in a xenograft mouse model to conceptually explore whether enhanced anti-tumor effects could be achieved. Additionally, the underlying mechanisms of synergistic effects and safety profiles were preliminarily investigated.RESULTS: OHSV2-DSTEFAP5/CD3 effectively targeted and eliminated fibroblasts in vitro while maintaining cytotoxicity and inducing immune activation compared to parental OHSV2. In vivo, dose adjustment combination therapy resulted in a remarkable antitumor effect compared to control treatments, leading to tumor regression in 40% of mice without significant toxicity to major organs. Mechanistically, rather than directly depleting fibroblasts, OHSV2-DSTEFAP5/CD3 played an essential role in priming T cell proliferation, infiltration, and activation, as well as inhibiting the supportive interaction between cancer cells and fibroblasts. CONCLUSIONS: This high-order combination represents a novel multiple-wave immunotherapeutic approach for HCC. Despite being a conceptual exploration, this strategy has demonstrated promising therapeutic efficacy and acceptable safety profiles.