Abstract
Hepatocellular carcinoma remains a life-threatening malignancy with limited therapeutic options following the failure of second-line treatments1,2. Oncolytic viruses selectively replicate in and lyse cancer cells, releasing neoantigens and stimulating systemic antitumour immunity3, offering a potential therapeutic option. Here we present the results of a multicentre phase 1 clinical trial evaluating VG161, an engineered oncolytic herpes simplex virus that expresses IL-12, IL-15, IL-15Rα and a PD-1–PD-L1-blocking fusion protein4, for safety and efficacy in patients with advanced liver cancer. VG161 was well tolerated, with no dose-limiting toxicities observed, and it demonstrated promising efficacy by reshaping the tumour immune microenvironment and re-sensitizing tumours that were previously resistant to systemic treatments. Notably, we also found that patients who had previously been sensitive to checkpoint inhibitor therapy showed enhanced efficacy with VG161 treatment. Furthermore, we developed an efficacy-prediction model based on differentially expressed genes, which successfully identified patients who were likely to benefit from VG161 and predicted prolonged overall survival. These findings position VG161 as a promising third-line therapeutic option for refractory hepatocellular carcinoma. This provides a new avenue for treatment and advances the field of oncolytic virus-based immunotherapies. ClinicalTrials.gov registration: NCT04806464.
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Data availability
The study protocol is available in the Supplementary Information. The datasets from the clinical trial can be requested 12 months after the publication of this article. Researchers wishing to access raw and analysed data should email the corresponding author, clearly stating the research purpose. Requests will be reviewed by the institutional review board, considering the risk of patient reidentification, and a response can be expected within 14 days. Individual deidentified data of the participants will be available to approved eligible applicants and investigators after signing a data access agreement. The raw sequence data reported in this study have been deposited in the Genome Sequence Archive (GSA-Human: HRA007839 (RNA sequence); GSA-Human: HRA007880 (scRNA-seq, scTCR-seq, scBCR-seq and spatial transcriptome)) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. Source data are provided with this paper.
Change history
23 April 2025
A Correction to this paper has been published: https://doi.org/10.1038/s41586-025-09022-x
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Acknowledgements
This work was supported by National Key Research and Development Program of China (2024YFA1306400 to T.L. and 2020YFA0804300 to Q.Z.); CNBG-Virogin Biotech (Shanghai); Virogin Biotech; the Joint Fund for Regional Innovation and Development of National Natural Science Foundation of China (U23A20462 to X.B.); National Natural Science Foundation of China (82071867 to X.B., 82103044 to Y.S., 92359304 to Q.Z., 32321002 to Q.Z., 82473461 to Q.Z., 82203699 to T.F. and 81902597 to L.W.); “Ling Yan” Research and Development Program of Department of Zhejiang Province Science and Technology (2024C03167 to X.B.); Natural Science Foundation of Zhejiang Province/Exploration Project (LY21H160037 to W.C. and LZ22H030003 to Y.Y.); and Medical Health Science and Technology Project of Zhejiang Province (2024KY958 to W.C.). The authors thank OE Biotech for providing technical support with RNA-seq, single-cell sequencing and spatial transcriptomics.
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Y.S., Q.T., R.Z. and T.L. designed the clinical trial. Y.S., X.B., Q.Z., X. Liang, S. Gu, G.S., Y.W., T.F., Y.L., L.W., X.S., W.C., Y.Y., Y.C. and X. Li were responsible for patient treatment and management. X.J. and G.W. performed ultrasound-guided puncture administration. Z. Zheng, J.S. and M.C. recruited patients and collected data. Y.S., A.Q., L.X., Y.Q., W.B., S.R., R.Z. and M.H. conducted statistical analysis. Z. Zhao, W.J., Z.W., Z.Y., S. Guo, D.L., F.W. and J.D. were responsible for the basic research work. W.S., M.S. and Y.M. performed bioinformatics analysis. Y.S. and T.L. wrote the manuscript. All authors read and approved the final draft of the paper.
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CNBG-Virogin Biotech (Shanghai) owns the patent rights for VG161 in mainland China. Virogin Biotech owns the patent rights for VG161 outside mainland China. The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Patient enrolment.
Of 92 patients who provided informed consent, 44 patients who matched the eligibility criteria were enrolled.
Extended Data Fig. 2 Exclusion of the possible effect of anti-HBV agents on VG161 replication in vitro and in vivo.
a, b, Comparison of the blood VG161 viral DNA concentration between patients who received (n = 6 biologically independent patients) or did not receive (n = 5 biologically independent patients) oral administration of anti-HBV agents within 24 h. c, Female NCG mice were implanted subcutaneously with Hep3B cells and gavaged with entecavir (0.1 mg/kg/day) or PBS (n = 3 biologically independent animals). Intratumoral injection of 1 × 107 PFU VG161 was performed 3 days after gavage. Hep3B tumors were excised at indicated time (0, 0.5, 1, 2, 3, and 7 days). The virus titers of tumor lysate were measured by plaque assay on Vero cells. d, The in vitro cytotoxicity of different anti-HBV agents on the tumor cells. Cells were treated with anti-HBV agents, and cell viability was measured using the CCK-8 assay at 65 h (6.7 × 104 cells examined over 4 independent experiments). e, Hep3B cells were treated with or without anti-HBV agents for 12 h, followed by replacement with fresh medium and infection with VG161. The cell viability upon different MOI and pretreatments was measured using the CCK-8 assay at 65 h (n = 6.7 × 104 cells examined over 4 independent experiments). f, The effect of anti-HBV agent treatment on VG161 replication ability in Hep3B cells. Hep3B cells were treated with or without anti-HBV agents for 12 h, followed by replacement with fresh medium and infection with VG161 for 24, 48, 72 h. The culture supernatant were collected, mixed, and lysed, then titrated by plaque assay (n = 0.8 × 106 cells examined over 3 independent experiments). For b, Fisher’s exact test was used for analysis. For d and e, No statistical tests were performed to compare differences. For a, c and f, Data are presented as the mean ± standard error and were analyzed using the unpaired two-sided Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and NS, not significant).
Extended Data Fig. 3 Immunological profiles in patients’ blood samples after VG161 administration.
a, Lymphocyte subset trends in peripheral blood (n = 9). b, Plasma cytokine changes in peripheral blood (n = 11). c, Changes of typical T cell markers representing immune state in peripheral blood (n = 11). The dosing regimen for each patient was as follows: Patient A1-101 received a single dose of 1.0 × 108 PFU. Patient A2-102 received daily doses of 1.0 × 108 PFU for 2 consecutive days, resulting in a total dose of 2.0 × 108 PFU. Patients A3-104, A3-105, and A3-108 each received daily doses of 1.0 × 108 PFU for 3 consecutive days, resulting in a total dose of 3.0 × 108 PFU. Patients A4-109, A4-110, and A4-202 were administered daily doses of 1.3 × 108 PFU for 3 consecutive days, resulting in a total dose of 4.0 × 108 PFU. Finally, patients A5-119, A5-120, and A5-205 received daily doses of 1.7 × 108 PFU for 3 consecutive days, resulting in a total dose of 5.0 × 108 PFU. The 1.3 × 108 PFU and 1.7 × 108 PFU dose represents a rounded value to one decimal place.
Extended Data Fig. 4 Pathological examination of patient no. A4-109 from Fig. 2.
a, Pathological results from biopsy samples taken before the patient received VG161. b, A puncture biopsy was performed at the injection site after the VG161 treatment, and the pathological results demonstrated typical tumor cell necrosis. Please note that these biopsies were obtained from a single clinical patient and could not be repeated due to ethical and procedural considerations.
Extended Data Fig. 5 Efficacy prediction model for VG161 in advanced HCC.
a, Graphic overview of the experimental strategy. Created in BioRender. Lin, D. (2025) https://biorender.com/m07m206. b, KM survival curves between low- and high-survival groups. c, Volcano plot for DEGs of low- (n = 10 biologically independent patients) and high-survival groups (n = 11 biologically independent patients), P values are calculated using two-sided Student’s t-test. Benjamini-Hochberg adjusted p value < 0.05 is calculated as a significance cutoff. d, GO and KEGG analysis of low- (n = 10 biologically independent patients) and high-survival groups (n = 11 biologically independent patients), p values are calculated using two-sided hypergeometric test. Benjamini-Hochberg adjusted p value < 0.05 is calculated as a significance cutoff. e, Univariate Cox regression analysis identification of the prognosis-associated DEGs. f and g, LASSO Cox regression analysis assessment of the prognostic and screen risk genes, data is the cross validation mean (cvm) ± cross validation standard error (cvsd) (Low-survival group, n = 10 biologically independent patients and high-survival group, n = 11 biologically independent patients). h, Multivariate Cox regression analysis of risk genes, data is the hazard ratio (HR), 95% confidence intervals (CIs), p values are calculated using two-sided log rank tests. i, KM survival curves based on the risk model. j, ROC curves based on the risk model. k, KM survival curves based on the risk model in the TCGA-LIHC cohort. For b, i and k, p values are calculated using two-sided log rank tests, the shadows on either side of the survival curves depict 95% CIs.
Extended Data Fig. 6 The contrast of KM curves between the VG161-C102 group vs. control group.
a, Overall OS in the VG161-C102 group vs. control group. b, Comparison of the OS between the VG161-C102 and control groups in the >3 m subgroup. c, OS of the non-PST and control groups in the VG161-C102 group. d, Comparison of the OS between the non-PST+PreCPI > 3 m patients in the VG161-C102 group and the PreCPI > 3 m patients in the control group. Subgroups were compared using the log-rank test.
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Shen, Y., Bai, X., Zhang, Q. et al. Oncolytic virus VG161 in refractory hepatocellular carcinoma. Nature 641, 503–511 (2025). https://doi.org/10.1038/s41586-025-08717-5
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DOI: https://doi.org/10.1038/s41586-025-08717-5
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