Kras-mutant oncolytic virus vaccine therapy for metastatic colorectal cancer: A preclinical study.

Abstract

e15547 Background: Kirsten rat sarcoma (KRAS) mutations lead to aberrant activation of the RAS/RAF/MEK/ERK signaling pathway, promoting cell proliferation and metastasis, and are among the most common oncogenic mutations in colorectal cancer (CRC) patients. Frequent point mutations include G12D, G12V, and G13D. Patients with these mutations often have poor prognosis and exhibit drug resistance. Immunotherapy may become a potential treatment to improve the current status.Oncolytic virus vaccines are genetically engineered viruses targeting tumor-specific antigens, which enhance tumor cytotoxicity and immune responses. This study aims to initially construct an oncolytic virus vaccine targeting KRAS mutations and validate its preclinical efficacy, addressing the lack of treatment options for advanced colorectal cancer and improving the poor response of CRC to immunotherapy. Methods: Based on the wild-type herpes simplex virus HSV-1 (F strain), we constructed KRAS-mutant oncolytic virus vaccine(KR10) by incorporating the KRAS mutation gene(G12D, G12C,etc). KR11, with GFP inserted, and KR12, with Flu-MP inserted, were used as control groups. Western blot was applied to verify the success of the construction. CCK-8 assay were performed to assess the selective cytotoxic effects of KR10 on normal cells and tumor cells.A subcutaneous xenograft tumor model (CT26 colorectal cancer) in mice was developed to evaluate the anti-tumor efficacy of KR10 at different viral doses. Pharmacokinetic and pharmacological studies were then conducted in large animal models(crab-eating macaques). Results: The KR10 was engineered successfully. Cytotoxicity assays showed the selective killing of tumor cells and the safety of KR10 on normal cells. In the CT26 (KRAS -G12D) model, KR10 significantly reduced tumor size and volume compared to the control groups, with effects increasing with dose within a certain range.Furthermore, re-administration of KR10 in mice demonstrated sustained anti-tumor effects. For crab-eating macaques, intravenous and subcutaneous KR10 administration showed no signs of severe toxicity, death, or distress, and there were no significant abnormalities in any safety indicators. Pharmacokinetic studies revealed dose-dependent viral clearance with minimal drug accumulation, and limited viral shedding via the respiratory/digestive tracts. Immunological assays indicated mild immune activation, evidenced by elevated IgG and IgM levels and complement consumption. Conclusions: This study is a preclinical investigation of KRAS-mutant oncolytic virus vaccine for the treatment of metastatic colorectal cancer. It is the first to construct an oncolytic virus (KR10) targeting the G12D mutation on KRAS, with in vitro efficacy validation and animal experiments. The funding for this study was supported by the Shanghai Shenkang Project (No.SHDC2022CRT009).