Recombinant Adenovirus Kh901 Anti-tumor Research

Background and Objective: KH901 is a tumor-specific, conditionally replicating oncolytic adenovirus in which the viral E1A gene is under the control of a genetically modified telomerase reverse transcriptase promoter. It can replicate largely in the tumor cell which have high level of telomerase activity and dissolve the cells, but can not replicate in normal cells. KH901 also carries a cDNA of human granulocyte macrophage colony stimulating factor (GM-CSF) in the E3 region replacing the MHC inhibitory gene. GM-CSF is considered the most potent cytokine that induces specific, long-lasting immunity to tumors. Thus, KH901 will not only produce local antitumor activity by direct killing of tumor cells at local sites through oncolytic effects, but also stimulate immune responses to tumor cells resulting in systemic antitumor immunity to distant tumor mess. This study was to show in vitro conditionally oncolytic specificity of KH901 by viral replication and cytotoxicity assays and in vivo anti-tumor efficacy by xenograft tumor model. In the meantime , the GM-CSF expression of KH901 and its biodistribution was measured.Methods: In vitro: A panel of tumor and normal cell lines was infected with KH901, then the GM-CSF expressing level and the titer of KH901 was determined and its cytotoxicity was assayed by MTT assay. In vivo: Nude mice were inoculated with human hepatocellular carcinoma Hep3B and prostate cancer LNcap cells and neck cancer SCC9 to establish tumors xenografts model. KH901 were intratumorally administered and then the Relative Tumor Growth Rate (T/C%) and Restraint Tumor Rate were calculated to observe the antitumor efficacy. C57 mice were inoculated with murine lung cancer CMT-64 cells to establish tumors xenografts model, then the antitumor efficacy was observed after KH901 had been administered . And the percent of CD11c+ cells were calculated in tumor flowing lymph node. KH901 were intratumorally administered in human lung cancer A549 xenografts tumors model in nude mouse, then the blood and tumor tissue of nude mouse were sampled at various time point and the rhGM-CSF content of the sample were to be measured with ELISA method. In the meantime, the biodistribution of KH901 in A549 xenografts tumors model and rhesus was measured by quantitative PCR.Conclusion: It was showed in vitro that KH901 was largely replicated in tumor cells(2526.4±136.8～2796.6±104.6TCID₅₀/Cell) and produced significant amount of GM-CSF [621.907±10.17～3924.497±17.79 IU/ (10⁶ cell·24h)] and killed tumor cells strongly (IC₅₀:0.31±0.06～0.19±0.01MOI) while it replicated poorly in non-permissive human normal cells (56.8±9.2～90.1±14.4TCID₅₀/Cell)and produced very small amount of GM-CSF [13.397±0.82 IU/ (10⁶ cell·24h)] and was attenuated in human primary cells killing (IC₅₀:92.33～121.20 MOD . In vivo studies showed that KH901 had a statistically significant antitumor efficacy in Hep3B and LNcap and SCC9 xenografts tumors model in nude mouse, Though it has a definite antitumor efficacy in CMT-64 xenografts tumors model in C57 mouse, but it has not the ability to induce the manifold of CD11c+ cell in murine tumor flowing lymph node. The adenovirus vector and the GM-CSF expression in the tumor and blood of A549 xenografts tumors model can be measured ,and they were gradually reduced as time process, there were less biodistribution in other tissues.Conclusion: In vitro and in vivo studies showed the selective replication, cytotoxicity, GM-CSF production of KH901, suggesting a potential utility of this oncolytic agent for the treatment of solid tumors.