AbstractObjectiveIn order to ensure the stable transcription of target genes, we constructed a eukaryotic high expression vector carrying an immune‐check inhibitor PD‐1v and a variety of cytokines, and studied their effects on activating immune response to inhibit tumor growth.MethodsA novel eukaryotic expression plasmid vector named pT7AMPCE containing T7RNA polymerase, T7 promoter, internal ribosome entry site (IRES), and poly A tailing signal was constructed by T4 DNA ligase, on which homologous recombination was used to clone and construct the vector carrying PD‐1v, IL‐2/15, IL‐12, GM‐CSF, and GFP. In vitro transfection of CT26 cells was performed, and the protein expression of PD‐1v, IL‐12 and GM‐CSF was detected by Western blot and ELISA after 48 h. Mice were subcutaneously inoculated with CT26‐IRFP tumor cells in the rib abdomen, and the tumor tissues were injected with PD‐1v, IL‐2/15, IL‐12, and GM‐CSF recombinant plasmids for treatment during the experimental period. The efficacy of the treatment was evaluated by assay tumor size and survival time of tumor‐bearing mice during the experiment. Expression levels of IFN‐γ, TNF, IL‐4, IL‐2, and IL‐5 in mouse blood were measured using the CBA method. Tumor tissues were extracted and immune cell infiltration in tumor tissues was detected by HE staining and the IHC method.ResultsThe recombinant plasmids carrying PD‐1v, IL‐2/15, IL‐12, and GM‐CSF were successfully constructed, and the Western blot and ELISA results showed that PD‐1v, IL‐12, and GM‐CSF were expressed in the supernatant of CT26 cells 48 h after in vitro cell transfection. The combined application of PD‐1v, IL‐2/15, IL‐12, and GM‐CSF recombinant plasmids significantly inhibited tumor growth in mice, and the tumor growth rate was significantly lower than that in the blank control group and GFP plasmid control group (p< 0.05). Cytometric bead array data suggested that the combination of PD‐1v and various cytokines can effectively activate immune cells. HE and IHC analysis revealed plenty of immune cell infiltrates in the tumor tissue, and a large proportion of tumor cells showed the necrotic phenotype in the combination treatment group.ConclusionThe combination of immune check blockade and multiple cytokine therapy can significantly activate the body's immune response and inhibit tumor growth.