Construction of a DNA vaccine encoding Flk-1 extracellular domain and C3d fusion gene and investigation of its suppressing effect on tumor growth

Although the critical role of complement component C3d as a molecular adjuvant in preventing virus infection is well established, its role in cancer prophylaxis and treatment is unclear. In this study, we constructed a recombinant plasmid encoding Flk-1 and C3d3 fusion proteins and investigated its transient expression in vitro in transfected eukaryotic cells and its antibody response in immunized mice. Subsequently, we investigated the vaccine's ability to elicit an immune response leading to suppression of angiogenesis and tumor growth in mice bearing bladder transitional cell carcinoma. Using Western blotting, immunocytochemistry, and flow cytometry, we detected the expression of Flk-1 and C3d3 fusion proteins in COS-7 cells transfected with these recombinant plasmids. Further binding experiment using CR2 (C3d receptor) positive Raji cells that were incubated with transfected COS-7 supernatant indicated that C3d was successfully fused to Flk-1. Although both vaccines elicited peak antibody levels at 5 weeks, Flk-1-specific antibody titer in pSG.SS.Flk-1(ECD).C3d3.YL-immunized mice was significantly higher when compared to pSG.SS.Flk-1(ECD).YL-immunized mice. The results of experiments with bladder tumor-bearing mice showed that the vaccine inhibited tumor growth significantly. These results suggest that C3d plays a critical role in tumor immunotherapy by promoting antibody response in Flk-1-based DNA vaccines. This approach may provide a new strategy for the rational design of anti-angiogenic therapies for the treatment of solid tumors and provide a basis for the further exploitation and application of the anti-angiogenesis DNA vaccines.