Research Of Therapeutic DNA Vaccine For Prostate Cancer Based On PSCA

In this research, we intend to enhance PSCA immunoresponse, which has been reported overexpression in prostate cancer tissues, by using human HSP70, thus move further toward the clinical applications of DNA vaccine for prostate cancer treatment. The immune responses and therapeutic efficacy of these vaccines were evaluated in mice model, and the value of PSCA as a potential target of prostate cancer diagnosis and therapy were confirmed. Furthermore, the optimal way to coupling antigen to HSP and the exact mechanism of HSP enhancing the potency of DNA vaccines were explored.Firstly, cDNA of DU145, a human prostate cancer cell line, was used as template, and the PSCA and HSP70 genes were amplified by PCR. Then the genes were cloned into pcDNA3.1(+) vector, to generate pc-PSCA,pc-HSP,pc-PSCA-HSP and pc-HSP-PSCA plasmids. The recombinant PSCA and HSP70 proteins were produced using E.coli system and were purified with chromatography methods. To evaluate the immune responses and therapeutic efficacy of these plasmids, MHC- restricted PSCA and HSP70-specific epitopes were predicted and a mouse prostate cancer cell line RM-PSCA was constructed. Furthermore, a mouse model with human PSCA-expressing tumor was also established. After injection with these plasmids, the PSCA-specific cellular and humoral immune responses were detected using ELISPOT assay, intracellular cytokines staining assay, cytotoxicity assay and ELISA assay. The result showed that mice vaccinated with pc-PSCA-HSP, pc-HSP-PSCA and pc-PSCA+pc-HSP all could induce a strong PSCA-specific cellular immune response. Furthermore, these vaccinations inhibited the growth of PSCA-expressing tumors and prolonged the mice's survival. In addition, coupling an antigen gene to the N-terminal of HSP70 may be the optimal fusion method to construct DNA vaccines, but the level of anti-PSCA antibody was similar among these groups.The PSCA or HSP70-specific CD4~+ T cell responses were detected to explore the immune mechanism of HSP70 molecule as an adjuvant. The results showed that mice injected with pc-PSCA, pc-PSCA-HSP, pc-HSP-PSCA and pc-PSCA+pc-HSP all generated stronger CD4~+T_h1 and CD4~+T_h2 responses than the PBS group. However, the differences among these groups were insignificant. It indicted that HSP70 did not enhance the antigen-specific humoral responses, and the enhancement of CD8~+ T cell response was independent of CD4~+ T cells. The detection of HSP-specific immune responses showed that mice injected with pc-HSP, pc-PSCA-HSP and pc-HSP-PSCA generated stronger HSP70-specific CD4~+T_H2 and anti-HSP antibody responses than pc-PSCA+pc-HSP group. However, the HSP-specific CD4~+ T_h1 cells were not activated.