Seminalbumin 17 (Sp17)-targeted Ovarian Cancer Tumor Vaccine
Song Kun Kong Beihua Song Kun, Department of Gynecology, Qilu Hospital, Shandong University
Carcinoma-testicular antigens (CT antigens) are a group of testicular tissue-specific proteins that can be aberrantly expressed in a variety of tumor cells and restricted in normal tissues outside the testis. Recently, Sp17 was found to be a member of the CT antigen family, which is a highly immunogenic human autoantigen containing a cytotoxic T lymphocyte (CTL) antigen determinant cluster in its structure, and therefore could be an ideal target molecule for tumor immunotherapy. To investigate the feasibility of Sp17 as a target molecule for ovarian cancer immunotherapy, tumor specimens from three ovarian cancer patients were selected for the study. The expression of Sp17 in the tumor specimens was detected by reverse transcription polymutase chain reaction (RT-PCR) and banding by agarose gel electrophoresis with bromoethidium. Single nucleated cells (PBMCs) were isolated from peripheral blood by density gradient centrifugation and grown in culture dishes containing RPMI 1640 plus 10% fetal bovine serum with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4), and the cells were harvested as dendritic cells (DCs) after 7 days of culture. The cationic liposome DOTAP was used to deliver the Sp17 conjugated protein obtained from E. coli to the DCs to make them Sp17 antigen-presenting cells. Fresh peripheral blood mononuclear cells were mixed with Sp17-carrying DCs in a 10:1 ratio and incubated with IL-2, self-serum and IL-7 at 37°C. IL-2 was added every 3-4 days and self PBMCs feeding cells and Sp17 co-protein were added weekly, and T lymphocytes were harvested for cytotoxicity assays after 4 cycles. The cytotoxicity of T lymphocytes stimulated by Sp17 antigen was assayed by standard 4-hour chromium release assay using EBV-transfected auto-lymphoblastoid cell line (LCL) expressing or not expressing Sp17 and auto-ovarian cancer cells as target cells. Anti-human leukocyte antigen-I (HLA-I) and anti-HLA-II antibodies were added to the culture medium to investigate the HLA-dependence of cytotoxicity. Flow cytometry technique was used to analyze the phenotype of T lymphocytes and their intracellular lymphokine content.
The results showed that 7 of 10 fresh ovarian cancer specimens were positive for Sp17, with a 70% Sp17 expression rate. Tumor tissues expressed abundant Sp17 molecules as well as normal testicular tissues. Sp17-specific T lymphocytes were grown through the delivery of Sp17 to PBMCs by DCs and the sensitizing effect of Sp17 co-protein, which were effective as effector cells in killing their own LCLs expressing Sp17 in cytotoxicity assays. lysis of LCL cells was Sp17-dependent, and LCLs not transfected with the Sp17 gene, i.e., not expressing Sp17, did not lyse ( P0.00001). In addition, the lysis of target cells was found to be restricted by HLA-Ⅰ, cytotoxic effect could be blocked by anti-HLA-Ⅰ monoclonal antibody, and anti-HLA-Ⅱ monoclonal antibody did not seem to have any effect on cytotoxic activity (P0.000001), suggesting that the T lymphocytes involved in cytotoxic effect are CD8 positive, i.e. cytotoxic T lymphocytes (CTL), and also suggesting the presence of the patient’s immune system In the cytotoxic assay with self-tumor cells as the target cells, tumor cells were killed in all three patients, and this effect was also blocked by anti-HLA-Ⅰ antibody without the effect of anti-HLA-Ⅱ antibody, again suggesting the existence of Sp17-specific CTL associated with HLA-Ⅰ in patients with Sp17-positive tumors, and also demonstrating that Sp17 co-protein can sensitize and recognize It was also demonstrated that Sp17 co-protein can sensitize and proliferate the CTL of Sp17 at concentrations and phenotypes similar to those of Sp17 on the surface of Sp17-positive tumor cells. CMA selectively inhibited the lysis of target cells in the Perforin pathway while Brefeldin A inhibited the Fas-mediated apoptotic pathway, thus demonstrating that Sp17-specific CTL mediates target cell lysis via the Perforin pathway. The flow cytometry results showed that the phenotype of the experimentally grown T lymphocytes was mostly CD8 positive, which was consistent with the cytotoxicity assay results, demonstrating that the effector T lymphocytes were CD8 positive, i.e., CTL. after re-stimulation of T lymphocytes with the Sp17 combination protein, the two-color flow cytometry technique revealed that CTL mainly produced IFN-γ and detectable amounts of IL-4, consistent with the helper T lymphocyte 1 (Th1) cytokine profile consistent with that of Th1.
With this study, it can be concluded that Sp17 is feasible to be used as a target molecule for ovarian cancer tumor vaccines.