Sp17 Targeted Monoclonal Antibodies And Their Adriamycin Immune Coupling Experiment Research For The Treatment Of Ovarian Cancer

Sp17is a spermatozoa-specific protein which is over expressed in multiple types of tumor, especially in gynecological cancers (ovarian, endometrial and cervical cancers) but is absent or expressed at low levels in most normal tissues. The function of Sp17is poorly understood, and it is thought to be involved in acrosomal reaction during fertilization, cell-cell interactions, and cell migration. With the rapid development of biotechnology in the medical field and the knowledge of pathogenesis from the molecular level, monoclonal antibody drugs has become one of hot topics of cancer targeted therapy. However, the experimental studies on the monoclonal antibody of Sp17(3C12, IgG1) for cancer have not been reported. Our previous studies showed that3C12targeted and accumulated in Sp17positive tumors in vivo, which prompted its capability for cancer target therapy. Thus, based on our previous work, we examined the antitumor effect, the target activity and the side effects of3C12and a novel immunoconjugate containing3C12linked to a chemotherapeutic agent doxorubicin against ovarian cancer cell lines and tumor models in vitro or in vivo. This study will set up the foundation for researching and developing antibody target drug for cancer with high efficient and low toxicity, and provide a new approach and theoretical foundation for therapy of Sp17postive tumor. The results are shown as below:1. Studies on the anti-tumor effects of anti-Sp17mAb3C12in vitroIn the first part of this paper, based on the previous works, anti-Spl7mAb (3C12) was prepared, purified and tested the titer by using the ELISA. Our researches investigated the degree of Sp17protein expression in three kinds of ovarian cancer cells by Western blot and immunofluorescence analyses, detected the directly toxicity of3C12to those ovarian cancer cells. Then, we examined the ADCC and CDC by using the CytoTox96Non-Radioactive Cytotoxicity Assay. We found the monoclonal antibody of Sp17(3C12, IgG1) had the titer of106, and intense Sp17expression in SKOV3cells and relatively lower expression in HO8910cells, whereas little Sp17expression in COC cells.3C12had significant higher binding to SKOV3or HO8910cells, and had little binding to COC2cells. The binding of3C12to SKOV3or HO8910was concentration-dependently. There was no direct cytotoxicity and significant difference in viability of SKOV3, HO8910, and COC2cells treated with3C12and nonspecific negative control antibody from the concentration of0.0001-10pμg/mL at any time point tested. The results indicated that3C12did not initiate the intracellular pathways of apoptosis, or Sp17expressed on cell surface was not a decisive role for tumor cell growth. To further discuss the antitumor effect and preliminary mechanism of3C12, we examined the cytotoxic action of3C12mediated by PBMC, as ADCC, or mediated by human serum, as CDC. We found the activity of antitumor cells was noted in investigation of SKOV3cells its maximum inhibition rate of approximately32%and50%, which indicated effect factor (peripheral blood mononuclear nuclear cells or complement), could improve the3C12monoclonal antibody cytotoxicity to tumor cells.2. Studies on the anti-tumor effects of Sp17immunoconjugate in vitro and in vivoAlthough3C12inhibit the growth of tumor cells in vitro by the effect of ADCC and CDC, the inhibition effect is limited. In this part, we investigated whether a novel immunoconjugate containing3C12linked to a chemotherapeutic agent DOX had antitumor activity against ovarian cancer cell lines and tumor models. The results showed the3C12-DOX immunoconjugate specifically bounded to Sp17-positive SKOV3but not to Sp17-negative cells and was internalized by SKOV3cells. Treatment with3C12-DOX decreased the viability of SKOV3cells in an Spl7-specific manner. Flow cytometry results were also found3C12-DOX immunoconjugates could induce SKOV3cells and HO8910cells apoptosis in vitro. In vivo, SKOV3cells (1×106) were inoculated subcutaneously into the right flanks of athymic nu/nu female mice and developed palpable tumors.3C12-DOX treatment, compared with control treatment, caused regression of established tumors of the mice with SKOV3cells. The antitumor effects of3C12-DOX therapy were significantly related to increased apoptosis of tumor cells. In addition,3C12-DOX showed no observable adverse effects or toxicity when compared with DOX alone in mice bearing ovarian tumor xenografts.In summary, this study explores the target antitumor activity of3C12and the immunoconjugate (3C12-DOX) from cellular and whole animal level. We found that effect factor could improve the3C12monoclonal antibody cytotoxicity to tumor cells; especially3C12-DOX conjugates had obvious inhibition effect of tumor, and its security was better than the single use of DOX. Based on these findings, we provided a powerful experimental basis that Sp17was an attractive target for tumor targeting therapy. Therefore,3C12-DOX has been identified as a potential antibody-drug conjugate for clinical development.