Anti-tumor Immune Response Induced By Xenogeneic Antigen Of Neu-Fc In Combination With GM-CSF/IFN-γ And BCG

IntroductionHuman epidermal growth factor receptor 2 is the second member of the epidermal growth factor receptor family. It is a transmembrane receptor protein that has tyrosine kinase activity. The protein is overpressed at 25%～30% breast cancer, but expressed very low at normal tissues. So it has been a perfect target for tumor immuno-biotherapy. HER2 is a self antigen. Immune system often shows tolerance to overpressed self antigen. It developes a deeply immunodepression microenvironment in the procession of tumor growth. The function of immune cells in the microenvironment is inhibited. For tumor immunotherapy, it is important to break the tolerance induced by tumor associated antigen and the immunodepression in tumor microenvironment, then to elicit effective anti-tumor immune response.ObjectiveTo discuss immunoregulation of xenogeneic antigen of neu-Fc protein in combination with hGM-CSG and BCG. To discuss anti-tumor immune response induced by xenogeneic antigen of neu-Fc protein in combination with IFN-γand BCG.MethodsThe rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc. The eukaryotic expression vector was constructed. The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column. The recombinant protein was identified by SDS-PAGE and Western Blot. PBMC were obtained by means of standard Ficoll separation from the blood of healthy donors. Neu-Fc-induced PBMC proliferation was tested by MTT. The production of IL-12 and IL-10 was measured by ELISA. The corresponding molecules on the surface of monocytes were detected by FACS. PBMC were stimulated by adjuvant and xenogeneic antigen and the lysis of target cells was tested by LDH. The mouse breast cancer cells EMT6 were transfected by pcDNA3.0/HER2 , then we obtained a cell strain EMT6/HER2 that expresses high level of HER2 molecules. Then we established a mouse animal model and observed tumor growth that influenced by adjuvant and xenogeneic antigen. Histochemistry stained tissue slices of tumors.ResultsThe relative molecular mass of neu-Fc protein is about 66 kDa. The level of IL-12 was decreased and IL-10 was increased after PBMC were incubated with MCF-7 cultural supernatant. 10 nM neu-Fc strongly induced the cell proliferation. Compared with neu-Fc or GM-CSF or BCG treatment alone, neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production. The corresponding molecules on monocytes surface were up-regulated when the cells were stimulated with adjuvant and xenogenetic antigen. The efficiency of lysis was enhanced after PBMC were affected by adjuvant and xenogenetic antigen. 99.59% cells of EMT6/HER2 express HER2. It was found that the tumor growth was inhibited in adjuvant and xenogenetic antigen group in animal experiment, and in the tissue slices stained by histochemistry, a large amount of infiltrating inflammatory cells surrounded the tumors and also diffusely infiltrated in the deeper tumor tissues in contrast to the control.ConclusionXenogenetic antigen neu-Fc protein in combination with hGM-CSG and BCG can regulate the immune response of Th1 and Th2 significantly. Xenogenetic antigen neu-Fc protein in combination with IFN-γand BCG can induce effective anti-tumor immune response.