Study Of Recombinant MUC1-MBP Fusion Protein Vaccine On Mechanism Of Anti-cancer

MUC1 is a member of mucin family, which expressed on epithelial cells and aberrantly overexpressed on epithelial-derived tumor cells including breast cancer, lung cancer, ovarian cancer, prostatic carcinoma and pancreatic cancer, et al. MUC1 have different forms in normal tissue and tumor tissue. It distributes in apical borders of secretory epithelia in normal tissue, which is hyperglycosylation and isolated from the immune cells. In contrast to hyperglycosylation of MUC1 in normal tissue, it is highly overexpressed on a wide variety of epithelial adenocarcinomas, and the extracellular domain of the protein that is underglycosylated in cancers, and is characterized by aberrantly glycosylated, resulting in exposure of cryptic peptide epitopes which makes it becoming the target of immune cells. In addition, it has been found that the PDTRP epitope of MUC1 protein core is recognized by specific MUC1 antibody and HLA-unrestricted CTL activity against MUC1. Hence MUC1 has long been considered as a target for anti-tumor immunotherapy. Currently, many MUC1-based cancer vaccines have been researched hot point for its universality and efficacy. Currently, MUC1-based cancer vaccines included carbohydrate vaccine, DNA vaccine, peptides and recombinant proteins vaccine and DC vaccine and several of them have entered clinical trial. The peptide and protein vaccine has a good prospect in variety of vaccine research. But most of protein vaccine have weak immunogenicity and hardly induced cellular immune response. Hence , we are faced with some question how to improve tumor vaccine immunogenicity.E. coli maltose-binding protein (MBP) is a gene products of malE. Some proteins are commonly fused to MBP to improve their yield and to facilitate their purification. Some scholars found that MBP protein had immunological regulation function. MBP protein have become a ideal couple protein because it could improve immunogenicity of interest protein that fusion expressed with MBP protein. Hence, we had cloned MUC1 tandem repeats sequence into an prokaryotic expression vector pMAL-p2 and constructed recombined pMAL-MUC1 plasmid, screened stable fusion expression MUC1-MBP of protein strain. We had established purification technology of MUC1-MBP fusion protein and prepared MUC1-MBP fusion protein.We had found that MUC1-MBP fusion protein could significantly inhibit the growth of moue Lewis lung cancer and human breast cancer. The immune regulation activity of MUC1-MBP fusion protein from specific immune response and nonspecific immune response would be observed in this study. To explore the mechanism of specific immune response in anti-tumor process and make a experimental foundation for human tumor vaccine, stable expression MUC1 of B16 cells animal model would be established .1. Preparation of MUC1-MBP fusion protein and MBP proteinThe pMAL-MUC1 and pMAL plasmid was transformed into E. coli DH5α. The MUC1-MBP fusion protein and MBP protein expressed upon induction with IPTG. The MUC1-MBP fusion protein and MBP protein was purified by Amylose resin affinity chromatography.2. Construction of stable expression MUC1 of B16 cellsMelanoma B16 cell was transfected with pcDNA3 plasmid containing full-length MUC1 cDNA with 22 tandem repeats or pcDNA3 plasmid. The B16 cell line stable expressing high levels of MUC1 and non-expression MUC1 was selected by G418. That MUC1 expressed on B16 cell was identified by fluorescence microscopy and FACS is 98.5%. The B16 cell line expressing high levels of MUC1 was named B16-MUC1 and the B16 cell line with non-expression MUC1 was named B16-neo.3. Immunization ProtocolThe mice were divided into PBS, BCG, MBP, MUC1-MBP or MUC1-MBP+BCG group. Mice received two or three weekly immunizations at s.c. PBS was injected in PBS group. The 3 mg BCG was administrated at the first and second immunization and PBS was administrated at the third immunization in BCG group. For each administration, 50μg of MUC1-MBP fusion protein or 34μg MBP protein was injected s.c. in MBP and MUC1-MBP groups. The 3 mg BCG and 50μg of MUC1-MBP were co-administrated at the first and second immunization and 50μg of MUC1-MBP was administrated at the third immunization in MUC1-MBP + BCG group.4. Humoral immunoresponse induced by MUC1-MBP fusion proteinFour days after the last immunization, the blood of the mice were collected and the serum MUC1 specific antibodies were tested by ELISA . The results showed that MUC1 specific antibody was found in MUC1-MBP and MUC1-MBP+BCG group. The antibody titer was ranged from 1280 to 2560 in MUC1-MBP group and from 320 to 2560 in MUC1-MBP+BCG group, respectively. The specific antibody was lower in the MUC1-MBP+BCG group than that of the MUC1-MBP group. The result showed that the MUC1-MBP protein alone and MUC1-MBP protein combined with BCG could induce specific humoral immunoresponse. The immunization induced by MUC1-MBP protein could promote Th2 cellular immunologic response. MUC1- MBP fusion protein combined with BCG could enhance Th1 cellular immunologic response and inhibit Th2 cellular immunologic response.5. Specific cellular immunologic response induced by MUC1-MBP fusion proteinFour days after the last immunization, the spleen cells were isolated and stimulated by the synthetic MUC1peptide for 5 or 6 days. MTS was used to detect the MUC1 specific lymphocyte proliferation. The levels of IL-4, IL-2 and IFN-γsecreted by the specific lymphocyte were detected by indirect ELISA assay. The lactate dehydrogenase (LDH) release assay was used to analyze the CTL activity. The results showed that the specific lymphocyte proliferation was be detected in the MUC1-MBP+BCG group, but not other groups.The levels of IL-2 secreted from induced lymphocyte were higher in MUC1-MBP and MUC1-MBP+BCG groups than those of the other groups (p<0.05), which was more higher in MUC1-MBP+BCG group than that of the MUC1-MBP group (p<0.05). However, the IFN-γonly found in the MUC1-MBP+BCG group (p<0.01). The levels of IL-4 were higher in MUC1-MBP and MUC1-MBP+BCG groups than those of the others (p<0.05), which was more higher in MUC1-MBP group than that of the MUC1-MBP+BCG (p<0.05). The results suggested that the activation of Th1 and Th2 cells was induced by MUC1-MBP fusion protein, while the levels of Th1 was increased and the level of Th2 was decreased combined with BCG .The CTL results showed that lymphocytes derived from the mice treated with MUC1-MBP combined with BCG could kill B16-MUC1 target cells. With the increase of the ratio of E : T, the cytotoxicity was gradually increased. The killing activity at E : T ratio of 50:1 was 44.34% , compared with that of control group (p<0.05), while the killing activity was not found in other groups. The result indicated that the MUC1-MBP in combination with BCG not only could induce the MUC1 specific Th1 activation, but also the MUC1 specific CTL activation.6. NK activation induced by MUC1-MBP fusion proteinFour days after the last immunization, the splenocyte were isolated as effector cells and incubated with YAC-1 as target cells for 5 hour. The NK cell activation was detected by LDH release. The results showed that NK cell activation was induced in BCG, MBP, MUC1-MBP an MUC1-MBP+BCG group and cytotoxicity activities to YAC-1 cells were 20.45%, 12.35%, 10.72% and 26.72% at E:T ratio of 100:1 in BCG, MBP, MUC1-MBP and MUC1-MBP+BCG group, respectively compared to PBS group (p<0.05). The frequency of NK cells in splenocyte increased in experiment group,compared to control group (p<0.05). The results indicated that NK cell was induced activation with both BCG and MBP protein. MUC1 coupled with MBP protein could induce NK activation , which the MBP protein was important. MBP protein could increase the immune activity of MUC1 by inducing the activation of NK cells. MUC1-MBP protein in combination with BCG induced NK cell activation was the combined effects of the BCG and MBP protein.7. Effect of MUC1-MBP fusion protein on mouse peritoneal macrophages and DCThe spleen cell from immunized mice was cultured for two hours following removed non- adherent cells, and then cultured for 3 to 4 days to observe the morphological changes of adherent cells. The number of large volume cell increased in BCG, MBP, MUC1-MBP and MUC1-MBP+BCG groups under the optical microscope. By Wright-Giemsa stain, it was found that some of cells in BCG, MBP, MUC1-MBP and MUC1-MBP+BCG groups gradually exhibited typical DC-like morphology with larger, irregular and burr-like protrusions.To further explore the activation of DC and macrophage, the surface markers of the DC and macrophage cell were tested by FACS. The results showed the CD14, CD68, CD86 and MHCⅡwere highly expressed in experiment group compared to PBS group, especially in MBP and MUC1-MBP group. CD11c and CD40 were highly expressed in BCG and MUC1-MBP+BCG group, especially in MUC1-MBP+BCG group. The results showed that macrophages could be activated by MUC1-MBP, MUC1-MBP+BCG, BCG and MBP, specifically MBP and MUC1-MBP. The enhanced DC activation by co-administration of BCG in MUC1-MBP group was observed, while decreased macrophage activation. The results indicated that MBP protein could enhance the immunogenicity of MUC1 by activation of macrophages and BCG could enhance the immunogenicity of MUC1-MBP fusion protein by activation of DC.To further explore the activation of macrophage, we detected the phagocytosis of mouse peritoneal macrophages derived from immunized mice by using chicken red cells. Our results showed that MBP and MUC1-MBP could significantly enhance phagocytosis of mouse peritoneal macrophages, compared with control. NO was an important active factor of monocytes and macrophages. After mouse peritoneal macrophages stimulated by MBP and MUC1-MBP, we detected levels of NO in cell culture supernatants by Griess methods. The results showed that MBP and MUC1-MBP could markedly promote the secretion of NO (p<0.05). The results suggested that MUC1 coupled with MBP protein could active macrophage to enhance the immune response by binding MBP protein.8,Anti-tumor effect of MUC1-MBP fusion proteinConstruction of tumor model The animal model was established using exoression MUC1 of B16-MUC1 cell or not expression of MUC1 of B16-neo cell .The mice were injected subcutaneously 2×106 B16-MUC1 or 2×106 B16-neo, respectively. The tumor could be touch at 7 to 9 days post-tumor challenge, tumor tissue was black and tumor formation rate was 100%.Tumor protection efficacy The mice were received three weekly intervals immunization. On day 4 day the last immunization, mice were injected B16-MUC1or B16-neo cells, respectively and monitored daily for tumor development. In the B16-MUC1 tumor model, the tumors grow slowly in BCG, MBP, MUC1-MBP and MUC1-MBP+BCG groups compared to controls within two weeks(p<0.05),among which the tumor was smallest in MUC1-MBP+BCG group and then in MUC1-MBP group. At 21 day post-tumor challenge, the mice were killed and the tumors were dissected. The weight and volume of tumors decreased dramatically in MUC1-MBP+BCG group compared to control group(p<0.05),while there was no statically significance in other groups. B16-neo cancer model showed that the tumor growth was inhibited slightly at the early stage in BCG, MBP, MUC1-MBP and MUC1-MBP+BCG groups. At 17 day post-tumor challenge,the mice were killed and the tumors were dissected. There was no difference of weight and volume of tumors compared to control group.Tumor therapy efficacy The mice were immunized s.c two weekly intervals immunization after 4 days challenged with B16-MUC1or B16-neo cells, respectively. B16-MUC1 cancer model showed that the tumors grow slowly within 13 day post-tumor challenge in experimental group. The volume of tumor decreased in MUC1-MBP+BCG group compared to control group after 18 day post-tumor challenge(p<0.05),while there was no difference in other groups compared to control group. B16-neo cancer model showed that the tumors grow slowly in experimental group, and there was no difference of volume of tumors compared to control group after 15 day of inoculation.Our results showed that there were noticeable preventive and therapeutic effects of MUC1- MBP+ BCG anti- B16-MUC1 cell, while no effect was found on B16-neo cell. The result showed that the specific cell immune induced by MUC1-MBP+BCG play an important role on anti-tumor effect .As MUC1-MBP+BCG not noly induce the activation of NK and macrophage cells, but also induce the activation of specific Th1 and CTL. The mild inhibition on two kinds of tumor cells indicate that the activation of no-specific immunity cell as NK and macrophage cells may play a role in experimental group at early stage because of BCG, MBP and MUC1-MBP could activate NK and macrophage cells but not CTL. The effect of MUC1-MBP anti- B16-MUC1 in tumor protection efficacy was better than BCG and MBP, suggesting that the humoral immune induced by MUC1-MBP could play an important role.In conclusion, MUC1-MBP only induced specific humoral immune response instead of cellular immune response, and MBP protein could enhance the immune activities of MUC1 by the activation of NK and macrophage cells. However, at the presence of BCG adjuvant, MUC1-MBP fusion protein not only induce MUC1-specific humoral immune response, but also induce MUC1-specific Th1, CTL response and activate DC, NK, macrophage cells. These mechanisms were involved in the anti-tumor effect of MUC1-MBP fusion protein, specially the specific cellular immune response at late stage.