Antitumor Effect Of Recombinant MUC1-MBP Fusion Protein Combined With CpG ODN1826

MUC1, as a kind of tumor associated antigen, is an attractive target for anti-tumor vaccine researches. There are various studies of MUC1-targeted anti-tumor peptide vaccines at home and abroad, and the keypoint of those researches in this field is to enhance immunogenicity of antigen peptide. In our previous studies, we successfully generated the highly purified recombinant MUC1-MBP fusion protein(MUC1-MBP) from E.Coli containing p MAL-c2 expression vectors inserted seven tandem repeats encoding the MUC1 gene. Improved anti-tumor effect of MUC1-MBP combined with BCG was observed, but potential problems of BCG in application drove us to find an alternative adjuvant which is much safer and more effective. In our adjuvant screening study of synthetic TLR agonists, murine B16-MUC1 melanoma model was used for the prophylactic anti-tumor experience. The results showed that Cp G ODN1826 inserted group had the best anti-tumor effects and slight side effects,then we were inspired to explore an improved immunization procedure for MUC1-MBP combined with Cp G ODN1826 and an optimum inserting dose of Cp G ODN1826, to observe anti-tumor effect and to discuss vaccine formulation. Our studies included the following contents:Preparation and identification of MUC1-MBPFor obtaining the anti-tumor peptide vaccine, antigen peptide was prepared and identified. p MAL-MUC1/DH5α was massively cultured, then the expression of recombinant MUC1-MBP fusion protein was induced by IPTG. After performing ultrasonication, cation exchange chromatography, ultrafiltration and concentration,recombinant MUC1-MBP fusion protein(Purity>95%, Endotoxin<2.5 EU/mg)was obtained and identified by SDS-PAGE and Western Blotting analysis.Improved immunization procedure of MUC1-MBP combined with Cp G ODN1826To investigate an improved immunization procedure of MUC1-MBP, fourvaccination procedures were designed, and Th1 and CTL immune responses were evaluated and compared. Spleen index was significantly increased in all four immunization procedures. The secretion levels of IFN-γ and IL-4 of MUC1-specific splenocytes were quantified by ELISA assay and NK/CTL cytotoxicity was detected by LDH assay. The results showed that the vaccination procedure of two times with an interval time of two weeks showed a significantly increased IFN-γ secretion level of MUC1-MBP combined with Cp G ODN1826 group. Nevertheless, there was a quite low IL-4 secretion level in all four immunization procedures with no significant difference in procedures and groups. Additionally, an enhanced MUC1-specific CTL cytotoxicity was observed, while there was no significant difference for NK cytotoxicity.Optimum inserting dose of Cp G ODN1826In order to conforming the optimum inserting dose of Cp G ODN1826,prophylaxis anti-tumor experiment based on murine B16-MUC1 melanoma model was conducted. The results showed that the enhanced inhibition effects of tumor incidence were observed under the gradually increasing inserting dose of Cp G ODN1826. When incorporating dose of Cp G ODN1826 was 50 μg per mouse and per time, lowest tumor incidence, 33.3%, and quite strong prevention effect of tumor growth were observed.Anti-tumor effect of MUC1-MBP combined with Cp G ODN1826To evaluate the anti-tumor effect of MUC1-MBP combined with Cp G ODN1826,prophylaxis and therapeutic anti-tumor experiments based on murine B16-MUC1 melanoma model were performed, and we monitored tumor volume, recorded survival time and calculated tumor incidence. MUC1-MBP combined with Cp G ODN1826 group showed an obviously decreased tumor incidence, a delayed mortality and an inhibition effect of tumor growth compared with other groups in prophylaxis anti-tumor experiment. Following therapeutic anti-tumor experiment, reduction effect of tumor volume was remarkable. These results illuminated Cp G ODN1826 could substitute BCG as an adjuvant of MUC1-MBP vaccine.Formulation of MUC1-MBP combined with Cp G ODN1826In order to improve stability of the vaccine, MF59 emulsion was prepared and added into the vaccine, then formulation of the vaccine was an oil-in-water emulsion.LDH assay was applied in detecting NK/CTL cytotoxicity. There was an strengthened MUC1-specific CTL cytotoxicity.ConclusionIn conclusion, improved immunization procedure, immuning two times with an interval time of two weeks, of MUC1-MBP combined with Cp G ODN1826(50 μg per mouse and per time) showed a robust Th1-biased and MUC1-specific CTL immune responses in mice with effects of reduction of murine B16-MUC1 melanoma incidence, prevention of tumor growth and delayed mortality. The oil-in-water formulation of MUC1-MBP combined with Cp G ODN1826 enhanced MUC1-specific CTL cytotoxicity. Eventually, our study conformed an improved immunization procedure and anti-tumor effect of MUC1-MBP combined with Cp G ODN1826 at an optimum inserting dose, meanwhile provided a reference for determining vaccine formulation and a basis for the vaccine’s application in clinical research.