Study On The Antitumor Effects Of Recombinant MUC1-MBP Fusion Protein Combined With Different Adjuvants

Mucin1(MUC1) as an oncogene, which is a transmembrane glycoprotein of themucin family, and is overexpressed in most adenocarcinomas with aberrantglycosylation and loss of apical expression, which make it to be an attractive targetfor anti-tumor immunotherapy. Up to date, most MUC1-based vaccines are still inphaseⅠandⅡclinical trials, and only a few have entered the phase III clinical trial.Our research group began to study on MUC1-based anti-tumor vaccines since2001.In our study, MUC1gene was inserted into pMAL-p2vector and Constructedrecombinant pMAL-MUC1that express recombinant MUC1-MBP fusion protein(MUC1-MBP). MUC1-MBP was induced by IPTG in E coli DH5α transformed bythe recombinant pMAL-MUC1and purified by a series of purification technology,which was used in the research of anti-tumor vaccines and had gain good biologicaleffects. In fact, the immunogenicity of protein-based vaccines is usually very weakand adjuvants are required to enhance the immune responses. In our previous study,MUC1-MBP/Bacillus Calmette-Guerin (BCG) anti-tumor vaccine, of which BCG asan adjuvant, induced a Th1immune profile and stimulated MUC1-specific cytotoxicT lymphocyte killing activity, and making it as a potential cancer vaccine for clinicalapplication. However, BCG as a live bacterial vaccine, is instable and cytotoxic whenimmunizated to mice, such as resulting in the loss of weight and necrosis at theinjection sites. Thus, finding a much safer and more effective adjuvant for theMUC1-MBP vaccine is the key point that we have to figure out.In this study, we aimed to find more effective adjuvants for our MUC1-MBPanti-tumor vaccine by detecting immunological activity and establishingtransplantation tumor models.1. To prepare MUC1-MBP, pMAL-MUC1/DH5α was cultured and induced theexpression of MUC1-MBP by IPTG. Then lysing bacterial cells by sonication,purifying MUC1-MBP by Resin affinity chromatography Amylose and ultrafiltration, and the result showed that the purity was up to95%and the endotoxin was less than2.5EU/mg, of which had meet the requirement of the2010Chinese Pharmacopoeia.Experimental groups and immunization processAccording to different administration drugs, C57BL/6mice were divided intoseven groups, including saline group and MUC1-MBP combined with differentadjuvant groups. The adjuvants are CpG-ODN1585, CpG-ODN1826, CpG-ODN2006,BCG, thymosin α1(Tα1) and R848，respectively. According to our previous results,the administration dose of MUC1-MBP, BCG, Tα1, and R848were50μg,1mg,30μg, and20μg per mouse, respectively, while CpG-ODN1585, CpG-ODN1826, andCpG-ODN2006were50μg per mouse. The vaccines were immuzated by SC once aweek for two weeks.2. A mouse melanoma transplantation model was established by thesubcutaneous injection of B16-MUC1cells.3. The occurrence of tumors was recorded until the end of the experiment,2/7(29.6%) in CpG-ODN1826group and0/7in the rest of the groups were tumor free.4. The incidence of tumors was recorded and it showed that the effect of tumorsuppression in CpG-ODN1826group was best, in which the tumor nodulesoccurrence at the latest, following by the R848group. The effects of tumorsuppression in NS and Tα1groups were worst, in which the tumor nodules firstappeared.5. By detecting the tumor size, the tumor volumes in CpG-ODN1826andCpG-ODN2006groups were smaller than other groups, and the tumor inCpG-ODN1826group was the smallest, of which was statistically significantcompared to NS group (P <0.05).6. By observing animal survival time, the results showed that the averagesurvival time were NS group (26days), Tα1group (28days), BCG group (28.7days),CpG-ODN2006group (32days), CpG-ODN1585group (33.4days), R848group(41.7days), and CpG-ODN1826group (66.4days), indicating that MUC1-MBPcombined with Tα1or BCG failed to prolong the survival of the mice. WhenMUC1-MBP was combined with CpG-ODN1826, a TLR9agonist, the effect was the best and was statistically significant compared to NS group (P <0.05) following byR848(TLR7/8agonist). Taken together, the results showed that MUC1-MBPcombined with TLR agonists were better than other adjuvants.7. Analysis of immune activities in different adjuvant combining groups1) Analysis of spleen index Mice were sacrificed3days after the lastimmunization. The body weight, spleen weight, and spleen weight/body weight wererecorded. The results showed that spleen index in all the groups were abviouslyupregulated (P <0.01) compared to NS group, and the spleen index in CpG-ODN1826was the highest.2) Analysis of lymphocyte proliferation The mouse spleen lymphocytes wereseparated and stimulated by ConA or MUC1-MBP, respectively, and the proliferationof MUC1-nonspecific and-specific lymphocytes was detected by MTT assay. Theresults showed that the proliferation in CpG-ODN1826group was most obviously (P<0.01) compared to NS group, and the proliferation in CpG-ODN1826,CpG-ODN2006, and R848groups were higher than BCG group, respectively.3) Analysis of cytokines The levels of MUC1-specific IFN-γ and IL-4in theculture supernatant of the lymphocytes were detected by indirect ELISA assay kit.The results showed that IFN-γ and IL-4were mutual antagonistic. The level of IFN-γin CpG-ODN1826group was the highest, following by R848group, when comparedto NS group, respectively (P <0.01); only in Tα1group, the level of IL-4wassignificant (P <0.05) compared to NS group. All these results indicating thatMUC1-MBP combined with CpG-ODN1826, CpG-ODN1585, CpG-ODN2006, BCG,and R848induced MUC1-specific Th1immune response, and the immune response inCpG-ODN1826and R848groups were higher, while MUC1-MBP combined withTα1induced the Th2immune response.4) Analysis of T lymphocyte subsets The T lymphocyte subsets were analysedby flow cytometry. The results showed that the ratio of T lymphocytes in MUC1-MBPcombined with adjuvant groups were increasd compared to NS group, of which wasConsistant with the proliferation results, suggesting that the increasing T lymphocytesin MUC1-MBP combined with adjuvants groups was due to the enhancement of cell proliferation. The CD4+T lymphocyte subsets in BCG group was the most obviously(p <0.01) compared to NS group, and in CpG-ODN1826, Tα1, and R848group theCD4+T lymphocyte subsets increased as well (p <0.05); The CD8+T lymphocytesubsets in BCG and Tα1groups were upregulated (p <0.05) compared to NS group,respectively.In summary, our study reveal that the anti-tumor effects of MUC1-MBPcombined with CpG-ODN1826is better than BCG, laying a foundation for furtherclinical research on MUC1-MBP anti-tumor vaccines.