Immunotherapy Of Liver Cancer In Mice By Recombinant Vegf From Dna-shuffling In Combination With Doxorubicin

Tumor cells induce tumor angiogenesis through producing vascular endothelial growth factor to provide oxygen and nutrients for tumor growth.Therefore,inhibition of VEGF can inhibit tumor growth.VEGF genes from human,chicken,rat,and mouse were randomly assembled to chimeric genes by DNA shuffling for constructing an expression library,and we found a new fusion VEGF protein antigen named as No.46,Mice were immunized subcutaneously with the No.46 recombinant protein emulsified with incomplete Freund’s adjuvant（IFA）,which could induce high level of antibodies recognizing VEGF in mice and inhibit tumor angiogenesis and growth.In order to further enhance immunotherapeutic effects,we explored the recombinant VEGF vaccine from six aspects:the immunogenicity,the immunological adjuvants used,route of administration,the dose of antigen inoculated,the times of immunization,and the combination of chemotherapeutic agents.The main methods and results are following:（1）Enhancing the immunogenicity of No.46 recombinant protein.In previous study,we constructed the recombinant Escherichia coli BL21-p ET28a-46,which could express No.46 recombinant protein with 6×HIS tag.In order to further enhance immunotherapeutic effects and remove the 6×HIS tag,we inserted the T cell epitope gene of tetanus toxin into the front of the 46 protein gene,and the BL21-p ET28a-TT46recombinant strain was constructed.The BL21-p ET28a-TT46 could express TT46recombinant protein in the form of inclusion bodies after cultured and induced,then the solubilized inclusion bodies were purified by DEAE-52 cellulose after Washed,dissolved,dialysis refolded.（2）Optimizing the expression of TT46 E.coli fermentation process.In this experiment,using BL21-p ET28a-TT46 as the research object,by means of a 12%SDS-PAGE analysis of a series of experiments,through single factor experiment modified M9 media,including glycerol concentration,sodium glutamate concentration,transit time,inducer（lactose）concentration,p H of the medium,the time post induction.To explore and establish preliminary fermentation process test,the optimum conditions were obtained by tests（transit 4.5 h,inducer（lactose）concentration was 2 mmol/L,incubation time post induction 8 h）.（3）Selecting adjuvant and administered route for immunization with TT46recombinant protein vaccine.In this study,we selected two routes of administration were subcutaneously（sc）and intramuscularly（im）,and the mice were immunized three doses with two immunological adjuvants（MF59 and Al（OH）₃）to test immunotherapeutic effects.One week after last immunization,blood samples were collected for determining anti-VEGF antibodies by ELISA.The results showed that TT46+PBS control group produced low titer antibody（800×）after immunized subcutaneously and intramuscularly,the antibody titers were 1600×and 800×for TT46+MF59 and TT46+Al（OH）₃group after immunized subcutaneously,he antibody titers were 6400×and 3200×for TT46+MF59and TT46+Al（OH）₃ group after immunized intramuscularly.Thus,the mice were immunized with adjuvants（MF59 and Al（OH）₃）could induce anti-m VEGF antibodies,especially the route of intramuscular administration combined with MF59 adjuvant could induce better immune effects and higher titer of anti-VEGF antibodies.（4）Determination of TT46 dose for immunization.mice were randomly divided into four doses groups（100μg/mouse,50μg/mouse,25μg/mouse,10μg/mouse）and PBS control group.The TT46 recombinant proteins were emulsified with MF59 adjuvant,then used for IM immunization of mice for 3 doses weekly.blood samples were collected from the first immunization for 29 weeks for determining anti-VEGF antibodies by ELISA.The results showed that TT46 recombinant protein could induce the specific antibodies in a certain period of time.The anti-VEGF antibody increased along with inoculated dose,and the specific antibody of 100μg/mouse dose group significantly higher than the other dose group,the highest antibody titer was 8960×,and the duration of antibody titer 2000×up to 12 weeks,no obvious side effects.In summary,The mice were immunized with 100μg/mouse dose could obtain better immune effect.（5）Anti-tumor effect of TT46 recombinant protein vaccine combined with doxorubicin（DOX）.TT46 or 46 recombinant protein were emulsified with MF59adjuvant,then used for IM immunization of mice at a dose of 100μg/mouse for 3 doses weekly.One week after last immunization,blood samples were collected from mice for determining anti-VEGF antibodies by ELISA.After that,tumor model was established by SC injection with H22 hepatocellular carcinoma cells in the right flank of mice.The results showed that the antibody titers were 6400×and 3200×for TT46（3）+MF59 group and 46（3）+MF59 group.Thereafter,6 mice in each group were killed for analysis of tumor weights on day 15 post challenge.The tumor inhibition rates was 46.2%for TT46（3）+MF59 group,which was significantly higher than 46（3）+MF59 group（18.3%）.Therefore,the tetanus toxin T cell epitopes could significantly improve the immunogenicity of No.46 recombinant protein,induce higher antibody,and obtain stronger immunotherapeutic effects.On the basis of this,mice were immunized intramuscularly with TT46 recombinant protein,then injected with 100μL DOX（1μg/μL）through the tail vein on day 2,6,10.The results showed that TT46 recombinant protein combined with DOX could significantly enhance the therapeutic effect on H22hepatocellular carcinoma,the tumor inhibition rate was 70.2%,which was significantly higher than TT46 alone group（46.2%）and DOX treatment group（49.9%）,indicating TT46 recombination protein combined with DOX can produce synergistic antitumor effects.Through HE staining and anti-CD31 immunohistochemistry,we found that TT46recombinant protein could significantly block the formation of blood vessels.By Western blotting analysis,We also found the antibodies in serum of TT46（3）+MF59 group could specifically bind with human VEGF protein,mouse VEGF protein and TT46 recombinant protein.（6）Increased immunization numbers of TT46 with an enhanced anti-tumor effect.On 14 day post challenge,We found the level of anti-VEGF antibodies in mice decreased to the control group level,With the rapid growth of the tumor,antitumor effects decreased.Therefore,we increased the times of immunization to enhance the antitumor effects.On21 day post challenge,the mice were immunized with one dose,The results showed that the antitumor effects of single-enhanced immune was not significant.We further increased the times of immunization and combined with lipopolysaccharide adjuvants（Iturin and Surfactin）,On day 7,14 and 21 post challenge,mice were immunized with three doses,the boost immune groups antibody titer dropped to the lowest level on day 7,then began to rise up and maintained high level of titers.The tumor volume of TT46（6）+MF59 group,TT59（6）+MF59+Iturin（3）group and TT46（6）+MF59+Surfactin（3）group were compared with TT46（3）+MF59 had significant difference（p<0.05）,but the tumor volume of TT46（6）+MF59 group,TT46（6）+MF59+Iturin（3）group and TT46（6）+MF59+Surfactin（3）group had no significant difference between the three groups（p>0.05）.On day 43 post challenge,all mice died in the TT46（3）+MF59 group,whereas the survival rates were still 22.2%,33.3%and 22.2%in the TT46（6）+MF59group,TT46（6）+MF59+Iturin（3）group and TT46（6）+MF59+Surfactin（3）group.The results showed that three times boosted immune antitumor effects was better than single boosted immunization,and the addition of Iturin or Surfactin did not further enhance the antitumor effect of TT46 recombinant protein.（7）TT46 recombinant protein vaccine combined with doxorubicin（DOX）can inhibit the growth of H22 liver in the therapeutic experiment.the mice were challenged with H22hepatocellular carcinoma as described previously at day-1,TT46 recombinant protein were emulsified with MF59 adjuvant,then used for IM immunization of mice at a dose of100μg/mouse for 6 doses weekly,then injected with DOX as described previously.The results showed that the tumor volume of TT46（6）+MF59+DOX group and TT46（6）+MF59 group had significant difference from the 17 days after tumor bearing（p<0.05）.The tumor volume of TT46（6）+MF59+DOX group and DOX group had significant difference（p<0.05）,On day 62 post challenge,all mice died in the TT46（6）+MF59 group,On day 66 post challenge,all mice died in the DOX group,whereas the survival rate was still 20%in the TT46（6）+MF59+DOX group.Therefore,compared with TT46 alone and DOX only,TT46 combined with DOX could significantly inhibit tumor growth and improve the survival rate of tumor-bearing mice.（8）TT46 recombinant protein vaccine combined with doxorubicin（DOX）can inhibit the metastasis of H22 liver cancer in the preventive experiment.TT46 recombinant protein were emulsified with MF59 adjuvant,then used for IM immunization of mice at a dose of 100μg/mouse for 6 doses,then injected with H22 cells through the tail vein on day 0 after third immunization.then injected with DOX as described previously.Thereafter,6 mice in each group were killed for analysis of tumor metastasis on day 21post challenge.The results showed that the average number of surface metastases was 38,18,6,12 for PBS+MF59,TT46（6）+MF59,TT46（6）+MF59+DOX,DOX respectively.The tumor inhibition rate calculated by tumor metastases was 52.2%,84.9%,68.6%for TT46（6）+MF59,TT46（6）+MF59+DOX,DOX respectively.Therefore,TT46 recombinant protein vaccine combined with DOX could significantly inhibit the lung metastasis of H22 hepatocellular carcinoma in vivo.On day 46 after post challenge,all mice died in the TT46（6）+MF59 group,On day 52 post challenge,all mice died in the DOX group,whereas the survival rate was still 22.2%in the TT46（6）+MF59+DOX group.（9）TT46 recombinant protein vaccine combined with doxorubicin（DOX）can inhibit the metastasis of H22 liver cancer in the therapeutic experiment.The mice were injected with H22 cells through the tail vein on day-1,TT46 recombinant protein were emulsified with MF59 adjuvant,then used for IM immunization of mice at a dose of 100μg/mouse for 6 doses weekly,then injected with DOX as described previously.Thereafter,6 mice in each group were killed for analysis of tumor metastasis on day 21 post challenge.The results showed that the average number of surface metastases was 38,25,9,14 for PBS+MF59,TT46（6）+MF59,TT46（6）+MF59+DOX,DOX respectively.The tumor inhibition rate calculated by tumor metastases was 34.2%,76.3%,63.2%for TT46（6）+MF59,TT46（6）+MF59+DOX,DOX respectively.Therefore,TT46 recombinant protein vaccine combined with DOX could significantly inhibit the lung metastasis of H22 hepatocellular carcinoma in vivo.On day 40 post challenge,all mice died in the TT46（6）+MF59 group,On day 47 post challenge,all mice died in the DOX group,whereas the survival rate was still 33.3%in the TT46（6）+MF59+DOX group.In summary,we used the tetanus toxin T cell epithelium to enhance the immunogenicity of recombinant protein 46,TT46 recombinant proteins were emulsified with MF59 adjuvant,then used for intramuscular immunization of mice at a dose of 100μg/mouse couled induce high titer anti-VEGF antibodies and inhibit tumor neovascularization and growth.TT46 recombinant protein vaccine combined with doxorubicin（DOX）could further enhance the prevention and treatment of H22hepatocellular carcinoma,significantly inhibit the growth and metastasis of tumor and prolong the survival time of tumor-bearing mice.These achievements provides new ideas and methods for cooperation of immunotherapy and chemotherapy.