Experimental Treatment Of PEP3 Tumor Vaccine Based On Adenovirus Vector In Malignant Glioma

The traditional therapeutics is greatly limited by the invasive growth of malignant glioma,the high rate of tumor recurrence,the blood brain barrier and so on.The tumor peptide vaccine depending on the immune system induces an immune response,which further specifically kills tumor cells.Due to the advantages of high specificity and safety,little damage for normal tissues,tumor peptide vaccine attracts widespread attention.The epidermal growth factor receptor class ? variant(EGFRv?)is present on the surface in a substantial proportion about 30%of malignant glioblastoma and other human cancers,yet completely absent in normal tissues.A variant receptor named EGFRv? was formed because of the in frame deletion of exons 2-7 in wild-type EGFR.This deletion causes the formation of a specific peptide in extracellular region of EGFRv?.The amino acids sequence of the tumor-specific epitope named PEP3 is LEEKKGNYVVTDH.The present studies suggest that the PEP3 has been used as a potential tumor specific antigen for immunotherapy of malignant gliomas.The immunogenicity and copy number of peptide are important factors for the immune efficiency.Because of the easily degradation and weak immunogenicity of chemical synthetic peptides in body,selecting a carrier to enhance the immunogenicity and improve the peptide-presenting copy numbers of tumor antigen is significant.For now,the common immune vectors are Virus-like particles(VLPs),viral vectors and so on.180 or 240 Hepatitis B virus core protein(HBc)monomers could form icosahedral HBc-VLPs which has a strong immunogenicity.When the peptide was inserted into the major immune region(MIR)at the top of HBc-VLPs,the strong humoral or cellular immune response would be induced by the epitope peptide displaying on the surface of VLPs,thus the immunogenicity of epitope peptide PEP3 could be enhanced.So HBc-VLPs could be the immune carrier for PEP3 to strengthen its vaccine efficiency.The adenovirus vector is wildly used as a kind of immune vector else.The adenovirus capsid consists of 240 hexons and 12 pentons.The exogenous peptide could be inserted into the HVR5 region,then expressed on the surface of capsid.Therefore,when the peptide PEP3 was inserted into HVR5 region,the adenovirus with high copy numbers of PEP3 on the capsid could induce effective immune response to PEP3 peptide.In summary,the different kind of tumor vaccine carrying PEP3 will be designed based on adenoviral vector in this project,then be evaluated for its therapeutic effect for malignant glioma therapy in tumor-bearing animal model.The results will provide a clue for vaccine designing and the choice for immunotherapy protocol of malignant glioma.The research project was carried out as follows.1.The construction,preparation and purification of the adenovirus carrying the epitope PEP3.(1)The preparation and purification of the adenovirus expressing the fusion protein HBc-PEP3 in E1 region.By means of molecular cloning,the HBc-PEP3 gene fragment was inserted into the adenovirus El shuttle vector pAd5-E1-CMV-MSC.The resultant plasmid was named pAd5-E1-CMV-HBc-PEP3.Then the shuttle vector digested by Pac I and backbone vector pAd5-E3-CMV-eGFP linearized by Pac I were co-transfected into HEK 293 cell.High titer adenovirus Ad5.E1-CMV-HBc-PEP3/E3-CMV-eGFP was achieved by propagation and purification,which was named 9Z1.(2)The preparation and purification of the adenovirus with PEP3 modification in hexon.By means of molecular cloning,the PEP3 gene fragment was inserted into HVR5 region of adenovirus backbone vector pAd5-HVR5-MSC/E3-CMV-luciferase-T2A-eGFP.The resultant vector was called pAd5-HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP.The adenovirus backbone vector pAd5-HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP vector linearized by Pac I was transfected into HEK 293 cell.High titer adenovirus Ad5.HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP was achieved by propagation and purification,which was named 9Z2.(3)The preparation and purification of the adenovirus with the fusion protein HBc-PEP3 expression in E1 region and PEP3 modification in hexon.By means of molecular cloning,adenovirus vector pAd5-El-CMV-HBc-PEP3/HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP were obtained by homologous recombination between backbone vector pAd5-HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP and E1 shuttle vector pAd5-E1-CMV-HBc-PEP3.Positive clone linearized by Pac I was transfected into HEK 293 cell.High titer adenovirus Ad5.E1-CMV-HBc-PEP3/HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP was achieved by propagation and purification,which was named 9Z3.(4)The preparation and purification of the adenovirus carrying a secreting fusion protein HBc-PEP3 expression in El region.By means of molecular cloning,the HBc-PEP3 gene fragment without stop code was inserted into the adenovirus El shuttle vector pAd5-El-CMV-H1H2-MSC,the obtained vector was called pAd5-E1-CMV-H1H2-HBc-PEP3.The shuttle vector digested by Pac I was co-transfected into HEK 293 cell with backbone vector pAd5-E3-CMV-eGFP linearized by Pac I.High titer adenovirus Ad5.El-CMV-H1H2-HBc-PEP3/E3-CMV-eGFP was achieved by propagation and purification,which was named 11H1.(5)The preparation and purification of the adenovirus carrying a secreting the fusion protein HBc-PEP3 in E1 region and PEP3 modification in hexon.By means of molecular cloning,adenovirus vector pAd5-E1-CMV-H1H2-HBc-PEP3/HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP were obtained by homologous recombination between backbone vector pAd5-HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP and E1 shuttle vector pAd5-E1-CMV-H1H2-HBc-PEP3.Positive clone linearized by Pac ? was transfected into HEK 293 cell.Then high titer adenovirus Ad5.E1-CMV-H1H2-HBc-PEP3/HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP was obtained by propagation and purification,which was named 11G1.2.The study of experimental therapy in the malignant glioma tumor-bearing animal model with adenovirus vector-based PEP3 tumor vaccines.(1)The evaluation of immune effect of the five kinds of adenoviruses in vivo.Mice were grouped randomly and injected with different adenoviruses respectively for 3 times,the immunizing dose is 2×1010vp each mice for one time.Then the level of antibodies against to PEP3 was evaluated by ELISA after immunization.(2)The study of experimental therapy for malignant glioma bearing mice of the adenovirus.After immunized by different adenoviruses,the animal tumor model was established by s.c inoculation with EGFRv?/G422 cell in Kunming mice.Then the tumor volume was measured and tumor growth curve was drawn,which was used for evaluation of the effect of tumor vaccine on malignant glioma tumor growth.This project achieved the following results.1.By conventional cloning and homologous recombination,five kind of recombinant adenoviruses were successfully constructed and prepared as follows.9Z1,Ad5.E1-CMV-HBc-PEP3/E3-CMV-eGFP,the virus titers was 1.9×1012 vp/mL.9Z2,Ad5.HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP,the virus titers was 1.1×1012 vp/mL.9Z3,Ad5.E1-CMV-HBc-PEP3/HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP,the virus titers was 2.6×1012 vp/mL.11H1,Ad5.E1-CMV-H1H2-HBc-PEP/E3-CMV-eGFP,the virus titers was 1.5×1012 vp/mL.11G1,Ad5-E1-CMV-H1H2-HBc-PEP3/HVR5-PEP3/E3-CMV-luciferase-T2A-eGFP,the virus titers was 1.6×1012 vp/mL.2.After immunization by each adenoviruses for three times,humoral immune response was induced by each adenovirus,the antibodies against to PEP3 could be detected in serum.The level of anti-PEP3 was highest in the mice which stimulatd by the adenovirus expressing a secreting HBc-PEP3 and PEP3 peptide modification in hexon.3.These adenoviruses could inhibit malignant glioma tumor growth,which show different degrees of inhibitory effect.The adenovirus expressing a secreting HBc-PEP3 and PEP3 peptide modification in hexon shows the most significant inhibitory effect on tumor growth,and the inhibitory effect of 9Z1 expressing HBc-PEP3 in E1 region on tumor growth was the weakest.In summary,this study designed five adenoviruses carrying epitope PEP3 in different ways.These five adenoviruses could induce the body to produce a humoral immune response against PEP3,also inhibit the malignant glioma growth of tumor-bearing mice.Among them,the recombinant adenovirus expressing a secreting HBc-PEP3 and PEP3 peptide modification in hexon showed the best immune and therapeutic effect.These results laid the foundation for the research of immunotherapy and clinical therapy for PEP3 vaccine in malignant glioma.