Immunotherapy For Lewis Lung Carcinoma By DCs Infected With CK19 Gene Recombinant Adenoviral Vectors

Lung cancer is the most malignant tumor with the highest morbidity and mortality. At present, the main means of treatment of lung cancer include surgery, radiotherapy, and chemotherapy and so on, but the treatment effect is not ideal. Dendritic cells are the most powerful antigen presenting cells, which have the ability to induce specific cytotoxic T lymphocytes. The study showed that the occurrence, development and prognosis of malignant tumor were related to the dendritic cells. The number of dendritic cells in tumor tissue and tumor adjacent tissue was decreased, which lead to the immune escape. We consider that DC cells play an important role in the immune escape mechanism of malignant lung tumor cells.The basic principle of tumor vaccine is to induce the passive or active immune response by tumor antigen, so as to achieve the goal of killing tumor cells. Vaccine therapy has the advantages of long duration of treatment and low toxicity. The superiority of DCs vaccine immunotherapy is the "Precision Medicine", which induce the CTL effect by tumor specific antigen as a signal. So the tumor specific antigen is the core of tumor immunotherapy. Most of the tumor antigens have been identified by MHC. Cytokeratin is widely distributed in the epithelial cells, which is one of the components of the cytoskeleton. The expression of CK19 in epithelial cancer cells is the most important in the diagnosis of malignant tumors, which makes CK19 as a reliable target for tumor detection and immunotherapy. Tumor vaccine therapy is a specific anti-tumor immune response that is induced by tumor antigens in vivo. Tumor vaccine research is currently the focus of cancer treatment with the advantages of specificity, and maintaining a long time.In the first part, we confirmed that expression of CK19 protein in Lewis lung cancer cells increased, that indicating the CK19 protein plays an important role in the development of lung cancer. CK19 protein is highly expressed in most tumor tissues, meanwhile it rarely expressed in most normal adult tissues. The CK19 should be a good target for immunotherapy. The effective method of gene transfer is the key to anti tumor immune therapy. In the second part, the recombinant adenovirus carrying human CK19 gene and enhanced green fluorescent protein gene was constructed by AdMax system. At present, transferring the target gene into DCs cell by using the defective recombinant adenovirus vector is the most effective method. The recombinant adenovirus carrying human CK19 gene was transfected into DCs cells, and the rAd-CK19-DCs vaccine was prepared to observe the therapeutic effect of anti mouse lung cancer xenograft in mice.Part ⅠThe expression of CK19 in mouse Lewis lung cancer cellsObjectiveIn the first part, it is confirmed that CK19 protein can be used as a target antigen for cellular immunotherapy, and it can prove that Lewis lung cancer cells can be used as the experimental cells for CK19 targeted immunotherapy.MethodsThe expression of CK19 protein in Lewis lung cancer cells was detected by Western-blot method, and the localization of CK19 protein in Lewis lung cancer cells were detected by immunofluorescence. Using the PCR method to detect the expression of mRNA Lewis in CK19 lung cancer cells, and the expression of CK19 in lung cancer cells was confirmed.ResultsIn this part, PCR Real-time, Western-blot and immunofluorescence were used to detect the expression of CK19 in Lewis lung cancer cells. Lewis lung cancer cells of CK19 mRNA positive expression. The fluorescence staining showed that CK19 protein is widely expressed in the cell membrane. And dendritic cells of CK19 protein negative expression, apparently in Lewis lung cancer cells CK19 protein expression level was significantly higher than that in the dendritic cells.ConclusionsThe results showed that CK19 protein can be used as a target antigen for tumor cell immunotherapy, which also proved that Lewis lung cancer cells can be used as CK19 targeted immunotherapy cells for in vivo and in vitro studies.Part IIConstruction of recombinant adenovirus vector as the basis for the construction of CK19 expression vector pTR-UF5/CK19ObjectiveBy using AdMax system constructed recombinant adenovirus carrying human CK19 vector for preparation of transfecting tumor immune cells with DC vaccine.MethodsRecombinant adenovirus associated virus transfer plasmid vector pTR-UF5 as the basis for the construction of CK19 expression vector pTR-UF5/CK19. Firstly, the human CK19 gene cDNA fragment was amplified by PCR using plasmid PTR-EGFP-CK19 as template. The shuttle plasmid pDC316-CK19 and the auxiliary plasmid BHGlox E1 (delta) P, were transfer into 293 cells using Ca CL2 method.ResultsThe results showed that the size of the Recombinant Adenovirus Shuttle Plasmid pDC316-cmv-EGFP-CK19 was detected by double enzyme digestion method. According to the reported gene sequencing Bank CK19cDNA, results are consistent with the expression of CK19 gene. Using AdMax system successfully constructed the recombinant adenovirus rAd-CK19, which could be the next step to carry out experimental to product DCS vaccine for immunotherapy of tumor.ConclusionsWe successfully constructed the recombinant virus containing lung cancer associated antigen CK19 gene carrier Ad/CK19.The RT-PCR test prove the expected size (138bp) fragments, which confirm that the recombinant virus was constructed successfully. The recombinant adenovirus Ad/CK19 was transfected into DCs cells for preparing vaccine therapy for lung cancer.Part IIICK19 expression analysis and phenotypic changes of dendritic cells transfect with recombinant adenovirus expressing CK19 geneObjectiveThe tumor targeting gene CK.19 was transferred into DCs cells by recombinant virus constructed in the part Ⅱ. Detect the phenotypic changes of DCs cells after transfection for preparation of DCs vaccine modified by CK19 gene.MethodsThe recombinant adenovirus vector was transferred into mouse DCs cells to prepare DCs vaccine modified by CK19 gene. DC cells were cultured in the culture plate and complete medium without antibiotic. The pTR-UF5/CK19 carrier was diluted in the culture medium without antibiotics. The Lipofectamine2000 was diluted in the culture medium without antibiotics, and continue incubated at room temperature. The culture medium of PBS or no serum medium was used to clean the cells for 2 times, and the compound was added to the culture hole. Than the shaking culture plate was used to make the composite distributed evenly. After 2 hours infection, the infected cells were collected and changed into the culture medium containing GM-CSF and IL-4 RMPI1640. The expression of CK19 in the infected DCs was used to detect by real-time PCR, Western blot and immunofluorescence. The detection of DCs cell phenotypes in the modified change (CD80, CD86 and MHC-Ⅱ).ResultsCK19 gene is rarely expression in DCs cells transfect with CK19 gene adenovirus vector, and empty adenovirus vector on DCs gene expression in CK19 cells did not affect. DCs transfect with CK19 gene adenovirus vector show that the high positive expression of CD80, CD86 and MHC, and empty adenovirus vector of DCs of CK19 protein expression did not influence. The expression of CD80, CD86 and MHC of DCs cells transfect with CK19 were higher than normal DCs. The experiments show that the CK19 expression was significantly increased, indicating that the DCs vaccine was done.ConclusionsIn this experiment, we construct CK19 in recombinant adenovirus vector by using homologous recombination principle and purified for CK19 gene as a target gene. Then, synthesis dendritic cells transfected with adenovirus carrying Ad/CK19 gene (DC), which made lung cancer vaccine for further experiments in vivo to test therapeutic effect of vaccine.Part IVAnti tumor activity of dendritic cells modified by CK19 gene in miceObjectiveAccording to the requirements of the guiding principles of drug efficacy and the non clinical research technical guidelines of cytotoxic drugs, the test specimens inhibitory effect on the growth of lung cancer in Lewis mice.MethodsThrough the application of in vitro carrying CK19 gene recombinant adenovirus infected DCS to made specific DCs vaccine (rAd-CK19-DCs).Vaccinating mice with Lewis lung cancer cells with DCs vaccine and observe the tumor growth rate (as measured by the tumor volume)to evaluate the tumor growth on mice lung cancer has no inhibitory effect and intensity. rAd-CK19-DCs vaccine can effectively inhibit the growth of lung cancer.ResultsrAd-CK19-DCs vaccine treatment can significantly inhibit the growth of Lewis lung cancer. In the observation period of 24 days, subcutaneous inoculation rAd-CK19-DCs treatment on tumor growth inhibitory effect is very obvious, showing the tumor growth delay. The group treated by rAd-CK19-DCs tumor volume is at least less than the control group 2 times. Using rAd-CK19-DCs vaccine can induce tumor specific immune response, which producing a strong effect of anti Lewis lung cancer.ConclusionsExperimental results show that the tumor vaccine (rAd-CK19-DCs) could inhibit the growth of lung cancer cells in mice. This result indicates that immunotherapy with tumor specific antigen is a good strategy.