Research Of Therapeutic Anti-tumor Adenovirus Vaccine Targeting Human Telomerase Reverse Transeriptase(hTERT)

Background： Recent data suggest that more than90%patients with malignantcancers died of tumor metastasis or recurrence. But the traditional treatment, whetheroperation or radiotherapy and chemotherapy, could not clear away all of the malignanttumor cells. Thus, it is urgent to develop a novel therapeutic strategy to cure metastasisand recurrence.Over the past decade, cancer gene therapy was considered that it has a brightapplication prospect to conquer cancer. The scepticism towards cancer gene therapy isnow focus on the development of therapeutic anti-tumor vaccine, which are capable ofinduction stronger and more efficient immune responses against specifictumour-associated antigens.Objective:In order to develop a novel anti-tumor adenovirus vaccine Ad5F11p-eTERTFcGB.This adenovirus vaccine could break the immune tolerance and accomplishImmunopotentiation through a variety of mechanisms.Methods:1. The gene of sig-eTERT-Fc-GPI-IRES-GM/B7was cloned into modifiedadenoviral vector AdEasy-1/F11p to construct the plasmid Ad5F11p-eTERTFcGB. Onthis basis, Ad5F11p-eTERTFcGB were transfected into293cells by lipofectamine2000. And then, primary adenovirus were harvested. A large amount of adenoviruswere produced in293cells. The Q Sepharose XL was used to purify recombinantadenovirus for experiments in vivo.2. hTERT gene(538aa-875aa) cDNA fragment was cloned into pET-42aprokaryotic expression vector. The hTERT fusion protein was expressed and purified.Then the fusion protein was identified by Western blot and ELISA. 3. The recombinant plasmids pIRES-neo-hTERT and pIRES-hyg3-Luc weretransfected into B16cells by lipofectamine2000. After screening culture byHygromycin B and G418, a stably transfected cell line B16-hTERT/Luc wasestablished. The expressions of hTERT and Luc gene was identified by Western blotand immunofluorescence assay. Then, we used B16-hTERT/Luc cells inoculated withC57BL/6mouse to established hTERT+, Luc+melanoma tumor-bearing mice model.4. B16-hTERT/Luc cells were inoculated into subcutaneous tissue or the lateraltail vein to establish the tumor-bearing mice model. C57BL/6mice were immunized byintramuscular injection using therapeutic anti-tumor adenovirus vaccineAd5F11p-eTERTFcGB. And then the anti-tumor activity was compared with thecontrol groups. Firstly, we observed and analysed the tumor growth and survivalanalysis. And then, we also studied the possible mechanism of vaccine-inducedhumoral and cellular immunity. ELISA test was used to detect the level of specificantibody and the Th1type cytokines in serum; LDH Assay was used to investigate thecytotoxicity of spleen lymphocytes against B16-hTERT/Luc target cells in vitro;ELISPOT assay was used to detect the amount of spleen lymphocytes which specificsecreted IFN-γ. Finally, percentages of CD4+T cells, CD8+T cells, NK cells in spleenwere quantitatively analyzed by flow cytometry. The infiltrated CD8+T cells in tumorwere observed by immunofluorescence staining. Histopathology stain (H&E) assay oftumors and testises was used to find the change of immunized mice’s tissues.Result：1.The plasmid Ad5F11p-eTERTFcGB was successfully constructed．The primaryAd5F11p-eTERTFcGB adenovirus was successfully harvested after being transfectedinto293cells. Then, the purified recombinant adenovirus for experiments in vivo wasobtained using Q Sepharose XL.The293cells after primary adenovirus infectionshowed successful expression eTERTFc, GM/CSF, B7.1.2. The prokaryotic expression vector pET-42a-hTERT was successfullyconstructed and the hTERT fusion protein about65KD was purified．Western blot andELISAdemonstrated the antigenicity of the purified hTERT fusion protein. 3. The eukaryotic expression vectors pIRES-neo-hTERT and pIRES-hyg3-Lucwere successfully constructed. A stably transfected cell line B16-hTERT/Luc wasestablished and the expresses rate of human TERT and Luc gene in theB16-hTERT/Luc cells were nearly100%. tumor-bearing mice models weresuccessfully established.4. After C57BL/6mice were immunized by intramuscular injection usingAd5F11p-eTERTFcGB, the time of tumor forming in adenovirus vaccine group was6days longer than the control groups. The adenovirus vaccine had inhibited tumourgrowth compared with the controls. The study of possible mechanism showed thehigher antibody level and Th1type cytokines levels were induced in adenovirusvaccine group. The hTERT antigen specific IFN-γ which released by splenocytes wasalso detected in adenovirus vaccine immunized mice about581±62SFU/106.Meanwhile, the cytotoxicity of spleen lymphocytes against B16-hTERT/Luc targetcells was61.4%,45.8%and35.3%when effector-target ratio was40:1、20:1and10:1.It had been observed that there were necrosis and infiltrated CD8+T cells in thetumors of adenovirus vaccine immunized mice. Meanwhile, there were nohistological evidences of infiltration by inflammatory cells and tissue damage intestises.Conclusion：In summary, we had constructed a therapeutic anti-tumor adenovirus vaccineAd5F11p-eTERTFcGB. This anti-tumor adenovirus vaccine can induce the higherhumoral and cellular immune responses and inhibit tumor growth after immunizationcompared with control groups. This kind of anti-tumor adenovirus vaccine providesnew exploration to Immunopotentiation in cancer biotherapy.