Enhanced Immunological Activity Of Adenovirus Vaccine For Renal Cell Carcinoma Using A Novel Double-targeted Fusion Protein

Background:Interest gene targeting dendritic cells is the current hot topic in the field of cancer therapeutic vaccines. Adenovirus type5vector vaccine has showed excellent capability of infecting DCs in vitro, but in vivo its efficiency is poor. Therefore, it is meanful to discuss interest gene targeting DCs mediated by adenoviral vector in vivo, furthermore, to enhance efficiency of therapeutic tmuor vaccine.Objective:Using molecular biology methods, to prepare a novel double-targeted protein(CFmDEC) which can both bind fiber knob of adenovirus type5and DEC205which is a receptor molecular exclusively expressed on the surface of DCs, then to investigate whether infection of adenovirus vaccine targeting DCs could be enhanced in vivo using CFmDEC.Method:Preparation and identification of double-targeted fusion protein:The gene encoding extracellular domain of type5adenovirus receptor CAR and bacteriophage T4fiber protein and single-chain antibody against mouse DEC205was optimized and spliced together to construct prokaryotic expression vector pGEX-CFmDEC. Then recombinant expression vector pGEX-CFmDEC was transformed to E. coli BL21(DE3) and express soluble recombinant protein by induction of IPTG. Then agarose gel was used to purify recombinant proteins and double-targeted fusion protein was obtained via removing GST label by PreScission Protease. Furthermore, we identified biological activity of CFmDEC.Construction and functional verification of RCC adenovirus vaccine in vitro:The complex gene sig-tG250-Fc-GPI-IRES-GMCSF-B7.1(tG250FcGB) was cloned into shuttle vector pDC316to construct eukaryotic plasmid pDC316-tG250FcGB. According to AdMax protocol, pDC316-tG250FcGB and adenovirus backbone plasmid pBHGlox (delta) E1,3Cre were recombind in HEK293cells to construct adenovirus RCC vaccine Ad5-tG250FcGB which can express RCC related antigen G250. Then, Western blot was used to identify expression of Ad5-tG250FcGB in eukaryotic cells.Study on synergistic anti-tumor activity of CFmDEC:CFmDEC and Ad5-tG250FcGB was binded together in vitro to immunize transgenic RCC Balb/c and effect of anti-tumor was compared with the controls. While serum antibody titers and cytokines secreted by spleen lymphocytes was analysed by ELISA. Cytotoxic T lymphocyte (CTL) was verified by lactate dehydrogenase (LDH) assay and production of specific IFN-y was assayed by ElispotResults:1. CFmDEC was successfully prepared. Double digestion and sequencing verified that prokaryotic expression vector pGEX-CFmDEC was constructed successfully. Western blot confirmed pGEX-CFmDEC can express CFmDEC in E. coli BL21(DE3). And ELISA analysis validated that CFmDEC can both bind its corresponding receptor efficaciously. Forthurmore, synergetic effect on adenovirus infection of DCs with CFmDEC was verified by FACS and immunofluorescence in vitro.2. Adenovirus vaccine Ad5-tG250FcGB was constructed successfully.First, plasmid pDC316-tG250FcGB was identified by double digestion and sequencing successfully. Then PCR and sequencing of viral genome confirmed that Ad5-tG250FcGB was constructed successfully. Interest antigen expressed by Ad5-tG250FcGB in293T cell could be detected by Western blot.3. Enhanced immunological activity of Ad5-tG250FcGB using CFmDEC. Mice immunized with Ad5-tG250FcGB/CFmDEC showed more significantly inhibitation of tumor growth compared with control groups, and prolonged survival time of7.2days. Compared to Ad5-tG250FcGB alone, Ad5-tG250FcGB combined CFmDEC can raise the levels of IFN-y (412pg/mL) and IL-2(89.3pg/mL) secreted by spleen lymphocytes, and serum antibody IgGl/IgG2a ratio reduced0.156compared with adenovirus vaccine alone. Meanwhile, the efficiency of CTL increased by13%. Conclusion:Double-targeted fusion protein can bind adenovirus vector effectively in vitro and improve adenovirus vaccine targeting to DCs in vivo, furthermore, induce higher level of specific anti-tumor immune response and slow the growth of renal cell carcinoma. This study might provide a meaningful reference with the use of adenovirus vectors targeting DCs in the field of therapeutic tmuor vaccine.