A Clinical Platform For Full-length Antibody Gene Therapy

Because of security in clinical trials and significant curative effect, monoclonal antibody became the most specific drug for tumor target therapy, which has a large market. However, monoclonal antibody is very expensive with which one therapy course would cost approximate two millions, and it is hard to afford for common peoples. Therefore, our lab first put forward full-length antibody gene therapy strategy in 2004. Tumor were treated with replication-deficient adenovirus-mediated gene therapy, the full-length antibody with function activity was transferred in vivo (major in liver) into the blood circulation. The strategy omitted the complex processes and procedures in preparation and purification of antibody, thereby significantly reduce the cost of treatment. However, because of defects of adenovirus itself,the strategy was still less effective in the level of antibody expression and express time.Adeno-associated Viruses (AAV) has a characteristic of mediating long-term efficient expression of foreign genes. As a vector for gene therapy, its effectivity in clinical trials would make it to be a preferable vector for full-length antibody gene therapy strategy. However, because of the replicative characteristic of AAV, the strategy of packaging rAAV with high efficiency and low cost has been a technological bottleneck in clinical application. Therefore, to prompt the efficient adenovirus-mediated full-length antibody gene therapy in clinical application, it is necessary to establish a stable and efficient rAAV production system.Baculovirus-insect cell system is a commonly used eukaryotic expression system. As an insect cell virus, Baculovirus can provide support for AAV replication as well as adenovirus or herpes simplex virus, but not have toxicity to human body. Baculovirus is significantly bigger than AAV, and can be purified easily. At the same time, insect cell can be cultured in suspension style, which would avail rAAV to package in a large scale. Therefore, baculovirus-insect cell system is expected to become effective starting point.Cetuximab is a full-length EGFR monoclonal antibody, and it has been used as a drug in the treatment of several types of malignant tumors which EGFR is over-expressed. rAAV8 as an ideal gene therapy vector is preferable to liver and has great gene transfer ability.Therefore,to explore and establish a full-length antibody gene therapy strategy in clinical treatment, we construct a vector of AAV8 carrying Cetuximab using baculovirus - insect cell system.Main methods:(1)Construction of three recombinational baculovirus vectors,they are BAPⅧ,pABV359-Cetuximab,pABV359-Cetuximab-WPRE.(2)Achieve the three baculovirus plasmids through DH10Bac recombination and blue-white screening;(3)Package and amplify Bac-AAV2RepiCap8i and Bac-Cetuximab in Sf9 cells, and determine the titeration using q-RT PCR.Dectect the expression level of Cetuximab in Sf9 and HEK293 cells using ELISA.(4)Package rAAV8-Cetuximab and rAAV8-EGFP in Sf9 cells,and to rAAV8-EGFP as control virus,dectect baculovirus inactivation and infect viability to HEK293 of rAAV8.Then,Determine the Cetuximab expression level of rAAV8-Cetuximab in HEK293 cells.The preliminary results show that rAAV8 was well prepared using baculovirus-insect cell system,and rAAV8 then subsequently extracted and purified by high performance liquid chromatography. The viral titer was determined through real-time quantitative PCR and infection viability of rAAV8 was examined by EGFP reporter in HEK293 cells,results show that the virus could effectively infect HEK 293 cells. Meanwhile,collect the proteins in the supernatants and cell protein 96h after rAAV8-Cetuximab infect HEK293 cells,and then dectect the expressiong level of Cetuximab using ELISA.Test results shows that the concentration of Cetuximab in the supernatants reached 49.8ng/mL,while 176ng/mL in cells.However,the amout of Cetuximab in the supernatants and cells is at the same level. Because of the low expression level of Cetuximab antibody in vitro,we should analyse the the reasons to enhance the expression level,Meanwhile,we should also ned to dectect the expression level in vivo.