Gene Therapy Of Tumor Using Non-replicative Adenovirus Coding For Full-length Antibody Cetuximab

For the advantages of targeting exactness, low toxin and high effect, the monoclonal have been applied to the treatments of kinds of disease, especially in the field of cancer therapy. More than 10 antibodies have been authorized to go on the market by US Food and Drug Administration (FDA) so far, used in the target tumor treatment, and are all full-length antibody. Although antibody has made an great progress in the field of the antitumor therapy, there is still a main problem to treat solid tumors. At present, the antibody production technology is far from the needs of the market, for the reason that the demand of antibody is much and the request of purity is high in clinic. The technology of extensive and high density mammal cell culture is a tough work around the world, and the technology was monopolized by few powerful companies. As a result, the cost of antibody production is very high which lead to only few of domestic patients can afford.In order to resolve the two main difficult problems, we design a new way that using the non-replicative adenovirus coding for the full-length monoclonal antibody gene which expression the antibody in the vitro which can avoid the costly and complicated course of production of antibody in the mammal cell. We choose the gene of the Cetuximab which targeting the HER1 exploited by ImClone Systems. Then, we examine the quality and quantity of the expressioned antibody. The over-expression of EGFR in many tumors plays a role in induction, growth, anti-drug and invasion. Luckily, the monoclonal antibody Cetuximab has a well therapy with it which had been authorized in the cure of head-neck carcinoma, and colorectal carcinoma, whether singleness or cooperation, and with low side effect.Methods and resultsFirst of all, we synthesize the light chain and high chain of Cetuximab, and add the gene of signal peptide in the front of them. For the sake of the balance and integrality of the light chain and high chain, we use the IRES to link them. In order to improve the expression level, we amend a part of the antibody code according to the code preference. Then, we add a AAV soured ITR sequence into the adenovirus which endow the adenovirus with replicative competent. The hybridvirus have the ability of high expression level and extend expression time. The construction of recombinant adenovirus Ad5-Her Ad5-C225,Ad5-NC225, Ad5- ITR-NC225 of expressing full-length antibody Cetuximab.The amended and un-amended genes of light chain and high chain of the monoclonal antibody Cetuximab were synthesized, and added to the secretion signal peptide on front end of the them, then singly cloned into the carrier pDC339-C225 and pDC311-ITR to generate new plasmids pDC339-C225, pDC339-NC225 and pDC311-ITR-NC225. Then, the plasmids pDC339-C225, pDC339-NC225 and pDC311-ITR-NC225 co-transfect with pBHGE3 into 293 cells to produce the new recombinant virus Ad5-C225,Ad5-NC225, Ad5- ITR-NC225. They were checked by PCR analysis using specific primers, propagated in 293 cells and purified by HPLC. Viral titer achieved with TCID50 and qRT-PCR methods.In vitro experimental studies on gene therapy system of expressioning full-length antibody Cetuximab.1. First of all, the 293 cell was infected by Ad-C225,Ad339-NC225 and Ad5-UDITR-NC225. 72 hours later, we got some culture liquid to examine the expressionion level of the antibody. The expression level of them are (25.2±3)ng/ml,317.6±5ng/ml and 425.9±5ng/ml. however, the recombined virus can not expression antibody in the LO2 cell.2. The examination of biological activity of expressed antibody, the indirect immunofluorescent assay (IFA) demonstrated that the antibody expressed by the recombined virus integrate well with HER1 which is over-expressing in lung cancer A431 cell. The fluorescence intensity was similar to that of the commercial Cetuximab, and can not been seen in HER1 negative SKBR-3 cell. This indicated that the Cetuximab expressed by recombined virus have fine specificity with HER1. Western Blot demonstrated that the light chain and heavy chain of the expressed antibody is balanceable, the relative molecular weight was similar to commercialized Cetuximab. Conclusion: we triumphantly construct the three recombined virus coding for full-length antibody Cetuximab, and the expressed antibody was active.