Generation Of Adenovirus-mediated Biospecific Antibody Fusion Protein Targeting To EGFR And HER2

Purpose:EGFR (epidermal growth factor receptor, EGFR) plays an important role in tumor proliferation and metastasis. It is over-expressed in many tumor cells and is served as a target for tumor therapies. Cetuximab is a full-length EGFR monoclonal antibody and is the first-line drugs during the treatment of several types of malignant tumors. However, the treatment with cetuximab is very expensive, and its monotherapy on a number of important tumors, including hepatocellular carcinoma is poor. HER2 is a member of EGFR family which is over-expressed in only a small number of tumors while expressed at a low level in most tumors. However, it functions centrally in EGFR family signaling network, so that its existence is important reason for treatment failure utilizing cetuximab on malignant tumors. Interesingly, an alternative HER-2 product, herstatin, consists of a segment of the ectodomain of p185HER-2 and an intron-encoded C-terminus, has the capability to negatively regulate combinations of interactions between group I receptor tyrosine kinases that confer synergistic growth signals with high affinity specific binding. Data were shown that the C terminal 79 amino acids (HERIN) of Herstatin can specially bind with HER2 and EGFR. Thus, in this study, we fused the coding sequence of cetuximab and HERIN, and subsequently cloned it into the genome of replication-defective adenovirus to establish a genetherapy system to express a bispecific antibody which can specially bind with EGFR and HER2. This system will improve therapy effencicy in treatment tumors in which EGFR and HER2 are double-positive, or EGFR positive, HER2 weak positive. Meaningwhile, this new system will significantly reduce the cost of cetuximab therapy.Experimental Design: Through dual luciferase reporting systems, we compared the regulation effect of different SV40 poly (A) signal sequence to upstream gene and bywhich to conduct an efficient full antibody expression system. We fused the coding sequence of cetuximab gene and HERIN through PCR, and cloned it into the adenovirus shuttle vector pDC339, and subsequently packaged a recombinant virus Ad5-AT2-Herin in 293 cells. Through ELISA, we quantitily detected the protein content of AT2-HERIN in vitro. Through Western blot analysis, we investigated the integrity of AT2-HERIN and expression balance between heavy chain and light chain. Through indirect immunofluorescence (IFA), we identified binding capacity and specificity of AT2-HERIN on EGFR (+) Her2 (-) A431 cells and EGFR (-) Her2 (+) SK-OV-3 cells.Results: It was showed that the effects of two kinds of SV40 polyA signals in regulating the upstream gene expression had no significance difference in 293 cells (P<0.01), while those in L-02 cells and Hela cells had significant difference(P<0.01). The expression value of the fused protein, AT2-HERIN in 293 cells was 1.66 ug / ml ~ 1.019 ug / ml. The molecular weight of AT2-HERIN was in line with expectation and two chains of antibody expressed in balance. The fused protein, AT2-HERIN could effectly bind to the embrance surface of A431 cell and SK-OV-3 cell.Conclusions: The two kinds of SV40 polyA signals have different effects in regulating the upstream gene expression according to varied cell lines. The recombinant adenovirus of Ad5-AT2-Herin can effectly express the fused protein of AT2-HERIN, which is obtained by fused the coding sequence of cetuximab and Hersatatin's C terminal 79 amino acids (HERIN). And AT2-HERIN is indeed a bispecific antibody protein, which can bind to EGFR and HER2 effectly.