Abstract 5478: A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2.

Abstract Esophageal cancer is one of the most common cancers, and the 5-year survival rate is less than 10% due to lack of effective therapeutic agents. This study was to evaluate antitumor activity of Ec-LDP-Hr-AE, a recently developed bispecific enediyne-energized fusion protein, on esophageal carcinoma, targeting both EGFR (epidermal growth factor receptor) and HER2 (human epidermal growth factor receptor 2), because both EGFR and HER2 have been shown overexpression in over 30% of esophageal cancers. The fusion protein Ec-LDP-Hr-AE was encoded by a gene that was genetically constructed by fusing the oligopeptides ligand Ec (22 amino acids of EGF COOH-terminal) and Hr (VH CDR3 region of anti-HER2 C6.5 antibody) with lidamycin (LDM, an enediyne antibiotic with potent cytotoxicity) via two (G4S)3-linker. The encoded 18.4 kDa fusion protein was expressed in Escherichia coli and characterized its binding affinity with esophageal squamous cell carcinoma (ESCC) cells by flow-cytometry-based immunofluorescence assay. The results showed that Ec-LDP-Hr-AE protein bound to ESCC cells EC9706 and KYSE150 with high affinity (the Kd values were 5.283μmol/L and 3.562μmol/L, respectively). The potency of Ec-LDP-Hr-AE was determined on four human ESCC cell lines with differential expression of EGFR and HER2 using MTT assay. The bispecific Ec-LDP-Hr-AE showed potent cytotoxicity to EC1, EC109, EC9706 and KYSE150 cells with IC50 values in the picomolar level. Moreover, the bispecific Ec-LDP-Hr-AE was more potent than the monospecific counterparts (Ec-LDP-AE and LDP-Hr-AE) and LDM. Cell cycle distribution assay revealed that Ec-LDP-Hr-AE caused G2-M arrest in EC9706 and KYSE150 cells, in which the G2-M phase cells reached the peak with 0.1nmol/L of Ec-LDP-Hr-AE treatment. Ec-LDP-Hr-AE also induced cancer cell apoptosis in EC9706 and KYSE150 cells and the induction of apoptosis was in a dosage-dependent manner. The apoptotic index in the EC9706 cells exposed to 0.1, 0.5, or 1 nmol/L of Ec-LDP-Hr-AE were 15.0 ± 0.3%, 38.1 ± 0.6%, and 50.0 ± 0.4%, respectively. Western blotting showed that Ec-LDP-Hr-AE promoted caspase-3 and caspase-7 activities as well as PARP cleavage, indicating that Ec-LDP-Hr-AE-induced apoptosis was associated with mitochondria-caspase cascade. Moreover, Ec-LDP-Hr-AE inhibited KYSE150 cancer cell proliferation via decreasing phosphorylation of EGFR and HER2, and further exerted inhibition of the activation of their downstream signaling molecules (e.g. AKT and ERK). In summary, the bispecific energized fusion protein Ec-LDP-Hr-AE exhibited potent cytotoxicity to ESCC cells in vitro, leading to cell cycle arrest and apoptosis via targeting EGFR and HER2. These results suggest that Ec-LDP-Hr-AE could be a promising candidate for esophageal cancer targeting therapy. Citation Format: Guo Xiaofang, Xiaofei Zhu, Yue Shang, Shenghua Zhang, Yongsu Zhen, Wancai Yang. A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2013-5478