Anti-cancer Effect Of The Melittin Mediated By The Single Chain Antibody In Osteosarcoma

Osteosarcoma (OS) is the most common sarcomas in children and adolescents.OS is the most frequent bone cancer occurring mostly in children and adolescentsbetween10and20years of age. It is thought to arise from mesenchymal boneforming tissue and its histological hallmark is the production of malignant osteoid.The metaphyseal growth regions of the long bones of the extremities are the mostcommon site of the primary bone tumor with peak incidences following theadolescent growth spurt and the seventh and eighth decades of life. Currentmultimodal therapy consisting of surgery and chemotherapy, achieves a5-yearsurvival rate of approximately60–70%in children while patients who present withmetastatic disease at diagnosis have a10–30%survival rate. The chemotherapy agentswhich have long demonstrated anti-tumor activity in OS include cisplatin,doxorubicin, ifosfamide, and high dose-methotrexate. The use of novel effectivetherapeutic approaches and treatment strategies in patients who are resistant to currenttherapy could provide an improvement in outcome in patients.In more than20years of drug development, agents targeting members of thehuman epidermal growth factor receptor family—EGFR (also known as HER-1,erbB1) and HER-2/neu (also known as erbB2)—have shown encouraging therapeuticefficacy. The first to be approved by the US Food and Drug Administration (FDA) in1998was trastuzumab (Herceptin) for the treatment of HER-2(erbB-2)-positivebreast cancer. Over the past few years, four EGFR specific agents have also receivedregulatory approval. Cetuximab (Erbitux) for metastatic colorectal cancer (mCRC)and squamous cell carcinoma of the head and neck (SCCHN), Gefitinib (Iressa) foradvanced or metastatic non-small-cell lung cancer (mNSCLC), Erlotinib (Tarceva) foradvanced or metastatic pancreatic cancer and non-small-cell lung cancer (NSCLC),and Panitumumab (Vectibix) for metastatic colorectal cancer (mCRC).Bee venom is a unique weapon for the bee colony and composed of melittin,apamin, adolapin, mast cell degranulating peptide, phospholipase A2, hyaluronidase,and some non-peptide components (histamine, dopamine, norepinephrine, etc.).Among them, melittin is the major component of bee venom (50–70%) and is acationic, hemolytic peptide. It is composed of26amino acid residues in which theamino-terminal region (residues1–20) is predominantly hydrophobic whereas the carboxyl-terminal region (residues21–26) is hydrophilic. Its amphiphilic propertymakes it water-soluble and membrane-active lytic peptide ideally suited formonitoring mechanisms of pore formation and lipid–protein interactions inmembranes. Above all, melittin shows several important medical effects includingenhancement of phospholipase A2activity, cytotoxic effects against cancer cells, andanti-inflammatory, anti-microbial, and anti-arthritic effects.In this study, we have fused the anti-EGFR single chain variable fragment (scFv)with melittin aiming at EGFR-targeted therapy for Osteosarcoma. The designed genesequence was optimized, then cloned and expressed in E. coli BL21strains. Inaddition, the expression of the fusion protein was optimized for its in vivo and in vitroantitumoral activity.