title: Construction and characterization of an anti-asialoglycoprotein receptor single-chain variable-fragment-targeted melittin.
abstract: Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 µg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.
authors.affiliation: Guangdong Key Laboratory of Molecular Epidemiology, School of Public Health, Guangdong Pharmaceutical University, Guangzhou, Guangdong, People's Republic of China.
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Zhao
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yu
authors.forename: Wentao
authors.initials: W
authors.lastname: Dai
authors.forename: Zhenjiang
authors.initials: Z
authors.lastname: Yao
authors.forename: Wei
authors.initials: W
authors.lastname: Zhou
authors.forename: Weiping
authors.initials: W
authors.lastname: Zhou
authors.forename: Junli
authors.initials: J
authors.lastname: Zhou
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Yang
authors.forename: Yinchang
authors.initials: Y
authors.lastname: Zhu
authors.forename: Sidong
authors.initials: S
authors.lastname: Chen
authors.forename: Limin
authors.initials: L
authors.lastname: Cao
mesh_terms: D037263:Asialoglycoprotein Receptor / Q000235:genetics / Q000276:immunology*; D001212:Asialoglycoproteins / Q000494:pharmacology; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics; D053878:Hemolytic Agents / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000188:drug therapy; D008555:Melitten / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D057127:Single-Chain Antibodies
chemical_list: D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D053878:Hemolytic Agents; D000922:Immunotoxins; D009961:Orosomucoid; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C017030:asialoorosomucoid; D008555:Melitten