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.

Melittin, a major peptide component of bee venom, and its conjugates in cancer therapy

“…Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was also confirmed. C1M kept the hemolytic activity of MEL and exhibited cytolytic capacity to HepG2 cells and the effects were greatly inhibited by co-administration with asialoorosomucoid, a natural ligand for ASGPR [83]. …”

Three Valuable Peptides from Bee and Wasp Venoms for Therapeutic and Biotechnological Use: Melittin, Apamin and Mastoparan

“…With regard to the first approach, a melittin-based recombinant immunotoxin obtained by fusion of genes that encoded an antibody fragment derived from the murine monoclonal antibody K121 with an oligonucleotide encoding melittin was tested successfully in vitro [ 78 ]. Another study was based on a recombinant immunotoxin of melittin fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (Ca) which conferred targeting and ASGPR-specific cytotoxicity to hepatocellular carcinoma cells [ 79 ]. Finally, a recent study characterized a CTLA-4-targeted scFv-melittin fusion protein as a potential immunosuppressive agent for organ transplant.…”

Immunotoxins Immunotherapy against Hepatocellular Carcinoma: A Promising Prospect

“…Conjugating melittin with scFv of ASGPR leads to killing specifically ASGPR-positive tumor cells. According to their analysis, a lack of C1M effect in coadministration with asialoorosomucoid (ASGPR ligand) confirmed its target-specific cytotoxicity [100].…”