Recombinant human IgG antibodies recognizing distinct extracellular domains of EGF receptor exhibit different degrees of growth inhibitory effects on human A431 cancer cells.

Abstract

Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.

• Authors: Chialun Chang, Atsushi Takayanagi, Tetsuhiko Yoshida, Nobuyoshi Shimizu
• Journal: Experimental cell research (Exp Cell Res) Vol. 319 Issue 8 Pg. 1146-55 (May 01 2013) ISSN: 1090-2422 [Electronic] United States
• PMID: 23499740 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2013 Elsevier Inc. All rights reserved.

Chemical References

• Antibodies, Monoclonal
• Antineoplastic Agents
• Epitopes
• Immunoglobulin G
• Recombinant Proteins
• ErbB Receptors

Topics

• Animals
• Antibodies, Monoclonal (metabolism, pharmacology, therapeutic use)
• Antibody Specificity
• Antineoplastic Agents (metabolism, pharmacology, therapeutic use)
• CHO Cells
• Cell Line, Tumor
• Cell Proliferation (drug effects)
• Cricetinae
• Cricetulus
• Epitopes (immunology, metabolism)
• ErbB Receptors (chemistry, immunology, metabolism)
• Extracellular Space
• Humans
• Immunoglobulin G (metabolism, pharmacology, therapeutic use)
• Immunotherapy (methods)
• Neoplasms (pathology, therapy)
• Protein Binding
• Protein Structure, Tertiary
• Recombinant Proteins (metabolism, pharmacology, therapeutic use)