Danesh Hassani1, Mahmood Jeddi-Tehrani2, Parisa Yousefi2, Samaneh Mansouri-Fard1, Maryam Mobini1, Hengameh Ahmadi-Zare2, Forough Golsaz-Shirazi1, Mohammad Mehdi Amiri3, Fazel Shokri4. 1. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 2. Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran. 3. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. m_amiri@tums.ac.ir. 4. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. fshokri@tums.ac.ir.
Abstract
PURPOSE: The therapeutic potential of targeting the human epidermal growth factor receptor-3 (ErbB3/HER3) has long been ignored due to impaired tyrosine kinase function and low expression level in tumor cells compared with EGFR and HER2. Although recent investigations have explored the potential benefit of HER3 targeting and several anti-HER3 agents have been developed, there is still a critical need to design and produce more efficient therapeutics. This study was designed to develop tumor inhibitory monoclonal antibodies (MAbs) against different extracellular subdomains of HER3. METHODS: Distinct extracellular subdomains of HER3 (DI+II and DIII+IV) were utilized to produce MAbs by hybridoma technology. Biochemical and functional characteristics of these MAbs were then investigated by various methodologies, including immunoblotting, flow cytometry, cell proliferation, cell signaling, and enzyme-linked immunosorbent assays. RESULTS: Four anti-DI+II and six anti-DIII+IV MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-DI+II and four anti-DIII+IV MAbs recognized the native form of HER3 by immunoblotting. Four MAbs recognized the membranous HER3 by flow cytometry leading to induction of different levels of receptor internalization and subsequent degradation. Results of cell proliferation assays using these MAbs indicated that they differentially inhibited proliferation of HER3-expressing cancer cells and showed considerable synergistic effects in combination with trastuzumab. Selected MAb with the highest inhibitory effect significantly inhibited the phosphorylation of AKT and ERK1/2 molecules. CONCLUSION: Some of the anti-HER3 MAbs produced in this study displayed tumor inhibitory function and may be considered promising candidates for future HER3-targeted cancer therapy.
PURPOSE: The therapeutic potential of targeting the human epidermal growth factor receptor-3 (ErbB3/HER3) has long been ignored due to impaired tyrosine kinase function and low expression level in tumor cells compared with EGFR and HER2. Although recent investigations have explored the potential benefit of HER3 targeting and several anti-HER3 agents have been developed, there is still a critical need to design and produce more efficient therapeutics. This study was designed to develop tumor inhibitory monoclonal antibodies (MAbs) against different extracellular subdomains of HER3. METHODS: Distinct extracellular subdomains of HER3 (DI+II and DIII+IV) were utilized to produce MAbs by hybridoma technology. Biochemical and functional characteristics of these MAbs were then investigated by various methodologies, including immunoblotting, flow cytometry, cell proliferation, cell signaling, and enzyme-linked immunosorbent assays. RESULTS: Four anti-DI+II and six anti-DIII+IV MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-DI+II and four anti-DIII+IV MAbs recognized the native form of HER3 by immunoblotting. Four MAbs recognized the membranous HER3 by flow cytometry leading to induction of different levels of receptor internalization and subsequent degradation. Results of cell proliferation assays using these MAbs indicated that they differentially inhibited proliferation of HER3-expressing cancer cells and showed considerable synergistic effects in combination with trastuzumab. Selected MAb with the highest inhibitory effect significantly inhibited the phosphorylation of AKT and ERK1/2 molecules. CONCLUSION: Some of the anti-HER3 MAbs produced in this study displayed tumor inhibitory function and may be considered promising candidates for future HER3-targeted cancer therapy.
Authors: Arjan Kol; Anton G T Terwisscha van Scheltinga; Hetty Timmer-Bosscha; Laetitia E Lamberts; Frederike Bensch; Elisabeth G E de Vries; Carolina P Schröder Journal: Pharmacol Ther Date: 2014-02-07 Impact factor: 12.310