Production and Characterization of Monoclonal Antibodies Against the Dimerization Domain of Human HER2

Mohammad Nadri,Mohsen Sisakhti,Tayebeh Kianoush,Mohammad Soukhtanloo,Amir Hosein Jafarian,Farnaz Zahedi Avval,Mahboobeh Alamdari

doi:10.17795/ajmb-29918

Abstract

Background: Human Epidermal Growth factor Receptor 2 (HER2), also known as ErbB2 is a 185 kDa protein belonging to the Human Epidermal Receptor (HER) family of tyrosine kinase receptors overexpressed in 20% - 30% of patients with breast cancer. Similar to other members of the HER family, HER2 glycoprotein comprises of multiple domains including an extracellular ligand-binding domain, a single transmembrane domain and a cytoplasmic domain with tyrosine kinase activity. The extracellular domain of HER2 with 632 amino acids is composed of four subdomains (I - IV); subdomains I and III form a ligand binding site, and cysteine-rich subdomains II and IV play an important role in dimerization of the receptor. Materials and Methods: Primarily, BALB/c mice were immunized with a 30-aminoacid peptide as a part of the human HER2 subdomain II. Splenocytes from hyperimmunized mice were fused with myeloma cells (SP2/0), selected in hypoxanthine-aminopterin-thymidine (HAT) medium, and screened by indirect Enzyme-Linked Immunosorbent Assay (ELISA). Secreted MAbs were characterized according to isotypes, reactions with the native HER2 in SKBR3 cells by western blotting, and in tissue sections from HER2 positive breast cancer specimens by Immunohistochemistry (IHC). Conclusions: The results of both western blotting and Immunohistochemistry showed that native HER2 can be detected with 1F1 monoclonal antibody.

Highlights

Human Epidermal Growth factor Receptor 2 (HER2), known as ErbB2 is a 185 kDa protein belonging to the Human Epidermal Receptor (HER) family of tyrosine kinase receptors overexpressed in 20% - 30% of patients with breast cancer
Serum samples were collected after four intraperitoneal injections, and different dilutions were evaluated for presence of anti-HER2 (266 - 296) peptide using indirect Enzyme-Linked Immunosorbent Assay (ELISA). (Negative Control: NC)
Western blot analysis showed that 1F1 monoclonal antibody reacts strongly with 185 kDa HER2 protein from SKBR3 cell line and bovine serum albumin (BSA) conjugated HER2 (266 - 296) peptide with molecular weight of around 70 kDa (Figure 2); whereas, 3L5 and 3G7 did not or weakly bounded to native HER2

Summary

Introduction

Human Epidermal Growth factor Receptor 2 (HER2), known as ErbB2 is a 185 kDa protein belonging to the Human Epidermal Receptor (HER) family of tyrosine kinase receptors overexpressed in 20% - 30% of patients with breast cancer. The extracellular domain of HER2 with 632 amino acids is composed of four subdomains (I - IV); subdomains I and III form a ligand binding site, and cysteine-rich subdomains II and IV play an important role in dimerization of the receptor. The extracellular domain (ECD) of HER2 with 632 amino acids is composed of four subdomains (I - IV), where subdomains I and III form a ligand-binding site, and cysteine-rich subdomains II and IV play an important role in dimerization of the receptor [3, 4]. Pertuzumab (2C4), the second humanized monoclonal antibody against different epitopes of HER2 ECD [13], in combination with trastuzumab and docetaxel, was approved by the FDA for the neoadjuvant treatment of HER2-positive patients [14, 15]

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