Epitope mapping of human HER2 specific mouse monoclonal antibodies using recombinant extracellular subdomains of HER2

Abstract

Background: Human epidermal growth factor receptor 2 (HER2) is overexpresed in several human malignancies. Numerous studies show that HER2 plays an important role in the development and maintenance of the malignant phenotype. Targetting of HER2 molecule by monoclonal antibodies (mAbs) is a promising therapeutic approach. Anti-HER2 mAbs affect cancer cells differently, targeting distinct epitopes of HER2. Methods: Reactivity of a panel of 8 mouse anti-HER2 mAbs was investigated by ELISA and Western blotting using different subdomains of the extracellular domain (ECD) of HER2. All subdomains of HER2, including I, II, III, IV, I+II, III+IV and full HER2-ECD were constructed and expressed in CHO cells. Cross-reactivity of the mAbs with other members of the human HER family and Cynomolgus HER2 was also studied by ELISA. The mAbs were also tested by immunohistochemistry (IHC) using HER2 positive breast cancer tissues. Results: Our results demonstrated that 3 out of 8 mAbs detected conformational epitopes (1T0, 2A8 and 1B5), while 5 mAbs identified linear epitopes (1F2, 1H9, 4C7, 1H6 and 2A9). Three of the mAbs recognized subdomain I, one mAb reacted with subdomain I+II, 2 mAbs recognized either subdomain III or IV and 2 mAbs recognized subdomain III+IV. However, none of our mAbs recognized subdomain II of HER2 alone. The mAbs displayed either inhibitory or stimulatory effect on HER2-overexpressing tumor cells and did not react with other members of the human HER family. The pattern of IHC results implies better reactivity of the linear epitopes recognizing mAbs. Conclusion: Our findings suggest that paired subdomains of HER2 are essential for mapping of mAbs recognizing conformational epitopes. Moreover, it seems to be no association between subdomain specificity and antitumor activity of our anti-HER2 mAbs.