Determination of critical epitope of PcMab-47 against human podocalyxin

Shunsuke Itai; Shinji Yamada; Mika K Kaneko; Yukinari Kato

doi:10.1016/j.bbrep.2018.04.003

Determination of critical epitope of PcMab-47 against human podocalyxin

Biochem Biophys Rep. 2018 Apr 24:14:78-82. doi: 10.1016/j.bbrep.2018.04.003. eCollection 2018 Jul.

Authors

Shunsuke Itai¹, Shinji Yamada¹, Mika K Kaneko¹, Yukinari Kato^{1

2}

Affiliations

¹ Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
² New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.

Abstract

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

Keywords: BSA, bovine serum albumin; DAB, 3,3-diaminobenzidine tetrahydrochloride; DMEM, Dulbecco's Modified Eagle's Medium; EDTA, ethylenediaminetetraacetic acid; Epitope mapping; FBS, fetal bovine serum; Monoclonal antibody; Oral cancer; PBS, phosphate-buffered saline; PODXL; Podocalyxin; SCC, squamous cell carcinoma; mAb, monoclonal antibody.