Development of novel monoclonal antibodies with specific binding affinity for denatured human CD26 in formalin-fixed paraffin-embedded and decalcified specimens.

Ryo Hatano,Satoshi Iwata,Haruna Otsuka,Nam H Dang,Taketo Yamada,Shuji Matsuoka,Takumi Itoh,Hiroto Yamazaki,Kei Ohnuma,Hiroko Madokoro,Yuka Narita,Chikao Morimoto,Eriko Komiya,Arun Rishi

doi:10.1371/journal.pone.0218330

Ryo Hatano, Satoshi Iwata + Show 12 more

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https://doi.org/10.1371/journal.pone.0218330

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Abstract

A 110-kDa type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 has a multitude of biological functions and plays an important role in the regulation of inflammatory responses and tumor biology. Our work has focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and we have recently developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which has promising safety profile and clinical activity in patients with malignant pleural mesothelioma. The development of an anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in formalin-fixed paraffin-embedded (FFPE) tissues in the clinical setting is therefore of the utmost importance. To develop novel anti-CD26 mAbs capable of binding to denatured CD26, we immunized mice with urea-treated CD26 protein. Hybridoma supernatants were screened for specific reactivity with human CD26 by immunostaining through the use of a set of FFPE human CD26-positive or negative tumor cell lines. This screening method enables us to develop novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in FFPE non-tumor and tumor tissue sections with reliable clarity and intensity. Specifically, these mAbs display strong binding affinity to denatured human CD26 rather than undenatured human CD26, and are capable of detecting denatured human CD26 in decalcified specimens. These novel anti-CD26 mAbs are potentially useful for the analysis of CD26 expression in cancer patients with bony metastasis, and may help decide the appropriateness of YS110 therapy for future cancer patients.

Highlights

CD26 is a homodimeric type II transmembrane glycoprotein with a molecular mass of 220– 240 kDa [1, 2]
CD26/dipeptidyl peptidase IV (DPPIV) function in cancer biology is not yet fully characterized, CD26 serves as a prognostic marker in multiple tumors such as colorectal cancer (CRC), gastrointestinal stromal tumor (GIST), thyroid carcinoma, urothelial carcinoma and prostate cancer [10,11,12,13,14]
formalin-fixed paraffin-embedded (FFPE) tissue specimens of normal liver, kidney, prostate, and malignant mesothelioma stained with 19–32 monoclonal antibody (mAb) exhibited reliable staining pattern and intensity [29], specimens from both CD26-positive MSTO-CD26 and JMN ctrl-short hairpin RNA (shRNA) cells as well as CD26-negative MSTO parent, JMN CD26-shRNA and A549 cells were all unexpectedly stained with 19–32 mAb (Fig 1A-ii)

Summary

Introduction

CD26 is a homodimeric type II transmembrane glycoprotein with a molecular mass of 220– 240 kDa [1, 2]. Human CD26 is composed of 766 amino acids (AAs), including a short cytoplasmic domain of 6 amino acid residues at the N-terminal end (AA 1–6), a transmembrane region of 22 amino acids (AA 7–28), and an extracellular domain, the predominant part of CD26 (AA 29–766) [3, 4] This C-terminal extracellular domain exhibits dipeptidyl peptidase IV (DPPIV) activity. We have previously demonstrated that antiCD26 monoclonal antibody (mAb) treatment resulted in both in vitro and in vivo inhibition of tumor cell growth, migration and invasion, and enhanced survival of mouse xenograft models inoculated with T-cell lymphoma, RCC or MPM via multiple mechanisms of action [19,20,21,22,23]. A subsequent phase II clinical trial of YS110 for MPM is currently in progress in Japan [25]

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