Date Published: June 13, 2019
Publisher: Public Library of Science
Author(s): Ryo Hatano, Taketo Yamada, Hiroko Madokoro, Haruna Otsuka, Eriko Komiya, Takumi Itoh, Yuka Narita, Satoshi Iwata, Hiroto Yamazaki, Shuji Matsuoka, Nam H. Dang, Kei Ohnuma, Chikao Morimoto, Arun Rishi.
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.
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. DPPIV belongs to the serine protease family, able to cleave dipeptides from polypeptides with N-terminal penultimate proline or alanine, and regulates biological activities of a number of mediators such as cytokines, chemokines, neuropeptides and incretin hormones . Although CD26 is expressed on various cell types, including epithelial cells (kidney proximal tubules, bile duct, prostate and intestine), endothelial cells as well as T lymphocytes [3, 4, 6], its function is dependent on cell types and the microenvironment, which influences the multiple biological roles of CD26.
Although it is difficult to develop anti-human CD26 mAbs that can clearly detect denatured CD26 in FFPE tissues, the anti-CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity. However, since treatment with targeted therapeutic agents is dependent on detection of the appropriate target antigens on clinical samples, uniformity of the diagnostic reagents is critical, suggesting that mAbs are desirable for diagnostic uses in the clinical setting. In the present study, we have improved the screening methods and succeeded in developing novel anti-human CD26 mAbs that can potentially be used as diagnostic reagents clinically.