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Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue

Ryo Hatano1, Taketo Yamada2, Shuji Matsuoka3, Satoshi Iwata1, Hiroto Yamazaki1, Eriko Komiya1, Toshihiro Okamoto1, Nam H Dang4, Kei Ohnuma1 and Chikao Morimoto1*

Author Affiliations

1 Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

2 Department of Pathology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

3 Department of Pathology & Oncology, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

4 Division of Hematology/Oncology, University of Florida, 1600 SW Archer Road- Box 100278, Room MSB M410A, Gainesville, FL 32610, USA

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Diagnostic Pathology 2014, 9:30  doi:10.1186/1746-1596-9-30

Published: 6 February 2014

Abstract

Background

A T cell costimulatory molecule with dipeptidyl peptidase IV (DPPIV) activity in its extracellular region, CD26 is a multifunctional molecule associated with various proteins such as adenosine deaminase, caveolin-1, CXCR4, collagen, and fibronectin, while playing an important role in the regulation of inflammatory responses and tumor biology. We have focused on CD26 as a novel therapeutic target for various tumors and immune disorders, and have developed a humanized anti-CD26 monoclonal antibody (mAb), YS110, which is currently being evaluated in a phase I clinical trial for patients with CD26-expressing tumors, including malignant mesothelioma. Since detection of tumor CD26 expression is required for determining potential eligibility for YS110 therapy, the development of anti-human CD26 mAb that can clearly and reliably detect the denatured CD26 molecule in the formalin-fixed paraffin-embedded tissues is critical.

Methods

To develop novel anti-CD26 mAbs capable of binding to the denatured CD26, we immunized mice with CD26 protein denatured in urea buffer. After the fusion of splenocytes and myeloma cells, the mAbs were screened for specific reactivity with human CD26 by flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemistry. The binding competitiveness of novel anti-CD26 mAbs with the humanized anti-CD26 mAb YS110 was also examined.

Results

We have succeeded in developing novel anti-human CD26 mAbs suitable for immunohistochemical staining of CD26 in formalin-fixed tissue sections with reliable clarity and intensity. Importantly, some of these mAbs exhibit no cross-reactivity with the humanized anti-CD26 mAb.

Conclusions

These novel mAbs are potentially useful as companion diagnostic agents to analyze CD26 expression in the clinical setting while advancing future CD26-related research.

Virtual slides

The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5987140221097729 webcite

Keywords:
CD26/dipeptidyl peptidase 4; Immunohistochemical staining; Companion diagnostic drug; Malignant mesothelioma; T cell costimulation