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

Our plain-English summary of Establishment of monoclonal anti-human CD26 antibodies suitable for immunostaining of formalin-fixed tissue, by R. Hatano, T. Yamada, S. Matsuoka, et al (Diagnostic Pathology, 2014).

The study

• Authors: R. Hatano, T. Yamada, S. Matsuoka, S. Iwata, H. Yamazaki, E. Komiya, T. Okamoto, N.H. Dang, K. Ohnuma, C. Morimoto
• Journal: Diagnostic Pathology
• Published: 2014;9:30
• Cited by: 8 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

In plain English

This laboratory study from Japan set out to make mouse antibodies that can detect the protein CD26 in formalin-fixed tissue samples, the kind pathology labs routinely keep. The authors explained that their humanized anti-CD26 antibody, YS110, was being tested in a phase I trial in patients with CD26-expressing tumors, including malignant mesothelioma, and that a reliable tissue test for CD26 is needed to judge who might be eligible. The study did not examine asbestos exposure. The authors immunized a mouse with CD26 protein denatured in urea buffer and screened the resulting antibodies. Of 31 clones evaluated, 6 stained CD26 in fixed tissue much more strongly than a commercial antibody. Two of them, clones 18 and 19, stained normal liver, kidney and prostate, two malignant mesothelioma cases and five other tumor types about as clearly as a commercial polyclonal antibody. YS110 hardly affected the binding of clones 1, 11, 16 and 19, but it completely blocked clone 18. The authors concluded that these antibodies are potentially useful as companion diagnostic agents in the clinical setting.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory study developing and testing mouse monoclonal antibodies (flow cytometry, ELISA, immunohistochemistry, cross-blocking and epitope mapping)
• Who was studied: Antibodies from an immunized female BALB/c mouse, tested on CD26-transfected Jurkat T cells and on formalin-fixed human tissues: normal liver, kidney and prostate, two cases of malignant mesothelioma, and autopsy specimens of hepatocellular carcinoma, renal cell carcinoma, prostate adenocarcinoma, colon adenocarcinoma and lung adenocarcinoma (Japan)
• Exposure: No asbestos or occupational exposure was studied, and the article does not mention asbestos. The experimental step was immunizing a mouse with soluble CD26 protein denatured in 8 M urea buffer.
• Main results:
  • Of 31 antibody-secreting clones evaluated, 6 (clones 1, 5, 11, 16, 18 and 19) stained CD26 in formalin-fixed tissues much more strongly than the commercial MBL monoclonal antibody.
  • The commercial MBL monoclonal antibody gave only weak staining of fixed tissues.
  • Clones 18 and 19 stained liver, kidney, prostate and malignant mesothelioma tissue with a pattern and intensity comparable to the R&D Systems polyclonal antibody.
  • When the antibodies were first mixed with soluble CD26, their binding to mesothelioma tissue was completely blocked, which supports specific binding to CD26. The authors judged 100 μg/ml an optimal concentration.
  • In hepatocellular, renal cell, prostate, colon and lung tumor tissues, clones 18 and 19 stained with clarity and intensity similar to the R&D Systems polyclonal antibody.
  • Pretreatment with the therapeutic antibody YS110 hardly affected binding of clones 1, 11, 16 and 19, partially inhibited clone 5, and completely inhibited clone 18.
  • Epitope mapping suggested that clone 19 binds within amino acids 1 to 247 of CD26, and clone 18 within amino acids 248 to 358, almost the same site as YS110.
• The authors' conclusion: The authors concluded that the new antibodies are potentially useful as companion diagnostic agents for analyzing CD26 expression in the clinical setting, and for future CD26 research.
• Limitations the authors state:
  • The authors did not compare antibodies raised against urea-treated protein with antibodies raised against untreated protein, so the role of the urea treatment is unclear.

Topics

Medical terms in this study, explained in our Lexicon: Monoclonal Antibody, Collagen, Clinical Trial, Mesothelioma, Myeloma, Immunohistochemistry.

Related studies

• Establishment of anti-mesothelioma monoclonal antibodies — N. Mizutani, M. Abe, et al. (2016)
• Differential expression of N-cadherin in pleural mesotheliomas and E-cadherin in lung adenocarcinomas in formalin-fixed, paraffin-embedded tissues — A.C. Han, A. Peralta-Soler, et al. (1997)
• Nuclear localization of CD26 induced by a humanized monoclonal antibody inhibits tumor cell growth by modulating of POLR2A transcription — K. Yamada, M. Hayashi, et al. (2013)
• CD9 negatively regulates CD26 expression and inhibits CD26-mediated enhancement of invasive potential of malignant mesothelioma cells — T. Okamoto, S. Iwata, et al. (2014)
• First-in-human phase 1 of YS110, a monoclonal antibody directed against CD26 in advanced CD26-expressing cancers — E. Angevin, N. Isambert, et al. (2017)
• Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization — H. Horn, J. Bausinger, et al. (2014)

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