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

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.

Development of a Novel CD26-Targeted Chimeric Antigen Receptor T-Cell Therapy for CD26-Expressing T-Cell Malignancies

“…This ubiquitous expression of CD26 in T-cell malignancies may result in high level of efficacy for CD26 CAR-T-cell therapy in the clinical setting. Of note, our immunostaining assays used the anti-human CD26 mouse monoclonal antibody U16-3, which was developed by our group with high sensitivity and specificity in formalin-fixed, paraffin-embedded samples [ 25 ].…”

“…Expression of CD26 on lymphoma cells in biopsy specimens was assessed by immunohistochemistry using the standard protocol. In brief, following deparaffinization and rehydration, biopsy specimens were incubated with anti-human CD26 mouse monoclonal antibody U16-3 as we previously described [ 25 ]. The CD26/anti-CD26 antibody immune complex on the tissue section was detected with the use of the second antibody conjugated with biotin, which was visualized with the 3,3′-diaminobenzidine peroxidase substrate kit (Vector Labs, Burlington, ON).…”

Antibody Identification for Antigen Detection in Formalin-Fixed Paraffin-Embedded Tissue Using Phage Display and Naïve Libraries

“…This means that a large collection of antibodies must be screened, and success is not always guaranteed. For instance, Morimoto’s group tested 13 home-made antibodies and 26 commercial monoclonal antibodies against CD26 without identifying a reliable monoclonal antibody for IHC [ 16 , 17 ].…”