Although anti-human CD26 mAbs which we have developed previously or commercially available mAbs cannot clearly detect denatured CD26 in formalin-fixed paraffin-embedded tissues, the anti-human CD26 pAb purchased from R&D Systems is able to stain CD26 with reliable clarity and intensity. However, it is of concern that the staining pattern and intensity may differ among different lots of the anti-CD26 pAb. Since treatment with targeted therapeutic agents depends on the ability to reliably detect the appropriate targets on clinical samples, uniformity of the diagnostic reagents is critical, suggesting that pAbs that are used as research reagents are not appropriate for diagnostic uses in the clinical setting. In the present study, we describe the successful development of novel anti-human CD26 mAbs by immunizing mice with CD26 protein denatured in urea buffer that can potentially be used as diagnostic reagents clinically.
In an attempt to improve diagnostic accuracy, markers used for immunohistochemistry have been studied, such as galectin-3, HBME-1 and CK-19 for diagnosis of benign and malignant thyroid lesions
[25, 26], and FAP-α and Calponin for diagnosing whether ductal carcinoma in situ has microinvasion
. CD26 is highly expressed on the surface of malignant mesothelioma cells especially tumors of the epitheloid and biphasic types, but not on benign mesothelial tissues
[16, 17]. It has been recently reported that the expression level of CD26 in prostate cancer tissues is higher than that of normal prostatic tissues and increased with prostate cancer stage advancement, and CD26 expression is correlated with prostate specific antigen, suggesting that CD26 may be a good marker for prostate cancer diagnosis
. Furthermore, the overall survival of patients with CD26-positive GISTs is worse than that of patients with CD26-negative GISTs, suggesting that CD26 appears to be a reliable biomarker of malignant GIST of the stomach
. These observations strongly suggest that immunohistochemical staining of CD26 in formalin-fixed tumor tissues is important for diagnosis and prognosis of various tumors.
Since several anti-human CD26 mAbs such as Ta1, 1F7, 5F8 and 14D10 that were already developed in our laboratory by immunizing mice with a CD26 positive human T cell line (EL156) or a PHA-stimulated Aotus trivirgatus T cell line or a murine pre-B human CD26 transfectant (300–19) cannot clearly detect CD26 in formalin-fixed tissues
[1, 8, 18], it was our hypothesis that utilizing human CD26 protein but not human CD26 positive cells as an immunogen would be important for the development of mAbs capable of recognizing the denatured CD26 molecule. Similar to CD26, only pAbs could react to the denatured HLA class I molecules in formalin-fixed paraffin-embedded tissues. Torigoe et al. recently succeeded in developing a novel anti-pan HLA class I mAb suitable for immunohistochemical staining of fixed tissues by immunizing a recombinant HLA-A protein denatured in urea buffer
. The exact role played by urea treatment of the CD26 protein in expanding the repertoire of the obtained anti-CD26 mAs is not yet clear, since we have not examined for potential differences in the characteristics of mAbs obtained after immunizing mice with urea-treated sCD26 protein or non-treated native sCD26 protein in this study. However, as shown in Figures
3, tissue specimens stained with anti-CD26 mAb purchased from MBL exhibited only a partially positive reaction with weak staining intensity, while this mAb showed higher absorbance to the urea treated sCD26 protein than the absorbance obtained from the novel anti-CD26 mAbs capable of staining CD26 with strong intensity in fixed tissues. These data strongly suggest that the structure of CD26 denatured by the method of antigen retrieval after formalin-fixation is quite different from that of CD26 denatured by urea buffer, and also suggest that anti-CD26 mAbs suitable for immunohistochemistry may be obtained more efficiently by immunizing mice with CD26 protein denatured by methods other than urea treatment, such as treatment with guanidine hydrochloride or sodium dodecyl sulfate (SDS), or with proteases such as trypsin or proteinase K, or by boiling. Further studies are needed to clarify the issue involving pretreatment of the immunogens and the characteristics of mAbs obtained after immunization.
In the present study, we have succeeded in developing novel anti-CD26 mAbs with a wide range of epitopes (Figures
6 and Additional file
1: Figure S3). Since most of these novel mAbs completely inhibited the binding of the anti-CD26 mAbs (4G8, 1F7, 5F8, 16D4B or 9C11) developed previously by our group, the epitopes defined by these novel mAbs are expected to be similar to those recognized by the earlier mAbs. However, these novel anti-CD26 mAbs are capable of detecting denatured CD26 in fixed tissues with strong intensity, unlike the previously developed mAbs. Similarly, while clone 18 and YS110 recognize the similar epitope on CD26 (Figure
4, Additional file
1: Figure S2 and Additional file
1: Figure S3), only clone 18 can stain CD26 clearly in fixed tissues with strong intensity, suggesting that slight differences in the recognized epitopes can determine whether mAb binding to its denatured antigen can occur.
Cross-blocking studies showed that, in contrast to clone 5 or 18, the binding of clone 1, 11, 16 or 19 to CD26 was hardly affected by the humanized anti-CD26 mAb YS110 (Figure
4, Additional file
1: Figure S2 and Additional file
1: Figure S3), suggesting that these 4 novel anti-CD26 mAbs are suitable for analyzing CD26 expression in clinical samples following YS110 therapy. Potential uses of these novel mAbs in the clinical setting may be to detect CD26 expression in formalin-fixed tissues, or on circulating cells in blood samples, or sCD26 levels in bodily fluids through such methods as immunohistochemistry, flow cytometry, or ELISA. Furthermore, these novel mAbs are potentially useful for analyzing CD26 expression in fixed tissues or on the surface of lymphocytes or tumors during or following the administration of humanized anti-CD26 mAb in animal disease models that involve inoculated human lymphocytes or tumors
[12, 16], and are expected to contribute to future CD26-related research effort.
Since we intend to utilize these novel anti-CD26 mAbs as companion diagnostic agents in the clinical setting, our current effort is focused on improving immunohistochemical staining methods by examining such issues as the condition of antigen retrieval or blocking, or the optimal concentration of the primary antibody (anti-CD26 mAb) that can maximize staining intensity while lowering background staining. Furthermore, we also identified the amino acid sequence of the variable region in both the heavy chain and light chain of clone 19 (data not shown), and will aim to refine the ability of this mAb to bind to CD26 through genetic engineering techniques.
In conclusion, 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. Furthermore, since some of these mAbs exhibit no cross-reactivity with the therapeutic humanized anti-CD26 mAb, they are potentially useful as companion diagnostic agents in the clinical setting while advancing future CD26-related research.