Epitope mapping of an anti-alpha thalassemia/mental retardation syndrome X-linked monoclonal antibody AMab-6

The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X chromosome. ATRX gene mutations were first discovered in pancreatic neuroendocrine tumors, and subsequently in other cancer subtypes, including gliomas. Molecular subgrouping of gliomas has been more important than conventional histological classifications. Mutations in the isocitrate dehydrogenase (IDH), telomerase reverse transcriptase (TERT) promoter, and ATRX and the codeletion of chromosomes 1p/19q are used as biomarkers for diagnosing the subtypes of diffuse gliomas. We recently developed a sensitive monoclonal antibody (mAb) AMab-6 against ATRX by immunizing mice with recombinant human ATRX. AMab-6 can help to detect ATRX mutations via Western blotting and immunohistochemical analyses. In this study, we characterized the binding epitope of AMab-6 using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical analysis, and found that Gln2368 of ATRX is critical for AMab-6 binding to ATRX. Our findings could be applied to the production of more functional anti-ATRX mAbs.


Introduction
The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X chromosome. ATRX gene mutations were first discovered in pancreatic neuroendocrine tumors [1], and subsequently in other cancer subtypes, including gliomas. Gliomas are the most frequently occurring brain tumors and have a heterogeneous molecular background [2]. Molecular subgrouping of gliomas using mutations in isocitrate dehydrogenase (IDH) 1/2, TERT promoter, and ATRX and codeletion of 1p/19q as biomarkers stratifies patients into distinct groups that are more prognostically assessed compared with conventional histological classifications [1,[3][4][5]. These molecular subtypes are clinically important because treatment strategies can be planned in accordance with molecular subtype along with the World Health Organization (WHO) tumor grading system. The 2016 WHO Classification of Tumors of the Central Nervous System (2016 WHO CNS) is both conceptually and practically more advanced than the 2007 WHO CNS [6,7]. The 2016 WHO CNS uses molecular parameters, in addition to histological assessments to define many tumor entities, presents major restructuring of diffuse gliomas, medulloblastomas, and other embryonal tumors; and incorporates new entities that are defined using both histology results and molecular features.
The loss of ATRX mRNA and protein in gliomas is caused by an ATRX mutation. Loss of the ATRX protein can be diagnosed by immunohistochemistry using anti-ATRX antibodies [8,9]; however, nearly all studies on ATRX protein have used polyclonal antibodies [10] because highly sensitive monoclonal antibodies (mAbs) against human ATRX protein had not been established. Recently, we established a novel anti-ATRX mAb, AMab-6, which is very useful in enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analyses [11]. In this study, we characterized the binding epitope of AMab-6 using ELISA, Western blot analysis, and immunohistochemical analyses.

Immunohistochemical analyses
This study examined one patient with oral cancer who underwent surgery at Tokyo Medical and Dental University. The Tokyo Medical and Dental University Institutional Review Board reviewed and approved the use of the human cancer tissues, and written informed consent was obtained from the patient. Histological sections (4-μm thick) were directly autoclaved for 20 min in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan). After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), the sections were incubated with 5 μg/mL AMab-6 or 5 μg/mL AMab-6 plus 5 μg/mL peptides for 1 h at room temperature and treated using an EnVision+ Kit (Agilent Technologies Inc.) for 30 min. Color was Fig. 1. Production of ATRX deletion mutants. Three ATRX C-terminal deletion mutants and three ATRX N-terminal deletion mutants were produced. Four ATRX peptides were also synthesized. Black bars, the deletion mutants or synthesized peptides, which were detected by AMab-6; white bar, the deletion mutants or synthesized peptides, which were not detected by AMab-6. developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
ATRX mutation has been reported in gliomas in which the ATRX protein is not detected by anti-ATRX antibodies via immunohistochemistry [8,9,11,22]. In contrast, the ATRX protein is usually detected in nearly all cancers, including oral cancers, via immunohistochemistry using anti-ATRX antibodies because ATRX mutations have not been reported in those cancers [22]. In this study, we first performed immunohistochemistry against oral cancers using AMab-6. The nuclei of oral cancer cells were strongly stained by AMab-6 ( Fig. 3). We next performed a blocking assay using immunohistochemistry against oral cancers. We found that the reaction of AMab-6 was neutralized by K2359A peptide (Fig. 3). In contrast, the Q2368A peptide did not block the reaction of AMab-6, thereby confirming the results of epitope mapping using ELISA and Western blotting.
In conclusion, Gln2368 of ATRX is critical for AMab-6 binding. Our findings can be applied for the production of more functional anti-ATRX mAbs.

Conflict of interest
The authors declare no conflicts of interest involving this article.   3. Immunohistochemistry using oral cancer tissue. Oral cancer tissues were autoclaved for 20 min in a citrate buffer. Sections were then incubated with 5 μg/ mL AMab-6 or 5 μg/mL AMab-6 plus 5 μg/mL peptides and stained using an EnVision+ kit. Color development was performed using 3,3′-diaminobenzidine tetrahydrochloride. Sections were then counterstained with hematoxylin. Notes: scale bar = 100 µm; HE, hematoxylin and eosin.