MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 3, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0007

Original Articles

Elucidation of the TMab-6 Monoclonal Antibody
Epitope Against Telomerase Reverse Transcriptase

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Mika K. Kaneko,1 Shinji Yamada,1 Shunsuke Itai,1,2 Yao-Wen Chang,1 Takuro Nakamura,1Miyuki Yanaka,1 Hiroyuki Harada,2 Hiroyoshi Suzuki,3 Yukinari Kato1,4Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, aswell as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal
antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, thebinding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent
assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed
that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT areespecially significant amino acids for TMab-6 recognition.
Keywords: TERT, monoclonal antibody, epitope mapping

Introduction

TMaterials and Methods

elomerase reverse transcriptase (TERT) is an important catalytic subunit of the telomerase holoenzyme
complex. In adults, diffuse gliomas are separated into threecomprehensive tumor groups with distinctive prognoses basedon the mutations of isocitrate dehydrogenase (IDH) 1 and 2,and 1p/19q-codeletion.(1) Mutation hotspots in the TERT promoter have recently been identified in human melanomas,(2,3)
and TERT promoter mutations were frequently and selectively observed in IDH wild-type glioblastomas and 1p/19qcodeleted oligodendrogliomas.(4) TERT promoter mutations
are associated with three distinctive glioma groups, whichindicates the involvement of TERT in the pathogenesis ofdiffuse gliomas.(1)Chang et al. also reported that mutations in the TERT promoter frequently occur in patients with oral cavity squamouscell carcinoma (SCC)(5); somatic TERT promoter mutations
could, therefore, play a vital role in the pathogenesis andprogression of oral cavity SCC.
Recently, we developed an antihuman TERT monoclonal
antibody (mAb), TMab-6 (IgM, kappa), which is useful for
immunohistochemistry in human tissues.(6) This study utilized
enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry and point mutants of TERT for investigating
the epitope of TMab-6.

Enzyme-linked immunosorbent assay

Synthesized TERT peptides (PEPScreen; Sigma-AldrichCorp., St. Louis, MO) were immobilized on Nunc Maxisorp
96-well immunoplates (Thermo Fisher Scientific, Inc., Waltham, MA) at 5 mg/mL for 30 minutes. After blocking withSuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with purified TMab-6(10 mg/mL), followed by peroxidase-conjugated antimouse
IgG (Agilent Technologies, Inc., Santa Clara, CA) diluted
1:2000. The enzymatic reaction was conducted using 1-Step
Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These
reactions were performed in a volume of 50–100 mL at 37C.
Immunohistochemical analyses

This study was approved by the ethical committee of Sendai
Medical Center. One patient with glioblastoma who underwent
surgery at the Sendai Medical Center was enrolled for analysis. Histological tumor sections (4-mm thick) were directly
autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc.,Tokyo, Japan) for 20 minutes, incubated with 5 mg/mL of
TMab-6 or 5 mg/mL of TMab-6 + 5 mg/mL of peptides for1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,Tokyo, Japan.
3
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2101

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KANEKO ET AL.

1 hour at room temperature, and treated using an Envision+ kit(Agilent Technologies, Inc.) for 30 minutes. Color development
was performed using 3,3-diaminobenzidine tetrahydrochloride(Agilent Technologies, Inc.) for 2 minutes. Sections were thencounterstained with hematoxylin (FUJIFILM Wako Pure
Chemical Industries Ltd., Osaka, Japan).

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Results and Discussion

In our previous study, BALB/c mice were immunizedagainst TERT by the intraperitoneal injection of human
TERT synthetic peptide (hTERT; 302–321 amino acids:
QHHAGPPSTSRPPRPWDTPC).(6) ELISA was utilized forscreening culture supernatants for binding to synthetic peptides
and recombinant proteins of hTERT purified from Escherichia
coli. We developed cloned TMab-6 (IgM, kappa), which isuseful for immunohistochemistry, using paraffin-embeddedtissues. Until now, the binding epitope remains to be elucidated.
In this study, we synthesized a series of hTERT peptides
from 302 to 320 amino acids in length using point mutations(Table 1). ELISA using TMab-6 detected Q302A, H303A,
H304A, A305G, G306A, P307A, R312A, P314A, R315A,P316A, T319A, and P320A. On the contrary, TMab-6 did notreact with T310A and S311A, moderately reacted with P308A,
S309A, P313A, and D318A, and weakly reacted with W317A,
which indicates that the TERT sequence PSTSRPPRPWD is a
critical epitope of TMab-6.
Next, we performed a peptide-blocking assay using immunohistochemistry against glioblastoma. TMab-6 reactedwith glioblastoma tissues (Fig. 1); however, this reaction wascompletely neutralized by Q302A. In contrast, S311A did not
block the TMab-6 reaction with glioblastoma tissues. Thesedata confirmed the ELISA-generated map of the TMab-6
epitope, which is summarized in Figure 2.
The information regarding the epitope of TMab-6 in thisstudy could be valuable for the development of sensitive andspecific mAbs against hTERT in immunohistochemistry.

Table 1. Determination of TMab-6 Epitopeby Enzyme-Linked Immunosorbent Assay
Mutation

Sequence

TMab-6

Q302A
H303A
H304A
A305G
G306A
P307A
P308A
S309A
T310A
S311A
R312A
P313A
P314A
R315A
P316A
W317A
D318A
T319A
P320A

AHHAGPPSTSRPPRPWDTP
QAHAGPPSTSRPPRPWDTPQHAAGPPSTSRPPRPWDTP
QHHGGPPSTSRPPRPWDTPQHHAAPPSTSRPPRPWDTP
QHHAGAPSTSRPPRPWDTPQHHAGPASTSRPPRPWDTP
QHHAGPPATSRPPRPWDTP
QHHAGPPSASRPPRPWDTP
QHHAGPPSTARPPRPWDTP
QHHAGPPSTSAPPRPWDTP
QHHAGPPSTSRAPRPWDTP
QHHAGPPSTSRPARPWDTP
QHHAGPPSTSRPPAPWDTP
QHHAGPPSTSRPPRAWDTPQHHAGPPSTSRPPRPADTP
QHHAGPPSTSRPPRPWATP
QHHAGPPSTSRPPRPWDAPQHHAGPPSTSRPPRPWDTA++++++
+++
+++
+++
+++++
++
+++++
+++
+++
++++++++++++

Bold ‘‘A’’ or ‘‘G’’ indicates the substitution from original amino acid.
+++, OD655S0.7; ++, 0.4 &OD655 < 0.7;+, 0.1&OD655 < 0.4; -, OD655 < 0.1.

FIG. 1. Immunohistochemistry using TMab-6 and point
mutants of TERT. Glioblastoma tissues were autoclaved incitrate buffer for 20 minutes. Sections were then incubated with
TMab-6, TMab-6 + Q302A, or TMab-6 + S311A, and treatedusing an Envision+ kit. Color development was performed
using 3,3-diaminobenzidine tetrahydrochloride. Sections werethen counterstained with hematoxylin. Scale bars = 100 mm.
HE, hematoxylin and eosin staining; TERT, telomerase reverse
transcriptase.

FIG. 2. Schematic illustration of TERT and TMab-6epitope. The critical epitope of TMab-6 is PSTSRPPRPWD.
Thr310 and Ser311 of TERT are especially significant
amino acids for TMab-6 recognition.

EPITOPE OF TMAB-6
Acknowledgments

We thank Noriko Saidoh, Saori Handa, and Yoshimi
Nakamura for their excellent technical assistance. This research was supported, in part, by AMED under Grant Numbers: JP17am0301010 (Y.K.), JP17am0101078 (Y.K.), andJP17ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number 17K07299 (M.K.K.) and Grant Number 16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp;
yukinari-k@bea.hi-ho.ne.jp
Received: February 2, 2018
Accepted: March 25, 2018