MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 4, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0019

Epitope Mapping of Anti-Telomerase Reverse
Transcriptase Monoclonal Antibodies

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Yoshikazu Furusawa,1,2 Shunsuke Itai,1 Shinji Yamada,1 Mika K. Kaneko,1 and Yukinari Kato1,2Telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase-associated protein machinery. Upregulation of TERT at the transcriptional level results in immortal cell phenotype associated withcancer. To date we have developed eight anti-TERT monoclonal antibodies (mAbs) (TMab-4, TMab-5, TMab6, TMab-7, TMab-9, TMab-10, TMab-11, and TMab-12) by immunizing mice with synthetic peptides(302-QHHAGPPSTSRPPRPWDTPC-321) of TERT. We further characterized those epitopes using enzymelinked immunosorbent assay, and here we discuss the critical epitope of an anti-TERT mAb, which is applicablefor immunohistochemical analysis.
Keywords: TERT, monoclonal antibody, epitope mappingof anti-TERT mAbs, which is applicable for immunohistochemical analysis.

Introduction

Telomerase reverse transcriptase (TERT) is themain catalytic subunit of telomerase-associated proteinmachinery. Mutation hotspots in the TERT promoter havebeen identified in human melanomas(1,2) and gliomas.(3,4)
Diffuse gliomas in adults are separated into three comprehensive tumor groups with distinctive prognoses based on themutations of isocitrate dehydrogenase (IDH) 1 and 2 and 1p/
19q-codeletion.(3) TERT promoter mutations were frequently
and selectively observed in IDH wild type glioblastomas
and 1p/19q-codeleted oligodendrogliomas.(4) TERT promoter mutations are associated with three distinctive glioma groups, which indicates the involvement of TERT in
the pathogenesis of diffuse gliomas.(3) Mutations in theTERT promoter have also been reported to occur frequently in patients with oral squamous cell carcinoma(OSCC)(5); therefore, somatic TERT promoter mutations
could play a vital role in the pathogenesis and progressionof OSCC. Therefore, a sensitive and specific anti-human
TERT monoclonal antibody (mAb) is very important forthe detection of TERT in those tumors.
To date, we have developed eight anti-TERT mAbs
(TMab-4, TMab-5, TMab-6, TMab-7, TMab-9, TMab-10,TMab-11, and TMab-12) by immunizing mice with synthetic peptides of TERT. We recently determined the epitope of TMab-6, which is useful for immunohistochemistryin human tissues,(6) using enzyme-linked immunosorbent
assay (ELISA) and immunohistochemistry.(7) In this study,
we further characterized epitopes of the other anti-TERTmAbs using ELISA, and here we discuss the critical epitope
12Materials and Methods
Cell lines

P3U1 was obtained from American Type Culture Collection (ATCC, Manassas, VA).(8) P3U1 cells were grown inRoswell Park Memorial Institute medium (Nacalai Tesque,Inc., Kyoto, Japan), supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc., Waltham,MA), 100 U/mL penicillin, 100 mg/mL streptomycin, 25 mg/mL
amphotericin B (Nacalai Tesque, Inc.).
The hybridoma cells were grown in Roswell Park MemorialInstitute medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific,Inc.), 10% heat-inactivated fetal bovine serum (Thermo FisherScientific, Inc.), 100 U/mL penicillin, 100 mg/mL streptomycin,
25 mg/mL amphotericin B (Nacalai Tesque, Inc.), and 5%
BriClone Hybridoma Cloning Medium (QED Bioscience, Inc.,San Diego, CA). Plasmocin (5 mg/mL; InvivoGen, San Diego,CA) was also used to prevent Mycoplasma contamination.
Cells were incubated at 37C in a humidified atmosphere of 5%
CO2 and 95% air.
Plasmids of TERTHuman TERT cDNA (Accession No. NM_001193376.1)
encoding a specific peptide composed of Glu281-Ala436
amino acid residues was obtained by PCR using a cDNA
derived from the HT1080 cell line (ATCC) as a template. The

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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FURUSAWA ET AL.

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Table 1. Epitope Mapping of Anti-Telomerase Reverse Transcriptase Monoclonal
Antibodies by Enzyme-Linked Immunosorbent Assay
MutationSequence

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

AHHAGPPSTSRPPRPWDTP
QAHAGPPSTSRPPRPWDTPQHAAGPPSTSRPPRPWDTP
QHHGGPPSTSRPPRPWDTPQHHAAPPSTSRPPRPWDTP
QHHAGAPSTSRPPRPWDTP
QHHAGPASTSRPPRPWDTP
QHHAGPPATSRPPRPWDTP
QHHAGPPSASRPPRPWDTP
QHHAGPPSTARPPRPWDTP
QHHAGPPSTSAPPRPWDTP
QHHAGPPSTSRAPRPWDTP
QHHAGPPSTSRPARPWDTP
QHHAGPPSTSRPPAPWDTP
QHHAGPPSTSRPPRAWDTPQHHAGPPSTSRPPRPADTP
QHHAGPPSTSRPPRPWATP
QHHAGPPSTSRPPRPWDAPQHHAGPPSTSRPPRPWDTA

TMab-4 TMab-5 TMab-6 TMab-7 TMab-9 TMab-10 TMab-11 TMab-12
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+

3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
1+

3+
3+
3+
3+
3+
3+
2+
2+
3+
2+
3+
3+
3+
1+
2+
3+
3+

3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+
3+

2+
1+
3+
2+
2+
3+
3+
3+
3+
3+2+3+2+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+3+
3+3+3+
3+3+3+3+3+3+
3+3+3+
3+
3+3+3+1+
3+3+
3+

3+, OD655S0.7; 2+, 0.4&OD655 < 0.7; 1+, 0.1&OD655 < 0.4; -, OD655 < 0.1.
TMab, anti-telomerase reverse transcriptase monoclonal antibodies.

primer set for TERT was as follows: 5¢-AAGGATTTCAGAATTCGAAGCCACCTCTTTGGA-3¢ (forward) and 5¢-TGCCGTCTCCGAATTCCGCCACAGAGCCCTGGG-3¢ (reverse). The PCR product was subcloned into an expressionvector, pMAL-c2 (New England Biolabs, Beverly, MA) withMAP tag (GDGMVPPGIEDK)(9) and PA tag (GVAMPGAEDDVV)(10) using In-Fusion HD Cloning Kit (Takara Bio,Inc., Shiga, Japan).

mice were fused with P3U1 cells using PEG1500 (RocheDiagnostics, Indianapolis, IN). The Animal Care and Use
Committee of Tohoku University approved the animal experiments about hybridoma production in this study.
Enzyme-linked immunosorbent assay

Synthesized TERT peptides (PEPScreen; Sigma-AldrichCorp.) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at 10 mg/mL for

Production of recombinant TERT

Competent Escherichia coli TOP-10 cells (Thermo FisherScientific, Inc.) were transformed with the plasmid pMALc2MAPhPAter/TERTepi and cultured overnight at 37C in
LB medium (Thermo Fisher Scientific, Inc.) containing
100 mg/mL ampicillin (Sigma-Aldrich Corp., St. Louis, MO).
Cell pellets were resuspended in phosphate-buffered saline(PBS; Nacalai Tesque, Inc.) with 1% Triton X-100 with50 mg/mL aprotinin (Sigma-Aldrich Corp.). After sonication,
the crude extracts were collected by centrifugation (9000 g,30 min, 4C). The supernatants were loaded onto amylose
resin. The loaded resins were washed extensively with column buffer consisting of 20 mM Tris hydrochloride (pH 7.4),
200 mM sodium chloride and 1 mM ethylenediaminetetraacetic acid, and the fusion proteins were eluted by column
buffer with 10 mM maltose.
Establishment of TERT-specific antibodies

BALB/c mice were immunized by intraperitoneal (i.p.)
injection of the synthetic peptide of TERT (302-QHHAGPPSTSRPPRPWDTPC-321; Sigma-Aldrich Corp.) conjugated with keyhole limpet hemocyanin together with Imject
Alum (Thermo Fisher Scientific, Inc.). After several additional
immunizations, a booster injection was given i.p. 2 days before
splenocytes were harvested. The splenocytes of immunized

FIG. 1. Epitope mapping of anti-TERT mAbs. Red characters, the most important amino acids detected by antiTERT mAbs; blue characters, the second most importantamino acids detected by anti-TERT mAbs. aa, aminoacids; mAbs, monoclonal antibodies; TERT, telomerase
reverse transcriptase.

EPITOPE OF ANTI-TERT MABS

30 min. After blocking with SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with purified anti-TERT mAbs (10 mg/mL) or culture
supernatant, followed by peroxidase-conjugated anti-mouse
immunoglobulin G (IgG) (Agilent Technologies, Inc., Santa
Clara, CA) diluted 1:2000. The enzymatic reaction wasconducted using 1-Step Ultra TMB-ELISA (Thermo FisherScientific, 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.

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Results and Discussion

In this study, BALB/c mice were immunized by the i.p. injection of human TERT synthetic peptide (302-QHHAGPPSTSRPPRPWDTPC-321). ELISA was utilized for screening
culture supernatants for binding to synthetic peptides and recombinant proteins of TERT. We herein developed sevenclones: TMab-4 (IgM, kappa), TMab-5 (IgM, kappa), TMab-7(IgG2b, kappa), TMab-9 (IgG2a, kappa), TMab-10 (IgG2a,kappa), TMab-11 (IgG2a, kappa), and TMab-12 (IgG1, kappa).
We next investigated whether the seven clones are applicable for Western blot and immunohistochemical analyses.
Unfortunately, none of the seven clones showed a specificband of TERT using glioblastoma cell lines, although they
showed a specific band using recombinant protein purified
from E. coli in Western blot analysis (data not shown). Inimmunohistochemical analysis, specific staining was notobserved in any of the seven clones (data not shown). In
contrast, we recently developed clone TMab-6 (IgM, kappa),
which is useful for immunohistochemistry in human tissues.(6) Because we also determined the epitope of TMab-6 in
a subsequent study,(7) we herein compare the epitope ofTMab-6 with those of the other anti-TERT mAbs.
In this study, we utilized a series of TERT peptides from
302 to 320 amino acids in length using point mutations(Table 1). ELISA using an immunohistochemistry-applicableanti-TERT mAb (TMab-6) did not react with T310A andS311A and weakly reacted with P313A and W317A, whichindicates that 310-TSRPPRPW-317 is a critical epitope ofTMab-6 (Fig. 1) and that Thr310 and Ser311 of TERT areespecially significant amino acids for TMab-6-recognition.
In contrast, the epitopes of TMab-4, TMab-5, and TMab-12
were determined to be 315-RPWD-318, 316-PWDTP-320, andTrp317 in C-terminus of immunized peptides, respectively.
The epitope of TMab-7, TMab-9, TMab-10, and TMab-11
was determined to be 303-HHAGP-307. These results indicatethat Thr310 and Ser311 of TERT are very important epitopes
for immunohistochemical analysis.
The information regarding the epitopes of anti-TERTmAbs in the present study could be valuable for the development of sensitive and specific mAbs against TERT inimmunohistochemical analysis.
Acknowledgments

The authors thank Takuro Nakamura, Miyuki Yanaka,
Saori Handa, and Yoshimi Nakamura for excellent technicalassistance. This research was supported in part by AMED
under Grant Nos.: JP18am0101078 (Y.K.), JP18am0301010(Y.K.), and JP18ae0101028 (Y.K.), and by JSPS KAKENHI
Grant Nos.: 17K07299 (M.K.K.) and 16K10748 (Y.K.).

187
Author Disclosure Statement

No competing financial interests exist.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: May 2, 2018
Accepted: June 2, 2018