ONCOLOGY LETTERS 20: 1961-1967, 2020

Establishment of C20Mab‑11, a novel anti‑CD20
monoclonal antibody, for the detection of B cells
YOSHIKAZU FURUSAWA1,2, MIKA KATO KANEKO1 and YUKINARI KATO1,2
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine;
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980‑8575, Japan

2

Received November 7, 2019; Accepted March 2, 2020
DOI: 10.3892/ol.2020.11753
Abstract. CD20 is one of several B‑lymphocyte antigens that has
been shown to be an effective target for the detection and treatment of B‑cell lymphomas. Sensitive and specific monoclonal
antibodies (mAbs) are required for every application used forthe diagnosis of B‑cell lymphoma. Although many anti‑CD20
mAbs have been established, the types of applications, thoseanti‑CD20 can be used in, are limited. In this study, we aimed
to establish novel anti‑CD20 mAbs to be used for broad
applications, such as flow cytometry, western blot, and immunohistochemical analyses, using the Cell‑Based Immunizationand Screening (CBIS) method. One of the established mAbs,
C20Mab‑11 (IgM, kappa), detected overexpression of CD20
in CHO‑K1 or LN229 cell lines, indicating that C20Mab‑11 is
specific for CD20. In western blot analyses, C20Mab‑11 detected
not only overexpression of CD20 in CHO‑K1 or LN229 celllines, but also CD20 of BALL‑1 and Raji cells with bothsensitivity and specificity. Furthermore, C20Mab‑11 strongly
stained B cells of the lymph follicle and B cell lymphomas inimmunohistochemical analyses. These results indicate that
C20Mab‑11 develped by CBIS method, is useful for the detection of CD20 in lymphoma tissues by flow cytometry, western
blot, and immunohistochemical analyses and potentially could
be beneficial for the treatment of B cell lymphomas.
Introduction
CD20 is an integral membrane protein with a molecularweight of 33‑37 kDa, which is expressed in high densities only

Correspondence to: Professor Yukinari Kato, New Industry
Creation Hatchery Center, Tohoku University, 2‑1 Seiryo‑machi,
Aoba‑ku, Sendai, Miyagi 980‑8575, JapanE‑mail: yukinarikato@med.tohoku.ac.jp
Abbreviations: mAb, monoclonal antibody; CBIS, Cell‑Based
Immunization and Screening; BSA, bovine serum albumin; DAB,
3,3'‑diaminobenzidine tetrahydrochloride
Key words: CD20, monoclonal antibody, western blotting, flow

cytometry, immunohistochemistryon B lymphocytes (1,2). CD20 has four membrane‑spanningdomains and consists of 297 amino acids (aa). The two
extracellular domains are located at 72‑80 and 142‑182 aa.
Homo‑oligomerization of CD20 into tetramers functions as
a calcium channel. CD20 is found expressed on B cells frompre‑B to mature B cell development and it is also detected on
many kinds of non‑Hodgkin lymphomas (NHL) (3). CD20
is detected on 50% of B‑lymphoblastic leukemia/lymphoma(B‑ALL/LBL) originating from pre‑B cells, but it is not detected
in terminally differentiated plasma cell malignancies (4,5).
The development of sensitive and specific monoclonal
antibodies (mAbs) are critical for diagnosis and chemotherapeutic treatment for many types of cancer (6). However, theproduction of sensitive and specific mAbs is known to be avery difficult procedure because it requires detecting theextracellular loop of multi‑pass transmembrane proteins (7).
As mentioned above, CD20 possesses two extracellulartransmembrane loops that are very small in size. Therefore,
compared to the production of mAbs against single‑pass transmembrane proteins, such as CD44 (8) or PD‑L1 (9), it is much
more difficult to develop sensitive and specific anti‑CD20
mAbs for use in multiple applications.
In our previous studies, we developed the Cell‑Based
Immunization and Screening (CBIS) method, in whichcell lines are exclusively used for both immunization andscreening. CBIS has previously been used for developing
several mAbs against various proteins, including the fivetransmembrane protein CD133 (7). Using the CBIS method,
we have successfully produced sensitive and specific mAbs
useful for not only flow cytometry, but also western blot andimmunohistochemical analyses (7‑17).
We have two objectives for developing anti‑CD20 mAbs
in this study. First, we employed CBIS to establish novelanti‑CD20 mAbs with a focus on improving this method fordeveloping advantageous mAbs against multiple‑pass transmembrane proteins. Second, we aimed to develop multi‑use
anti‑CD20 mAbs that can be used for flow cytometric, western
blot, and immunohistochemical analyses.
Materials and methods
Cell lines. P3X63Ag8U.1 (P3U1), Chinese hamster ovary(CHO)‑K1, Lec1, Lec2, Lec8, and LN229 cells were obtained
from the American Type Culture Collection. Raji and BALL‑1

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FURUSAWA et al: ESTABLISHMENT OF AN ANTI-CD20 mAb

were obtained from the Cell Resource Center for Biomedical
Research (Institute of Development, Aging and Cancer, Tohoku
University, Miyagi, Japan). DNA encoding the CD20 gene
(IRAL012D02) was provided by the RIKEN BRC throughthe National BioResource Project of MEXT, Japan. The
open reading frame of CD20 plus an N‑terminal PA tag was
subcloned into a pCAG‑Neo or pCAG‑Ble vector (FUJIFILM
Wako Pure Chemical Corporation). CHO/CD20 andCHO/mock were produced by transfecting pCAG‑Neo/CD20
and pCAG‑Neo into CHO‑K1 cells, respectively, using a Gene
Pulser Xcell electroporation system (Bio‑Rad Laboratories,Inc.). LN229/CD20 and LN229/mock were produced by transfecting pCAG‑Ble/CD20 and pCAG‑Ble into LN229 cells,
respectively, using a Neon transfection system (Thermo Fisher
Scientific, Inc.). Lec1/CD20 (N‑glycan‑deficient), Lec2/CD20(sialic acid‑deficient), and Lec8/CD20 (galactose‑deficient)were produced by transfecting pCAG‑Ble/CD20 into Lec1,
Lec2, and Lec8 cells, respectively, using a Neon transfection system. Lec1/mock, Lec2/mock, and Lec8/mock were
produced by transfecting pCAG‑Ble into Lec1, Lec2, andLec8 cells, respectively, using a Neon transfection system.
The cell line BALL‑1/CD20‑KO (BINDS‑24) was generated
by transfecting CRISPR/Cas9 plasmids for CD20 (Thermo
Fisher Scientific, Inc.) using a Neon transfection system. Stabletransfectants were established using SH800 (Sony Corp.).
P3U1, CHO ‑K1, CHO/CD20, CHO/mock, Lec1,
Lec1/CD20, Lec1/mock, Lec2, Lec2/CD20, Lec2/mock,Lec8, Lec8/CD20, Lec8/mock, Raji, BALL‑1, and BINDS‑24
were cultured in Roswell Park Memorial Institute (RPMI)
1640 medium (Nacalai Tesque, Inc.). LN229, LN229/CD20,
and LN229/mock were cultured using Dulbecco's modified Eagle's medium (DMEM; Nacalai Tesque, Inc.). The
media were supplemented with 10% heat‑inactivated fetal
bovine serum (Thermo Fisher Scientific Inc.), 100 units/mlof penicillin, 100 µg/ml of streptomycin, and 25 µg/ml ofamphotericin B (Nacalai Tesque, Inc.). L-proline (0.04 mg/ml;
MP Biomedicals, LLC) was added to Lec1, Lec2, and Lec8.
The cells were grown in an incubator at 37˚C with humidity
and 5% CO2 and 95% air atmosphere.
Reverse transcription‑PCR (RT‑PCR). Total RNAs wereprepared from cell lines using an RNeasy mini prep kit(Qiagen Inc.). The initial cDNA strand was synthesizedusing SuperScript IV Reverse Transcriptase (Thermo Fisher
Scientific, Inc.) by priming nine random oligomers and an
oligo(dT) primer according to the manufacturer's instructions. We performed 35 cycles of PCR for amplification using
HotStarTaq DNA Polymerase (Qiagen Inc.) with 0.2 mM ofprimer sets: CD20 sense (5'‑ATG​ACA​ACA​CCC​AGA​A AT​
TC‑3'), CD20 antisense (5'‑TTA ​ AGG​ AGA​G CT ​ GTC​ ATT​TTC‑3'), GAPDH sense (5'‑CAA​T GA​CCC​ C TT ​ CAT ​ T GA​
CC‑3'), and GAPDH antisense (5'‑GTC​T TC​TGG ​GTG ​G CA​
GTG​AT‑3').
Animals. All animal experiments were performed in accordancewith relevant guidelines and regulations to minimize animal
suffering and distress in the laboratory. Animal experiments
described in the hybridoma production were approved by the
Animal Care and Use Committee of Tohoku University (Permit
no: 2016MdA‑153). Mice were monitored for health every day.

The duration of the experiment was four weeks. A body weightloss exceeding 25% of total body weight were defined as a humane
endpoint. Mice were euthanized by cervical dislocation, and thedeath was verified by respiratory arrest and cardiac arrest.
Hybridoma production. Two female BALB/c mice (6 weeksold) were purchased from CLEA Japan. Animals were housed
under specific pathogen‑free conditions. Animal experiments
described in the hybridoma production were approved by
the Animal Care and Use Committee of Tohoku University(Permit no: 2016MdA‑153). Briefly, LN229/CD20 cells(1x108 cells) were immunized into two BALB/c mice using
intraperitoneal injection together with Imject Alum (Thermo
Fisher Scientific, Inc.). After three additional immunizations, abooster injection was administered two days before harvesting
spleen cells. These spleen cells were fused with P3U1 cells
using polyethylene glycol 1500 (Roche Diagnostics), and thenhybridomas were grown in an RPMI medium supplemented
with sodium hypoxanthine, aminopterin, and thymidine(Thermo Fisher Scientific, Inc.). The culture supernatants wereused for hybridoma screening by flow cytometry.
Flow cytometry. Cells were harvested by brief exposure to
0.25% trypsin with 1 mM ethylenediaminetetraacetic acid
(Nacalai Tesque, Inc.). After washing with phosphate bufferedsaline (PBS) containing 0.1% bovine serum albumin (BSA),
the cells were treated with 10 µg/ml of C20Mab‑11 for 30 min
at 4˚C, followed by treatment with Alexa Fluor 488‑conjugatedanti‑mouse IgG (1:2,000; Cell Signaling Technology, Inc.).
Fluorescence data were collected using Spectral Cell Analyzer
SA3800 (Sony Corp.).
Western blot analysis. Cell lysates were prepared by 1% Triton
X‑100 and cell debris was removed by centrifugation. Cell
lysates were boiled in sodium dodecyl sulfate sample buffer
with a reducing reagent (Nacalai Tesque, Inc.). These proteins(10 µg) were electrophoresed on 5‑20% polyacrylamide gels
(FUJIFILM Wako Pure Chemical Corporation) and transferred
onto polyvinylidene difluoride membranes (Merck KGaA). After
blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes
were incubated with 10 µg/ml of C20Mab‑11, 1 µg/ml of NZ‑1
(anti‑PA tag) or 1 µg/ml of anti‑β ‑actin for control (clone
AC‑15; Sigma‑Aldrich Corp.), followed by incubation withsecondary antibody peroxidase‑conjugated anti‑mouse immunoglobulin (1:1,000; Agilent Technologies Inc., Santa Clara,
CA, USA) or anti‑rat IgG (1:10,000; Sigma‑Aldrich Corp.),respectively. Finally, proteins were detected with ImmunoStar
LD (FUJIFILM Wako Pure Chemical Corporation) using the
Sayaca‑Imager (DRC Co. Ltd.).
Immunohistochemical analyses. One formalin‑fixed paraffin‑­embedded (FFPE) tissue sample from an oropharyngeal
squamous cell carcinoma patient who underwent surgery at
Sendai Medical Center was used for this study (13). Written
informed consent was obtained from the patient for sample
procurement and subsequent data analyses. Tissue microarray(CC00‑10‑001) including lymphomas, normal lymph node,and normal thyroid was purchased from Cybrdi, Inc.
The 4‑µm thick parafﬁn‑embedded tissue sections weredirectly autoclaved in EnVision FLEX Target Retrieval Solution

ONCOLOGY LETTERS 20: 1961-1967, 2020

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Figure 1. Production of anti‑CD20 monoclonal antibodies. Procedure of Cell‑Based Immunization and Screening method. LN229/CD20 cells were immunized
into BALB/c mice using intraperitoneal injection. Screening was performed using flow cytometry.

High pH (Agilent Technologies, Inc.) for 20 min. After blocking
with the SuperBlock T20 (PBS) Blocking Buffer (Thermo
Fisher Scientific, Inc.), tissue sections were incubated with
C20Mab‑11 (5 µg/ml) for 1 h at room temperature and treatedwith the Envision+ Kit for mouse (Agilent Technologies, Inc.)for 30 min. Color was developed using 3,3'‑diaminobenzidine
tetrahydrochloride (Agilent Technologies, Inc.) for 2 min, andcounterstaining was performed using hematoxylin (FUJIFILM
Wako Pure Chemical Corporation).
Statistical analysis. Statistical analysis was conducted usingt‑test with GraphPad Prism 6 (GraphPad Software, Inc.).
P<0.05 was considered to indicate a statistically significant
difference. All data are expressed as the mean ± SEM.
Results
Establishment of anti‑CD20 monoclonal antibodies. In
this study, we employed the CBIS method (Fig. 1). First, we
immunized two mice with LN229/CD20 cells and their spleencells were harvested and grown as hybridomas in cell culture.
Next, supernatants from cultured hybridomas positive for
CHO/CD20 and negative for CHO‑K1, were selected by flow
cytometry. We detected strong signals from CHO/CD20 andweak or no signals from CHO‑K1 in 14 of the 960 wells (1.5%).
Further screening using western blotting and immunohistochemistry techniques led to the establishment of C20Mab‑11(IgM, kappa).
CD20 expression was investigated by RT‑PCR. CD20 was
not detected in CHO‑K1/mock, LN229/mock, Lec1/mock,Lec2/mock, and Lec8/mock (Fig. 2A). Then, overexpression of CD20 was confirmed in CHO/CD20, LN229/CD20,
Lec1/CD20, and Lec2/CD20, Lec8/CD20. We used thesestable transfectants of CD20 in flow cytometry and western
blot analyses to characterize C20Mab‑11.
Flow cytometric analyses. Using flow cytometry analysis,
we found that C 20Mab‑11 reacted with CHO/CD20 cells,
but not with CHO‑K1 cells (Fig. 2B). Similarly, C20Mab‑11
reacted with LN229/CD20 cells, but not with LN229
cells. Additionally, we performed flow cytometry using
CD20‑stable transfectants of glycan‑deficient CHO celllines (Lec1, Lec2, and Lec8), although it was previously
reported that CD20 is not glycosylated (18). C 20Mab‑11
reacted with Lec1/CD20 (N‑glycan‑deficient), Lec2/CD20(sialic acid‑deficient), and Lec8/CD20 (galactose‑deficient)cells, but not with Lec1, Lec2, and Lec8 cells, indicatingthat the binding epitope of C 20Mab‑11 is independent of1964

FURUSAWA et al: ESTABLISHMENT OF AN ANTI-CD20 mAb

Figure 2. Detection of CD20 by C20Mab‑11 using flow cytometry. (A) Reverse transcription‑PCR for CD20. Overexpression of CD20 was confirmed in
CHO/CD20, LN229/CD20, Lec1/CD20, and Lec2/CD20, and Lec8/CD20 cell lines. GAPDH was used as an internal control. (B) Detection of CD20 by C20Mab‑11.
CHO‑K1, CHO/CD20, LN229, LN229/CD20, Lec1 (N‑glycan‑deficient), Lec1/CD20, Lec2 (sialic acid‑deficient), Lec2/CD20, and Lec8 (galactose‑deficient),and Lec8/CD20 cells were treated with C20Mab‑11 (red line) at a concentration of 10 µg/ml or 0.1% bovine serum albumin in PBS (gray) for 30 min, followed
by incubation with secondary antibodies. (C) Fluorescence intensity was quantified. **P<0.01. n.s., not significant. All data are presented as the mean ± SEM.

glycans. These results indicate that C20Mab‑11 is specific forCD20. Fluorescence intensity was quantitatively analyzed(Fig. 2C).

Western blot analysis. Next, we performed western blot using
C20Mab‑11. C20Mab‑11 detected CD20 with a 45‑kDa band in
CHO/CD20 and a 37‑kDa band in BALL‑1 and Raji cells; itONCOLOGY LETTERS 20: 1961-1967, 2020

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Figure 3. Detection of CD20 by C20Mab‑11 by western blotting. Cell lysatesof CHO‑K1, CHO/CD20, BALL‑1, BINDS‑24 and Raji cells were electrophoresed and transferred onto PVDF membranes. These membranes weretreated with C20Mab‑11 (left panel), NZ‑1 (anti‑PA tag; middle panel) or
anti‑β‑actin (right panel), followed by incubation with secondary antibodies.

Figure 5. Immunohistochemical analyses using C 20Mab‑11 for B‑cell
lymphomas and B‑cells. (A and B) Consecutive tissue sections of BCL wereincubated with C20Mab‑11, followed by an Envision+ kit. Counterstaining
was performed using hematoxylin. (C and D) Consecutive tissue sectionsof BCL were incubated with blocking buffer, followed by an Envision+ kit.
Counterstaining was performed using hematoxylin. (E and F) Consecutive
tissue sections of thyroid were incubated with C 20Mab‑11, followed by
an Envision+ kit. Counterstaining was performed using hematoxylin.
(G and H) Consecutive tissue sections of thyroid were incubated with blocking
buffer, followed by an Envision+ kit. Counterstaining was performed using
hematoxylin. Scale bar, 100 µm.

Figure 4. Immunohistochemical analyses using C20Mab‑11 for OSCC or
normal lymph node. (A and B) Consecutive tissue sections of OSCC wereincubated with C20Mab‑11, followed by an Envision+ kit. Counterstaining
was performed using hematoxylin. (C and D) Consecutive tissue sections of
OSCC were incubated with blocking buffer, followed by an Envision+ kit.
Counterstaining was performed using hematoxylin. (E and F) HE staining
was also performed using consecutive OSCC tissues. (G and H) Consecutive
tissue sections of normal lymph node tissue were incubated with C20Mab‑11,
followed by an Envision+ kit. Counterstaining was performed using hematoxylin. Scale bar, 100 µm. OSCC, oropharyngeal squamous cell carcinoma;HE, hematoxylin and eosin.

did not detect CD20 in CHO‑K1 cells and CD20‑knockout
BALL‑1 (BIND‑24) cells (Fig. 3), indicating that C20Mab‑11 is
specific for CD20. An anti‑PA tag mAb (NZ‑1) detected CD20
at a 45‑kDa band without detecting any bands in CHO‑K1,
BALL‑1, BINDS‑24, and Raji cells. The difference ofmolecular size between CHO/CD20 and BALL‑1 cells might
be due to N‑terminal PA‑tag of CD20. Although additional
bands of 30 and 20 kDa were detected by C20Mab‑11 and ananti‑PA tag mAb (NZ‑1), respectively, in CHO/CD20 cells,
we postulated that they might be degraded fragments of theCD20 protein.
Immunohistochemical analyses. We further investigated theimmunohistochemical utility of C20Mab‑11 in a lymph node oforopharyngeal squamous cell carcinoma patient. C20Mab‑11strongly stained the lymph follicle and weakly stained thecortex (Fig. 4A and B). No staining was observed without the
primary antibody (Fig. 4C and D). Hematoxylin and eosin

1966

FURUSAWA et al: ESTABLISHMENT OF AN ANTI-CD20 mAb(HE) staining was also performed using consecutive oropharyngeal squamous cell carcinoma tissues (Fig. 4E and F).
C20Mab‑11 weakly stained B cells of normal lymph node(Fig. 4G and H) and B cell‑lymphomas (Fig. 5A and B). No
staining was observed in B cell‑lymphomas without the
primary antibody (Fig. 5C and D). C20Mab‑11 (Fig. 5E and F)
and control (Fig. 5G and H) did not stain normal thyroid tissues.
These results indicate that C20Mab‑11 is useful for detecting
B cells in immunohistochemical analyses using FFPE tissues.
Discussion
In this study, we successfully established a sensitive andspecific anti‑CD20 mAb, C20Mab‑11, using the CBIS method,previously developed by our laboratory (Fig. 1) (7). Because
this CBIS method does not need purified proteins for immunization and screening, we can develop mAbs against multi‑passtransmembrane proteins more effectively. Previously, we developed anti‑CD133 mAbs using CBIS method without purifying
CD133 proteins (7). The anti‑CD133 mAbs were determined
to be useful for every application, including for flow cytometry,western blot, and immunohistochemical analyses. While there
are several anti‑CD133 mAbs are commercially available, theapplications of those mAbs are usually limited (19); therefore,
we have to use several mAbs or polyclonal antibodies fordifferent experimental procedures.
We can also obtain many anti‑CD20 mAbs commerciallyas shown in Table SI. As listed, anti‑CD20 mAbs are usuallyapplicable for only one or two experimental techniques.
Although a few mAbs are useful for at least three applications, including flow cytometry, they do not react with theextracellular domains of CD20 because only the intracellular domains were used as the immunogen. Rituximab[a mouse‑human chimeric mAb; the original mouse clone is
2B8 (20)] is known as the first anti‑CD20 mAb approved by
the US FDA and is used for the treatment of B‑cell NHL or
B‑cell chronic lymphocytic leukemia (21). Rituximab showshigh sensitivity in flow cytometry as well as exerting highantibody‑dependent cell‑mediated cytotoxicity and complement dependent cytotoxicity (ADCC/CDC) and anti‑tumor
activities. However, rituximab could not detect CD20 protein
in western blot analysis in our study (data not shown). In
contrast, C20Mab‑11 could detect endogenous CD20 protein,which is expressed in BALL‑1 and Raji cells, very sensitively(Fig. 3). Furthermore, C20Mab‑11 could stain normal B‑cells
or B‑cell lymphoma cells in immunohistochemical analyses
using FFPE tissues (Figs. 4 and 5). Because mAbs possess
different epitopes, it might be preferable to use the same mAbof the accordant epitope for both diagnosis and therapy. In thisstudy, we used one oropharyngeal squamous cell carcinoma
tissue, one B‑cell lymphoma tissue, and two normal humantissues for C20Mab‑11 immunostaining. In our future study, it
would be useful to further validate our results in more tissues
and with different degrees of disease progression.
C 20Mab‑11 could be also advantageous for targeting
CD20‑expressing B‑cell lymphomas. Our future studies will
focus on changing the subclass of C20Mab‑11 (mouse IgM)
into mouse IgG2a or human IgG1 and determine whether
these mAbs also demonstrate ADCC/CDC and anti‑tumor
activities (22).

Anti‑CD20 mAbs in combination with rituximab,
I‑Tositumomab (radio‑immunotherapy) (23), ibritumomab
tiuxetan (radio‑immunotherapy) (24), ofatumumab (a fully
human mAb) (25), or obinutuzumab (a humanized glycoengineered mAb) (26), have brought significant survival benefits
to B‑cell lymphoma patients. In follicular lymphoma, thefour‑year overall survival rate was significantly improved withthe combination of cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) therapy and an anti‑CD20 mAb (91%)
compared with CHOP alone (69%) (27). In elderly patients(60‑80 years old) with diffuse large B‑cell lymphoma, overall
survival was improved by 13% following treatment with rituximab plus CHOP compared with CHOP alone (27). Althoughanti‑CD20 mAbs have been shown to be very effective for thetreatment of B‑cell lymphoma, many patients do not have aclinical response to anti‑CD20 mAbs (28). Furthermore, 60%of NHL relapse patients, who initially responded to rituximab,
have no subsequent clinical response to rituximab due tothe loss of CD20 expression (28). To overcome resistance toanti‑CD20 mAbs, the development of more effective first‑line
lymphoma treatments is needed. Future studies will investigate
whether C20Mab‑11 may be used as an antibody‑containing
drug to treat CD20‑expressing lymphomas.
131

Acknowledgements
The authors would like to thank Mr. Takuro Nakamura, Ms.
Miyuki Yanaka and Ms. Saori Handa (Tohoku University) for
technical assistance of in vitro experiments.
Funding
The present study was supported in part by Japan Agency
for Medical Research and Development (AMED) [grantnos. JP19am0401013 (YK), JP19am0101078 (YK) andJP19ae0101028 (YK)] and by Japan Society for the Promotionof Science Grants‑in‑Aid for Scientific Research (KAKENHI)[grant. nos. 17K07299 (MKK) and 19K07705 (YK)].
Availability of data and materials
The datasets used and/or analyzed during the study are available from the corresponding author on reasonable request.
Authors' contributions
YF performed experiments. MKK analyzed experimental
data. YK designed the present study. YF and YK wrote themanuscript.
Ethics approval and consent to participate
Animal experiments described in the hybridoma production
were approved by The Animal Care and Use Committee of
Tohoku University (permit no. 2016MdA‑153). A patient withoropharyngeal squamous cell carcinoma who underwent surgery
at Sendai Medical Center was recruited for examinations, andThe Ethical Committee of Sendai Medical Center approved thepresent study. Written informed consent was obtained from thepatient for sample procurement and subsequent data analyses.

ONCOLOGY LETTERS 20: 1961-1967, 2020

Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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