ONCOLOGY LETTERS 19: 2809-2816, 2020

A novel anti‑EGFR monoclonal antibody (EMab‑17) exerts
antitumor activity against oral squamous cell carcinomas via
antibody‑dependent cellular cytotoxicity andcomplement‑dependent cytotoxicity
JUNKO TAKEI1,2, MIKA KATO KANEKO1, TOMOKAZU OHISHI3,
MANABU KAWADA3, HIROYUKI HARADA2 and YUKINARI KATO1,4
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Aoba‑ku, Sendai,
Miyagi 980‑8575; 2Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University, Bunkyo‑ku, Tokyo 113‑8510; 3Institute of Microbial Chemistry (BIKAKEN),
Numazu, Microbial Chemistry Research Foundation, Numazu‑shi, Shizuoka 410‑0301; 4New Industry
Creation Hatchery Center, Tohoku University, Aoba‑ku, Sendai, Miyagi 980‑8575, Japan
Received November 6, 2019; Accepted January 21, 2020
DOI: 10.3892/ol.2020.11384
Abstract. The epidermal growth factor receptor (EGFR) is amember of the human epidermal growth factor receptor (HER)
family of receptor tyrosine kinases; it is a transmembrane receptor
involved in cell growth and differentiation. EGFR homodimers
or heterodimers in combination with other HER members,
such as HER2 and HER3, activate downstream signalingcascades in many types of cancer, including oral squamous cell
carcinoma (OSCC). The present study produced novel anti‑EGFR
monoclonal antibodies (mAbs) possessing antibody‑dependent
cellular cytotoxicity (ADCC) and complement‑dependent
cytotoxicity (CDC), and investigated antitumor activity. Mice
were immunized with an EGFR‑overexpressed glioblastoma
cell line, LN229 (LN229/EGFR), after which ELISA was
performed using recombinant EGFR. mAbs were subsequently

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: EGFR, epidermal growth factor receptor; HER,

human epidermal growth factor receptor; OSCC, oral squamous cell
carcinoma; mAbs, monoclonal antibodies; ADCC, antibody‑dependent
cellular cytotoxicity; CDC, complement‑dependent cytotoxicity;
HNC, head and neck cancer; PD‑1, programmed cell death‑1; VEGF,
vascular endothelial growth factor; DMEM, Dulbecco's Modified
Eagle's medium; ELISA, enzyme‑linked immunosorbent assay; FBS,
fetal bovine serum; EDTA, ethylenediaminetetraacetic acid; BSA,
bovine serum albumin; PBS, phosphate‑buffered saline

Key words: epidermal growth factor receptor, monoclonal antibody,

antibody‑dependent cellular cytotoxicity, complement‑dependent
cytotoxicity, oral squamous cell carcinoma

selected according to their efficacy for LN229/EGFR, as
determined via flow cytometry. After determining the subclassof mAbs, the EMab‑17 (IgG2a, kappa) clone exhibited ADCC
and CDC activities against two OSCC cell lines, HSC‑2 andSAS. Furthermore, EMab‑17 exerted antitumor activities againstmouse xenograft models using HSC‑2 and SAS, indicatingthat EMab‑17 may be used in an antibody‑based therapy for
EGFR‑expressing OSCC.
Introduction
Oral squamous cell carcinoma (OSCC) is usually treatedby surgical removal; it can be complemented by chemotherapy, including cisplatin (CDDP), 5‑fluorouracil (5‑FU),and docetaxel (1,2), and/or radiotherapy, particularly atadvanced stages. As an antibody drug, cetuximab, which is amouse‑human (IgG1) chimeric antibody against the epidermal
growth factor receptor (EGFR), was approved for the treatmentof head and neck cancer (HNC), including oral cancer (1). The
effectiveness of cetuximab against locoregionally advanced
head and neck squamous cell carcinoma (HNSCC) or recurrent and/or metastatic (R/M) HNSCC was reported in various
clinical studies (1,3‑5). Recently, nivolumab‑a fully human
IgG4 monoclonal antibody (mAb) against programmed cell
death‑1 (PD‑1)‑was approved for the treatment of R/M HNC
previously treated with platinum‑based chemotherapy (6).
Furthermore, bevacizumab, which is a mouse‑human IgG1chimeric antibody against vascular endothelial growth factor
first approved for colorectal cancer treatment, was the subjectof clinical trials involving R/M HNSCC patients (7). Moleculartargeting drugs that are clinically applicable for oral cancers
are limited; therefore, novel drugs with greater efficacy andlower toxicity are required.
EGFR is a member of the human epidermal growth
factor receptor (HER) family of receptor tyrosine kinases
and involved in cell growth and differentiation (8‑10). EGFR

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TAKEI et al: mAbs AGAINST EGFR

homodimers or heterodimers in conjunction with other HER
members (such as HER2 and HER3) activate downstream
signaling cascades. These pathways are frequently dysregulated via the overexpression of EGFR in many malignant
tumors, including colorectal, lung, and breast cancers, brain
tumors, head and neck cancers, pancreatic, kidney, and prostate cancers, and ovarian, bladder, and oral cancers (11).
In the previous research of this study, mice were immunizedwith purified recombinant EGFR to produce an EMab‑134
monoclonal antibody (mAb; IgG1, kappa), which reacted withthe endogenous EGFR of oral cancers in flow cytometry,
Western blotting, and immunohistochemistry (12). In immunohistochemical analysis, EMab‑134 stained 36 of 38 (94.7%)
oral cancer specimens. The minimum epitope of EMab‑134
was found to be the 377‑RGDSFTHTPP‑386 sequence (13).
Although EMab‑134 is a very useful mAb‑targeting EGFR,
the subclass was determined to be mouse IgG1, which did notexhibit antibody‑dependent cellular cytotoxicity (ADCC) andcomplement‑dependent cytotoxicity (CDC) activities. This
study develops novel anti‑EGFR mAbs possessing the ADCC
and CDC activities of mouse IgG2a or the IgG2b subclass andinvestigates the antitumor activity.
Materials and methods
Cell lines. HSC‑2 and SAS were obtained from the JapaneseCollection of Research Bioresources Cell Bank. Chinese
hamster ovaries (CHO)‑K1, P3X63Ag8U.1 (P3U1), and LN229
were obtained from the American Type Culture Collection.
LN229/EGFR (a stable transfectant) was previously producedby transfecting pCAG/PA‑EGFR‑RAP‑MAP (14) into LN229
cells using the Neon Transfection System (Thermo Fisher
Scientific, Inc.), and EGFR upregulation was demonstrated
by Western blot analysis using anti‑EGFR mAb, clone
EMab‑51 (15). P3U1 was cultured in Roswell Park Memorial
Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.), while
LN229, LN229/EGFR, HSC‑2, and SAS were cultured in
Dulbecco's Modified Eagle's medium (DMEM) (Nacalai
Tesque, Inc.) supplemented with 10% heat‑inactivatedfetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.),
100 units/ml of penicillin, 100 µg/ml of streptomycin, and25 µg/ml of amphotericin B (Nacalai Tesque, Inc.) at 37˚C in ahumidified atmosphere containing 5% CO2 and 95% air.
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
number: 2016MdA‑153). Mice were monitored for health everyday. Animal studies for the antitumor activity were approvedby the institutional committee for experiments of the Instituteof Microbial Chemistry (Permit number: 2019‑014). Mice weremonitored for health and weight every 3 or 4 days. The duration of the experiment was three weeks. A body weight loss
exceeding 25% of total body weight and a maximum tumor
size exceeding 3,000 mm3 were defined as a humane endpoint.
Hybridoma production. One four‑week‑old female BALB/cmouse was purchased from CLEA Japan and housed under

specific pathogen‑free conditions. Anti‑EGFR hybridomas
were produced, as previously mentioned (15). The ectodomain of EGFR with N‑terminal PA tag (16), C‑terminal RAP
tag (17), and MAP tag (14) (EGFRec) was purified from thesupernatant of LN229/EGFRec using the anti‑RAP tag previously described (17).
One BALB/c mouse was immunized by intraperitoneal
injections of LN229/EGFR together with Imject Alum (Thermo
Fisher Scientific, Inc.). After several additional immunizations,a booster injection was intraperitoneally administered 2 days
before harvesting spleen cells. Spleen cells were then fusedwith P3U1 cells using GenomONE‑CF (Ishihara Sangyo
Kaisha, Ltd.). The resulting hybridomas were cultured in an
RPMI medium supplemented with hypoxanthine, aminopterin,
and thymidine selection medium supplement (Thermo Fisher
Scientific, Inc.). Culture supernatants were screened using
enzyme‑linked immunosorbent assays (ELISA) with a recombinant EGFR‑extracellular domain. mAbs were purified fromthe supernatants of hybridomas, cultured in Hybridoma‑SFM
medium (Thermo Fisher Scientific, Inc.) using Protein G
Sepharose 4 Fast Flow (GE Healthcare UK, Ltd.).
Enzyme‑linked immunosorbent assay. Recombinant proteins
were immobilized on Nunc MaxiSorp 96‑well immunoplates(Thermo Fisher Scientific, Inc.) at 1 µg/ml for 30 min. After
blocking using a SuperBlock buffer (Thermo Fisher ScientificInc.), the plates were incubated with primary antibodies,
followed by 1:2,000 diluted peroxidase‑conjugated anti‑mouse
IgG (Agilent Technologies). The enzymatic reaction was
produced using a 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.).
Flow cytometry. Cells were harvested by brief exposure to
0.25% trypsin/1‑mM ethylenediaminetetraacetic acid (EDTA)(Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine
serum albumin (BSA)/phosphate‑buffered saline (PBS) andtreated with 1 µg/ml of anti‑EGFR mAbs for 30 min at 4˚C,
followed by Alexa Fluor 488‑conjugated anti‑mouse IgG(1:1,000; Cell Signaling Technology, Inc.). Fluorescence data
was collected using EC800 Cell Analyzers (Sony Corp.).
Determination of the binding affinity using flow cytometry.
SAS (2x105 cells) was suspended in 100 µl of serially diluted
mAbs (6 ng/ml‑100 µg/ml); Alexa Fluor 488‑conjugatedanti‑mouse IgG (1:1,000) (Cell Signaling Technology, Inc.)
was then added, and fluorescence data was collected using acell analyzer (EC800) (Sony Corp.). The dissociation constants(K D) were computed by fitting the binding isotherms using
the built‑in one‑site binding models in GraphPad Prism 6(GraphPad Software, Inc.).
ADCC. Six six‑week‑old female BALB/c nude mice werepurchased from Charles River, and spleens were removed
aseptically, and single‑cell suspensions were obtained bydispersing the spleens using a syringe and pressing throughstainless steel mesh. Erythrocytes were effectively lysed by
10‑s exposure to ice‑cold distilled water. Splenocytes were
washed with DMEM and resuspended in DMEM with 10%
FBS as effector cells. Target cells were labeled with 10‑µg/ml

ONCOLOGY LETTERS 19: 2809-2816, 2020

Calcein AM (Thermo Fisher Scientific, Inc.) and resuspended
in the medium. The target cells (2x104 cells/well) were placedin 96‑well plates and mixed with effector cells, anti‑EGFR
antibodies, or control IgG (mouse IgG2a) (Sigma‑AldrichCorp.). After a 4‑h incubation period, the Calcein AM releaseof supernatant from each well was measured. The fluorescence intensity was determined at an excitation wavelengthof 485 nm and an emission wavelength of 538 nm using a
microplate reader (Power Scan HT) (BioTek Instruments).
Cytolytic activity (as % of lysis) was calculated using thefollowing formula: % lysis=(E‑S)/(M‑S) x100 (where E is thefluorescence released in the experimental cultures of target
and effector cells, S is the spontaneous fluorescence releasedin cultures containing only target cells, and M is the maximum
fluorescence obtained by adding a lysis buffer containing 0.5%
Triton X‑100, 10 mM Tris‑HCl (pH 7.4), and 10 mM of EDTA
to the target cells in order to lyse all cells).
CDC. HSC‑2 and SAS cells were placed in 96‑well platesof 2x104 cells/well in DMEM supplemented with 10% FBS.
Cells were incubated with either anti‑EGFR antibodies orthe control IgG (mouse IgG2a) (Sigma‑Aldrich Corp.) and
10% of rabbit complement (Low‑Tox‑M Rabbit Complement)(Cedarlane Laboratories) for 5 h at 37˚C. To assess cell
viability, MTS [3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxym‑
ethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium; inner salt]
assay was performed using a CellTiter 96 AQueous assay kit(Promega Corp.).
EGF stimulation assay. HSC‑2 and SAS cells were washedwith DMEM lacking FBS to eliminate the growth factors
present in the enriched medium. Then, the cells were plated
in 96‑well culture plates at a density of 2,000 cells per well in
100 µl of 0.1% dialyzed FBS with or without 50 ng/ml of EGF(PeproTech). MTS assay was performed after 12, 24, and 36 h.
Antitumor activity of Anti‑EGFR antibodies. Thirty‑twosix‑week‑old female BALB/c nude mice were purchasedfrom Charles River and used in experiments at 7 weeks ofage. HSC‑2 or SAS (0.3 ml of 1.33x108/ml in RPMI) was
mixed with 0.5 ml of BD Matrigel Matrix Growth Factor
Reduced (BD Biosciences). A 100‑µl suspension (containing
5x106 cells) was injected subcutaneously into the left flanks ofnude mice. After day 1, 100 µg of EMab‑17 and control mouse
IgG (Sigma‑Aldrich Corp.) in 100 µl of PBS was injected into
the peritoneal cavity of each mouse, followed by additional
antibody injections on days 7 and 14. Mice were monitored for
health and weight every 3 or 4 days. The diameter and volumeof the tumor were determined as previously described (18), andthe mice were euthanized 21 days after cell implantation. The
duration of the experiment was three weeks. A body weightloss exceeding 25% of total body weight was defined as ahumane endpoint. All data was expressed as mean ± SEM, andstatistical analysis was conducted using Tukey‑Kramer's test;
P<0.05 was considered to indicate a statistically significant
difference.
Statistical analyses. Statistical analysis was conducted using
ANOVA followed by Tukey‑Kramer's test. P<0.05 was considered to indicate a statistically significant difference. All data2811

was expressed as mean ± SEM and analyzed using GraphPad
Prism 6 (GraphPad Software, Inc.).
Results
Production and characterization of Anti‑EGFR mAbs. In
this study, one mouse was immunized with LN229/EGFR,
and culture supernatants of hybridoma were screened forbinding to purified EGFRec using ELISA. Flow cytometry
was used as a second screening to assess reactions with LN229
and LN229/EGFR cells. LN229 cells express endogenous
EGFR (15); therefore, a stronger reaction against LN229/EGFR
was required. One clone was obtained‑EMab‑17 of IgG2a
subclass‑although almost all mAbs were determined to be amouse IgG1 subclass like EMab‑51 (15) or EMab‑134 (12).
Flow cytometry was used to demonstrate a stronger
reaction with EMab‑17 LN229/EGFR than with endogenous
EGFR‑expressing LN229 brain tumor cells (Fig. 1A), whichindicated that EMab‑17 is EGFR‑specific. As a positive
control, EMab‑51 demonstrated a similar reaction with LN229
and LN229/EGFR (Fig. 1B). Endogenous HSC‑2 and HSC‑3
OSCC cell lines were also identified with both EMab‑17
and EMab‑51 (Fig. 1C and D). Flow cytometry was again
applied to determine the binding affinity of EMab‑17 forSAS cells (Fig. 2) and calculated K D values for EMab‑17 of
5.0x10‑9 M against SAS. Similarly, KD values were determined
for EMab‑51 as 6.3x10 ‑9 M against SAS (Fig. 2), revealingthat both EMab‑17 and EMab‑51 possess a high affinity for
EGFR‑expressing cell lines.
ADCC and CDC activities against OSCC cell lines. This
study examined whether EMab‑17 induced ADCC and CDC
in EGFR‑expressing OSCC cell lines. EMab‑17 was determined to be a mouse IgG2a subclass that might possess both
ADCC and CDC (although mouse IgG1 such as EMab‑51 andEMab‑134 does not) (12,15). As detailed in Fig. 3A, EMab‑17
exhibited high ADCC activity against HSC‑2 and SAS.
Furthermore, high CDC activity was also observed for HSC‑2and SAS by EMab‑17 (Fig. 3B), suggesting that EMab‑17
might exert antitumor activities in vivo. Although we added
EGF to SAS and HSC‑2, these cell lines did not grow wellcompared to control cells by responding to EGF stimulation(data not shown), indicating that EMab‑17 could not neutralize
EGF‑EGFR axis.
Antitumor activities against OSCC. HSC‑2 cells were subcutaneously implanted into the flanks of nude mice in orderto study the antitumor activity of EMab‑17 on cell growthin vivo. EMab‑17 and the control mouse IgG were injected (ondays 1, 7, and 14 after the cell injections) three times into theperitoneal cavity. Tumor formation was observed in mice fromthe control and EMab‑17‑treated groups in HSC‑2 xenograftmodels. EMab‑17 demonstrated significant reduction in tumor
development of the HSC‑2 xenograft compared with that in thecontrol mouse IgG group on days 7, 10, 14, 17, and 21 (Fig. 4A).
Mice treated with EMab‑17 had significantly lower tumor
weights compared to the control mouse IgG group in HSC‑2
xenograft models (Fig. 4B). The resected tumors of HSC‑2
xenografts are shown in Fig. 4C. The body weights of the
HSC‑2 xenograft mice were recorded for 21 days (Fig. S1A).

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TAKEI et al: mAbs AGAINST EGFR

Figure 1. Flow cytometry of EMab‑17. (A) LN229 and LN229/EGFR cells were treated with 1 µg/ml (A) EMab‑17 (red line) and (B) EMab‑51 (blue line)
followed by Alexa Fluor 488‑conjugated anti‑mouse IgG. (C) HSC‑2 and SAS were treated with 1 µg/ml of (C) EMab‑17 (red line) and (D) EMab‑51 (blue line),followed by Alexa Fluor 488‑conjugated anti‑mouse IgG. Black line, PBS treated control.

Body weight did not vary significantly between the two groups
in the HSC‑2 xenograft models.
Identical experiments were performed using SAS xenograft models. EMab‑17 demonstrated significantly reduced

tumor development in the SAS xenograft group comparedwith that in the control mouse IgG group on days 7, 10, 14,17, and 21 (Fig. 4D). The tumor weight of EMab‑17‑treatedmice was significantly lower compared to the control mouse

ONCOLOGY LETTERS 19: 2809-2816, 2020

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Figure 2. Binding affinities of anti‑EGFR antibodies were determined using flow cytometry. SAS cells were suspended in 100 µl serially diluted EMab‑17 or
EMab‑51 (0.006‑100 µg/ml), after which secondary antibodies were added. Fluorescence data was collected using a cell analyzer. GeoMean, geometric meanof fluorescence intensity.

Figure 3. ADCC and CDC activities. For ADCC activity, cells were incubated with splenocytes from mice that were treated with the indicated antibodies at
100 µg/ml for 4 h. Cell lysis was determined using Calcein AM. For CDC activity, cells were incubated with 10% rabbit complement in the presence of theindicated antibodies for 5 h. Cell lysis was determined using an MTS assay. (A) ADCC against HSC‑2 and SAS. (B) CDC against HSC‑2 and SAS. **P<0.01 as
indicated. ADCC, antibody‑dependent cellular cytotoxicity; CDC, complement‑dependent cytotoxicity.

IgG group in the SAS xenograft models (Fig. 4E); the resected
tumors of the SAS xenografts are illustrated in Fig. 4F. The
body weights of the SAS xenograft mice were recorded for
21 days (Fig. S1B). The body weight did not vary significantly
between the two SAS xenograft model groups.
Combined results confirm that EMab‑17 exerted an antitumor activity against HSC‑2 and SAS xenograft models via
ADCC and CDC activities.

Discussion
EGFR is the first receptor target using mAbs that has been developed for cancer treatment (19‑21). This includes necitumumab (afully human mAb; IgG1) for non‑small cell lung cancers, panitumumab (a fully human mAb; IgG2) for colorectal cancers, andcetuximab (a human‑mouse chimeric mAb; IgG1) for colorectal,
head, and neck cancers. Anti‑EGFR mAbs possess several

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TAKEI et al: mAbs AGAINST EGFR

Figure 4. Antitumor activity of EMab‑17 against HSC‑2 and SAS. (A) The tumor volumes of HSC‑2 xenografts were recorded. HSC‑2 cells were subcutaneously injected into female nude mice. The indicated antibodies (100 µg/day; 5 mg/kg) were administered intraperitoneally 1, 7 and 14 days after cancer cell
inoculation. Tumor volume was measured at the indicated time points. (B) Tumor weight of HSC‑2 xenografts (day 21). (C) Comparison of HSC‑2 tumor size(day 21). Scale bar, 1 cm. (D) Tumor volume of SAS xenografts. SAS cells were injected subcutaneously into female nude mice. The indicated antibodies(100 µg/day; 5 mg/kg) were administered intraperitoneally 1, 7 and 14 days after cancer cell inoculation. Tumor volume was measured at the indicated time
points. (E) Tumor weight of SAS xenografts (day 21). (F) Comparison of SAS tumor size (day 21). Scale bar, 1 cm. Data are presented as the mean ± standard
error of the mean. **P<0.01 vs. the control group or as indicated.

functional mechanisms, including ADCC, CDC, blocking
dimerization, and EGFR endocytosis. For investigating ADCC
and CDC in mouse xenograft models, the subclass of mAbs
should be IgG2a or IgG2b of mouse IgG (22), IgG2a or IgG2b of rat
IgG (23), IgG1 of human IgG (24), or type B of canine IgG (25).
For the previous production of anti‑EGFR mAbs (suchas EMab‑51 (15) or EMab‑134 (12)), purified recombinant
EGFR was immunized into mice. This methodology usingcancer cell lines (such as LN229) for producing immunogenwas previously identified as a CasMab method (26). Because
almost all mAbs were determined to be a mouse IgG1 subclasslike EMab‑51 (15) or EMab‑134 (12), it was not possible toinvestigate the antitumor activities of anti‑EGFR mAbs thatwere produced using CasMab methods. Therefore, EMab‑17
is the first anti‑EGFR mAb of IgG2a or IgG2b that has beendeveloped using the CasMab method.

ONCOLOGY LETTERS 19: 2809-2816, 2020

In the present study, we added EGF to HSC‑2 and SAS
cell lines; however, these cell lines did not respond to EGF
stimulation and did not grow well (data not shown), indicatingthat EMab‑17 could not neutralize EGF‑EGFR axis. Taken
together, anti‑tumor activities by EMab‑17 were exerted by
ADCC and CDC activities.
Unfortunately, EMab‑17 was not suitable for Western blot
and immunohistochemical analyses (data not shown); therefore, EMab‑51 or EMab‑134 should be used for diagnosing
EGFR expression in cancer patients. In subsequent futurestudies, the subclasses of EMab‑51 and EMab‑134 will be
converted into the mouse IgG2a subclass, and comparisons
between ADCC/CDC activities and EMab‑17 will be made.
In conclusion, this study successfully developed ananti‑EGFR mAb of an IgG2a subclass‑EMab‑17‑which demonstrated the antitumor activity via ADCC/CDC activities.
EMab‑17 could potentially be used for antibody‑based therapy
for EGFR‑expressing OSCC.
Acknowledgements
The authors would like thank Ms. Akiko Harakawa (Instituteof Microbial Chemistry) for technical assistance during animal
experiments. The authors would also like to acknowledge Ms.
Miyuki Yanaka, Ms. Saori Handa, Ms. Kayo Hisamatsu and
Mr. Takuro Nakamura (Tohoku University) for in vitro experiment technical assistance.
Funding
The present study was supported by the Japanese
Agency for Medical Research and Development (grantnos. JP19am0401013, JP19am0101078 and JP19ae0101028)
and the Japanese Society for the Promotion of Science Grants in
Aid for Scientific Research (KAKENHI; grant nos. 17K07299
and 19K07705).
Availability of data and materials
The datasets used and/or analyzed during the present study areavailable from the corresponding author on reasonable request.
Authors' contributions
JT and TO performed experiments. MKK analyzed experimental
data. MK, HH and YK designed the current study and wrote themanuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal experiments used for hybridoma production wereapproved by the Animal Care and Use Committee of Tohoku
University (permit no. 2016MdA‑153). Animal studies for antitumor activity were approved by the Institutional Committee
for experiments of the Institute of Microbial Chemistry(permit no. 2019‑014).
Patient consent for publication
Not applicable.

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Competing interests
The authors declare that they have no competing interests.
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