ONCOLOGY REPORTS 44: 1949-1960, 2020

A defucosylated anti‑CD44 monoclonal antibody
5‑mG2a‑f exerts antitumor effects in mouse xenograftmodels of oral squamous cell carcinoma
JUNKO TAKEI1,2*, MIKA K. KANEKO1*, TOMOKAZU OHISHI3*, HIDEKI HOSONO1,
TAKURO NAKAMURA1, MIYUKI YANAKA1, MASATO SANO1, TEIZO ASANO1,YUSUKE SAYAMA1, 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;
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi 980‑8575, Japan
Received May 14, 2020; Accepted July 23, 2020
DOI: 10.3892/or.2020.7735
Abstract. CD44 is widely expressed on the surface of mosttissues and all hematopoietic cells, and regulates many
genes associated with cell adhesion, migration, proliferation,

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*

Contributed equally

Abbreviations: AAL, Aleuria aurantia lectin; ABTU, Antibody

Bank of Tohoku University; ADCC, antibody‑dependent cellularcytotoxicity; ATCC, American Type Culture Collection; BSA, bovine
serum albumin; CasMab, cancer‑specific mAb; CBIS, cell‑based
immunization and screening; CDC, complement‑dependent
cytotoxicity; CD44s, CD44 standard; CD44v, CD44 variant; CHO,
Chinese hamster ovary; Con A, concanavalin A; DMEM, Dulbecco's
modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid;
ELISA, enzyme‑linked immunosorbent assay; FBS, fetal bovine
serum; FDA, Food and Drug Administration; FGF, fibroblast growth
factor; HNSCC, head and neck squamous cell carcinoma; HB‑EGF,
heparin‑binding epidermal growth factor; JCRB, Japanese Collectionof Research Bioresources Cell Bank; mAb, monoclonal antibody;
OSCC, oral squamous cell carcinoma; PBS, phosphate‑bufferedsaline; PDPN, podoplanin; PhoSL, Pholiota squarrosa lectin;
PODXL, podocalyxin; PVDF, polyvinylidene difluoride; RTKs,
receptor tyrosine kinases; RT‑PCR, reverse transcription‑polymerase
chain reaction; SCC, squamous cell carcinoma; SDS, sodium
dodecyl sulfate; SEM, standard error of the mean; VEGFR, vascularendothelial growth factor receptor

Key words: CD44, monoclonal antibody, antibody‑dependent
cellular cytotoxicity, complement‑dependent cytotoxicity, antitumor
activity, oral cancer

differentiation, and survival. CD44 has also been studied as atherapeutic target in several cancers. Previously, an anti‑CD44
monoclonal antibody (mAb), C44Mab‑5 (IgG1, kappa) wasestablished by immunizing mice with CD44‑overexpressing
Chinese hamster ovary (CHO)-K1 cells. C44Mab‑5 recognized all
CD44 isoforms, and showed high sensitivity for flow cytometry
and immunohistochemical analysis in oral cancers. However,as the IgG1 subclass of C44Mab‑5 lacks antibody‑dependent
cellular cytotoxicity (ADCC) and complement‑dependent
cytotoxicity (CDC), the antitumor activity of C44Mab‑5 could
not be determined. In the present study, we converted themouse IgG1 subclass antibody C44Mab‑5 into an IgG2a subclassantibody, 5‑mG2a, and further produced a defucosylated
version, 5‑mG 2a ‑f, using FUT8‑deficient ExpiCHO‑S(BINDS‑09) cells. Defucosylation of 5‑mG2a‑f was confirmed
using fucose‑binding lectins, such as AAL and PhoSL. The
dissociation constants (K D) for 5‑mG2a‑f against SAS and
HSC‑2 oral cancer cells were determined through flow cytometry to be 2.8x10‑10 M and 2.6x10‑9 M, respectively, indicatingthat 5‑mG2a‑f possesses extremely high binding affinity.
Furthermore, immunohistochemical staining using 5‑mG2a‑f
specifically stained the membranes of oral cancer cells.
In vitro analysis demonstrated that 5‑mG2a‑f showed moderate
ADCC and CDC activities against SAS and HSC‑2 oral cancer
cells. In vivo analysis revealed that 5‑mG2a‑f significantly
reduced tumor development in SAS and HSC‑2 xenografts incomparison to control mouse IgG, even after injection sevendays post‑tumor inoculation. Collectively, these results suggestthat treatment with 5‑mG2a‑f may represent a useful therapy
for patients with CD44‑expressing oral cancers.
Introduction
Oral cancers account for about 2% of all cancer cases diagnosed
worldwide (1). More than 350,000 individuals are diagnosed
with oral cancer every year, and oral cancers prove fatal for
approximately 170,000 of these people. Major risk factors for

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TAKEI et al: ANTITUMOR ACTIVITIES OF AN ANTI-CD44s mAboral cancer include the use of alcohol and tobacco (2). Althoughdecreased drinking and smoking have resulted in a decline in
the incidence of oral cancer, recent studies have reported anincrease in the number of young patients diagnosed with these
diseases (3,4).
CD44 is known to be expressed in many cell types,
including epithelial cells, fibroblasts, endothelial cells, andleukocytes (5). CD44 plays important roles in cell proliferation, adhesion, and migration (6). The CD44 gene consists of
20 exons (7). The smallest isoform is the standard form of CD44
(CD44s), which possesses 10 exons; other possible isoforms
are categorized as CD44 variants (CD44v), which are generated by alternatively spliced transcripts (8). Post‑translational
modifications such as N‑ and O‑glycosylation and heparansulfate modification also augment the diversity of CD44 (9,10).
Both CD44s and CD44v are overexpressed in many cancers;
however, a pattern of expression remains to be elucidated.
One of the CD44 variants, CD44v6, was first identified
as contributing to cancer metastasis, and CD44v6‑specificmonoclonal antibodies (mAbs) were found to inhibit metastasis of rat pancreatic cancers (11,12). Some CD44v6 isoforms
act as co‑receptors for receptor tyrosine kinases (RTKs) suchas MET and vascular endothelial growth factor receptor(VEGFR)‑2 (13‑15). The transfection of CD44v4‑7 cDNA
confers a metastatic phenotype in non‑metastatic cells (16).
Another CD44 variant, CD44v3, binds to several heparansulfate‑binding growth factors such as fibroblast growth factors(FGFs) and heparin‑binding epidermal growth factor (HB‑EGF),
and induces tumor progression (17,18). Several CD44 variants
were also reported as prognostic markers in head and neck,lung, colorectal, breast, and hepatocellular cancers (19‑23).
Many mAbs have been developed to target CD44 (24‑26).
mAbs that neutralize contact between hyaluronic acid andCD44 have been shown to inhibit anchorage‑independent
growth of murine mammary carcinoma cells and humancolon carcinoma cells (24). Anti‑CD44 mAbs were also found
to exhibit significant antitumor activity in mouse xenograftmodels of human cancers (25,26). Previously, we establishedclone C44Mab‑5 (IgG1, kappa) using Cell‑Based Immunizationand Screening (CBIS) (27). C44Mab‑5 recognized both CD44s
and CD44v isoforms, and demonstrated high sensitivity
for flow cytometry and immunohistochemical analysis inoral cancers. Because the IgG1 subclass of C44Mab‑5 lacks
antibody‑dependent cellular cytotoxicity (ADCC) andcomplement‑dependent cytotoxicity (CDC), antitumor activityof C44Mab‑5 could not be determined.
In this study, we converted the IgG1 subclass C44Mab‑5 intoa mouse IgG2a subclass mAb, 5‑mG2a, and further produced
a defucosylated version, 5‑mG2a‑f, using FUT8‑deficient
ExpiCHO‑S cells (28). We then investigated whether 5‑mG2a‑fexhibited ADCC, CDC and antitumor activities against oral
cancers.
Materials and methods
Cell lines. Oral squamous carcinoma cell lines including
HSC‑2 (oral cavity) and SAS (tongue) were obtained fromthe Japanese Collection of Research Bioresources Cell Bank(JCRB; Osaka, Japan). Chinese hamster ovary (CHO)-K1
was obtained from the American Type Culture Collection(ATCC). CD44v3‑10 plus N‑terminal PA16 tag‑overexpressed CHO‑K1 (CHO/PA16‑CD44v3‑10) was generated
by transfection of pCAG/PA16‑CD44v3‑10 to CHO‑K1
cells using the Neon Transfection System (Thermo Fisher
Scientific, Inc.). The PA16 tag consists of 16 amino acids(GLEGGVAMPGAEDDVV) (27). HSC‑2 and SAS cells were
cultured in Dulbecco's modified Eagle's medium (DMEM;
Nacalai Tesque, Inc.), and CHO‑K1 and CHO/PA16‑CD44v3‑10
were cultured in RPMI‑1640 medium (Nacalai Tesque, Inc.),
supplemented with 10% heat‑inactivated fetal bovine serum(FBS; Thermo Fisher Scientific Inc.), 100 units/ml of penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin
B (Nacalai Tesque, Inc.) at 37˚C in a humidified atmosphere
containing 5% CO2.
Antibodies. Mouse anti‑CD44s mAb C44Mab‑5 (IgG1, kappa)
was developed as previously described (27). Mouse IgG
was purchased from Sigma‑Aldrich Corp. (Merck KGaA).
To generate recombinant C44Mab‑5 (recC44Mab‑5), cDNAs
of C44Mab‑5 heavy and light chains were subcloned intopCAG‑Neo and pCAG‑Ble vectors (FUJIFILM Wako Pure
Chemical Corporation), respectively. To generate 5‑mG2a‑f,
appropriate VH cDNA of mouse C44Mab‑5 and CH of mouse
IgG2a were subcloned into pCAG‑Neo vector, and light chain of
C44Mab‑5 was subcloned into pCAG‑Ble vector. Vectors weretransfected into ExpiCHO‑S or BINDS‑09 (FUT8‑deficient
ExpiCHO‑S cells) using the ExpiCHO Expression System (28).
recC 44Mab‑5 and 5‑mG2a ‑f were purified using Protein
G‑Sepharose (GE Healthcare Bio‑Sciences).
Animals. All animal experiments were performed inaccordance with relevant guidelines (e.g. ARRIVE guidelines)
and regulations (e.g. 3R regulations) to minimize animal
suffering and distress in the laboratory (29,30). Seventy
female BALB/c nude mice (6 weeks old, 15‑18 g) werepurchased from Charles River (Kanagawa, Japan). Animalstudies for ADCC and antitumor activity were approved by
the Institutional Committee for Experiments of the Institute ofMicrobial Chemistry (permit number: 2020‑003). Mice weremaintained in a pathogen‑free environment (23±2˚C, 55±5%
humidity) on 11 h light/13 h dark cycle with food and watersupplied ad libitum during the experimental period. Mice weremonitored for health and weight every 1 or 5 days. Experiment
duration was three weeks. A bodyweight loss exceeding 25%
and a maximum tumor size exceeding 3,000 mm3 were identified as humane endpoints. Mice were euthanized by cervicaldislocation, and the death was verified by respiratory arrest
and cardiac arrest.
Enzyme‑linked immunosorbent assay (ELISA). C44Mab‑5 and5‑mG2a‑f were immobilized on Nunc Maxisorp 96‑well immunoplates (Thermo Fisher Scientific Inc.) at 1 µg/ml for 30 min.
After blocking using SuperBlock buffer (Thermo Fisher
Scientific Inc.) containing 0.5 mM CaCl 2, the plates wereincubated with biotin‑labeled lectins, such as Aleuria aurantia
lectin (AAL; Vector Laboratories), Pholiota squarrosa
lectin (PhoSL; J‑OIL MILLS, Inc.) (31), and concanavalin A(ConA; Vector Laboratories), followed by 1:3,000 diluted
peroxidase‑conjugated streptavidin (Agilent Technologies).
The enzymatic reaction was produced using a 1‑Step Ultra

ONCOLOGY REPORTS 44: 1949-1960, 2020

TMB‑ELISA (Thermo Fisher Scientific Inc.). The opticaldensity 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.). After washing with 0.1% bovine serum
albumin (BSA) in phosphate‑buffered saline (PBS), cells weretreated with primary mAbs for 30 min at 4˚C and subsequently
with Alexa Fluor 488‑conjugated anti‑mouse IgG (1:1,000; Cell
Signaling Technology, Inc.). Fluorescence microscopy data
were collected using an EC800 Cell Analyzer (Sony Corp.).
Immunohistochemical analyses. Histologic sections (4‑µm
thick) of an oral cancer tissue microarray (catalogue number:
OR481; US Biomax Inc.) were directly autoclaved in citratebuffer (pH 6.0; Agilent Technologies Inc.) for 20 min. Sections
were then incubated with 1 µg/ml primary mAbs for 1 h atroom temperature and treated using an Envision+ Kit (Agilent
Technologies) for 30 min. Color was developed using 3,3'‑diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies
Inc.) for 2 min, and sections were then counterstained withhematoxylin (FUJIFILM Wako Pure Chemical Corporation).
Hematoxylin and eosin (H&E) staining (FUJIFILM Wako
Pure Chemical Corporation) was performed using consecutive
tissue sections. Leica DMD108 (Leica Microsystems GmbH)was used to examine the sections and obtain images.
Determination of the binding affinity. Cells were suspended in
100 µl of serially diluted mAbs (0.3 ng/ml‑5 µg/ml), followedby the addition of Alexa Fluor 488‑conjugated anti‑mouse
IgG (1:200; Cell Signaling Technology, Inc.). Fluorescencemicroscopy data were collected using an EC800 Cell Analyzer(Sony Corp.). The dissociation constant (KD) was calculated byfitting binding isotherms to built‑in one‑site binding models in
GraphPad PRISM 8 (GraphPad Software, Inc.).
Western blot analysis. Cell lysates (10 µg) were boiled in
sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque,Inc.). Proteins were separated on 5‑20% polyacrylamidegels (FUJIFILM Wako Pure Chemical Corporation) andtransferred onto polyvinylidene difluoride (PVDF) membranes(Merck KGaA). After blocking with 4% skim milk (Nacalai
Tesque, Inc.) in PBS with 0.05% Tween 20, the membranes
were incubated with 10 µg/ml of an anti‑CD44 mAb [clone
C44Mab‑46 (mouse IgG1, kappa)]; available from Antibody
Bank of Tohoku University (ABTU; Miyagi, Japan); http://www.
med‑tohoku‑antibody.com/topics/001_paper_antibody_PDIS.
htm#antiCD44) or 1 µg/ml of anti‑ β ‑actin (clone AC‑15;
cat. no. A5441; Sigma‑Aldrich Corp.; Merck KGaA). This wasfollowed by incubation with peroxidase‑conjugated anti‑mouse
immunoglobulins (Agilent Technologies Inc.). Finally, protein
bands were detected with ImmunoStar LD (FUJIFILM
Wako Pure Chemical Corporation) using a Sayaca‑Imager(DRC Co., Ltd.).
Reverse transcription‑polymerase chain reaction (RT‑PCR).
Total RNAs were prepared from cell lines using an RNeasy
Mini Prep Kit (Qiagen Inc.). The initial cDNA strand wassynthesized using SuperScript IV Reverse Transcriptase

1951(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 µM
of primer sets: Human CD44 sense (5'‑GAA​AGG​AGC​AGC​
ACT​TCA​GG‑3'), human CD44 antisense (5'‑ACT​GCA​ATG​
CAA​ACT​G CA​AGC‑3'), GAPDH sense (5'‑CAA​TGA​CCC​
CTT​CAT ​TGA​CC‑3'), and GAPDH antisense (5'‑GTC​T TC​
TGG​GTG​GCA​GTG​AT‑3').
ADCC. Six six‑week‑old female BALB/c nude mice werepurchased from Charles River (Kanagawa, Japan). Aftereuthanization by cervical dislocation, spleens were removed
aseptically and single‑cell suspensions obtained by forcing
spleen tissues through a sterile cell strainer (352360,
BD Falcon, Corning, Inc.) using a syringe. Erythrocytes
were lysed with a 10‑sec exposure to ice‑cold distilled water.
Splenocytes were washed with DMEM and resuspended
in DMEM with 10% FBS and used as effector cells. Target
cells were labeled with 10‑µg/ml Calcein AM (Thermo Fisher
Scientific, Inc.) and resuspended in the same medium. The
target cells (2x104 cells/well) were plated in 96‑well plates andmixed with effector cells, anti‑CD44s antibodies, or control
IgG (mouse IgG2a) (Sigma‑Aldrich Corp.; Merck KGaA). Aftera 5‑h incubation, the Calcein AM release of supernatant fromeach well was measured. Fluorescence intensity was determined using a microplate reader (Power Scan HT) (BioTek
Instruments) with an excitation wavelength of 485 nm and anemission wavelength of 538 nm. Cytolytic activity (as % oflysis) was calculated using the equation: % lysis=(E‑S)/(M‑S)
x100, where E is the fluorescence of the combined target andeffector cells, S is the spontaneous fluorescence of the target
cells only, and M is the maximum fluorescence measured after
lysing all cells with a buffer containing 0.5% Triton X‑100,
10 mM Tris‑HCl (pH 7.4), and 10 mM of EDTA.
CDC. Cells in DMEM supplemented with 10% FBS wereplated in 96‑well plates (2x10 4 cells/well), and incubatedfor 5 h at 37˚C with either anti‑CD44s antibodies or control
IgG (mouse IgG2a) (Sigma‑Aldrich Corp.; Merck KGaA) and
10% rabbit complement (Low‑Tox‑M Rabbit Complement;
Cedarlane Laboratories). To assess cell viability, an MTS[3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium; inner salt] assay was
performed using a CellTiter 96®AQueous assay kit (Promega
Corp.).
3D cell proliferation assay. 3D cell proliferation was measured
with the CellTiter‑Glo® 3D cell viability assay (Promega
Corp.) according to the manufacturer's instructions. Briefly,
the cells were plated (2,000 cells/100 µl/well) in triplicate in
96‑well ultra low attachment plates (Corning Inc.) with PBS or
100 µg/ml of mouse IgG2a and an anti‑CD44 mAb (5‑mG2a‑f) in
DMEM containing 10% FBS. The cell viability was measured
after 48 h of incubation. The CellTiter‑Glo® 3D reagent wasadded into wells in a 1:1 dilution (100 µl volume in well:100 µlof reagent) and then the plates were shaken for 5 min on
an orbital shaker and incubated at room temperature for an
additional 25 min. The luminescent signal was read using an
EnSpire multi‑plate reader (Perkin Elmer). Images were taken

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TAKEI et al: ANTITUMOR ACTIVITIES OF AN ANTI-CD44s mAb

using an Evolution MP camera (Media Cybernetics). The
proliferation rate was calculated relative to the control (PBS
was added instead of the antibodies).
Antitumor activity of 5‑mG 2a ‑f in the xenografts of oral
cancers. Sixty‑four six‑week‑old female BALB/c nude mice
were purchased from Charles River (Kanagawa, Japan) andused at 10 weeks of age. HSC‑2 and SAS cells (0.3 ml of
1.33x108 cells/ml in DMEM) were mixed with 0.5 ml BD
Matrigel Matrix Growth Factor Reduced (BD Biosciences).
One hundred microliters of this suspension (5x10 6 cells)
was injected subcutaneously into the left flank. After day 1(protocol‑1) or day 7 (protocol‑2), 100 µg of 5‑mG2a‑f andcontrol mouse IgG (Sigma‑Aldrich Corp.; Merck KGaA) in
100 µl PBS were injected intraperitoneally (i.p.) into treatedand control mice, respectively. Additional antibodies werethen injected on days 7 and 14 (protocol‑1) or on days 14
and 21 (protocol‑2). Nineteen days (protocol‑1) or 27 days
(protocol‑2) after cell implantation, all mice were euthanizedby cervical dislocation and tumor diameters and volumes were
determined as previously described (32).
Statistical analyses. All data are expressed as mean ± standard
error of the mean (SEM). Statistical analysis was carried out
using ANOVA following Tukey‑Kramer's test for ADCC and
CDC. Sidak's multiple comparisons test was used for tumor
volume and mouse weight, or Welch's t test for tumor weightand 3D cell proliferation assay using GraphPad Prism 7(GraphPad Software, Inc.). P<0.05 was adopted as a level ofstatistical significance.
Results
Produ ct ion a n d ch a ra cteriza t ion of 5 ‑ m G 2 a ‑f, acore‑fucose‑deficient mouse IgG2a‑type anti‑CD44 antibody.
As mouse IgG2a possesses high ADCC and CDC activities (33),
we first produced a mouse IgG2a version of mouse IgG1
C44Mab‑5 by subcloning appropriate VH cDNA of C44Mab‑5
and CH of mouse IgG2a into pCAG‑Neo vector, and light chain of
C44Mab‑5 into pCAG‑Ble vector. This IgG2a‑type of C44Mab‑5
is henceforth referred to as 5‑mG2a. We additionally produceda core‑fucose‑deficient type of 5‑mG2a, henceforth referred toas 5‑mG2a‑f, using the BINDS‑09 cell line (FUT8‑knockout
Expi‑CHO‑S cell line) (28). Defucosylation of 5‑mG2a‑f
was confirmed using lectins such as Aleuria aurantia lectin(AAL, fucose binder) (34) and Pholiota squarrosa lectin(PhoSL, core fucose binder) (31). Concanavalin A (ConA,
mannose binder) (35) was used as a control. Both C44Mab‑5
and 5‑mG2a‑f were detected using ConA (Fig. 1A). C44Mab‑5,
but not 5‑mG2a‑f, was detected using AAL (Fig. 1B) or PhoSL(Fig. 1C), indicating that 5‑mG2a‑f was defucosylated.
We examined the sensitivity of 5‑mG 2a ‑f in CHO
cells expressing CD44v3‑10 plus N‑terminal PA16 tag(CHO/PA16‑CD44v3‑10) and in oral squamous cell carcinoma(OSCC) cell lines (SAS and HSC‑2) using flow cytometry. BothC44Mab‑5 and 5‑mG2a‑f reacted with CHO/PA16‑CD44v3‑10
cells (Fig. 2A), but not with CHO‑K1 cells (Fig. 2B). BothC44Mab‑5 and 5‑mG2a‑f reacted with SAS cells (Fig. 2C) and
HSC‑2 cells (Fig. 2D), indicating that both mAbs showed highsensitivity against SAS and HSC‑2 cells.

Figure 1. Confirmation of defucosylation of 5‑mG2a‑f by enzyme‑linked
immunosorbent assay (ELISA) using lectins. (A) C44Mab‑5 and 5‑mG2a‑f
were immobilized and incubated with biotin‑labeled concanavalin A (ConA), followed by peroxidase‑conjugated streptavidin. The enzymatic reaction
was produced using a 1‑Step Ultra TMB‑ELISA. (B) C44Mab‑5 and 5‑mG2a‑f
were immobilized and incubated with biotin‑labeled Aleuria aurantia lectin(AAL), followed by peroxidase‑conjugated streptavidin. The enzymatic
reaction was produced using a 1‑Step Ultra TMB‑ELISA. (C) C44Mab‑5
and 5‑mG 2a ‑f were immobilized and incubated with biotin‑labeled
Pholiota squarrosa lectin (PhoSL), followed by peroxidase‑conjugatedstreptavidin. The enzymatic reaction was produced using a 1‑Step Ultra
TMB‑ELISA.

As shown in Fig. S1A, CD44 was not detected by ananti‑CD44s mAb (C44Mab‑46) in both SAS and HSC‑2 cells
presumably because the CD44 expression level in those cells
might be low for the detection in western blot analysis. Then,we performed RT‑PCR analysis for detection of CD44. As
shown in Fig. S1B, the multiple bands of CD44v were detectedin SAS and HSC‑2 cells using PCR, indicating that CD44v isexpressed in SAS and HSC‑2 cells.

ONCOLOGY REPORTS 44: 1949-1960, 2020

1953

Figure 3. Immunohistochemical analysis using anti‑CD44 mAbs against oral
squamous cell carcinomas (OSCCs). (A and B) Consecutive tissue sections of
OSCC were incubated with 1 µg/ml of C44Mab‑5 for 1 h at room temperature
followed by treatment with an Envision+ kit for 30 min. Color was developed
using DAB for 2 min, and sections were then counterstained with hematoxylin. (C and D) Consecutive tissue sections of OSCC were incubated with
1 µg/ml of 5‑mG2a‑f for 1 h at room temperature followed by treatment withan Envision+ kit for 30 min. Color was developed using DAB for 2 min, andsections were then counterstained with hematoxylin. (E and F) Hematoxylin
and eosin (HE) staining of consecutive tissue sections of OSCC. Scale bar,
100 µm. mAbs, monoclonal antibodies.

Figure 2. Flow cytometry using anti‑CD44 mAbs. (A) CHO/PA16‑CD44v3‑10
cells were treated with C 44Mab‑5 and 5‑mG2a‑f (1 µg/ml), followed bysecondary antibodies. (B) CHO‑K1 cells were treated with C44Mab‑5 and5‑mG2a‑f (1 µg/ml), followed by secondary antibodies. (C) SAS cells weretreated with C44Mab‑5 and 5‑mG2a‑f (1 µg/ml), followed by secondary antibodies. (D) HSC‑2 cells were treated with C44Mab‑5 and 5‑mG2a‑f (1 µg/ml),
followed by secondary antibodies. The black line represents the negative
control. mAbs, monoclonal antibodies.

Next, we performed immunohistochemical analysis on oral
cancer cell lines. Representative images are shown in Fig. 3. BothC44Mab‑5 (Fig. 3A and B) and 5‑mG2a‑f (Fig. 3C and D) stained
the plasma membrane of oral cancer cells. The sensitivity of
5‑mG2a‑f was similar with that of C44Mab‑5. Both C44Mab‑5 and5‑mG2a‑f stained 33/38 cases (86.8%) of OSCCs of the tissue
microarray. Hematoxylin & eosin (H&E) staining of consecutive tissue sections of OSCC is shown in Fig. 3E and F.
We performed a kinetic analysis of the interactions of
recC44Mab‑5 and 5‑mG2a‑f with SAS and HSC‑2 oral cancer

cell lines using flow cytometry. As shown in Fig. 4A, thedissociation constant (KD) for recC44Mab‑5 in SAS cells was
2.4x10‑10 M. In contrast, the KD for 5‑mG2a‑f in SAS cells was
2.8x10‑10 M (Fig. 4A). The binding affinity of 5‑mG2a‑f in SAS
cells was very similar to that of recC44Mab‑5. Likewise, theKD for recC44Mab‑5 against HSC‑2 was 2.3x10‑9 M (Fig. 4B).
In contrast, the KD for 5‑mG2a‑f in HSC‑2 cells was 2.6x10‑9 M(Fig. 4B). The binding affinity of 5‑mG2a‑f in HSC‑2 cells wasvery similar to that of recC44Mab‑5. The binding affinity of
5‑mG2a‑f in SAS was 9.3‑fold higher than that against HSC‑2.
ADCC and CDC activities of 5‑mG2a‑f in oral cancer celllines. Because the mouse IgG1 subclass of C44Mab‑5 does notpossess ADCC or CDC activities, we synthesized a mouse
IgG2a subclass mAb, and further defucosylated it to augment
those activities. In this study, we examined whether the developed 5‑mG2a‑f induced ADCC and CDC in CD44‑expressing
oral cancer cell lines, such as SAS and HSC‑2 cells. As shownin Fig. 5A, 5‑mG2a‑f exhibited higher ADCC (16% cytotoxicity) in SAS cells compared with that of control PBS (3.4%
cytotoxicity; P<0.01) and control mouse IgG2a treatment (4.2%
cytotoxicity; P<0.01). Similarly, 5‑mG2a‑f exhibited higher
ADCC (18% cytotoxicity) against HSC‑2 cells compared withthat of control PBS (3.1% cytotoxicity; P<0.01) and control
mouse IgG2a treatment (5.2% cytotoxicity; P<0.01), indicatingthat ADCC in SAS cells is similar with that in HSC‑2 cells,

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TAKEI et al: ANTITUMOR ACTIVITIES OF AN ANTI-CD44s mAb

Figure 4. Determination of the binding affinity of anti‑CD44 mAbs for oral cancer cells using flow cytometry. (A) SAS cells were suspended in 100 µl ofserially diluted mAbs (0.3 ng/ml‑5 µg/ml), followed by the addition of Alexa Fluor 488‑conjugated anti‑mouse IgG. Fluorescence data were collected using
an EC800 Cell Analyzer. (B) HSC‑2 cells were suspended in 100 µl of serially diluted mAbs (0.3 ng/ml‑5 µg/ml), followed by the addition of Alexa Fluor
488‑conjugated anti‑mouse IgG. Fluorescence data were collected using an EC800 Cell Analyzer. KD, dissociation constants; mAbs, monoclonal antibodies.

despite the binding affinity of 5‑mG2a‑f in SAS cells being
9.3‑fold higher than in HSC‑2 cells (Fig. 4). As shown in
Fig. 5B, 5‑mG2a ‑f exhibited slightly higher CDC (33%
cytotoxicity) in SAS cells compared with control PBS (21%cytotoxicity; P<0.01) and control mouse IgG2a treatment (22%cytotoxicity; P<0.01). Similarly, 5‑mG2a‑f exhibited slightly
higher CDC (30% cytotoxicity) in HSC‑2 cells compared with
control PBS (18% cytotoxicity; P<0.01) and control mouse
IgG2a treatment (19% cytotoxicity; not significant). Although
ADCC/CDC activities of 5‑mG2a‑f in oral cancer cells are notoutstanding, 5‑mG2a‑f may exert antitumor activity against
oral cancer cells in vivo.
The influence of 5‑mG 2a ‑f in oral cancer cell lines inanchorage‑independent condition. Next, we investigatedwhether 5‑mG2a‑f inhibits cell growth of SAS and HSC‑2 cells
in anchorage‑independent condition. As shown in Fig. S2A,
both SAS and HSC‑2 cells grew in anchorage‑independentcondition for 48 h. In contrast, an anti‑CD44 mAb (5‑mG2a‑f)
did not inhibit the growth of SAS or HSC‑2 compared tocontrol mouse IgG2a (Fig. S2B), indicating that 5‑mG2a‑f
did not affect the cell growth of oral cancer cell lines inanchorage‑independent condition.

Antitumor activities of 5‑mG2a ‑f in the mouse xenograftsof SAS oral cancer cells. SAS cells were subcutaneously
implanted into the flanks of nude mice. In protocol‑1, 5‑mG2a‑f(100 µg) and control mouse IgG (100 µg) were injected i.p.
three times into the mice, on days 1, 7, and 14 after SAS cell
injections. Tumor volume was measured on days 6, 12, 15,
and 19. Tumor development was significantly reduced in the
5‑mG2a‑f‑treated mice on days 12, 15, and 19 in comparison to
the IgG‑treated control mice (Fig. 6A). Tumor volume on day
19 was reduced by 27% after 5‑mG2a‑f treatment. Tumors from
5‑mG2a‑f‑treated mice weighed significantly less than tumors
from IgG‑treated control mice (16.9% reduction, Fig. 6B).
Resected tumors on day 19 are depicted in Fig. 6C. Control
and 5‑mG2a‑f‑treated SAS xenograft mice are shown on day 19
in Fig. S3A and B, respectively. Total body weights did not
significantly differ between the two groups (Fig. S3C).
In protocol‑2 of the SAS xenograft models, tumor formationof 16 SAS‑bearing mice was observed on day 7. Then, these
16 SAS‑bearing mice were divided into a 5‑mG2a‑f‑treatedgroup and a control group. On days 7, 14, and 21 after SAS cell
injections into the mice, 5‑mG2a‑f (100 µg) and control mouse
IgG (100 µg) were injected i.p. into the mice. Tumor formation was observed in mice in both treated and control groups.

ONCOLOGY REPORTS 44: 1949-1960, 2020

1955

Figure 5. Evaluation of ADCC and CDC activities by 5‑mG2a‑f. (A) ADCC activities by 5‑mG2a‑f, control mouse IgG2a, and control PBS in SAS and HSC‑2
cells. (B) CDC activities by 5‑mG2a‑f, control mouse IgG2a, and control PBS in SAS and HSC‑2 cells. Values are mean ± SEM. Asterisk indicates statisticalsignificance (**P<0.01; n.s., not significant; ANOVA and Tukey‑Kramer's test). ADCC, antibody‑dependent cellular cytotoxicity; CDC, complement‑dependent
cytotoxicity.

Tumor volume was measured on days 7, 12, 15, 19, 22, and 27.
5‑mG2a‑f‑treated mice displayed significantly reduced tumor
development on days 22 and 27 in comparison to IgG‑treatedcontrol mice (Fig. 7A). Tumor volume reduction by 5‑mG2a‑f
was 43% on day 27. Tumors from the 5‑mG2a‑f‑treated miceweighed significantly less than tumors from the IgG‑treatedcontrol mice (27.1% reduction, Fig. 7B). Resected tumors on
day 27 are depicted in Fig. 7C. Control and 5‑mG2a‑f‑treated
SAS xenograft mice are shown on day 27 in Fig. S4A and B,
respectively. Total body weights did not significantly differ
between the two groups (Fig. S4C). These results indicate that
5‑mG2a‑f reduced the growth of SAS xenografts effectively,
even when 5‑mG2a ‑f was injected 7 days post‑SAS cell
injections in mice.
Antitumor activities of 5‑mG2a‑f in mouse xenografts of HSC‑2
oral cancer cells. In a second xenograft model of oral cancers,
HSC‑2 cells were subcutaneously implanted into the flanks ofnude mice. In protocol‑1 of HSC‑2 xenograft models, 5‑mG2a‑f(100 µg) and control mouse IgG (100 µg) were injected i.p.
three times into the mice, on days 1, 7, and 14 after HSC‑2cell injections into the mice. Tumor volume was measured
on days 6, 12, 15, and 19. 5‑mG2a‑f‑treated mice displayed

significantly reduced tumor development on days 12, 15, and
19 in comparison to IgG‑treated control mice (Fig. 8A). Tumor
volume reduction by 5‑mG2a‑f was 53% on day 19. Tumors
from 5‑mG2a‑f‑treated mice weighed significantly less thanHSC‑2 tumors from IgG‑treated control mice (44.1% reduction, Fig. 8B). Resected tumors on day 19 are depicted inFig. 8C. Control and 5‑mG2a‑f‑treated HSC‑2 xenograft mice
are shown on day 19 in Fig. S5A and B, respectively. Total
body weights did not significantly differ between the twogroups (Fig. S5C).
In protocol‑2 of the HSC‑2 xenograft models, tumor
formation of 16 HSC‑2‑bearing mice was observed on day
7. Then, these 16 HSC‑2‑bearing mice were divided into a5‑mG2a‑f‑treated group and a control group. On days 7, 14, and21 after cell injections into the mice, 5‑mG2a‑f (100 µg) andcontrol mouse IgG (100 µg) were injected i.p. into the mice.
Tumor volume was measured on days 7, 12, 15, 19, 22, and27. 5‑mG2a‑f‑treated mice displayed significantly reduced
tumor development on days 22 and 27 in comparison to
IgG‑treated control mice (Fig. 9A). Tumor volume reductionin 5‑mG2a‑treated mice was 32% on day 27. Tumors from
5‑mG2a‑f‑treated mice weighed significantly less than tumors
from IgG‑treated control mice (27.1% reduction, Fig. 9B).

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TAKEI et al: ANTITUMOR ACTIVITIES OF AN ANTI-CD44s mAb

Figure 6. Evaluation of antitumor activity of 5‑mG2a‑f (from day 1) in SAS
xenografts. (A) SAS cells (5x106 cells) were injected subcutaneously into theleft flank. After day 1, 100 µg of 5‑mG2a‑f and control mouse IgG in 100 µl
PBS were injected i.p. into treated and control mice, respectively. Additional
antibodies were then injected on days 7 and 14. Tumor volume was measured
on days 6, 12, 15, and 19. Values are mean ± SEM. Asterisk indicatesstatistical significance (**P<0.01; n.s., not significant; ANOVA and Sidak's
multiple comparisons test). (B) Tumors of SAS xenografts were resectedfrom 5‑mG2a‑f and control mouse IgG groups. Tumor weight on day 19 was
measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (*P<0.05, Welch's t test). (C) Resected tumorsof SAS xenografts from 5‑mG2a‑f and control mouse IgG groups on day 19.
Scale bar, 1 cm.

Resected tumors on day 27 are depicted in Fig. 9C. Control and
5‑mG2a‑f‑treated HSC‑2 xenograft mice are shown on day 27
in Fig. S6A and B, respectively. Total body weights did not
significantly differ between the two groups (Fig. S6C). These
results indicate that 5‑mG2a‑f reduced the growth of HSC‑2
xenografts effectively, even when 5‑mG2a‑f was injected 7 days
post‑HSC‑2 cell injections in mice.
Discussion
In the present study, we investigated whether anti‑CD44
mAbs are advantageous for the treatment of oral cancers. We
previously developed a sensitive and specific anti‑CD44 mAb,
C44Mab‑5, but were unable to demonstrate antitumor activity
because the IgG1 subclass does not possess ADCC/CDC
activities (27). In this study, we developed this antibody into an
IgG2a subclass antibody, and augmented ADCC activity using
a defucosylated variant. Oral cancers comprise several histological tumor types, such as squamous cell carcinoma (SCC),
adenocarcinoma, mucoepidermoid carcinoma, and adenoid

Figure 7. Evaluation of antitumor activity of 5‑mG2a‑f (from day 7) in SAS
xenografts. (A) SAS cells (5x106 cells) were injected subcutaneously into theleft flank. After day 7, 100 µg of 5‑mG2a‑f and control mouse IgG in 100 µl
PBS were injected i.p. into treated and control mice, respectively. Additional
antibodies were then injected on days 14 and 21. Tumor volume was measured
on days 7, 12, 15, 19, 22, and 27. Values are mean ± SEM. Asterisk indicatesstatistical significance (**P<0.01; n.s., not significant; ANOVA and Sidak's
multiple comparisons test). (B) Tumors of SAS xenografts were resectedfrom 5‑mG2a‑f and control mouse IgG groups. Tumor weight on day 27 was
measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (*P<0.05, Welch's t test). (C) Resected tumorsof SAS xenografts from 5‑mG2a‑f and control mouse IgG groups on day 27.
Scale bar, 1 cm.

cystic carcinoma. Among these, SCC accounts for over 90%
of all oral cancers (36). Therefore, we used SAS and HSC‑2cell lines, which are derived from SCC, and investigated
ADCC/CDC and antitumor activities.
The most effective treatment of OSCC depends upon its
clinical stage. Stage‑I and ‑II (early stages) are treated via
surgery or radiotherapy alone. In contrast, stage‑III and ‑IV(advanced stages) require a combination of surgery, radiotherapy, and chemotherapy (37). For chemotherapy of OSCCs,
cisplatin is mainly used, and is often combined with docetaxel
and 5‑fluorouracil (38,39). Other anticancer agents, such as
paclitaxel, carboplatin, and methotrexate can be also used for
OSCCs (40), but effective molecular targeting drugs, such as
antibody therapies, are limited.
Recently, cetuximab, a mouse‑human chimeric mAb(IgG1) that targets epidermal growth factor receptor (EGFR),
was approved by the Food and Drug Administration (FDA)
in the USA for treatment of oral cancers (41). Cetuximab has
been shown effective against locoregionally advanced head

ONCOLOGY REPORTS 44: 1949-1960, 2020

Figure 8. Evaluation of antitumor activity of 5‑mG2a‑f (from day 1) in HSC‑2
xenografts. (A) HSC‑2 cells (5x106 cells) were injected subcutaneously into
the left flank. After day 1, 100 µg of 5‑mG2a‑f and control mouse IgG in 100 µl
PBS were injected i.p. into treated and control mice, respectively. Additional
antibodies were then injected on days 7 and 14. Tumor volume was measured
on days 6, 12, 15, and 19. Values are mean ± SEM. Asterisk indicatesstatistical significance (**P<0.01; n.s., not significant, ANOVA and Sidak's
multiple comparisons test). (B) Tumors of HSC‑2 xenografts were resectedfrom 5‑mG2a‑f and control mouse IgG groups. Tumor weight on day 19 was
measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (**P<0.01, Welch's t test). (C) Resected tumors of
HSC‑2 xenografts from 5‑mG2a‑f and control mouse IgG groups on day 19.
Scale bar, 1 cm.

and neck cancer and recurrent or metastatic squamous cell
carcinoma of the head and neck (41‑43). Although advancesin diagnosis and therapeutic techniques have improved theoverall 5‑year survival rate to 70%, the 5‑year survival rate
in stage IV is only 40%; therefore, further treatments must
be developed (44). In our recent study, we also developed asensitive and specific mAb (EMab‑17) against EGFR, anddemonstrated its ADCC/CDC and antitumor activity against
SAS and HSC‑2 xenografts (32). Although we showed that
EMab‑17 could potentially be used for antibody‑based therapy
for EGFR‑expressing OSCC, the difference between cetuximab and EMab‑17 has not been clarified. Several studies
characterizing EMab‑17, including epitope mapping and signal
induction in OSCC cells, are currently ongoing.
In another recent study, HER2 was shown to be expressedin oral cancers, and an anti‑HER2 mAb (H2Mab‑19) demonstrated antitumor activity (45). Therefore, anti‑HER2 therapies
using trastuzumab could be effective for the treatment oforal cancers. HER2 expression was reported in only 1.4% of1957

Figure 9. Evaluation of antitumor activity of 5‑mG2a‑f (from day 7) in HSC‑2
xenografts. (A) HSC‑2 cells (5x106 cells) were injected subcutaneously into
the left flank. After day 7, 100 µg of 5‑mG2a‑f and control mouse IgG in 100 µl
PBS were injected i.p. into treated and control mice, respectively. Additional
antibodies were then injected on days 14 and 21. Tumor volume was measured
on days 7, 12, 15, 19, 22, and 27. Values are mean ± SEM. Asterisk indicatesstatistical significance (**P<0.01; n.s., not significant; ANOVA and Sidak's
multiple comparisons test). (B) Tumors of HSC‑2 xenografts were resectedfrom 5‑mG2a‑f and control mouse IgG groups. Tumor weight on day 27 was
measured from excised xenografts. Values are mean ± SEM. Asterisk indicates statistical significance (*P<0.05, Welch's t test). (C) Resected tumors of
HSC‑2 xenografts from 5‑mG2a‑f and control mouse IgG groups on day 27.
Scale bar, 1 cm.

immunohistochemical analyses of oral cancer (46), although it
is expressed in 10.4% of breast cancers (47). Therefore, targeting
only HER2 may be insufficient for conquering oral cancers.
As antitumor effects of combined gefitinib and trastuzumab orcetuximab and trastuzumab treatment on head and neck SCC(HNSCC) were demonstrated in vitro (48,49), those few oral
cancer patients displaying HER2 overexpression/amplification
may possibly benefit from anti‑HER2 therapy.
Furthermore, we previously investigated whether podocalyxin (PODXL) may be a therapeutic target in OSCC usinganti‑PODXL mAbs (50). We engineered an anti‑PODXL mAb
of IgG1 subclass (PcMab‑47) into a mouse IgG2a‑type mAb(47‑mG2a) to increase ADCC. We further developed 47‑mG2a‑f,
a core fucose‑deficient variant of 47‑mG2a to further augment
its ADCC. In vivo analysis revealed that 47‑mG2a‑f, but not
47‑mG2a, exhibited antitumor activity in SAS and HSC‑2 xenograft models at a dose of 100 µg/mouse/week administered
three times. Although both 47‑mG2a and 47‑mG2a‑f exhibited

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TAKEI et al: ANTITUMOR ACTIVITIES OF AN ANTI-CD44s mAb

antitumor activity in HSC‑2 xenograft models at a dose of
500 µg/mouse/week administered twice, 47‑mG2a‑f also demonstrated higher antitumor activity than 47‑mG2a, indicating thata core fucose‑deficient anti‑PODXL mAb could be useful for
antibody‑based therapy against PODXL‑expressing OSCCs.
Therefore, we used the core‑fucose‑deficient anti‑CD44 mAb(5‑mG2a‑f) for treating CD44‑expressing oral cancers.
In this study, we demonstrated that 5‑mG2a ‑f exerts
ADCC/CDC activities in vitro, and antitumor activities in vivo.
Importantly, 5‑mG2a‑f effectively reduced the growth of SAS
and HSC‑2 xenografts, even when 5‑mG2a‑f was injected 7 days
after cell implantations into the mice. However, tumor volume
reduction of SAS and HSC‑2 on day 27 by 5‑mG2a‑f was still
only 43 and 32%, respectively, indicating that anti‑CD44
therapy might not be robust enough for conquering most oral
cancers. One potential reason for this weak antitumor activity
is the lower ADCC activity and CDC activity of 5‑mG2a‑f,
despite high binding activity in SAS cells (KD: 2.8x10 ‑10 M)
and HSC‑2 cells (KD: 2.6x10‑9 M).
In our previous report, C44Mab‑5 detected CD44s (27).
Although the binding epitope of C44Mab‑5 may potentially
be located between exon‑1 and exon‑5, we have not been able
to determine the exact binding epitope of C44Mab‑5, likely
because C44Mab‑5 recognizes the tertiary structure of CD44
rather simple peptides or glycans. Because the binding epitopeis critical for ADCC/CDC activities of mAbs, other anti‑CD44
mAbs of various epitopes will need to be developed in futurestudies.
Targeting multiple targets, such as EGFR, HER2,PODXL, and CD44 may be needed for effective therapy toconquer oral cancers. Another important goal is the targetingof cancer‑specific antigens using a cancer‑specific mAb(CasMab) because EGFR, HER2, PODXL, and CD44 arewidely expressed in normal tissues. We previously established
CasMab against podoplanin (PDPN), which is expressed inmany cancers, including oral cancers (51‑54). In xenograftmodels with HSC‑2 cells, a mouse‑human chimeric mAb,
chLpMab‑23, exerted antitumor activity using human naturalkiller cells, indicating that chLpMab‑23 may be useful for
antibody therapy against PDPN‑expressing oral cancers (54).
In the future study, cancer‑specific anti‑CD44 mAbs may also
be developed that can reduce the adverse effects of traditional
antibody therapy.
Acknowledgements
We thank Ms. Saori Handa and Mr. Yu Komatsu (Departmentof Antibody Drug Development, Tohoku University Graduate
School of Medicine) for technical assistance concerning thein vitro experiments, and Ms. Akiko Harakawa [Instituteof Microbial Chemistry (BIKAKEN), Numazu, Microbial
Chemistry Research Foundation] for technical assistanceregarding the animal experiments.
Funding
This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under grantnos. JP20am0401013 (YK), JP20am0101078 (YK), andJP20ae0101028 (YK), and by the Japan Society for the

Promotion of Science (JSPS) Grants‑in‑Aid for Scientific
Research (KAKENHI) grant nos. 17K07299 (MKK),
19K07705 (YK), and 20K16322 (MS).
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
HHo, JT, TO, TN, MS, TA, YS, and MY performed the experiments. MKK analyzed the experimental data. MK, HHa, andYK designed the current study and wrote the manuscript. All
authors read and approved the manuscript and agree to beaccountable for all aspects of the research in ensuring that theaccuracy or integrity of any part of the work are appropriately
investigated and resolved.
Ethics approval and consent to participate
Animal studies for ADCC and the antitumor activity wereapproved by the Institutional Committee for Experiments of
the Institute of Microbial Chemistry (Numazu‑shi, Shizuoka,
Japan) (permit no. 2020‑003).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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