International Journal of

Molecular Sciences
ArticleAnti-CD44 Variant 10 Monoclonal Antibody Exerts Antitumor
Activity in Mouse Xenograft Models of Oral Squamous
Cell Carcinomas
Kenichiro Ishikawa 1,2 , Hiroyuki Suzuki 1, * , Tomokazu Ohishi 3,4 , Guanjie Li 1 , Tomohiro Tanaka 1 ,Manabu Kawada 4 , Akira Ohkoshi 2 , Mika K. Kaneko 1 , Yukio Katori 2 and Yukinari Kato 1, *
1

2

3

4

*

Citation: Ishikawa, K.; Suzuki, H.;
Ohishi, T.; Li, G.; Tanaka, T.; Kawada,
M.; Ohkoshi, A.; Kaneko, M.K.; Katori,
Y.; Kato, Y. Anti-CD44 Variant 10
Monoclonal Antibody Exerts
Antitumor Activity in Mouse
Xenograft Models of Oral Squamous
Cell Carcinomas. Int. J. Mol. Sci. 2024,
25, 9190. https://doi.org/10.3390/
ijms25179190
Academic Editor: Norio Sogawa
Received: 2 August 2024

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan; ken.ishikawa.r3@dc.tohoku.ac.jp (K.I.);
clownair716@gmail.com (G.L.); tomohiro.tanaka.b5@tohoku.ac.jp (T.T.);mika.kaneko.d4@tohoku.ac.jp (M.K.K.)
Department of Otolaryngology, Head and Neck Surgery, Tohoku University Graduate School of Medicine,
1-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan; ohkoshia@hotmail.com (A.O.);
yukio.katori.d1@tohoku.ac.jp (Y.K.)
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
18-24 Miyamoto, Numazu-shi 410-0301, Shizuoka, Japan; ohishit@bikaken.or.jp
Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology, Microbial Chemistry Research
Foundation, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan; kawadam@bikaken.or.jp
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)

Abstract: CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considereda tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44
variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas
compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb)therapy. We previously developed an anti-CD44v10 mAb, C44 Mab-18 (IgM, kappa), to recognize
the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44 Mab-18(C44 Mab-18-mG2a ) was generated to evaluate the antitumor activities against CD44-positive cells
compared with the previously established anti-pan CD44 mAb, C44 Mab-46-mG2a . C44 Mab-18mG2a exhibited higher reactivity compared with C44 Mab-46-mG2a to CD44v3–10-overexpressed
CHO-K1 (CHO/CD44v3–10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow
cytometry. C44 Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC)
against CHO/CD44v3–10. In contrast, C44 Mab-46-mG2a showed a superior complement-dependent
cytotoxicity (CDC) against CHO/CD44v3–10. A similar tendency was observed in ADCC and
CDC against HSC-2 and SAS. Furthermore, administering C44 Mab-18-mG2a or C44 Mab-46-mG2asignificantly suppressed CHO/CD44v3–10, HSC-2, and SAS xenograft tumor growth comparedwith the control mouse IgG2a . These results indicate that C44 Mab-18-mG2a could be a promisingtherapeutic regimen for CD44v10-positive tumors.

Revised: 20 August 2024
Accepted: 22 August 2024

Keywords: monoclonal antibody therapy; CD44v10; ADCC; CDC; oral cancer

Published: 24 August 2024

1. Introduction
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

CD44 is involved in tumor malignant progression through the promotion of tumor cellproliferation, migration, invasiveness, and stemness [1,2]. The variety of CD44 function ismediated by the alternative splicing of 20 exons [3–5]. CD44 standard (CD44s), the shortest
isoform of CD44, is generated by the first five (1–5) and the last five (16–20) exons andexpressed in a broad range of tissues [6–8]. The central (6–15) exons are alternatively spliced
and inserted between the first and last five exons of CD44s. The variant exon-containing
CD44 is designated as the CD44 variant (CD44v) isoform [9–12].

Int. J. Mol. Sci. 2024, 25, 9190. https://doi.org/10.3390/ijms25179190

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The CD44 ectodomain includes a hyaluronic acid (HA)-binding domain (HABD) [13–15].
The HABD is present in both CD44s and CD44v isoforms. Upon HA binding, CD44s andCD44v transduce the intracellular signaling through the cytoplasmic domain, which promotes
cell migration and proliferation [16]. The variant exon-encoding regions possess a variety
of functions. The v3-encoded region can be attached to a heparan sulfate side chain, which
recruits heparin-binding growth factors and stimulates the signal transduction through activation of the receptors [4,17,18]. The v6-encoded region potentiates the MET signaling pathway
through the formation of a ternary complex with its ligand, hepatocyte growth factor [19–22].
Moreover, the v8–10-encoded region regulates the intracellular reduced glutathione levels
through the promotion of a cystine–glutamate transporter function [23]. These functions
are essential for tumor cell proliferation, invasiveness, and survival to oxidative stress andchemotherapeutic drugs [24–29]. Therefore, CD44 has been considered as an essential target
for tumor therapy [14,30].
Monoclonal antibodies (mAbs) against CD44 have been evaluated in clinical trials [31,32].
RG7356, a humanized anti-pan-CD44 mAb, exhibited the antitumor effect for B cell leukemia
but no cytotoxicity on normal B cells [33]. In a human chronic lymphocytic leukemia-engraftedmouse model, RG7356 administration resulted in complete clearance of engrafted leukemia
cells [33]. In acute myeloid leukemia [34] and advanced CD44-positive solid tumors [35],phase I clinical trials were conducted. Although RG7356 exhibited an acceptable safety profile,the studies were terminated due to the lack of dose–response relationship with RG7356 inboth clinical and pharmacodynamic aspects [35].
Since CD44v expression is restricted in epithelial tissue and carcinomas, anti-CD44v
mAbs were developed and evaluated in clinical studies. Humanized anti-CD44v6 mAbs
(BIWA-4 and BIWA-8) labeled with 186 Re showed antitumor efficacy in head and necksquamous cell carcinoma (SCC) xenograft-bearing mice [36]. Moreover, the antibody–drugconjugate (ADC) of BIWA-4, bivatuzumab–mertansine was developed and evaluated inclinical trials [32]. However, the clinical trials were terminated due to severe toxicity in theskin, probably due to the efficient accumulation of mertansine in the skin [32,37]. Therefore,anti-CD44 mAbs with more potent efficacy and lower toxicity to normal cells are desired.
The Fc region of therapeutic mAb binds to FcγRs on dendritic cells, macrophages, andneutrophils, which influences the adaptive immune responses through antigen presentationand cytokine production [38]. Moreover, the FcγR binding results in the activation ofnatural killer (NK) cells [39] and macrophages [40], which mediates antibody-dependent
cellular cytotoxicity (ADCC). The complement-dependent cellular cytotoxicity (CDC) isalso considered as an essential effector function in tumor immunotherapy [41]. The Fc
region of therapeutic mAbs binds to complement C1q, facilitating the assembly of active
C1 complex (C1q, C1r, and C1s). The reaction of the complement cascade finally promotes
the assembly of the pore-forming membrane attack complex (MAC or C5b–C9) on thetumor cell membrane, which results in the terminal cell lysis [41]. The involvement of CDC
in the antitumor effect was first recognized in the treatment of B cell lymphomas by ananti-CD20 mAb, rituximab [42,43]. Furthermore, the cytolytic capacity by complement
has been shown in anti-CD38 and CD52 immunotherapies for multiple myeloma andchronic lymphocytic leukemia, respectively [43–45]. Moreover, a growing body of evidence
suggests that complement plays critical roles in not only tumor cell lysis but also in several
immunologic functions in antitumor immunity [46,47]. In the immunotherapy against solid
tumors, an anti-HER2 bispecific and biparatopic antibody zanidatamab exerted more potent
CDC against HER2-positive breast cancers compared with clinically approved anti-HER2
mAb trastuzumab [48].
The Cell-Based Immunization and Screening method is a strategy to obtain mAbs
against a membrane protein comprehensively. We immunized mice with the CD44v3–10overexpressed cells or CD44v3–10 ectodomain and established various anti-CD44 mAbs
and determined their epitopes. C44 Mab-5 [49] and C44 Mab-46 [50] are anti-pan-CD44
mAbs, which have the epitopes within the constant exon 2- and 5-encoded sequences, respectively [51,52]. We also established various CD44v-specific mAbs. C44 Mab-6 recognizes

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variant exon 3-encoded sequences and is referred to as anti-CD44v3 mAb [53]. Furthermore,
C44 Mab-3 (an anti-CD44v5 mAb) [54], C44 Mab-9 (an anti-CD44v6 mAb) [55], C44 Mab-34(an anti-CD44v7/8 mAb) [56], C44 Mab-1 (an anti-CD44v9 mAb) [57], and C44 Mab-18 (ananti-CD44v10 mAb) [58] were established. C44 Mab-108 is an anti-CD44v4 mAb, established by peptide immunization [59]. These mAbs cover almost all variant exons and areapplicable to flow cytometry, Western blotting, and immunohistochemistry.
In this study, we produced an IgG2a -type C44 Mab-18 (C44 Mab-18-mG2a ) and investigated the antitumor efficacy against CHO/CD44v3–10 and oral SCC (OSCC) xenografts bycomparing anti-pan-CD44 mAb, C44 Mab-46-mG2a .
2. Results
2.1. Flow Cytometric Analysis against CHO/CD44v3–10, HSC-2, and SAS Cells Using
C44 Mab-18-mG2a and C44 Mab-46-mG2a
We previously established an anti-CD44v10 mAb (C44 Mab-18, IgM, kappa) andshowed the availability for flow cytometry, Western blotting, and immunohistochemistry
against OSCC tissues [58]. In the present study, we cloned the VH cDNA of C44 Mab18 and combined it with the CH cDNA of mouse IgG2a . We also cloned the VL cDNAof C44 Mab-18 and combined it with the CL cDNA of the mouse kappa light chain. Finally, a class-switched C44 Mab-18 (C44 Mab-18-mG2a ) was produced (Figure 1A). We also
used mouse IgG2a -type C44 Mab-46 (C44 Mab-46-mG2a ) as an anti-pan CD44 mAb [60]. In
reduced conditions, we confirm the purity of original and recombinant mAbs by SDSPAGE (Supplementary Figure S1). We next confirmed the reactivity of C44 Mab-18-mG2aand C44 Mab-46-mG2a using CHO/CD44v3–10 and CHO/CD44s cells. Both C44 Mab-18mG2a and C44 Mab-46-mG2a detected CHO/CD44v3–10 cells in a concentration-dependent
manner (Figure 1B). The reactivity of C44 Mab-18-mG2a to CHO/CD44v3–10 was superior to that of C44 Mab-46-mG2a (Figure 1B). In contrast, C44 Mab-18-mG2a did not reactwith CHO/CD44s cells compared to C44 Mab-46-mG2a (Figure 1C). Both C44 Mab-18-mG2aand C44 Mab-46-mG2a did not respond with CHO-K1 cells (Supplementary Figure S2).
These results confirmed that C44 Mab-18-mG2a recognizes only CHO/CD44v3–10 and that
C44 Mab-46-mG2a recognizes both CHO/CD44v3–10 and CHO/CD44s.
We next investigated the reactivity of C44 Mab-18-mG2a and C44 Mab-46-mG2a against
endogenous CD44-expressing OSCC cell lines, HSC-2 and SAS. Both C44 Mab-18-mG2aand C44 Mab-46-mG2a reacted with HSC-2 (Figure 2A) and SAS (Figure 2B) cells in aconcentration-dependent manner. The reactivity of C44 Mab-18-mG2a to these cells was
superior to that of C44 Mab-46-mG2a (Figure 2). We also used PMab-231 as a control mouse
IgG2a, which did not react with CHO/CD44v3–10, HSC-2, and SAS cells (Supplementary
Figure S3).
2.2. Induction of ADCC and CDC by C44 Mab-18-mG2a and C44 Mab-46-mG2a against
CHO/CD44v3–10 Cells
Next, we performed in vitro ADCC and CDC assays to examine the cytotoxicity of
C44 Mab-18-mG2a and C44 Mab-46-mG2a against CHO/CD44v3–10 cells. As shown in
Figure 3A, C44 Mab-18-mG2a significantly exerted ADCC against CHO/CD44v3–10 cells in
the presence of mouse splenocytes (30% cytotoxicity, p < 0.01) compared with control mouse
IgG2a (PMab-231, 11% cytotoxicity). Although a tendency of increased ADCC comparedwith the control was observed by C44 Mab-46-mG2a , the difference was not significant.
In contrast, C44 Mab-46-mG2a significantly exerted CDC against CHO/CD44v3–10 cells
in the presence of complements (27% cytotoxicity, p < 0.05) compared with the control (12%cytotoxicity). We did not observe a significant difference in CDC between C44 Mab-18-mG2aand the control (Figure 3B)
These results indicated that C44 Mab-18-mG2a and C44 Mab-46-mG2a possess different
properties for exerting ADCC and CDC against CHO/CD44v3–10 cells.

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Figure 1.
using
C44C
Mab-18-mG
C44 Mab-46-mG
mouse mouse
2a and2a . (A)2aClass-switched
Figure1. Flow
Flowcytometry
cytometryusing
44Mab-18-mG
2a andC44Mab-46-mG. (A) Class-switched
IgG
mAbs,
C
Mab-18-mG
andC
Mab-46-mG
weregeneratedfromC
Mab-18(mouse
IgM)
2a
44
2a,
44
2a
44
IgG2a mAbs, C44Mab-18-mG2a, and C44Mab-46-mG2a were generated from C44Mab-18 (mouse
IgM)
and
C
Mab-46(mouseIgG),
respectively.
CHO/CD44v3–10(B)
and
CHO/CD44s
(C)
cells
were
44Mab-46 (mouse IgG
1 1), respectively. CHO/CD44v3–10 (B) and CHO/CD44s (C) cells were
and C44
treated with
(black)or 10–0.01µg/mL
of C44of
Mab-18-mG
C44
Mab-46-mG
2a and 2a2a (red). 2a (red).
treatedwithbuffer
buﬀercontrol
control(black)or 10–0.01µg/mL
C44Mab-18-mG
andC44Mab-46-mG
The cells
cells were
with
Alexa
Fluor
488-conjugatedanti-mouse
IgG. Fluorescence
data
The
werefurther
furthertreatedtreated
with
Alexa
Fluor
488-conjugatedanti-mouse
IgG. Fluorescence
data
were
analyzedusing
theSA3800Cell
Analyzer.
were analyzed using the SA3800 Cell Analyzer.

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Mol.Sci.
Sci. 2024,
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Figure2.2.Flow
Flowcytometryusing
C44 Mab-18-mG
C44 Mab-46-mG
againstoral squamous
Figurecytometryusing
C44Mab-18-mG
2a and2a
C44and
Mab-46-mG
2a against2a
oral
squamous
cell carci- cell
noma
(OSCC)
cell lines.
HSC-2
and SAScells
werewith buffer
control
10–0.01
carcinoma(OSCC)
celllines.(A)HSC-2(A)(B)
andSAS(B)treatedcells weretreatedwith (black)buffer orcontrol(black)44Mab-18-mG2a and C44Mab-46-mG2a (red). The cells were further treated with Alexa Fluor
µg/mL
of
C
or 10–0.01 µg/mL of C44 Mab-18-mG2a and C44 Mab-46-mG2a (red). The cells were further treatedInt. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
6 of 17488-conjugated anti-mouse IgG. Fluorescence data were analyzed using the SA3800 Cell Analyzer.
with Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were analyzed using the SA3800
Cell Analyzer.

2.2. Induction of ADCC and CDC by C44Mab-18-mG2a and C44Mab-46-mG2a against
CHO/CD44v3–10 Cells

Next, we performed in vitro ADCC and CDC assays to examine the cytotoxicity of
C44Mab-18-mG2a and C44Mab-46-mG2a against CHO/CD44v3–10 cells. As shown in Figure
3A, C44Mab-18-mG2a significantly exerted ADCC against CHO/CD44v3–10 cells in thepresence of mouse splenocytes (30% cytotoxicity, p < 0.01) compared with control mouse
IgG2a (PMab-231, 11% cytotoxicity). Although a tendency of increased ADCC comparedwith the control was observed by C44Mab-46-mG2a, the diﬀerence was not significant.
In contrast, C44Mab-46-mG2a significantly exerted CDC against CHO/CD44v3–10 cells
in the presence of complements (27% cytotoxicity, p < 0.05) compared with the control(12% cytotoxicity). We did not observe a significant diﬀerence in CDC between C44Mab18-mG2a and the control (Figure 3B)
These results indicated that C44Mab-18-mG2a and C44Mab-46-mG2a possess diﬀerent
properties for exerting ADCC and CDC against CHO/CD44v3–10 cells.

Figure3.3.Evaluation
EvaluationofofADCC
ADCCand
andCDC
CDCactivity
activity
C44 Mab-46-mG
44 Mab-18-mG
2a and2a against
Figureofof
C44CMab-18-mG
2a andC44Mab-46-mG
2a against
CHO/CD44v3–10
cells.
The
ADCC
(A)
and
CDC
(B)
inducedbyC
Mab-18-mG,
C
Mab-46-mG
CHO/CD44v3–10 cells. The ADCC (A) and CDC (B) induced by C44Mab-18-mG
2a, C2a44Mab-46-mG
2a,
44
44
2a ,
or
2a2a
(mIgG
2a, 2aPMab-231)
against
CHO/CD44v3–10
cells.cells.
Valuesare shownasor control
controlmouse
mouseIgG
IgG(mIgG, PMab-231)
against
CHO/CD44v3–10
Valuesare shownas
mean
Asterisks
indicate
statisticalsignificance(** p(**< 0.01and and* p <*0.05;
one-way
ANOVA
mean±±SEM.
SEM.
Asterisks
indicate
statisticalsignificancep < 0.01
p < 0.05;
one-way
ANOVAwithtest).
withTukey’s
Tukey’smultiple
multiplecomparisonscomparisonstest).

2.3. Antitumor Eﬀects of C44Mab-18-mG2a and C44Mab-46-mG2a in the Mouse Xenografts of
CHO/CD44v3–10
We next evaluated the in vivo antitumor eﬀect of C44Mab-18-mG2a and C44Mab-46mG2a against CHO/CD44v3–10 xenograft tumors inoculated in nude mice. C44Mab-18mG2a, C44Mab-46-mG2a, or control mouse IgG2a (100 µg/mouse) were intraperitoneally injected into mice on days 7, 14, and 21 following the inoculation. The tumor volume wasmeasured on days 7, 14, 17, 21, 24, and 28. The C44Mab-18-mG2a and C44Mab-46-mG2a ad-

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6 of 162.3. Antitumor Effects of C44 Mab-18-mG2a and C44 Mab-46-mG2a in the Mouse Xenografts of
CHO/CD44v3–10
We next evaluated the in vivo antitumor effect of C44 Mab-18-mG2a and C44 Mab-46mG2a against CHO/CD44v3–10 xenograft tumors inoculated in nude mice. C44 Mab-18mG2a , C44 Mab-46-mG2a , or control mouse IgG2a (100 µg/mouse) were intraperitoneallyinjected into mice on days 7, 14, and 21 following the inoculation. The tumor volume
was measured on days 7, 14, 17, 21, 24, and 28. The C44 Mab-18-mG2a and C44 Mab-46mG2a administration resulted in a significant reduction in tumor volume on days 17(p < 0.05 in C44 Mab-18-mG2a ), 21 (p < 0.01), 24 (p < 0.01), and 28 (p < 0.01) compared withthat of the control mouse IgG2a (Figure 4A). The C44 Mab-18-mG2a and C44 Mab-46-mG2aInt. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
7 of 17 compared with theadministration resulted in 56% and 50% reductions in tumor volume
control mouse IgG2a on day 28, respectively.

Figure4. Antitumor
activityof C442aMab-18-mG
C44 Mab-46-mG
CHO/CD44v3–10
2a and2a against
Figure4. Antitumor
activity of
C44Mab-18-mG
and C44Mab-46-mG
2a against
CHO/CD44v3–10
xen6 cells) wereograft.
(A)
Tumor
volumeof
CHO/CD44v3–10
xenograft.
CHO/CD44v3–10
cells
(5
×
10
xenograft. (A) Tumor volume of CHO/CD44v3–10 xenograft. CHO/CD44v3–10 cells (5 × 106 cells)
injected into mice subcutaneously. On days 7, 14, and 21, 100 µg of C44Mab-18-mG2a (n = 8), C44Mabwere injected into mice subcutaneously. On days 7, 14, and 21, 100 µg of C44 Mab-18-mG2a
46-mG2a (n = 8), or control mouse IgG2a (mIgG2a, PMab-231) (n = 8) was injected into mice intraperi(n = 8),
C44 Mab-46-mG8),measured
or control
mouse
PMab-231)
toneally(arrows).
The tumor volume
wason days7, 14,IgG
17, 21,24, and 28
the (n = 8) was injected
2a (n =
2a (mIgG
2a ,following
inoculation.
are presented as(arrows).
the mean ±The
SEM.tumor* p < 0.05
and ** pwas
< 0.01(two-way ANOVA
into miceValues
intraperitoneallyvolumemeasuredon days 7, 14, 17, 21, 24, andwith Tukey’s multiple comparisons test). The weight (B) and appearance (C) of excised
28 followingxenografts
the inoculation.
Valuespresented
as themean
SEM.
* p < 0.05 and ** p < 0.01
CHO/CD44v3–10
on day 28. Values
arearepresented
as the mean
± SEM.
* p <±0.05(one-way
ANOVA
with ANOVA
Tukey’s multiple
comparisonstest). The
body weight (D)andTheappearance(two-waywith Tukey’s
multiplecomparisonstest).
weight (E)(B)ofand appearance (C) of
CHO/CD44v3–10 xenograft-bearing mice treated with C44Mab-18-mG2a, C44Mab-46-mG2a, or control
excised CHO/CD44v3–10 xenografts on day 28. Values are presented as the mean ± SEM. * p < 0.05
mIgG2a. Scale bar, 1 cm.
(one-way ANOVA with Tukey’s multiple comparisons test). The body weight (D) and appearance

2.4.
of ADCC and CDC
by C44Mab-18-mG2amice
and Ctreated
44Mab-46-mG
HSC-2 and(E)Inductionof CHO/CD44v3–10
xenograft-bearing
with2aCagainst
44 Mab-18-mG
2a , C44 Mab-46-mG2a , or
SAS
Cells
control mIgG . Scale bar, 1 cm.
2a

Next, we performed in vitro ADCC and CDC assays to examine the cytotoxicity of
C44Mab-18-mG2a and C44Mab-46-mG2a against HSC-2 and SAS cells. Both C44Mab-18-mG2aand C44Mab-46-mG2a significantly exerted ADCC against HSC-2 cells [Figure 5A, 17% (p <0.01) and 13% (p < 0.05) cytotoxicity, respectively] and SAS cells [Figure 5C, 19% (p < 0.01)
and 14% (p < 0.05) cytotoxicity, respectively] compared with the control.

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The weight of CHO/CD44v3–10 tumors treated with C44 Mab-18-mG2a was significantly lower than those treated with the control mouse IgG2a (Figure 4B,C, 62% reduction;p < 0.05). Although a 53% reduction in tumor weight was observed by C44 Mab-46-mG2atreatment, it is not statistically different compared to the control (p = 0.0626). The loss ofbody weight was not observed in the CHO/CD44v3–10 tumor-implanted mice during thetreatments (Figure 4D,E).
2.4. Induction of ADCC and CDC by C44 Mab-18-mG2a and C44 Mab-46-mG
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
8 of 172a against HSC-2 and
SAS Cells

Next, we performed in vitro ADCC and CDC assays to examine the cytotoxicity of
C44Mab-18-mGand
2a and C44Mab-46-mG2a significantly exerted CDC against HSC-2
C44BothMab-18-mG
C44 Mab-46-mG2a against HSC-2 and SAS cells. Both C44 Mab-182acells [Figure 5B, 27% (p < 0.05) and 40% (p < 0.01) cytotoxicity, respectively] compared withmG
andC
Mab-46-mG
exerted ADCC against HSC-2 cells [Figure 5A,
2a44
2a significantly
the control. In SAS cells, C44Mab-46-mG
2a significantly exerted CDC [Figure 5D, 26% (p <17%cytotoxicity](p < 0.01)comparedand 13%with(p <cytotoxicity,and SAScells [Figure 5C, 19%
0.05)
the0.05)
control.
We could notrespectively]observe the significant
diﬀerence
CDC against
SAS(p
cells
between
C44Mab-18-mG
2a and the control(Figure 5D).
(p <of0.01)
and 14%< 0.05)
cytotoxicity,respectively]
comparedwith the control.

Figure5. Evaluationof ADCCand CDCand
activityof activity
C44Mab-18-mG
C44Mab-46-mG
2a against
Figure5. Evaluationof ADCCCDC
of C2a44and
Mab-18-mG
C44 Mab-46-mG2a against
2a and
HSC-2 and SAS cells. The ADCC (A,C) and CDC (B,D) induced by C44Mab-18-mG2a, C44Mab-46HSC-2 and SAS cells. The ADCC (A,C) and CDC (B,D) induced by C44 Mab-18-mG2a , C44 Mab-46mG2a, or control mouse IgG2a (mIgG2a, PMab-231) against HSC-2 (A,B) and SAS (C,D) cells. ValuesmG
mouse
IgG2aindicate(mIgG
againstandSAS (C,D) cells. Values
are
shown
as control
mean ± SEM.
Asterisksstatisticalsignificance(** p < HSC-2
0.01 and (A,B)* p < 0.05;
one2a , or
2a , PMab-231)
way
ANOVA
with
Tukey’smultiplecomparisonstest).
are shown as mean ± SEM. Asterisks indicate statistical significance (** p < 0.01 and * p < 0.05;

one-way ANOVA with Tukey’s multiple comparisons test).

2.5. Antitumor Eﬀects of C44Mab-18-mG2a and C44Mab-46-mG2a in the Mouse Xenografts of
HSC-2 and SAS

Both C44 Mab-18-mG2a and C44 Mab-46-mG2a significantly exerted CDC against HSC-2
In the HSC-2
and SAS xenograft models, C44Mab-18-mG2a, C44Mab-46-mG2a, or control
cells[Figure5B, 27% (p < 0.05) and 40% (p < 0.01) cytotoxicity, respectively] comparedmouse IgG
2a (500 µg/mouse) were intraperitoneally administrated into mice on days 7 andwiththe control.
In SAS
cells,
Cvolume
CDC [Figure 5D, 26%
14,
following
the inoculation.
The
tumor
was measured
on days 7, 14,exerted
and 17. The
44 Mab-46-mG
2a significantlyC(p44Mab-18-mG
2a and C44Mab-46-mG
2a administration
resulted
in a significant
reductionin< 0.05) cytotoxicity]
comparedwith thecontrol.
We could
not observe
the significant
both tumor volume on day 14 (p < 0.01) and day 17 (p < 0.01) compared with that of thedifference of CDC against SAS cells between C44 Mab-18-mG2a and the control (Figure 5D).
control mouse IgG2a (Figure 6A,B). The C44Mab-18-mG2a and C44Mab-46-mG2a administration resulted in 43% and 36% reduction in HSC-2 tumor volume, respectively, comparedwith that treated with the control mouse IgG2a on day 17. The C44Mab-18-mG2a and C44Mab-

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2.5. Antitumor Effects of C44 Mab-18-mG2a and C44 Mab-46-mG2a in the Mouse Xenografts of
HSC-2 and SAS
In the HSC-2 and SAS xenograft models, C44 Mab-18-mG2a , C44 Mab-46-mG2a , orcontrol
mouse IgG2a (500 µg/mouse) were intraperitoneally administrated
into mice on
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
9 of 17days 7 and 14, following the inoculation. The tumor volume was measured on days 7, 14,
and 17. The C44 Mab-18-mG2a and C44 Mab-46-mG2a administration resulted in a significant
46-mG
2a administration
also resulted
in a 33%
and 32%
SASdaytumor
reductionin both tumor
volumeon day14 (preduction< 0.01) inand
17 volume,(p < 0.01) compared withrespectively, compared with that treated with the control mouse IgG2a on day 17.
that of the control mouse IgG2a (Figure 6A,B). The C44 Mab-18-mG2a and C44 Mab-46-mG2a
The weight of HSC-2 tumors treated with C44Mab-18-mG2a and C44Mab-46-mG2a wasadministration
resultedin 43%
andreduction
HSC-2 tumor volume, respectively,
significantly
lower thanthat treatedwiththe 36%
control
mouse IgGin
2a [Figure 6C, 40% (p <comparedwiththat
treatedwiththecontrolmouse
IgG
17.withThe C44 Mab-18-mG2a0.01) and 30% (p < 0.05) reduction, respectively]. The weight of SAS tumors
treated
2a on dayCand
44Mab-18-mG
2a and C44Mab-46-mG
2a was significantly
lower
than
that
treatedwiththe reduction in SAS
C44 Mab-46-mG
administration
also
resulted
in
a
33%
and
32%
2a
controlmouse
IgG2a [Figure
6D, 23% compared(p < 0.01) andwith
19% (pthat< 0.05)
reduction,
respectively].
tumor
volume,respectively,
treatedwiththe control mouse IgG2a on
The loss of body weight was not observed in the HSC-2 and SAS tumor-implanted miceday 17.
during the treatments (Figure 6E,F).

Figure 6. Antitumor activity of C44Mab-18-mG2a and C44Mab-46-mG2a against HSC-2 and SAS xenoFigure 6. Antitumor activity of C44 Mab-18-mG2a and C44 Mab-46-mG2a against HSC-2 and SAS
graft. (A,B) Tumor volume in HSC-2 (A) and SAS (B) xenograft. HSC-2 and SAS cells (5 × 106 cells)
xenograft.
Tumor volume
in 7HSC-2(A)
SAS (B) 2axenograft.
HSC-2 and SAS cells
were injected into(A,B)
mice subcutaneously.
On daysand 14, 500µgandof C44Mab-18-mG(n = 8), C44Mab46-mG(n6=cells)8), or control
IgG2ainto(mIgG
2a, PMab-231)(n = 8) was injected
into miceintraperito(5 ×2a10
were mouse
injectedmice
subcutaneously.
On days
7 and
14, 500 µg of C44 Mab-18neally (arrows). The tumor volume was measured on days 7, 14, and 17 following the inoculation.
mG are(n= 8), C44theMab-46-mG(n = 8), or control mouse IgG (mIgG2a , PMab-231) (n = 8) was
Values2apresented asmean ± SEM.2a** p < 0.01 (two-way ANOVA with Tukey’s2amultiple comparinjected
into The
mice
intraperitoneallytumor
volumemeasuredisonstest). (C,D)
weightand appearance of(arrows).
the excisedThe
HSC-2(C) and
SAS (D) was
xenografts
on day on days 7, 14, and 17
17.
Values are presented
as the meanValues± SEM. *arep < presented
0.05 and ** p <as0.01(one-wayfollowing
the inoculation.
themean ANOVA± SEM.with** pTukey’s< 0.01 (two-way ANOVA

with Tukey’s multiple comparisons test). (C,D) The weight and appearance of the excised HSC-2 (C)and SAS (D) xenografts on day 17. Values are presented as the mean ± SEM. * p < 0.05 and ** p < 0.01
(one-way ANOVA with Tukey’s multiple comparisons test). (E,F) The body weight of HSC-2 (E) andSAS (F) xenograft-bearing mice treated with C44 Mab-18-mG2a , C44 Mab-46-mG2a , or control mIgG2a .
Scale bar, 1 cm.

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9 of 16

The weight of HSC-2 tumors treated with C44 Mab-18-mG2a and C44 Mab-46-mG2a
was significantly lower than that treated with the control mouse IgG2a [Figure 6C, 40%(p < 0.01) and 30% (p < 0.05) reduction, respectively]. The weight of SAS tumors treated with
C44 Mab-18-mG2a and C44 Mab-46-mG2a was significantly lower than that treated with thecontrol mouse IgG2a [Figure 6D, 23% (p < 0.01) and 19% (p < 0.05) reduction, respectively].
The loss of body weight was not observed in the HSC-2 and SAS tumor-implanted mice
during the treatments (Figure 6E,F).
3. Discussion
Among CD44v, v10-containing isoforms include the most abundant CD44v, suchas CD44v3–10, CD44v6–10, and CD44v8–10 [2,61]. Therefore, anti-CD44v10 mAbs including C44 Mab-18-mG2a can target the broad range of CD44v-expressing tumor cells.
In this study, we evaluated the antitumor activities against CD44-positive cells compared with an anti-pan-CD44 mAb, C44 Mab-46-mG2a . C44 Mab-18-mG2a exhibited thehigher reactivity to CHO/CD44v3–10 and OSCC cells compared with C44 Mab-46-mG2a(Figures 1 and 2). C44 Mab-18-mG2a exhibited a superior ADCC against CHO/CD44v3–10(Figure 3) and OSCC (Figure 5) cells. In contrast, C44 Mab-46-mG2a showed a superior
CDC against those cells. Furthermore, C44 Mab-18-mG2a or C44 Mab-46-mG2a similarly
inhibited CHO/CD44v3–10 and OSCC xenograft growth compared with the control mouse
IgG2a (Figures 4 and 6). These results indicate that C44 Mab-18-mG2a could be a promisingtherapeutic regimen for CD44v10-positive tumors.
OSCC arises from the oral cavity and is a type of head and neck squamous cell carcinoma (HNSCC). HNSCC has been revealed to be associated with alcohol, smoking, andhuman papillomavirus types 16 and 18 infection [62]. In the Pan-Cancer Atlas, HNSCC has
been shown as the second-highest CD44-expressing tumor type [63]. The overexpressionof CD44 is associated with resistance to therapy and unfavorable outcomes [64–66]. Furthermore, CD44-high cancer stem cells (CSCs) from HNSCC exhibited increased migration,
invasiveness, and stemness [67]. In immunodeficient mice, the CD44-high CSCs could form
more lung metastatic foci than CD44-low cells [68]. Therefore, CD44 is considered an important target for mAb therapies. Since C44 Mab-18 is available to immunohistochemistry [58],
C44 Mab-18 could be used for diagnosis and therapy of OSCC.
In CHO/CD44v3–10 cells, C44 Mab-18-mG2a and C44 Mab-46-mG2a recognized a common target but mainly exerted ADCC and CDC activity, respectively (Figure 3). The epitopeof C44 Mab-46 was previously determined as the 174- TDDDV-178 sequence in the constantexon 5-encoded region [51], which is relatively apart from transmembrane domain compared to variant exon 10-encoded region recognized by C44 Mab-18 [58]. To activate theclassical pathway of complement, an ordered hexamer formation of IgG mAb is required
to bind to the hexavalent complement C1q [69,70]. The structure of the C44 Mab-46-mG2a –CD44v3–10 complex may provide the appropriate space to form the hexameric structure of
the mAb–C1q complex and recruit the pore-forming membrane attack complex to exert
CDC. In contrast, C44 Mab-18-mG2a showed a higher reactivity to CHO/CD44v3–10 compared with C44 Mab-46-mG2a in flow cytometry (Figure 1). The difference in the reactivity
and the epitope would influence the ADCC activity. Further studies are required to reveal
the relationship among the affinity of mAb, epitope, and ADCC activity.
The limitation of this study is that both C44 Mab-46-mG2a and C44 Mab-18-mG2a are
effective in human xenograft models of nude mice. To apply human tumor therapy, a classswitch to human IgG1 is essential. We previously generated humanized IgG1 mAbs andevaluated the antitumor activity with injections of human NK cells [71,72]. We will produce
humanized mAbs from C44 Mab-46 and C44 Mab-18 and evaluate the ADCC and CDC in
the presence of human NK cells and complement, respectively. Furthermore, the antitumor
effects should be investigated with injections of human NK cells.
Near-infrared photoimmunotherapy (NIR-PIT) uses a targeted mAb conjugated witha photoactivatable dye such as IRDye700DX (IR700) [73–76]. When the mAb binds to theantigen-expressed target cells, IR700 induces plasma membrane rupture and immunogenic

Int. J. Mol. Sci. 2024, 25, 9190

10 of 16cell death by NIR-light exposure. Preclinical studies of anti-pan-CD44 mAb-based NIR-PIT(IM7-IR700) were conducted. In the syngeneic mouse model of OSCC, IM7-IR700 administration and the NIR light exposure to OSCC tumors resulted in a significant reduction
but failed to induce durable antitumor responses [77]. Because IM7 is a pan-CD44 mAb,
IM7 might target not only tumor cells but also CD44s-positive immune cells which are
involved in the antitumor immunity. The expression of CD44v is low in hematopoietic cells
compared with CD44s [2]. We previously showed that C44 Mab-18 can distinguish tumor
cells from stromal tissues in immunohistochemistry. In contrast, C44 Mab-46 stained both tumor and stromal tissue including fibroblasts and leukocytes [58]. Therefore, anti-CD44v10
mAbs such as C44 Mab-18 might be a promising mAb for NIR-PIT without affecting thehost immune cells in the tumor microenvironment.
Since both CD44s and CD44v are expressed in normal cells, there is a concern about
adverse effects due to the recognition of normal cells by mAbs. In fact, clinical trialsof the anti-CD44v6 mAb–ADC to advanced solid tumors were discontinued because of
the skin toxicities [32,37]. Therefore, cancer-specific antibodies are desired to reduce theadverse effects. A cancer-specific anti-CD44v6 mAb (clone 4C8) recognizes aberrantly
O-glycosylated Tn (GalNAcα1-O-Ser/Thr) antigen in the variant exon 6-encoded region.
The 4C8 mAb was further developed for chimeric antigen receptor (CAR)-T cells, which exhibited target-specific in vitro cytotoxicity and significant tumor regression in vivo [78]. We
have developed cancer-specific mAbs (CasMabs) against various tumor antigens, including
HER2 (clones H2 Mab-214 [79] and H2 Mab-250 [80]), and reported the antitumor effect inmouse xenograft models using recombinant mouse IgG2a or human IgG1 mAbs [71,72].
These anti-HER2 mAbs were screened by the reactivity to cancer and normal cells in flow
cytometry. H2 Mab-214 was revealed to recognize a locally misfolded structure in the
Cys-rich HER2 extracellular domain 4, which usually forms a β-sheet [79]. H2 Mab-250 also
shows a specific reactivity against HER2-positive tumor cells, which has been developed
as CAR-T-cell therapy. The phase I study has been conducted in the US (NCT06241456).
We have developed CasMabs against CD44s or CD44v by comparing the reactivity against
tumor and normal cells. The anti-CD44 CasMabs could contribute to developing novel
modalities such as ADCs and CAR-T cells.
4. Materials and Methods
4.1. Cell Lines and Cell Culture
CHO-K1 was obtained from the American Type Culture Collection (ATCC, Manassas,
VA, USA). OSCC cell lines HSC-2 and SAS were obtained from the Japanese Collection of
Research Bioresources (Osaka, Japan). CHO/CD44s and CHO/CD44v3–10 were previously
established by transfecting pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3–10 into
CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc., Waltham,
MA, USA) [50,58].
HSC-2 and SAS were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Nacalai
Tesque, Inc., Kyoto, Japan) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS,
Thermo Fisher Scientific Inc.), 100 U/mL of penicillin (Nacalai Tesque, Inc.), 100 µg/mL
streptomycin (Nacalai Tesque, Inc.), and 0.25 µg/mL amphotericin B (Nacalai Tesque, Inc.).
CHO/CD44v3–10 was cultured in Roswell Park Memorial Institute (RPMI)-1640
medium (Nacalai Tesque, Inc.) supplemented with 10% (v/v) FBS, antibiotics as mentioned
above, and 5 mg/mL Zeocin (InvivoGen, San Diego, CA, USA). All cells were grown in ahumidified incubator at 37 ◦ C with 5% CO2 .
4.2. Antibodies
An anti-pan-CD44 mAb (C44 Mab-46) and an anti-CD44v10 mAb (C44 Mab-18) werepreviously established [50,58]. A recombinant mouse IgG2a -type mAb, C44 Mab-46-mG2a,
was generated previously [60]. To generate a recombinant mouse IgG2a -type mAb from
C44 Mab-18 (IgM, kappa), VH cDNAs of C44 Mab-18 and CH of mouse IgG2a were cloned
into the pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). VL

Int. J. Mol. Sci. 2024, 25, 9190

11 of 16and mouse kappa light chain (CL ) cDNA of C44 Mab-18 was also cloned into the pCAG-Neo
vector (FUJIFILM Wako Pure Chemical Corporation). Using the ExpiCHO Expression
System (Thermo Fisher Scientific, Inc.), the vectors were transfected into BINDS-09 cells(http://www.med-tohoku-antibody.com/topics/001_paper_cell.htm, accessed on 23 August 2024) and the supernatants were collected. C44 Mab-18-mG2a were purified using
Ab-Capcher (ProteNova Co., Ltd., Kagawa, Japan). PMab-231 (a control mouse IgG2a ) was
previously described [80].
4.3. Flow Cytometry
CHO/CD44v3–10, HSC-2, and SAS were obtained using 1 mM ethylenediamine
tetraacetic acid (EDTA; Nacalai Tesque, Inc.) and 0.25% trypsin treatment. The cells
were treated with C44 Mab-18-mG2a , C44 Mab-46-mG2a , PMab-231, or blocking buffer [0.1%bovine serum albumin (BSA; Nacalai Tesque, Inc.) in phosphate-buffered saline (PBS)](control) for 30 min at 4 ◦ C. Subsequently, the cells were incubated in Alexa Fluor 488conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA)for 30 min at 4 ◦ C. Fluorescence data were collected using the SA3800 Cell Analyzer (SonyCorp., Tokyo, Japan) and analyzed using SA3800 software ver. 2.05 (Sony Corp.) [50].
4.4. ADCC
Animal studies for ADCC were approved by the Institutional Committee for Experiments of the Institute of Microbial Chemistry (permit no. 2024-038). The ADCC activityof C44 Mab-18-mG2a and C44 Mab-46-mG2a was measured as follows. The target cells(CHO/CD44v3–10, HSC-2, and SAS) were labeled with 10 µg/mL Calcein AM (Thermo
Fisher Scientific, Inc.) and plated in 96-well plates (1 × 104 cells/well). The calceinlabeled target cells were mixed with the effector splenocyte (effector to target ratio, 50:1)
from female BALB/c nude mice (Jackson Laboratory Japan, Inc., Kanagawa, Japan) with
100 µg/mL of C44 Mab-18-mG2a , C44 Mab-46-mG2a , or control mouse IgG2a (PMab-231).
After a 4 h incubation at 37 ◦ C, the calcein release into the medium was measured using a
microplate reader (Power Scan HT; BioTek Instruments, Inc., Winooski, VT, USA).
The cytolyticity (% lysis) was determined: % lysis is calculated as (E − S)/(M − S) ×100, where “E” indicates the fluorescence in effector and target cell cultures, “S” means the
spontaneous fluorescence of only target cells, and “M” indicates the maximum fluorescence
after treatment with a lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5% Triton
X-100] [49].
4.5. CDC
The calcein-labeled target cells were mixed with rabbit complement (final concentration 15%, Low-Tox-M Rabbit Complement; Cedarlane Laboratories, Hornby, ON, Canada)
and 100 µg/mL of C44 Mab-18-mG2a , C44 Mab-46-mG2a , or control mouse IgG2a (PMab-231).
After a 4 h incubation at 37 ◦ C, the calcein release into the medium was measured as
described previously [60].
4.6. Antitumor Activity of C44 Mab-18-mG2a and C44 Mab-46-mG2a in Xenografts of
CHO/CD44v3–10, HSC-2, and SAS
The animal study protocol was approved (approval nos. 2024-013) by the Institutional
Committee for Experiments of the Institute of Microbial Chemistry (Numazu, Japan). The
animal study was performed as described previously [49]. CHO/CD44v3–10, HSC-2, orSAS cells (5 × 106 cells) suspended with BD Matrigel Matrix Growth Factor Reduced (BDBiosciences, Franklin Lakes, NJ, USA) were inoculated into the left flank of female BALB/cnude mice subcutaneously. On day 7 after the inoculation, 100 µg of C44 Mab-18-mG2a(n = 8), C44 Mab-46-mG2a (n = 8), or control mouse IgG2a (PMab-231) (n = 8) in 100 µL PBS
was injected intraperitoneally. Additional antibody injections were performed on days 14
and 21. The tumor volume was measured on the indicated days.

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In the HSC-2 or SAS xenograft experiment, 500 µg of C44 Mab-18-mG2a (n = 8), C44 Mab46-mG2a (n = 8), or control mouse IgG2a (PMab-231) (n = 8) in 100 µL PBS was injected
intraperitoneally on days 7 and 14 after the inoculation. The tumor volume was measured
on the indicated days. The xenograft tumors were carefully removed from the sacrificed
mice and weighed immediately.
The tumor volume was calculated using the following formula: Volume = W2 × L/2,
where W is the short diameter and L is the long diameter. All data are expressed as the mean
± standard error of the mean (SEM). In tumor weight measurement, one-way ANOVA
with Tukey’s multiple comparisons test was conducted. Two-way ANOVA with Tukey’s
multiple comparisons test was utilized for tumor volume and mice weight. GraphPad
Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) was used for all calculations. p < 0.05
was considered to indicate a statistically significant difference.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25179190/s1.
Author Contributions: Conceptualization, M.K., M.K.K. and Y.K. (Yukinari Kato); methodology,
T.O.; formal analysis, T.T.; investigation, K.I., H.S., T.O., G.L. and T.T.; data curation, H.S. and Y.K.
(Yukinari Kato); writing—original draft preparation, K.I. and H.S.; writing—review and editing, Y.K.
(Yukinari Kato); supervision, A.O. and Y.K. (Yukio Katori); project administration, Y.K. (YukinariKato); funding acquisition, H.S., T.T., M.K.K. and Y.K. (Yukinari Kato). All authors have read andagreed to the published version of the manuscript.
Funding: This research was supported in part by the Japan Agency for Medical Research andDevelopment (AMED) under Grant Numbers 24am0521010 (to Y.K. (Yukinari Kato)), JP23ama121008(to Y.K. (Yukinari Kato)), JP23am0401013 (to Y.K. (Yukinari Kato)), JP23bm1123027 (to Y.K. (YukinariKato)), and JP23ck0106730 (to Y.K. (Yukinari Kato)) and by the Japan Society for the Promotionof Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant nos. 22K06995 (to H.S.),
21K20789 (to T.T.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K. (Yukinari Kato)).
Institutional Review Board Statement: The Institutional Committee for Experiments of the Instituteof Microbial Chemistry approved animal experiments (approval nos. 2024-013 and 2024-038).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article andSupplementary Material.
Acknowledgments: The authors thank Shun-ichi Ohba and Akiko Harakawa (Institute of Microbial
Chemistry [BIKAKEN], Numazu, Microbial Chemistry Research Foundation) for technical assistancewith the animal experiments.
Conflicts of Interest: The authors have no conflicts of interest to declare.

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