Article

A Novel Anti-CD44 Variant 9 Monoclonal Antibody C44Mab-1
Was Developed for Immunohistochemical Analyses against
Colorectal Cancers
Mayuki Tawara 1,† , Hiroyuki Suzuki 1, *,† , Nohara Goto 1 , Tomohiro Tanaka 1 , Mika K. Kaneko 1,2
and Yukinari Kato 1,2, *
1

2

*
†

Citation: Tawara, M.; Suzuki, H.;
Goto, N.; Tanaka, T.; Kaneko, M.K.;
Kato, Y. A Novel Anti-CD44 Variant 9
Monoclonal Antibody C44 Mab-1 Was
Developed for Immunohistochemical
Analyses against Colorectal Cancers.
Curr. Issues Mol. Biol. 2023, 45,
3658–3673. https://doi.org/10.3390/
cimb45040238
Academic Editors: Irina
V. Kondakova, Liudmila V. Spirina
and Natalya V. Yunusova
Received: 30 March 2023

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan; tawara.mayuki.p8@dc.tohoku.ac.jp (M.T.); s1930550@s.tsukuba.ac.jp (N.G.);
tomohiro.tanaka.b5@tohoku.ac.jp (T.T.); k.mika@med.tohoku.ac.jp (M.K.K.)
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1
Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
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.)
These authors contributed equally to this work.

Abstract: Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has beenshown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing
variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness,invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of
the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains thevariant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers.
CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promisingtarget for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal
antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed Chinese
hamster ovary-K1 (CHO/CD44v3–10) cells. We first determined their critical epitopes using enzymelinked immunosorbent assay and characterized their applications as flow cytometry, western blotting,and immunohistochemistry. One of the established clones, C44 Mab-1 (IgG1 , kappa), reacted witha peptide of the variant 9-encoded region, indicating that C44 Mab-1 recognizes CD44v9. C44 Mab-1
could recognize CHO/CD44v3–10 cells or colorectal cancer cell lines (COLO201 and COLO205) inflow cytometric analysis. The apparent dissociation constant (KD ) of C44 Mab-1 for CHO/CD44v3–
10, COLO201, and COLO205 was 2.5 × 10−8 M, 3.3 × 10−8 M, and 6.5 × 10−8 M, respectively.
Furthermore, C44 Mab-1 was able to detect the CD44v3–10 in western blotting and the endogenous
CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that
C44 Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in
immunohistochemistry against colorectal cancers.
Keywords: CD44; CD44v9; monoclonal antibody; colorectal cancer

Revised: 17 April 2023
Accepted: 19 April 2023
Published: 20 April 2023

1. Introduction
Copyright: © 2023 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/).

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein, and its
variety of isoforms are expressed in various types of cells [1]. The alternative splicing ofCD44 mRNA mediates the variety of isoforms [2]. The CD44 standard (CD44s) isoform, thesmallest isoform of CD44, is expressed in most vertebrate cells. CD44s mRNA is assembled
by the first five (1 to 5) and the last five (16 to 20) constant region exons [3]. The CD44
variant (CD44v) isoforms are assembled by the alternative splicing of middle variant exons(v1–v10) in various combinations with the standard exons of CD44s [4]. Both CD44s andCD44v (pan-CD44) bind to hyaluronic acid (HA), which plays critical roles in cellularadhesion, migration, homing, and proliferation [5].

Curr. Issues Mol. Biol. 2023, 45, 3658–3673. https://doi.org/10.3390/cimb45040238

https://www.mdpi.com/journal/cimb

Curr. Issues Mol. Biol. 2023, 45

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The CD44 protein is further modified using a variety of glycosyltransferases [6]. Dueto the post-translational modifications, including N-glycans, O-glycans, and glycosaminoglycans (heparan sulphate, etc.), the molecular weight of CD44s is enlarged to 80–100 kDa,
and some CD44v isoforms surpass 200 kDa due to a high level of glycosylation [7].
Several isoforms of the CD44 are associated with malignant progression in various
tumors [8], including head and neck squamous cell carcinomas (HNSCCs) [9], pancreaticcancers [10,11], breast cancers [12], gliomas [13,14], prostate cancers [15], and colorectal
cancers (CRC) [16]. CD44 is also known as a cell surface marker of cancer stem-like cells(CSCs) in various carcinomas [17]. Specific monoclonal antibodies (mAbs) to CD44s orCD44v are utilized for sorting CD44high CSCs [17]. The CD44high population exhibited theincreased stemness property, drug resistance, and tumor formation in vivo [17]. Therefore,
development of anti-CD44 mAbs, which recognize each variant, is important for the further
characterization of CSCs in various cancers.
The functions of CD44v have been reported in the promotion of tumor invasion,
metastasis, CSC properties [18], and resistance to chemotherapy and radiotherapy [8,19].
The v3-encoded region is modified by heparan sulfate, which promotes the binding toheparin-binding growth factors, including fibroblast growth factors and heparin-binding
epidermal growth factor-like growth factor. Therefore, the v3-encoded region functions
as a co-receptor of receptor tyrosine kinases and potentiates their signal transduction [20].
Furthermore, the v6-encoded region is essential for the activation of c-MET through ternarycomplex formation with the ligand hepatocyte growth factor [21]. The v8–10-encoded
region could bind to and stabilize a cystine–glutamate transporter (xCT), which promotes
the defense to reactive oxygen species (ROS) via cystine uptake-mediated glutathione
synthesis [22]. The regulation of redox status depends on the expression of CD44v8–10
that is associated with the xCT function and links to the poor prognosis of patients [23].
Therefore, the establishment and characterization of mAbs, which recognize each CD44v,
are essential for understanding each variant function and development of CD44-targetingcancer therapy. However, the function and distribution of the variant 9-encoded region intumors have not been fully understood.
We previously developed an anti-pan-CD44 mAb, C44 Mab-5 (IgG1 , kappa) [24], using
the Cell-Based Immunization and Screening (CBIS) method. Furthermore, another antipan-CD44 mAb, C44 Mab-46 (IgG1 , kappa) [25], was established by immunizing mice with
CD44v3–10 ectodomain. We showed that both C44 Mab-5 and C44 Mab-46 could be applied
to flow cytometry and immunohistochemistry in oral [24] and esophageal SCCs [25]. We
also determined the epitopes of C44 Mab-5 and C44 Mab-46 within the standard exons (1 to5)-encoding regions [26–28]. Furthermore, we produced a defucosylated version (5-mG2a -f)
using FUT8-deficient ExpiCHO-S cells (BINDS-09) and investigated the antitumor effects of
5-mG2a -f in mouse xenograft models of oral SCC [29]. Recently, we have established various
CD44v mAbs, including C44 Mab-108 (v4) [30], C44 Mab-3 (v5) [31], C44 Mab-9 (v6) [32], andC44 Mab-34 (v7/8) [33].
In this study, we established a novel anti-CD44v9 mAb, C44 Mab-1 (IgG1 , kappa),
using the CBIS method and evaluated its applications for flow cytometry, western blotting, and immunohistochemical analyses of oral squamous cell carcinoma and colorectal
adenocarcinomas.
2. Materials and Methods
2.1. Cell Lines
COLO201 (a human colorectal cancer cell line), P3X63Ag8U.1 (P3U1; a mouse multiple
myeloma), and Chinese hamster ovary (CHO)-K1 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). COLO205 (a human colorectal
cancer cell line) was obtained from the Cell Resource Center for Biomedical ResearchInstitute of Development, Aging, and Cancer at Tohoku University (Miyagi, Japan). To
cultivate these cell lines, we used Roswell Park Memorial Institute (RPMI)-1640 medium(Nacalai Tesque, Inc., Kyoto, Japan), which is supplemented with 10% heat-inactivated fetal

Curr. Issues Mol. Biol. 2023, 45

3660

bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA). We further added
the antibiotics, including 100 µg/mL streptomycin, 100 U/mL penicillin, and 0.25 µg/mL
amphotericin B (Nacalai Tesque, Inc.). All cell lines were grown in a humidified incubator
at 37◦ C with 5% CO2 .
We amplified CD44s cDNA from LN229 cDNA using the HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany). We obtained CD44v3–10 ORF from the
RIKEN BRC. CD44v3–10 and CD44s cDNAs were cloned into a pCAG-Ble-ssPA16 vector,
which possesses the signal sequence and the N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [24,34–37], which can be detected by an anti-human podoplanin (PDPN) mAb(NZ-1) [38–53]. Using a Neon transfection system (Thermo Fisher Scientific, Inc.), twostable transfectants, such as CHO/CD44v3–10 and CHO/CD44s, were established by introducing pCAG-Ble/PA16-CD44v3–10 and pCAG-Ble/PA16-CD44s into CHO-K1 cells,
respectively.
2.2. Production of Hybridoma Cells
The 6-week-old female BALB/c mice were purchased from CLEA Japan (Tokyo,
Japan). Mice were housed under specific pathogen-free conditions. To minimize animal
suffering and distress in the laboratory, all mice experiments were performed according
to relevant guidelines and regulations. Our animal experiments were approved by the
Animal Care and Use Committee of Tohoku University (Permit number: 2019NiA-001).
Mice were monitored every day for health during the period of experiments. Mice wereintraperitoneally immunized with CHO/CD44v3–10 (1 × 108 cells) with Imject Alum
(Thermo Fisher Scientific Inc.) as an adjuvant. We performed additional immunizationsof CHO/CD44v3–10 (1 × 108 cells, three times) and performed a booster injection of
CHO/CD44v3–10 (1 × 108 cells) 2 days before harvesting the spleen cells. We used
polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA) to fuse thesplenocytes and P3U1 cells. The hybridoma supernatants, which are negative for CHO-K1
cells and positive for CHO/CD44v3–10 cells, were selected using SA3800 Cell Analyzer(Sony Corp. Tokyo, Japan).
2.3. ELISA
Fifty-eight peptides, which cover the extracellular domain of CD44v3–10 [26], wereobtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). We immobilized them on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific Inc) at 1 µg/mL for 30 min at
37 ◦ C. The palate washing was performed using the HydroSpeed Microplate Washer (Tecan,
Zürich, Switzerland) with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20(PBST; Nacalai Tesque, Inc.). After the blocking with 1% (w/v) bovine serum albumin (BSA)in PBST for 30 min at 37 ◦ C, C44 Mab-1 (10 µg/mL) was added to each well. Then, the wells
were further incubated with anti-mouse immunoglobulins peroxidase-conjugate (1:2000
diluted; Agilent Technologies Inc., Santa Clara, CA, USA) for 30 min at 37 ◦ C. One-Step
Ultra TMB (Thermo Fisher Scientific Inc.) was used for enzymatic reactions. An iMark
microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) was used to measure theoptical density at 655 nm.
2.4. Flow Cytometry
CHO/CD44v3–10 and CHO-K1 cells were prepared using 0.25% trypsin and 1 mM
ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). COLO201 and COLO205
were obtained by pipetting. The cells (1 × 105 cells/sample) were incubated with C44 Mab-1,
C44 Mab-46, or blocking buffer (0.1% BSA in PBS; control) for 30 min at 4 ◦ C. Then, the cells
were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:2000; Cell Signaling
Technology, Inc.) for 30 min at 4 ◦ C. Fluorescence data were collected and analyzed using
the SA3800 Cell Analyzer and SA3800 software (ver. 2.05, Sony Corp.), respectively.

Curr. Issues Mol. Biol. 2023, 45

3661

2.5. Determination of Apparent Dissociation Constant (KD ) by Flow Cytometry
In CHO/CD44v3–10 cells, we prepared from 260 to 0.016 nM (diluted by 1/2) of
C44 Mab-1. In COLO201 and COLO205 cells, we prepared from 1300 to 0.08 nM (dilutedby 1/2) of C44 Mab-1. The serially diluted C44 Mab-1 was suspended with 2 × 105 cells.
Then, those cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:200).
Fluorescence data were collected and analyzed as indicated above. GraphPad Prism 8 (thefitting binding isotherms to built-in one-site binding models; GraphPad Software, Inc., La
Jolla, CA, USA) was used to determine the apparent dissociation constant (KD ).
2.6. Western Blot Analysis
Cell lysates were prepared using NP-40 lysis buffer (20 mM tris-HCl [pH 7.5], 150 mM
NaCl, 1% NP-40, and 50 µg/mL of aprotinin) and were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.). The 10 µg of cell lysates were subjected to SDSpolyacrylamide gel for electrophoresis using polyacrylamide gels (5–20%; FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan) and electrotransferred onto polyvinylidene
difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The blocking was
performed using 4% skim milk (Nacalai Tesque, Inc.) in PBST. The membranes wereincubated with 10 µg/mL of C44 Mab-1, 10 µg/mL of C44 Mab-46, or 1 µg/mL of an antiisocitrate dehydrogenase 1 (IDH1; RcMab-1; rat IgG2a ) [54,55] and then incubated withperoxidase-conjugated anti-mouse immunoglobulins (diluted 1:1000; Agilent Technologies,Inc.) or peroxidase-conjugated anti-rat immunoglobulins (diluted 1:10,000; Sigma-AldrichCorp.). Finally, the signals were enhanced using a chemiluminescence reagent, ImmunoStar
LD (FUJIFILM Wako Pure Chemical Corporation), and detected using a Sayaca-Imager(DRC Co. Ltd., Tokyo, Japan).
2.7. Immunohistochemical Analysis
The formalin-fixed paraffin-embedded (FFPE) oral SCC tissues were obtained as
described previously [56]. We purchased a colorectal carcinoma tissue array (CO483a)from US Biomax Inc. (Rockville, MD, USA). We used a cat rectum paraffin tissue section(Zyagen; FP-312) as a negative tissue control [57]. The sections were autoclaved in EnVision
FLEX Target Retrieval Solution High pH (Agilent Technologies, Inc.) for 20 min. After
blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), we incubated the tissue
sections with C44 Mab-1 (1 µg/mL) and C44 Mab-46 (1 µg/mL) for 1 h. For isotype control,
we used PMab-44 (mouse IgG1 ), an anti-bovine PDPN mAb [58]. The peptide blocking
assay was performed as described previously [30]. The sections were further treated with
the EnVision+ Kit for mouse (Agilent Technologies Inc.) for 30 min at room temperature.
The chromogenic reaction was conducted using 3,30 -diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies Inc.). The counterstaining was performed using hematoxylin(FUJIFILM Wako Pure Chemical Corporation). To examine the sections and obtain images,
we used Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany).
3. Results
3.1. Establishment of an Anti-CD44v9 mAb, C44 Mab-1
Figure 1A shows the structure of CD44s and representative CD44v. For the CBIS
method, we prepared the CD44v3–10-overexpressed CHO-K1 cells (CHO/CD44v3–10) as
an immunogen. Mice were immunized with CHO/CD44v3–10 cells (Figure 1B), and hybridomas were produced and seeded into 96-well plates (Figure 1C). Then, the supernatants,
which were positive to CHO/CD44v3–10 cells and negative to CHO-K1, were selected byhigh throughput screening using flow cytometry (Figure 1D). After cloning by the limiting
dilution, anti-CD44 mAb-producing clones were finally established (Figure 1E). We next
performed the ELISA to determine the epitope of each mAb. Among them, C44 Mab-1 (IgG1 ,kappa) was shown to recognize the CD44p471–490 peptide (STSHEGLEEDKDHPTTSTLT),
which corresponds to the variant 9-encoded sequence (Supplementary Table S1). In con-

Curr. Issues Mol. Biol. 2023, 45

the limiting dilution, anti-CD44 mAb-producing clones were finally established1E). We next performed the ELISA to determine the epitope of each mAb. Amon
C44Mab-1 (IgG1, kappa) was shown to recognize the CD44p471–490
3662(STSHEGLEEDKDHPTTSTLT), which corresponds to the variant 9-encoded se(Supplementary Table S1). In contrast, C44Mab-1 never recognized other CD44v3
tracellularregions.
results
indicated
that
C44Mab-1regions.
specificallyth
trast, C44 Mab-1
never These
recognizedotherCD44v3–10
extracellular
These recognizes
resultsvariant
9-encodedsequence.
indicated that C44 Mab-1 specifically recognizes the CD44 variant 9-encoded sequence.

Figure 1.1.AAschematic
representationof anti-humanCD44 mAbs
production.
(A) Structure of(A)CD44.
Figureschematic
representationof anti-human
CD44
mAbs production.
Structure o
The
CD44s
mRNA
is
assembled
by
the
first
five
(1
to
5)
and
the
last
five
(16
to
20)
exons
and
translates
The CD44s mRNA is assembled by the first five (1 to 5) and the last five (16 to 20) exons an
CD44s.CD44s.
The mRNAs
the CD44
are produced
the produced
alternative splicing
middle variant
lates
The ofmRNAsofvariant
the CD44
variantbyareby theofalternative
splicing ofexons and translate multiple CD44v such as CD44v3–10, CD44v4–10, CD44v6–10, and CD44v8–10.
variant exons and translate multiple CD44v such as CD44v3–10, CD44v4–10, CD44v6–(B) CHO/CD44v3–10 cells were intraperitoneally injected into BALB/c mice. (C) Hybridomas wereCD44v8–10. (B) CHO/CD44v3–10 cells were intraperitoneally injected into BALB/c mice.
produced by fusion of the splenocytes and P3U1 cells. (D) The screening was performed by flow
bridomas were produced by fusion of the splenocytes and P3U1 cells. (D) The screening w
cytometry using CHO/CD44v3–10 and parental CHO-K1 cells. (E) After cloning and additional
formedby flow cytometry using CHO/CD44v3–10 and parental CHO-K1 cells. (E) After clon
screening, a clone C44 Mab-1 (IgG1 , kappa) was established. Furthermore, we used peptides that
additional
screening,a clone
C44Mab-1(IgG1, kappa)
wasFurthermore,cover the extracellulardomainof CD44v3–10(SupplementaryTableS1)established.
and determined
the binding we us
tides
that
cover
the
extracellulardomainof
CD44v3–10(Supplementary
Table
S1) and det
epitopes of each mAb using enzyme-linked immunosorbent assay (ELISA).
the binding epitopes of each mAb using enzyme-linked immunosorbent assay (ELISA).

3.2. Flow Cytometric Analysis of C44Mab-1 to CD44-Expressing Cells

We next investigated the reactivity of C44Mab-1 against CHO/CD44v3–
CHO/CD44s cells using flow cytometry. C44Mab-1 recognized CHO/CD44v3–10 c

Curr. Issues Mol. Biol. 2023, 45

3663

Curr. Issues Mol. Biol. 2023, 3, FOR PEER REVIEW

6

3.2. Flow Cytometric Analysis of C44 Mab-1 to CD44-Expressing Cells
We next investigated the reactivity of C44Mab-1 against CHO/CD44v3–10 and CHO/CD44s

dose-dependent
manner C(Figure2A). In contrast, C44Mab-1 never recognized CHO/CD44s
cells using flow cytometry.
44 Mab-1 recognized CHO/CD44v3–10 cells in a dose-dependent(Figure2B)
or
CHO-K1(Figure2C)
cells.
We
confirmedthat a pan-CD44
mAb,
C44Mab-46
manner (Figure 2A). In contrast, C44Mab-1
neverrecognized
CHO/CD44s(Figure 2B)
or CHO[25],recognized
both
CHO/CD44v3–10
and CHO/CD44s
cells (Supplementary
Figure S1A
K1 (Figure2C) cells.
We
confirmed that a pan-CD44
mAb, C44Mab-46[25], recognized both
CHO/CD44v3–10
and CHO/CD44s
cells (Supplementary
Figure S1A
and B,S1C).
respectively),
andB, respectively),
but not CHO-K1
cells (Supplementary
FigureFurthermore,
not CHO-K1(Supplementary
Figure S1C).
Furthermore,
Mab-1 could
recognize
Cbut
44Mab-1
couldcells
recognize
endogenous
CD44v9in bothC44
COLO201(Figure2D) andendogenous
CD44v9inboth
COLO201(Figure2D)
andCOLO205(Figure
2E)
cells
in
a
doseCOLO205 (Figure 2E) cells in a dose-dependent manner.
dependent manner.

Figure 2.
2. Flow
Flow cytometry
cytometry using
Mab-1.
(B),(B),
CHO-K1FigureusingCC
Mab-1. CHO/CD44v3–10
CHO/CD44v3–10(A),(A),CHO/CD44s
CHO/CD44s
CHO-K1 (C),4444
(C), COLO201
COLO205
weretreatedtreatedwith
with 0.01–10
0.01–10 µg/mL
byby treatCOLO201(D),(D),
andandCOLO205(E)(E)
wereµg/mL ofofCC4444Mab-1,
Mab-1,followed
followed
treatment
Alexa
Fluor
488-conjugatedanti-mouseIgG
IgG (Red(Red line).
represents
thethe negment
withwith
Alexa
Fluor
488-conjugatedanti-mouseline).The
Theblack
blackline
line
represents
negative
control(blockingbuffer).
ative
control(blockingbuffer).

We next performed the flow cytometry-based measurement of the apparent binding
affinity of C44Mab-1 to CHO/CD44v3–10, COLO201, and COLO205 cells. As shown in Figure 3, the dissociation constant (KD) of C44Mab-1 for CHO/CD44v3–10 (Figure 3A),

Curr. Issues Mol. Biol. 2023, 453664

We next performed the flow cytometry-based measurement of the apparent binding
affinity of C44 Mab-1 to CHO/CD44v3–10, COLO201, and COLO205 cells. As shown in
Figure 3, the dissociation constant (KD ) of C44 Mab-1 for CHO/CD44v3–10 (Figure 3A),
Curr. Issues Mol. Biol. 2023, 3, FOR PEER
REVIEW(Figure 3B), and COLO205 (Figure 3C) was 2.5 × 10−8 M, 3.3 × 10−8 M, and
COLO201
6.5 × 10−8 M, respectively. The results indicated that C44 Mab-1 possesses a moderatebinding affinity for CD44v3–10 or endogenous CD44v9-expressing cells.

Figure 3. The determination of the binding affinity of C Mab-1. Serially diluted C Mab-1 at

44
44
Figure 3. The determination of the binding affinityof C44Mab-1. Serially diluted
C44Mab-1
indicated concentrations was treated with CHO/CD44v3–10 (A), COLO201 (B), and COLO205 (C).
cated concentrations was treated with CHO/CD44v3–10 (A), COLO201 (B), and COLO205 (CThen, cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488. Fluorescence data
cells
were treated with anti-mouse IgG conjugated with Alexa Fluor 488. Fluorescence dawere collected, followed by the calculation of the apparent dissociation constant (KD ) by GraphPad
collected,
PRISM 8. followed by the calculation of the apparent dissociation constant (KD) by Gr
PRISM 8.

3.3. Western Blot Analysis
We next performed
western blot analysis to assess the sensitivity of C44 Mab-1. Total
3.3. Western
Blot Analysis

cell lysates of CHO-K1, CHO/CD44s, and CHO/CD44v3–10 were analyzed. As shown inWeperformed western blot analysis to assess the sensitivity of C44MabFigure
4, Cnext
44 Mab-1 detected CD44v3–10 at more than 180-kDa and ~75 kDa bands mainly.

cell lysates of CHO-K1, CHO/CD44s, and CHO/CD44v3–10 were analyzed. As sh
Figure 4, C44Mab-1 detected CD44v3–10 at more than 180-kDa and ~75 kDa bands m
However, C44Mab-1 never detected any bands from lysates of CHO/CD44s and C
cells (Figure 4A). An anti-pan-CD44 mAb, C44Mab-46, recognized CD44s (~75 kD

Curr. Issues Mol. Biol. 2023, 45

3665

However, C44 Mab-1 never detected any bands from lysates of CHO/CD44s and CHOK1 cells (Figure 4A). An anti-pan-CD44 mAb, C44 Mab-46, recognized CD44s (~75 kDa)
and CD44v3–10 (>180 kDa) bands in the lysates of CHO/CD44s and CHO/CD44v3–10,
Curr. Issues Mol. Biol. 2023, 3, FOR PEER REVIEW
8
respectively (Figure 4B). The loading control, IDH1 was observed in each lane (Figure 4C).
These results indicated that C44 Mab-1 is able to detect exogenous CD44v3–10.

FigureWesternblot
blot
analysisby
C44 Mab-1.
Thecelltotal
cell lysates(10µg of protein)
were separated
Figure 4.4.Western
analysisby C
44Mab-1.
The total
lysates(10 µg ofprotein)were separated
and transferredtransferred onto
difluoride(PVDF)
membranes.
The Themembranes
werewereincu-incubatedandontopolyvinylidene
polyvinylidene
difluoride(PVDF)
membranes.
membranesbated with 10 µg/mL of C44Mab-1 (A), 10 µg/mL of C44Mab-46 (B), or 1 µg/mL of RcMab-1 (C), folwith 10 µg/mL of C44 Mab-1 (A), 10 µg/mL of C44 Mab-46 (B), or 1 µg/mL of RcMab-1 (C), followed
lowed by incubation with peroxidase-conjugated anti-mouse (for C44Mab-1 and C44Mab-46) or antiby
withperoxidase-conjugatedanti-mouse(for
C44kDa).
Mab-46)or anti-rat (for44 Mab-1 andrat incubation(for RcMab-1)
immunoglobulins.
The red arrows
indicate
theCCD44v3–10(>180
The black
RcMab-1)
immunoglobulins.
The
redarrowsindicatetheCD44v3–10
(>180
kDa).
The
black arrow
arrow indicates CD44s (~75 kDa). The white arrow indicates the lower molecular weight band recognized
by
C
44
Mab-1
in
CHO/CD44v3–10
lysate(~75
kDa).
indicates CD44s (~75 kDa). The white arrow indicates the lower molecular weight band recognizedby C44 Mab-1 in CHO/CD44v3–10 lysate (~75 kDa).
3.4. Immunohistochemical Analysis Using C44Mab-1 against Tumor Tissues

3.4. Immunohistochemical
AnalysisUsingcould
C44 Mab-1
againstTumor Tissues
We next examined whether
C44Mab-1
be usedfor immunohistochemical
analyses using
FFPE
sections. Because
has been
revealed
as forthe immunohistochemicalsecond highest CD44- analyWe next
examined
whetherHNSCC
C44 Mab-1
could
be usedexpressing
cancertype
inthe
Pan-Cancer
Atlas
[9],
we
first
examinedthesecondreactivityof CD44ses using FFPE sections. Because HNSCC has been revealed as thehighestC
44Mab-1 and C44Mab-46 in an oral SCC tissue, as a positive tissue control. As shown in
expressing cancer type in the Pan-Cancer Atlas [9], we first examined the reactivity ofSupplementary Figure S2, C44Mab-1 exhibited a clear membranous staining and was able
C
44 Mab-1 and C44 Mab-46 in an oral SCC tissue, as a positive tissue control. As shown in
to clearly distinguish tumor cells from stromal tissues. In contrast, C 44Mab-46 stained
Supplementary Figure S2, C44 Mab-1 exhibited a clear membranous staining and was ableboth. The reactivity of C44Mab-1 was completely blocked by the epitope peptide
toclearly distinguish tumor cells from stromal tissues. In contrast, C44 Mab-46
stained both.
CD44p471–490 (Supplementary Figure S3A). Isotype control antibody (PMab-44,
mouse
The
C44stain
Mab-1
completely
blocked by theFigure
epitope
peptideCD44p471–490
IgG1,reactivity
kappa) didofnot
the wasoral SCC
tissue (Supplementary
S3B).
C 44Mab-1
did(Supplementary
Figurecontrol
antibody (PMab-44,
mouse IgG1 , kappa) did
not stain a negative
tissue S3A).
controlIsotype(cat rectum,
Supplementary
Figure S3C).
not stain
the investigatedoral SCC tissue(Supplementary
FigureC44 Mab-1
notstainara negative
We thenthe reactivityof C44Mab-1
andS3B).
C44Mab-46
in thedid
CRC
tissuetissue(cat rectum,
Supplementary
FigureS3C). staining throughout CRC
ray. C44control
Mab-1 showedstrongmembranous andcytoplasmic
cells (Figure
5A).
C44Mab-46 thesimilarly
stainedthe
CRC cells(Figure5B). Ininsome
We theninvestigatedreactivityof C
and
C44 Mab-46
the CRC
CRCtistissue array.
44 Mab-1
sues,
both
C
44Mab-1 andC
44Mab-46
stainedthebasolateralsurface
of
CRC
cells
(FigureC
Mab-1
showedstrongmembranousandcytoplasmic
staining
throughout
CRC cells
44
5C,D). In5A).
contrast,
neither similarly
C44Mab-1 nor
C44Mab-46
evercells
stainedCRC 5B).
cells Inin some(FigureC44 Mab-46
stainedthe CRC(FiguresomeCRC
CRC tissues,
tissuesC(Figure
5E,F). In addition, stromal staining by C44Mab-46 was also observed in sevboth
44 Mab-1 and C44 Mab-46 stained the basolateral surface of CRC cells (Figure 5C,D).
eral tumor tissues (Figure 5F). In normal colon epithelium, epithelial cells were rarelyCurr. Issues Mol. Biol. 2023, 45

3666

In contrast,
Curr. Issues Mol. Biol. 2023, 3, FOR PEER
REVIEW neither C44 Mab-1 nor C44 Mab-46 ever stained CRC cells in some CRC
9 tissues(Figure 5E,F). In addition, stromal staining by C44 Mab-46 was also observed in several
tumor tissues (Figure 5F). In normal colon epithelium, epithelial cells were rarely stainedby
C44 Mab-1(Figure(Figure5G). In
contrast,
C44 Mab-46
mainlystainedin normal
stainedby C44Mab-1
5G).
In contrast,
C44Mab-46
mainlystainedstromal
stromal tissues
tissues incolon
epithelium(Figure5H). 5H).
normalcolon epithelium(FigureFigure 5.
5. Immunohistochemical
analysisusing
C44Mab-1
and Cand
44Mab-46
against against
CRC tissues.
FigureImmunohistochemical
analysisusing
C44 Mab-1
C44 Mab-46
CRCAftertissues. After
antigenretrieval,
serialsectionsofCRC
tissuearrays(CO483a)wereincubatedwith1
antigen retrieval, serial sections of CRC tissue arrays (CO483a) were incubated µg/mL
with 1ofµg/mL of
C44Mab-1 or C44Mab-46 followed by treatment with the Envision+ kit. The color was developed usCing
or C44 Mab-46tetrahydrochloride
followed by treatment
theEnvision+kit. The colorwith
washedeveloped
44 Mab-1
3,3’-diaminobenzidine(DAB), withand thesections
were counterstainedusing
3,3’-diaminobenzidine
tetrahydrochloride(DAB),
andthesections
were
counterstainedwithmatoxylin. Scale bar = 100 µm. CRC (A–F); normal colon epithelium (G,H).
hematoxylin. Scale bar = 100 µm. CRC (A–F); normal colon epithelium (G,H).
We summarized the data of the immunohistochemical analyses in Table 1; C44Mab-1
stainedout of 40 cases
(40%)
CRC.
results indicated that
C44Mab-1
useful1;forWe16summarized
thedatainof
the These
immunohistochemical
analysesinisTable
C44 Mab-1
immunohistochemical
analysisof FFPE
tumor
sections.
stained16 out of 40 cases(40%)
in CRC.
These
results indicated that C44 Mab-1 is useful for

immunohistochemical analysis of FFPE tumor sections.

Curr. Issues Mol. Biol. 2023, 45

3667

Table 1. Immunohistochemical analysis using C44 Mab-1 against colorectal carcinoma tissue array.
No.

Age

Sex

Organ

Pathology Diagnosis

Grade

Stage

Type

C44 Mab-1

C44 Mab-46

1

67

M

Colon

Adenocarcinoma

2

48

M

Colon

Adenocarcinoma

1-

Malignant

+

+

1

IIA

Malignant

-

-

3

58

M

Colon

4

75

M

Colon

Adenocarcinoma

1–2

IIA

Malignant++Adenocarcinoma

1

IV

Malignant

-

++

5

86

M

6

55

M

Colon

Adenocarcinoma

2

II

Malignant

-

+

Colon

Adenocarcinoma

2

IIIC

Malignant

-

-

7

38

8

52

M

Colon

Adenocarcinoma

1

I

Malignant

-

++

M

Colon

Adenocarcinoma

1

IIIB

Malignant

+

-

9

46

M

Colon

Adenocarcinoma

2

IIIB

Malignant

++

+

10

61

M

Colon

Mucinous
adenocarcinoma

2

IIIB

Malignant

+

++

11

55

M

Colon

Mucinous
adenocarcinoma withnecrosis

2

IIA

Malignant

-

++

12

55

M

Colon

Adenocarcinoma

1

IIIB

Malignant

+

-

13

44

M

Colon

Adenocarcinoma

1-

Malignant

-

-

14

31

M

Colon

Adenocarcinoma

2

IIIB

Malignant

-

+

15

74

F

Colon

Adenocarcinoma

2

IIIB

Malignant

+

+

16

61

M

Colon

Adenocarcinoma

2

II

Malignant

++

++

17

45

M

Colon

Adenocarcinoma

2

III

Malignant

+

+

18

58

M

Colon

Adenocarcinoma

2

IIIB

Malignant

-

++

19

58

M

Colon

Adenocarcinoma

2

IIA

Malignant

+++

+++

20

69

M

Colon

Adenocarcinoma

3-

Malignant

-

-

21

64

F

Colon

Adenocarcinoma

2

IIIC

Malignant

++

++

22

82

M

Colon

Adenocarcinoma

2

IIIB

Malignant

-

-

23

34

M

Colon

Adenocarcinoma

2

IIIB

Malignant

++

++

24

50

F

Colon

Adenocarcinoma

2

IIB

Malignant

-

-

25

34

F

Colon

Adenocarcinoma

1

IIB

Malignant

-

+

26

52

F

Colon

Adenocarcinoma

2

IIA

Malignant

-

+

27

53

F

Colon

Adenocarcinoma

2

IIIB

Malignant

-

-

28

58

F

Colon

Adenocarcinoma

2

I

Malignant

-

+

29

59

F

Colon

Adenocarcinoma

2

IIA

Malignant

++

++

30

67

M

Colon

Adenocarcinoma

2

IIIB

Malignant

-

++

31

31

M

Colon

Adenocarcinoma

2

IIIB

Malignant

+++

+++

32

54

F

Colon

Adenocarcinoma

2

IIB

Malignant

-

+

33

54

F

Colon

Adenocarcinoma

2

IIIB

Malignant

-

-

34

62

M

Colon

Adenocarcinoma

2-

Malignant

-

+

35

67

F

Colon

Adenocarcinoma

2-

Malignant

+

-

36

52

F

Colon

Adenocarcinoma

2

IIA

Malignant

-

-

37

52

F

Colon

Adenocarcinoma

3

IIIB

Malignant

-

-

38

75

M

Colon

Adenocarcinoma

2-

Malignant

-

-

39

57

F

Colon

Adenocarcinoma

2

IIB

Malignant

+

+++

40

38

M

Colon

Mucinous
adenocarcinoma

3

I

Malignant---, No stain; +, Weak intensity; ++, Moderate intensity; +++, Strong intensity.

Curr. Issues Mol. Biol. 2023, 453668

4. Discussion
Using the CBIS method, we developed C44 Mab-1 (Figure 1) and determined its epitope
as a variant 9-encoded region using ELISA (Supplementary Table S1). Then, we showed themultiple applications of C44 Mab-1 for flow cytometry (Figures 2 and 3), western blotting(Figure 4), and immunohistochemistry using OSCC (Supplementary Figure S2) and CRC
tissues (Figure 5 and Table 1).
Ishimoto et al. [22] demonstrated that CD44v interacts with xCT, a glutamate-cystinetransporter, and regulates the level of reduced glutathione (GSH) in gastric cancer cells.
As a result, CD44v contributes to the reduction of intracellular ROS. The knockdown of
CD44 reduced the cell surface expression of xCT and suppressed tumor growth in a mousegastric cancer model. Furthermore, they showed that the v8–10 region of CD44v is required
for the specific interaction between CD44v and xCT, and CD44v8–10 (S301A), an N-linked
glycosylation site mutant, failed to interact with xCT. These results showed an importantfunction for CD44v in the regulation of ROS defense and tumor growth.
Ishimoto et al. [22] also established a rat mAb (clone RV3) against CD44v8–10 byimmunizing CD44v8–10-expressed RH7777 cells. The epitope of the mAb was determined
as a variant 9-encoded region using the recombinant CD44v9 protein by ELISA. RV3 wasmainly used in immunohistochemistry and revealed a predictive marker for recurrenceof gastric [59] and urothelial [60] cancers, predicting survival outcome in hepatocellular
carcinomas [61], and an indicator for identifying a cisplatin-resistant population in urothelial cancers [62]. Therefore, CD44v9 is a critical biomarker to evaluate the malignancy
and prognosis of tumors. Furthermore, sulfasalazine, an xCT inhibitor, was shown tosuppress the survival of CD44v9-positive CSCs both in vitro [63–65] and in vivo [66]. A
dose-escalation clinical study in patients with advanced gastric cancers revealed that sulfasalazine reduced the population of CD44v9-positive cells in tumors [67], suggesting thatCD44v9 is a biomarker for patient selection and efficacy of xCT inhibitors.
As mentioned above, RV3 recognized the recombinant CD44v9 protein using ELISA.
Therefore, RV3 is thought to recognize the peptide or glycopeptide structure of CD44v9.
However, the detailed binding epitope of RV3 has not been determined. As shown in
Supplementary Table S1, C44 Mab-1 recognized a synthetic peptide (CD44p471–490; STSHEGLEEDKDHPTTSTLT) that possesses multiple predicted and confirmed O-glycan sites [68].
As shown in Figure 4A, C44 Mab-1 recognized a ~75kDa band in CHO/CD44v3–10 lysate,
which is approximately identical to the predicted molecular weight of CD44v3–10 basedon the amino acid length. Therefore, C44 Mab-1 could recognize CD44v3–10 regardless ofthe glycosylation. The detailed epitope mapping and the influence of the glycosylation on
C44 Mab-1 recognition should be investigated in future studies.
Using large-scale genomic analyses, CRCs were classified into four subtypes: microsatellite instability immune, canonical, metabolic, and mesenchymal types [69]. Since
the CD44v9 was upregulated in 40% of CRC tissues (Figure 5 and Table 1), the relationshipto the subtypes should be determined. Additionally, the mechanism of CD44v9 upregulation, including the transcription and the v9 inclusion by alternative splicing, should be
investigated. Wielenga et al. [70] demonstrated that CD44 is a target gene of Wnt/β-cateninin a mice intestinal tumor model, suggesting that β-catenin signaling pathway could upregulate CD44 transcription. However, the mechanism of the variant 9 inclusion during the
CRC development remains to be determined.
In immunohistochemical analysis, we observed CD44v9 expression throughout CRC
cells (Figure 5A) and on the basolateral surface of CRC cells (Figure 5C). The basolateralexpression of CD44 was previously observed and shown to be co-localized with HA [71],
EpCAM-Claudin-7 complex [72], and Annexin II [73]. Therefore, the basolateral expression of CD44 may function to promote HA/adhesion-mediated signal transduction andcontribute CRC tumorigenesis.
Clinical trials of anti-pan CD44 and CD44v6 mAbs have been conducted [74]. RG7356,
an anti-pan CD44 mAb, exhibited an acceptable safety profile. However, the trial was terminated because of no clinical and dose-response relationship with RG7356 [75]. Clinical trials

Curr. Issues Mol. Biol. 2023, 453669

of an antibody-drug conjugate (ADC), an anti-CD44v6 mAb bivatuzumab−mertansine,
were conducted. However, it failed due to the high toxicity to skin [76,77]. The anti-CD44v6
mAb is further developed to chimeric antigen receptor T (CAR-T) cell therapy. The CD44v6
CAR-T showed antitumor effects against primary human multiple myeloma and acute
myeloid leukemia [78]. Furthermore, the CD44v6 CAR-T also suppressed the xenograft
tumor growth of lung and ovarian carcinomas [79], which is expected for the application
against solid tumors. Although CD44v9 is rarely detected in normal colon epithelium by
C44 Mab-1, CD44v9 could be detected in other normal tissues, including oral squamous
epithelium (Supplementary Figure S2). For the development of the therapeutic use of
C44 Mab-1, further investigations are required to reduce the toxicity to the above tissues.
Because anti-CD44 mAbs could have side effects by affecting normal tissues, theclinical applications of anti-CD44 mAbs are still limited. We previously developed PDPNtargeting cancer-specific mAbs (CasMabs) [80–83] and podocalyxin-targeting CasMabs [84],which are currently being applied to CAR-T therapy in mice models [39,40,48]. These
CasMabs recognize cancer-specific aberrant glycosylation of the target proteins [83]. It
is worthwhile to establish cancer-specific anti-CD44 mAbs using the CasMab method.
Anti-CD44 CasMabs production can be applicable as a basis for designing and optimizing
potent immunotherapy modalities, including ADCs and CAR-T therapies.
5. Conclusions
An anti-CD44v9 mAb, C44 Mab-1 is useful for detecting CD44v9 in flow cytometry,western blotting, and immunohistochemistry.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cimb45040238/s1. Figure S1: Conformation of the recognitionof CHO/CD44s and CHO/CD44v3–10 by C44Mab-46 by flow cytometry. CHO/CD44v3–10 (A),CHO/CD44s (B), and CHO-K1 (C) were treated with 0.01-10 µg/mL of C44Mab-46, followed bytreatment with Alexa Fluor 488-conjugated antimouse IgG (Red line). The black line represents thenegative control (blocking buffer). Figure S2: Immunohistochemical analysis using C44Mab-1 andC44Mab-46 against oral squamous cell carcinoma tissues. After antigen retrieval, the sections wereincubated with 1 µg/mL of C44Mab-1 (A) and 1 µg/mL of C44Mab-46 (B), followed by treatment with
the Envision+ kit. The color was developed using 3,30 -diaminobenzidine tetrahydrochloride (DAB),
and the sections were counterstained with hematoxylin. Scale bar = 100 µm. Figure S3: The blocking
assay by an epitope peptide, isotype control, and negative tissue control in immunohistochemistry.
(A) Blocking of the C44Mab-1 reactivity to oral SCC tissue (positive tissue control) by the CD44 peptide(aa 471–490) containing the C44Mab-1 epitope. After antigen retrieval, sections were incubated withC44Mab-1 (1 µg/mL) or C44Mab-1 (1 µg/mL) plus human CD44 peptide (aa 471–490, 10 µg/mL).
(B) The oral SCC tissue section was incubated with an isotype control mAb, PMab-44 (1 µg/mL).
(C) Anegative control tissue section (cat rectum) was incubated with C44Mab-1 (1 µg/mL). The
tissues were further treated with the Envision+ kit. The color was developed using DAB, and sections
were counterstained with hematoxylin. Scale bar = 100 µm. Table S1: The determination of thebinding epitope of C44Mab-1 by ELISA.
Author Contributions: M.T., N.G. and T.T. performed the experiments. M.K.K. and Y.K. designed
the experiments. M.T. and H.S. analyzed the data. M.T., H.S. and Y.K. wrote the manuscript. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.), JP22bm1004001(to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.) and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) under Grant Numbers
21K20789 (to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal
Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving animals.
Informed Consent Statement: Not applicable.

Curr. Issues Mol. Biol. 2023, 45

3670

Data Availability Statement: All related data and methods are presented in this paper. Additional
inquiries should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

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