International Journal of

Molecular Sciences
ArticleDevelopment of a Novel Anti-CD44 Variant 6 Monoclonal
Antibody C44Mab-9 for Multiple Applications against
Colorectal Carcinomas
Ryo Ejima 1,† , Hiroyuki Suzuki 1, *,† , Tomohiro Tanaka 1 , Teizo Asano 2 , Mika K. Kaneko 2
and Yukinari Kato 1,2, *
1

2

*
†

Citation: Ejima, R.; Suzuki, H.;
Tanaka, T.; Asano, T.; Kaneko, M.K.;
Kato, Y. Development of a Novel
Anti-CD44 Variant 6 Monoclonal
Antibody C44 Mab-9 for Multiple
Applications against Colorectal

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan
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: CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative
splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v)
are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts
poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion,
proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target
for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal
antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary(CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow
cytometry, western blotting, and immunohistochemistry. One of the established clones (C44 Mab9; IgG1 , kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44 Mab-9
recognizes CD44v6. Furthermore, C44 Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines(COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD ) of C44 Mab-9
for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2.3 × 10−8 M,
respectively. C44 Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalinfixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44 Mab-9 is useful for
detecting CD44v6 in various applications.

Carcinomas. Int. J. Mol. Sci. 2023, 24,4007. https://doi.org/10.3390/

Keywords: CD44; CD44v6; monoclonal antibody; colorectal cancer

ijms24044007
Academic Editor: Donatella Delle
Cave
Received: 5 January 2023
Revised: 2 February 2023
Accepted: 14 February 2023
Published: 16 February 2023

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/).

1. Introduction
Colorectal cancer (CRC) has become the second cancer type for the estimated deaths
in men and women combined in the United States, 2023 [1]. The development of CRC
is classically explained by Fearon and Vogelstein model; the sequential genetic changes
including APC (adenomatous polyposis coli), KRAS, DCC (deleted in colorectal cancer,
chromosome 18q), and TP53 lead to CRC progression [2]. However, CRC exhibits heterogeneous outcomes and drug responses. Therefore, the large-scale data analysis by
an international consortium classified the CRC into four consensus molecular subtypes,
including the microsatellite instability immune, the canonical, the metabolic, and the mesenchymal types [3]. In addition, various marker proteins have been investigated for theprediction of prognosis and drug responses of CRC [4,5]. Among them, recent studies
suggest that CD44 plays a critical role in tumor progression through its cancer-initiating
and metastasis-promoting properties [6].
CD44 is a polymorphic integral membrane protein, which binds to hyaluronic acid
(HA), and contributes to cell-matrix adhesion, cell proliferation, migration, and tumor

Int. J. Mol. Sci. 2023, 24, 4007. https://doi.org/10.3390/ijms24044007

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metastasis [7]. When the CD44 is transcribed, its pre-messenger RNA can be received
alternative splicing and maturated into mRNAs that encode various CD44 isoforms [8].
The mRNA assembles with ten standard exons and the sixth variant exon encodes CD44v6,
which plays critical roles in cell proliferation, migration, survival, and angiogenesis [9,10].
Functionally, CD44v6 can interact with HA via the standard exons-encoded region [11].
Furthermore, the v6-encoded region functions as a co-receptor of various receptors for
epidermal growth factor, hepatocyte growth factor, C-X-C motif chemokine 12, and osteopontin [12]. Therefore, the receptor tyrosine kinase or G protein-coupled receptor signaling
pathways are potentiated in the presence of CD44v6 [13]. These functions are essential forhomeostasis or regeneration in normal tissues. Importantly, CD44v6 overexpression playsa critical role in CRC progression. For instance, CD44v6 is involved in colorectal carcinoma
invasiveness, colonization, and metastasis [14]. Therefore, CD44v6 is a promising target forcancer diagnosis and therapy.
The clinical significance of CD44v6 in CRC deserves consideration. Anti-CD44v6
therapies mainly include the blocking of the v6-encoded region by monoclonal antibody(mAb) [12]. First, humanized anti-CD44v6 mAbs (BIWA-4 and BIWA-8) labeled with
186 Re exhibited therapeutic efficacy in head and neck squamous cell carcinoma (SCC)
xenograft-bearing mice [15]. Furthermore, the humanized anti-CD44v6 mAb, bivatuzumabmertansine (anti-tubulin agent) conjugate, was evaluated in clinical trials [16]. However,the clinical trials were discontinued due to severe skin toxicity, including a case of lethal
epidermal necrolysis [17]. The efficient accumulation of mertansine was most likely responsible for the high toxicity [17,18]. Therefore, the development of anti-CD44v6 mAbs withmore potent and fewer side effects is desired.
We established the novel anti-CD44 mAbs, C44 Mab-5 (IgG1 , kappa) [19] and C44 Mab46 (IgG1 , kappa) [20] by Cell-Based Immunization and Screening (CBIS) method and immunization of CD44v3-10 ectodomain, respectively. Both C44 Mab-5 and C44 Mab-46 recognize
the first five standard exons-encoding sequences [21–23]. Therefore, they can recognize
both CD44s and CD44v (pan-CD44). Furthermore, C44 Mab-5 and C44 Mab-46 exhibitedhigh reactivity for flow cytometry and immunohistochemical analysis in oral [19] andesophageal [20] SCCs. C44 Mab-5 reacted with oral cancer cells such as Ca9-22, HO-1-u-1,
SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry [19]. Moreover, immunohistochemical analysis revealed that C44 Mab-5 detected 166/182 (91.2%) of oral cancers [19]. In
contrast, C44 Mab-46 reacted with esophageal squamous cell carcinoma (ESCC) cell lines(KYSE70 and KYSE770) using flow cytometry [20]. In immunohistochemical analyses using
C44 Mab−46 against ESCC tissue microarrays, C44 Mab−46 stained 63 of 67 (94.0%) cases of
ESCC [20].
We also examined the antitumor effects of C44 Mab-5 in mouse xenograft models [24].
We converted the mouse IgG1 subclass antibody (C44 Mab-5) into an IgG2a subclass antibody(5-mG2a ), and further produced a defucosylated version (5-mG2a -f) using FUT8-deficient
ExpiCHO-S (BINDS-09) cells. In vitro analysis demonstrated that 5-mG2a -f showed moderate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity activities against HSC-2 and SAS oral cancer cells. In vivo analysis revealed that
5-mG2a -f significantly reduced tumor growth in HSC-2 and SAS xenografts in comparison
to control mouse IgG, even after injection seven days post-tumor inoculation. These results
suggested that treatment with 5-mG2a -f may represent a useful therapy for patients with
CD44-expressing oral cancers.
For epitope mapping of C44 Mab-5, we employed the RIEDL tag system (“RIEDL”
peptide and LpMab-7 mAb) [23]. We inserted the “RIEDL” peptide into the CD44 proteinfrom the 21st to 41st amino acids. The transfectants produced were stained by LpMab-7
and C44 Mab-5 in flow cytometry. C44 Mab-5 did not react with the 30th-36th amino acidsof the deletion mutant of CD44. Further, the reaction of C44 Mab-5 to RIEDL tag-inserted
CD44 from the 25th to 36th amino acids was lost, although LpMab-7 detected most of the
RIEDL tag-inserted CD44 from the 21st to 41st amino acids. These results indicated that

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Int. J. Mol. Sci. 2023, 24, 4007

3 of 16RIEDL tag-inserted CD44 from the 21st to 41st amino acids. These results indicated thatthe epitope of C44Mab-5 for CD44 was determined to be the peptide from the 25th to 36th
amino acids of CD44 using the RIEDL insertion for epitope mapping (REMAP) method.
the epitopeof Cdeveloped
determinedmAb,
to be thepeptide(IgG
from1, the25th to44 Mab-5 foraCD44
In thisstudy, wenovelwasanti-CD44v6
C44Mab-9
kappa)
by36th
amino
acids
of
CD44
using
the
RIEDL
insertionfor
epitopemapping(REMAP)
method.
CBIS method, and evaluated its applications, including flow cytometry, western blotting,In this study, wedeveloped a novel anti-CD44v6 mAb, C44 Mab-9 (IgG1 , kappa) by
and immunohistochemical
analyses.

CBIS method, and evaluated its applications, including flow cytometry, western blotting,and immunohistochemical analyses.

2. Results

2.1. Establishment
2. Resultsof Anti-CD44v6 mAb, C44Mab-9
Establishmentof Anti-CD44v6
mAb, C44anti-CD44
Mab-9
We2.1.
employed
the CBIS
method to develop
mAbs. In the CBIS method, we
We employed
the CBIS
to develop
anti-CD44
mAbs. Inthehigh
CBISthroughmethod, we
prepared a stabletransfectant
as anmethod
immunogen.
Then,
we performed
thepreparedastabletransfectant
asan
immunogen.
Then,weperformedthehighthroughput
put hybridoma screening using flow cytometry (Figure 1). In this study, mice were
imhybridoma
screening
using
flow
cytometry(Figure1).
In
thisstudy,
mice
wereimmumunized with CHO/CD44v3-10 cells. Hybridomas were seeded into 96-well plates, andnized with CHO/CD44v3-10
cells. Hybridomas
wereselected.
seeded into
plates,CHO/CD44v3-10-positive
and CHO-K1-negative
wells were
After96-welllimiting
dilu-andCHO/CD44v3-10-positive
and
CHO-K1-negative
wells
were
selected.
Afterlimiting
tion, anti-CD44 mAb-producing clones were finally established. We next performed an dilution, anti-CD44 mAb-producing clones were finally established. We next performed
enzyme-linked immunosorbent assay (ELISA) to determine the epitope of each mAb.
an enzyme-linked immunosorbent assay (ELISA) to determine the epitope of each mAb.
Among them, C44Mab-9 (IgG1, kappa) was shown to recognize the only CD44p351–370
Among them, C44 Mab-9 (IgG1 , kappa) was shown to recognize the only CD44p351–370
peptide peptide(EETATQKEQWFGNRWHEGYR),
which is corresponding to variant 6-encoded(EETATQKEQWFGNRWHEGYR), which is corresponding to variant 6-encoded
sequence(Table1).
sequence (Table 1).

Figure 1.Figure
A schematic
illustrationof ant-humanCD44 CD44
mAbsmAbs
production.
A BALB/cmouse
was was
1. A schematic
illustrationof ant-human
production.
A BALB/c
mouse
intraperitoneallyimmunized
with
CHO/CD44v3-10
cells.
Hybridomas
were
producedbythefusionintraperitoneally immunized with CHO/CD44v3-10 cells. Hybridomas were produced by the fusionof the splenocytes
and P3U1
screening
was performedby flowcytometryusingof the splenocytes
andcells.
P3U1Then,cells. theThen,
the screening
was performedby flowcytometryusing
parental CHO-K1 and CHO/CD44v3-10 cells. After cloning and additional screening, a clone
parental CHO-K1 and CHO/CD44v3-10 cells. After cloning and additional screening, a clone C44 MabC44Mab-9 (IgG1, kappa) was established. Finally, the binding epitopes were determined by enzyme9 (IgG1 , kappa) was established. Finally, the binding epitopes were determined by enzyme-linked
linked immunosorbent
assay (ELISA) using peptides that cover the extracellular domain of CD44v3immunosorbent assay (ELISA) using peptides that cover the extracellular domain of CD44v3-10.
10.

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Table 1. The determination of the binding epitope of C44 Mab-9 by ELISA.
PeptideCoding Exon *Sequence

C44 Mab-9

CD44p21–40

2

QIDLNITCRFAGVFHVEKNG−

CD44p31–50

2

AGVFHVEKNGRYSISRTEAA−

CD44p41–60

2

RYSISRTEAADLCKAFNSTL−

CD44p51–70

2

DLCKAFNSTLPTMAQMEKAL−

CD44p61–80

2/3

PTMAQMEKALSIGFETCRYG−

CD44p71–90

2/3

SIGFETCRYGFIEGHVVIPR−

CD44p81–100

3

FIEGHVVIPRIHPNSICAAN−

CD44p91–110

3

IHPNSICAANNTGVYILTSN−

CD44p101–120

3

NTGVYILTSNTSQYDTYCFN−

CD44p111–130

3/4

TSQYDTYCFNASAPPEEDCT−

CD44p121–140

3/4

ASAPPEEDCTSVTDLPNAFD−

CD44p131–150

4/5

SVTDLPNAFDGPITITIVNR−

CD44p141–160

4/5

GPITITIVNRDGTRYVQKGE−

CD44p151–170

5

DGTRYVQKGEYRTNPEDIYP−

CD44p161–180

5

YRTNPEDIYPSNPTDDDVSS−

CD44p171–190

5

SNPTDDDVSSGSSSERSSTS−

CD44p181–200

5GSSSERSSTSGGYIFYTFST−

CD44p191–210

5

GGYIFYTFSTVHPIPDEDSP−

CD44p201–220

5

VHPIPDEDSPWITDSTDRIP−

CD44p211–230

5/v3

WITDSTDRIPATSTSSNTIS−

CD44p221–240

5/v3

ATSTSSNTISAGWEPNEENE−

CD44p231–250

v3

AGWEPNEENEDERDRHLSFS−

CD44p241–260

v3

DERDRHLSFSGSGIDDDEDF−

CD44p251–270

v3/v4

GSGIDDDEDFISSTISTTPR−

CD44p261–280

v3/v4

ISSTISTTPRAFDHTKQNQD−

CD44p271–290

v4

AFDHTKQNQDWTQWNPSHSN −CD44p281–300

v4

WTQWNPSHSNPEVLLQTTTR−

CD44p291–310

v4/v5

PEVLLQTTTRMTDVDRNGTT−

CD44p301–320

v4/v5

MTDVDRNGTTAYEGNWNPEA−

CD44p311–330

v5

AYEGNWNPEAHPPLIHHEHH−

CD44p321–340

v5

HPPLIHHEHHEEEETPHSTS−

CD44p331–350

v5/v6

EEEETPHSTSTIQATPSSTT−

CD44p341–360

v5/v6

TIQATPSSTTEETATQKEQW−

CD44p351–370

v6

EETATQKEQWFGNRWHEGYR+

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Table 1. Cont.
PeptideCoding Exon *Sequence

C44 Mab-9

CD44p361–380

v6

FGNRWHEGYRQTPREDSHST−CD44p371–390

v6/v7

QTPREDSHSTTGTAAASAHT−

CD44p381–400

v6/v7

TGTAAASAHTSHPMQGRTTP−

CD44p391–410

v7

SHPMQGRTTPSPEDSSWTDF−

CD44p401–420

v7

SPEDSSWTDFFNPISHPMGR−

CD44p411–430

v7/v8

FNPISHPMGRGHQAGRRMDM−

CD44p421–440

v7/v8

GHQAGRRMDMDSSHSTTLQP−

CD44p431–450

v8

DSSHSTTLQPTANPNTGLVE−

CD44p441–460

v8

TANPNTGLVEDLDRTGPLSM−

CD44p451–470

v8/v9

DLDRTGPLSMTTQQSNSQSF−

CD44p461–480

v8/v9

TTQQSNSQSFSTSHEGLEED−

CD44p471–490

v9

STSHEGLEEDKDHPTTSTLT−CD44p481–500

v9/v10KDHPTTSTLTSSNRNDVTGG−

CD44p491–510

v9/v10SSNRNDVTGGRRDPNHSEGS−

CD44p501–520

v10

RRDPNHSEGSTTLLEGYTSH−

CD44p511–530

v10

TTLLEGYTSHYPHTKESRTF−

CD44p521–540

v10

YPHTKESRTFIPVTSAKTGS−

CD44p531–550

v10

IPVTSAKTGSFGVTAVTVGD−

CD44p541–560

v10

FGVTAVTVGDSNSNVNRSLS−

CD44p551–570

v10/16SNSNVNRSLSGDQDTFHPSG−

CD44p561–580

v10/16GDQDTFHPSGGSHTTHGSES−

CD44p571–590

16/17

GSHTTHGSESDGHSHGSQEG−

CD44p581–600

16/17

DGHSHGSQEGGANTTSGPIR−

CD44p591–606

17

GANTTSGPIRTPQIPEAAAA−+, OD655 = 0.3; −, OD655 < 0.1. * The CD44 exons are illustrated in Figure 1.

2.2. Flow Cytometric Analysis of C44 Mab-9 to CD44-Expressing Cells
We next confirmed the reactivity of C44 Mab-9 against CHO/CD44v3-10 and CHO/CD44s
cells by flow cytometry. As shown in Figure 2A, C44 Mab-9 recognized CHO/CD44v310 cells in a dose-dependent manner, but neither CHO/CD44s (Figure 2B) nor CHO-K1(Figure 2C) cells. The CHO/CD44s cells were recognized by a pan-CD44 mAb, C44 Mab46 [20] (Supplemental Figure S1). Furthermore, C44 Mab-9 also recognized endogenous
CD44v6 in CRC cell lines as it reacted with both COLO201 (Figure 2D) and COLO205(Figure 2E) in a dose-dependent manner.
Next, we determined the binding affinity of C44 Mab-9 with CHO/CD44v3-10, COLO201,
and COLO205 using flow cytometry. The dissociation constant (KD ) of C44 Mab-9 for
CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and2.3 × 10−8 M, respectively, indicating that C44 Mab-9 possesses a moderate affinity forCD44v3-10 or endogenous CD44v6-expressing cells (Figure 3).

Int. J. Mol. Sci. 2023, 24, 4007

CHO/CD44s cells by flow cytometry. As shown in Figure 2A, C44Mab-9 rec
CHO/CD44v3-10 cells in a dose-dependent manner, but neither CHO/CD44s (Fignor CHO-K1 (Figure 2C) cells. The CHO/CD44s cells were recognized by a pa
mAb, C44Mab-46 [20] (Supplemental Figure S1). Furthermore, C44Mab-9 also recendogenous CD44v6 in CRC cell lines as it reacted with both COLO201(Figure6 of 16
COLO205 (Figure 2E) in a dose-dependent manner.

Figure 2. Flow cytometry to CD44-expressing cells using C44 Mab-9. CHO/CD44v3-10 (A),Figure 2. Flow cytometry to CD44-expressing cells using C44Mab-9. CHO/CD44v3
CHO/CD44s (B), CHO-K1 (C), COLO201 (D), and COLO205 (E) were treated with 0.01–10 µg/mL of
CHO/CD44s (B), CHO-K1 (C), COLO201 (D), and COLO205 (E) were treated with 0.01–1
C44 Mab-9, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red line). The
of C44Mab-9, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red l
black line represents the negative control (blocking buffer).

black line represents the negative control (blocking buffer).

Next, we determined the binding affinity of C44Mab-9 with CHO/CD
COLO201, and COLO205 using flow cytometry. The dissociation constant (KD) of C
for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10−9 M, 1.7 × 10−8 M, and 2

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Int. J. Mol. Sci. 2023, 24, 4007

6 o

16M, respectively, indicating that C44Mab-9 possesses a moderate affinity for7 of
CD44v3-10
endogenous CD44v6-expressing cells (Figure 3).

Figure 3.

The determination of the binding affinity of C44 Mab-9 to CD44-expressing cells.

Figure3. The determinationthebinding
C44Mab-9ce
CHO/CD44v3-10(A), COLO201 (B),ofand
COLO205(C) affinity
cells were ofsuspendedin 100toµLCD44-expressingof serially diCHO/CD44v3-10(A), COLO201(B), andCOLO205
cellswithwere
suspendedin 100 µL of seria
luted C44 Mab-9 at indicatedconcentrations.
Then,cells were(C)treatedAlexa
Fluor 488-conjugatedat indicatedconcentrations.
cells
were bytreatedwith Alexa
dilutedC44Mab-9
anti-mouse
IgG. Fluorescencedata were
subsequently Then,collected,
followed
the calculationof the Fluor 4
apparent dissociation
constant) by GraphPad data
PRISMwere8.
conjugatedanti-mouse
IgG.(KDFluorescence
subsequently collected, followed by
calculation of the apparent dissociation constant (KD) by GraphPad PRISM 8.

2.3. Western Blot Analysis

We next performed western blot analysis to assess the sensitivity of C44Mab-9. To

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7 of

2.3. Western Blot Analysis

performed western blot analysis to assess the sensitivity of C44 Mab-9. Total
FigureWe4, next
C44Mab-9
detected CD44v3-10 as a more than 180-kDa band. However, C44Ma
cell lysates of CHO-K1, CHO/CD44s, and CHO/CD44v3-10 were analyzed. As shown in
9 Figure
did not
any bands from lysates of CHO-K1 and CHO/CD44s cells. An anti-pa
4, Cdetect
44 Mab-9 detected CD44v3-10 as a more than 180-kDa band. However, C44 Mab-9
CD44
mAb,
C
44
Mab-46,
recognizedlysates
both CHO/CD44s(~75 kDa) a
did not detect any bands fromlysates ofthe
CHO-K1
andfrom
CHO/CD44s
cells. An anti-panCHO/CD44v3-10(>180 kDa).
These
results
C44Mab-9(~75specificallyCD44 mAb, C44 Mab-46,
recognizedthelysatesindicatedfrom boththat
CHO/CD44s
kDa) anddetects
CHO/CD44v3-10
(>180
kDa).
These
results
indicated
that
C
Mab-9
specificallydetects
ogenous CD44v3-10.
44exogenous CD44v3-10.

Figure 4.

Western blotting by C Mab-9.

The cell lysates of CHO-K1, CHO/CD44s, andFigure 4. Western blotting by44 C44Mab-9. The cell lysates of CHO-K1, CHO/CD44s, a
CHO/CD44v3-10 (10 µg of protein) were electrophoresed and transferred onto polyvinylidene difluoCHO/CD44v3-10 (10 µg of protein) were electrophoresed and transferred onto polyvinylide
ride (PVDF) membranes. The membranes were incubated with 10 µg/mL of C44 Mab-9, 10 µg/mL ofdifluoride (PVDF) membranes. The membranes were incubated with
10 µg/mL of C44Mab-9,
C44 Mab-46, and 1 µg/mL of anti-β-actin mAb, followed by incubation with peroxidase-conjugatedµg/mL of C44Mab-46, and 1 µg/mL of anti-β-actin mAb, followed by incubation with peroxida
anti-mouse immunoglobulins. The black arrow indicates the CD44s (~75 kDa). The red arrows
conjugatedanti-mouse immunoglobulins. The black arrow indicates the CD44s (~75 kDa). The
indicate the CD44v3-10 (>180 kDa).
arrows indicate the CD44v3-10 (>180 kDa).
2.4. Immunohistochemical Analysis Using C44 Mab-9 against Tumor Tissues
We next examined whether
C44 Mab-9
be usedfor immunohistochemical
analy2.4. Immunohistochemical
AnalysisUsingcould
C44Mab-9
againstTumor Tissues

ses using formalin-fixed paraffin-embedded (FFPE) sections. Since previous anti-CD44v6
We next examined whether C44Mab-9 could be used for immunohistochemical anmAbs could detect CD44v6 in SCC tissues at a high frequency, we first stained an oral SCC
yses
using
formalin-fixedparaffin-embedded(FFPE) sections. Since previous an
tissue.
As shown
in Figure 5A, C
44 Mab-9 exhibited clear membranous staining, and could
CD44v6
mAbs could
detectin SCCtissues
at a highfrequency,
wefirst stain
clearly distinguishtumor
cells CD44v6
from stromal
tissues.
In contrast,
C44 Mab-46
stainedboth
5B). Wenext investigatedCRC
sections.
membranous
stainingan(Figureoral SCC
tissue.
As shown in
Figure
5A,CC4444Mab-9
Mab-9showed
exhibitedclear membranoussta
in CRCbut
not stromal
tissues (Figure
contrast,
C44 Mab-46
alsoInstained
bothC44Mab
ing,
and cells,
could
clearly
distinguishtumor5C).
cellsInfrom
stromaltissues.
contrast,(Figure both
5D). These
results
that C44 Mab-9 CRC
is usefulfor immunohistochemical
stained(Figure5B). indicatedWe next investigatedsections.
C44Mab-9 showed memb
analysis of FFPE tumor sections.

nous staining in CRC cells, but not stromal tissues (Figure 5C). In contrast, C44Mab-46 astained both (Figure 5D). These results indicated that C44Mab-9 is useful for immunohis
chemical analysis of FFPE tumor sections.

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FigureMab-9 and C44Mab-46. (A,B) Oral SCC sections
Figure 5.
5. Immunohistochemical
Immunohistochemical analysis
analysis using
using C
C44
44 Mab-9 and C44 Mab-46. (A,B) Oral SCC sections
44Mab-9 (A) and C44Mab-46 (B). (C,D) CRC sections were incuwere
incubated
with
1
µg/mL
of
C
were incubated with 1 µg/mL of C44 Mab-9 (A) and C44 Mab-46 (B). (C,D) CRC sections were incubated with 1 µg/mL of C44Mab-9 (C) and C44Mab-46 (D), followed by treatment with the Envision+bated with 1 µg/mL of C44 Mab-9 (C) and C44 Mab-46 (D), followed by treatment with the Envision+
kit. The color was developed
using DAB, and sections were counterstained with hematoxylin. Scale
kit. The color was developed using DAB, and sections were counterstained with hematoxylin. Scalebar = 100 µm.
bar = 100 µm.

3.
3.Discussion
Discussion
In
Inthisthisstudy,
study, wewe developed
developed C
C4444Mab-9
Mab-9using
usingthetheCBIS
CBISmethod
method(Figure(Figure1),1),andanddeterdeterminedvariant 66 encoded
encodedregion
region(Table(Table1).
1).Then,Then,we
weshowed
showedthe
theusefulness
usefulness
mined its
its epitope
epitope as
as variant
of
of
C
44
Mab-9
for
multipleapplications,
includingflow
cytometry(Figures2
and3),
western
C44 Mab-9 for multiple applications, including flow cytometry (Figures 2 and 3), western
blottingblotting(Figure(Figure 4),4), andand immunohistochemistry
immunohistochemistry (Figure(Figure 5).
5).
Anti-CD44v6
mAbs(clones
2F10,
VFF4,
VFF7,
and
VFF18)
werepreviously
develAnti-CD44v6 mAbs (clones 2F10, VFF4, VFF7, andVFF18)
werepreviously
developed,
oped,
and
mainly
used
for
tumor
diagnosis
andtherapy.
The
2F10
was
establishedtheand mainly used for tumor diagnosis and therapy. The 2F10 was established by the byimmuimmunizationof CD44v3-10-Fc
producedbycells.
COS1Thecells.
The
exon specificityofnization of CD44v3-10-Fc
proteinprotein
producedby COS1
exon
specificityof the 2F10
thewas determined
byimmunofluorescentindirect immunofluorescent
stainingof COS1
cells transwas2F10
determinedby indirect
staining of
COS1 cells
transfectedwithfectedhuman
CD44v
cDNAs,
including
CD44v3-10,
CD44v6-10,
CD44v7-10,
humanwith
CD44v
cDNAs,
including
CD44v3-10,
CD44v6-10,
CD44v7-10,
CD44v8-10,
andCD44v8-10,
andTherefore,
CD44v10the[25].
Therefore,
theto2F10
is thought
to recognize
the peptideorCD44v10 [25].
2F10
is thought
recognize
the peptide
or glycopeptide
strucglycopeptide
structureofCD44v6.
However,thedetailed
binding
epitope
of
2F10
has
not
ture of CD44v6. However, the detailed binding epitope of 2F10 has not been determined.
been determined.
The VFF series mAbs were established by the immunization of bacterial-expressed
The VFF
seriesmAbs
were establishedby theimmunization
bacterial-expressed
CD44v3-10
fused
with
glutathione
S-transferase[26,27].
Afterward,ofVFF4
and VFF 7 wereused in thefused
immunohistochemical
analysis [28],and VFF18
was VFF4
humanized
as 7BIWACD44v3-10
with glutathione S-transferase[26,27].
Afterward,
and VFF
were

Int. J. Mol. Sci. 2023, 24, 4007

10 of 16

4 [15], and developed to bivatuzumab-mertansine drug conjugate for clinical trials [17,18].
The VFF18 bound only to the fusion proteins, containing a variant 6-encoded region.
Furthermore, the VFF18 recognized several synthetic peptides, spanning the variant 6encoded region in ELISA, and the WFGNRWHEGYR peptide was determined as the
epitope [26]. As shown in Table 1, C44 Mab-9 also recognized a synthetic peptide (CD44p351–
370), which possesses the above sequence. In contrast, a synthetic peptide (CD44p361–380)
possesses the FGNRWHEGYR sequence, which is not recognized by C44 Mab-9. Therefore,
C44 Mab-9 and VFF18 recognize CD44v6 with a similar variant 6-encoded region. Detailed
epitope mapping for C44 Mab-9 is required in the future.
A mutated version of BIWA-4, called BIWA-8, was constructed for improving binding
affinity. This was achieved by two amino acid mutations of the light chain without changing
the humanized heavy chain [15]. The BIWA-8 was further engineered to chimeric antigen
receptors (CARs). The CD44v6 CAR-T exhibited antitumor effects against primary humanacute myeloid leukemia and multiple myeloma cells in immunocompromised mice [29].
Furthermore, the CD44v6 CAR-T also showed efficacy in xenograft models of lung andovarian carcinomas [30], which is expected for a wider development toward solid tumors.
However, Greco et al. demonstrated that the N-glycosylation of CD44v6 protects tumor cells
from the CD44v6 CAR-T targeting [31]. This phenomenon is probably due to the maskingof CD44v6 CAR binding by the N-glycosylation because the original VFF18 was establishedby bacterial-expressed CD44v3-10 immunization and recognized the peptidic epitope
lacking the N-glycosylation [26]. In contrast, C44 Mab-9 was established by immunization of
CHO/CD44v3-10 cells, but recognizes a synthetic peptide (Table 1). Meanwhile, C44 Mab-9
could detect more than 180 kDa, heavily glycosylated CD44v3-10 in western blot analysis(Figure 4). Further studies are required to reveal whether the N-glycosylation affects therecognition by C44 Mab-9 for future application to CAR-T therapy.
The clinical significance of CD44v6 expression in patients with CRC using immunohistochemical analysis remains controversial. The elevated expression has been associatedwith poor prognosis, linked to adverse prognosis [32,33]. However, others have reported
that CD44v6 expression is associated with a favorable outcome [34,35]. Various clones ofanti-CD44v6 mAbs appeared to influence the outcome of the clinical significance. Amongthese clinical studies, Saito et al. used VFF18 and showed similar staining patterns of
C44 Mab-9 (Figure 5). They also found that CD44v6 expression was observed in poorly differentiated CRC without E-cadherin expression. Furthermore, the high CD44v6 expression
exhibited a significant inverse correlation with E-cadherin expression and was found to be
an independent poor prognostic factor in disease-free survival and overall survival [36]. In
the future, we should evaluate the clinical significance of the C44 Mab-9-positive CRC with
E-cadherin expression.
Large-scale genomic analyses of CRCs defined 4 subtypes: (1) microsatellite instability
immune; (2) canonical; (3) metabolic; (4) mesenchymal types [3]. Since the CD44v6 expression was observed in a part of CRC tissues (Figure 5), the relationship to the subtypes shouldbe evaluated. In addition, the mechanism of CD44v6 upregulation including the transcriptional regulation and the v6 inclusion by alternative splicing should be determined. The
inclusion of CD44 variant exons was reported to be promoted by the ERK-Sam68 axis [37].
Moreover, CD44v6 forms a ternary complex with MET and HGF, which is essential forthe c-MET activation [38]. This positive feedback is a potential mechanism to promote thevariant exon inclusion.
CD44v6-positive CRC cells exhibited cancer-initiating cell properties [39]. Cytokines,
HGF, C-X-C motif chemokine 12, and osteopontin, secreted from tumor-associated fibroblasts, promote the CD44v6 expression in the cancer-initiating cells, which promotes
migration and metastasis of CRC cells [14]. Clinically, circulating-tumor cells (CTCs), which
express EpCAM, MET, and CD44, identify a subset with increased metastasis-initiating phenotype [40], suggesting that CD44v6 plays an important role in cancer-initiating cell property cooperating with MET. In addition, CTC culture methods, including two-dimensional(2D) expansion, 3D organoids/spheroids culture, and xenograft formation in mice, haveInt. J. Mol. Sci. 2023, 24, 4007

11 of 16

been developed to evaluate the character of CTCs [41]. Therefore, the biological property
to affect cell proliferation and invasiveness by C44 Mab-9 should be investigated because
CD44v6 can potentiate the MET signaling by forming the ternary complex with HGF [38].
Therefore, it would be valuable to examine the effect of C44 Mab-9 on CTC proliferationin vitro and metastasis in vivo.
To evaluate the in vivo effect, we previously converted the IgG1 subclass of mAbs
into a mouse IgG2a , and produced a defucosylated version. These defucosylated IgG2amAbs exhibited potent ADCC in vitro, and reduced tumor growth in mouse xenograftmodels [24,42–48]. Therefore, the production of a class-switched and defucosylated version
of C44 Mab-9 is required to evaluate the antitumor activity in vivo.
4. Materials and Methods
4.1. Cell Lines
Mouse multiple myeloma P3X63Ag8U.1 (P3U1) and CHO-K1 cell lines were obtained
from the American Type Culture Collection (ATCC, Manassas, VA, USA). These cells
were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Nacalai Tesque,Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS;
Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/mL penicillin, 100 µg/mL
streptomycin, and 0.25 µg/mL amphotericin B (Nacalai Tesque, Inc.). Human colorectal
cancer cell lines, COLO201 and COLO205, were obtained from ATCC and the Cell Resource
Center for Biomedical Research Institute of Development, Aging, and Cancer at Tohoku
University, respectively. The COLO201 and COLO205 were cultured in RPMI-1640 medium,supplemented with 10% heat-inactivated FBS, 100 units/mL of penicillin, and 100 µg/mL
streptomycin (Nacalai Tesque, Inc.). All the cells were grown in a humidified incubator at
37 ◦ C with 5% CO2 .
CD44s cDNA was amplified using HotStar HiFidelity Polymerase Kit (Qiagen Inc.,
Hilden, Germany) using LN229 cDNA as a template. CD44v3-10 ORF was obtained
from the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan.
CD44s and CD44v3-10 cDNAs were subcloned into pCAG-Ble-ssPA16 vector possessing
signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [19,49–52], which
is detected by NZ-1 [53–68]. CHO/CD44s and CHO/CD44v3-10 were established bytransfecting pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3-10 into CHO-K1 cells
using a Neon transfection system (Thermo Fisher Scientific, Inc.).
4.2. Hybridoma Production
The female BALB/c mice (6-weeks old) were purchased from CLEA Japan (Tokyo,
Japan). Animals were housed under specific pathogen-free conditions. All animal experiments were also conducted according to relevant guidelines and regulations to minimize
animal suffering and distress in the laboratory. The Animal Care and Use Committee of
Tohoku University (Permit number: 2019NiA-001) approved animal experiments. The
mice were monitored daily for health during the full four-week duration of the experiment. A reduction of more than 25% of the total body weight was defined as a humane
endpoint. During sacrifice, the mice were euthanized through cervical dislocation, after
which death was verified through respiratory and cardiac arrest. The mice were intraperitoneally immunized with CHO/CD44v3-10 (1 × 108 cells) and Imject Alum (Thermo
Fisher Scientific Inc.) as an adjuvant, which stimulates a nonspecific immune response formixed antigens using this formulation of aluminum hydroxide and magnesium hydroxide. After three additional immunizations of CHO/CD44v3-10 (1 × 108 cells), a booster
injection of CHO/CD44v3-10 was intraperitoneally administered 2 days before harvesting
the spleen cells. The splenocytes were fused with P3U1 cells using polyethylene glycol
1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA). The supernatants, which are
positive for CHO/CD44v3–10 cells and negative for CHO-K1 cells, were selected by theflow cytometry-based high throughput screening using SA3800 Cell Analyzers (Sony Corp.,
Tokyo, Japan).

Int. J. Mol. Sci. 2023, 24, 4007

12 of 164.3. ELISA
Fifty-eight synthesized peptides (Sigma-Aldrich Corp., St. Louis, MO, USA), which
cover the CD44v3-10 extracellular domain [21], were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific Inc) at a concentration of 1 µg/mL for 30 min
at 37 ◦ C. After washing with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween
20 (PBST; Nacalai Tesque, Inc.) using Microplate Washer, HydroSpeed (Tecan, Zürich,
Switzerland), wells were blocked with 1% (w/v) bovine serum albumin (BSA)-containing
PBST for 30 min at 37 ◦ C. C44 Mab-9 was added to each well, and then incubated withperoxidase-conjugated anti-mouse immunoglobulins (1:2000 diluted; Agilent Technologies
Inc., Santa Clara, CA, USA). Enzymatic reactions were performed using 1 Step Ultra TMB(Thermo Fisher Scientific Inc.). The optical density at 655 nm was measured using an iMark
microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).
4.4. Flow Cytometry
CHO-K1 and CHO/CD44v3-10 were isolated using 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.) treatment. COLO201 and COLO205
were isolated by brief pipetting. The cells were treated with primary mAbs or blocking
buffer (0.1% bovine serum albumin (BSA; Nacalai Tesque, Inc.) in phosphate-bufferedsaline [PBS]; control) for 30 min at 4 ◦ C. Subsequently, the cells were incubated in Alexa
Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc.) for 30 min at
4 ◦ C. Fluorescence data were collected using the SA3800 Cell Analyzer and analyzed using
SA3800 software ver. 2.05 (Sony Corporation).
4.5. Determination of Dissociation Constant (KD ) by Flow Cytometry
Serially diluted C44 Mab-9 was suspended with CHO/CD44v3-10, COLO201, and
COLO205 cells. The cells were further treated with Alexa Fluor 488-conjugated anti-mouse
IgG (1:200). Fluorescence data were collected using BD FACSLyric and analyzed using BD
FACSuite software version 1.3 (BD Biosciences). To determine the dissociation constant(KD ), GraphPad Prism 8 (the fitting binding isotherms to built-in one-site binding models;GraphPad Software, Inc., La Jolla, CA, USA) was used.
4.6. Western Blot Analysis
The cell lysates (10 µg of protein) were separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and transferred onto polyvinylidene
difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking (4%skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20), the membranes were incubated with 10 µg/mL of C44 Mab-9, 10 µg/mL of C44 Mab-46 or 1 µg/mL of an anti-β-actinmAb (clone AC-15; Sigma-Aldrich Corp.), and then incubated with peroxidase-conjugatedanti-mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc., Santa Clara, CA,
USA). Finally, the signals were detected with a chemiluminescence reagent, ImmunoStar
LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co., Ltd.,Tokyo, Japan).
4.7. Immunohistochemical Analysis
The FFPE oral SCC tissue was obtained from Tokyo Medical and Dental University [69].
FFPE sections of colorectal carcinoma tissue array (Catalog number: CO483a) were purchased from US Biomax Inc. (Rockville, MD, USA). The sections were autoclaved in citratebuffer (pH 6.0; Nichirei biosciences, Inc., Tokyo, Japan) for 20 min. After blocking with
SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C44 Mab-9(1 µg/mL) and C44 Mab-46 (1 µg/mL) for 1 h at room temperature and then treated with
the EnVision+ Kit for mouse (Agilent Technologies, Inc.) for 30 min. The color was developed using 3,30 -diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.)for 2 min. Hematoxylin (FUJIFILM Wako Pure Chemical Corporation) was used for theInt. J. Mol. Sci. 2023, 24, 4007

13 of 16counterstaining. Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used
to examine the sections and obtain images.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24044007/s1.
Author Contributions: R.E., T.T. and T.A. performed the experiments. M.K.K. and Y.K. designed theexperiments. R.E. and H.S. analyzed the data. R.E., 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) grantnos. 21K20789 (to T.T.), 22K06995 (to H.S.), 21K15523 (to T.A.), 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.
Data Availability Statement: All related data and methods are presented in this paper. Additional
inquiries should be addressed to the corresponding authors.
Acknowledgments: The authors would like to thank Saori Okuno, and Saori Handa (Department of
Antibody Drug Development, Tohoku University Graduate School of Medicine) for technical assistance.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

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