International Journal of

Molecular Sciences
ArticleA Novel Anti-CD44 Variant 3 Monoclonal Antibody C44Mab-6
Was Established for Multiple Applications
Hiroyuki Suzuki 1,2, * , Kaishi Kitamura 1 , Nohara Goto 1 , Kenichiro Ishikawa 1 , Tsunenori Ouchida 1,2 ,Tomohiro Tanaka 1,2 , Mika K. Kaneko 1,2 and Yukinari Kato 1,2, *1

2*

Citation: Suzuki, H.; Kitamura, K.;
Goto, N.; Ishikawa, K.; Ouchida, T.;
Tanaka, T.; Kaneko, M.K.; Kato, Y.
A Novel Anti-CD44 Variant 3
Monoclonal Antibody C44 Mab-6 Was
Established for Multiple Applications.

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan; kitamura.kaishi.s7@dc.tohoku.ac.jp (K.K.); s1930550@s.tsukuba.ac.jp (N.G.);
ken.ishikawa.r3@dc.tohoku.ac.jp (K.I.); tsunenori.ouchida.d5@tohoku.ac.jp (T.O.);
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.: +81-29-853-3944 (H.S. & Y.K.)

Abstract: Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment ofgrowth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple
isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and uniquefunctions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor isessential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s
and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3–10-overexpressed
cells. In this study, we established C44 Mab-6 (IgG1 , kappa), which recognized the CD44 variant 3-encoded
region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44 Mab-6 reacted with
CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10) or some cancer
cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44 Mab-6 for CHO/CD44v3–10,
COLO205, and HSC-3 was 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M, respectively. C44 Mab-6
could detect the CD44v3–10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor
sections in immunohistochemistry. These results indicate that C44 Mab-6 is useful for detecting CD44v3 invarious experiments and is expected for the application of tumor diagnosis and therapy.
Keywords: CD44; CD44 variant 3; monoclonal antibody; flow cytometry; immunohistochemistryInt. J. Mol. Sci. 2023, 24, 8411.
https://doi.org/10.3390/
ijms24098411

1. Introduction
Academic Editors: Elena Levantini
and Daniela Sanchez Bassères
Received: 15 April 2023
Revised: 4 May 2023
Accepted: 5 May 2023
Published: 7 May 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/

The cell surface glycoprotein known as cluster of differentiation 44 (CD44) is broadly
expressed by epithelial, mesenchymal, and hematopoietic cells and is involved in adhesion
to the extracellular matrix (ECM), lymphocyte homing, and lymphocyte activation [1].
A growing body of evidence reveals the critical roles of CD44 in tumor progression andmetastasis [2,3]. The human CD44 gene consists of 19 exons, 10 of which are constant inall variants, and makes up the standard form of CD44 (CD44s) [4]. Furthermore, a large
number of CD44 variants (CD44v) are generated due to alternative splicing. The CD44v
consists of 10 constant exons in combination with the remaining 9 variant exons.
The translated CD44 usually receives post-translational modifications, such as N-/O-linked
glycosylation or proteoglycans, including chondroitin sulfate, keratan sulfate, and heparansulfate, which lead to further diversity in CD44 function [5–8]. Therefore, the molecular weights
of CD44s and CD44v are 75–95 kDa and 100~250 kDa, respectively [5]. These CD44 isoforms
have both overlapping and unique functions. Both CD44s and CD44v (pan-CD44) possess
hyaluronic acid (HA)-binding motifs that promote interaction with the microenvironment,
which mediates cellular homing, migration, adhesion, and proliferation [9].

4.0/).

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CD44v is overexpressed in tumors, and it plays critical roles in the promotion of tumor
invasion, metastasis, cancer-initiating properties [10], and resistance to therapies [2,11].
CD44v has the ability to bind to heparin-binding growth factors, including fibroblast growth
factors (FGFs) [7]. These growth factors bind to a heparan sulfate side chain attached to
the SGSG sequence encoded by variant exon 3 [7,12]. Heparan sulfate proteoglycans play
critical roles in cell proliferation and motility through presenting the growth factors toreceptors. Therefore, the CD44 variant exon 3-containing isoform (CD44v3) can recruit
heparin-binding growth factors to their receptor and promote growth-factor-receptormediated signal transduction [13–15]. Furthermore, the v6-encoded region forms a complex
with hepatocyte growth factor and its receptor MET, which is essential for activation [16].
The v8–10-encoded region interacts with a cystine–glutamate transporter (xCT) subunit
and mediates the oxidative stress resistance through the regulation of the intracellular
redox state [17].
Cancer stem cells (CSCs) exhibit important properties, such as self-renewal, resistanceto therapy, and the promotion of tumor metastasis [18–20]. Several cell surface and intracellular proteins have been reported as CSC markers in hematopoietic malignancy andsolid tumors [21,22]. Among them, CD44 has been identified as a CSC marker in several
solid tumors. In breast cancer, the CD44+ CD24−/low Lineage− population was first shown
to be 10- to 50-fold enriched with the ability to form tumors in immunodeficient micerelative to unfractionated tumor cells [23]. In head and neck squamous cell carcinoma(HNSCC), the CD44-high CSCs from HNSCC exhibited elevated migration, invasiveness,and stemness [24,25] and could form metastatic foci in the lungs of immunodeficient mice.
In contrast, the CD44-low populations failed to form the metastatic proliferation [26]. In theabove studies, anti-pan-CD44 monoclonal antibodies (mAbs) were used to isolate the CSCs
from cultured cells and patient-derived tumor tissues. Furthermore, several CD44v-specificmAbs were reported to separate CSCs from colorectal cancer by using anti-CD44v6 [27] andanti-CD44v9 [28] mAbs. Therefore, specific mAbs against CD44s and CD44v are required
for the isolation of CSCs and the analysis of their properties in detail.
By using the Cell-Based Immunization and Screening (CBIS) method, we establishedan anti-pan-CD44 mAb, namely, C44 Mab-5 (IgG1 , kappa) [29]. We further establishedanother anti-pan-CD44 mAb, namely, C44 Mab-46 (IgG1 , kappa), via the immunization of
CD44v3–10 ectodomain (CD44ec) [30]. Both C44 Mab-5 and C44 Mab-46 have been revealed
to recognize the standard exon (1 to 5)-encoding sequences at the N-terminus [31–33].
Furthermore, both C44 Mab-5 and C44 Mab-46 are available for flow cytometry and immunohistochemical analyses in oral squamous cell carcinomas (OSCC) [29] and esophageal
squamous cell carcinomas [30]. We further 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) by using fucosyltransferase 8-deficient ExpiCHO-S (BINDS-09) cells.
The 5-mG2a -f exhibited in vitro antibody-dependent cellular cytotoxicity (ADCC) activity
against OSCC cell lines (HSC-2 and SAS). Furthermore, the 5-mG2a -f suppressed the growthof the HSC-2 and SAS xenograft [34].
Recently, we established an anti-CD44v5 mAb [35] and an anti-CD44v6 mAb [36] via
the CBIS method, an anti-CD44v7/8 mAb [37] via the immunization of CD44ec, and ananti-CD44v4 mAb via peptide immunization [38]. In this study, we developed a novelanti-CD44v3 mAb, namely, C44 Mab-6 (IgG1 , kappa), via the CBIS method and evaluatedits applications, such as flow cytometry, Western blot, and immunohistochemical analyses.
2. Results
2.1. Development of C44 Mab-6 as an anti-CD44v3 mAb
The CBIS method involves the immunization of antigen-overexpressed cells and highthroughput hybridoma screening by using flow cytometry. We prepared CD44v3–10-overexpressed
Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10), as an immunogen (Figure 1). The cells
were immunized into mice, and hybridomas were plated into 96-well plates. We next performed
flow-cytometry-based, high-throughput screening to select the supernatants, which were positive

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for CHO/CD44v3–10 cells and negative for CHO-K1 cells. After the limiting dilution, anti-CD44
mAb-producing clones were finally established. Among them, C44Mab-6 (IgG1, kappa) was
shown to recognize CD44 p231–250 peptide (AGWEPNEENEDERDRHLSFS), which corresponds
to variant-3-encoded sequence (Figure 2 and Supplementary Table S1). In contrast, C44Mab-6
4 of 18
never recognized other extracellular region peptides of CD44v3-10.
These results indicated that
C44Mab-6 specifically recognizes the CD44 variant 3-encoded sequence.

Figure 1. Anti-human CD44 mAbs production. (A) The CD44 structure. CD44s mRNA is assembled
Figure1. andAnti-human
mAbs
The
CD44
structure.
by the first five
(1 to 5)the last five CD44
(16 to 20)
exonsproduction.
and translates (A)CD44s.
CD44v
mRNAs
are CD44s mRNA is assembled bygenerated by
thealternative
splicingofvariant
exons
andtranslatemultipleCD44v
isoforms,
such
the first five (1 to 5) and the last five (16 to 20) exons and translates CD44s. CD44v mRNAs are generated
as CD44v3-10, CD44v4-10, CD44v6-10, and CD44v8-10. (B) BALB/c mice were intraperitoneallyalternative splicing
of variant
exons
translatemultiple
CD44v isoforms, such as CD44v3-10,
immunized bywiththeCHO/CD44v3–10
cells. (C) The
hybridomas
wereandproduced
via fusionof the splenocytes andCD44v4-10,
P3U1 cells. (D)The flow cytometry-mediatedscreening
was conductedby using
CD44v6-10,
and CD44v8-10.
(B) BALB/cmice wereintraperitoneally immunized withparental CHO-K1 and CHO/CD44v3–10 cells. (E) After cloning and additional screening, a clone
CHO/CD44v3–10
cells.
(C)The
hybridomas
were
producedvia
fusionof the splenocytes and P3U1
C44Mab-6 (IgG1, kappa) was established. Finally, the binding epitope was determined via enzymelinked immunosorbent
by using peptides, which screening
cover the extracellulardomain of by using parental CHO-K1 andcells. (D)assay
The(ELISA)
flow cytometry-mediatedwas conducted
CD44v3–10.
CHO/CD44v3–10 cells. (E) After cloning and additional screening, a clone C44 Mab-6 (IgG1 , kappa)

was established. Finally, the binding epitope was determined via enzyme-linked immunosorbent assay(ELISA) by using peptides, which cover the extracellular domain of CD44v3–10.

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Figure2.2.Determination
Determination ofof C
C44Mab-6
epitope by ELISA. The synthesized peptides, which cover the
Figure44 Mab-6 epitope by ELISA. The synthesized peptides, which cover theCD44v3–10 extracellular domain, were immobilized on immunoplates. The plates were incubatedCD44v3–10 extracellular domain, were immobilized on immunoplates. The plates were incubatedwith C44Mab-6, followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins.
with
C44 Mab-6,
followedby incubation
anti-mouse
immunoglobulins.
Opticaldensity
was measured
at with655 peroxidase-conjugated
nm. The CD44 p231–250
sequence(AGWEPOpticaldensitywas
measured
at
655
nm.
The
CD44
p231–250
sequence(AGWEPNEENEDERNEENEDERDRHLSFS) corresponds to the variant 3-encoded sequence. ELISA: enzyme-linked imDRHLSFS)
corresponds
the variant
3-encoded
ELISA: enzyme-linked immunosorbent
munosorbent
assay. NC:tonegative
control(solvent;sequence.
DMSO in PBS).
assay. NC: negative control (solvent; DMSO in PBS).

2.2. The Reactivity of C44Mab-6 to CD44-Expressing Cells in Flow Cytometry

2.2. The Reactivity of C44 Mab-6 to CD44-Expressing Cells in Flow Cytometry

The reactivity of C44Mab-6 to CHO/CD44v3–10, CHO/CD44s, and CHO-K1 cells was
The reactivity
C44 Mab-6
CHO/CD44v3–10,
CHO/CD44s,
and CHO-K1
cells wasinvestigated
by of
using
flow tocytometry.
C44Mab-6
dose-dependently
recognizedinvestigatedby using
cytometry.
C44 Mab-6
dose-dependently
recognized
CHO/
CHO/CD44v3–10
cells flow
(Figure3A). In contrast,
C44Mab-6
recognized neither
CHO/CD44s
CD44v3–10(Figure (Figure
3A). In 3C)
contrast,
Mab-6 which
recognizedneither CHO/CD44s(Figure 3B) cells
nor CHO-K1
cells. CC
4444
Mab-46,
is an anti-pan-CD44
mAb(Figure3B) nor CHO-K1(Figure 3C) cells.
C44
Mab-46, which
an anti-pan-CD44mAb[30], recognizedboth CHO/CD44v3–10
and
CHO/CD44s
cells is(Supplementary
FigureS1).[30],
recognizedboth CHO/CD44v3–10
CHO/CD44s
cells (Supplementary
S1). WeWe next examined
the reactivity ofand
C44Mab-6
to a colorectal
cancer cell line Figure(COLO205)
andexamined
an OSCC thecell reactivity
line (HSC-3).
wasselected incancer
this study
fromvarious cancer
nextof CCOLO205
a colorectal
cell line(COLO205)
and an44 Mab-6 tocell lines
becauseC44Mab-6
showedvery
high reactivity
to it.from
Furthermore,
HSC-3cell
was
OSCC
cell line(HSC-3).
COLO205wasselectedin this study
various cancer
lines
selectedCbecause
shownbe the second
highest CD44-expressing
becauseshowedwas
veryhigh toreactivity
to it. Furthermore,
HSC-3 wascancer
selected
44 Mab-6HNSCC
type in HNSCC
the Pan-Cancer
Atlasto[39].
C44Mab-6
recognize
a colorectal cancer
cancer typecell line
becausewas shown
be thesecondcould
highestCD44-expressingin theCOLO205 (Figure3D) andoral squamouscell lineaHSC-3(Figure3E) incell
a dose-dependPan-Cancer
Atlas [39].
C44an
Mab-6
could recognize
colorectal
cancerline COLO205
ent manner.
(Figure3D) and an oral squamous cell line HSC-3 (Figure 3E) in a dose-dependent manner.

2.3. The Binding Affinity of C44 Mab-6 to CD44-Expressing Cells
The binding affinity of C44 Mab-6 to CHO/CD44v3–10, COLO205, and HSC-3 wasdetermined by using flow cytometry. As shown in Figure 4, the KD of CHO/CD44v3–10
COLO205 and HSC-3 was determined as 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M,
respectively. These results indicated that C44 Mab-6 possesses a high affinity for CD44v3–10
and endogenous CD44v3-expressing cells.

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6 o

Figure 3. The reactivity of C44 Mab-6 to CD44-expressing cells in flow cytometry. CHO/CD44v3–10 (A),Figure3. The(B),reactivityof C
44Mab-6 to CD44-expressing cells in flow cytometry. CHO/CD44v3
CHO/CD44s
CHO-K1 (C),COLO205(D), and HSC-3 (E) cells were treated with C44 Mab-6 at 0.01–10(A),
CHO/CD44s
CHO-K1
COLO205(D), and with
HSC-3
(E)Fluor
cells488
werewith C44Maµg/mL,
followed by(B),treatmentwith(C),anti-mouse
IgG conjugated
Alexa(Redtreatedline). Black
atline:0.01–10µg/mL,
followedbytreatment
withanti-mouse
IgG
conjugated
with
Alexa
Fluor 488 (negative control (blocking buffer).
line). Black line: negative control (blocking buffer).

2.3. The Binding Affinity of C44Mab-6 to CD44-Expressing Cells

The binding affinity of C44Mab-6 to CHO/CD44v3–10, COLO205, and HSC-3 wastermined by using flow cytometry. As shown in Figure 4, the KD of CHO/CD44v3
COLO205 and HSC-3 was determined as 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M,
spectively. These results indicated that C44Mab-6 possesses a high affinity for CD44v3
and endogenous CD44v3-expressing cells.

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Figure 4. The binding affinity of C Mab-6 to CD44-expressing cells. CHO/CD44v3–10 (A),44
Figure 4. The binding affinity of C44Mab-6 toCD44-expressing cells. CHO/CD44v3–10 (A),COLO205 (B), and HSC-3 (C) cells were suspended in C44 Mab-6 at the indicated concentrations.
COLO205 (B), and HSC-3 (C) cells were suspended in C44Mab-6 at the indicated concentrations.
Cells were incubated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data were
Cells were incubated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data werecollected, and the apparent dissociation constant (KD ) was calculated by using GraphPad Prism 8.
collected, and the apparent dissociation constant (KD) was calculated by using GraphPad Prism 8.
2.4. Western Blot Analysis

2.4. Western Blot Analysis
Western blot analysis was preformed to investigate the specificity of C44 Mab-6. As
in Figure 5A, C44 Mab-6 detected CD44v3–10 as showing more than 180-kDa bands.
Western shown
blot analysis
was preformed to investigate the specificity of C44Mab-6. As
However, C44 Mab-6 did not detect any bands from lysates of CHO-K1 and CHO/CD44s
shown in Figure
5A, C44Mab-6 detected CD44v3–10 as showing more than 180-kDa bands.
cells at more than 48-kDa. An anti-pan-CD44 mAb, namely, C44 Mab-46, recognized the
However, C44Mab-6
did not
any bands
fromandlysatesof CHO-K1 (>180
and CHO/CD44s
lysates from
bothdetect
CHO/CD44s(~75 kDa)
CHO/CD44v3–10
kDa) (Figure 5B).
cells at more than
48-kDa.
An
anti-pan-CD44
mAb,
namely,C
44
Mab-46,
recognized
thebut
These results indicated that C44 Mab-6 specifically detects exogenous
CD44v3–10
not CD44s.
lysates from both
CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa) (Figure 5B).
These results indicated that C44Mab-6 specifically detects exogenous CD44v3–10 but not
CD44s.

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Figure 5. Western blot analysis by using C Mab-6. The cell lysates of CHO-K1, CHO/CD44s, andFigure 5. Western blot analysis by using C44
44Mab-6. The cell lysates of CHO-K1, CHO/CD44s, andCHO/CD44v3–10(10(10µµg)wereelectrophoresedelectrophoresedand
andtransferredtransferredonto
ontopolyvinylidene
polyvinylidenefluoride
fluoridememmemCHO/CD44v3–10g) werebranes.
Themembranes
were
incubated
with
10
µg/mL
of
C
Mab-6
(A),
10
µg/mL
of
C
Mab-4644
44
branes. The membranes were incubated with 10 µ g/mL of C44Mab-6(A), 10 µ g/mL of C44Mab-46(B),(B), and1 µg/mLan anti-β-actin
mAb
Then,
membranes
wereincubatedincubatedwith
withanti-mouseanti-mouseand1 µ g/mLof anofanti-β-actin
mAb(C).(C).
Then,
thethemembranes
wereimmunoglobulins
conjugatedwith
withperoxidase.
peroxidase.The
Theblack
black
arrow
indicates
CD44s
(~75
kDa).
immunoglobulins conjugatedarrow
indicates
thethe
CD44s
(~75
kDa).
TheThered
redarrowsindicate
the CD44v3–10
arrowsindicate
the CD44v3–10(>180(>180kDa).kDa).

2.5. Immunohistochemical
ImmunohistochemicalAnalysis
Analysis by
by Using
Using CC4444Mab-6
Mab-6against
againstTumor
TumorTissues
Tissues
2.5.
Immunohistochemical analysis
analysis against
againstthe
theformalin-fixed
formalin-fixedparaffin-embedded
paraffin-embedded(FFPE)(FFPE)
Immunohistochemical
sections
of
OSCC
was
conducted
toassesstheavailabilityofC
Mab-6.
We
used
sequential
sections of OSCC was conducted to assess the availability of C4444Mab-6. We used sequential
sections ofof OSCC
OSCC tissue
tissue microarraymicroarray andand compared
compared thethe staining
staining patterns
patterns ofof C
C44
Mab-6 andand
44Mab-6sectionsC
Mab-46.
Clear
membranous
staining
wasobserved
for
C
Mab-6
and
C
Mab-46in aa
44Mab-6 and C44
44Mab-46 in
C4444Mab-46. Clear membranous staining was observed for C44
well-differentiatedOSCC
section(Figure6A,B).
Figure6C,D
showedan
OSCC
sectionwithwell-differentiated OSCC section (Figure 6A,B). Figure 6C,D showed an OSCC section
the stromal
invadedphenotype.
C44 Mab-6
stronglystrongly
stained stromal-invaded
OSCC andOSCC
could
with
the stromal
invadedphenotype.
C44Mab-6
stained stromal-invaded
clearly
distinguish
tumor
cells
from
the
surrounding
stromacells(Figure6C).
In
contrast,
and could clearly distinguish tumor cells from the surrounding stroma cells (Figure 6C).
C44
Mab-46 stained
bothstained
invadedboth
tumor
and stromal
In(Figure
Figure 6E,F,
Mab-6
In
contrast,
C44Mab-46
invadedtumorcellsand(Figure
stromal6D).
cells
6D). CIn44Figure
partially
stained
tumor
cells
but
not
stromalcells(Figure6E).
Incontrast,C
Mab-46
mainly44
6E and F, C44Mab-6 partially stained tumor cells but not stromal cells (Figure 6E). In constained stromal cells (Figure 6F). We summarized the data of immunohistochemical analysis of
trast, C44Mab-46 mainly stained stromal cells (Figure 6F). We summarized the data of imCD44 expression in tumor cells in Table 1; C44 Mab-6 stained 44 out of 50 (88%) cases of OSCC.
munohistochemical analysis of CD44 expression
in tumor cells in Table 1; C44Mab-6
We also stained FFPE sections of colorectal cancer tissue microarray and found that C44 Mab-6
stained 44 out of 50 (88%) cases of OSCC. We also stained FFPE sections of colorectal canstained 7 out of 40 (18%) cases (Supplementary Table S2). However, the C44 Mab-6 reactivity
cer tissue microarray and found that C44Mab-6 stained 7 out of 40 (18%)
cases (Supplewas faint and partly localized compared to that of C44 Mab-46 (Supplementary Figure S2).
mentary Table S2). However, the C44Mab-6 reactivity was faint and partly localized comThese results indicated that C Mab-6 is useful for the immunohistochemical analysis of FFPE
pared to that of C44Mab-4644(Supplementary Figure S2). These results indicated thattumor sections.
C44Mab-6 is useful for the immunohistochemical analysis of FFPE tumor sections.

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FigureFigure6.6.Immunohistochemical
Immunohistochemicalanalysis
analysisby
byusingusingCC
Mab-6and
andCC4444Mab-46
Mab-46 against
against OSCC
OSCC tissues.
tissues.
4444Mab-6
Afterantigen
antigenretrieval,retrieval,
serialsectionsof OSCCtissuearray(Catalog
number:
OR601c)incuAfter
serialsectionsof OSCCtissuearray(Catalog
number:
OR601c)were wereincubatedbated1 with
1 μg/mLof C44Mab-6(A,C,E)
1 μg/mLof C44Mab-46(B,D,F),followed
followedby
bytreatment
treatment
with
µg/mL
of C44Mab-6(A,C,E)or 1 orµg/mL
of C44Mab-46(B,D,F),
0 -diaminobenzidine
withthetheEnvision+
Envision+kit.
kit.The
Thechromogenic
chromogenicreaction
reactionwas
wasconductedconductedby
byusingusing3,3
3,3′-diaminobenzidine
withtetrahydrochloride(DAB).
The
counterstaining
wasperformedbyusing
hematoxylin.
Scale bar =tetrahydrochloride (DAB). The counterstaining was performed by using hematoxylin.
100μm.
Scale bar = 100 µm.

Table1.1.Immunohistochemical
Immunohistochemical
analysisusing
44Mab-6 and C44Mab-46 against OSCC tissue arTable
analysis
by by
using
C44C
Mab-6
and C44 Mab-46 against OSCC tissue arrays.
rays.
No.

No.
11
22
33
44
55
66
7
7
8
8
9
10

Age

Age
7878
4040
7575
3535
6161
4141
64
64
76
76
50
44

Sex

Anatomic Site

Sex Anatomic Site
TongueM M Tongue
M
TongueM
TongueTongueF F Tongue
F F Tongue
TongueM M Tongue
TongueF F Tongue
TongueM
TongueM
TongueM
TongueM
TongueF
TongueM
TonguePathology Diagnosis

Pathology Diagnosis
of tongue
SCCSCCof tongue
SCC
of tongue
SCC of tongueof tongue
SCCSCCof tongue
SCCSCCof tongue
of tongue
SCCSCCof tongue
of tongue
SCCSCCof tongue
of tongue
SCC of right tongue
SCC of right tongue
SCC of tongue
SCC of tongue
SCC of tongue
SCC of tongue

TNM

TNM
T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N0M0T2N2M0T2N2M0T1N0M0T1N0M0T2N0M0T2N1M0

Grade

Grade
11
11
11
11
11
1
1
1
1
1
1
1
1

Type

Type
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant

C44 Mab-6

C44Mab-6
++
++
++
++
++
++
++
+++
+++
+
+
++
++
++
++
++
+++

C44 Mab-46

C44Mab-46
++
++
++
++
++
++
+++
+++
+
+
++
++
++
++
++
+++

Int. J. Mol. Sci. 2023, 24, 8411

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Table 1. Cont.
No.

Age

Sex

Anatomic Site

Pathology Diagnosis

TNM

Grade

Type

9

50

F

TongueSCC of tongue

T2N0M0

1

Malignant

++

++

10

44

M

TongueSCC of tongue

T2N1M0

1

Malignant

+++

+++

11

53

F

TongueSCC of tongue

T1N0M0

1

Malignant

++

++

12

46

F

TongueSCC of tongue

T2N0M0

1

Malignant

+

+

13

50

M

TongueSCC of root of tongue

T3N1M0

1

Malignant

+++

+

14

36

F

TongueSCC of tongue

T1N0M0

1

Malignant

+++

+++

15

63

F

TongueSCC of tongue

T1N0M0

1

Malignant

++

+

16

46

M

TongueSCC of tongue

T2N0M0

1

Malignant

+++

-

17

58

M

TongueSCC of tongue

T2N0M0

1

Malignant

+

+

18

64

M

Lip

SCC of lower lip

T1N0M0

1

Malignant

+++

+++

19

57

M

Lip

SCC of lower lip

T2N0M0

1

Malignant

+++

+++

20

61

M

Lip

SCC of lower lip

T1N0M0

1

Malignant

++

++

21

60

M

Gum

SCC of gum

T3N0M0

1

Malignant

+

+

22

60

M

Gum

SCC of gum

T1N0M0

1

Malignant

+++

+++

23

69

M

Gum

SCC of upper gum

T3N0M0

1

Malignant

++

++

24

53

M

Bucca cavioris

SCC of bucca cavioris

T2N0M0

1

Malignant

+

+

25

55

M

Bucca cavioris

SCC of bucca cavioris

T1N0M0

1

Malignant

++

+

26

58

M

TongueSCC of base of tongue

T1N0M0

1

Malignant

++

++

27

63

M

Oral cavity

SCC

T1N0M0

1

Malignant

++

++

28

48

F

TongueSCC of tongue

T1N0M0

1–2

Malignant

++

+

29

80

M

Lip

SCC of lower lip

T1N0M0

1–2

Malignant

+++

+++

30

77

M

TongueSCC of base of tongue

T2N0M0

1–2

Malignant

+++

++

31

59

M

TongueSCC of tongue

T2N0M02

Malignant

+

-

32

77

F

TongueSCC of tongue

T1N0M02

Malignant

++

++

33

56

M

TongueSCC of root of tongue

T2N1M02

Malignant

+

+

34

60

M

TongueSCC of tongue

T2N1M02

Malignant

++

++

35

62

M

TongueSCC of tongue

T2N0M02

Malignant

+++

++

36

67

F

TongueSCC of tongue

T2N0M02

Malignant

+++

++

37

47

F

TongueSCC of tongue

T2N0M02

Malignant

+++

+++

38

37

M

TongueSCC of tongue

T2N1M02

Malignant

-

-

39

55

F

TongueSCC of tongue

T2N0M02

Malignant

++

+

40

56

F

Bucca cavioris

SCC of bucca cavioris

T2N0M02

Malignant

+++

+

41

49

M

Bucca cavioris

SCC of bucca cavioris

T1N0M02

Malignant

-

-

42

45

M

Bucca cavioris

SCC of bucca cavioris

T2N0M02

Malignant

-

-

43

42

M

Bucca cavioris

SCC of bucca cavioris

T3N0M02

Malignant

+++

++

44

44

M

Jaw

SCC of right drop jaw

T1N0M02

Malignant

++

+++

45

40

F

TongueSCC of base of tongue

T2N0M02

Malignant

-

++

46

49

M

Bucca cavioris

SCC of bucca cavioris

T1N0M02

Malignant

+++

+++

47

56

F

TongueSCC of base of tongue

T2N0M03

Malignant

-

+

48

42

M

Bucca cavioris

SCC of bucca cavioris

T3N0M03

Malignant

+++

+++

49

87

F

Face

SCC of left face

T2N0M03

Malignant

+

+

50

50

M

Gum

SCC of gum

T2N0M03

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

C44 Mab-6

C44 Mab-46

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3. Discussion
In this study, we developed C44Mab-6 by using the CBIS method (Figure 1) and determined
its epitope as a variant 3-encoded region of CD44 (Figure 2 and Supplemental Table S1). Then,
we showed the usefulness of C44Mab-6 for multiple applications, including flow cytometry(Figures 3 and 4), Western blotting (Figure 5), and immunohistochemistry (Figure 6).
An anti-CD44v3 mAb (clone 3G5) was previously developed and widely used forvarious applications [40]. The 3G5 was developed by the immunization of COS1-produced
CD44v3-10-Fc protein. The specificity to the exon was determined via indirect immunofluorescent staining of COS1 cells which expressed CD44v3–10, CD44v6–10, CD44v7–10,
CD44v8–10, and CD44v10 [40]. Therefore, the 3G5 is thought to recognize the peptide
or glycopeptide structure of CD44v3. However, the detailed binding epitope of 3G5
has not been determined. As shown in Supplementary Table S1, C44 Mab-6 recognized
CD44 p231–250 peptide (AGWEPNEENEDERDRHLSFS) but not CD44 p241–260 peptides (DERDRHLSFSGSGIDDDEDF). The underlined SGSG sequence is a heparan sulfatemodified sequence in the variant-3-encoded region [7,12]. Therefore, the recognition of
C44 Mab-6 is probably not affected by the heparan sulfate modification.
Head and neck cancers are derived mainly from the oral cavity, larynx, pharynx,
and nasal cavity [41]. SCC is the common type. As shown in Figure 6, C44 Mab-6 clearlystained the membrane of OSCC and recognized a human OSCC cell line, namely, HSC-3(Figures 3 and 4). The CD44v3-high and aldehyde dehydrogenase-1 (ALDH1)-high population of HSC-3 exhibited a potent tumorigenic potential in immunodeficient NOD/SCID
mice [42]. The population showed increased stemness-related transcriptional factors,
including OCT4, SOX2, and NANOG [42]. In a future study, we will investigate the application of C44 Mab-6 to isolate cancer stem-like cells from cancer cell lines and/or OSCC tissues.
We will further establish the strategy to deplete the cancer stem-like cells for tumor therapy.
We have just started the cDNA cloning of C44 Mab-6 heavy and light chains for therapeutic
application. We have investigated the antitumor activity by using class-switched anddefucosylated IgG2a mAbs [34,43–49]. The defucosylated IgG2a mAbs can be produced byfucosyltransferases 8-deficient CHO-K1 cells, exhibited potent ADCC activity in vitro, andsuppressed the xenograft growth [34,43–49]. Therefore, the production of defucosylated
C44 Mab-6 is one of the strategies to evaluate antitumor activity in vivo.
The HNSCC treatments include surgery, chemotherapy, radiotherapy, molecular targeted therapy, immunotherapy, or a combination of these modalities [50]. Despite theprogress of the therapies, drug resistance and metastasis are still the main causes ofdeath [51]. In a preclinical study, a pan-CD44 mAb was applied to the novel modalities,
including near-infrared photoimmunotherapy (NIR-PIT). The CD44 mAb–photoactivatable
dye IRDye700DX conjugate exhibited significant antitumor effects after the NIR-light exposure against CD44-expressing OSCC [52]. However, a pan-CD44 mAb, namely, C44 Mab-46,
recognized not only tumor cells but also stromal tissues (Figure 6D,F) and probably immune cells, which are important for antitumor immunity. Therefore, CD44v is a morerational tumor antigen for NIR-PIT, which could be a new modality for OSCC with locoregional recurrence.
Zen et al. established a unique mAb (clone C3H7), which recognized the basolateral
membranes of epithelium and inhibited both the adhesion of epithelial cells to immobilized
CD11b/CD18 and the transepithelial migration of leukocytes [53]. CD11b/CD18, also
known as Macrophage-1 antigen, is a leukocyte integrin that is essential for firm adhesion
to epithelial cells and the transepithelial migration of leukocytes [54]. However, the receptor of CD11b/CD18 on epithelial cells has not been identified. They revealed that the
antigen of C3H7 is CD44v3, which specifically binds to CD11b/CD18 through its heparansulfate moieties [53]. The C3H7 antigen was increased via treatment with pro-inflammatory
cytokine, including interferon-γ and tumor necrosis factor-α in epithelial monolayers [53],which supports the previous finding that CD44v3 is increased in inflammatory diseases,
including ulcerative colitis [55]. C44 Mab-6 also recognized the basolateral surface of colorectal cancer cells (Supplementary Figure S2A). Further investigations are required for theInt. J. Mol. Sci. 2023, 24, 8411

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relationship between CD44v3 expression and the transepithelial migration of leukocytes.
The study could provide the basis for the development of novel therapeutic applications ofanti-CD44v3 mAbs, including C44 Mab-6.
Chimeric antigen receptor T-cell (CAR-T) therapies have been developed for a varietyof hematopoietic malignances and solid tumors [56]. CAR-T cells have demonstrated
remarkable success in treating CD19+ B cell leukemias [57]. However, CAR-T therapy
for acute myeloid leukemia (AML) has been elusive because of target restriction andphenotypic heterogeneity [58]. Mutations of the FMS-like tyrosine kinase 3 (FLT3) andDNA methyltransferase 3A (DNMT3A) genes were identified as common driver mutations
associated with poor prognosis of AML patients [59]. Tang et al. showed that AML cells
expressed high levels of CD44 mRNA, and the expression of AML-derived FLT3 andDNMT3A mutants promote the transcription of CD44 mRNA through suppression of
CpG island methylation in the CD44 promoter [60]. They also found that AML patientswith FLT3 or DNMT3A mutations had higher expression of CD44v6 compared to normal
specimens. Furthermore, they showed that CD44v6 CAR-T cells exhibited potent antileukemic effects [60]. Therefore, CD44v6 is thought to be a rational antigen of CAR-T
therapy for AML with FLT3 or DNMT3A mutations.
Since CD44 mRNA is upregulated in AML, there is a possibility that other CD44
variants are also transcribed and expressed in AML. In a humanized mouse model ofchronic myeloid leukemia (CML) progression from chronic phase to blast crisis, a CD44
variant (CD44v8–10) was elevated, which is required for the maintenance of stemness [61].
Although we have examined the reactivity of C44 Mab-6 against cell lines derived fromhematopoietic malignancy and found increased reactivity in several cell lines, further
studies are required for the selective expression of CD44v3 in leukemia cells, but not inhematopoietic stem cells, to ensure its safety as a CAR-T antigen.
4. Materials and Methods
4.1. Cell Lines
COLO205 (a human colorectal cancer cell line) was obtained from the Cell Resource
Center for Biomedical Research Institute of Development, Aging and Cancer at Tohoku
University (Sendai, Japan). HSC-3 (a human OSCC cell line) and LN229 (a human glioblastoma
cell line) were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan).
P3X63Ag8U.1 (P3U1: a mouse multiple myeloma) and CHO-K1 cell lines were obtained
from the American Type Culture Collection (ATCC, Manassas, VA, USA). HSC-3 and LN229
were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai Tesque, Inc., Kyoto,
Japan), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Thermo Fisher
Scientific, Inc., Waltham, MA, USA), 100 U/mL of penicillin, 100 µg/mL streptomycin, and0.25 µg/mL amphotericin B. CHO-K1, COLO205, and P3U1 were cultured in Roswell Park
Memorial Institute (RPMI)-1640 medium (Nacalai Tesque, Inc.) supplemented with 10% FBS
and antibiotics, as indicated above. All the cells were grown in a humidified incubator at
37 ◦ C with 5% CO2 .
4.2. Construction of Expression Plasmids and Stable Transfectants
By using LN229 cDNA as a template, CD44s cDNA was amplified by using HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany). We obtained CD44v3–10
cDNA from the RIKEN BRC through the National Bio-Resource Project of the MEXT,
Japan. The CD44s and CD44v3–10 cDNAs were cloned into pCAG-Ble-ssPA16 vector
with signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [29,62–65],
which is detected by NZ-1, which was originally developed as an anti-human podoplaninmAb [66–81]. The pCAG-Ble/PA16-CD44s and pCAG-Ble/PA16-CD44v3–10 vectors weretransfected into CHO-K1 cells by using a Neon transfection system (Thermo Fisher Scientific, Inc.), and CHO/CD44s and CHO/CD44v3–10 were finally established, as described previously [36].

Int. J. Mol. Sci. 2023, 24, 8411

12 of 174.3. Production of Hybridomas
All animal experiments were approved by the Animal Care and Use Committee of
Tohoku University (Permit number: 2019NiA-001). The female BALB/c mice (CLEA Japan,
Tokyo, Japan) were intraperitoneally immunized with CHO/CD44v3–10 (1 × 108 cells) with
Imject Alum (Thermo Fisher Scientific Inc.) as an adjuvant. The three additional immunizations per week and a booster injection were performed two days before the harvest of thespleen cells. The hybridomas were produced via the fusion of splenocytes and P3U1 cells
by using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA).
The supernatants, which were positive for CHO/CD44v3–10 cells and negative for CHO-K1
cells, were selected by using the SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).
4.4. ELISA
We obtained fifty-eight peptides, which cover the extracellular domain of CD44v3–10 [31],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. Immunoplate washing was performed by using 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, C44Mab-6 (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 the optical density at 655 nm.
4.5. Flow Cytometry
CHO-K1, CHO/CD44s, CHO/CD44v3–10, COLO205, and HSC-3 cells were obtained byusing 0.25% trypsin and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.).
The cells were treated with C44 Mab-6, C44 Mab-46, or blocking buffer (control) (0.1% BSA in
PBS) for 30 min at 4 ◦ C. Then, the cells (1 × 105 cells/sample) were treated with anti-mouse
IgG conjugated with Alexa Fluor 488 (1:2000; Cell Signaling Technology, Inc., Danvers, MA,
USA) for 30 min at 4 ◦ C. The data were analyzed by using the SA3800 Cell Analyzer and
SA3800 software ver. 2.05 (Sony Corp.).
4.6. Determination of Dissociation Constant (KD ) via Flow Cytometry
In CHO/CD44v3–10 cells, we prepared from 130 to 0.008 nM (diluted by 1/2) of
C44 Mab-6. In COLO201 and HSC-3 cells, we prepared from 1300 to 0.08 nM (diluted by1/2) of C44 Mab-6. The serially diluted C44 Mab-6 was suspended with 2 × 105 cells. Then,the cells were incubated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:200). BD
FACSLyric and BD FACSuite software version 1.3 (BD Biosciences, Franklin Lakes, NJ,
USA) were used for the fluorescence data analyses. The KD was determined by the fitting
binding isotherms to built-in one-site binding models of GraphPad Prism 8 (GraphPad
Software, Inc., La Jolla, CA, USA).
4.7. Western Blot Analysis
The 10 µg of cell lysates were subjected to SDS-polyacrylamide gel for electrophoresis
by using polyacrylamide gels (5–20%; FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan). The separated proteins were transferred onto polyvinylidene difluoride (PVDF)
membranes (Merck KGaA, Darmstadt, Germany). The blocking was performed by using 4%skim milk (Nacalai Tesque, Inc.) in PBST. The membranes were incubated with 10 µg/mL of
C44 Mab-6, 10 µg/mL of C44 Mab-46, or 1 µg/mL of an anti-β-actin mAb (clone AC-15; SigmaAldrich Corp.) and then incubated with peroxidase-conjugated anti-mouse immunoglobulins(diluted 1:1000; Agilent Technologies, Inc.). Finally, the signals were enhanced by using achemiluminescence reagent, ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation),
and were detected by a Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan).

Int. J. Mol. Sci. 2023, 24, 8411

13 of 174.8. Immunohistochemical Analysis
FFPE sections of OSCC tissue array (OR601c) and colorectal carcinoma tissue array(CO483a) were purchased from US Biomax Inc. (Rockville, MD, USA). The tissue arrays
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.), thesections were incubated with C44 Mab-6 (1 µg/mL) and C44 Mab-46 (1 µg/mL) for 1 h at room
temperature. The sections were further incubated with the EnVision+ Kit for mouse (Agilent
Technologies Inc.) for 30 min. Then, a chromogenic reaction using 3,30 -diaminobenzidine
tetrahydrochloride (DAB; Agilent Technologies Inc.) was conducted. Hematoxylin (FUJIFILM
Wako Pure Chemical Corporation) was used for the counterstaining. To examine the sections
and obtain images, we used Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany).
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/ijms24098411/s1. We listed the information of anti-CD44 mAbs in our original “Antibody Bank”(http://www.med-tohoku-antibody.com/topics/001_paper_antibody_PDIS.htm#CD44 (accessedon 5 May 2023)).
Author Contributions: H.S., K.K., N.G., K.I., T.O. and T.T. performed the experiments. M.K.K. and
Y.K. designed the experiments. H.S. and Y.K. analyzed the data. 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 the Japan Agency for Medical Research andDevelopment (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.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal Careand Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving animals.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and supplementary material.
Conflicts of Interest: The authors have no conflict of interest to declare.

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