antibodies
ArticleDevelopment of a Novel Anti-CD44 Variant 5 Monoclonal
Antibody C44Mab-3 for Multiple Applications against
Pancreatic Carcinomas
Yuma Kudo 1 , Hiroyuki Suzuki 1,2, * , Tomohiro Tanaka 1,2 , Mika K. Kaneko 1,2
12*

Citation: Kudo, Y.; Suzuki, H.;
Tanaka, T.; Kaneko, M.K.; Kato, Y.
Development of a Novel Anti-CD44

and Yukinari Kato 1,2, *Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, Japan
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Miyagi, 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: Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers
and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker
and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing
variants are overexpressed in many carcinomas and play essential roles in the cancer stemness,invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44
variant’s (CD44v) function and distribution in carcinomas is essential for the establishment of CD44targeting tumor therapy. In this study, we immunized mice with CD44v3–10-overexpressed Chinese
hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs).
One of the established clones (C44 Mab-3; IgG1 , kappa) recognized peptides of the variant-5-encoded
region, indicating that C44 Mab-3 is a specific mAb for CD44v5. Moreover, C44 Mab-3 reacted with
CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The
apparent KD of C44 Mab-3 for CHO/CD44v3–10 and PK-1 was 1.3 × 10−9 M and 2.6 × 10−9 M,
respectively. C44 Mab-3 could detect the exogenous CD44v3–10 and endogenous CD44v5 in Western
blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal
pancreatic epithelial cells in immunohistochemistry. These results indicate that C44 Mab-3 is useful for
detecting CD44v5 in various applications and is expected to be useful for the application of pancreaticcancer diagnosis and therapy.

Variant 5 Monoclonal Antibody
C44 Mab-3 for Multiple Applications

Keywords: CD44; CD44 variant 5; monoclonal antibody; flow cytometry; immunohistochemistry

against Pancreatic Carcinomas.
Antibodies 2023, 12, 31. https://doi.org/10.3390/antib12020031
Academic Editor: Christian Kellner
Received: 30 January 2023
Revised: 24 March 2023
Accepted: 10 April 2023
Published: 28 April 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
Pancreatic cancer has become the third leading cause of death in men and women
combined in the United States in 2023 [1]. The development of pancreatic cancer has
been explained by four common oncogenic events, including KRAS, CDKN2A, SMAD4,
and TP53 [2,3]. However, pancreatic cancer shows a heterogeneity in drug response andclinical outcomes [4]. Therefore, detailed understanding of pancreatic cancers has beenrequired to improve patient selection for current therapies and to develop novel therapeuticstrategies. An integrated genomic analysis of pancreatic ductal adenocarcinomas (PDAC)
was performed and defined four subtypes, including squamous, pancreatic progenitor,
immunogenic, and aberrantly differentiated endocrine exocrine (ADEX), which correspondto the histopathological characteristics [5]. Additionally, various marker proteins havebeen investigated for the early diagnostic and drug responses of pancreatic cancers [6].
Studies have suggested that CD44 plays important roles in malignant progression of tumors
through its cancer stemness and metastasis-promoting properties [7,8].
CD44 is a type I transmembrane glycoprotein that is expressed as a wide variety of
isoforms in various types of cells. [9]. The variety of isoforms is produced by the alternative

Antibodies 2023, 12, 31. https://doi.org/10.3390/antib12020031

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

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splicing of CD44 mRNA. The CD44 standard isoform (CD44s) is the smallest isoform of
CD44 (85–95 kDa); it is presented on the membrane of most vertebrate cells. CD44s mRNA
is assembled by the first five and the last five constant region exons [10]. The CD44 variant
isoforms (CD44v) are produced by the alternative splicing of middle variant exons (v1–v10)
and the standard exons of CD44s [11]. CD44v is heavily glycosylated, leading to various
molecular weights (~250 kDa) owing to N-glycosylation and O-glycosylation [12]. Both
CD44s and CD44v (pan-CD44) are known as hyaluronic acid (HA) receptors that mediate
cellular homing, migration, adhesion, and proliferation [13].
CD44v is overexpressed in carcinomas and induce metastatic properties [14,15]. A
growing body of evidence suggests that CD44v plays critical roles in the promotion oftumor invasion, metastasis, cancer-initiating properties [16], and resistance to chemo- andradiotherapy [7,17]. Reports indicated the important functions of each variant’s exonencoded region. The v3-encoded region functions as a co-receptor for receptor tyrosine
kinases [18]. Since the v3-encoded region possesses heparan sulfate moieties, it can recruit
to heparin-binding epidermal growth factor-like growth factor (HB-EGF) and fibroblast
growth factors (FGFs). Furthermore, the v6-encoded region forms a ternary complex with
HGF and its receptor c-MET, which is essential for its activation [19]. Additionally, oxidative
stress resistance is mediated by the v8–10-encoded region through binding with a cystine–
glutamate transporter (xCT) subunit [20]. Therefore, establishment and characterization ofmAbs that recognize each CD44v is thought to be essential for understanding each variant’s
function and development of CD44-targeting tumor diagnosis and therapy. However,the function and distribution of the variant-5-encoded region in tumors has not been
fully understood.
Our group established the novel anti-pan-CD44 mAbs, C44 Mab-5 (IgG1 , kappa) [21]and C44 Mab-46 (IgG1 , kappa) [22] using the Cell-Based Immunization and Screening(CBIS) method and immunization with the CD44v3–10 ectodomain, respectively. Both
C44 Mab-5 and C44 Mab-46 have epitopes within the standard exon (1 to 5)-encoding
sequences [23–25]. Furthermore, we showed that both C44 Mab-5 and C44 Mab-46 areapplicable to flow cytometry and immunohistochemistry in oral [21] and esophageal
squamous cell carcinomas (SCC) [22]. We have also investigated the antitumor effectsof core-fucose-deficient C44 Mab-5 in mouse xenograft models of oral SCC [26]. In thisstudy, we developed a novel anti-CD44v5 mAb, C44 Mab-3 (IgG1 , kappa), by the CBIS
method and evaluated its applications, including flow cytometry, Western blotting, andimmunohistochemical analyses.
2. Materials and Methods
2.1. Cell Lines
Chinese hamster ovary (CHO)-K1 and mouse multiple myeloma P3X63Ag8U.1 (P3U1)
cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA,
USA). The human pancreas cancer cell lines PK-1 and PK-8 were obtained from the Cell
Resource Center for Biomedical Research Institute of Development, Aging and Cancer at
Tohoku University. These cells were cultured in Roswell Park Memorial Institute (RPMI)1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 100 U/mL penicillin,
100 µg/mL streptomycin, 0.25 µg/mL amphotericin B (Nacalai Tesque, Inc.), and 10%
heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA,
USA). All the cells were grown in a humidified incubator at 37 ◦ C with 5% CO2 .
2.2. Plasmid Construction and Establishment of Stable Transfectants
CD44v3–10 open reading frame was obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. CD44s cDNA was amplified using the
HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) and LN229 (a glioblastoma cell line) cDNA as a template. CD44v3–10 and CD44 cDNAs were subcloned into
pCAG-Ble-ssPA16 vectors with a signal sequence and N-terminal PA16 tag of 16 aminoacids (GLEGGVAMPGAEDDVV) [21,27–30]; this can be detected by NZ-1, which was orig-

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inally developed as an anti-human podoplanin mAb [31–46]. The pCAG-Ble/PA16-CD44s
and pCAG-Ble/PA16-CD44v3–10 vectors were transfected into CHO-K1 cells using a Neontransfection system (Thermo Fisher Scientific, Inc.), which offers an innovative electroporation method that utilizes a proprietary biologically compatible pipette tip chamber togenerate a more uniform electric field for a significant increase in transfection efficiency
and cell viability. By the limiting dilution method, CHO/CD44s and CHO/CD44v3–10
clones were finally established.
2.3. Hybridomas
The female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). All
animal experiments were approved by the Animal Care and Use Committee of Tohoku
University (Permit number: 2019NiA-001) and performed according to relevant guidelines
and regulations to minimize animal suffering and distress in the laboratory. The mice
were intraperitoneally immunized with CHO/CD44v3–10 (1 × 108 cells) and Imject Alum
(Thermo Fisher Scientific Inc.) as an adjuvant. After the three additional immunizations
per week, a booster injection was performed two days before harvesting the spleen cells of
immunized mice. The hybridomas were established by the fusion of splenocytes and P3U1
cells using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA).
RPMI-1640 supplemented with hypoxanthine, aminopterin, and thymidine (HAT; Thermo
Fisher Scientific Inc.) was used for the selection of hybridomas. The supernatants, which
are negative for CHO-K1 cells and positive for CHO/CD44v3–10 cells, were selected byflow cytometry using SA3800 Cell Analyzers (Sony Corp. Tokyo, Japan).
2.4. Enzyme-Linked Immunosorbent Assay (ELISA)
Fifty-eight synthesized peptides, covering the CD44v3–10 extracellular domain [23],were synthesized by Sigma-Aldrich Corp. (St. Louis, MO, USA). The peptides (1 µg/mL)
were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher ScientificInc.). Plate washing was performed with phosphate-buffered saline (PBS) containing
0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.). After blocking with 1% (w/v) bovine
serum albumin (BSA) in PBST, C44 Mab-3 (10 µg/mL) was added to each well. Then, thewells were further incubated with peroxidase-conjugated anti-mouse immunoglobulins(1:2000 dilution; Agilent Technologies Inc., Santa Clara, CA, USA). 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 mesure the opticaldensity at 655 nm.
2.5. Flow Cytometry
CHO-K1, CHO/CD44v3–10, PK-1, and PK-8 were obtained using 0.25% trypsin and
1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells wereincubated with C44 Mab-3, C44 Mab-46, or blocking buffer (control) (0.1% BSA in PBS) for
30 min at 4 ◦ C. Then, the cells were treated with Alexa Fluor 488-conjugated secondary
antibody (Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4 ◦ C. The data
were analyzed using the SA3800 Cell Analyzer and SA3800 software ver. 2.05 (Sony Corp.).
2.6. Determination of Dissociation Constant (KD ) via Flow Cytometry
CHO/CD44v3–10 and PK-1 cells were treated with serially diluted C44 Mab-3(0.01–10 µg/mL). Then, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody. Fluorescence data were analyzed using BD FACSLyric and BD FACSuite
software version 1.3 (BD Biosciences, Franklin Lakes, NJ, USA). The KD was determinedby the fitting binding isotherms to built-in one-site binding models of GraphPad Prism 8(GraphPad Software, Inc., La Jolla, CA, USA).

Antibodies 2023, 12, 31

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2.7. Determination of KD via Surface Plasmon Resonance (SPR)
Measurement of KD between C44 Mab-3 and the epitope peptide was performed using
SPR. C44 Mab-3 was immobilized on the sensor chip CM5 according to the manufacturer’s
protocol by Cytiva (Marlborough, MA, USA). C44 Mab-3 (10 µg/mL in acetate buffer (pH 4.0;
Cytiva)) was immobilized using an amine coupling reaction. The surface of the flow cell 2of the sensor chip CM5 was treated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
and N-hydroxysuccinimide (NHS), followed by the injection of C44 Mab-3. The KD between
C44 Mab-3 and the epitope peptide (CD44p311–330) was determined using Biacore X100
(Cytiva). A single cycle kinetics method was used to measure the binding signals. The data
were analyzed by 1:1 binding kinetics to determine the association rate constant (ka) anddissociation rate constant (kd) and KD using Biacore X100 evaluation software (Cytiva).
2.8. Western Blot Analysis
The total cell lysates (10 µg of protein) were separated on 5–20% polyacrylamide gels
(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The separated proteins weretransferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt,
Germany). The blocking was performed with 4% skim milk (Nacalai Tesque, Inc.) in PBST.
The membranes were incubated with 10 µg/mL of C44 Mab-3, 10 µg/mL of C44 Mab-46,
0.5 µg/mL of NZ-1, or 1 µg/mL of an anti-β-actin mAb (clone AC-15; Sigma-Aldrich Corp.)
and then incubated with peroxidase-conjugated anti-mouse immunoglobulins (diluted
1:1000; Agilent Technologies, Inc.) for C44 Mab-3, C44 Mab-46, and anti-β-actin. Anti-rat
immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.) conjugated to peroxidase
was used for NZ-1. The chemiluminescence signals were obtained with ImmunoStar
LD (FUJIFILM Wako Pure Chemical Corporation) and detected using a Sayaca-Imager(DRC Co., Ltd., Tokyo, Japan).
2.9. Immunohistochemical Analysis
One formalin-fixed paraffin-embedded (FFPE) oral SCC tissue was obtained from
Tokyo Medical and Dental University [47]. FFPE sections of pancreatic carcinoma tissue arrays (Catalog number: PA241c and PA484) were purchased from US Biomax Inc. (Rockville,
MD, USA). Pancreas adenocarcinoma tissue microarray with adjacent normal pancreas
tissue (PA241c) contains 6 cases of pancreas adenocarcinoma with matched adjacent normal pancreas tissue, with quadruple cores per case. One oral SCC tissue was autoclavedin citrate buffer (pH 6.0; Nichirei biosciences, Inc., Tokyo, Japan), and pancreatic carcinoma 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.), the sections were incubated with C44 Mab-3 (1 µg/mL) and C44 Mab-46(1 µg/mL) for 1 h at room temperature. Then, the sections were incubated 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.). Hematoxylin(FUJIFILM Wako Pure Chemical Corporation) was used for the counterstaining. A Leica
DMD108 (Leica Microsystems GmbH, Wetzlar, Germany) was used to examine the sections
and obtain images.
3. Results
3.1. Development of an Anti-CD44v5 mAb, C44 Mab-3
In the CBIS method, we used a stable transfectant (CHO/CD44v3–10 cells) as an
immunogen (Figure 1). Mice were immunized with CHO/CD44v3–10 cells, and hybridomas were seeded into 96-well plates. The supernatants, which are negative for CHO-K1
cells and positive for CHO/CD44v3–10 cells, were selected using flow-cytometry-based
high throughput screening. By limiting dilution, anti-CD44-mAb-producing clones were
finally established. Among them, C44 Mab-3 (IgG1 , kappa) was shown to recognize both
CD44p311–330 (AYEGNWNPEAHPPLIHHEHH) and CD44p321–340 peptides (HPPLIHHEHHEEEETPHSTS), which correspond to the variant-5-encoded sequence (Table 1

Antibodies 2023, 12, 31

munogen (Figure 1). Mice were immunized with CHO/CD44v3–10 cells, and hybridomas
were seeded into 96-well plates. The supernatants, which are negative for CHO-K1 cells
and positive for CHO/CD44v3–10 cells, were selected using flow-cytometry-based highthroughput screening. By limiting dilution, anti-CD44-mAb-producing clones were finally established. Among them, C44Mab-3 (IgG1, kappa) was shown to recognize 5bothof 16
CD44p311–330 (AYEGNWNPEAHPPLIHHEHH) and CD44p321–340 peptides (HPPLIHHEHHEEEETPHSTS), which correspond to the variant-5-encoded sequence (Table 1 andSupplementary Figure S1). In contrast, C44Mab-3 did not recognize other CD44v3–10 exand Supplementary Figure S1). In contrast, C44 Mab-3 did not recognize other CD44v3–10
tracellular regions. These results indicated that C44Mab-3 specifically recognizes the CD44
extracellular regions. These results indicated that C44 Mab-3 specifically recognizes thevariant-5-encoded sequence.
CD44 variant-5-encoded sequence.

Figureof anti-humanCD44 mAbs
mAbs production.
(A) Structure
Structure ofof CD44.
CD44.
Figure 1.
1. A
A schematic
schematic illustration
illustration ofanti-human CD44
production. (A)
CD44s
mRNA
is
assembled
by
the
first
five
(1
to
5)
and
the
last
five
(16
to
20)
exons
and
translates
CD44s mRNA is assembled by the first five (1 to 5) and the last five (16 to 20) exons and translates
CD44s. The mRNAs
mRNAs of CD44 variants are produced by the alternative
alternative splicing of middle
middle variant
variant
exons and translate multiple CD44v such as CD44v3–10, CD44v4–10, CD44v6–10, and CD44v8–10.
CD44v8–10.
(B) CHO/CD44v3–10 cells were intraperitoneally injected into BALB/c mice. (C) The splenocytes
and P3U1 cells were fused and the hybridomas were produced. (D) The screening was conducted byflow cytometry using parental CHO-K1 and CHO/CD44v3–10 cells. (E) After cloning and additional
screening, a clone (C44 Mab-3 (IgG1 , kappa)) was established. Furthermore, the binding epitopewas determined by enzyme-linked immunosorbent assay (ELISA) using peptides that cover the
extracellular domain of CD44v3–10.

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Table 1. Determination of the binding epitope of C44 Mab-3 by ELISA.
PeptideCoding Exon *Sequence

C44 Mab-3

CD44p21–40
CD44p31–50
CD44p41–60
CD44p51–70
CD44p61–80
CD44p71–90
CD44p81–100
CD44p91–110
CD44p101–120
CD44p111–130
CD44p121–140
CD44p131–150
CD44p141–160
CD44p151–170
CD44p161–180
CD44p171–190
CD44p181–200
CD44p191–210
CD44p201–220
CD44p211–230
CD44p221–240
CD44p231–250
CD44p241–260
CD44p251–270
CD44p261–280
CD44p271–290
CD44p281–300
CD44p291–310
CD44p301–320
CD44p311–330
CD44p321–340
CD44p331–350
CD44p341–360
CD44p351–370
CD44p361–380
CD44p371–390
CD44p381–400
CD44p391–410
CD44p401–420
CD44p411–430
CD44p421–440
CD44p431–450
CD44p441–460
CD44p451–470
CD44p461–480
CD44p471–490
CD44p481–500
CD44p491–510
CD44p501–520
CD44p511–530
CD44p521–540
CD44p531–550
CD44p541–560
CD44p551–570
CD44p561–580
CD44p571–590
CD44p581–600
CD44p591–606

2
2
2
2
2/3
2/3
3
3
3
3/4
3/4
4/5
4/5
5
5
5
5
5
5
5/v3
5/v3
v3
v3
v3/v4
v3/v4
v4
v4
v4/v5
v4/v5
v5
v5
v5/v6
v5/v6
v6
v6
v6/v7
v6/v7
v7
v7
v7/v8
v7/v8
v8
v8
v8/v9
v8/v9
v9
v9/v10v9/v10
v10
v10
v10
v10
v10
v10/16v10/16
16/17
16/17
17

QIDLNITCRFAGVFHVEKNG
AGVFHVEKNGRYSISRTEAA
RYSISRTEAADLCKAFNSTLDLCKAFNSTLPTMAQMEKALPTMAQMEKALSIGFETCRYG
SIGFETCRYGFIEGHVVIPR
FIEGHVVIPRIHPNSICAANIHPNSICAANNTGVYILTSNNTGVYILTSNTSQYDTYCFN
TSQYDTYCFNASAPPEEDCTASAPPEEDCTSVTDLPNAFD
SVTDLPNAFDGPITITIVNRGPITITIVNRDGTRYVQKGEDGTRYVQKGEYRTNPEDIYPYRTNPEDIYPSNPTDDDVSSSNPTDDDVSSGSSSERSSTSGSSSERSSTSGGYIFYTFSTGGYIFYTFSTVHPIPDEDSP
VHPIPDEDSPWITDSTDRIPWITDSTDRIPATSTSSNTISATSTSSNTISAGWEPNEENEAGWEPNEENEDERDRHLSFSDERDRHLSFSGSGIDDDEDF
GSGIDDDEDFISSTISTTPRISSTISTTPRAFDHTKQNQD
AFDHTKQNQDWTQWNPSHSNWTQWNPSHSNPEVLLQTTTRPEVLLQTTTRMTDVDRNGTTMTDVDRNGTTAYEGNWNPEAAYEGNWNPEAHPPLIHHEHH
HPPLIHHEHHEEEETPHSTS
EEEETPHSTSTIQATPSSTTTIQATPSSTTEETATQKEQW
EETATQKEQWFGNRWHEGYR
FGNRWHEGYRQTPREDSHSTQTPREDSHSTTGTAAASAHTTGTAAASAHTSHPMQGRTTP
SHPMQGRTTPSPEDSSWTDFSPEDSSWTDFFNPISHPMGR
FNPISHPMGRGHQAGRRMDMGHQAGRRMDMDSSHSTTLQPDSSHSTTLQPTANPNTGLVETANPNTGLVEDLDRTGPLSMDLDRTGPLSMTTQQSNSQSF
TTQQSNSQSFSTSHEGLEEDSTSHEGLEEDKDHPTTSTLT
KDHPTTSTLTSSNRNDVTGG
SSNRNDVTGGRRDPNHSEGSRRDPNHSEGSTTLLEGYTSH
TTLLEGYTSHYPHTKESRTFYPHTKESRTFIPVTSAKTGSIPVTSAKTGSFGVTAVTVGDFGVTAVTVGDSNSNVNRSLSSNSNVNRSLSGDQDTFHPSG
GDQDTFHPSGGSHTTHGSESGSHTTHGSESDGHSHGSQEG
DGHSHGSQEGGANTTSGPIRGANTTSGPIRTPQIPEAAAA

−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−

+, OD655 ≥ 0.3; −, OD655 < 0.1. * The CD44 exon-encoded regions are illustrated in Figure 1.

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3.2. Flow Cytometric Analysis of C44 Mab-3 to CD44-Expressing Cells
We next investigated the reactivity of C44 Mab-3 against CHO/CD44v3–10 and
CHO/CD44s cells by flow cytometry. C44 Mab-3 recognized CHO/CD44v3–10 cells in adose-dependent manner (Figure 2A) but do not recognize either CHO/CD44s(Figure 2B) or CHO-K1 (Figure 2C) cells. An anti-pan-CD44 mAb, C44 Mab-46 [22], recognized CHO/CD44s cells (Supplementary Figure S2). Furthermore, C448Mab-3
Antibodies 2023, 12, x FOR PEER REVIEW
of 17 also recognized pancreatic cancer cell lines, such as PK-1 (Figure 2D) and PK-8 (Figure 2E), in adose-dependent manner.

Figure2. 2.
Flowcytometryusing
C44Mab-3
againstagainst
CD44-expressing
cells. CHO/CD44v3–10(A),
FigureFlowcytometryusing
C44 Mab-3
CD44-expressing
cells. CHO/CD44v3–10(A),
CHO/CD44s(B), (B),CHO-K1(C), PK-1(D), andcells(E)weretreated0.01–10of µg/mL of
CHO/CD44s
CHO-K1(C), PK-1(D),PK-8
and(E)
PK-8
cells
werewithtreatedwithμg/mL
0.01–10C44Mab-3, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red line). The
C44 Mab-3, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG (Red line). The
blackline represents the negative control (blocking buffer).

black line represents the negative control (blocking buffer).

3.3. Determination of the Binding Affinity of C44Mab-3 by Flow Cytometry to CD44-Expressing
Cells and SPR with the Epitope Peptide

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Next, we determined the binding affinity of C44Mab-3 to CHO/CD44v3–10 and8PK-1
of 16using flow cytometry. As shown in Figure 3, the KD of CHO/CD44v3–10 and PK-1 was 1.3
× 10−9 M and 2.6 × 10−9 M, respectively, indicating that C44Mab-3 possesses high affinity forCD44v3–10 and endogenous CD44v5-expressing cells.
3.3. Determination of the Binding Affinity of C44 Mab-3 by Flow Cytometry to CD44-Expressing
the KDPeptideof C44Mab-3 with the epitope peptide (CD44p311–330) using
Cells Weand also
SPRmeasured
with the Epitope
Biacore X100. The binding kinetics and measured values are summarized in SupplemenNext, we determined the binding affinity of C Mab-3
to CHO/CD44v3–10 and PK-1
tary Figure S3. The KD of CD44p311–330 was 5.5 × 44
10−6 M.
using flow cytometry. As shown in Figure 3, the KD of CHO/CD44v3–10 and PK-1 was
1.3 × 10−9 M and 2.6 × 10−9 M, respectively, indicating that C44 Mab-3 possesses highaffinity for CD44v3–10 and endogenous CD44v5-expressing cells.

Figure 3.
3. TheMab-3
cells.
CHO/CD44v3–10(A) (A)and and
PKFigure
The binding
binding affinity
affinityof
ofCC4444
Mab-3totoCD44-expressing
CD44-expressing
cells.
CHO/CD44v3–10
1 (B) (B)
cells
were
suspended
in in
100
μLµLofofserially
Mab-3
PK-1
cells
were
suspended
100
seriallydiluted
dilutedCC4444
Mab-3atatthe
theindicatedindicated concentrations.
concentrations.
Then, cells
cells were
were treatedtreated with
with Alexa
Alexa Fluor
Fluor 488-conjugated488-conjugated secondary
secondary antibody.
antibody. Fluorescence
Fluorescence data
data
Then,
were collected and the apparent dissociation constant (KD) was calculated using GraphPad PRISM
were collected and the apparent dissociation constant (KD ) was calculated using GraphPad PRISM 8.
8. Error bars represent means ± SDs.
Error bars represent means ± SDs.

3.4. Western Blot Analysis
We also measured the KD of C44 Mab-3 with the epitope peptide (CD44p311–330) using
WeX100.
next The
performed
blot measured
analysis toinvestigate
the sensitivityof C44Mab-3.
BiacorebindingWestern
kinetics andvaluesare summarizedin Supplementary
−
6
Total
cell
lysatesfrom
CHO-K1,
CHO/CD44s,
CHO/CD44v3–10,
PK-1,
and
PK-8 were
Figure S3. The KD of CD44p311–330 was 5.5 × 10 M.

Antibodies 2023, 12, 31

9 of 16

Antibodies 2023, 12, x FOR PEER REVIEW

3.4. Western Blot Analysis

10 of 17

We next performed Western blot analysis to investigate the sensitivity of C44 Mab-3.
Total cell lysates from CHO-K1, CHO/CD44s, CHO/CD44v3–10, PK-1, and PK-8 wereanalyzed. As shown in Figure 4A, an anti-pan-CD44 mAb, C44Mab-46, recognized the lyanalyzed. As shown in Figure 4A, an anti-pan-CD44 mAb, C44 Mab-46, recognized thesates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa). C44Mab-3 delysates from both CHO/CD44s (~75 kDa) and CHO/CD44v3–10 (>180 kDa). C44 Mab-3
tected CD44v3–10 as bands of more than 180-kDa. Furthermore, C44Mab-3 detected endetected CD44v3–10 as bands of more than 180-kDa. Furthermore, C44 Mab-3 detecteddogenous CD44v5-containing CD44v in PK-1 and PK-8 cells. However, C44Mab-3 did not
endogenous CD44v5-containing CD44v in PK-1 and PK-8 cells. However, C44 Mab-3 diddetect any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 4B). An anti-PA16
not detect any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 4B). An
tag mAb (NZ-1) recognized the lysates from both CHO/CD44s (~75 kDa) andanti-PA16 tag mAb (NZ-1) recognized the lysates from both CHO/CD44s (~75 kDa) andCHO/CD44v3–10 (>180 kDa) (Figure 4C). These results indicated that C44Mab-3 specifiCHO/CD44v3–10 (>180 kDa) (Figure 4C). These results indicated that C44 Mab-3 specifically
cally detects exogenous CD44v3–10 and endogenous CD44v5-containing CD44v.
detects exogenous CD44v3–10 and endogenous CD44v5-containing CD44v.

FigureMab-3.
cell
Figure 4.
4. Western
Westernblot
blotanalysis
analysis using
using CC4444
Mab-3.The
The
celllysates
lysatesofofCHO-K1,
CHO-K1,CHO/CD44s,
CHO/CD44s,
CHO/CD44v3–10,
were electrophoresedelectrophoresed andandtransferredtransferredonto
ontopolyvinylidene
polyvinyliCHO/CD44v3–10,PK-1,
PK-1,and
andPK-8
PK-8 (10(10 μg)µg) weredene
fluoride(PVDF)
membranes.
Themembranes
were
incubated
with
10
μg/mL
of
44Mab-46
fluoride (PVDF) membranes. The membranes were incubated with 10 µg/mL of C44CMab-46(A),(A), 10 μg/mL of C44Mab-3 (B), 0.5 μg/mL of an anti-PA16 tag mAb (NZ-1) (C), and 1 μg/mL of an
10 µg/mL of C44 Mab-3 (B), 0.5 µg/mL of an anti-PA16 tag mAb (NZ-1) (C), and 1 µg/mL of ananti-β-actin mAb (D). Then, the membranes were incubated with anti-mouse immunoglobulins conanti-β-actinmAb (D). Then,
membranes
werewithanti-mouse
immunoglobulins
jugatedwith peroxidase
for C44theMab-46,C44Mab-3,
andincubatedanti-β-actin.
Anti-rat
immunoglobulins
conconjugatedwithperoxidaseforC
Mab-46,C
Mab-3,
andanti-β-actin.
Anti-rat
immunoglobulins
44 red arrows indicate CD44s (~75 kDa).
jugated with peroxidase were used44for NZ-1. The
The black
conjugated
withCD44v3–10
peroxidaseor
wereused (>180
for NZ-1.
arrowsindicate
CD44v5kDa).The red arrows indicate CD44s (~75 kDa). The blackarrows indicate CD44v3–10 or CD44v5 (>180 kDa).

3.5. Immunohistochemical Analysis Using C44Mab-3 against Tumor Tissues

Antibodies 2023, 12, 31

10 of 16

3.5. Immunohistochemical Analysis Using C44 Mab-3 against Tumor Tissues
We next examined whether C44 Mab-3 could be used for immunohistochemical analyses using FFPE sections. We first examined the reactivity of C44 Mab-3 and C44 Mab-46
in an oral SCC tissue. As shown in Supplementary Figure S4, C44 Mab-3 exhibited a clear
membranous staining and could clearly distinguish tumor cells from stromal tissues. In
contrast, C44 Mab-46 stained both. We then investigated the reactivity of C44 Mab-3 andC44 Mab-46 in pancreatic carcinoma tissue arrays. Although we performed the antigen
retrieval using citrate buffer (pH 6.0) for pancreatic carcinoma tissue arrays in the same
way as with an oral SCC tissue, weak staining was observed. Therefore, we next used
EnVision FLEX Target Retrieval Solution High pH for the antigen retrieval procedure;
C44 Mab-3 showed clear membranous staining in pancreatic carcinoma cells with a relatively larger cytoplasm (Figure 5A). C44 Mab-46 also stained the same type of pancreatic
carcinoma cells (Figure 5B). The staining intensity of C44 Mab-3 was much stronger thanthat of C44 Mab-46 (Figure 5A,B). Furthermore, diffusely spread tumor cells in the stroma
were stained by both C44 Mab-3 and C44 Mab-46 (Figure 5C,D). In contrast, both C44 Mab-3
and C44 Mab-46 did not stain the typical ductal structure of PDAC (Figure 5E,F). In addition,
stromal staining using C44 Mab-46 was observed in several tissues (Figure 5F). Importantly,
normal pancreatic epithelial cells were not stained by C44 Mab-3 (Figure 5G). A similarstaining pattern was also observed in another tissue array (Supplementary Figure S5).
We summarized the data of immunohistochemical analyses in Table 2; C44 Mab-3 stained
8 out of 20 cases (40%) (PA484, Figure 5) and 2 out of 6 cases (33%) (PA241c, Supplementary
Figure S5) of pancreatic carcinomas. These results indicated that C44 Mab-3 could be usefulfor immunohistochemical analysis of FFPE tumor sections and could recognize a specific
type of pancreatic carcinoma.
Table 2. Immunohistochemical analysis using C44 Mab-3 against pancreatic carcinoma tissue arrays.
Tissue Array

Age

Sex

Organ

Pathology Diagnosis

TNM

Grade

Stage

Type

C44 Mab-3

PA241c

66
66
54
54
44
44
59
59
63
63
53
53

F
F
F
F
M
M
M
M
F
F
F
F

Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas

Adenocarcinoma
Adjacent normal pancreas tissue
Adenocarcinoma
Adjacent normal pancreas tissue
Adenocarcinoma
Adjacent normal pancreas tissue
Adenocarcinoma
Adjacent normal pancreas tissue
Adenocarcinoma
Adjacent normal pancreas tissue
Adenocarcinoma
Adjacent normal pancreas tissue

T2N0M01

Imalignant

T3N0M02IImalignant

T3N0M02IImalignant

T2N0M03

Imalignant

T2N0M03

Imalignant

T3N0M03IImalignant

+
–
–
–
–
–
–
–
+
–
–
–

35
38
38
60
68
54
42
65
75
57
44
47
41
64
58
47
78
49
53
60
57
61
69

M
F
M
M
F
F
F
M
F
M
M
M
M
F
F
F
M
M
F
M
F
M
M

Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas

Normal pancreas tissue
Normal pancreas tissue
Normal pancreas tissue
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Mucinous adenocarcinoma
Undifferentiated carcinoma

T3N0M0T2N0M0
T3N0M0
T3N0M0
T3N0M0
T3N0M1
T3N0M0
T3N0M0
T3N0M0
T4N1M0
T3N0M0
T3N0M0
T3N1M0T2N0M0
T3N0M0
T3N0M0T2N0M0T2N0M0
T3N0M1
T2N0M0

2
2
2
2
2
2
3
3
2
2
3
3
3
2
3
3
3
2
-

II
I
II
II
II
IV
II
II
II
III
II
II
III
I
II
II
I
I
IV
I

normalnormalnormalmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignant

PA484+, OD655 ≥ 0.3; −, OD655 < 0.1.

–
–
–
–
+
–
–
–
–
+
–
–
–
–
–
+
+
+
+
+
–
–
+

49
53
60
57
Antibodies 2023, 12, 31
61
69

M
F
M
F
M
M

Pancreas
Pancreas
Pancreas
Pancreas
Pancreas
Pancreas

Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Mucinous adenocarcinoma
Undifferentiated carcinoma

T3N0M0
T3N0M0T2N0M0T2N0M0
T3N0M1
T2N0M0

2
3
3
3
2
-

II
II
I
I
IV
I

malignantmalignantmalignantmalignantmalignantmalignant+++–
11 of 16–+

Figure 5. Immunohistochemical analysis using C44 Mab-3 and C44 Mab-46 against pancreatic adenocarcinomas and normal pancreatic tissues. After antigen retrieval, serial sections of pancreatic
carcinoma tissue arrays (Catalog number: PA484) were incubated with 1 µg/mL of C44 Mab-3 or
C44 Mab-46, 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. (A–F) pancreatic adenocarcinomas; (G,H) normal pancreas tissues.

Antibodies 2023, 12, 31

12 of 16

4. Discussion
PDAC is the most common type of pancreatic cancer and has extremely poor prognosis,
with a 5-year survival rate of approximately 10% [48]. Advances in therapy have only
achieved incremental improvements in overall outcome but can provide notable benefits
for undefined subgroups of patients. PDACs are heterogenous neoplasms with various
histology [4] and heterogenous molecular landscapes [5]. Therefore, the identificationof early diagnostic markers and therapeutic targets in each group has been desired. In
this study, we developed C44 Mab-3 using the CBIS method (Figure 1) and determined its
epitope as variant-5-encoded region of CD44 (Table 1). Then, we showed the usefulness of
C44 Mab-3 for multiple applications, including flow cytometry (Figures 2 and 3), Western
blotting (Figure 4), and immunohistochemistry of PDAC (Figure 5).
An anti-CD44v5 mAb (clone VFF-8) was previously developed and is mainly used forthe immunohistochemical analyses of tumors [49]. The epitope of VFF-8 was determined
as IHHEHHEEEETPHSTST in the v5-encoded region by ELISA [50]. As shown in Table 1,
C44 Mab-3 recognized both CD44p311–330 and CD44p321–340 peptides, which commonly
possess the HPPLIHHEHH sequence. The epitope of C44 Mab-3 partially shares that of
VFF-8. Further investigation of the detailed epitope mapping is required. In addition, CD44
is known to be heavily glycosylated [12], and the glycosylation pattern is thought to dependon the host cells. Since the epitope of C44 Mab-3 does not contain serine or threonine, therecognition of C44 Mab-3 is thought to be independent of the glycosylation.
Immunohistochemistry using VFF-8 and conventional RT-PCR analyses were performed against PDAC [49]. VFF-8 recognized PDAC but not normal pancreas cells. Furthermore, the RT-PCR analysis revealed that the exon v5 appeared in the chain containing
at least v4–10 in 80% of PDACs and the cell lines tested. The authors discussed that oneof the major differences between normal and PDAC was the linkage of CD44v5 to theCD44v6-containing chain [49]. Our immunohistochemical analysis also support this finding (Figure 5A,C,G). Furthermore, we found that C44 Mab-3 could detect atypical types of
PDAC, including metaplastic and diffusely invaded tumor cells (Figure 5A,C). In contrast,
C44 Mab-3 did not stain a typical ductal structure of PDAC (Figure 5E) and normal pancreatic epithelial cells (Figure 5G). In addition to conventional PDAC, the World HealthOrganization has classified nine histological subtypes of PDAC, which further highlight themorphologic heterogeneity of PDAC [4]. It is worthwhile to investigate whether CD44v5 isexpressed in a specific subtype of PDAC in a future study.
Large-scale genomic analyses of PDACs defined four subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) ADEX, which correlate with histopathologicalcharacteristics [5]. Among them, the squamous subtype is characterized as being enriched
for TP53 and KDM6A mutations and having upregulation of the ∆Np63 transcriptional
network, hypermethylation of pancreatic endodermal determinant genes, and a poor prognosis [5]. ∆Np63 is known as a marker of basal cells of stratified epithelium and SCC [51]; itis also reported to regulate HA metabolism and signaling [52]. Specifically, ∆Np63 directly
regulates the expression of CD44 through p63-binding sites that are located in the promoter
region and in the first intron of CD44 gene [52]. Therefore, CD44 transcription could be
upregulated in ∆Np63-positive PDAC. However, the mechanism of the variant 5 inclusion
during alternative splicing remains to be determined.
Clinical trials of anti-pan-CD44 and variant-specific CD44 mAbs have been conducted [53].
An anti-pan-CD44 mAb, RG7356, exhibited an acceptable safety profile in patients with advanced solid tumors expressing CD44. However, the study was terminated due to no evidenceof a clinical and pharmacodynamic dose-response relationship with RG7356 [54]. A clinical
trial of a humanized anti-CD44v6 mAb bivatuzumab−mertansine drug conjugate was conducted. However, it failed due to severe skin toxicities [55,56]. The efficient accumulation of
mertansine was most likely responsible for the high toxicity [55,56]. Although CD44v5 is notdetected in normal pancreatic epithelium by C44 Mab-3 (this study) and VFF-8 [49], CD44v5
could be detected in normal lung, skin, gastric, and bladder epithelium by VFF-8 [50]. For theAntibodies 2023, 12, 31

13 of 16development of the therapeutic use of C44 Mab-3, further investigations are required to reduce
the toxicity to the above tissues.
We previously converted a mouse IgG1 subclass of mAbs into IgG2a mAb and produced defucosylated mAbs using fucosyltransferase-8-deficient CHO-K1 cells. The defucosylated IgG2a mAbs showed potent antibody-dependent cellular cytotoxicity in vitro
and suppressed tumor xenograft growth [26,57–63]. Therefore, the production of a classswitched and defucosylated version of C44 Mab-3 is required to evaluate the antitumor
activity in vivo.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antib12020031/s1, Figure S1, Determination of the binding epitopeof C44 Mab-3 by ELISA. Figure S2, Recognition of CHO/CD44s and CHO/CD44v3–10 by C44 Mab-46
using flow cytometry. Figure S3, Measurement of dissociation constants (KD ) between C44 Mab-3
and the epitope peptide using SPR. Figure S4, Immunohistochemical analysis using C44 Mab-3 andC44 Mab-46 against oral squamous cell carcinoma tissue. Figure S5, Immunohistochemical analysis
using C44 Mab-3 and C44 Mab-46 against pancreatic adenocarcinomas and normal pancreatic tissues.
Author Contributions: Y.K. (Yuma Kudo), H.S. and T.T. performed the experiments. M.K.K. and
Y.K. (Yukinari Kato) designed the experiments. H.S. and Y.K. (Yuma Kudo) analyzed the data. Y.K.
(Yuma Kudo), H.S. and Y.K. (Yukinari Kato) 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.), 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.
Data Availability Statement: The data presented in this study are available in the article andsupplementary material.
Conflicts of Interest: The authors have no conflict of interest to declare.

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