Article

Establishment of a Novel Anti-CD44 Variant 10 Monoclonal
Antibody C44Mab-18 for Immunohistochemical Analysis
against Oral Squamous Cell Carcinomas
Kenichiro Ishikawa 1,† , Hiroyuki Suzuki 1,2, *,† , Mika K. Kaneko 1,2 and Yukinari Kato 1,2, *
1

2

*
†

Citation: Ishikawa, K.; Suzuki, H.;
Kaneko, M.K.; Kato, Y. Establishmentof a Novel Anti-CD44 Variant 10
Monoclonal Antibody C44 Mab-18 for
Immunohistochemical Analysis
against Oral Squamous Cell
Carcinomas. Curr. Issues Mol. Biol.
2023, 45, 5248–5262. https://doi.org/
10.3390/cimb45070333

Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai 980-8575, Japan; ken.ishikawa.r3@dc.tohoku.ac.jp (K.I.); k.mika@med.tohoku.ac.jp (M.K.K.)
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-22-717-8207 (H.S. & Y.K.)
These authors contributed equally to this work.

Abstract: Head and neck squamous cell carcinoma (HNSCC) is the most common type of headand neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44
has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor
malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressedin cancers and considered a promising target for cancer diagnosis and therapy. We developed
monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed
PANC-1 cells. Among the established clones, C44 Mab-18 (IgM, kappa) reacted with CHO/CD44v3–10,
but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping
using peptides that cover variant exon-encoded regions revealed that C44 Mab-18 recognized theborder sequence between variant 10 and the constant exon 16-encoded sequence. These results
suggest that C44 Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore,
C44 Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow
cytometry. The apparent dissociation constant (KD ) of C44 Mab-18 for CHO/CD44v3–10 and HSC-3
was 1.6 × 10−7 M and 1.7 × 10−7 M, respectively. Furthermore, C44 Mab-18 detected CD44v3–10
but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry
using OSCC tissues. These results indicate that C44 Mab-18 is useful for detecting CD44v10 in flow
cytometry and immunohistochemistry.
Keywords: CD44; CD44v10; monoclonal antibody; oral squamous cell carcinoma; immunohistochemistry

Academic Editors: Violeta Popovici
and Emma Adriana Ozon
Received: 25 May 2023
Revised: 14 June 2023
Accepted: 19 June 2023
Published: 21 June 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
Head and neck cancer is the seventh most common cancer type globally, and exhibits a
profound impact on patients and their quality of life after surgical ablation and therapies [1].
Head and neck squamous cell carcinoma (HNSCC) is the most common type of head andneck cancer. The treatment of HNSCC includes surgery, chemotherapy, radiation therapy,
immunotherapy, molecular targeted therapy, or a combination of those modalities [2]. Although survival can be improved through the development of treatments, cancer metastasis
and resistance to drugs remain the main causes of death [3]. The rate of 5-year survival
remains stagnant at approximately 50% [4].
CD44 is a multifunctional type I transmembrane glycoprotein that mediates metastasis
and drug resistance in tumor cells. HNSCC is the second-highest CD44-expressing tumor
in the Pan-Cancer Atlas [5]. The alternative splicing of CD44 mRNA produces the various
isoforms [6]. The constant exons including the first five (1 to 5) and the last five (16 to 20)
are present in all CD44 variants and make up the standard isoform (CD44s). The CD44

Curr. Issues Mol. Biol. 2023, 45, 5248–5262. https://doi.org/10.3390/cimb45070333

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

Curr. Issues Mol. Biol. 2023, 455249

variant (CD44v) isoforms are generated by the alternative splicing of variant exons (v1 tov10) with the constant exons of CD44s [7]. Both CD44s and CD44v (pan-CD44) attach to
the extracellular matrix, including hyaluronic acid (HA), and facilitate the activation ofmetastasis-associated intracellular signaling pathways [8].
Tumor metastasis includes multiple processes called the invasion–metastasis cascade. The processes contain (i) dissemination from primary sites, (ii) the acquisition of
migration/invasion phenotype, (iii) intra/extravasation, (iv) survival in circulation, and(v) adaptation and colonization in distant organs [9]. Moreover, (vi) cancer-associatedfibroblasts and tumor-infiltrating lymphocytes in the tumor microenvironment involve inthe promotion of the invasion–metastasis cascade [10]. CD44 mediates the multiple stepsof the invasion–metastasis cascade through interaction with HA [11] and CD44v-specificfunctions [12].
CD44 has been studied as a marker of cancer stem-like cells (CSCs) in tumors [13]. AntiCD44s or CD44v monoclonal antibodies (mAbs) are used to collect the CD44-high CSCs [13].
The CD44-high population showed the increased self-renewing property, resistance todrugs, and metastatic colonization in vivo [13]. CD44 is the first applied CSC marker toisolate CSCs derived from HNSCC [14]. Notably, CD44-high CSCs from HNSCC showed
the features of epithelial to mesenchymal transition (EMT). The EMT program activation
confers tumor cells the stemness and the ability to migrate, invade, and extravasate [15].
Moreover, CD44-high cells could make colonization in the lungs of immunodeficient mice,compared to CD44-low, which failed to form the metastatic colonization [16].
Furthermore, CD44v8–10 mediates the resistance to treatment. The v8–10-encoded
region binds to and stabilizes a cystine–glutamate transporter (xCT), which enhances cystine uptake and glutathione synthesis [17]. The elevation of reduced glutathione (GSH)
mediates the defense to reactive oxygen species (ROS) [17], radiation [18], and chemotherapeutic drugs [19]. The expression of CD44v8–10 is associated with the xCT-mediatedredox status and the poor prognosis of patients [18]. Therefore, the establishment of eachCD44v-specific mAb is essential to reveal the function and develop CD44-targeting cancer
therapy. However, the tissue distribution or function of the variant 10-containing CD44 has
not been fully understood.
In our previous work, we developed an anti-pan-CD44 mAb, C44 Mab-5 (IgG1 , kappa) [20]using the Cell-Based Immunization and Screening (CBIS) method. Additionally, anotheranti-pan-CD44 mAb, C44 Mab-46 [21], was established by immunizing mice with theCD44v3–10 ectodomain. Both C44 Mab-5 and C44 Mab-46 have the epitopes within theconstant exon 2- and 5-encoding sequences [22–24] and could be applied to immunohistochemistry in oral squamous cell carcinomas (OSCC) [20] and esophageal SCC [21],respectively. Furthermore, we produced a class-switched and defucosylated type of recombinant C44 Mab-5 (5-mG2a -f) using fucosyltransferase 8 (Fut8)-deficient ExpiCHO-S cells
and investigated the antitumor activity in OSCC xenograft-transplanted mice [25]. We havedeveloped various anti-CD44v mAbs, including C44 Mab-6 (an anti-CD44v3 mAb) [26],
C44 Mab-108 (an anti-CD44v4 mAb) [27], C44 Mab-3 (an anti-CD44v5 mAb) [28], C44 Mab-9(an anti-CD44v6 mAb) [29], C44 Mab-34 (an anti-CD44v7/8 mAb) [30], and C44 Mab-1 (ananti-CD44v9 mAb) [31]. The generation of anti-CD44 mAbs, which recognize all variant
exons, is important for the comprehensive analysis of human tumors.
In this study, we established a novel anti-CD44v10 mAb, C44 Mab-18 (IgM, kappa),
using the CBIS method, and evaluated its applications via flow cytometry, Western blotting,and immunohistochemical analyses of OSCC tissues.
2. Materials and Methods
2.1. Cell Lines
A human pancreatic cancer cell line (PANC-1, the Cell Resource Center for Biomedical
Research Institute of Development, Aging Sendai, Japan), a mouse multiple myeloma
P3x63Ag8U.1 (P3U1) and Chinese hamster ovary (CHO)-K1 cell lines (the American
Type Culture Collection, Manassas, VA, USA) were cultured using RPMI-1640 medium

Curr. Issues Mol. Biol. 2023, 45

5250(Nacalai Tesque, Inc., Kyoto, Japan) containing 10% heat-inactivated fetal bovine serum(FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B). A human OSCC cell line
(HSC-3, the Japanese Collection of Research Bioresources, Osaka, Japan) was cultured in
Dulbecco’s Modified Eagle Medium (DMEM) (Nacalai Tesque, Inc., Kyoto, Japan) supplemented as indicated above. All cell lines were grown in a humidified incubator at 37 ◦ Cwith 5% CO2 .
The cDNAs of CD44v3–10 and CD44s were obtained as described previously [20].
The cDNAs were cloned into pCAG-zeo-ssPA16 and pCAG-neo-ssPA16 vectors with asignal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV). The PA16 tag canbe detected by NZ-1 mAb, which was originally developed as an anti-human podoplanin(PDPN) mAb [32]. Stable transfectants including PANC-1/CD44v3–10, CHO/CD44v3–10,
and CHO/CD44s were established by introducing corresponding vectors into the cells
using a Neon transfection system (Thermo Fisher Scientific, Inc.).
2.2. Production of Hybridoma Cells
PANC-1/CD44v3–10 (1 × 108 cells) was intraperitoneally administrated into the 6week-old female BALB/c mice (CLEA Japan, Tokyo, Japan) with Imject Alum (Thermo
Fisher Scientific Inc.). Additional three times immunizations of PANC-1/CD44v3–10(1 × 108 cells) and a booster injection of PANC-1/CD44v3–10 (1 × 108 cells) two days before the sacrifice was performed. Hybridomas were produced as described previously [28].
The supernatants were selected by flow cytometer (SA3800 Cell Analyzer) and SA3800
software (ver. 2.05, Sony Corp. Tokyo, Japan).
2.3. Enzyme–Linked Immunosorbent Assay (ELISA)
Thirty-four peptides, which cover the variant region of CD44v3–10 [22], were obtained
from Sigma-Aldrich Corp. (St. Louis, MO, USA), and immobilized on Nunc Maxisorp
immunoplates (Thermo Fisher Scientific Inc.) at 20 µg/mL. After the blocking with 1%(w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.05%(v/v) Tween 20 (PBST; Nacalai Tesque, Inc.), C44 Mab-18 (1 µg/mL) was added to each well.
The wells were further treated with anti-mouse immunoglobulins peroxidase-conjugate(1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA, USA). The enzymatic reaction
was performed using an ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.) The opticaldensity (655 nm) was measured using an iMark microplate reader (Bio-Rad Laboratories,
Inc., Berkeley, CA, USA).
2.4. Flow Cytometry
CHO/CD44v3–10, CHO-K1, and HSC-3 cells (1 × 105 cells/sample) were incubatedwith C44 Mab-18, C44 Mab-46, or blocking buffer (0.1% BSA in PBS; control) for 30 min at 4 ◦ C.
The cells were further treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:2000;
Cell Signaling Technology, Inc. Danvers, MA, USA), and analyzed as indicated above.
2.5. Determination of Apparent Dissociation Constant (KD ) via Flow Cytometry
The serially diluted C44 Mab-18 at the indicated concentrations was suspended with
2 × 105 of HSC-3 and CHO/CD44v3–10 cells. The cells were further treated with antimouse IgG conjugated with Alexa Fluor 488 (1:200). Fluorescence data were analyzed, andthe apparent dissociation constant (KD ) was determined by fitting binding isotherms tobuilt-in one-site binding models of GraphPad Prism 8 (GraphPad Software, Inc., La Jolla,
CA, USA).
2.6. Western Blot Analysis
The SDS-polyacrylamide gel for electrophoresis and transfer onto polyvinylidene
difluoride membranes was achieved as described previously [28]. After the blocking in
PBST containing 4% skim milk (Nacalai Tesque, Inc.), the membranes were incubatedCurr. Issues Mol. Biol. 2023, 45

5251

with 10 µg/mL of C44 Mab-46, 10 µg/mL of C44 Mab-18, or 0.5 µg/mL of an anti-β-actinmAb (AC-15; Sigma-Aldrich Corp.). The membranes were further treated with peroxidaseconjugated anti-mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.). Finally, the chemiluminescence signal was obtained using ImmunoStar LD (FUJIFILM Wako
Pure Chemical Corporation, Osaka, Japan) and was detected using a Sayaca-Imager (DRC
Co., Ltd., Tokyo, Japan).
2.7. Immunohistochemical Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Tissues
Antigen retrieval of an OSCC tissue array (OR601c; US Biomax Inc., Rockville, MD,USA) was performed using EnVision FLEX Target Retrieval Solution High pH (Agilent
Technologies, Inc.). SuperBlock T20 (Thermo Fisher Scientific, Inc.) was used for blocking.
The sections were incubated with 1 µg/mL of C44 Mab-18 and 1 µg/mL of C44 Mab-46 atroom temperature for 1 h. The sections were further treated with the EnVision+ Kit fora mouse (Agilent Technologies Inc.) at room temperature for 30 min. The chromogenic
reaction and counterstaining were performed using 3,30 -diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) and hematoxylin (FUJIFILM Wako Pure Chemical
Corporation), respectively.
3. Results
3.1. Establishment of an Anti-CD44 mAbs via Immunization of PANC-1/CD44v3–10 Cells
In our previous work, we have established anti-CD44 mAbs, including C44 Mab-5(pan-CD44) [20], C44 Mab-6 (v3) [26], C44 Mab-3 (v5) [28], C44 Mab-9 (v6) [29], and C44 Mab-1(v9) [31], using CHO/CD44v3–10 cells as an immunogen. In this study, we establishedanother stable transfectant (PANC-1/CD44v3–10 cells) (Figure 1A). Mice were immunizedwith PANC-1/CD44v3–10 cells (Figure 1B), and hybridomas were produced via fusion
between the splenocyte and P3U1 cells (Figure 1C). The supernatants, which were reactive
to CHO/CD44v3–10 cells, but not to CHO-K1, were selected via flow cytometry-based
high throughput screening (Figure 1D). After cloning by limiting dilution, anti-CD44
mAb-producing clones were finally established (Figure 1E).
3.2. Flow Cytometric Analysis of C44 Mab-18- to CD44-Expressing Cells
In this study, established clones, the epitope of which includes CD44v10, were mainly
determined to be IgM, although all mAbs against other CD44 variants are IgG [26–31].
Among those clones, we examined the reactivity of C44 Mab-18 (IgM, kappa) against
CHO/CD44v3–10 and CHO/CD44s cells via flow cytometry. C44 Mab-18 dose-dependently
recognized CHO/CD44v3–10 cells (Figure 2A). In contrast, C44 Mab-18 recognized neither CHO/CD44s (Figure 2B) nor CHO-K1 (Figure 2C) cells. We confirmed that an
anti-pan-CD44 mAb, C44 Mab-46 [21], recognized CHO/CD44s cells, but not CHO-K1
cells (Supplementary Figure S1). Furthermore, C44 Mab-18 could recognize HSC-3 cells(Figure 2D) in a dose-dependent manner. These results indicated that C44 Mab-18 recognizes the variant exon-encoded region between v3 and v10 (Figure 1A).
3.3. Epitope Mapping of C44 Mab-18 by ELISATo determine the epitope of C44 Mab-18, we performed the ELISA using synthetic
peptides, which cover the variant exon-encoded region between v3 and v10 [22]. As
shown in Figure 3, C44 Mab-18 recognized the CD44 p551–570 peptide (SNSNVNRSLSGDQDTFHPSG), which corresponds to variant 10 and constant exon 16-encoded sequence(Supplementary Table S1). In contrast, C44 Mab-18 never recognized other v3- and v10encoded peptides. This and the results in Figure 2 indicate that C44 Mab-18 specificallyrecognizes the variant 10-containing CD44.

Curr.
Curr. Issues
IssuesMol.
Mol.Biol.
Biol.2023,
2023,3,45FOR PEER REVIEW

55252

Figure1.1.AA schematic
schematic illustration
illustration ofanti-human
CD44
mAbs.
(A)(A)
StrucFigureof thetheCBIS
CBISmethod
methodtotoestablish
establish
anti-human
CD44
mAbs.
ture of CD44.
The
CD44s
mRNA
(1 to
to5)
5)and
and(16(16toto
20).
The
CD44v
Structureof CD44.
The
CD44s
mRNAcontains
containsthe
theconstantconstant exons (1
20).
The
CD44v
including
CD44v6–10,
andCD44v8–10
areareproducedviaviathethealternative
includingCD44v3–10,
CD44v3–10,CD44v4–10,
CD44v4–10,
CD44v6–10,
andCD44v8–10
producedalternative
splicing(B)(B)
PANC-1/CD44v3–10
cellscells
were
injected
intointo
BALB/cmice miceintraperitosplicingofofvariant
variantexons.
exons.
PANC-1/CD44v3–10
wereinjected
BALB/cintraperineally.
(C)(C)
Hybridomas
were
produced
via via
the thefusionof thesplenocytes
andandP3U1
cells(D)(D)
TheThetoneally.
Hybridomas
were
producedfusionof thesplenocytes
P3U1
cells
screening was performed via flow cytometry using CHO/CD44v3–10 and parental CHO-K1 cells.
screening was performed via flow cytometry using CHO/CD44v3–10 and parental CHO-K1 cells.
(E) A clone C44Mab-18 (IgM, kappa) was established after cloning.
(E) A clone C44 Mab-18 (IgM, kappa) was established after cloning.

recognized CHO/CD44v3–10 cells (Figure 2A). In contrast, C44Mab-18 recognized neither
CHO/CD44s (Figure 2B) nor CHO-K1 (Figure 2C) cells. We confirmed that an anti-panCD44 mAb, C44Mab-46 [21], recognized CHO/CD44s cells, but not CHO-K1 cells (Supplementary Figure S1). Furthermore, C44Mab-18 could recognize HSC-3 cells (Figure 2D) in a
Curr. Issues Mol.
Biol. 2023, 45
dose-dependent
manner. These results indicated that C44Mab-18 recognizes the variant
exon-encoded region between v3 and v10 (Figure 1A).

5253

Figure 2. Flow cytometry
C44Mab-18.
CHO/CD44v3–10(A), CHO/CD44s(B), CHO-K1
Figure 2. using
Flow cytometryusing
C44 Mab-18. CHO/CD44v3–10(A), CHO/CD44s(B),(C),
CHO-K1 (C),cells werewithand HSC-3 (D) cells
treatedwith
0.01–10of C44Mab-18.
andwereHSC-3(D) cells
weretreatedµg/mL
with 0.01–10µg/mL ofThen,
C44 Mab-18.
Then,treatedcells weretreated with
Alexa Fluor 488-conjugatedanti-mouse
IgG anti-mouse(Red line). IgG
The(Redblack
lineTherepresents
negativethe
controlAlexa Fluor
488-conjugatedline).
black linethe
represents
negative control(blocking buﬀer). (blocking buffer).

3.4. Determination of the Apparent Binding Affinity of C44 Mab-18 via Flow Cytometry
We measured the apparent binding affinity of C44 Mab-18 to CHO/CD44v3–10 and
HSC-3 cells. The apparent dissociation constant (KD ) of C44 Mab-18 for CHO/CD44v3–10(Figure 4A) and HSC-3 (Figure 4B) was 1.6 × 10−7 M and 1.7 × 10−7 M, respectively. These
results indicated that C44 Mab-18 possesses a moderate binding affinity for CD44v3–10 orendogenous CD44v10-expressing cells.

Curr. Issues Mol. Biol. 2023, 45

tides, which cover the variant exon-encoded region between v3 and v10 [22]. As shown in
Figure3,
C44Mab-18
recognizedtheCD44
p551–570
peptide(SNSNVNRSLSGDQDTFHPSG), which corresponds to variant 10 and constant exon 16encoded sequence (Supplementary Table S1). In contrast, C44Mab-18 never recognized
Mabother v3- and v10-encoded peptides. This and the results in Figure 2 indicate that C445254
18 specifically recognizes the variant 10-containing CD44.

Figure 3. Determination of C44 Mab-18 epitope using ELISA. The synthesized peptides, that cover

23, 3, FOR PEER REVIEW

Figure 3. Determination of C44Mab-18 epitope using ELISA. The synthesized peptides, that cover
the variant exon-encoded region between v3 and v10, were immobilized on immunoplates. The
the variant exon-encoded region between v3 and v10, were immobilized on immunoplates. The
plates were incubated with C44 Mab-18, followed by incubation with anti-mouse immunoglobulins
plates were incubated with C44Mab-18, followed by incubation with anti-mouse immunoglobulins
8-conjugated with peroxidase. Optical density (655 nm) was measured. The CD44 p551–570 se-conjugated with peroxidase. Optical density (655 nm) was measured. The CD44 p551–570 sequence
quence (SNSNVNRSLSGDQDTFHPSG) corresponds to variant 10 and the constant exon 16-encoded(SNSNVNRSLSGDQDTFHPSG) corresponds to variant 10 and the constant exon 16-encoded sesequence. Error bars represent means ± SDs. NC, negative control (0.1% DMSO [solvent] in PBS).
quence. Error bars represent means ± SDs. NC, negative control (0.1% DMSO [solvent] in PBS).

3.4. Determination of the Apparent Binding Aﬃnity of C44Mab-18 via Flow Cytometry
We measured the apparent binding aﬃnity of C44Mab-18 to CHO/CD44v3–10 and
HSC-3 cells. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3–10(Figure 4A) and HSC-3 (Figure 4B) was 1.6 × 10−7 M and 1.7 × 10−7 M, respectively. These
results indicated that C44Mab-18 possesses a moderate binding aﬃnity for CD44v3–10 orendogenous CD44v10-expressing cells.

Figure of
4. Theof the apparent
dissociation(KD ) of CC
C44was
Mab-18Figure 4. The determination
thedetermination
apparent dissociation
constant(KD)constantof C44Mab-18.
44Mab-18
44 Mab-18.
wastreatedwith
CHO/CD44v3–10
atindicatedconcentrations
(A)
and
with
HSC-3
(B).
The
treated with CHO/CD44v3–10 at indicated concentrations (A) and with HSC-3 (B). The cells werecells
were treated with anti-mouse IgG conjugated with Alexa Fluor 488. Fluorescence data were collected,
treated with anti-mouse
IgG conjugated with Alexa Fluor 488. Fluorescence data were collected,
followed by the calculation of KD using GraphPad PRISM 8.
PRISM 8.
followed by the calculation of KD using GraphPad

3.5. Western Blot Analysis
To assess the sensitivity of C44Mab-18 in Western blot analysis, we analyzed the celllysates from CHO-K1, CHO/CD44s, and CHO/CD44v3–10. C44Mab-18 mainly detectedtreated with CHO/CD44v3–10 at indicated concentrations (A) and with HSC-3 (B). The
treated with anti-mouse IgG conjugated with Alexa Fluor 488. Fluorescence data wer
followed by the calculation of KD using GraphPad PRISM 8.
Curr. Issues Mol. Biol. 2023, 45

3.5. Western Blot Analysis

5255

To assess the sensitivity of C44Mab-18 in Western blot analysis, we analyz
lysatesfrom
CHO/CD44s, and CHO/CD44v3–10. C44Mab-18 mainly
3.5. Western
Blot CHO-K1,
Analysis
To assess thesensitivity
C44 Mab-18andin Western
blotbands.
analysis,
we analyzed
cellCD44v3–10
as morethanof180-kDa~70-kDa
However,C44theMab-18didlysatesfrom
CHO-K1,
CHO/CD44s,
andCHO/CD44v3–10.
C
Mab-18mainlydetected44
any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 5A). An antiCD44v3–10 as more than 180-kDa and ~70-kDa bands. However, C44 Mab-18 did not detectmAb,
C44Mab-46, recognized both CD44v3–10 (>180 kDa) and CD44s (~75 kDa
any bands from lysates of CHO-K1 and CHO/CD44s cells (Figure 5A). An anti-pan-CD44
thelysatesof CHO/CD44v3–10
and CHO/CD44s,
respectively(Figure5B). We
mAb,
C44 Mab-46,
recognized both CD44v3–10(>180 kDa) andCD44s (~75 kDa)
bands
in theof CHO/CD44v3–10
and5C).
CHO/CD44s,
respectively(Figure5B).CWeusedtin
aslysates
a loadingcontrol (Figure
These results
indicatethat
44Mab-18canβ-actinasaloading
control(Figure5C).
These
results
indicatethatC
Mab-18candetect44
ogenous CD44v3–10.
exogenous CD44v3–10.

Figure 5. Western blot analysis using C44 Mab-18. The total cell lysates (10 µg of protein) were

Figure5. Western blot analysis using C44Mab-18. The total cell lysates (10 µg of protein)
separated and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were
rated
and
transferredonto
polyvinylidene
difluoride(PVDF)
membranincubated with
10 µg/mL of
C44 Mab-18(A), 10 µg/mL
of C44 Mab-46(B), or membranes.
0.5 µg/mL of anTheanti-βcubatedwith
10 µg/mLof C44Mab-18(A), 10 µg/mLanti-mouseof C44Mab-46(B), or 0.5Theµg/mL oactin mAb (C),followedby incubation
with peroxidase-conjugatedimmunoglobulins.
actin
mAb
(C), followed
by incubation
withperoxidase-conjugatedanti-mouse
red arrows
indicate
the CD44v3–10(>180 kDa). The
blackarrow indicates the CD44s (~75
kDa). The immun
whiteredarrow
indicates
a lower molecularweight band
recognizedby C44Mab-18in CHO/CD44v3–10
The
arrowsindicate
the CD44v3–10(>180kDa). The
black
arrow
indicates the CD44
lysate (~70
kDa).
The
white
arrow indicates a lower molecular weight band recognized by C44
CHO/CD44v3–10
lysate(~70 kDa).
3.6. Immunohistochemical
AnalysisUsing C44 Mab-18 against Tumor Tissues
Since HNSCC is revealed as the second highest CD44-expressing tumor in the PanCancer Atlas [5], we examined the reactivity of C44 Mab-18 and C44 Mab-46 in immunohistochemical analyses using FFPE sections of OSCC tissue array. As shown in Figure 6,
C44 Mab-18 was able to distinguish tumor cells from stromal tissues. In contrast, C44 Mab46 stained both. We summarized the data of immunohistochemical analyses in Table 1;
C44 Mab-18 stained 41 out of 50 cases (82%) in OSCC. These results indicate that C44 Mab-18
applies to the immunohistochemical analysis of FFPE tumor sections.

Since HNSCC is revealed as the second highest CD44-expressing tumor in the PanCancer Atlas [5], we examined the reactivity of C44Mab-18 and C44Mab-46 in immunohistochemical analyses using FFPE sections of OSCC tissue array. As shown in Figure 6,
C44Mab-18 was able to distinguish tumor cells from stromal tissues. In contrast, C44Mab46 stained both. We summarized the data of immunohistochemical analyses in Table 1;
5256
C44Mab-18 stained 41 out of 50 cases (82%) in OSCC. These results indicate that C44Mab18 applies to the immunohistochemical analysis of FFPE tumor sections.

Curr. Issues Mol. Biol. 2023, 45

Figureand C44Mab-46 against FFPE OSCC tisFigure 6.
6. Immunohistochemical
Immunohistochemicalanalysis
analysisusing
usingC44
CMab-1844 Mab-18 and C44 Mab-46 against FFPE OSCC
sues.
(A–F)
Serialsections
of
the
OSCC
tissuearray(OR601c)µg/mL oftissues. (A–F) Serial sections of the OSCC tissue array (OR601c)were
were incubatedincubated with
with 11 µg/mL
of
C44Mab-18 or C44Mab-46 followed by treatment with the Envision+ kit. The chromogenic reaction
C44 Mab-18 or C44 Mab-46 followed by treatment with the Envision+ kit. The chromogenic reaction andand counterstaining were performed using0 3,3′-diaminobenzidine tetrahydrochloride and hematoxcounterstaining were performed using 3,3 -diaminobenzidine tetrahydrochloride and hematoxylin,
ylin, respectively. Scale bar = 100 µm.
respectively. Scale bar = 100 µm.
Table 1. Immunohistochemical analysis using C44 Mab-18 against OSCC tissue array.
No.

Age

Sex

Organ
/Anatomic Site

Pathology Diagnosis

TNM

C44 Mab-18

C44 Mab-46

1

78

M

TongueSCC of tongue

T2N0M0

+

+

2

40

M

TongueSCC of tongue

T2N0M0

+

++

3

75

F

TongueSCC of tongue

T2N0M0-+Curr. Issues Mol. Biol. 2023, 45

5257

Table 1. Cont.
No.

Age

Sex

Organ
/Anatomic Site

Pathology Diagnosis

TNM

C44 Mab-18

C44 Mab-46

4

35

F

TongueSCC of tongue

T2N0M0

++

++

5

61

M

TongueSCC of tongue

T2N0M0

++

+++

6

41

F

TongueSCC of tongue

T2N0M0

+

+

7

64

M

TongueSCC of right tongue

T2N2M0

++

++

8

76

M

TongueSCC of tongue

T1N0M0

++

++

9

50

F

TongueSCC of tongue

T2N0M0

++

++

10

44

M

TongueSCC of tongue

T2N1M0

++

+++

11

53

F

TongueSCC of tongue

T1N0M0

+

++

12

46

F

TongueSCC of tongue

T2N0M0

++

+

13

50

M

TongueSCC of root of tongue

T3N1M0

++

+

14

36

F

TongueSCC of tongue

T1N0M0

++

+++

15

63

F

TongueSCC of tongue

T1N0M0

+

+

16

46

M

TongueSCC of tongue

T2N0M0

+

-

17

58

M

TongueSCC of tongue

T2N0M0

+

+

18

64

M

Lip

SCC of lower lip

T1N0M0

+

+++

19

57

M

Lip

SCC of lower lip

T2N0M0

+

+++

20

61

M

Lip

SCC of lower lip

T1N0M0

+

++

21

60

M

Gum

SCC of gum

T3N0M0

++

+

22

60

M

Gum

SCC of gum

T1N0M0

+++

+++

23

69

M

Gum

SCC of upper gum

T3N0M0

++

++

24

53

M

Bucca cavioris

SCC of bucca cavioris

T2N0M0

++

+

25

55

M

Bucca cavioris

SCC of bucca cavioris

T1N0M0

+++

+

26

58

M

TongueSCC of base of tongue

T1N0M0

++

++

27

63

M

Oral cavity

SCC

T1N0M0

+++

++

28

48

F

TongueSCC of tongue

T1N0M0

+

+

29

80

M

Lip

SCC of lower lip

T1N0M0

+++

+++

30

77

M

TongueSCC of base of tongue

T2N0M0

++

++

31

59

M

TongueSCC of tongue

T2N0M0

+

-

32

77

F

TongueSCC of tongue

T1N0M0

+

++

33

56

M

TongueSCC of root of tongue

T2N1M0

+

+

34

60

M

TongueSCC of tongue

T2N1M0

++

++

35

62

M

TongueSCC of tongue

T2N0M0

+

++

36

67

F

TongueSCC of tongue

T2N0M0

-

++

37

47

F

TongueSCC of tongue

T2N0M0

+++

+++

38

37

M

TongueSCC of tongue

T2N1M0

-

-

39

55

F

TongueSCC of tongue

T2N0M0

+

+

40

56

F

Bucca cavioris

SCC of bucca cavioris

T2N0M0

+

+

41

49

M

Bucca cavioris

SCC of bucca cavioris

T1N0M0

-

-

42

45

M

Bucca cavioris

SCC of bucca cavioris

T2N0M0--Curr. Issues Mol. Biol. 2023, 45

5258

Table 1. Cont.
No.

Age

Sex

Organ
/Anatomic Site

Pathology Diagnosis

TNM

C44 Mab-18

C44 Mab-46

43

42

M

Bucca cavioris

SCC of bucca cavioris

T3N0M0

+++

++

44

44

M

Jaw

SCC of right drop jaw

T1N0M0

+

+++

45

40

F

TongueSCC of base of tongue

T2N0M0

-

++

46

49

M

Bucca cavioris

SCC of bucca cavioris

T1N0M0

++

+++

47

56

F

TongueSCC of base of tongue

T2N0M0

-

+

48

42

M

Bucca cavioris

SCC a of bucca cavioris

T3N0M0

+++

+++

49

87

F

Face

SCC a of left face

T2N0M0

-

+

50

50

M

Gum

SCC of gum

T2N0M0---, No stain; +, Weak intensity; ++, Moderate intensity; +++, Strong intensity.

4. Discussion
In our previous work, we established anti-CD44 mAbs using CHO/CD44v3–
10 [20,26,28,29,31] and purified CD44v3–10 ectodomain [21,30] as immunogens. In thisstudy, we used PANC-1/CD44v3–10 as another immunogen. We have compiled a list withthis information in “Antibody Bank” (see Supplementary Materials). In this study, we listed
a novel anti-CD44v antibody C44 Mab-18, which recognizes the border sequence between
variant 10 and constant exon 16 (Figure 3). Furthermore, C44 Mab-18 could recognize
CHO/CD44v3–10, but not CHO/CD44s in flow cytometry (Figure 2) and Western blot
analyses (Figure 5). Moreover, C44 Mab-18 could stain tumor cells, but not stromal tissues,
which could be stained by C44 Mab-46, an anti-pan-CD44 mAb (Figure 6). These results
indicate that C44 Mab-18 is an anti-CD44v10 mAb.
The VFF series anti-human CD44v mAbs were previously established via the immunization of glutathione S-transferase fused CD44v3–10 produced by bacteria [33,34].
The clones, VFF-8 (anti-CD44v5), VFF-18 (anti-CD44v6), VFF-9 (anti-CD44v7), VFF-17(anti-CD44v7/8), and VFF-14 (anti-CD44v10) have been used for various applications [35].
Although VFF-14 was shown to apply to immunohistochemistry [36], the detailed binding
epitope of VFF-14 has not been reported. In this study, we determined the epitope of
C44 Mab-18 as the CD44 p551–570 peptide (SNSNVNRSLSGDQDTFHPSG), which corresponds to the variant 10 (underlined) and constant exon 16-encoded region. In contrast,
C44 Mab-18 never recognizes the p541–560 peptide (FGVTAVTVGDSNSNVNRSLS) in thevariant 10 region. Therefore, C44 Mab-18 could have the epitope in the border region, but
the inclusion of variant 10 is essential for the recognition.
Since the CD44 protein is modified by a variety of N-glycans and O-glycans, themolecular weight of CD44v isoforms surpasses 200-kDa [37]. C44 Mab-18 recognized bothmore than 180-kDa and ~70-kDa bands (Figure 5A) in the lysate from CHO/CD44v3–10.
The 70 kDa is approximately identical to the predicted molecular weight of CD44v3–10 fromthe amino acid sequence. Therefore, C44 Mab-18 could recognize CD44v3–10 regardlessof the glycosylation. The detailed epitope mapping and the influence of glycosylation on
C44 Mab-18 recognition should be investigated in future studies.
CD44v8–10 was shown to interact with xCT, a glutamate–cystine transporter, andregulate the level of reduced glutathione in tumor cells. The interaction is important forthe stabilization of xCT on the cell surface, which promotes the defense against reactive
oxygen species [17]. Furthermore, the interaction failed in CD44v8–10 (S301A), an Nlinked glycosylation consensus motif (Asn-X-Ser/Thr) mutant in the variant 10-encoded
region [17]. Therefore, it is worthwhile to investigate whether C44 Mab-18 interferes with
the interaction between CD44v8–10 and xCT in future studies. Furthermore, several
studies have revealed that CD44v9 is used as a predictive marker for recurrence [38] anda biomarker for patient selection and efficacy of xCT inhibitors, sulfasalazine in gastricCurr. Issues Mol. Biol. 2023, 455259

cancer [39]. Further investigations are also required to clarify the clinical significance ofCD44v10 expression using C44 Mab-18.
The mAbs against CD44 have been considered a therapeutic option for solid tumors and leukemia [12]. However, anti-pan-CD44 mAbs can affect normal tissues suchas the epithelium and hematopoiesis. In a preclinical study using a murine thymoma
model, a comparative study between an anti-pan-CD44 mAb (IM-7) and an anti-murine
CD44v10 mAb (K926) was conducted in CD44v10-transfected EL4 thymoma (EL4-v10) [40].
The results showed that a blockade of CD44v10 by K926 was superior to that of IM-7
in intra-marrow EL4-v10 growth retardation. Furthermore, K926 hardly disturbed thehematopoietic stem cell (HSC) interaction with the bone marrow stroma. In contrast, IM-7strongly affected the embedding of HSC in the bone marrow stroma [40]. These results
indicated that the therapeutic use of anti-pan-CD44 mAbs should be avoided in favor of
CD44v-specific mAbs as far as leukemic cells express CD44v isoforms.
In a humanized mouse model, CD44v8–10 was elevated during chronic myeloid
leukemia progression from chronic phase to blast crisis [41]. Furthermore, increased transcription of CD44 mRNA was observed in human acute myeloid leukemia (AML) patientswith FLT3 or DNMT3A mutations through the suppression of CpG islands methylationin the promoter [42]. An anti-CD44v6 mAb (BIWA-8) derived from VFF-18 [43] was engineered to develop chimeric antigen receptors (CARs) for AML with FLT3 or DNMT3A
mutations. The CD44v6 CAR-T cells exhibited potent anti-leukemic effects [42]. We haveestablished class-switched and defucosylated IgG2a recombinant mAbs and evaluated theantitumor activity in xenograft models [44]. Therefore, the production of class-switchedand defucosylated C44 Mab-18 is an important strategy to evaluate the antitumor effect inpreclinical models.
Since anti-pan-CD44 and anti-CD44v mAbs still have the possibility of causing side effects by affecting normal tissues, the clinical applications are limited. This study used tumor
cell-expressed CD44v3–10 as an immunogen. This strategy is critical for the developmentof cancer-specific mAbs (CasMabs). We developed podocalyxin-targeting CasMabs [45]and PDPN-targeting CasMabs [46], which react with the aberrantly glycosylated targets
selectively expressed in cancer [47]. Anti-PDPN-CasMabs have been applied to CAR-T therapy in preclinical studies [48–50]. For CasMab development, we should perform a further
selection of our established anti-CD44 mAbs by comparing the reactivity against normal
cells and tissues. Anti-CD44 CasMabs could be applicable for designing the modalities,
including antibody-drug conjugates and CAR-T.
5. Conclusions
In this study, we established an anti-CD44v10 mAb (C44 Mab-18). We also establishedan anti-CD44v8 mAb (C44 Mab-94) (manuscript submitted, see Supplementary Materials).
Therefore, we have established an anti-CD44 mAb library that covers almost all CD44
variants. This library could contribute to the diagnosis of not only carcinoma, but also
hematopoietic malignancies. Since we have already cloned the VH and VL cDNA ofanti-CD44 mAbs, the production of recombinant mAbs or CARs could contribute to thedevelopment of novel tumor therapies.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cimb45070333/s1. Supplementary Figure S1, Recognition of
CHO/CD44s and CHO/CD44v3–10 by C44 Mab-46 by flow cytometry. Supplementary Table S1, Thedetermination of the binding epitope of C44 Mab-18 by ELISA. The information on anti-CD44 mAbs
in our laboratory is available in “Antibody Bank” [http://www.med-tohoku-antibody.com/topics/
001_paper_antibody_PDIS.htm#CD44 (accessed on 14 June 2023)].
Author Contributions: K.I. and H.S. performed the experiments. M.K.K. and Y.K. designed theexperiments. K.I. and H.S. analyzed the data. K.I., H.S. and Y.K. wrote the manuscript. All authors
have read and agreed to the published version of the manuscript.

Curr. Issues Mol. Biol. 2023, 45

5260

Funding: This research was supported in part by the Japan Agency for Medical Research andDevelopment (AMED) under grant numbers: JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.), andJP23ck0106730 (to Y.K.); and by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid
for Scientific Research (KAKENHI) grant numbers 22K06995 (to H.S.), 21K07168 (to M.K.K.), and22K07224 (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: All related data and methods are presented in this paper. Additional
inquiries should be addressed to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest involving this article.

References
1.
2.

3.
4.
5.

6.
7.
8.
9.
10.
11.
12.
13.
14.

15.

16.
17.

18.

Mody, M.D.; Rocco, J.W.; Yom, S.S.; Haddad, R.I.; Saba, N.F. Head and neck cancer. Lancet 2021, 398, 2289–2299. [CrossRef]
Xing, D.T.; Khor, R.; Gan, H.; Wada, M.; Ermongkonchai, T.; Ng, S.P. Recent Research on Combination of Radiotherapy withTargeted Therapy or Immunotherapy in Head and Neck Squamous Cell Carcinoma: A Review for Radiation Oncologists. Cancers
2021, 13, 5716. [CrossRef]
Muzaffar, J.; Bari, S.; Kirtane, K.; Chung, C.H. Recent Advances and Future Directions in Clinical Management of Head and Neck
Squamous Cell Carcinoma. Cancers 2021, 13, 338. [CrossRef]
Johnson, D.E.; Burtness, B.; Leemans, C.R.; Lui, V.W.Y.; Bauman, J.E.; Grandis, J.R. Head and neck squamous cell carcinoma. Nat.
Rev. Dis. Prim. 2020, 6, 92. [CrossRef]
Ludwig, N.; Szczepanski, M.J.; Gluszko, A.; Szafarowski, T.; Azambuja, J.H.; Dolg, L.; Gellrich, N.C.; Kampmann, A.; Whiteside,
T.L.; Zimmerer, R.M. CD44(+) tumor cells promote early angiogenesis in head and neck squamous cell carcinoma. Cancer Lett.
2019, 467, 85–95. [CrossRef] [PubMed]
Ponta, H.; Sherman, L.; Herrlich, P.A. CD44: From adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 2003, 4,
33–45. [CrossRef] [PubMed]
Chen, C.; Zhao, S.; Karnad, A.; Freeman, J.W. The biology and role of CD44 in cancer progression: Therapeutic implications.
J. Hematol. Oncol. 2018, 11, 64. [CrossRef] [PubMed]
Slevin, M.; Krupinski, J.; Gaffney, J.; Matou, S.; West, D.; Delisser, H.; Savani, R.C.; Kumar, S. Hyaluronan-mediated angiogenesis
in vascular disease: Uncovering RHAMM and CD44 receptor signaling pathways. Matrix Biol. 2007, 26, 58–68. [CrossRef]
Valastyan, S.; Weinberg, R.A. Tumor metastasis: Molecular insights and evolving paradigms. Cell 2011, 147, 275–292. [CrossRef][PubMed]de Visser, K.E.; Joyce, J.A. The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer Cell
2023, 41, 374–403. [CrossRef]
Zöller, M. CD44, Hyaluronan, the Hematopoietic Stem Cell, and Leukemia-Initiating Cells. Front. Immunol. 2015, 6, 235.
[CrossRef] [PubMed]
Hassn Mesrati, M.; Syafruddin, S.E.; Mohtar, M.A.; Syahir, A. CD44: A Multifunctional Mediator of Cancer Progression.
Biomolecules 2021, 11, 1850. [CrossRef] [PubMed]
Zöller, M. CD44: Can a cancer-initiating cell profit from an abundantly expressed molecule? Nat. Rev. Cancer 2011, 11, 254–267.
[CrossRef] [PubMed]
Prince, M.E.; Sivanandan, R.; Kaczorowski, A.; Wolf, G.T.; Kaplan, M.J.; Dalerba, P.; Weissman, I.L.; Clarke, M.F.; Ailles, L.E.
Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc. Natl.
Acad. Sci. USA 2007, 104, 973–978. [CrossRef]
Yang, J.; Antin, P.; Berx, G.; Blanpain, C.; Brabletz, T.; Bronner, M.; Campbell, K.; Cano, A.; Casanova, J.; Christofori, G.; et al.
Guidelines and definitions for research on epithelial-mesenchymal transition. Nat. Rev. Mol. Cell Biol. 2020, 21, 341–352.
[CrossRef] [PubMed]
Davis, S.J.; Divi, V.; Owen, J.H.; Bradford, C.R.; Carey, T.E.; Papagerakis, S.; Prince, M.E. Metastatic potential of cancer stem cells
in head and neck squamous cell carcinoma. Arch. Otolaryngol. Head Neck Surg. 2010, 136, 1260–1266. [CrossRef] [PubMed]
Ishimoto, T.; Nagano, O.; Yae, T.; Tamada, M.; Motohara, T.; Oshima, H.; Oshima, M.; Ikeda, T.; Asaba, R.; Yagi, H.; et al. CD44
variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth.
Cancer Cell 2011, 19, 387–400. [CrossRef]
Kagami, T.; Yamade, M.; Suzuki, T.; Uotani, T.; Tani, S.; Hamaya, Y.; Iwaizumi, M.; Osawa, S.; Sugimoto, K.; Baba, S.; et al. Highexpression level of CD44v8-10 in cancer stem-like cells is associated with poor prognosis in esophageal squamous cell carcinoma
patients treated with chemoradiotherapy. Oncotarget 2018, 9, 34876–34888. [CrossRef]

Curr. Issues Mol. Biol. 2023, 45

19.

20.
21.
22.

23.
24.
25.

26.
27.
28.
29.
30.
31.

32.

33.

34.

35.
36.
37.
38.
39.

40.
41.

42.

5261

Hagiwara, M.; Kikuchi, E.; Tanaka, N.; Kosaka, T.; Mikami, S.; Saya, H.; Oya, M. Variant isoforms of CD44 involves acquisition ofchemoresistance to cisplatin and has potential as a novel indicator for identifying a cisplatin-resistant population in urothelial
cancer. BMC Cancer 2018, 18, 113. [CrossRef]
Yamada, S.; Itai, S.; Nakamura, T.; Yanaka, M.; Kaneko, M.K.; Kato, Y. Detection of high CD44 expression in oral cancers using thenovel monoclonal antibody, C(44)Mab-5. Biochem. Biophys. Rep. 2018, 14, 64–68. [CrossRef]
Goto, N.; Suzuki, H.; Tanaka, T.; Asano, T.; Kaneko, M.K.; Kato, Y. Development of a Novel Anti-CD44 Monoclonal Antibody for
Multiple Applications against Esophageal Squamous Cell Carcinomas. Int. J. Mol. Sci. 2022, 23, 5535. [CrossRef] [PubMed]
Takei, J.; Asano, T.; Suzuki, H.; Kaneko, M.K.; Kato, Y. Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C44Mab-46)
Using Alanine-Scanning Mutagenesis and Surface Plasmon Resonance. Monoclon. Antib. Immunodiagn. Immunother. 2021, 40,
219–226. [CrossRef] [PubMed]
Asano, T.; Kaneko, M.K.; Takei, J.; Tateyama, N.; Kato, Y. Epitope Mapping of the Anti-CD44 Monoclonal Antibody (C44Mab-46)
Using the REMAP Method. Monoclon. Antib. Immunodiagn. Immunother. 2021, 40, 156–161. [CrossRef]
Asano, T.; Kaneko, M.K.; Kato, Y. Development of a Novel Epitope Mapping System: RIEDL Insertion for Epitope Mapping
Method. Monoclon. Antib. Immunodiagn. Immunother. 2021, 40, 162–167. [CrossRef] [PubMed]
Takei, J.; Kaneko, M.K.; Ohishi, T.; Hosono, H.; Nakamura, T.; Yanaka, M.; Sano, M.; Asano, T.; Sayama, Y.; Kawada, M.; et al. A
defucosylated antiCD44 monoclonal antibody 5mG2af exerts antitumor effects in mouse xenograft models of oral squamous cell
carcinoma. Oncol. Rep. 2020, 44, 1949–1960. [CrossRef]
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 C(44)Mab-6 Was Established for Multiple Applications. Int. J. Mol. Sci. 2023, 24, 8411. [CrossRef]
Suzuki, H.; Tanaka, T.; Goto, N.; Kaneko, M.K.; Kato, Y. Development of a Novel Anti-CD44 Variant 4 Monoclonal Antibody
C44Mab-108 for Immunohistochemistry. Curr. Issues Mol. Biol. 2023, 45, 1875–1888. [CrossRef]
Kudo, Y.; Suzuki, H.; Tanaka, T.; Kaneko, M.K.; Kato, Y. Development of a Novel Anti-CD44 variant 5 Monoclonal Antibody
C44Mab-3 for Multiple Applications against Pancreatic Carcinomas. Antibodies 2023, 12, 31. [CrossRef]
Ejima, R.; Suzuki, H.; Tanaka, T.; Asano, T.; Kaneko, M.K.; Kato, Y. Development of a Novel Anti-CD44 Variant 6 Monoclonal
Antibody C(44)Mab-9 for Multiple Applications against Colorectal Carcinomas. Int. J. Mol. Sci. 2023, 24, 4007. [CrossRef]
Suzuki, H.; Ozawa, K.; Tanaka, T.; Kaneko, M.K.; Kato, Y. Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody,
C44Mab-34, for Multiple Applications against Oral Carcinomas. Biomedicines 2023, 11, 1099. [CrossRef]
Tawara, M.; Suzuki, H.; Goto, N.; Tanaka, T.; Kaneko, M.K.; Kato, Y. A Novel Anti-CD44 Variant 9 Monoclonal Antibody
C44Mab-1 was Developed for Immunohistochemical Analyses Against Colorectal Cancers. Curr. Issues Mol. Biol. 2023, 45,
3658–3673. [CrossRef] [PubMed]
Kato, Y.; Kaneko, M.K.; Kuno, A.; Uchiyama, N.; Amano, K.; Chiba, Y.; Hasegawa, Y.; Hirabayashi, J.; Narimatsu, H.; Mishima, K.;
et al. Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its plateletaggregation-stimulating domain. Biochem. Biophys. Res. Commun. 2006, 349, 1301–1307. [CrossRef] [PubMed]
Heider, K.H.; Sproll, M.; Susani, S.; Patzelt, E.; Beaumier, P.; Ostermann, E.; Ahorn, H.; Adolf, G.R. Characterization of ahigh-affinity monoclonal antibody specific for CD44v6 as candidate for immunotherapy of squamous cell carcinomas. Cancer
Immunol. Immunother. 1996, 43, 245–253. [CrossRef]
Heider, K.H.; Mulder, J.W.; Ostermann, E.; Susani, S.; Patzelt, E.; Pals, S.T.; Adolf, G.R. Splice variants of the cell surface
glycoprotein CD44 associated with metastatic tumour cells are expressed in normal tissues of humans and cynomolgus monkeys.
Eur. J. Cancer 1995, 31a, 2385–2391. [CrossRef] [PubMed]
Gansauge, F.; Gansauge, S.; Zobywalski, A.; Scharnweber, C.; Link, K.H.; Nussler, A.K.; Beger, H.G. Differential expression of
CD44 splice variants in human pancreatic adenocarcinoma and in normal pancreas. Cancer Res. 1995, 55, 5499–5503.
Beham-Schmid, C.; Heider, K.H.; Hoefler, G.; Zatloukal, K. Expression of CD44 splice variant v10 in Hodgkin's disease isassociated with aggressive behaviour and high risk of relapse. J. Pathol. 1998, 186, 383–389. [CrossRef]
Mishra, M.N.; Chandavarkar, V.; Sharma, R.; Bhargava, D. Structure, function and role of CD44 in neoplasia. J. Oral Maxillofac.
Pathol. 2019, 23, 267–272. [CrossRef]
Hirata, K.; Suzuki, H.; Imaeda, H.; Matsuzaki, J.; Tsugawa, H.; Nagano, O.; Asakura, K.; Saya, H.; Hibi, T. CD44 variant 9
expression in primary early gastric cancer as a predictive marker for recurrence. Br. J. Cancer 2013, 109, 379–386. [CrossRef]
Shitara, K.; Doi, T.; Nagano, O.; Imamura, C.K.; Ozeki, T.; Ishii, Y.; Tsuchihashi, K.; Takahashi, S.; Nakajima, T.E.; Hironaka, S.;et al. Dose-escalation study for the targeting of CD44v(+) cancer stem cells by sulfasalazine in patients with advanced gastriccancer (EPOC1205). Gastric Cancer 2017, 20, 341–349. [CrossRef]
Erb, U.; Megaptche, A.P.; Gu, X.; Büchler, M.W.; Zöller, M. CD44 standard and CD44v10 isoform expression on leukemia cells
distinctly influences niche embedding of hematopoietic stem cells. J. Hematol. Oncol. 2014, 7, 29. [CrossRef]
Holm, F.; Hellqvist, E.; Mason, C.N.; Ali, S.A.; Delos-Santos, N.; Barrett, C.L.; Chun, H.J.; Minden, M.D.; Moore, R.A.; Marra,
M.A.; et al. Reversion to an embryonic alternative splicing program enhances leukemia stem cell self-renewal. Proc. Natl. Acad.
Sci. USA 2015, 112, 15444–15449. [CrossRef] [PubMed]
Tang, L.; Huang, H.; Tang, Y.; Li, Q.; Wang, J.; Li, D.; Zhong, Z.; Zou, P.; You, Y.; Cao, Y.; et al. CD44v6 chimeric antigen receptor T
cell specificity towards AML with FLT3 or DNMT3A mutations. Clin. Transl. Med. 2022, 12, e1043. [CrossRef] [PubMed]

Curr. Issues Mol. Biol. 2023, 45

43.

44.

45.
46.
47.
48.

49.

50.

5262

Verel, I.; Heider, K.H.; Siegmund, M.; Ostermann, E.; Patzelt, E.; Sproll, M.; Snow, G.B.; Adolf, G.R.; van Dongen, G.A. Tumortargeting properties of monoclonal antibodies with different affinity for target antigen CD44V6 in nude mice bearing head-andneck cancer xenografts. Int. J. Cancer 2002, 99, 396–402. [CrossRef] [PubMed]
Li, G.; Suzuki, H.; Ohishi, T.; Asano, T.; Tanaka, T.; Yanaka, M.; Nakamura, T.; Yoshikawa, T.; Kawada, M.; Kaneko, M.K.; et al.
Antitumor activities of a defucosylated anti-EpCAM monoclonal antibody in colorectal carcinoma xenograft models. Int. J. Mol.
Med. 2023, 51, 18. [CrossRef]
Kaneko, M.K.; Ohishi, T.; Kawada, M.; Kato, Y. A cancer-specific anti-podocalyxin monoclonal antibody (60-mG(2a)-f) exerts
antitumor effects in mouse xenograft models of pancreatic carcinoma. Biochem. Biophys. Rep. 2020, 24, 100826. [CrossRef]
Kato, Y.; Kaneko, M.K. A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci. Rep. 2014,
4, 5924. [CrossRef] [PubMed]
Suzuki, H.; Kaneko, M.K.; Kato, Y. Roles of Podoplanin in Malignant Progression of Tumor. Cells 2022, 11, 575. [CrossRef]
Ishikawa, A.; Waseda, M.; Ishii, T.; Kaneko, M.K.; Kato, Y.; Kaneko, S. Improved anti-solid tumor response by humanizedanti-podoplanin chimeric antigen receptor transduced human cytotoxic T cells in an animal model. Genes Cells 2022, 27, 549–558.
[CrossRef]
Chalise, L.; Kato, A.; Ohno, M.; Maeda, S.; Yamamichi, A.; Kuramitsu, S.; Shiina, S.; Takahashi, H.; Ozone, S.; Yamaguchi, J.;et al. Efficacy of cancer-specific anti-podoplanin CAR-T cells and oncolytic herpes virus G47∆ combination therapy against
glioblastoma. Mol. Ther.-Oncolytics 2022, 26, 265–274. [CrossRef]
Shiina, S.; Ohno, M.; Ohka, F.; Kuramitsu, S.; Yamamichi, A.; Kato, A.; Motomura, K.; Tanahashi, K.; Yamamoto, T.; Watanabe,R.; et al. CAR T Cells Targeting Podoplanin Reduce Orthotopic Glioblastomas in Mouse Brains. Cancer Immunol. Res. 2016, 4,
259–268. [CrossRef]

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