biomedicines
ArticleDevelopment of a Novel Anti-CD44 Variant 7/8 Monoclonal
Antibody, C44Mab-34, for Multiple Applications against
Oral Carcinomas
Hiroyuki Suzuki *,† , Kazuki Ozawa † , Tomohiro Tanaka, Mika K. Kaneko

and Yukinari Kato *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.)† These authors contributed equally to this work.

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

Abstract: Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker
as it plays critical roles in tumor malignant progression. The splicing variants are overexpressedin many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotionof tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore,
each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for theestablishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a
CD44 variant (CD44v3–10) ectodomain and established various anti-CD44 monoclonal antibodies(mAbs). One of the established clones (C44 Mab-34; IgG1 , kappa) recognized a peptide that covers
both variant 7- and variant 8-encoded regions, indicating that C44 Mab-34 is a specific mAb for
CD44v7/8. Moreover, C44 Mab-34 reacted with CD44v3–10-overexpressed Chinese hamster ovary-K1(CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The
apparent KD of C44 Mab-34 for CHO/CD44v3–10 and HSC-3 was 1.4 × 10−9 and 3.2 × 10−9 M,
respectively. C44 Mab-34 could detect CD44v3–10 in Western blotting and stained the formalin-fixedparaffin-embedded OSCC in immunohistochemistry. These results indicate that C44 Mab-34 is usefulfor detecting CD44v7/8 in various applications and is expected to be useful in the application of
OSCC diagnosis and therapy.

Variant 7/8 Monoclonal Antibody,
C44 Mab-34, for Multiple Applications
against Oral Carcinomas.

Keywords: CD44; CD44 variant 7/8; monoclonal antibody; flow cytometry; immunohistochemistry

Biomedicines 2023, 11, 1099.
https://doi.org/10.3390/
biomedicines11041099
Academic Editor: Vui
King Vincent-Chong
Received: 27 February 2023
Revised: 29 March 2023
Accepted: 3 April 2023
Published: 5 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
Head and neck cancers mainly arise from the oral cavity, pharynx, larynx, and nasal
cavity. These tumors exhibit strong associations with smoking tobacco products, alcohol,
and infection with human papillomavirus (HPV) types 16 and 18 [1]. The estimated numberof new cases in the oral cavity and pharynx in the United States increased from 35,310 in2008 to 54,540 in 2023 due to rising HPV-positive cases [2–4]. Mortality rates continue toincrease for the oral cavity cancers associated with HPV infection (cancers of the tongue,
tonsil, and oropharynx) by about 2% per year in men and 1% per year in women [2].
Although many different histologies exist in head and neck cancers, head and necksquamous cell carcinoma (HNSCC) is the common type. The treatment options for HNSCC
include surgery, chemo-radiation, molecular targeted therapy, immunotherapy, or a combination of these modalities [5]. Despite the development in cancer treatment, metastasis anddrug resistance remain the main causes of death [6]. Although survival can be improved,
the impairment due to surgery and the toxicities of treatments deteriorate the patient’s
quality of life. Thus, the 5-year survival rate remains stagnant at approximately 50% [1].
Cancer stem cells (CSCs) play critical roles in tumor development through their important properties, including self-renewal, resistance to therapy, and tumor metastasis [7–9].

Biomedicines 2023, 11, 1099. https://doi.org/10.3390/biomedicines11041099

https://www.mdpi.com/journal/biomedicinesBiomedicines 2023, 11, 1099

2 of 15

Studies have reported the importance of CSCs in HNSCC development [10] and regulation
by both intrinsic and extrinsic mechanisms in the tumor microenvironment [11]. Several
cell surface receptors and intracellular proteins have been reported as applicable CSC
markers in HNSCC [12,13]. Among them, cluster of differentiation 44 (CD44) is one of theimportant CSC markers in solid tumors, and it was first applied to study HNSCC-derived
CSCs [14]. Notably, CD44-high CSCs from HNSCC tumors exhibited the properties of
epithelial to mesenchymal transition, including elevated migration, invasiveness, and stemness [15]. Furthermore, CD44-high cells could form lung metastases in immunodeficient
mice, in contrast to CD44-low cells, which failed to exhibit a similar metastatic proliferationof cancer cells [16]. Therefore, specific monoclonal antibodies (mAbs) against CD44 arerequired for the isolation of CD44-high CSCs and the analysis of their properties in detail.
CD44 is a multifunctional transmembrane protein that binds to the extracellular matrix,
including hyaluronic acid (HA) [17]. Human CD44 has 19 exons, 10 of which are constant orpresent in all variants and make up the standard form of CD44 (CD44s). The CD44 variants(CD44v) are produced by alternative splicing and consist of the 10 constant exons in any
combination with the remaining nine variant exons [18]. The CD44 isoforms have both
overlapping and unique roles. Both CD44s and CD44v (pan-CD44) possess HA-binding
motifs that promote interaction with the microenvironment and facilitate the activation ofvarious signaling pathways [19].
Overexpression of CD44v has been observed in many types of carcinomas and is considered
a promising target for tumor diagnosis and therapy [20,21]. There is growing evidence thatCD44v plays important roles in the promotion of tumor metastasis, the acquisition of CSC
properties [22], and resistance to chemotherapy and radiotherapy [23,24]. Several variant exonencoded regions have been reported to promote tumorigenesis through their interacting
proteins. The v3-encoded region is modified by heparan sulfate, which promotes therecruitment of heparin-binding growth factors such as fibroblast growth factors. Thus, thev3-encoded region functions as a co-receptor of receptor tyrosine kinases [25]. Furthermore,
the v6-encoded region has been reported to be essential for the activation of c-MET throughthe formation of ternary complexes with HGF [26]. Moreover, the v8–10-encoded region
mediates oxidative stress resistance through the regulation of intracellular redox states. [27].
Therefore, CD44v-specific mAbs are required not only for the understanding of each variant
function but also for CD44v-targeting tumor diagnosis and therapy. However, the function
and distribution of the variant-encoded region in tumors have not been fully understood.
Our group has developed the Cell-Based Immunization and Screening (CBIS) method
and established a novel anti-pan-CD44 mAb, C44 Mab-5 (IgG1 , kappa) [28]. We also established another anti-pan-CD44 mAb, C44 Mab-46 (IgG1 , kappa) [29], using the immunizationof CD44v3–10 ectodomain (CD44ec). We determined the epitopes of C44 Mab-5 and C44 Mab46 in the standard exons (1 to 5)-encoding sequences [30–32]. We further showed that both
C44 Mab-5 and C44 Mab-46 are available for flow cytometry, Western blot, and immunohistochemistry in oral SCC (OSCC) [28] and esophageal SCC [29]. Furthermore, we have also
investigated the antitumor effects using recombinant C44 Mab-5 in mouse xenograft modelsof oral OSCC [33]. We converted the mouse IgG1 subclass antibody (C44 Mab-5) into an
IgG2a subclass antibody (5-mG2a ) and further produced a defucosylated version (5-mG2a f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. The 5-mG2a -f showed moderate
in vitro ADCC and CDC activities against HSC-2 and SAS OSCC cell lines. Furthermore,
the 5-mG2a -f significantly suppressed the xenograft growth of HSC-2 and SAS compared tocontrol mouse IgG [33]. Here, we have developed a novel anti-CD44v7/8 mAb, C44 Mab-34(IgG1 , kappa), and examined its applications to flow cytometry, Western blotting, andimmunohistochemical analyses.
2. Materials and Methods
2.1. Cell Lines
Chinese hamster ovary (CHO)-K1, a human glioblastoma cell line (LN229), and mouse
multiple myeloma P3X63Ag8U.1 (P3U1) cell lines were obtained from the American Type

Biomedicines 2023, 11, 1099

3 of 15

Culture Collection (ATCC, Manassas, VA, USA). The human OSCC cell line, HSC-3, wasobtained from the Japanese Collection of Research Bioresources (Osaka, Japan). CHOK1 and P3U1 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-inactivatedfetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA).
LN229 and HSC-3 were cultured in Dulbecco’s modified Eagle medium (DMEM)(Nacalai Tesque, Inc.), supplemented with 10% (v/v) FBS, 100 U/mL of penicillin (Nacalai
Tesque, Inc.), 100 µg/mL streptomycin (Nacalai Tesque, Inc.), and 0.25 µg/mL amphotericin
B (Nacalai Tesque, Inc.). LN229/CD44ec was cultured in the presence of 0.5 mg/mL of
G418 (Nacalai Tesque, Inc.).
All the cells were grown in a humidified incubator at 37 ◦ C with 5% CO2 .
2.2. Plasmid Construction and Establishment of Stable Transfectants
Human CD44v3–10 open reading frame (ORF) was obtained from the RIKEN BRC
through the National Bio-Resource Project of the MEXT, Japan. CD44s cDNA was amplified
using a HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany) using LN229
cDNA as a template. The CD44s and CD44v3–10 ORFs were subcloned into a pCAGBle-ssPA16 vector possessing signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV) [28,34–37], which is detected by NZ-1, which was originally developed as ananti-human podoplanin mAb [38–53].
CHO/CD44s and CHO/CD44v3–10 were established by transfecting the plasmids into
CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc.). CD44ecsecreting LN229 (LN229/CD44ec) was established by transfecting pCAG-Neo/PA-CD44ecRAP-MAP into LN229 cells using the Neon transfection system. The amino acid sequencesof the tag system in this study were as follows: PA tag [43,47,51], 12 amino acids (GVAMPGAEDDVV); RAP tag [54,55], 12 amino acids (DMVNPGLEDRIE); and MAP tag [56,57], 12
amino acids (GDGMVPPGIEDK).
2.3. Purification of CD44ec
The purification of CD44ec from the culture supernatant of LN229/CD44ec was performed
using an anti-RAP tag mAb (clone PMab-2) and a RAP peptide (GDDMVNPGLEDRIE) [54,55].
The culture supernatant (5 L) was passed through a 2 mL bed volume of PMab-2-sepharose,and the process was repeated three times. After washing the beads with 100 mL ofphosphate-buffered saline (PBS, Nacalai Tesque, Inc.), CD44ec was eluted with 0.1 mg/mLof a RAP peptide in a step-wise manner (2 mL × 10). The purity of CD44ec was determinedby Coomassie Brilliant Blue (CBB) staining using the Bio-Safe CBB G-250 Stain (Bio-Rad
Laboratories, Inc., Berkeley, CA, USA) (Supplemental Figure S1A).
2.4. Hybridomas
Female BALB/c mouse was purchased from CLEA Japan (Tokyo, Japan). The Animal
Care and Use Committee of Tohoku University approved the animal experiments (permit number: 2019NiA-001). The immunization of CD44ec was performed as described
previously [29].
The splenic cells were fused with P3U1 cells using polyethylene glycol 1500 (PEG1500;
Roche Diagnostics, Indianapolis, IN, USA). The culture supernatants of hybridomas werescreened using an enzyme-linked immunosorbent assay (ELISA) against CD44ec. The
supernatants were further screened using CHO/CD44v3–10 and parental CHO-K1 cells by
flow cytometry using SA3800 Cell Analyzers (Sony Corp. Tokyo, Japan).
C44 Mab-34 was purified from the cultured supernatants of C44 Mab-34-producing
hybridomas using Ab-Capcher ExTra (ProteNova Co., Ltd., Kagawa, Japan). The purity of
C44 Mab-34 was determined by CBB staining (Supplemental Figure S1B).

Biomedicines 2023, 11, 1099

4 of 15

2.5. ELISA
Fifty-eight synthesized peptides, which cover the CD44v3–10 extracellular domain [30],were synthesized by Sigma-Aldrich Corp (St. Louis, MO, USA). The peptides (1 µg/mL)or CD44ec were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher
Scientific Inc) for 30 min at 37 ◦ C. The immunoplate washing was performed with 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, C44 Mab-34 (10 µg/mL) was added toeach well. Then, the wells were further incubated with anti-mouse immunoglobulins
peroxidase-conjugate (1:2000 diluted; 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.) was used to measure the opticaldensity at 655 nm.
2.6. Flow Cytometry
CHO/CD44v3–10, CHO-K1, and HSC-3 were harvested using 0.25% trypsin and
1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai Tesque, Inc.). The cells were treatedwith C44 Mab-34, C44 Mab-46, or blocking buffer (control) (0.1% BSA in PBS) for 30 min at
4 ◦ C. Then, the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488(1:2000; Cell Signaling Technology, Inc, Danvers, MA, USA) for 30 min at 4 ◦ C. The SA3800
Cell Analyzer and SA3800 software ver. 2.05 (Sony Corporation) were used for fluorescencedata collection and analysis, respectively.
2.7. Dissociation Constant (KD ) Determination by Flow Cytometry
Serially diluted C44 Mab-34 was treated with CHO/CD44v3–10 and HSC-3 cells. Then,the cells were treated with anti-mouse IgG conjugated with Alexa Fluor 488 (1:200). BD
FACSLyric and BD FACSuite software version 1.3 (BD Biosciences) were used for fluorescence data collection and analysis, respectively. The GeoMean of each histogram, including
primary mAb (C44 Mab-34) + secondary Ab (Alexa Fluor 488-conjugated anti-mouse IgG)
and only secondary Ab (for background), was determined. We further withdrew the background from each data and determined the dissociation constant (KD ) by GraphPad Prism
8 (the fitting binding isotherms to built-in one-site binding models; GraphPad Software,Inc., La Jolla, CA, USA).
2.8. Western Blot Analysis
The total cell lysates (10 µg of protein) were denatured by sodium dodecyl sulfate (SDS)
sample buffer (Nacalai Tesque, Inc.) in the presence of 2-mercaptoethanol and separated on
7.5% or 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA,
Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.) in PBST,
the membranes were incubated with 10 µg/mL of C44 Mab-34, 10 µg/mL of C44 Mab-46,
1 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-34, C44 Mab-46, and anti-β-actin. 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
Formalin-fixed paraffin-embedded (FFPE) sections of the OSCC tissue array (OR601c)
were purchased from US Biomax Inc. (Rockville, MD, USA). The OSCC tissue array was
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-34 (10 µg/mL) and C44 Mab-46 (1 µg/mL) for 1 h at room
temperature and then treated with the EnVision+ Kit for mouse (Agilent Technologies Inc.)for 30 min. The chromogenic reaction was conducted using 3,30 -diaminobenzidine tetrahy-

Biomedicines 2023, 11, 1099

5 of 15drochloride (DAB; Agilent Technologies Inc.). The counterstaining was performed using
hematoxylin (FUJIFILM Wako Pure Chemical Corporation). To examine the sections andobtain images, we used a Leica DMD108 (Leica Microsystems GmbH, Wetzlar, Germany).
3. Results
3.1. Development of an Anti-CD44v7/8 mAb, C44 Mab-34
In this study, we purified human CD44ec as an immunogen (Figure 1). One mouse
was immunized with CD44ec, and hybridomas were seeded into 96-well plates. The
supernatants were first screened by the reactivity to CD44ec by ELISA. Subsequently, thesupernatants, which were positive for CHO/CD44v3–10 cells and negative for CHO-K1
cells, were further selected using flow cytometry. Finally, anti-CD44 mAb-producing clones
were established by limiting dilution. Among them, C44 Mab-34 (IgG1 , kappa) was shown
to recognize CD44p421–440 (GHQAGRRMDMDSSHSTTLQP), which corresponds to thevariant 7- and variant 8-encoded sequence (Supplementary Table S1). In contrast, C44 Mab34 never recognized other CD44v3–10 extracellular regions. These results indicate that
C44 Mab-34 specifically recognizes the border region between variants 7 and 8.
A. Immunization of purified CD44ec (CD44v3-10 ectodomain)
CD44s
CD44v3-10
Ectodomaln (CD44ec)

Purified
CD44v3-10
Ectodomaln_.--_�
4)-

B. Production of hybridomas

Myelomas!Spleen

Fusion(to 96 well plates):m

C. Screening of supernatantsI· ·· _.... '"" I I

2 nd screening: Flow cytometry@@ >r @� @

-(9@

l� �

-uJD
.

Splenocytes(B cells)��

D. Cloning of Hybridomas

Positive well

@� �

00 �

Establishment of anti-CD44 mAb-producing clones,,,,,,,,,,@,,,,,,,,,,@

Determination of epitope(ELISA)

Figure 1. A schematic illustration of anti-human CD44 mAb production. (A) Purified CD44v3–10
ectodomain was intraperitoneally injected into BALB/c mouse. (B) Hybridomas were produced
by fusion of the splenocytes and P3U1 cells. (C) The screening was performed by enzyme-linked
immunosorbent assay (ELISA) and flow cytometry using parental CHO-K1 and CHO/CD44v3–
10cells. (D) After cloning and additional screening, a clone C44 Mab-34 (IgG1 , kappa) was established.
Furthermore, the binding epitopes were determined by ELISA using peptides, which cover the
extracellular domain of CD44v3–10.

Biomedicines 2023, 11, 1099

6 of 15

3.2. Flow Cytometric Analysis of C44 Mab-34 to CD44-Expressing Cells
We next investigated the reactivity of C44Mab-34 against CHO/CD44v3–10 and CHO/
CD44s cells by flow cytometry. C44Mab-34 recognized CHO/CD44v3–10cells in a dosedependent manner (Figure 2A) but not CHO/CD44s (Figure 2B) or CHO-K1 (Figure 2C)
cells. The CHO/CD44 cells were recognized by an anti-pan-CD44 mAb, C44 Mab-46 [29](Supplemental Figure S2). C44 Mab-34 also recognized the OSCC cell line HSC-3 (Figure 2D)
in a dose-dependent manner.

Figure 2. Flow cytometry using C44 Mab-34 against CD44-expressing cells. CHO/CD44v3–10 (A),CHO/CD44s (B), CHO-K1 (C), and HSC-3 (D) cells were treated with C44 Mab-34, followed bytreatment with anti-mouse IgG conjugated with Alexa Fluor 488 (red line). The black line represents
the negative control (blocking buffer).

We next determined the binding affinity of C44 Mab-34 with CHO/CD44v3–10 and
HSC-3 using flow cytometry. The KD of CHO/CD44v3–10 and HSC-3 was 1.4 × 10−9 and3.2 × 10−9 M, respectively. These results indicate that C44 Mab-34 possesses a moderateaffinity for CD44v3–10-expressing cells (Figure 3).

Biomedicines 2023, 11, 1099

7 of 15

Figure 3. The binding affinity of C44 Mab-34 to CD44-expressing cells. CHO/CD44v3–10 (A) andHSC-3 (B) cells were suspended in serially diluted C44 Mab-34 at indicated concentrations. Then, cells
were treated with anti-mouse IgG conjugated with Alexa Fluor 488. Fluorescence data were collected,
followed by the calculation of the dissociation constant (KD ) by GraphPad PRISM 8.

3.3. Western Blot Analysis
We next performed Western blot analysis to assess the sensitivity of C44 Mab-34. As
shown in Figure 4A, an anti-pan-CD44 mAb, C44 Mab-46, recognized the lysates from both
CHO/CD44s (75~100 kDa) and CHO/CD44v3–10 (>180 kDa). C44 Mab-34 mainly detected
CD44v3–10 as more than 180-kDa bands. However, C44 Mab-34 did not detect any bands
from the lysates of CHO-K1 and CHO/CD44s cells (Figure 4B). These results indicate that
C44 Mab-34 can detect CD44v3–10.

Figure 4. Western blot analysis using C44 Mab-34. The cell lysates from CHO-K1, CHO/CD44s,
and CHO/CD44v3–10 (10 µg) were electrophoresed and transferred onto polyvinylidene fluoride(PVDF) membranes. The membranes were incubated with 10 µg/mL of C44 Mab-46 (A), 10 µg/mL
of C44 Mab-34 (B), and 1 µg/mL of an anti-β-actin mAb (C). Then, the membranes were incubatedwith anti-mouse immunoglobulins conjugated with peroxidase for C44 Mab-46, C44 Mab-34, and ananti-β-actin mAb. The red arrows indicate the CD44s (75~100 kDa). The black arrows indicate theCD44v3–10.

Biomedicines 2023, 11, 1099

Figure 4. Western blot analysis using C44Mab-34. The cell lysates from CHO-K1, CHO/CD44s, andCHO/CD44v3–10 (10 µg) were electrophoresed and transferred onto polyvinylidene fluoride(PVDF) membranes. The membranes were incubated with 10 µg/mL of C44Mab-46 (A), 10 µg/mL of
C44Mab-34 (B), and 1 µg/mL of an anti-β-actin mAb (C). Then, the membranes were incubated with
anti-mouse immunoglobulins conjugated with peroxidase for C44Mab-46, C44Mab-34, and an anti-βactin mAb. The red arrows indicate the CD44s (75~100 kDa). The black arrows indicate the CD44v3–
10.

8 of 15

3.4. Immunohistochemical Analysis against Tumor Tissues Using C44 Mab-34

3.4. Immunohistochemical
againstTumor Tissues
C44Mab-34
We next examinedAnalysis
whether
C44 Mab-34
could Using
be usedfor immunohistochemical analy-

ses using
FFPE
sections.
We usedsequential
sections
an OSCC tissue microarray.
In acould beused forof
immunohistochemical
analWe next
examined
whether
C44Mab-34
yses
using FFPE sections.
Wesection,used sequential
sectionsof an OSCC
tissue microarray.
In aobserved
well-differentiated
OSCC
the clearmembranous
staining
in OSCC waswell-differentiatedsection,(Figure
the clear5A,B).
membranous
staining
in OSCC
by C44 Mab-34 andOSCC
C44 Mab-46
In an OSCC
sectionwithwastheobserved
stromal-invaded
by
C44Mab-34C
andC
44Mab-46(Figure5A,B).
Inan
OSCC
sectionwiththestromal-invaded
phenotype,Mab-34strongly
stainedstromal-invaded
OSCC
and
could
clearly distin44
phenotype,C44cells
Mab-34strongly
stainedstromal-invaded
and could
distinguish tumor
fromstromaltissues(Figure 5C).OSCC
In contrast,
C44clearly
Mab-46stained bothstained
both
guish
tumor
cellscells
fromandstromaltissues (Figure
5C).
In contrast,
C44Mab-46
invadedtumor
surrounding
stromacells(Figure 5D).
In Figure
5E,F,
C44 Mab-34
invaded tumor cells and surrounding stroma cells (Figure 5D). In Figure 5E,F, C44Mab-34
and C Mab-46 never stained tumor tissue, but clear stromal staining was observed by
Mab-46 never stained tumor tissue, but clear stromal staining was observed by
and C4444
C Mab-46 (Figure 5F). We have summarized the data of the immunohistochemical analysis
C4444Mab-46 (Figure 5F). We have summarized the data of the immunohistochemical analof CD44
expression
in in
tumor
Mab-34 stained 42 out of 49 (86%) cases
ysisof CD44expression
tumorcells
cellsininTable
Table 1;
1; C
C44
44Mab-34 stained 42 out of 49 (86%)
of
OSCC.
These
results
indicatethatC
Mab-34
usefulthe immunohistochemical
44
cases of OSCC. These results indicate that C44Mab-34 is isusefulfor forthe immunohistochemanalysis
of
FFPE
tumor
sections.
ical analysis of FFPE tumor sections.

Figure 5. Immunohistochemical analysis using C44Mab-34 and C44Mab-46 against OSCC tissues. After
antigen retrieval, serial sections of OSCC tissue array (Catalog number: OR601c) were incubated with
10 µg/mL of C44Mab-34 (A, C, E) or 1 µg/mL of C44Mab-46 (B, D, F), followed by treatment with theEnvision+ kit. The chromogenic reaction was conducted using 3,30 -diaminobenzidine tetrahydrochloride(DAB). The counterstaining was performed using hematoxylin. Scale bar = 100 µm.

Biomedicines 2023, 11, 1099

9 of 15

Table 1. Immunohistochemical analysis using C44 Mab-34 and C44 Mab-46 against OSCC.
No

Age

Sex

Organ/Anatomic Site

Pathology Diagnosis

TNM

C44 Mab-34

1

78

M

TongueSquamous cell carcinoma of tongue

T2N0M0+C44 Mab-46
+

2

40

M

TongueSquamous cell carcinoma of tongue

T2N0M0

+

++

3

35

F

TongueSquamous cell carcinoma of tongue

T2N0M0

+++

++

4

61

M

TongueSquamous cell carcinoma of tongue

T2N0M0

+++

+++

5

41

F

TongueSquamous cell carcinoma of tongue

T2N0M0

+

+

6

64

M

TongueSquamous cell carcinoma of right side of tongue

T2N2M0

+

++

7

76

M

TongueSquamous cell carcinoma of tongue

T1N0M0

+

++

8

50

F

TongueSquamous cell carcinoma of tongue

T2N0M0

+++

++

9

44

M

TongueSquamous cell carcinoma of tongue

T2N1M0

+++

++

10

53

F

TongueSquamous cell carcinoma of tongue

T1N0M0

+

++

11

46

F

TongueSquamous cell carcinoma of tongue

T2N0M0

-

+

12

50

M

TongueSquamous cell carcinoma of root of tongue

T3N1M0

+++

+

13

36

F

TongueSquamous cell carcinoma of tongue

T1N0M0

+++

+++

14

63

F

TongueSquamous cell carcinoma of tongue

T1N0M0

+

+

15

46

M

TongueSquamous cell carcinoma of tongue

T2N0M0

++

-

16

58

M

TongueSquamous cell carcinoma of tongue

T2N0M0

+

+

17

64

M

Lip

Squamous cell carcinoma of lower lip

T1N0M0

+++

+++

18

57

M

Lip

Squamous cell carcinoma of lower lip

T2N0M0

++

+++

19

61

M

Lip

Squamous cell carcinoma of lower lip

T1N0M0

+++

++

20

60

M

Gum

Squamous cell carcinoma of gum

T3N0M0

+

+

21

60

M

Gum

Squamous cell carcinoma of gum

T1N0M0

+++

+++

22

69

M

Gum

Squamous cell carcinoma of upper gum

T3N0M0

+

++

23

53

M

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T2N0M0

++

+

24

55

M

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T1N0M0

++

+

25

58

M

TongueSquamous cell carcinoma of base of tongue

T1N0M0

+

++

26

63

M

Oral cavity

Squamous cell carcinoma

T1N0M0

++

++

27

48

F

TongueSquamous cell carcinoma of tongue

T1N0M0

++

+

28

80

M

Lip

Squamous cell carcinoma of lower lip

T1N0M0

+++

+++

29

77

M

TongueSquamous cell carcinoma of base of tongue

T2N0M0

+++

++

30

59

M

TongueSquamous cell carcinoma of tongue

T2N0M0

++

-

31

77

F

TongueSquamous cell carcinoma of tongue

T1N0M0

+

++

32

56

M

TongueSquamous cell carcinoma of root of tongue

T2N1M0

+

+

33

60

M

TongueSquamous cell carcinoma of tongue

T2N1M0

+

++

34

62

M

TongueSquamous cell carcinoma of tongue

T2N0M0

+++

++

35

67

F

TongueSquamous cell carcinoma of tongue

T2N0M0

+++

++

36

47

F

TongueSquamous cell carcinoma of tongue

T2N0M0

+++

+++

37

37

M

TongueSquamous cell carcinoma of tongue

T2N1M0

-

-

38

55

F

TongueSquamous cell carcinoma of tongue

T2N0M0

++

++

39

56

F

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T2N0M0

++

+

40

49

M

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T1N0M0

-

-

41

45

M

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T2N0M0

-

-

42

42

M

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T3N0M0

++

++

43

44

M

Jaw

Squamous cell carcinoma of right drop jaw

T1N0M0

+

+++

44

40

F

TongueSquamous cell carcinoma of base of tongue

T2N0M0

-

++

45

49

M

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T1N0M0

+++

+++

46

56

F

TongueSquamous cell carcinoma of base of tongue

T2N0M0

-

-

47

42

M

Bucca cavioris

Squamous cell carcinoma of bucca cavioris

T3N0M0

+++

+++

48

87

F

Face

Squamous cell carcinoma of left side of face

T2N0M0

+

+

49

50

M

Gum

Squamous cell carcinoma of gum

T2N0M0--Biomedicines 2023, 11, 1099

10 of 15

4. Discussion
Head and neck cancer is the seventh most common type of cancer worldwide, and it
exhibits aggressive development in clinical settings [58]. Head and neck cancer remains acomplex disease with a profound impact on patients and their quality of life after surgicalablation and therapies. Knowledge of the disease has been accumulated with regard totumor biology and prevention, and therapeutic options have been simultaneously developed [58]. HNSCC is the most common type of head and neck cancer, and it has been
revealed as the second-highest CD44-expressing cancer type in the Pan-Cancer Atlas [59].
CD44 overexpression is associated with poor prognosis and resistance to therapy [60–62].
Reduced CD44 expression leads to the growth suppression of tumor cells [17,63]. Therefore,
CD44 is considered an important target for mAb therapies. In this study, we developed anovel anti-CD44v7/8 mAb, C44 Mab-34, and showed multiple applications to flow cytometry (Figures 2 and 3), Western blotting (Figure 4), and the immunohistochemistry of OSCC
(Figure 5).
An anti-CD44v7/8 mAb (clone VFF-17) was previously developed, and it has been
mainly used for the immunohistochemistry of normal tissue and tumors [64,65]. The
epitope of VFF-17 mAb was determined by binding studies with fusion proteins encoding v7 or v8 exons, either alone or in combination [66]. However, a detailed amino acidsequence of the epitope has not been determined. As shown in Supplementary Table
S1, C44 Mab-34 recognizes CD44p421–440 [GHQAGRRMD (included in v7) + MDSSHSTTLQP (included in v8)]. In contrast, C44 Mab-34 has never recognized CD44p411–430(FNPISHPMGRGHQAGRRMD (included in v7) + M (included in v8)) or CD44p431–450(DSSHSTTLQPTANPNTGLVE (included in v8)). These results suggest that C44 Mab-34 recognizes the border sequence between v7 and v8. In addition, CD44 is known to be heavily
glycosylated [67], and the glycosylation pattern is thought to depend on the host cells. Since
the epitope of C44 Mab-34 contains predicted and confirmed O-glycan sites [67], further
studies are needed on whether the recognition of C44 Mab-34 is affected by glycosylation.
Among many CD44v types, CD44v8–10, CD44v6–10, CD44v4–10, and CD44v3–10
were mainly detected in SCC cells by semi-quantitative RT-PCR analysis (manuscript submitted). Since C44 Mab-34 recognizes the border sequence between v7 and v8 (Supplemental
Table S1), C44 Mab-34 can distinguish CD44v8–10 and the longer CD44v (v6-10, v4-10, andv3–10). Furthermore, the inclusion of these variants (from v8-10 to the longer variants) is
promoted by EGF signaling [68,69]. If the expression of CD44v8-10 and the longer variants
are differently regulated in normal and tumor cells, C44 Mab-34 could contribute to tumor
diagnosis and therapy. We are now investigating the C44 Mab-34 reactivity against othertumor tissues together with the epitope analyses.
∆Np63 is known as a marker of basal cells of stratified epithelium and SCC [70].
∆Np63 mediates HA metabolism and signaling [71]. Specifically, ∆Np63 directly binds
to the p63-binding sequence on the promoter/enhancer region of the CD44 gene [71]. In
whole-exome sequencing data analysis from 74 HNSCC–normal pairs, the ∆Np63-encoded
gene, TP63, was identified as a significantly mutated gene that results in the activation of the∆Np63 pathway [72]. The relationship between ∆Np63 activation and CD44 transcription
should be investigated in future studies. Furthermore, the mechanism of the variant 7/8
inclusion by alternative splicing remains to be determined.
An anti-pan CD44 mAb, RG7356, demonstrated some efficacy and an acceptablesafety profile in the phase I study. However, the study was terminated due to no evidenceof a clinical and dose–response relationship with RG7356 [73]. Furthermore, a variant
6-specific CD44 mAb-drug conjugate (bivatuzumab–mertansine) was also evaluated inclinical trials. However, lethal epidermal necrolysis halted further development. The
efficient accumulation of mertansine in the skin was most likely responsible for the hightoxicity [74,75]. Therefore, the therapeutic effects of CD44 mAbs have been disappointing
until now.
Near-infrared photoimmunotherapy (NIR-PIT) is a novel tumor therapy that uses atargeted mAb–photoabsorber conjugate (APC) [76]. The mAb binds to the targeted cellBiomedicines 2023, 11, 1099

11 of 15

surface antigen, and the photoactivatable dye IRDye700DX (IR700) induces the disruption of the cellular membrane after NIR-light exposure. Since NIR-light exposure can be
performed at tumor sites locally, APC can exert antitumor effect selectivity while minimizing damage to the surrounding tissue [77,78]. Preclinical studies indicate that NIR-PIT
induces tumor necrosis and immunogenic cell death through the induction of innate andadaptive immunity [79]. A first-in-human phase I and II trial of NIR-PIT with RM-1929 (ananti-epidermal growth factor receptor mAb, cetuximab–IR700 conjugate) in patients withinoperable HNSCC was conducted and exhibited the efficacy [80].
A preclinical study of anti-CD44 mAb-based NIR-PIT has been reported [81]. The
study used an anti-mouse/human pan-CD44 mAb, IM7, conjugated with IR700 (CD44–
IR700) in a syngeneic mouse model of OSCC. The CD44–IR700 can induce significant
antitumor responses after a single injection of the conjugate and NIR-light exposure inCD44-expressing OSCC tumors [81]. As shown in Figure 5D,F, a pan-CD44 mAb, C44 Mab46, recognized not only tumor cells but also stromal tissue and probably immune cells,
which are important for antitumor immunity. Therefore, CD44v is a promising tumor
antigen for NIR-PIT, which could be a new modality for OSCC with locoregional recurrence.
We have previously produced recombinant antibodies that are converted to a mouse
IgG2a subclass from mouse IgG1 . Furthermore, we produced defucosylated IgG2a mAbs
using fucosyltransferase 8-deficient CHO-K1 cells to potentiate antibody-dependent cellularcytotoxicity. The defucosylated mAbs showed potent antitumor activity in mouse xenograftmodels [33,82–88]. Therefore, a class-switched and defucosylated version of C44 Mab-34 isrequired to evaluate the antitumor activity in vivo.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biomedicines11041099/s1, Table S1, The determination of thebinding epitope of C44 Mab-34 by ELISA. Figure S1, CBB staining of CD44ec and C34 Mab-34. Figure S2,
Recognition of CHO/CD44s and CHO/CD44v3–10 by C44 Mab-46 by flow cytometry.
Author Contributions: K.O., H.S. and T.T. performed the experiments. M.K.K. and Y.K. designed theexperiments. H.S. and K.O. analyzed the data. K.O., H.S. and Y.K. wrote the manuscript. All authors
have read and agreed to 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
Care and Use Committee of Tohoku University (permit number: 2019NiA-001, approved on 1 April2019) 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.

References
1.
2.
3.

4.

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]
Siegel, R.L.; Miller, K.D.; Wagle, N.S.; Jemal, A. Cancer statistics, 2023. CA Cancer J. Clin. 2023, 73, 17–48. [CrossRef] [PubMed]
Kang, J.J.; Yu, Y.; Chen, L.; Zakeri, K.; Gelblum, D.Y.; McBride, S.M.; Riaz, N.; Tsai, C.J.; Kriplani, A.; Hung, T.K.W.; et al. Consensuses, controversies, and future directions in treatment deintensification for human papillomavirus-associated oropharyngeal
cancer. CA Cancer J. Clin. 2023, 73, 164–197. [CrossRef]
Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Murray, T.; Thun, M.J. Cancer statistics, 2008. CA Cancer J. Clin. 2008, 58, 71–96.
[CrossRef] [PubMed]

Biomedicines 2023, 11, 1099

5.

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

15.

16.
17.
18.
19.

20.
21.
22.
23.
24.
25.

26.
27.

28.
29.
30.

31.
32.

12 of 15

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] [PubMed]
Muzaffar, J.; Bari, S.; Kirtane, K.; Chung, C.H. Recent advances and future directions in clinical management of head and necksquamous cell carcinoma. Cancers 2021, 13, 338. [CrossRef]
Maitland, N.J.; Collins, A.T. Cancer stem cells—A therapeutic target? Curr. Opin. Mol. Ther. 2010, 12, 662–673.
Prince, M.E.; Ailles, L.E. Cancer stem cells in head and neck squamous cell cancer. J. Clin. Oncol. 2008, 26, 2871–2875. [CrossRef]
Ailles, L.E.; Weissman, I.L. Cancer stem cells in solid tumors. Curr. Opin. Biotechnol. 2007, 18, 460–466. [CrossRef]
Keysar, S.B.; Le, P.N.; Miller, B.; Jackson, B.C.; Eagles, J.R.; Nieto, C.; Kim, J.; Tang, B.; Glogowska, M.J.; Morton, J.J.; et al.
Regulation of head and neck squamous cancer stem cells by PI3K and SOX2. J. Natl. Cancer Inst. 2017, 109, djw189. [CrossRef]de Miranda, M.C.; Melo, M.I.A.; Cunha, P.D.S.; Gentilini, J.J.; Faria, J.; Rodrigues, M.A.; Gomes, D.A. Roles of mesenchymal
stromal cells in the head and neck cancer microenvironment. Biomed. Pharmacother. 2021, 144, 112269. [CrossRef]
Yu, S.S.; Cirillo, N. The molecular markers of cancer stem cells in head and neck tumors. J. Cell Physiol. 2020, 235, 65–73. [CrossRef]
Tahmasebi, E.; Alikhani, M.; Yazdanian, A.; Yazdanian, M.; Tebyanian, H.; Seifalian, A. The current markers of cancer stem cell inoral cancers. Life Sci. 2020, 249, 117483. [CrossRef]
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]
Lee, Y.; Shin, J.H.; Longmire, M.; Wang, H.; Kohrt, H.E.; Chang, H.Y.; Sunwoo, J.B. CD44+ cells in head and neck squamous cell
carcinoma suppress T-cell-mediated immunity by selective constitutive and inducible expression of PD-L1. Clin. Cancer Res. 2016,
22, 3571–3581. [CrossRef]
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]
Ponta, H.; Sherman, L.; Herrlich, P.A. CD44: From adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 2003, 4,
33–45. [CrossRef]
Yan, Y.; Zuo, X.; Wei, D. Concise review: Emerging role of CD44 in cancer stem cells: A promising biomarker and therapeutictarget. Stem Cells Transl. Med. 2015, 4, 1033–1043. [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][PubMed]Naor, D.; Wallach-Dayan, S.B.; Zahalka, M.A.; Sionov, R.V. Involvement of CD44, a molecule with a thousand faces, in cancer
dissemination. Semin. Cancer Biol. 2008, 18, 260–267. [CrossRef] [PubMed]
Günthert, U.; Hofmann, M.; Rudy, W.; Reber, S.; Zöller, M.; Haussmann, I.; Matzku, S.; Wenzel, A.; Ponta, H.; Herrlich, P. A newvariant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 1991, 65, 13–24. [CrossRef]
Guo, Q.; Yang, C.; Gao, F. The state of CD44 activation in cancer progression and therapeutic targeting. Febs J. 2021. [CrossRef]
Mesrati, M.H.; Syafruddin, S.E.; Mohtar, M.A.; Syahir, A. CD44: A multifunctional mediator of cancer progression. Biomolecules
2021, 11, 1850. [CrossRef]
Morath, I.; Hartmann, T.N.; Orian-Rousseau, V. CD44: More than a mere stem cell marker. Int. J. Biochem. Cell Biol. 2016, 81,
166–173. [CrossRef]
Bennett, K.L.; Jackson, D.G.; Simon, J.C.; Tanczos, E.; Peach, R.; Modrell, B.; Stamenkovic, I.; Plowman, G.; Aruffo, A. CD44
isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor. J. Cell Biol. 1995, 128, 687–698.
[CrossRef]
Orian-Rousseau, V.; Chen, L.; Sleeman, J.P.; Herrlich, P.; Ponta, H. CD44 is required for two consecutive steps in HGF/c-Met
signaling. Genes Dev. 2002, 16, 3074–3086. [CrossRef]
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]
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]
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]
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] [PubMed]
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]

Biomedicines 2023, 11, 1099

33.

34.

35.
36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.
48.

49.

50.

51.
52.

13 of 15

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] [PubMed]
Kato, Y.; Yamada, S.; Furusawa, Y.; Itai, S.; Nakamura, T.; Yanaka, M.; Sano, M.; Harada, H.; Fukui, M.; Kaneko, M.K. PMab-213:
A monoclonal antibody for immunohistochemical analysis against pig podoplanin. Monoclon. Antib. Immunodiagn. Immunother.
2019, 38, 18–24. [CrossRef] [PubMed]
Furusawa, Y.; Yamada, S.; Itai, S.; Sano, M.; Nakamura, T.; Yanaka, M.; Fukui, M.; Harada, H.; Mizuno, T.; Sakai, Y.; et al. PMab-210:
A monoclonal antibody against pig podoplanin. Monoclon. Antib. Immunodiagn. Immunother. 2019, 38, 30–36. [CrossRef]
Furusawa, Y.; Yamada, S.; Itai, S.; Nakamura, T.; Yanaka, M.; Sano, M.; Harada, H.; Fukui, M.; Kaneko, M.K.; Kato, Y. PMab-219:
A monoclonal antibody for the immunohistochemical analysis of horse podoplanin. Biochem. Biophys. Rep. 2019, 18, 100616.
[CrossRef]
Furusawa, Y.; Yamada, S.; Itai, S.; Nakamura, T.; Takei, J.; Sano, M.; Harada, H.; Fukui, M.; Kaneko, M.K.; Kato, Y. Establishmentof a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin. Biochem. Biophys.
Rep. 2019, 18, 100631. [CrossRef]
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]
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 G47Delta combination therapy against
glioblastoma. Mol. Ther. Oncolytics 2022, 26, 265–274. [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]
Tamura-Sakaguchi, R.; Aruga, R.; Hirose, M.; Ekimoto, T.; Miyake, T.; Hizukuri, Y.; Oi, R.; Kaneko, M.K.; Kato, Y.; Akiyama, Y.;
et al. Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion. Acta
Crystallogr. D Struct. Biol. 2021, 77, 645–662. [CrossRef]
Kaneko, M.K.; Ohishi, T.; Nakamura, T.; Inoue, H.; Takei, J.; Sano, M.; Asano, T.; Sayama, Y.; Hosono, H.; Suzuki, H.; et al.
Development of core-fucose-deficient humanized and chimeric anti-human podoplanin antibodies. Monoclon. Antib. Immunodiagn.
Immunother. 2020, 39, 167–174. [CrossRef]
Fujii, Y.; Matsunaga, Y.; Arimori, T.; Kitago, Y.; Ogasawara, S.; Kaneko, M.K.; Kato, Y.; Takagi, J. Tailored placement of aturn-forming PA tag into the structured domain of a protein to probe its conformational state. J. Cell Sci. 2016, 129, 1512–1522.
[CrossRef]
Abe, S.; Kaneko, M.K.; Tsuchihashi, Y.; Izumi, T.; Ogasawara, S.; Okada, N.; Sato, C.; Tobiume, M.; Otsuka, K.; Miyamoto, L.; et al.
Antitumor effect of novel anti-podoplanin antibody NZ-12 against malignant pleural mesothelioma in an orthotopic xenograftmodel. Cancer Sci. 2016, 107, 1198–1205. [CrossRef]
Kaneko, M.K.; Abe, S.; Ogasawara, S.; Fujii, Y.; Yamada, S.; Murata, T.; Uchida, H.; Tahara, H.; Nishioka, Y.; Kato, Y. Chimericanti-human podoplanin antibody NZ-12 of lambda light chain exerts higher antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity compared with NZ-8 of kappa light chain. Monoclon. Antib. Immunodiagn. Immunother. 2017,36, 25–29. [CrossRef]
Ito, A.; Ohta, M.; Kato, Y.; Inada, S.; Kato, T.; Nakata, S.; Yatabe, Y.; Goto, M.; Kaneda, N.; Kurita, K.; et al. A real-time near-infrared
fluorescence imaging method for the detection of oral cancers in mice using an indocyanine green-labeled podoplanin antibody.
Technol. Cancer Res. Treat. 2018, 17, 1533033818767936. [CrossRef]
Tamura, R.; Oi, R.; Akashi, S.; Kaneko, M.K.; Kato, Y.; Nogi, T. Application of the NZ-1 fab as a crystallization chaperone for PA
tag-inserted target proteins. Protein Sci. 2019, 28, 823–836. [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]
Kuwata, T.; Yoneda, K.; Mori, M.; Kanayama, M.; Kuroda, K.; Kaneko, M.K.; Kato, Y.; Tanaka, F. Detection of circulating tumor
cells (CTCs) in malignant pleural mesothelioma (MPM) with the “universal” CTC-chip and an anti-podoplanin antibody NZ-1.2.
Cells 2020, 9, 888. [CrossRef]
Nishinaga, Y.; Sato, K.; Yasui, H.; Taki, S.; Takahashi, K.; Shimizu, M.; Endo, R.; Koike, C.; Kuramoto, N.; Nakamura, S.; et al.
Targeted phototherapy for malignant pleural mesothelioma: Near-infrared photoimmunotherapy targeting podoplanin. Cells
2020, 9, 1019. [CrossRef]
Fujii, Y.; Kaneko, M.; Neyazaki, M.; Nogi, T.; Kato, Y.; Takagi, J. PA tag: A versatile protein tagging system using a super highaffinity antibody against a dodecapeptide derived from human podoplanin. Protein Expr. Purif. 2014, 95, 240–247. [CrossRef]
Kato, Y.; Kaneko, M.K.; Kunita, A.; Ito, H.; Kameyama, A.; Ogasawara, S.; Matsuura, N.; Hasegawa, Y.; Suzuki-Inoue, K.; Inoue,O.; et al. Molecular analysis of the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to theC-type lectin-like receptor CLEC-2. Cancer Sci. 2008, 99, 54–61. [CrossRef] [PubMed]

Biomedicines 2023, 11, 1099

53.

54.
55.

56.
57.
58.
59.

60.

61.
62.

63.
64.
65.

66.

67.
68.
69.
70.
71.

72.
73.

74.

75.

76.

14 of 15

Kato, Y.; Vaidyanathan, G.; Kaneko, M.K.; Mishima, K.; Srivastava, N.; Chandramohan, V.; Pegram, C.; Keir, S.T.; Kuan, C.T.;
Bigner, D.D.; et al. Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas. Nucl. Med. Biol.
2010, 37, 785–794. [CrossRef] [PubMed]
Miura, K.; Yoshida, H.; Nosaki, S.; Kaneko, M.K.; Kato, Y. RAP tag and PMab-2 antibody: A tagging system for detecting andpurifying proteins in plant cells. Front. Plant Sci. 2020, 11, 510444. [CrossRef] [PubMed]
Fujii, Y.; Kaneko, M.K.; Ogasawara, S.; Yamada, S.; Yanaka, M.; Nakamura, T.; Saidoh, N.; Yoshida, K.; Honma, R.; Kato,
Y. Development of RAP tag, a novel tagging system for protein detection and purification. Monoclon. Antib. Immunodiagn.
Immunother. 2017, 36, 68–71. [CrossRef]
Fujii, Y.; Kaneko, M.K.; Kato, Y. MAP tag: A novel tagging system for protein purification and detection. Monoclon. Antib.
Immunodiagn. Immunother. 2016, 35, 293–299. [CrossRef]
Wakasa, A.; Kaneko, M.K.; Kato, Y.; Takagi, J.; Arimori, T. Site-specific epitope insertion into recombinant proteins using the MAP
tag system. J. Biochem. 2020, 168, 375–384. [CrossRef]
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]
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]
Boxberg, M.; Götz, C.; Haidari, S.; Dorfner, C.; Jesinghaus, M.; Drecoll, E.; Boskov, M.; Wolff, K.D.; Weichert, W.; Haller, B.; et al.
Immunohistochemical expression of CD44 in oral squamous cell carcinoma in relation to histomorphological parameters andclinicopathological factors. Histopathology 2018, 73, 559–572. [CrossRef]
Chen, J.; Zhou, J.; Lu, J.; Xiong, H.; Shi, X.; Gong, L. Significance of CD44 expression in head and neck cancer: A systemic review
and meta-analysis. BMC Cancer 2014, 14, 15. [CrossRef]
de Jong, M.C.; Pramana, J.; van der Wal, J.E.; Lacko, M.; Peutz-Kootstra, C.J.; de Jong, J.M.; Takes, R.P.; Kaanders, J.H.; van derLaan, B.F.; Wachters, J.; et al. CD44 expression predicts local recurrence after radiotherapy in larynx cancer. Clin. Cancer Res. 2010,
16, 5329–5338. [CrossRef]
Zöller, M. CD44: Can a cancer-initiating cell profit from an abundantly expressed molecule? Nat. Rev. Cancer 2011, 11, 254–267.
[CrossRef]
Woerner, S.M.; Givehchian, M.; Dürst, M.; Schneider, A.; Costa, S.; Melsheimer, P.; Lacroix, J.; Zöller, M.; Doeberitz, M.K.
Expression of CD44 splice variants in normal, dysplastic, and neoplastic cervical epithelium. Clin. Cancer Res. 1995, 1, 1125–1132.
Dall, P.; Heider, K.H.; Hekele, A.; von Minckwitz, G.; Kaufmann, M.; Ponta, H.; Herrlich, P. Surface protein expression andmessenger RNA-splicing analysis of CD44 in uterine cervical cancer and normal cervical epithelium. Cancer Res. 1994, 54,
3337–3341.
Dall, P.; Hekele, A.; Ikenberg, H.; Göppinger, A.; Bauknecht, T.; Pfleiderer, A.; Moll, J.; Hofmann, M.; Ponta, H.; Herrlich, P.
Increasing incidence of CD44v7/8 epitope expression during uterine cervical carcinogenesis. Int. J. Cancer 1996, 69, 79–85.
[CrossRef]
Mereiter, S.; Martins, Á.M.; Gomes, C.; Balmaña, M.; Macedo, J.A.; Polom, K.; Roviello, F.; Magalhães, A.; Reis, C.A. O-glycan
truncation enhances cancer-related functions of CD44 in gastric cancer. FEBS Lett. 2019, 593, 1675–1689. [CrossRef]
Matter, N.; Herrlich, P.; König, H. Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 2002, 420,
691–695. [CrossRef]
Weg-Remers, S.; Ponta, H.; Herrlich, P.; König, H. Regulation of alternative pre-mRNA splicing by the ERK MAP-kinase pathway.
Embo J. 2001, 20, 4194–4203. [CrossRef]
Rothenberg, S.M.; Ellisen, L.W. The molecular pathogenesis of head and neck squamous cell carcinoma. J. Clin. Investig. 2012, 122,
1951–1957. [CrossRef]
Compagnone, M.; Gatti, V.; Presutti, D.; Ruberti, G.; Fierro, C.; Markert, E.K.; Vousden, K.H.; Zhou, H.; Mauriello, A.; Anemone,L.; et al. ∆Np63-mediated regulation of hyaluronic acid metabolism and signaling supports HNSCC tumorigenesis. Proc. Natl.
Acad. Sci. USA 2017, 114, 13254–13259. [CrossRef] [PubMed]
Stransky, N.; Egloff, A.M.; Tward, A.D.; Kostic, A.D.; Cibulskis, K.; Sivachenko, A.; Kryukov, G.V.; Lawrence, M.S.; Sougnez, C.;
McKenna, A.; et al. The mutational landscape of head and neck squamous cell carcinoma. Science 2011, 333, 1157–1160. [CrossRef]
Menke-van der Houven van Oordt, C.W.; Gomez-Roca, C.; van Herpen, C.; Coveler, A.L.; Mahalingam, D.; Verheul, H.M.; vander Graaf, W.T.; Christen, R.; Rüttinger, D.; Weigand, S.; et al. First-in-human phase I clinical trial of RG7356, an anti-CD44
humanized antibody, in patients with advanced, CD44-expressing solid tumors. Oncotarget 2016, 7, 80046–80058. [CrossRef][PubMed]Riechelmann, H.; Sauter, A.; Golze, W.; Hanft, G.; Schroen, C.; Hoermann, K.; Erhardt, T.; Gronau, S. Phase I trial with theCD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma. Oral. Oncol. 2008, 44,
823–829. [CrossRef] [PubMed]
Tijink, B.M.; Buter, J.; de Bree, R.; Giaccone, G.; Lang, M.S.; Staab, A.; Leemans, C.R.; van Dongen, G.A. A phase I dose escalation
study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck oresophagus. Clin. Cancer Res. 2006, 12, 6064–6072. [CrossRef]
Mitsunaga, M.; Ogawa, M.; Kosaka, N.; Rosenblum, L.T.; Choyke, P.L.; Kobayashi, H. Cancer cell-selective in vivo near infrared
photoimmunotherapy targeting specific membrane molecules. Nat. Med. 2011, 17, 1685–1691. [CrossRef]Biomedicines 2023, 11, 1099

77.
78.
79.

80.

81.
82.

83.

84.

85.

86.

87.

88.

15 of 15

Maruoka, Y.; Wakiyama, H.; Choyke, P.L.; Kobayashi, H. Near infrared photoimmunotherapy for cancers: A translational
perspective. EBioMedicine 2021, 70, 103501. [CrossRef]
Kato, T.; Wakiyama, H.; Furusawa, A.; Choyke, P.L.; Kobayashi, H. Near infrared photoimmunotherapy; A review of targets forcancer therapy. Cancers 2021, 13, 2535. [CrossRef]
Ogawa, M.; Tomita, Y.; Nakamura, Y.; Lee, M.J.; Lee, S.; Tomita, S.; Nagaya, T.; Sato, K.; Yamauchi, T.; Iwai, H.; et al. Immunogenic
cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity. Oncotarget 2017, 8,10425–10436. [CrossRef]
Cognetti, D.M.; Johnson, J.M.; Curry, J.M.; Kochuparambil, S.T.; McDonald, D.; Mott, F.; Fidler, M.J.; Stenson, K.; Vasan, N.R.;
Razaq, M.A.; et al. Phase 1/2a, open-label, multicenter study of RM-1929 photoimmunotherapy in patients with locoregional,
recurrent head and neck squamous cell carcinoma. Head Neck 2021, 43, 3875–3887. [CrossRef]
Nagaya, T.; Nakamura, Y.; Okuyama, S.; Ogata, F.; Maruoka, Y.; Choyke, P.L.; Allen, C.; Kobayashi, H. Syngeneic mouse modelsof oral cancer are effectively targeted by anti-CD44-based NIR-PIT. Mol. Cancer Res. 2017, 15, 1667–1677. [CrossRef]
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, 1–14. [CrossRef]
Nanamiya, R.; Takei, J.; Ohishi, T.; Asano, T.; Tanaka, T.; Sano, M.; Nakamura, T.; Yanaka, M.; Handa, S.; Tateyama, N.; et al.
Defucosylated anti-epidermal growth factor receptor monoclonal antibody (134-mG(2a)-f) exerts antitumor activities in mouse
xenograft models of canine osteosarcoma. Monoclon. Antib. Immunodiagn. Immunother. 2022, 41, 1–7. [CrossRef]
Kawabata, H.; Suzuki, H.; Ohishi, T.; Kawada, M.; Kaneko, M.K.; Kato, Y. A defucosylated mouse anti-CD10 monoclonal
antibody (31-mG(2a)-f) exerts antitumor activity in a mouse xenograft model of CD10-overexpressed tumors. Monoclon. Antib.
Immunodiagn. Immunother. 2022, 41, 59–66. [CrossRef]
Kawabata, H.; Ohishi, T.; Suzuki, H.; Asano, T.; Kawada, M.; Suzuki, H.; Kaneko, M.K.; Kato, Y. A defucosylated mouse anti-CD10
monoclonal antibody (31-mG(2a)-f) exerts antitumor activity in a mouse xenograft model of renal cell cancers. Monoclon. Antib.
Immunodiagn. Immunother. 2022, 41, 320–327. [CrossRef]
Asano, T.; Tanaka, T.; Suzuki, H.; Li, G.; Ohishi, T.; Kawada, M.; Yoshikawa, T.; Kaneko, M.K.; Kato, Y. A defucosylated
anti.-EpCAM monoclonal antibody (EpMab-37-mG(2a)-f) exerts antitumor activity in xenograft model. Antibodies 2022, 11, 74.
[CrossRef]
Tateyama, N.; Nanamiya, R.; Ohishi, T.; Takei, J.; Nakamura, T.; Yanaka, M.; Hosono, H.; Saito, M.; Asano, T.; Tanaka, T.; et al.
Defucosylated anti-epidermal growth factor receptor monoclonal antibody 134-mG(2a)-f exerts antitumor activities in mouse
xenograft models of dog epidermal growth factor receptor-overexpressed cells. Monoclon. Antib. Immunodiagn. Immunother. 2021,
40, 177–183. [CrossRef]
Takei, J.; Ohishi, T.; Kaneko, M.K.; Harada, H.; Kawada, M.; Kato, Y. A defucosylated anti-PD-L1 monoclonal antibody 13-mG(2a)f exerts antitumor effects in mouse xenograft models of oral squamous cell carcinoma. Biochem. Biophys. Rep. 2020, 24, 100801.
[CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.