Article

Development of a Novel Anti-CD44 Variant 4 Monoclonal
Antibody C44Mab-108 for Immunohistochemistry
Hiroyuki Suzuki 1,2,*, Tomohiro Tanaka 1, Nohara Goto 1, Mika K. Kaneko 1,2 and Yukinari Kato 1,2,*
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-Machi, Aoba-Ku, Sendai 980-8575, Japan
2 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-Machi, Aoba-Ku, Sendai 980-8575, Japan* Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.);
Tel.: +81-29-853-3944 (H.S. & Y.K.)
1

Citation: Suzuki, H.; Tanaka, T.;
Goto, N.; Kaneko, M.K.; Kato, Y.

Abstract: CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic
functions in many cancers. The splicing variants play critical roles in the malignant progression ofcancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- andradiotherapy. To understand each CD44 variant (CD44v) function is essential to know the propertyof cancers and the establishment of the therapy. However, the function of the variant 4-encoded
region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4
are indispensable for basic research, tumor diagnosis, and therapy. In this study, we establishedanti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry
to characterize them. One of the established clones (C44Mab-108; IgG1, kappa) reacted with CD44v310-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The KD of C44Mab-108 for
CHO/CD44 v3-10 was 3.4 × 10−7 M. In western blot analysis, C44Mab-108 detected CD44v3-10 in thelysate of CHO/CD44v3-10 cells. Furthermore, C44Mab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that
C44Mab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.

Development of a Novel Anti-CD44
Variant 4 Monoclonal Antibody

Keywords: CD44; CD44 variant 4; monoclonal antibody; flow cytometry; immunohistochemistry

C44Mab-108 for
Immunohistochemistry. Curr. Issues
Mol. Biol. 2023, 45, 1875–1888.
https://doi.org/10.3390/

1. Introduction

cimb45030121
Academic Editors: Guido Giordano
and Pietro Parcesepe
Received: 3 February 2023
Revised: 17 February 2023
Accepted: 22 February 2023
Published: 25 February 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution

(CC

BY)

license(https://creativecommons.org/licenses/by/4.0/).

CD44 is a type I transmembrane glycoprotein, which is widely distributed in normal
tissues [1]. CD44 consists of several exons. CD44 standard isoform (CD44s) is the shortest
isoform of CD44 (85–95 kDa) which is produced by assembling the first five and the last
five constant region exons [2]. The middle variant exons (v1–v10) can be alternatively
spliced and assembled with the first five and the last five exons of CD44s. They are defined
as CD44 variant isoforms (CD44v) [3]. It attracted considerable interest when it was discovered that CD44v induced metastatic properties in tumor cells [4]. A growing body ofevidence suggests the importance of CD44v in the malignant progression of tumors [5],as well as cancer-initiating properties [6].
Both CD44s and CD44v can bind to hyaluronic acid (HA) and are involved in celladhesion, proliferation, and migration [7,8]. CD44s is widely expressed in normal tissues
and plays important roles in hematopoiesis, the immune system, and organogenesis [9].
CD44v plays critical roles in the promotion of tumor invasion, metastasis, stemness, andresistance to chemo- and radiotherapy [10,11]. CD44v3-10 can bind to heparin-binding
epidermal growth factor-like growth factor (HB-EGF) and fibroblast growth factors(FGFs) via v3-encoded region, and functions as a co-receptor of receptor tyrosine kinases[12]. Moreover, the v6-encoded region is essential for the recruitment of hepatocyte

Curr. Issues Mol. Biol. 2023, 45, 1875–1888. https://doi.org/10.3390/cimb45030121

www.mdpi.com/journal/cimb

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growth factor (HGF) to its receptor, c-MET [13]. In addition, the v8-10-encoded region
confers oxidative stress resistance [14]. Therefore, understanding the function of each variant is essential to identify the properties of carcinomas. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific antibodies against
CD44v4 are indispensable for basic research, tumor diagnosis, and therapy.
We previously established the novel anti-CD44 mAbs, C44Mab-5 (IgG1, kappa) [15],and C44Mab-46 (IgG1, kappa) [16] using Cell-Based Immunization and Screening (CBIS)
method and immunization of CD44v3-10 ectodomain, respectively. Both C44Mab-5 andC44Mab-46 have epitopes in the first five exons-encoding sequences [17–19]. Therefore,
they can recognize both CD44s and CD44v (pan-CD44). Furthermore, they showed highsensitivity for flow cytometry and immunohistochemical analysis in oral [15] and esophageal tumors [16]. We have also investigated the antitumor effects in mouse xenograftmodels of oral squamous cell carcinomas (OSCC) [20].
In this study, we developed a novel anti-CD44v4 mAb, C44Mab-108 (IgG1, kappa) by
peptide immunization of the v4-encoded region, and evaluated its applications, including
flow cytometry, western blotting, and immunohistochemical analyses.
2. Materials and Methods
2.1. Cell Lines
Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1 (P3U1, mouse multiple myeloma) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Esophageal squamous cell carcinoma cell lines, KYSE70 and KYSE770
were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan).
CD44s open reading frame (ORF) was amplified from LN229 cDNA using HotStar HiFidelity Polymerase Kit (Qiagen Inc., Hilden, Germany). CD44v3-10 ORF was provided by
the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. CD44s
and CD44v3-10 ORFs were subcloned into pCAG-Ble-ssPA16 vector possessing signal sequence and the PA16 tag (GLEGGVAMPGAEDDVV) [15,21–24], which is detected by NZ1 [25–35]. CHO/CD44s and CHO/CD44v3-10 were established by transfecting pCAGBle/PA16-CD44s and pCAG-Ble/PA16-CD44v3-10 into CHO-K1 cells using a Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
CHO-K1 and P3U1 cells were cultured in Roswell Park Memorial Institute (RPMI)1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 μg/mL streptomycin,
100 U/mL penicillin, 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.), and 50 μg/mL
plasmocin prophylactic (InvivoGen, San Diego, CA, USA). KYSE70 and KYSE770 were
cultured in Dulbecco’s Modified Eagle Medium (DMEM; 4.5 g/L glucose), containing LGln and without sodium pyruvate (Nacalai Tesque, Inc.), 10% FBS, 100 U/mL of penicillin,
100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, and 50 μg/mL plasmocin prophylactic. All the cells were cultured in a humidified incubator at 37°C with 5% CO2.
2.2. Peptides
Sigma-Aldrich (St. Louis, MO, USA) synthesized a partial sequence of the humanCD44 variant 4 (273-DHTKQNQDWTQWNPSHSNP-291) plus C-terminal cysteine. Subsequently, the keyhole limpet hemocyanin (KLH) was conjugated at the C-terminus of thepeptide. CD44 peptides, which cover the extracellular domain of CD44v3-10, were described previously [17].
2.3. Hybridoma Production
The female BALB/c mice were purchased from CLEA Japan (Tokyo, Japan). All animal experiments were conducted according to the guidelines and regulations to minimize
animal suffering and distress in the laboratory. The Animal Care and Use Committee of
Tohoku University (Permit number: 2019NiA-001) approved animal experiments. The

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mice were housed under specific pathogen-free conditions and monitored daily for healthduring the duration of the experiment. To develop mAbs against CD44v4, we intraperitoneally immunized mice with the KLH-conjugated CD44v4 peptide (100 μg) plus an adjuvant (Imject Alum; Thermo Fisher Scientific Inc.). The procedure included three additional
weekly immunization (100 μg/mouse). A final booster injection (100 μg/mouse) was performed two days before harvesting spleen cells. The harvested spleen cells were subsequently fused with P3U1 cells, using PEG1500 (Roche Diagnostics, Indianapolis, IN, USA).
The hybridomas were selected in the presence of hypoxanthine, aminopterin, and thymidine (HAT; Thermo Fisher Scientific Inc.). The supernatants were screened using enzymelinked immunosorbent assay (ELISA) with the CD44v4 peptide [1 μg/mL in phosphatebuffered saline (PBS)], followed by flow cytometry using CHO/CD44v3-10 and CHO-K1
cells. CHO/CD44v3-10-reactive and parental CHO-K1-non-reactive supernatants were
determined to be positive for CD44v3-10. To establish single clones, limiting dilution was
performed.
2.4. ELISA
The synthesized peptide (DHTKQNQDWTQWNPSHSNP), which is included in
CD44v4 plus C-terminal cysteine, was immobilized at a concentration of 1 μg/mL in PBS
on Nunc Maxisorp 96 well immunoplates (Thermo Fisher Scientific Inc.) for 30 min at 37°C. This peptide might be dimerized using the disulfide bond. The plates were washedwith 0.05% Tween 20 in PBS (PBST; Nacalai Tesque, Inc.), and the wells were blocked with
1% bovine serum albumin (BSA) in PBST for 30 min at 37°C. Then, the plates were incubated with the supernatants of hybridomas, followed by peroxidase-conjugated antimouse immunoglobulins (1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA,
USA). ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.) was used for enzymatic reactions. The optical density at 655 nm was measured using an iMark microplate reader (BioRad Laboratories, Inc., Berkeley, CA, USA).
In Supplementary Table S1, CD44v3−10 ectodomain [16] was immobilized at a concentration of 1 μg/mL for 30 min at 37°C. After blocking with 1% BSA in PBST for 30 min
at 37°C, the plates were incubated with the supernatant of C44Mab-108, followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins, anti-mouse heavy
chains (IgG1, IgG2a, IgG2b, IgG3, and IgM; SouthernBiotech, Birmingham, AL, USA), or
anti-mouse light chains (kappa and lambda; SouthernBiotech). The enzymatic reactions
were conducted and the optical density was measured. C44Mab-108 isotype wasdetermined by the reactivity of secondary antibodies.
In Supplementary Figure S2, fifty-eight synthesized peptides [17] were immobilized
at a concentration of 10 μg/mL for 30 min at 37°C. After blocking with 1% BSA in PBST
for 30 min at 37°C, the plates were incubated with C44Mab-108 (1 μg/mL), followed byincubation with peroxidase-conjugated anti-mouse immunoglobulins. The enzymatic reactions were conducted and the optical density was measured.
2.5. Flow Cytometry
The cells were isolated using 0.25% trypsin and 1 mM ethylenediaminetetraacetic
acid (Nacalai Tesque, Inc.). The cells were washed with blocking buffer (0.1% BSA in PBS)
and treated with C44Mab-108 or C44Mab-46 for 30 min at 4°C. Subsequently, the cells weretreated with Alexa Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 4°C. Fluorescence data were collected using
the SA3800 Cell Analyzer and analyzed using SA3800 software ver. 2.05 (Sony Corporation, Tokyo, Japan) and FlowJo (BD Biosciences, Franklin Lakes, NJ, USA).
2.6. Determination of Dissociation Constant (KD) via Flow Cytometry
Serially diluted C44Mab-108 was suspended with CHO/CD44v3-10 cells. The cells
were further treated with Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell

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Signaling Technology, Inc.). Fluorescence data were collected using BD FACSLyric andanalyzed using BD FACSuite software version 1.3 (BD Biosciences). To determine the dissociation constant (KD), GraphPad Prism 8 (the fitting binding isotherms to built-in onesite binding models; GraphPad Software, Inc., San Diego, CA, USA) was used.
2.7. Western Blot Analysis
The cell lysates (10 μg of protein) were prepared using sodium dodecyl sulfate (SDS)
sample buffer (Nacalai Tesque, Inc.), and separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The proteins were transferred
onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany).
The membranes were incubated with 10 μg/mL of C44Mab-108, 1 μg/mL of an anti-PA16
tag mAb (NZ-1), or 1 μg/mL of anti-β-actin mAb (clone AC-15; Sigma-Aldrich Corp.) in
4% skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20. Then, the membranes
were incubated with anti-mouse immunoglobulins conjugated with peroxidase (diluted
1:1000; Agilent Technologies, Inc.) for C44Mab-108 and anti-β-actin. The anti-rat immunoglobulins (diluted 1:10000; Sigma-Aldrich Corp.) conjugated with peroxidase were used
for NZ-1. Finally, the signals were detected with a chemiluminescence reagent, ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC
Co. Ltd., Tokyo, Japan).
2.8. Immunohistochemical Analysis
The formalin-fixed paraffin-embedded (FFPE) OSCC tissue microarray (ProductCode: OR601c, US Biomax Inc., Rockville, MD, USA) and the esophageal tissue microarray(Product Code: BC02011, US Biomax Inc.) were deparaffinized in xylene (Sigma-AldrichCorp.) and rehydrated. The FFPE OSCC tissue for peptide blocking assay was obtained
from Tokyo Medical and Dental University [36]. The tissues were autoclaved in citratebuffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min for antigen retrieval.
After blocking with SuperBlock T20 (Thermo Fisher Scientific, Inc.), the sections were incubated with C44Mab-108 (10 μg/mL) and C44Mab-46 (1 μg/mL), or without the primary
antibody (control) for 1 h at room temperature and then treated with the EnVision+ Kit
for mouse (Agilent Technologies Inc.) for 30 min. The color was developed using 3,3′diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min. Counterstaining was performed with hematoxylin (FUJIFILM Wako Pure Chemical Corporation). Hematoxylin and eosin (HE) staining (FUJIFILM Wako Pure Chemical Corporation)
was performed using consecutive tissue sections. Leica DMD108 (Leica Microsystems
GmbH, Wetzlar, Germany) was used to examine the sections and obtain images.
3. Results
3.1. Development of Anti-CD44v4 mAbs by Peptide Immunization
To develop anti-CD44v4 mAbs, mice were immunized with the KLH-conjugatedCD44v4 peptide (Figure 1A). The splenocytes were fused with myeloma P3U1 cells (Figure 1B). The developed hybridomas were subsequently seeded into 96-well plates andcultured for six days. The positive wells for the naked CD44v4 peptide were selected using
ELISA, followed by the selection of CHO/CD44v3-10-reactive and parental CHO-K1-nonreactive supernatants using flow cytometry (Figure 1C). After the limiting dilution, 12
clones were established. After several additional screenings, including flow cytometry
and immunohistochemistry, an anti-CD44v4 mAb (clone C44Mab-108; mouse IgG1, kappa)
was finally selected and investigated in this study (Figure 1D and Supplementary Table
S1). We confirmed that C44Mab-108 recognized only a CD44v4-containing peptide (aa 271–
290) among peptides, which cover the extracellular domain of CD44v3-10 [17] (Supplementary Figure S1).

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Figure 1. A schematic procedure of anti-CD44v4 mAbs production. (A) The mice wereintraperitoneally immunized with the CD44v4 peptide conjugated with keyhole limpet hemocyanin(KLH). (B) The spleen cells were fused with P3U1 cells, and hybridomas were produced. (C) The
supernatants were subsequently screened using enzyme-linked immunosorbent assay (ELISA) with
the CD44v4 peptide, followed by flow cytometry using CHO/CD44v3-10 and CHO-K1 cells.
CHO/CD44v3-10-reactive and parental CHO-K1-non-reactive supernatants were determined to bepositive for CD44v3-10. (D) To establish single clones, limiting dilution was performed. A clone
C44Mab-108 (IgG1, kappa) was finally established.

3.2. Flow Cytometric Analysis
We conducted flow cytometry using C44Mab-108 against CHO/CD44s,
CHO/CD44v3-10, and CHO-K1 cells. C44Mab-108 recognized CHO/CD44v3-10 cells dosedependently at 10, 1, and 0.1 μg/mL. C44Mab-108 did not recognize parental CHO-K1 cells
even at 10 μg/mL (Figure 2A). Furthermore, C44Mab-108 never recognized CHO/CD44s
cells, which were recognized by a previously established anti-pan-CD44 mAb, C44Mab-46[16] at 10 μg/mL (Figure 2B). These results indicated that C44Mab-108 specifically recognizes CD44v3-10, but not CD44s by flow cytometry.
We next determined the KD of C44Mab-108 with CHO/CD44v3-10 cells using flow cytometry. As shown in Figure 2C, the KD of C44Mab-108 for CHO/CD44v3-10 was determined as 3.4 × 10−7 M.

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Figure 2. Flow cytometry to CD44 expressing cells using C44Mab-108. (A) The CHO/CD44v3-10 and
CHO-K1 cells were treated with 0.01–10 μg/mL of C44Mab-108, followed by treatment with Alexa
Fluor 488-conjugated anti-mouse IgG. The red lines show the cells with the C44Mab-108 treatment.
The black lines represent the negative control. (B) The CHO/CD44s cells were treated with 10 μg/mL
of C44Mab-108 and C44Mab-46, followed by treatment with Alexa Fluor 488-conjugated anti-mouse
IgG. The red lines show the cells with the mAbs treatment. The black lines represent the negative
control. (C) The CHO/CD44v3-10 were suspended in 100 μL of serially diluted C44Mab-108 (0.08–1303 nM). The cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG. The fluorescencedata were collected using a BD FACSLyric, following the calculation of the dissociation constant(KD) by GraphPad PRISM 8.

3.3. Western Blot Analysis
Western blotting was performed to further assess the sensitivity of C44Mab-108. The
lysates of CHO-K1, CHO/CD44s, and CHO/CD44v3-10 cells were probed. As shown in
Figure 3A, C44Mab-108 detected CD44v3-10 as a ~120-kDa band. However, C44Mab-108

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did not detect any bands from the lysates of CHO-K1 and CHO/CD44s cells. An anti-PA16
tag mAb, NZ-1, recognized the lysates from CHO/CD44s (~75 kDa) and CHO/CD44v3-10(~120 kDa), both of which possess the PA16 tag at their N-terminus. Next, we examinedthe detection of endogenous CD44v4 using lysates from KYSE70 and KYSE770 cells. As
shown in Figure 3B, C44Mab-108 could detect CD44v4 as 120 kDa-band from lysates of
KYSE770 cells. These results indicated that C44Mab-108 can detect exogenous CD44v3-10
and endogenous CD44v4.

Figure 3. Western blotting by C44Mab-108. (A) The cell lysates of 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-108, 1 μg/mL of antiPA16 tag mAb (NZ-1), and 1 μg/mL of anti-β-actin mAb. Then, the membranes were incubated with
anti-mouse immunoglobulins conjugated with peroxidase for C44Mab-108 and anti-β-actin. The
anti-rat immunoglobulins conjugated with peroxidase was used for NZ-1. The black arrow indicates
the CD44s (~75 kDa). The red arrows indicate the CD44v3-10 (~120 kDa). (B) The cell lysates of
KYSE70 and KYSE770 (10 μg) were electrophoresed and transferred onto PVDF membranes. The
membranes were incubated with 10 μg/mL of C44Mab-108 and 1 μg/mL of anti-β-actin and subsequently with peroxidase-conjugated anti-mouse immunoglobulins. The red arrow indicates theCD44v4 (~120 kDa).

3.4. Immunohistochemical Analysis Using C44Mab-108 against OSCC Tissues
To investigate whether C44Mab-108 can be used for immunohistochemical analyses
using paraffin-embedded tumor sections, we used sequential sections of an OSCC tissue
microarray. In a well-differentiated OSCC section (Figure 4A–H), a clear membrane-staining in OSCC was observed by C44Mab-46 (Figure 4C,D), but hardly detected by C44Mab108 (Figure 4A,B). In an OSCC section with the stromal invaded phenotype (Figure 4I–P),
C44Mab-108 (Figure 4I,J) strongly stained stromal invaded OSCC and could clearly distinguish tumor cells from stromal tissues. In contrast, C44Mab-46 (Figure 4K,L) stained both.
The reactivity of C44Mab-108 was eliminated completely by a CD44 peptide (aa 271–290),
which contains the C44Mab-108 epitope (Figure 4Q,R). We summarized the data of immunohistochemical analysis in Table 1; C44Mab-108 stained 20 out of 50 (40%) cases of
OSCC. Similar staining patterns were also observed in esophageal SCC tissues. C44Mab108 also stained tumors selectively, and could clearly distinguish tumor cells from stroma(Supplementary Figure S2). These results indicated that C44Mab-108 is useful for immunohistochemical analysis of paraffin-embedded tumor sections.

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Figure 4. Immunohistochemical analysis using C44Mab-108 and C44Mab-46 against oral squamouscell carcinoma (OSCC) tissues. After antigen retrieval, the sections were incubated with 10 μg/mL
of C44Mab-108 (A,B,I,J), 1 μg/mL of C44Mab-46 (C,D,K,L), and without the primary antibody (control) (E,F,M,N) followed by treatment with the Envision+ kit. The color was developed using 3,3′diaminobenzidine tetrahydrochloride (DAB), and the sections were counterstained with hematoxylin. (G,H,O,P) Hematoxylin and eosin (HE) staining. (Q,R) Blocking of the C44Mab-108 reactivity
to OSCC tissue by the CD44 peptide (aa 271–290) containing the C44Mab-108 epitope. After antigen
retrieval, sections were incubated with C44Mab-108 (10 μg/mL) or C44Mab-108 (10 μg/mL) plus human CD44 peptide (aa 271–290, 10 μg/mL) followed by treatment with the Envision+ kit. The color
was developed using DAB, and sections were counterstained with hematoxylin. Scale bar = 100 μm.

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Table 1. Immunohistochemical analysis using C44Mab−108 against OSCC tissues.
No.
1
2
3
4
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
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

Age
78
40
75
35
61
41
64
76
50
44
53
46
50
36
63
46
58
64
57
61
60
60
69
53
55
58
63
48
80
77
59
77
56
60
62
67
47
37
55
56
49
45
42
44
40
49
56
42
87
50

Sex Organ/Anatomic Site
Pathology Diagnosis
TNM
C44Mab-108 C44Mab-46
MTongueSquamous cell carcinoma of tongue
T2N0M0+M
TongueSquamous cell carcinoma of tongue
T2N0M0++F
TongueSquamous cell carcinoma of tongue
T2N0M0+++F
TongueSquamous cell carcinoma of tongue
T2N0M0++++M
TongueSquamous cell carcinoma of tongue
T2N0M0++++F
TongueSquamous cell carcinoma of tongue
T2N0M0+++M
TongueSquamous cell carcinoma of right tongue
T2N2M0+M
TongueSquamous cell carcinoma of tongue
T1N0M0++F
TongueSquamous cell carcinoma of tongue
T2N0M0+++M
TongueSquamous cell carcinoma of tongue
T2N1M0+++F
TongueSquamous cell carcinoma of tongue
T1N0M0+++F
TongueSquamous cell carcinoma of tongue
T2N0M0++M
TongueSquamous cell carcinoma of root of tongue
T3N1M0+++F
TongueSquamous cell carcinoma of tongue
T1N0M0++++F
TongueSquamous cell carcinoma of tongue
T1N0M0++M
TongueSquamous cell carcinoma of tongue
T2N0M0+++M
TongueSquamous cell carcinoma of tongue
T2N0M0+++M
Lip
Squamous cell carcinoma of lower lip
T1N0M0+++M
Lip
Squamous cell carcinoma of lower lip
T2N0M0+++M
Lip
Squamous cell carcinoma of lower lip
T1N0M0+++M
Gum
Squamous cell carcinoma of gum
T3N0M0+++M
Gum
Squamous cell carcinoma of gum
T1N0M0++++M
Gum
Squamous cell carcinoma of upper gum
T3N0M0+++M
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T2N0M0+M
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T1N0M0++M
TongueSquamous cell carcinoma of base of tongue
T1N0M0++M
Oral cavity
Squamous cell carcinoma
T1N0M0++F
TongueSquamous cell carcinoma of tongue
T1N0M0++M
Lip
Squamous cell carcinoma of lower lip
T1N0M0+++M
TongueSquamous cell carcinoma of base of tongue
T2N0M0+++M
TongueSquamous cell carcinoma of tongue
T2N0M0++F
TongueSquamous cell carcinoma of tongue
T1N0M0++++M
TongueSquamous cell carcinoma of root of tongue
T2N1M0++M
TongueSquamous cell carcinoma of tongue
T2N1M0++++M
TongueSquamous cell carcinoma of tongue
T2N0M0+++++F
TongueSquamous cell carcinoma of tongue
T2N0M0+++++F
TongueSquamous cell carcinoma of tongue
T2N0M0++++M
TongueSquamous cell carcinoma of tongue
T2N1M0F
TongueSquamous cell carcinoma of tongue
T2N0M0++F
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T2N0M0++++M
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T1N0M0M
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T2N0M0++M
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T3N0M0+++M
Jaw
Squamous cell carcinoma of right drop jaw
T1N0M0++++F
TongueSquamous cell carcinoma of base of tongue
T2N0M0+++M
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T1N0M0++++F
TongueSquamous cell carcinoma of base of tongue
T2N0M0+M
Bucca cavioris
Squamous cell carcinoma of bucca cavioris
T3N0M0++++F
Face
Squamous cell carcinoma of left face
T2N0M0+M
Gum
Squamous cell carcinoma of gum
T2N0M0++-, No stain; +, Weak intensity; ++, Moderate intensity; +++, Strong intensity.

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4. Discussion
CD44v have been implicated as a marker of cancer-initiating cells and plays pro-tumorigenic functions in many carcinomas [37]. The v3-encoded region possesses heparansulfate moieties that recruit to HB-EGF and FGFs [12]. The v6-encoded region forms aternary complex with MET and HGF, which is essential for the c-MET activation [13]. Furthermore, the CD44 intracellular domain is required for the recruitment of the ezrin, radixin, and moesin complex, which potentiates the c-MET signal transduction [38]. These
functions of CD44v are important for the malignant progression of tumors to potentiate
proliferation, invasiveness, and metastatic spread. Furthermore, CD44v interacts with acystine–glutamate transporter (xCT) subunit through a v8-10-encoded region [14], whichmediates oxidative stress and antitumor drug resistance in several carcinomas [39]. Therefore, the establishment and characterization of mAbs, which recognize CD44v are thought
to be essential for the development of CD44-targeting tumor diagnosis and therapy.
In contrast, the function of the v4-encoded region has not been elucidated. Therefore,
specific antibodies against CD44v4 have been desired. In this study, we developed
C44Mab-108, which can recognize the v4 region. We showed the usefulness of flow cytometry (Figure 2), western blotting (Figure 3), and immunohistochemistry (Figure 4 and Supplementary Figure S2). In Supplementary Figure S3, we showed the homology of the v4
region between human, mouse, rat, and Chinese hamster sequences. In our preliminary
epitope mapping analysis, the alanine-substitution of each amino acid in the human v4
region, which is identical to the mouse sequence, reduced the reactivity of C44Mab-108(manuscript preparation). The result suggests that C44Mab-108 recognizes both humanand mouse CD44v4.
As shown in Figure 2C, the affinity of C44Mab-108 for CHO/CD44v3-10 was not high.
Although high affinity is the general goal for antibody generation, recent reports showedthat low rather than high affinity exhibits elevated activity through inducing clustering ofreceptors. For instance, low-affinity variants of an anti-PD-1 mAb nivolumab mediatedmore potent signaling and affected T-cell activation. These findings reveal a new paradigm for antibody-mediated receptor signaling [40]. Since CD44 is involved in intracellular signaling, the relationship between the antibody affinity and the effect of cellular signaling should be investigated in future studies.
Most CD44v mAbs have been developed by the immunization of recombinant or cellsurface expressed CD44v, which received the glycosylation. CD44 is known to be heavily
glycosylated [41], and the glycosylation pattern is thought to depend on the host cells.
Moreover, it is not clear whether the available anti-CD44 mAbs recognize the peptide or
glycopeptide structures of CD44v. Since C44Mab-108 was established by the peptide immunization, C44Mab-108 can recognize the definite peptide structure of the variant 4-encoded region. Since the region is also expected to receive the glycosylation [41], further
studies are required to reveal whether the glycosylation affects the recognition by C44Mab108. In the immunohistochemical analysis (Figure 4, Table 1), C44Mab-108 could detect
endogenous CD44v4 in some OSCC tissues. Although an anti-pan-CD44 mAb, C44Mab-46
recognized not only OSCC tissues, but also stromal tissues, C44Mab-108 stained the tumor
tissues selectively. Furthermore, C44Mab-108 recognized a limited population of OSCC
tissues compared to C44Mab-46 (Figure 4, Table 1). In the future, we should evaluate theclinical significance of the expression pattern and histology of C44Mab-108-positive OSCC.
The information could help the future diagnostic and therapeutic applications of C44Mab108 in OSCC.
Clinical trials of anti-CD44 mAbs have been conducted [42]. A humanized antiCD44v6 mAb BIWA4 (bivatuzumab)−mertansine drug conjugate was evaluated but discontinued due to severe skin toxicities [43,44]. An anti-pan CD44 mAb, RG7356, exhibitedan acceptable safety profile in patients with advanced CD44-expressing solid tumors.
However, the study was terminated due to no evidence of a clinical and pharmacodynamic dose-response relationship with RG7356 [45]. Therefore, the development of antiCD44 mAbs with more potent and fewer side effects is required.

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We previously established cancer-specific mAbs (CasMabs) against podoplanin [46–48] and podocalyxin [49], which are expressed in many cancers including OSCC [50]. The
strategy also contributes to the development of various types of mAbs [51–58]. An antipodoplanin CasMab, LpMab-2 can recognize the cancer-type aberrant glycosylation ofThr55 and/or Ser56 with surrounding peptide, which is not expressed in normal cells [48].
LpMab-2 has also been applied to chimeric antigen receptor T-cell therapy in mice models[59]. We should understand the cancer-type glycosylation of CD44v, which could support
the development of cancer-specific anti-CD44v mAbs.
Supplementary Materials: The following supporting information can be downloaded at:https://www.mdpi.com/article/10.3390/cimb45030121/s1, Table S1, Determination of C44Mab-108
isotype by ELISA. Figure S1, Conformation of C44Mab-108 epitope by ELISA using peptides which
cover the CD44v3-10 extracellular domain. Figure S2, Immunohistochemical analysis using C44Mab108 and C44Mab-46 against esophageal squamous cell carcinoma tissues. Figure S3, An alignment of
human, mouse, rat, and Chinese hamster (Cham) CD44 variant 4 sequences.
Author Contributions: H.S., N.G. and T.T. performed the experiments. M.K.K. and Y.K. designed
the experiments. H.S., N.G. and Y.K. analyzed the data. H.S. and Y.K. wrote the manuscript. All
authors have read and agreed to the published version of the manuscript.
Funding: This research was supported in part by Japan Agency for Medical Research and Development (AMED) under Grant Numbers: JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), and JP21am0101078 (to Y.K.), and by the Japan
Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grantnos. 21K20789 (to T.T.), 22K06995 (to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal
Care and Use Committee of Tohoku University (Permit number: 2019NiA-001) for studies involving
animals.
Data Availability Statement: The data presented in this study are available in the article and supplementary material.
Acknowledgments: The authors would like to thank Saori Okuno and Saori Handa (Department of
Antibody Drug Development, Tohoku University Graduate School of Medicine) for technical assistance.
Conflicts of Interest: The authors have no conflict of interest to declare.

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