Article

Development of a Novel Anti−CD44 Monoclonal Antibody for
Multiple Applications against Esophageal Squamous
Cell Carcinomas
Nohara Goto 1,2, Hiroyuki Suzuki 1,*, Tomohiro Tanaka 3, Teizo Asano 3, Mika K. Kaneko 3 and Yukinari Kato 1,3,*
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980−8575, Japan; s1930550@s.tsukuba.ac.jp
2 Ph.D. Program in Human Biology, School of Integrative and Global Majors, University of Tsukuba,
Tsukuba 305−8575, Japan
3 Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai 980−8575, Japan; tomohiro.tanaka.b5@tohoku.ac.jp (T.T.);
teizo.asano.a7@tohoku.ac.jp (T.A.); k.mika@med.tohoku.ac.jp (M.K.K.)* Correspondence: hiroyuki.suzuki.b4@tohoku.ac.jp (H.S.); yukinari.kato.e6@tohoku.ac.jp (Y.K.)
1

Citation: Goto, N.; Suzuki, H.;
Tanaka, T.; Asano, T.; Kaneko, M.K.;
Kato, Y. Development of a NovelAnti−CD44 Monoclonal Antibody
for Multiple Applications against
Esophageal Squamous Cell

Abstract: CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressedon cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, andchemo−resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti−CD44 monoclonal antibodies (mAbs) by immunizing mice witha CD44 variant (CD44v3−10) ectodomain and screening using enzyme−linked immunosorbent assay.
We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. Oneof the established clones (C44Mab−46; IgG1, kappa) reacted with CD44 standard isoform (CD44s)−overexpressed Chinese hamster ovary−K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma(ESCC) cell lines (KYSE70 and KYSE770). The apparent KD of C44Mab−46 for CHO/CD44s, KYSE70,
and KYSE770 was 1.1 × 10−8 M, 4.9 × 10−8 M, and 4.1 × 10−8 M, respectively. C44Mab−46 detected CD44s
of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore,
C44Mab−46 strongly stained the formalin−fixed paraffin−embedded ESCC tissues in immunohistochemistry. Collectively, C44Mab−46 is very useful for detecting CD44 in various applications.

Carcinomas. Int. J. Mol. Sci. 2022, 23,5535. https://doi.org/

Keywords: CD44; monoclonal antibody; esophageal cancer

10.3390/ijms23105535
Academic Editor: Anne Poupon
Received: 11 April 2022

1. Introduction

Accepted: 13 May 2022

CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. [1]. The CD44 gene is comprised of 19 exons in human [2]. The first
five and the last five exons are constant and encode the shortest isoform of CD44 (85–95
kDa), called the CD44 standard isoform (CD44s). CD44s is widely distributed and plays
critical roles in normal homeostasis, including hematopoiesis and immune system, and organogenesis [3]. The middle nine exons can be alternatively spliced and assembled with theten exons contained in CD44s. They are referred to as CD44 variant isoforms (CD44v) [4].
For instance, CD44v3−10 is expressed in epithelial tissues and overexpressed in carcinomas[3]. Furthermore, CD44v3−10 functions as a co−receptor of receptor tyrosine kinases bybinding to heparin−binding epidermal growth factor-like growth factor and fibroblast
growth factors (via v3−encoded region) [5] and hepatocyte growth factor (via v6−encoded
region) [6], and confers oxidative stress resistance via v8−10−encoded region [7]. Moreover,both CD44s and CD44v are major receptors for hyaluronic acid (HA), which is involved in

Published: 16 May 2022
Publisher’s Note: MDPI stays neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Int. J. Mol. Sci. 2022, 23, 5535. https://doi.org/10.3390/ijms23105535

www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 5535

2 of 16cell adhesion, migration, and proliferation [8]. They also play important roles in tumor progression, metastasis, stemness, and resistance to chemo− and radiotherapy [9,10]. Therefore,
CD44 is a promising target for cancer diagnosis and therapy.
Several CD44−targeting monoclonal antibodies (mAbs) have been developed for preclinical research applications. IM7 mAb was shown to inhibit HA−induced vascular endothelial growth factor production in human vascular endothelial cells [11] and significantly
decrease cell migration and invasion in breast cancer cells [12]. Another mAb, H4C4 mAb,
was found to reduce tumor growth, metastasis, and post−radiation recurrence [13]. A humanized mAb specific for CD44, RG7356, has been found to be directly cytotoxic for leukemia B cells, but with no effect on normal B cells. Administration of RG7356 to immune−deficient mice engrafted with human chronic lymphocytic leukemia cells was found to result
in complete clearance of engrafted leukemia cells [14]. Finally, CD44v6−specific humanizedmAbs (BIWA−4 and BIWA−8) labeled with 186Re showed the therapeutic efficacy in headand neck SCC xenograft bearing nude mice in another study [15].
Sulfasalazine is an inhibitor of xCT, the subunit of cystine–glutamate transporter.
CD44v has been found to interact with the xCT and promotes oxidative stress resistant incancer cells [7,16]. The clinical trials of sulfasalazine have been evaluated in combination
with cisplatin in patients with CD44v−expressing advanced gastric cancer refractory tocisplatin [17], and the reduction of the CD44v−positive gastric cancer cells was observedin sulfasalazine treated patients [18]. These results suggest that CD44v is an important
marker to predict and evaluate the sensitivity of sulfasalazine.
Previously, we established the novel mAb, C44Mab−5 (IgG1, kappa) against CD44 using
the Cell−Based Immunization and Screening method [19]. C44Mab−5 showed high sensitivity
for flow cytometry and immunohistochemical analysis in oral cancers. In this study, we developed a novel anti−CD44 mAb, C44Mab−46 (IgG1, kappa), and evaluated it for various applications, including flow cytometry, Western blotting, and immunohistochemical analyses.
2. Results
2.1. Flow Cytometric Analysis of C44Mab−46 to CD44 Expressing Cells
In this study, C44Mab−46 was established by immunizing one mouse with humanCD44v3−10 ectodomain (Figure 1). We already confirmed the epitope of C44Mab−46 as
174−TDDDV−178 [20,21] encoded within the first five exons, indicating that C44Mab−46 recognizes CD44 standard isoforms (CD44s). Therefore, we confirmed the reactivity of C44Mab−46
against CHO/CD44s by flow cytometry. As shown in Figure 2A, C44Mab−46 recognized
CHO/CD44s cells in a dose dependent manner, but not CHO−K1 cells (Figure 2B). The
recognition of CHO/CD44s by C44Mab−46 was strongly inhibited by CD44 peptide (aminoacids 161−180, WT), which contains 174−TDDDV−178 sequence. However, the D175A mutant
peptide, which could not be recognized by C44Mab−46 [21], never inhibited (Figure 2C). Furthermore, C44Mab−46 also recognized endogenous CD44 in the context of ESCC cell lines as
it reacted with both KYSE 70 and KYSE770 in a dose−dependent manner (Figure 2D,E).

Int. J. Mol. Sci. 2022, 23, 5535

3 of 16

1. Immunization of purified CD44ec (CD44v3-10 ectodomain)
CD44s
CD44v3-10

Purified CD44v3-10
ectodomain
Spleen2. Hybridomas production

Myelomas

Fusion(to 96 well plates)

Splenocytes(B cells)

3. Screening of surpernatants by flow cytometry
1st screening: ELISA

Positive wells

2nd screening:
Flow cytometry

CD44v3-10-expressing cells

4. Cloning of hybridomas
Establishment of anti-CD44 mAb-producing clones

Figure 1. A schematic procedure of anti−human CD44 mAbs production. A BALB/c mouse wasintraperitoneally immunized with the purified CD44v3−10 ectodomain. The screening was thenconducted by ELISA using the purified CD44v3−10 ectodomain, flow cytometry using parental andCD44v3−10−overexpressed CHO−K1 cells.

Int. J. Mol. Sci. 2022, 23, 5535

4 of 16

Figure 2. Flow cytometry to CD44 expressing cells using C44Mab−46. (A,B) CHO/CD44s (A) andCHO−K1 (B) cells were treated with 0.01–10 µg/mL of C44Mab−46, followed by treatment with Alexa
Fluor 488−conjugated anti−mouse IgG. (C) C44Mab−46 (1 µg/mL), C44Mab−46 (1 µg/mL) plus humanCD44 peptide (161−180 amino acids, WT) or alanine−substituted peptide (D175A) (10 µg/mL), andcontrol (blocking buffer) were reacted with CHO/CD44 cells for 30 min at 4 °C, followed by treatment with Alexa Fluor 488−conjugated anti−mouse IgG. (D,E) KYSE70 (D) and KYSE770 (E) cells
were treated with 0.01–10 µg/mL of C44Mab−46, followed by treatment with Alexa Fluor
488−conjugated anti−mouse IgG. The black line represents the negative control (blocking buffer).

Int. J. Mol. Sci. 2022, 23, 5535

5 of 16

Next, we assessed the binding affinity of C44Mab−46 with CHO/CD44s, KYSE70, andKYSE770 using flow cytometry. The apparent KD of C44Mab−46 for CHO/CD44s, KYSE70,
and KYSE770 was 1.1 × 10−8 M, 4.9 × 10−8 M, and 4.1 × 10−8 M, respectively, indicating that
C44Mab−46 possesses moderate affinity for CD44s−expressing cells (Figure 3).

Figure 3. The determination of the binding affinity of C44Mab−46. CHO/CD44s (A), KYSE70 (B), andKYSE770 (C) cells were suspended in 100 µL serially diluted C44Mab−46 (0.08 to 1300 nM). Then, cells
were treated with Alexa Fluor 488−conjugated anti−mouse IgG. Fluorescence data were subsequently
collected, followed by the calculation of the apparent dissociation constant (KD) by GraphPad PRISM 8.

2.2. Western Blot Analysis
Western blotting was performed to further assess the sensitivity of C44Mab−46. Lysates of CHO−K1 and CHO/CD44s cells were probed. As shown in Figure 4A, C44Mab−46
detected CD44s as a ~85 kDa band. However, C44Mab−46 did not detect any band from

Int. J. Mol. Sci. 2022, 23, 5535

6 of 16

lysates of CHO−K1 cells. These results indicated that C44Mab−46 specifically detect exogenous CD44s. Next, we examined the detection of endogenous CD44 using lysates fromKYSE70 and KYSE770 cells. As shown in Figure 4B, C44Mab−46 could detect CD44 as 85
and 48kDa double bands from lysates of KYSE70 cells. In contrast, C44Mab−46 could detect
CD44 as more than 100 kDa triplet bands, probably CD44v isoforms, from lysates of
KYSE770 cells. We also performed the peptide blocking experiment and found that 85 kDaband in CHO/CD44s (Figure 4C), 85 and 48 kDa bands in KYSE70, and CD44v isoforms
in KYSE770 (Figure 4D) were blocked in the presence of the CD44 peptide (amino acids
161−181, WT). These results suggest that C44Mab−46 recognizes endogenous CD44s andCD44v(s) in the esophageal cancer cell lines.

Figure 4. Western blotting by C44Mab−46. (A) Cell lysates of CHO−K1 and CHO/CD44s (10 µg) were
electrophoresed and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes
were incubated with 1 µg/mL of C44Mab−46 and 1 µg/mL of anti−β−actin and subsequently withperoxidase−conjugated anti−mouse immunoglobulins. (B) Cell lysates of KYSE70 and KYSE770 (10µg) were electrophoresed and transferred onto PVDF membranes. The membranes were incubatedwith 1 µg/mL of C44Mab−46 and 1 µg/mL of anti−β−actin and subsequently with peroxidase−conjugated anti−mouse immunoglobulins. (C) Cell lysates of CHO−K1 and CHO/CD44s (10 µg) were
electrophoresed and transferred onto PVDF membranes. The membranes were incubated with 1µg/mL of C44Mab−46 and C44Mab−46 (1 µg/mL) plus the CD44 peptide (10 µg/mL, WT), and subsequently with peroxidase−conjugated anti−mouse immunoglobulins. (D) Cell lysates of KYSE70 andInt. J. Mol. Sci. 2022, 23, 5535

7 of 16

KYSE770 (10 µg) were electrophoresed and transferred onto PVDF membranes. The membranes
were incubated with 1 µg/mL of C44Mab−46 and C44Mab−46 (1 µg/mL) plus the CD44 peptide (10µg/mL, WT), and subsequently with peroxidase−conjugated anti−mouse immunoglobulins. Black
arrows indicate the predicted size of CD44s (~85 kDa). The red arrow indicates the CD44 variants.

2.3. Immunohistochemical Analysis Using C44Mab−46 against ESCC Tissues
To investigate whether C44Mab−46 can be used for immunohistochemical analyses using paraffin−embedded tumor sections, we used tissue microarray of ESCC. In a well differentiated ESCC section (Figure 5A–F), C44Mab−46 strongly stained ESCC cells (Figure 5A).
A clear membrane−staining in ESCC was observed (Figure 5B). Hematoxylin and eosin (HE)staining was performed using the serial sections (Figure 5E,F). In an ESCC section with stromal invaded phenotype (Figure 5G–L), C44Mab−46 strongly stained stromal invaded ESCC
(Figure 5G,H). HE staining was also performed using the serial sections (Figure 5K,L). Additionally, C44Mab−46 also stained at basal layer and several layers above in normal squamous epithelium of esophagus (Supplemental Figure S1). We also performed the peptide
blocking experiment and found that the recognition of ESCC tissues by C44Mab−46 was completely blocked by CD44 peptide (amino acids 161−180, WT) (Supplemental Figure S2). In
immunohistochemical analyses using C44Mab−46 against ESCC tissue microarrays (Table 1),
C44Mab−46 stained 63 of 67 (94.0%) cases of ESCC. These results indicated that C44Mab−46 isuseful for immunohistochemical analysis of paraffin−embedded tumor sections.

Int. J. Mol. Sci. 2022, 23, 5535

8 of 16

Figure 5. Immunohistochemical analysis using C44Mab−46 against ESCC tissues. (A–D,G–J) After antigen retrieval, sections were incubated with 5 µg/mL of C44Mab−46 or negative control (blocking buffer)
followed by treatment with the Envision+ kit. Color was developed using DAB, and sections were counterstained with hematoxylin. (E,F,K,L) Hematoxylin and eosin (HE) staining. Scale bar = 100 µm.

Int. J. Mol. Sci. 2022, 23, 5535

9 of 16

Table 1. Immunohistochemical analysis using C44Mab−46 against ESCC tissues.

Case No.
Age Sex Organ/Anatomic Site
1
52 M
Esophagus
2
62 F
Esophagus
3
54 M
Esophagus
459 M
Esophagus
5
60 M
Esophagus
6
66 M
Esophagus
7
36 M
Esophagus
8
55 F
Esophagus
959 F
Esophagus
10
48 M
Esophagus
11
41 M
Esophagus
12
58 M
Esophagus
13
56 M
Esophagus
14 (Figure 5G–L) 72 F
Esophagus
15
41 M
Esophagus
16
50 M
Esophagus
17
48 M
Esophagus
18
55 M
Esophagus
19
61 F
Esophagus
20
35 M
Esophagus
21
72 M
Esophagus
22
70 M
Esophagus
23
42 F
Esophagus
24
53 M
Esophagus
25
54 M
Esophagus
26 (Figure 5A–F) 54 F
Esophagus
27
65 F
Esophagus
28
63 F
Esophagus
29
62 M
Esophagus
30
63 M
Esophagus
31
65 M
Esophagus
32
64 F
Esophagus
33
71 M
Esophagus
34
55 M
Esophagus
35
57 F
Esophagus
36
56 M
Esophagus
37
60 M
Esophagus
38
61 F
Esophagus
39
61 M
Esophagus
40
50 M
Esophagus
41
66 M
Esophagus
42
45 M
Esophagus
43
68 M
Esophagus
44
58 M
Esophagus
45
57 M
Esophagus
46
54 F
Esophagus

Pathology diagnosis
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Smooth muscle and fatty tissue
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma

Grade
Type
IntensityImalignant++Imalignant++Imalignant++Imalignant+IImalignant++−malignant+Imalignant++Imalignant−I − II malignant++
I − II malignant++
Imalignant++Imalignant++Imalignant++Imalignant+++I − II malignant+++
Imalignant−Imalignant+I − II malignant−
IImalignant+Imalignant++Imalignant++−malignant+++Imalignant++Imalignant++I − II malignant++
I − II malignant+++
I − II malignant++
I − II malignant+++
IImalignant+++IImalignant++IImalignant+++IImalignant++IImalignant+++IImalignant+++IImalignant+++IImalignant+++II − III malignant+++
IImalignant++II − III malignant+++−
malignant+++IImalignant+++IImalignant++IImalignant+++IImalignant+++II − III malignant++
IImalignant+Int. J. Mol. Sci. 2022, 23, 5535

47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67

48
68
54
70
72
62
63
49
53
61
61
59
62
56
73
57
64
60
66
67
75

10 of 16

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

Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus
Esophagus

Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma
Squamous cell carcinoma

II
II
II
III
III
III
II − IIIII − IIIII − III
III
II
III
II − IIIIII
II − IIIII − IIIIII
II − IIIIII
II − IIIIII

malignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignantmalignant

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

3. Discussion
CD44 have functions in many processes in normal cells (hematopoietic, immune system, and organogenesis), and in pathological situations (inflammation and cancer) [3].
CD44 exhibits passive ligand binding, including adhesion to hyaluronan and other components of the extracellular matrix. Furthermore, CD44 acts as a co−receptor for growth
factors and as a scaffold for enzymes. Therefore, establishment and characterization ofanti−CD44 mAbs are thought to be important for the development of CD44 targeting therapy and diagnosis.
In this study, we developed C44Mab−46, which can recognize all isoforms of CD44,
and shows utility in flow cytometry, Western blotting, and immunohistochemistry applications. As shown in Figure 2, C44Mab−46 similarly recognized endogenous CD44 of
KYSE70 and KYSE770 cells by flow cytometry; however, the expression pattern of CD44s
and CD44v was different in Western blot analysis (Figure 4). Furthermore, we detected a
48 kDa band in KYSE70 cells, which is also blocked by the CD44 peptide (Figure 4D). By
the radio labeled CD44s pulse chasing assay, a CD44s precursor form (about 48 kDa) was
first translated, after which the precursor received N− and O−glycosylation and reached
85 kDa mature form [22]. This result suggests that the 48 kDa band is a non−glycosylated
precursor form of CD44 in KYSE70 cells. We previously determined the epitope of
C44Mab−46 as 174−TDDDV−178 by enzyme−linked immunosorbent assay using synthetic
peptides [21]. Although the Thr174 of CD44 have been confirmed as an O−glycan site [23],C44Mab−46 could recognize the epitope in the absence of glycosylation. In the future, it
should be determined whether the 48 kDa CD44 is exposed on cell surface, and O−glycosylation at Thr174 affect the recognition by C44Mab−46. In KYSE770 cells, C44Mab−46 could
recognize the triple bands, suggesting the expression of variant isoforms of CD44. Compared to previously established CD44 mAb C44Mab−5, C44Mab−46 tended to show the highreactivity to detect endogenous CD44 in Western blotting.
CD44s and CD44v interact with various proliferation−, migration−, and invasion−promoting membrane proteins. CD44 binds to platelet−derived growth factor β−re-Int. J. Mol. Sci. 2022, 23, 5535

11 of 16

ceptor and transforming growth factor−β type I receptor [24], and modulates their functions. CD44s also interacts with podoplanin (PDPN) and colocalizes at cell−surface protrusions. PDPN−induced migration requires CD44 in MDCK cells, and knockdown of
CD44 and PDPN in oral SCC cells affect cell spreading, suggesting that CD44 directlyinteracts with PDPN and modulates the intracellular signaling and cell migration [25]. It
is interesting that C44Mab−46 inhibits these protein–protein interactions and influences
their functions.
CD44v6 is highly expressed in many cancers, and acts as co−receptor for at least three
receptor tyrosine kinases (c−Met [26,27], EGFR [28], and VEGFR−2 [29]), which contributeto the oncogenic functions of CD44v6. Therefore, humanized anti−CD44v6 mAb BIWA4(bivatuzumab)−mertansine drug conjugate, was evaluated in clinical trials [30]. However,the clinical trials were discontinued because of the severe skin toxicities [31,32]. A
first−in−human phase I clinical trial with RG7356, targeting to the constant region of CD44,
showed an acceptable safety profile in patients with advanced CD44−expressing solid tumors. However, the study was terminated due to no evidence of a clinical and/or pharmacodynamic dose–response relationship with RG7356, but not due to safety concerns[33]. Therefore, the development of anti−CD44 targeting mAbs with more potent andfewer side effects is desired.
We previously established cancer−specific mAbs (CasMabs) targeting to PDPN [34–37] and podocalyxin [38], which are expressed on many cancers, including ESCC [39–42].
These CasMabs recognize cancer−specific aberrant glycosylation of the target proteins[43]. It is worthwhile to develop a cancer−specific anti−CD44 antibody using the CasMabmethod. We used the glioblastoma LN229−secreted CD44v3−10 ectodomain as an antigen
and generated many mAbs other than C44Mab−46. We selected the mAbs, which react withcancer cells, but not with normal cells by flow cytometry and immunohistochemistry. Furthermore, we previously converted the IgG1 subclass of C44Mab−5 into a mouse IgG2a,
named 5−mG2a, and produced a defucosylated version, 5−mG2a−f. The 5−mG2a−f exhibited
ADCC/CDC activities in vitro and significantly reduced tumor growth in oral cancer cells[44]. Therefore, the production of a class switched and defucosylated version of
C44Mab−46 is warranted to evaluate the in vivo anti−tumor activity. Since KYSE70 wasused in this study, it was reported to be tumorigenic in athymic nude mice [45]; the cellline can be used to evaluate the anti−tumor activity as in the in vivo model.
4. Materials and Methods
4.1. Cell Lines
Mouse multiple myeloma P3X63Ag8U.1 (P3U1), a glioblastoma cell line (LN229) [46],and CHO−K1 cell lines were obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA). Esophageal squamous cell carcinoma (ESCC) cell lines, KYSE70 and
KYSE770 were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan).
Human CD44s ORF was amplified from LN229 cDNA using a HotStar HiFidelity
Polymerase Kit (Qiagen Inc., Hilden, Germany). CD44s ORF was subcloned intopCAG−Ble−ssPA16 vector possessing signal sequence and N−terminal PA16 tag (GLEGGVAMPGAEDDVV) [19,47–51]. CHO/CD44s was established by transfecting
pCAG−Ble/PA16−CD44s into CHO−K1 cells using a Neon transfection system (Thermo
Fisher Scientific, Inc., Waltham, MA, USA). CD44v3−10 ectodomain (CD44ec)−secreting
LN229(LN229/CD44ec)
wasestablishedbytransfecting
pCAG−Neo/PA−CD44ec−RAP−MAP into LN229 cells using the Neon transfection system.
The amino acid sequences of the tag system in this study were as follows: PA tag [52–54],
12 amino acids (GVAMPGAEDDVV); RAP tag [55,56], 12 amino acids (DMVNPGLEDRIE); and MAP tag [57,58], 12 amino acids (GDGMVPPGIEDK).
LN229, KYSE70, and KYSE770 were cultured in Dulbecco’s Modified Eagle Medium(DMEM) complete medium, containing DMEM (4.5 g/L glucose) with L−Gln and without

Int. J. Mol. Sci. 2022, 23, 5535

12 of 16

sodium pyruvate (Nacalai Tesque, Inc., Kyoto, Japan), 10% (v/v) heat−inactivated fetal bovine serum (FBS, Thermo Fisher Scientific Inc.), 100 U/mL of penicillin (Nacalai Tesque,Inc.), 100 µg/mL streptomycin (Nacalai Tesque, Inc.), 0.25 µg/mL amphotericin B (Nacalai
Tesque, Inc.). LN229/CD44ec was cultured in DMEM complete medium, containing 0.5
mg/mL of G418 (Nacalai Tesque, Inc.).
P3U1 and CHO−K1 were cultured in Roswell Park Memorial Institute (RPMI)−1640
medium (Nacalai Tesque, Inc.) supplemented with 10% (v/v) FBS, 100 U/mL penicillin,
100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. CHO/CD44s was cultured in
RPMI complete medium, including 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA, USA).
All cells were grown in a humidified incubator at 37 °C with 5% CO2.
4.2. Purification of CD44ec
After LN229/CD44ec was cultured using DMEM complete medium without G418,
CD44ec was purified from the supernatants using RAP tag system, comprised of ananti−RAP tag mAb (clone PMab−2) and a RAP peptide (GDDMVNPGLEDRIE) [55,56].
The filtered culture supernatant (5 L) was passed through PMab−2−Sepharose (2 mL bed
volume), and the same process was repeated three times. The beads were then washedwith 100 mL of phosphate−buffered saline (PBS, Nacalai Tesque, Inc.), and eluted with
0.1 mg/mL of RAP peptide in a step−wise manner (2 mL × 10).
4.3. Hybridoma Production
Female BALB/c mice (6−weeks old) were purchased from CLEA Japan (Tokyo, Japan). The animals were housed under specific pathogen−free conditions. The Animal Careand Use Committee of Tohoku University approved all animal experiments.
A BALB/c mouse was immunized with CD44ec (150 µg) intraperitoneally (i.p.) with
Imject Alum (Thermo Fisher Scientific Inc.). The procedure included three additional immunizations with CD44ec (2nd immunization, 150 µg; 3rd immunization, 100 µg; 4th immunization, 50 µg) followed by a final booster injection of CD44ec (50 µg) two days prior
to the harvest of splenic cells.
Subsequently, splenic cells were fused with P3U1 cells using polyethylene glycol
1500 (PEG1500; Roche Diagnostics, Indianapolis, IN, USA). The hybridomas were thengrown in RPMI media supplemented with hypoxanthine, aminopterin, and thymidine(HAT) for selection (Thermo Fisher Scientific Inc.). The culture supernatants werescreened for the anti−CD44ec antibody production using enzyme−linked immunosorbent
assay (ELISA).
4.4. ELISA
CD44ec was immobilized on Nunc Maxisorp 96−well immunoplates (Thermo Fisher
Scientific Inc) at a concentration of 1 µg/mL for 30 min at 37 °C. After washing with PBS
containing 0.05% (v/v) Tween 20 (PBST; Nacalai Tesque, Inc.), wells were blocked with 1%(w/v) bovine serum albumin (BSA)−containing PBST for 30 min at 37 °C. Culture
supernatants were added to each well, followed by peroxidase−conjugated anti−mouse
immunoglobulins (1:2000 diluted; Agilent Technologies Inc., Santa Clara, CA, USA).
Enzymatic reactions were conducted using 1 Step Ultra TMB (Thermo Fisher ScientificInc.) followed by the measurement of the optical density at 655 nm, using an iMark
microplate reader (Bio−Rad Laboratories, Inc., Berkeley, CA, USA).
4.5. Flow Cytometry
Exponentially growing cells were collected following a brief exposure to 0.25% (w/v)
trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells werethen washed with 0.1% (w/v) BSA in PBS and treated with C44Mab−46 for 30 min at 4 °C.
After incubation, cells were treated with Alexa Fluor 488−conjugated anti−mouse IgG(1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA). In the peptide blocking assay,

Int. J. Mol. Sci. 2022, 23, 5535

13 of 16

C44Mab−46 (1 µg/mL) was incubated with 10 µg/mL of human CD44 peptide (amino acids
161−180, WT) or the D175A mutant peptide [21] for 30 min at 4 °C. CHO/CD44s cells weretreated with C44Mab−46 + each peptide, and further treated with Alexa Fluor 488−conjugatedanti−mouse IgG. Fluorescence data were collected using SA3800 Cell Analyzer (Sony Corp.)
and analyzed using FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).
4.6. Determination of Dissociation Constant (KD) by Flow Cytometry
CHO/CD44s, KYSE70, and KYSE770 cells were suspended in 100 µL serially−dilutedanti−C44Mab−46 mAbs, after which 50 µL Alexa Fluor 488−conjugated anti−mouse IgG(1:200; Cell Signaling Technology, Inc.) was added. Fluorescence data were collected, using EC800 (Sony Corp., CHO/CD44s) or BD FACSLyric (KYSE70 and 770). The apparent
KD was calculated by fitting saturation binding curves to the built−in, one−site binding
models in GraphPad PRISM 8 (GraphPad Software, Inc., La Jolla, CA, USA).
4.7. Western Blot Analysis
Cell lysates (10 µg) were boiled in sodium dodecyl sulfate (SDS) sample buffer(Nacalai Tesque, Inc.). Proteins were separated on 5–20% polyacrylamide gels (FUJIFILM
Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride(PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% (w/v)
skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% (v/v) Tween 20, membranes were incubated with 1 µg/mL C44Mab−46 or 1 µg/mL anti−β−actin (clone AC−15; Sigma–AldrichCorp., St. Louis, MO, USA). Membranes were then incubated with peroxidase−conjugatedanti−mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.) to detect
C44Mab−46 and anti−β−actin. Finally, protein bands were detected with a chemiluminescence reagent, ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a
Sayaca−Imager (DRC Co. Ltd., Tokyo, Japan).
4.8. Immunohistochemical Analysis
Paraffin−embedded ESCC tissue microarray (Product Code: BC02011, US Biomax
Inc., Rockville, MD, USA) were deparaffinized in xylene and rehydrated. Then, they wereautoclaved in citrate buffer (pH 6.0; Agilent Technologies Inc.) for 20 min. After blocking
with SuperBlock T20 (Thermo Fisher Scientific, Inc.), sections were incubated withC44Mab−46 (5 µg/mL) for 1h at room temperature and then treated with the EnVision+ Kit
for mouse (Agilent Technologies Inc.) for 30 min. 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.
Supplementary Materials: The following supporting information can be downloaded at:www.mdpi.com/article/10.3390/ijms23105535/s1.
Author Contributions: N.G., T.T. and T.A. performed the experiments. M.K.K. and Y.K. designed
the experiments. N.G. analyzed the data. N.G., H.S. and Y.K. wrote the manuscript. All authors haveread and agreed to the published version of the manuscript.
Funding: This research was supported in part by AMED (grant numbers: JP22ama121008,
JP21am0401013 (to Y.K.), JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), JP21am0101078 (to Y.K.)).
Institutional Review Board Statement: The animal study protocol was approved by the Animal Careand Use Committee of Tohoku University (Permit number: 2019NiA−001) for studies involving animals.
Conflicts of Interest: The authors declare no conflicts of interest involving this article.

Int. J. Mol. Sci. 2022, 23, 5535

14 of 16

References
1.
2.
3.
4.
5.

6.
7.

8.

9.
10.
11.

12.

13.

14.

15.

16.
17.

18.

19.
20.
21.

22.

23.

Fox, S.B.; Fawcett, J.; Jackson, D.G.; Collins, I.; Gatter, K.C.; Harris, A.L.; Gearing, A.; Simmons, D.L. Normal human tissues, inaddition to some tumors, express multiple different CD44 isoforms. Cancer Res. 1994, 54, 4539–4546.
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. https://doi.org/10.5966/sctm.2015-0048.
Ponta, H.; Sherman, L.; Herrlich, P.A. CD44: From adhesion molecules to signalling regulators. Nat. Rev. Mol. Cell Biol. 2003, 4,
33–45. https://doi.org/10.1038/nrm1004.
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. https://doi.org/10.1186/s13045-018-0605-5.
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. https://doi.org/10.1083/jcb.128.4.687.
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. https://doi.org/10.1101/gad.242602.
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. https://doi.org/10.1016/j.ccr.2011.01.038.
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.
https://doi.org/10.1016/j.matbio.2006.08.261.
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. https://doi.org/10.1016/j.biocel.2016.09.009.
Hassn Mesrati, M.; Syafruddin, S.E.; Mohtar, M.A.; Syahir, A. CD44: A multifunctional mediator of cancer progression. Biomolecules 2021, 11, 1850. https://doi.org/10.3390/biom11121850.
Murphy, J.F.; Lennon, F.; Steele, C.; Kelleher, D.; Fitzgerald, D.; Long, A.C. Engagement of CD44 modulates cyclooxygenase
induction, VEGF generation, and proliferation in human vascular endothelial cells. FASEB J. 2005, 19, 446–448.
https://doi.org/10.1096/fj.03-1376fje.
Uchino, M.; Kojima, H.; Wada, K.; Imada, M.; Onoda, F.; Satofuka, H.; Utsugi, T.; Murakami, Y. Nuclear beta-catenin and CD44
upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells. BMC Cancer 2010, 10, 414.
https://doi.org/10.1186/1471-2407-10-414.
Li, L.; Hao, X.; Qin, J.; Tang, W.; He, F.; Smith, A.; Zhang, M.; Simeone, D.M.; Qiao, X.T.; Chen, Z.N.; et al. Antibody against
CD44s inhibits pancreatic tumor initiation and postradiation recurrence in mice. Gastroenterology 2014, 146, 1108–1118.
https://doi.org/10.1053/j.gastro.2013.12.035.
Zhang, S.; Wu, C.C.; Fecteau, J.F.; Cui, B.; Chen, L.; Zhang, L.; Wu, R.; Rassenti, L.; Lao, F.; Weigand, S.; et al. Targeting chronic
lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44. Proc. Natl. Acad. Sci. USA 2013, 110, 6127–
6132. https://doi.org/10.1073/pnas.1221841110.
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. https://doi.org/10.1002/ijc.10369.
Nagano, O.; Okazaki, S.; Saya, H. Redox regulation in stem-like cancer cells by CD44 variant isoforms. Oncogene 2013, 32, 5191–
5198. https://doi.org/10.1038/onc.2012.638.
Shitara, K.; Doi, T.; Nagano, O.; Fukutani, M.; Hasegawa, H.; Nomura, S.; Sato, A.; Kuwata, T.; Asai, K.; Einaga, Y.; et al. Phase
1 study of sulfasalazine and cisplatin for patients with CD44v-positive gastric cancer refractory to cisplatin (EPOC1407). Gastric
Cancer 2017, 20, 1004–1009. https://doi.org/10.1007/s10120-017-0720-y.
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. https://doi.org/10.1007/s10120-016-0610-8.
Yamada, S.; Itai, S.; Nakamura, T.; Yanaka, M.; Kaneko, M.K.; Kato, Y. Detection of high CD44 expression in oral cancers using
the novel monoclonal antibody, C(44)Mab-5. Biochem. Biophys. Rep. 2018, 14, 64–68. https://doi.org/10.1016/j.bbrep.2018.03.007.
Hayatsu, N.; Ogasawara, S.; Kaneko, M.K.; Kato, Y.; Narimatsu, H. Expression of highly sulfated keratan sulfate synthesized inhuman glioblastoma cells. Biochem. Biophys. Res. Commun. 2008, 368, 217–222. https://doi.org/10.1016/j.bbrc.2008.01.058.
Furusawa, Y.; Yamada, S.; Itai, S.; Sano, M.; Nakamura, T.; Yanaka, M.; Handa, S.; Mizuno, T.; Maeda, K.; Fukui, M.; et al. Establishment of monoclonal antibody PMab-202 against horse podoplanin. Monoclon. Antib. Immunodiagn. Immunother. 2018, 37, 233–
237. https://doi.org/10.1089/mab.2018.0030.
Furusawa, Y.; Yamada, S.; Itai, S.; Sano, M.; Nakamura, T.; Yanaka, M.; Fukui, M.; Harada, H.; Mizuno, T.; Sakai, Y.; et al. PMab210: A monoclonal antibody against pig podoplanin. Monoclon. Antib. Immunodiagn. Immunother. 2019, 38, 30–36.
https://doi.org/10.1089/mab.2018.0038.
Kato, Y.; Yamada, S.; Furusawa, Y.; Itai, S.; Nakamura, T.; Yanaka, M.; Sano, M.; Harada, H.; Fukui, M.; Kaneko, M.K. PMab213: A Monoclonal Antibody for Immunohistochemical Analysis Against Pig Podoplanin. Monoclon. Antib. Immunodiagn. Immunother. 2019, 38, 18–24. https://doi.org/10.1089/mab.2018.0048.

Int. J. Mol. Sci. 2022, 23, 5535

24.

25.

26.
27.

28.

29.
30.

31.
32.
33.

34.
35.
36.

37.

38.

39.

40.

41.
42.

43.
44.

45.

46.

15 of 16

Furusawa, Y.; Yamada, S.; Itai, S.; Nakamura, T.; Yanaka, M.; Sano, M.; Harada, H.; Fukui, M.; Kaneko, M.K.; Kato, Y. PMab219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin. Biochem. Biophys. Rep. 2019, 18, 100616.
https://doi.org/10.1016/j.bbrep.2019.01.009.
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. https://doi.org/10.1016/j.bbrep.2019.100631.
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. https://doi.org/10.1002/pro.3580.
Fujii, Y.; Matsunaga, Y.; Arimori, T.; Kitago, Y.; Ogasawara, S.; Kaneko, M.K.; Kato, Y.; Takagi, J. Tailored placement of a turnforming PA tag into the structured domain of a protein to probe its conformational state. J. Cell Sci. 2016, 129, 1512–1522.
https://doi.org/10.1242/jcs.176685.
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.
https://doi.org/10.1016/j.pep.2014.01.009.
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. https://doi.org/10.3389/fpls.2020.510444.
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. https://doi.org/10.1089/mab.2016.0052.
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. https://doi.org/10.108.89/mab.2016.0039.
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. https://doi.org/10.1093/jb/mvaa054.
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. https://doi.org/10.1089/mab.2021.0028.
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. https://doi.org/10.1089/mab.2021.0012.
Lokeshwar, V.B.; Bourguignon, L.Y. Post-translational protein modification and expression of ankyrin-binding site(s) in GP85
(Pgp-1/CD44) and its biosynthetic precursors during T-lymphoma membrane biosynthesis. J. Biol Chem 1991, 266, 17983–17989.
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.
https://doi.org/10.1002/1873-3468.13432.
Porsch, H.; Mehić, M.; Olofsson, B.; Heldin, P.; Heldin, C.H. Platelet-derived growth factor β-receptor, transforming growth
factor β type I receptor, and CD44 protein modulate each other's signaling and stability. J. Biol. Chem. 2014, 289, 19747–19757.
https://doi.org/10.1074/jbc.M114.547273.
Martín-Villar, E.; Fernández-Muñoz, B.; Parsons, M.; Yurrita, M.M.; Megías, D.; Pérez-Gómez, E.; Jones, G.E.; Quintanilla, M.
Podoplanin associates with CD44 to promote directional cell migration. Mol. Biol. Cell 2010, 21, 4387–4399.
https://doi.org/10.1091/mbc.E10-06-0489.
Orian-Rousseau, V.; Morrison, H.; Matzke, A.; Kastilan, T.; Pace, G.; Herrlich, P.; Ponta, H. Hepatocyte growth factor-induced
Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Mol. Biol. Cell 2007, 18, 76–83.
https://doi.org/10.1091/mbc.e06-08-0674.
Van der Voort, R.; Taher, T.E.; Wielenga, V.J.; Spaargaren, M.; Prevo, R.; Smit, L.; David, G.; Hartmann, G.; Gherardi, E.; Pals,
S.T. Heparan sulfate-modified CD44 promotes hepatocyte growth factor/scatter factor-induced signal transduction through thereceptor tyrosine kinase c-Met. J. Biol. Chem. 1999, 274, 6499–6506. https://doi.org/10.1074/jbc.274.10.6499.
Wang, S.J.; Bourguignon, L.Y. Role of hyaluronan-mediated CD44 signaling in head and neck squamous cell carcinoma progression and chemoresistance. Am. J. Pathol. 2011, 178, 956–963. https://doi.org/10.1016/j.ajpath.2010.11.077.
Tremmel, M.; Matzke, A.; Albrecht, I.; Laib, A.M.; Olaku, V.; Ballmer-Hofer, K.; Christofori, G.; Héroult, M.; Augustin, H.G.;
Ponta, H.; et al. A CD44v6 peptide reveals a role of CD44 in VEGFR-2 signaling and angiogenesis. Blood 2009, 114, 5236–5244.
https://doi.org/10.1182/blood-2009-04-219204.
Orian-Rousseau, V.; Ponta, H. Perspectives of CD44 targeting therapies. Arch. Toxicol. 2015, 89, 3–14.
https://doi.org/10.1007/s00204-014-1424-2.
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. https://doi.org/10.1016/j.oraloncology.2007.10.009.
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. https://doi.org/10.1158/1078-0432.Ccr-06-0910.
Menke-van der Houven van Oordt, C.W.; Gomez-Roca, C.; van Herpen, C.; Coveler, A.L.; Mahalingam, D.; Verheul, H.M.; Van
der Graaf, W.T.; Christen, R.; Rüttinger, D.; Weigand, S.; et al. First-in-human phase I clinical trial of RG7356, an anti-CD44

Int. J. Mol. Sci. 2022, 23, 5535

47.

48.

49.
50.

51.

52.

53.

54.

55.
56.
57.

58.

16 of 16

humanized antibody, in patients with advanced, CD44-expressing solid tumors. Oncotarget 2016, 7, 80046–80058.
https://doi.org/10.18632/oncotarget.11098.
Kaneko, M.K.; Nakamura, T.; Kunita, A.; Fukayama, M.; Abe, S.; Nishioka, Y.; Yamada, S.; Yanaka, M.; Saidoh, N.; Yoshida, K.;
et al. ChLpMab-23: Cancer-Specific Human-Mouse Chimeric Anti-Podoplanin Antibody Exhibits Antitumor Activity via Antibody-Dependent
Cellular
Cytotoxicity.
Monoclon. Antib.
Immunodiagn.
Immunother.
2017,
36,
104–112.
https://doi.org/10.1089/mab.2017.0014.
Kaneko, M.K.; Yamada, S.; Nakamura, T.; Abe, S.; Nishioka, Y.; Kunita, A.; Fukayama, M.; Fujii, Y.; Ogasawara, S.; Kato, Y.
Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via antibody-dependent cellular cytotoxicity. Cancer Med. 2017, 6, 768–777. https://doi.org/10.1002/cam4.1049.
Kato, Y.; Kaneko, M.K. A cancer-specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci. Rep. 2014,
4, 5924. https://doi.org/10.1038/srep05924.
Miyazaki, A.; Nakai, H.; Sonoda, T.; Hirohashi, Y.; Kaneko, M.K.; Kato, Y.; Sawa, Y.; Hiratsuka, H. LpMab-23-recognizing cancer-type podoplanin is a novel predictor for a poor prognosis of early stage tongue cancer. Oncotarget 2018, 9, 21156–21165.
https://doi.org/10.18632/oncotarget.24986.
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.
https://doi.org/10.1016/j.bbrep.2020.100826.
Kaneko, M.K.; Itai, S.; Yamada, S.; Kato, Y. 47-mG(2a): A Mouse IgG(2a)-Type of PcMab-47 Useful for detecting podocalyxin inesophageal cancers by immunohistochemistry. Monoclon. Antib. Immunodiagn. Immunother. 2018, 37, 158–161.
https://doi.org/10.1089/mab.2018.0003.
Tanaka, M.; Kijima, H.; Shimada, H.; Makuuchi, H.; Ozawa, S.; Inokuchi, S. Expression of podoplanin and vimentin is correlated
withprognosis
inesophageal
squamouscellcarcinoma.
Mol.
Med.
Rep.
2015,
12,
4029–4036.
https://doi.org/10.3892/mmr.2015.3966.
Nakashima, Y.; Yoshinaga, K.; Kitao, H.; Ando, K.; Kimura, Y.; Saeki, H.; Oki, E.; Morita, M.; Kakeji, Y.; Hirahashi, M.; et al.
Podoplanin is expressed at the invasive front of esophageal squamous cell carcinomas and is involved in collective cell invasion.
Cancer Sci. 2013, 104, 1718–1725. https://doi.org/10.1111/cas.12286.
Rahadiani, N.; Ikeda, J.; Makino, T.; Tian, T.; Qiu, Y.; Mamat, S.; Wang, Y.; Doki, Y.; Aozasa, K.; Morii, E. Tumorigenic role of podoplaninin esophageal squamous-cell carcinoma. Ann. Surg. Oncol. 2010, 17, 1311–1323. https://doi.org/10.1245/s10434-009-0895-5.
Suzuki, H.; Kaneko, M.K.; Kato, Y. Roles of podoplanin in malignant progression of tumor. Cells 2022, 11, 575.
https://doi.org/10.3390/cells11030575.
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 squamouscell carcinoma. Oncol. Rep. 2020, 44, 1949–1960. https://doi.org/10.3892/or.2020.7735.
Shimada, Y.; Imamura, M.; Wagata, T.; Yamaguchi, N.; Tobe, T. Characterization of 21 newly established esophageal cancer celllines. Cancer 1992, 69, 277–284. https://doi.org/10.1002/1097-0142(19920115)69:2<277::aid-cncr2820690202>3.0.co;2-c.