ONCOLOGY REPORTS 52: 147, 2024

Antitumor activities of anti‑CD44 monoclonal antibodies
in mouse xenograft models of esophageal cancer
KENICHIRO ISHIKAWA1,2, HIROYUKI SUZUKI1, TOMOKAZU OHISHI3,4, TAKURO NAKAMURA1,MIYUKI YANAKA1, GUANJIE LI1, TOMOHIRO TANAKA1, AKIRA OHKOSHI2, MANABU KAWADA4,MIKA K. KANEKO1, YUKIO KATORI2 and YUKINARI KATO1
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Aoba‑ku, Sendai, Miyagi 980‑8575, Japan;
2

Department of Otolaryngology, Head and Neck Surgery, Tohoku University Graduate School of Medicine, Aoba‑ku, Sendai,

Miyagi 980‑8575, Japan; 3Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation,
Numazu, Shizuoka 410‑0301, Japan; 4Institute of Microbial Chemistry (BIKAKEN), Laboratory of Oncology,
Microbial Chemistry Research Foundation, Shinagawa‑ku, Tokyo 141‑0021, Japan

Received April 18, 2024; Accepted July 23, 2024
DOI: 10.3892/or.2024.8806
Abstract. CD44 is a type I transmembrane glycoprotein
associated with poor prognosis in various solid tumors. Since
CD44 plays a critical role in tumor development by regulatingcell adhesion, survival, proliferation and stemness, it has been
considered a target for tumor therapy. Anti‑CD44 mono‑clonal antibodies (mAbs) have been developed and applied to
antibody‑drug conjugates and chimeric antigen receptor‑T celltherapy. Anti-pan‑CD44 mAbs, C44Mab‑5 and C44Mab‑46,
which recognize both CD44 standard (CD44s) and variant
isoforms were previously developed. The present study gener‑
ated a mouse IgG2a version of the anti‑pan‑CD44 mAbs (5‑mG2aand C44Mab‑46‑mG2a) to evaluate the antitumor activities
against CD44‑positive cells. Both 5‑mG2a and C44Mab‑46‑mG2arecognized CD44s‑overexpressed CHO‑K1 (CHO/CD44s)
cells and esophageal tumor cell line (KYSE770) in flow
cytometry. Furthermore, both 5‑mG2a and C44Mab‑46‑mG2acould activate effector cells in the presence of CHO/CD44s
cells and exhibited complement-dependent cytotoxicity

Correspondence to: Dr Yukinari Kato, Department of Antibody
Drug Development, Tohoku University Graduate School of
Medicine, 2‑1, Seiryo‑machi, Aoba‑ku, Sendai, Miyagi 980‑8575,
Japan
E‑mail: yukinari.kato.e6@tohoku.ac.jp

Abbreviations: CD44s, CD44 standard; CD44v, CD44 variant;
HA, hyaluronic acid; mAb, monoclonal antibody; ADCC, antibodydependent cellular cytotoxicity; CDC, complement‑dependent
cytotoxicity; FcγR, Fcγ receptor; CAR, chimeric antigen receptor;
FUT8, fucosyl‑transferase 8; CHO, Chinese hamster ovary; CBIS,
Cell‑Based Immunization and Screening; ESCC, esophageal
squamous cell carcinoma; PBS, phosphate‑buffered saline
Key words: CD44, mAb, ADCC, CDC, esophageal tumor,
antitumor activity

against both CHO/CD44s and KYSE770 cells. Furthermore,
the administration of 5‑mG2a and C44Mab‑46‑mG2a signifi‑
cantly suppressed CHO/CD44s and KYSE770 xenograft
tumor development compared with the control mouse IgG2a.
These results indicate that 5‑mG2a and C44Mab‑46‑mG2a could
exert antitumor activities against CD44‑positive cancers andbe a promising therapeutic regimen for tumors.
Introduction
Overexpression of CD44 is observed in various solid tumors,which are involved in the tumor malignant progression throughthe promotion of cellular proliferation, invasiveness and stem‑
ness via specific signaling pathways (1). The diversity of CD44
molecular function is mediated by the alternative splicing (2).
CD44 is encoded in 20 exons. The first five (1‑5) and the last
five (16‑20) are constant exons that generate the shortest CD44
standard (CD44s) isoform. The exons 6‑15 are alternatively
spliced and inserted into the CD44s as variant exons (3). The
CD44 splice variants with variant exons are designated CD44
variant (CD44v) isoforms. The inclusion of CD44v exons invarious combinations is regulated by receptor tyrosine kinasesignaling and splicing activators of pre‑mRNA (1).
CD44 possesses an extracellular domain (ectodomain),a transmembrane domain, and an intracellular cytoplasmic
domain (4). The ectodomain contains a hyaluronic acid
(HA)‑binding domain (HABD) that mediates cellular homing,
adhesion, migration and proliferation (4). Both CD44s andCD44v isoforms have the HABD, and the HA binding causes
conformational changes of the CD44, which results in the
promotion of intracellular signaling pathways to regulate cell
migration and proliferation (5). In CD44v isoforms, the variant
exons‑encoding sequences form the stem region, which
provides a co‑receptor for various growth factors and cyto‑kines. These functions activate specific signaling pathways
to promote invasion and stemness (6). Therefore, different
isoforms of CD44v exhibit different functions according to theinserted variant exons in the CD44 ectodomain.

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ISHIKAWA et al: ANTITUMOR ACTIVITY OF ANTI-CD44 MAB

The relationship between CD44 expression and prognosis
in patients with cancer has been evaluated and showed bothpoor and favorable outcomes (7). Currently, increased evidence
suggests that overexpression of CD44 and its isoforms is
an unfavorable indicator in patients with cancer (7). In an
analysis of multiple studies, including 583 pancreatic cancer
cases, overexpression of CD44 was predictive of poor overall
survival, a more advanced stage and more lymph node inva‑
sion (8). Furthermore, another meta‑analysis investigated theprognostic significance of cancer stem cell markers, including
CD44, from 52 studies of ovarian cancer. The study concluded
that CD44 overexpression was predictive of worse disease‑freesurvival and resistance to chemotherapy (9).
Several anti‑CD44 monoclonal antibodies (mAbs) havebeen developed for preclinical and clinical research fortumor therapy. An anti‑pan‑CD44 mAb (H4C4) reduced
tumor growth, metastasis and post‑radiation recurrence ina human pancreatic tumor xenograft model (10). A human‑
ized anti‑pan‑CD44 mAb, RG7356, showed the cytotoxicity
for B cell leukemia but no cytotoxicity on normal B cells.
Administration of RG7356 to immunodeficient mice engraftedwith chronic lymphocytic leukemia cells resulted in complete
clearance of engrafted leukemia cells (11). Phase I clinical
trials with RG7356 were conducted in patients with acute
myeloid leukemia (12) and advanced CD44‑positive solid
tumor patients (13). Although RG7356 showed an acceptablesafety profile, the studies were not continued due to the lack ofa clinical and/or pharmacodynamic dose‑response relationshipwith RG7356 (13).
Therapies using anti‑CD44v6 mAbs were consideredsince CD44v6 plays critical roles in the malignant progres‑sion of tumors (14). Humanized anti‑CD44v6 mAbs, including
BIWA‑4 and BIWA‑8, were developed. These mAbs labeledwith 186Re were evaluated in head and neck squamous cell
carcinoma xenograft‑bearing mice and exhibited therapeutic
efficacy (15). Furthermore, the BIWA‑4 was developed into ahumanized version‑drug conjugate, bivatuzumab‑mertansine(anti‑tubulin agent), which was evaluated in clinical trials (16).
However, the clinical trials were terminated because of thesevere skin toxicity, such as lethal epidermal necrolysis (17).
Since CD44v6 is expressed in normal skin epithelium, thetoxicity of mertansine in the skin was most likely responsible
for the high toxicity (17,18). Previously, a mutated version of
BIWA‑4, called BIWA‑8, was developed for increasing binding
affinity by two amino acid substitutions of the light chain (15).
The BIWA‑8 was further developed to chimeric antigen recep‑
tors (CARs). The BIWA‑8 CAR‑T showed antitumor activities
against multiple myeloma or acute myeloid leukemia engraftedwith immunodeficient mice (19). Furthermore, the BIWA‑8
CAR‑T exhibited efficacy in lung and ovarian carcinomas
xenograft models (20), which is expected for an application
toward solid tumors.
In our previous studies, an anti‑pan‑CD44 mAb,
C44Mab‑5 (IgG1, kappa) was developed using the Cell‑Based
Immunization and Screening (CBIS) method (21). AnothermAb (C44Mab‑46) (22) was created by immunization of theCD44v3‑10 ectodomain. Both C 44Mab‑5 and C 44Mab‑46
have the epitopes within the constant exon 2‑ and 5‑encoded
sequences (23‑25) and could be applied to immunohis‑
tochemistry in oral squamous cell carcinoma (21) andesophageal squamous cell carcinoma (ESCC) (22), respectively.
Furthermore, various anti‑CD44v mAbs have been developed,
such as C44Mab‑6 (an anti‑CD44v3 mAb) (26), C44Mab‑108(an anti‑CD44v4 mAb) (27), C 44Mab‑3 (an anti‑CD44v5
mAb) (28), C44Mab‑9 (an anti‑CD44v6 mAb) (29), C44Mab‑34(an anti‑CD44v7/8 mAb) (30), C44Mab‑1 (an anti‑CD44v9
mAb) (31) and C44Mab‑18 (an anti‑CD44v10 mAb) (32). The
combinational use of the anti‑CD44 mAbs is essential forcomprehensively analyzing human tumors.
In the present study, a mouse IgG2a type of recombinant
C 44Mab‑5 (5‑mG 2a) and C 44Mab‑46 (C 44Mab‑46‑mG 2a)
was produced using fucosyl‑transferase 8 (FUT8)‑deficient
ExpiCHO‑S cells and the antitumor activity in xenograftbearing mice was investigated.
Materials and methods
Cell lines. ESCC cell line KYSE770 was obtained from theJapanese Collection of Research Bioresources. Chinese
hamster ovary (CHO)‑K1 was obtained from the American
Type Culture Collection. CHO/CD44s was previously estab‑lished by transfecting pCAG‑Ble/PA16‑CD44s into CHO‑K1
cells (22). KYSE770 was cultured in Dulbecco's Modified
Eagle's Medium (DMEM; Nacalai Tesque, Inc.) supplemented
with 10% (v/v) heat‑inactivated fetal bovine serum (FBS;
Thermo Fisher Scientific, Inc.), 0.25 µg/ml amphotericin B,
100 µg/ml streptomycin, and 100 U/ml penicillin (Nacalai
Tesque, Inc.). CHO‑K1 was cultured in Roswell Park Memorial
Institute (RPMI)‑1640 medium (Nacalai Tesque, Inc.) supple‑
mented with 10% (v/v) FBS, 0.25 µg/ml amphotericin B,
100 µg/ml streptomycin and 100 U/ml penicillin (RPMI‑1640
complete medium). CHO/CD44s were cultured in RPMI‑1640
complete medium containing 0.5 mg/ml Zeocin (InvivoGen).
All cells were cultured in a humidified incubator at 37˚C with
5% CO2.
Antibodies. Anti‑pan‑CD44 mAbs, C44Mab‑5 and C44Mab‑46
were previously established (21,22). To generate recombinantmAbs, VH cDNAs of C44Mab‑5 or C44Mab‑46 and CH of mouse
IgG2a were cloned into the pCAG‑Ble vector (FUJIFILM
Wako Pure Chemical Corporation). VL cDNA of C44Mab‑5 or
C44Mab‑46 and CL cDNA of mouse kappa light chain werealso subcloned into the pCAG‑Neo vector (FUJIFILM Wako
Pure Chemical Corporation). The vectors were transduced
into BINDS‑09 (FUT8‑knockout ExpiCHO‑S) cells (33).
5‑mG2a and C44Mab‑46‑mG2a were purified using Ab‑Capcher(ProteNova Co., Ltd.). 281‑mG2a (an anti‑hamster podoplaninmAb, control mouse IgG2a) was previously described (34).
Flow cytometry. The cells, obtained using 0.25% trypsin
and 1 mM ethylenediamine tetraacetic acid (EDTA; Nacalai
Tesque, Inc.), were treated with 5‑mG2a and C44Mab‑46‑mG2a,or control blocking buffer [phosphate‑buffered saline (PBS)
containing 0.1% bovine serum albumin (Nacalai Tesque,Inc.)] for 30 min at 4˚C. Subsequently, the cells were treatedwith anti‑mouse IgG conjugated with Alexa Fluor 488(1:2,000; Cell Signaling Technology, Inc.) for 30 min at 4˚C.
Fluorescence data were collected using the SA3800 Cell
Analyzer (Sony Corp.) and analyzed using SA3800 softwarever. 2.05 (Sony Corp.).

ONCOLOGY REPORTS 52: 147, 2024

Antibody-dependent cellular cytotoxicity (ADCC) reporter
bioassay. An ADCC Reporter Bioassay kit (Promega
Corporation) was used for the ADCC reporter bioassay.
The human Fc γRIIIa receptor and a nuclear factor of
activated T‑cells (NFAT) response element driving firefly
luciferase‑expressed Jurkat cells were used as effector cells.
The target CHO/CD44s and KYSE770 cells (12,500 cells perwell) were seeded into a 96‑well white solid plate. The seriallydiluted 5‑mG2a and C44Mab‑46‑mG2a were added to the target
cells. The effector cells (75,000 cells in 25 µl) were then added
and co‑cultured with antibody‑treated target cells at 37˚C for
6 h. Luminescence was measured with a GloMax luminometer(Promega Corporation).
Complement-dependent cytotoxicity (CDC). The target
CHO/CD44s and KYSE770 cells were labeled using 10 µg/ml
Calcein AM (Thermo Fisher Scientific, Inc.). The target cells(1x10 4 cells/well) were mixed with 100 µg/ml of control
281‑mG2a, 5‑mG2a, or C44Mab‑46‑mG2a and rabbit complement(final dilution 1:10, Low‑Tox‑M Rabbit Complement; Cedarlane
Laboratories). The calcein release to the medium was measured
after a 4‑h incubation. The maximum fluorescence of themedium was also measured after lysing all cells with a buffer
containing 0.5% Triton X‑100, 10 mM Tris‑HCl (pH 7.4), and10 mM EDTA. Cytotoxicity (% of lysis) was calculated as %lysis=(E‑S)/(M‑S) x100. E is the fluorescence of combined
target and effector cells. S is the spontaneous fluorescence oftarget cells only. M is the maximum fluorescence measured.
Statistical analyses were performed using GraphPad PRISM 6
software (GraphPad Software, Inc.; Dotmatics).
Antitumor activity of 5‑mG2a and C44Mab‑46‑mG2a in xenografts of CHO/CD44s and KYSE770. All animal experiments
were performed following regulations and guidelines tominimize animal distress and suffering in the laboratory by
the Institutional Committee for Experiments of the Instituteof Microbial Chemistry (Numazu, Japan). The animal study
protocol was approved (approval nos. 2023‑037 and 2023‑054)
by the Institutional Committee for Experiments of the Instituteof Microbial Chemistry (Numazu, Japan). BALB/c nude mice
(5‑week‑old female, a total of 72 mice) were purchased from
Jackson Laboratories, Inc. and maintained on an 11‑h light/13‑hdark cycle at a temperature of 23±2˚C and 55±5% humidity ina specific pathogen‑free environment, across the experimental
period. Food and water were supplied ad libitum. The weightof the mice was monitored twice per week and their health wasmonitored three times per week. CHO/CD44s and KYSE770
cells (5x106 cells) suspended with BD Matrigel Matrix Growth
Factor Reduced (BD Biosciences) were inoculated into theleft flank of the mice subcutaneously. On day 7 after the
inoculation, 100 µg of 5‑mG2a (n=8), C44Mab‑46‑mG2a (n=8),
or control 281‑mG2a (n=8) in 100 µl PBS were injected intra‑
peritoneally. Additional antibody injections were performedon days 14 and 21. The tumor volume was measured on days
7, 9, 14, 17, 21 and 23. In the KYSE770 xenograft experiment(500 µg dosage), 500 µg of 5‑mG2a (n=8), C44Mab‑46‑mG2a(n=8), or control 281‑mG2a (n=8) in 100 µl PBS were injected
intraperitoneally on days 8 and 13 after the inoculation. The
tumor volume was measured on days 8, 12, and 19. The tumor
volume was calculated using the formula: Volume=W2 x L/2,

3

where W is the short diameter and L is the long diameter.
The loss of original body weight was determined to a point>25% (35) and/or a maximum tumor size >3,000 mm3 and/or
significant changes in the appearance of tumors as humane
endpoints for euthanasia. Cervical dislocation was used foreuthanasia. Mice death was confirmed by respiratory arrest
and rigor mortis. The xenograft tumors were carefully removed
from the sacrificed mice and weighed immediately.
Immunohistochemical analysis. The paraffin‑embedded
xenograft tumors were autoclaved in citrate buffer (pH 6.0;
Nichirei Biosciences, Inc.) for 20 min. After blocking with
SuperBlock T20 (Thermo Fisher Scientific, Inc.), sections(4 µm) were incubated with 1 µg/ml of C 44Mab‑46 andmPMab‑1 [a mouse‑rat chimeric antibody against mouse
podoplanin (36)] for 1 h at room temperature and then treatedwith the EnVision+ Kit for mouse (Agilent Technologies,Inc.) for 30 min. The color was developed using 3,3'‑diami‑
nobenzidine tetrahydrochloride (DAB; Agilent Technologies,Inc.). Counterstaining was performed with hematoxylin(Merck KGaA). Hematoxylin and eosin (H&E) staining was
performed using hematoxylin and eosin (FUJIFILM Wako
Pure Chemical Corporation). Leica DMD108 fluorescencemicroscope (Leica Microsystems GmbH) was used to examine
the sections and obtain images.
Statistical analyses. All data are expressed as the mean ± stan‑
dard error of the mean (SEM). Two‑way ANOVA with Tukey's
multiple comparisons test and two‑way ANOVA with Sidak's
multiple comparisons tests were conducted in CDC andtumor weight measurement, respectively. Two‑way ANOVA
with Sidak's multiple comparisons test was utilized for tumor
volume and mice weight. GraphPad Prism 6 (GraphPad
Software, Inc.; Dotmatics) was used for all calculations. P<0.05
was considered to indicate a statistically significant difference.
Results
Flow cytometric analysis against CHO/CD44s and KYSE770
cells using 5‑mG2a and C44Mab‑46‑mG2a. In our previous
study, anti‑pan‑CD44 mAbs (C44Mab‑5 and C44Mab‑46) were
established and were shown to be available for flow cytometry
and immunohistochemistry (21,22). In the present study, amouse IgG2a type of C44Mab‑5 and C44Mab‑46 (5‑mG2a and
C44Mab‑46‑mG2a) were produced by combining V H and V Lof both mAbs with CH and CL of mouse IgG2a, respectively(Fig. 1A). The reactivity of CD44s‑overexpressed CHO‑K1
cells (CHO/CD44s) and an endogenous CD44‑expressing
esophageal tumor cell line (KYSE770) was first confirmed.
5‑mG2a and C 44Mab‑46‑mG2a detected CHO/CD44s in
a concentration‑dependent manner (Fig. 1B), but did notdetect parental CHO‑K1 cells (negative control, Fig. S1).
5‑mG2a and C44Mab‑46‑mG2a also detected KYSE770 cells
in a concentration‑dependent manner (Fig. 1C). KYSE770
cells were also recognized by anti‑CD44v mAbs including
C44Mab‑6 (an anti‑CD44v3 mAb), C44Mab‑3 (an anti‑CD44v5
mAb), C44Mab‑9 (an anti‑CD44v6 mAb), and C 44Mab‑18(an anti‑CD44v10 mAb) (Fig. S2). These results indicatedthat 5‑mG2a and C44Mab‑46‑mG2a recognize exogenous andendogenous CD44.

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ISHIKAWA et al: ANTITUMOR ACTIVITY OF ANTI-CD44 MAB

Figure 1. Flow cytometry using 5‑mG2a and C44Mab‑46‑mG2a. (A) Class‑switched and defucosylated mouse IgG2a mAbs, 5‑mG2a and C44Mab‑46‑mG2a, weregenerated from C44Mab‑5 and C44Mab‑46 (mouse IgG1), respectively. (B) CHO/CD44s and (C) KYSE770 cells were treated with 10‑0.01 µg/ml of 5‑mG2a and
C44Mab‑46‑mG2a (red) or buffer control (black), followed by Alexa Fluor 488‑conjugated anti‑mouse IgG. Fluorescence data were analyzed using the SA3800
Cell Analyzer. mAb, monoclonal antibody.

ONCOLOGY REPORTS 52: 147, 2024

5

Figure 2. Evaluation of ADCC activity. The ADCC reporter assay by 5‑mG2a and C44Mab‑46‑mG2a in the presence of (A) CHO/CD44s and (B) KYSE770 cells.
Values are presented as the mean ± SD. N.D., not determined; ADCC, antibody‑dependent cellular cytotoxicity; mAb, monoclonal antibody.

5‑mG 2a and C 44 Mab‑46‑mG 2a ‑mediated ADCC pathway
activation in the presence of CHO/CD44s and KYSE770 cells.
To compare the ADCC pathway activation by 5‑mG2a and
C44Mab‑46‑mG2a, the ADCC reporter bioassay was performed
to measure the biological activity of the FcγRIIIa‑mediatedpathway activation by mAbs (37). CHO/CD44s cells weretreated with diluted mAbs and then incubated with effector cells,
which possess the human FcγRIIIa receptor and Firefly lucif‑
erase regulated by an NFAT response element. As demonstrated
in Fig. 2A, 5‑mG2a and C44Mab‑46‑mG2a activated the effector
in a concentration‑dependent manner (EC50; 6.4x10‑8 g/ml and5.4x10‑8 g/ml, respectively). In the presence of KYSE770 cells,
5‑mG2a activated the effector (EC50; 2.5x10 ‑7 g/ml, Fig. 2B).
However, C44Mab‑46‑mG2a did not (Fig. 2B).
The CDC by 5‑mG 2a and C 4 4 Mab‑ 46 ‑mG 2a against
CHO/CD44s and KYSE770 cells. The CDC mediated by
5‑mG2a and C 44Mab‑46‑mG2a against CHO/CD44s andKYSE770 cells was next examined. As shown in Fig. 3,
both 5‑mG2a and C 44Mab‑46‑mG2a significantly exerted
CDC against CHO/CD44s cells (67 and 88% cytotoxicity,respectively) and KYSE770 (27 and 31% cytotoxicity, respec‑tively) compared with those induced by control mouse IgG2a(281‑mG2a). These results demonstrated that 5‑mG2a and
C44Mab‑46‑mG2a exhibited potent CDC activities against
CHO/CD44s and KYSE770 cells.

Figure 3. Evaluation of CDC activity by 5‑mG2a and C 44Mab‑46‑mG2aagainst CHO/CD44s and KYSE770 cells. The CDC induced by 5‑mG2a,
C44Mab‑46‑mG2a, or control mouse IgG2a (281‑mG2a) against (A) CHO/CD44s
and (B) KYSE770 cells. Values are shown as the mean ± SEM. *P<0.05 and**P<0.01; one‑way ANOVA with Tukey's multiple comparisons test. CDC,
complement‑dependent cytotoxicity; mAb, monoclonal antibody.

Antitumor effects of 5‑mG2a and C44Mab‑46‑mG2a against
CHO/CD44s xenograft. In the CHO/CD44s xenograft tumors,
100 µg of 5‑mG2a, C44Mab‑46‑mG2a, or control mouse IgG2awere injected into mice intraperitoneally on days 7, 14 and21, following CHO/CD44s inoculation. On days 7, 9, 14, 17,
21 and 23, the tumor volume was measured. The 5‑mG2a and
C44Mab‑46‑mG2a administration resulted in a significant
reduction in tumor volume on days 17 (P<0.05), 21 (P<0.01)
and 23 (P<0.01) compared with that of the control mouse

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ISHIKAWA et al: ANTITUMOR ACTIVITY OF ANTI-CD44 MAB

Figure 4. Antitumor activity of 5‑mG2a and C44Mab‑46‑mG2a against CHO/CD44s xenograft tumor. (A) Measurement of tumor volume in CHO/CD44s
xenograft. CHO/CD44s cells (5x106 cells) were injected into mice subcutaneously. On day 7, 100 µg of 5‑mG2a and C44Mab‑46‑mG2a or control mouse IgG2a(281-mG2a) were injected into mice intraperitoneally. On days 14 and 21, additional antibodies were injected. On days 7, 9, 14, 17, 21 and 23 following the inocu‑
lation, the tumor volume was measured. Values are presented as the mean ± SEM. *P<0.05 and **P<0.01; two‑way ANOVA with Sidak's multiple comparisonstest. (B) The weight (left) and appearance (right) of excised CHO/CD44s xenografts on day 23. Values are presented as the mean ± SEM; one‑way ANOVA
with Tukey's multiple comparisons test. (C) The body weight (left) and appearance (right) of control mouse IgG2a, 5‑mG2a, and C44Mab‑46‑mG2a‑treatedmice. Scale bar, 1 cm. mAb, monoclonal antibody.

IgG2a (Fig. 4A). The 5‑mG2a and C44Mab‑46‑mG2a adminis‑
tration resulted in 46 and 48% reductions in tumor volume
compared with that of the control mouse IgG2a on day 23,

respectively. The histological analyses of tumors are demon‑
strated in Fig. S3. In the necrotic area of tumors, leukocytes
and erythrocytes were infiltrated. Membranous staining of

ONCOLOGY REPORTS 52: 147, 2024

CD44 in tumor and podoplanin‑positive macrophage werealso detected. However, a significant difference among control,
5‑mG2a and C44Mab‑46‑mG2a‑treated tumors could not be
observed.
The weight of CHO/CD44s tumors treated with 5‑mG2aand C44Mab‑46‑mG2a was lower than that of tumors treatedwith the control mouse IgG2a (53 and 55% reduction, respec‑tively; P=0.11 and P=0.09; Fig. 4B). Loss of body weight was
not observed in the CHO/CD44s tumor‑implanted mice during
the treatments (Fig. 4C).
Antitumor effects of 5‑mG 2a and C 44 Mab‑46‑mG 2a on
KYSE770 xenografts. In the KYSE770 xenograft models,
100 µg of 5‑mG2a and C44Mab‑46‑mG2a and control mouse
IgG2a were injected into mice on days 7, 14 and 21, following
KYSE770 inoculation. Although the tendency of the reduc‑tion of tumor volume by the treatment with 5‑mG2a and
C44Mab‑46‑mG2a was observed, significant differences were
not obtained (Fig. S4). The dosage (500 µg) was next increased
and the antitumor effects were investigated. As revealed inFig. 5, 500 µg of 5‑mG2a, C44Mab‑46‑mG2a and control mouse
IgG2a were injected into mice on days 8 and 13, following
KYSE770 inoculation. On days 8, 12 and 19, the tumor volume
was measured. The 5‑mG2a and C44Mab‑46‑mG2a administra‑tion resulted in a significant reduction in tumor volume on day
19 (P<0.01) compared with that of the control mouse IgG2a(Fig. 5A). The 5‑mG2a and C44Mab‑46‑mG2a administration
resulted in 51 and 37% reduction of tumor volume comparedwith that of the control mouse IgG2a on day 19, respectively.
The histological analyses of tumors are demonstrated inFig. S5. Membranous staining of CD44 in tumors andpodoplanin‑positive macrophages at the tumor periphery wereobserved. Infiltrated podoplanin‑positive macrophages into
tumors were not observed compared with KYSE770 xenograft.
The weight of KYSE770 tumors treated with 5‑mG2a and
C44Mab‑46‑mG2a was significantly lower than that of tumors
treated with the control mouse IgG2a [47% (P<0.05) and 40%(P<0.05) reduction, respectively; Fig. 5B]. Loss of body weight
was not observed in the KYSE770 tumor‑implanted mice
during the treatments (Fig. 5C).
Discussion
In the present study, mouse IgG2a type anti‑pan‑CD44
mAbs (5‑mG2a and C 44Mab‑46‑mG2a) was produced andantitumor activity against CHO/CD44s and KYSE770
xenograft tumors was evaluated. Although 5‑mG2a possesses
superior affinity to CD44‑expressing cells compared withC44Mab‑46‑mG2a (22,38), both 5‑mG2a and C44Mab‑46‑mG2ashowed comparable ability of effector activation (Fig. 2A),
CDC (Fig. 3A) and antitumor activity against CHO/CD44s(Fig. 4). Against KYSE770 cells, 5‑mG2a exhibited superior
reactivity in flow cytometry (Fig. 1C) and effector activation(Fig. 2B) compared with C44Mab‑46‑mG2a. However, bothmAbs showed similar antitumor activity to KYSE770 xeno‑
graft in high dosage (Fig. 5).
After the phase I trial of RG7356 in solid tumors, thedeveloper Roche group reported that the CD44s expression
is associated with HA production and predicts response totreatment with RG7356 in both xenograft tumor models

7

and clinical response (39). The group also identified thatoverexpression of CD44s stimulates the HA production (39).
These results suggest the close interplay between CD44s and
HA and a potential biomarker to enrich patient responses toanti‑CD44 mAb therapy in the clinic. In the present study,
CD44s‑overexpressed CHO‑K1 cells and KYSE770 cells wereused, which mainly express CD44v (Fig. S2) (22). Due to thelower levels of total CD44 (Fig. 1) and the CD44v expression
in KYSE770 cells, a high dosage (500 µg) of mAbs was needed
to suppress the KYSE770 xenograft.
As revealed in Fig. 2B, C44Mab‑46‑mG2a did not activate
the effector cells in the presence of KYSE770 but exerted
the antitumor effect in vivo (Fig. 5). The CDC is a crucial
antitumor mechanism by mAbs (40,41). Some tendency wasobserved that C44Mab‑46‑mG2a showed a more significant
CDC effect compared with 5‑mG2a (Fig. 3). The difference
could be sufficient to exert similar antitumor effects in vivoby 5‑mG2a and C44Mab‑46‑mG2a. There was less infiltrated
podoplanin‑positive macrophage into KYSE770 xenograftcompared with CHO/CD44 (Figs. S3 and S5). Since theinfiltrated effector cells exert ADCC activity (42), this may
be one of the reasons why ADCC did not contribute mark‑edly in KYSE770 cells. One more possibility is their epitope.
5‑mG2a recognizes the N‑terminal part of CD44 [amino acids
25‑36, (25)], and C44Mab‑46‑mG2a recognizes the central
part of CD44 [amino acids 174‑178, (23,24)]. Furthermore,
IgG antibodies can form ordered hexamers upon binding totheir antigen on cell surfaces. These hexamers efficiently bindthe hexavalent complement component C1q, the first step inthe classical pathway of complement activation (43,44). The
structure of C44Mab‑46‑mG2a‑CD44 complex may provide theadequate access of complements to exert CDC.
Complement has been considered as an adjunctive compo‑
nent that potentiates the antibody‑mediated cytolytic effects.
However, complement is currently considered an essential
effector of tumor cytotoxic responses of antibody‑based
immunotherapy, which is guiding new therapeutic options (41).
An increasing body of evidence suggests that complement
plays critical roles in not only mAb‑mediated tumor cytolysis
but also several immunomodulatory functions in tumor
immunosurveillance and antitumor immunity (45,46). The
complicated crosstalk of complement effectors with cellularpathways that drive B cell and T cell responses influences
T helper/effector T cell survival, differentiation and B cell
activation. Therefore, the involvement of complement in our
experimental system and/or immunocompetent mouse models
should be investigated in future studies.
Most therapeutic mAbs exhibit adverse effects by recog‑
nizing antigens in normal cells (47). Clinical trials of ananti‑CD44v6 mAb (BIWA‑4) bivatuzumab‑mertansine drug
conjugate to solid tumors failed because of the skin toxici‑
ties (17,18). Therefore, cancer‑selective or specific mAbs would
reduce the adverse effects. Cancer‑specific mAbs (CasMabs)
against HER2 [H 2Mab‑214 (48) and H 2Mab‑250 (49)] havebeen developed by the authors and the reactivity to cancer
and normal cells has been evaluated using flow cytometry.
The antitumor effect in mouse xenograft models has been also
reported using a mouse IgG2a or human IgG1 types recombi‑
nant mAbs (50). Some anti‑CD44 mAbs which exhibit cancer
specificity have been reported (51). Among them, the 4C8

8

ISHIKAWA et al: ANTITUMOR ACTIVITY OF ANTI-CD44 MAB

Figure 5. Antitumor activity of 5‑mG2a and C44Mab‑46‑mG2a against KYSE770 xenograft tumor. (A) Measurement of tumor volume in KYSE770 xenograft.
KYSE770 cells (5x106 cells) were injected into mice subcutaneously. On day 8, 500 µg of 5‑mG2a and C44Mab‑46‑mG2a or control mouse IgG2a (281‑mG2a) were
injected into mice intraperitoneally. On day 13, additional antibodies were injected. On days 8, 12, and 19 following the inoculation, the tumor volume wasmeasured. Values are presented as the mean ± SEM. **P<0.01; two‑way ANOVA with Sidak's multiple comparisons test. (B) The weight (left) and appearance(right) of the excised KYSE770 xenografts on day 19. Values are presented as the mean ± SEM. *P<0.05; one‑way ANOVA with Tukey's multiple comparisonstest. (C) The body weight (left) and appearance (right) of control mouse IgG2a, 5‑mG2a and C44Mab‑46‑mG2a‑treated mice. Scale bar, 1 cm. mAb, monoclonal
antibody.

ONCOLOGY REPORTS 52: 147, 2024

mAb recognizes aberrantly O‑glycosylated CD44v6 with Tn(GalNAca1‑O‑Ser/Thr) antigen. The 4C8 chimeric antigen
receptor (CAR)‑T cells demonstrated target‑specific cyto‑
toxicity in vitro, significant tumor regression, and prolonged
survival in vivo (52). In our CasMab development against
CD44, already established anti‑CD44 mAbs were screened
by comparing the reactivity against cancer and normal cells.
The CasMabs against CD44 could be applicable for designingmodalities such as antibody‑drug conjugates and CAR‑T cells.
Acknowledgements
The authors thank Mr Shun‑ichi Ohba and Ms Akiko
Harakawa [Institute of Microbial Chemistry (BIKAKEN),
Numazu, the Microbial Chemistry Research Foundation] fortechnical assistance with animal experiments.
Funding
The present study was supported in part by Japan Agency
for Medical Research and Development (AMED) (grantnos. JP23ama121008, JP24am0521010, JP23bm1123027 andJP23ck0106730) and by the Japan Society for the Promotionof Science (JSPS) Grants‑in‑Aid for Scientific Research(KAKENHI) (grant nos. 22K06995, 21K20789, 21K07168 and
22K07224).
Availability of data and materials
The data generated in the present study are included in the
figures and/or tables of this article.
Authors' contributions
KI, HS, TO, TN, MY, GL and TT performed the experiments.
MK, MKK and YKato designed the experiments. KI, HS, AO,
YKatori and YKato engaged the analysis and interpretation ofdata. KI, HS, and YKato wrote the manuscript. HS and YKato
confirm the authenticity of all the raw data. All authors readand approved the final version of the manuscript.
Ethics approval and consent to participate
Animal experiments were approved (approval nos. 2023‑037
and 2023‑054) by the Institutional Committee for Experimentsof the Institute of Microbial Chemistry (Numazu, Japan).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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