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Oncotarget, 2018, Vol. 9, (No. 32), pp: 22480-22497
Research Paper

Anti-podocalyxin antibody exerts antitumor effects via antibodydependent cellular cytotoxicity in mouse xenograft models oforal squamous cell carcinoma
Shunsuke Itai1,2, Tomokazu Ohishi3, Mika K. Kaneko1, Shinji Yamada1, Shinji Abe4,5,
Takuro Nakamura1, Miyuki Yanaka1, Yao-Wen Chang1, Shun-Ichi Ohba3, Yasuhiko
Nishioka5, Manabu Kawada3, Hiroyuki Harada2 and Yukinari Kato1,6
1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi 9808575, Japan2

Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental
University, Bunkyo-ku, Tokyo 113-8510, Japan

3

Institute of Microbial Chemistry, BIKAKEN, Numazu, Microbial Chemistry Research Foundation, Numazu-shi, Shizuoka 4100301, Japan

4

Department of Clinical Pharmacy Practice Pedagogy, Graduate School of Biomedical Sciences, Tokushima University,
Tokushima 770-8505, Japan

5

Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University,
Tokushima 770-8503, Japan

6

New Industry Creation Hatchery Center, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8575, Japan

Correspondence to: Yukinari Kato, email: yukinarikato@med.tohoku.ac.jp, yukinari-k@bea.hi-ho.ne.jp
Keywords: podocalyxin; PODXL; monoclonal antibody; antibody-dependent cellular cytotoxicity; oral squamous cell carcinoma
Received: October 26, 2017     Accepted: March 24, 2018     Published: April 27, 2018
Copyright: Itai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0(CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited.

ABSTRACT
Podocalyxin (PODXL) overexpression is associated with progression, metastasis,and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal
antibody (mAb) PcMab-47 (IgG1, kappa). Herein, we engineered PcMab-47 into 47mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity(ADCC). We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a
to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using
PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma(OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and
47-mG2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples,
respectively. 47-mG2a-f also detected PODXL in OSCCs at a similar frequency as 47mG2a. In vitro analysis revealed that both 47-mG2a and 47-mG2a-f exhibited strong
complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast,
47-mG2a-f exhibited much stronger ADCC than 47-mG2a against OSCC cells, indicatingthat ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis
revealed that both 47-mG2a and 47-mG2a-f exerted antitumor activity in CHO/hPODXL
xenograft models at a dose of 100 μg or 500 μg/mouse/week administered twice.
47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and HSC-2 xenograftmodels at a dose of 100 μg/mouse/week administered three times. Although both
47-mG2a and 47-mG2a-f exerted antitumor activity in HSC-2 xenograft models at a doseof 500 μg/mouse/week administered twice, 47-mG2a-f also showed higher antitumor
activity than 47-mG2a. These results suggested that a core fucose-deficient anti-PODXL
mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.

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INTRODUCTIONPODXL has been suggested to promote tumor growth,invasion, and metastasis [30, 31]; accordingly, high
PODXL expression could have adverse effects on overall
survival (OS), disease-specific survival (DSS), anddisease-free survival (DFS) in several cancers. PODXL isoverexpressed in the aforementioned cancers, identifying
the protein as a potential target for antibody therapy.
Despite the development of anti-PODXL monoclonal
antibodies (mAbs) [32, 33], the efficacy of these treatments
against oral cancers remains to be fully elucidated. We
previously immunized mice with recombinant PODXL,
which was purified from the culture supernatant of LN229/
ectodomain-PODXL cells [17]. One clone, PcMab-47(mouse IgG1, kappa), was successfully produced. We further
produced chPcMab-47 from PcMab-47 and investigated its
antitumor activity against colorectal cancers [34]. In thosestudies, we injected human NK cells around the tumors toinvestigate antitumor activity because chPcMab-47 could
not induce ADCC activity using mouse NK cells. Although
chPcMab-47 significantly reduced tumor development
compared with the effects of control human IgG, its
antitumor activity might not be sufficient for antibodybased target therapy. We used human NK cells from one
donor; therefore, the cause of the low antitumor activity is
due to differences in the sources of cells. Because we had to
inject human NK cells around the subcutaneous tumors of
xenograft models several times, we experienced difficulties
in measuring tumor diameters because the tumor shapesometimes changed post-injection. Furthermore, mouse
IgG1 does not induce ADCC and CDC, and PcMab-47 wasdetermined to be IgG1. In this study, we established the
47-mG2a, a chimeric anti-PODXL antibody by combining
variable region of PcMab-47 and constant region of mouse
IgG2a. We further produced 47-mG2a-f, a core fucosedeficient 47-mG2a to analyze antitumor activity in xenograftmodels [35].

In total, 300,000 cases of oral cancer are reported
annually, constituting approximately 2% of all cancer
cases globally [1], and several histological tumor types
exist, including squamous cell carcinoma (OSCC),
adenoid cystic carcinoma, mucoepidermoid carcinoma,
and osteosarcoma. Among them, almost 90% of oral
cancers are OSCCs [2]. The most common location of
OSCC is the tongue, accounting for approximately 40%
of OSCCs [3, 4]. Despite improvements in diagnostic
technology and therapeutic techniques, the survival rateof OSCC has improved by only 5% over the past 20 years.
Consequently, the 5-year survival rate is 60% [5], and theincidence of OSCC has increased globally [4, 6].
OSCC is mainly treated via surgical removal,
which can be complemented by radiotherapy and/or
chemotherapy, especially in advanced stages. Many
types of anticancer agents, including cisplatin (CDDP),
5-fluorouracil (5-FU), and docetaxel, are used forchemotherapy [7, 8]. In contrast, the availability of
approved molecular targeting drugs with efficacy against
OSCC is limited. Recently, cetuximab, a mouse-human(IgG1) chimeric antibody against epidermal growth
factor receptor (EGFR), was approved for treating headand neck cancer (HNC) including oral cancer. In several
clinical studies, cetuximab was found to be effective
against locoregionally advanced head and neck squamouscell carcinoma (HNSCC) or recurrent and/or metastatic(R/M) HNSCC [7, 9-11]. Furthermore, nivolumab, a fully
human IgG4 mAb against programmed cell death-1, wasalso approved for the treatment of R/M HNC, which has
been treated with platinum-based chemotherapy [12]. In
addition, bevacizumab, which is a mouse-human IgG1chimeric antibody against vascular endothelial growth
factor (VEGF) and first approved for colorectal cancer,
had some clinical trial in which R/M HNSCC patientswere enrolled [13]. Because molecular targeting drugs thatare clinically applicable for oral cancers are limited, novel
drugs with greater efficacy and lower toxicity are required.
Therefore, we have been investigating several membraneproteins, and we developed several mAbs against thosetargets, including HER2 [14], EGFR [15], podoplanin[16], and PODXL [17].
Podocalyxin (PODXL) is a type I transmembraneprotein with a molecular weight of 150,000–200,000[18-20], and it is also known as PCLP, MEP21, Gp200,
Gp135, thrombomucin, GCTM2, TRA-1-60 antigen, andTRA-1-81 antigen. PODXL is highly glycosylated with
N- and O-glycans [21]. It can be found in kidney, heart,pancreatic, and breast tissues, and it plays important rolesin the development of some tissues [22]. PODXL is alsoa pluripotent stem cell marker [23-26]. Furthermore, it isa diagnostic marker and a prognostic indicator for certain
cancers such as brain tumors [21], colorectal cancer[27], renal cancer [28], and oral cancer [29]. Moreover,
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RESULTS
Production of the mouse IgG2a-type antibody
PcMab-47
In this study, we first produced a mouse IgG2a-typeversion of PcMab-47 by subcloning appropriate VH and
VL cDNAs of PcMab-47 and CH and CL of mouse IgG2a
into pCAG vectors because mouse IgG2a possess high
ADCC and CDC activities (Figure 1) [36]. The IgG2a-type
PcMab-47 was designated 47-mG2a.
We further produced 47-mG2a-f (Figure 1).
Defucosylation was confirmed using lectins such as
Aleuria aurantia lectin (AAL, fucose binder) [37] andPholiota squarrosa lectin (PhoSL, core fucose binder) [38].
Concanavalin A (ConA, mannose binder) [39] was used asa control. Both 47-mG2a and 47-mG2a-f were detected using
ConA (Figure 2A). 47-mG2a, but not 47-mG2a-f, was detectedusing AAL and PhoSL (Figure 2A), indicating that 47-mG2a-f
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The binding affinity of mouse IgG2a-type
PcMab-47

was defucosylated. We also confirmed the defucosylation
using a lectin microarray (Figure 2B). Although 47-mG2a wasrecognized by core fucose binders such as Aspergillus oryzae
lectin (AOL) [40], AAL, and Pisum sativum agglutinin (PSA)[41], these binders did not detect 47-mG2a-f. 47-mG2a wasstrongly detected using Lens culinaris agglutinin (LCA,
core fucose and agalactosylated N-glycan binder) [42]; in
contrast, 47-mG2a-f was moderately detected. Both 47-mG2aand 47-mG2a-f were detected using ConA. These results
indicate that 47-mG2a-f was defucosylated. All anti-PODXL
antibodies reacted with CHO/hPODXL cells, but not withparental CHO-K1 cells (Figure 2C).
We confirmed the PODXL expression in OSCC
cell lines such as HSC-2, HSC-3, HSC-4, Ca9-22, HO1-u-1, and SAS cells using RT-PCR (data not shown). We
examined the sensitivity of 47-mG2a against these OSCC
cell lines using flow cytometry. As shown in Figure 3A,
IgG1-type PcMab-47 recognized endogenous PODXL,
which is expressed in OSCC cell lines such as HSC-2, HSC3, HSC-4, Ca9-22, HO-1-u-1, and SAS cells. PcMab-47 has
weaker reactivity against HO-1-u-1 cells than against theother cell lines. The mouse-human chimeric chPcMab-47
reacted with OSCC cells similarly as PcMab-47 (Figure
3B). Furthermore, 47-mG2a and 47-mG2a-f exhibited similar
reactivity against OSCC cell lines (Figure 3C and 3D). 47mG2a and 47-mG2a-f exhibited greater reactivity against HO1-u-1 cells, indicating that 47-mG2a and 47-mG2a-f are moresensitive for PODXL than PcMab-47. Polyclonal antibody
against PODXL reacted with all OSCC cell lines although
the reactivity was lower than PcMab-47 (Figure 3E).
Another anti-PODXL mAb (clone 53D11) reacted them inthe similar pattern with PcMab-47.

We performed a kinetic analysis of theinteractions of PcMab-47, chPcMab-47, 47-mG2a, and47-mG2a-f with OSCC cells using flow cytometry. As
shown in Figure 4, the dissociation constant (KD) for
PcMab-47 against Ca9-22 was 5.1 × 10-8 M. In contrast,
those for chPcMab-47, 47-mG2a, and 47-mG2a-f were
2.1 × 10−8, 1.5 × 10−8, and 2.2 × 10−8 M, respectively.
The binding affinities of chPcMab-47, 47-mG2a, and47-mG2a-f against Ca9-22 were 2.4-, 3.4-, and 2.3fold higher, respectively, than that of PcMab-47. We
further determined the KD for PcMab-47 against HSC-2
and SAS cells. The results were summarized in Table
1. The binding affinities of chPcMab-47 and 47-mG2afor HSC-2 cells were 1.1- and 1.5-fold higher than thatof PcMab-47, respectively, although 47-mG2a-f had
lower binding affinity for HSC-2 cells than PcMab-47.
The binding affinities of chPcMab-47, 47-mG2a, and47-mG2a-f for SAS cells were 1.7-, 1.8-, and 1.1-foldhigher than that of PcMab-47, respectively, indicatingthat all chimeric antibodies, including chPcMab-47,
47-mG2a, and 47-mG2a-f, possess higher affinity for
PODXL than the original PcMab-47. The binding
affinities of 47-mG2a for HSC-2, SAS, and Ca9-22
cells were 3.1-, 1.6-, and 1.5-fold higher than those of
47-mG2a-f, respectively, indicating that 47-mG2a has
greater binding affinity than 47-mG2a-f for OSCC celllines. The difference of binding affinity among celllines was observed probably because the glycosylationof PODXL might be different in those OSCC cell lines.

Figure 1: Schematic illustration of PcMab-47, 47-mG2a and 47-mG2a-f. 47-mG2a was produced by subcloning appropriate VH

and VL cDNAs of PcMab-47 and CH and CL of mouse IgG2a into pCAG vectors. 47-mG2a-f was further produced by defucosylating 47-mG2a.
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Figure 2: Confirmation of the defucosylation of 47-mG2a-f by lectins. (A) Enzyme-linked immunosorbent assay (ELISA) using

lectins. 47-mG2a and 47-mG2a-f were immobilized and incubated with biotin-labeled lectins such as Aleuria aurantia lectin (AAL), Pholiota
squarrosa lectin (PhoSL), and concanavalin A (Con A) followed by peroxidase-conjugated streptavidin. The enzymatic reaction was
produced using a 1-Step Ultra TMB-ELISA. (B) Lectin microarray. AOL, Aspergillus oryzae lectin; PSA, Pisum sativum agglutinin; LCA,
Lens culinaris agglutinin. (C) Flow cytometry using anti-PODXL antibodies. Cells were treated with PcMab-47 (1 μg/mL), chPcMab-47(1 μg/mL), 47-mG2a (1 μg/mL), 47-mG2a-f (1 μg/mL), polyclonal anti-PODXL antibody (10 μg/mL), or 53D11 (10 μg/mL) followed bysecondary antibodies. Black line, negative control. pAb, polyclonal antibody.

Figure 3: Flow cytometry using anti-PODXL antibodies. Cells were treated with PcMab-47 (1 μg/mL) (A), chPcMab-47 (1 μg/mL) (B), 47-mG2a (1 μg/mL) (C), 47-mG2a-f (1 μg/mL) (D), polyclonal anti-PODXL antibody (10 μg/mL) (E), or 53D11 (10 μg/mL) (F)followed by secondary antibodies. Black line, negative control.
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Table 1: KD of anti-PODXL mAbs against OSCC cell lines
PcMab-47

chPcMab-47

47-mG2a47-mG2a-f

HSC-2

2.3 × 10-8 M

2.1 × 10-8 M

1.5 × 10-8 M

4.6 × 10-8 M

SAS

8.5 × 10-9 M

4.9 × 10-9 M

4.6 × 10-9 M

7.4 × 10-9 M

Ca9-22

5.1 × 10-8 M

2.1 × 10-8 M

1.5 × 10-8 M

2.2 × 10-8 M

Immunohistochemical analysis of PcMab-47, 47mG2a, and 47-mG2a-f in OSCC tissues

3+ in 15/197 (7.6%) samples, indicating that 47-mG2a ismore sensitive than PcMab-47 in immunohistochemical
analyses of OSCCs. All staining results for PcMab-47 and
47-mG2a are shown in Supplementary Table 1.
We also performed immunohistochemical analysis
using PcMab-47 and 47-mG2a against normal tongue,
and checked the PODXL expression in normal squamous
epithelium. As shown in Figure 6, PODXL was notdetected in normal squamous epithelium. In contrast,
PODXL was detected in normal endothelial cells. The
staining intensity of 47-mG2a was also higher than thatof PcMab-47 in normal endothelial cells. We further
checked the cross-reactivity of PcMab-47 against mouse
PODXL using flow cytometry and immunohistochemistry.
However, no reaction was observed against mouse
PODXL (data not shown), indicating that anti-PODXL
antibodies might not affect the tumor angiogenesis or
tumor microenvironment in mouse xenograft model.

We next performed immunohistochemical analysis
in OSCCs using PcMab-47, 47-mG2a, and 47-mG2a-f. As
shown in Figure 5A and 5F, PcMab-47 stained OSCCs ina cytoplasmic staining pattern; contrarily, no signals wereobserved for the negative control (no primary mAb) in a
serial section of OSCCs (Figure 5D and 5I). In addition,
47-mG2a and 47-mG2a-f also stained OSCCs sensitively(Figure 5B, 5C, 5G, and 5H). Of interest, the concentration
used for 47-mG2a and 47-mG2a-f in immunohistochemical
analysis was 0.5 μg/mL, which was 10-fold lower than thatfor PcMab-47, indicating that 47-mG2a and 47-mG2a-f are
extremely sensitive for use in OSCC tissues. PcMab-47,
47-mG2a, and 47-mG2a-f also stained endothelial cells
around tumor cells (Figure 5F, 5G, and 5H). The
sensitivity of 47-mG2a and 47-mG2a-f was extremely
similar in immunohistochemical analysis, and we used
PcMab-47 and 47-mG2a for further immunohistochemical
studies.
We stained 201 OSCC samples using PcMab-47
and 47-mG2a and summarized our findings in Table 2.
PcMab-47 stained 163/201 (81.1%) OSCC samples.
Among them, the intensity of staining was 3+ in 11/163(6.7%) samples. In contrast, 47-mG2a stained 197/201(98.0%) OSCC samples. Among them, the intensity was

Functional analysis of PODXL in OSCC cells in
vitro and in vivo
Next, we investigated whether PODXL is
associated with tumor phenotype in OSCC cell lines. We
selected SAS cells for this study because they express
PODXL at higher levels than other OSCC cells (Figure3) and they have been reported to grow extremely wellFigure 4: Binding affinities of anti-PODXL antibodies were determined using flow cytometry. Ca9-22 cells were suspended
in 100 μL of serially diluted antibodies (6 ng/mL to 100 μg/mL), and secondary antibodies were then added. Fluorescence data werecollected using a cell analyzer. GeoMean, geometric mean of fluorescence intensity.
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Table 2: Summary of anti-PODXL immunostaining in OSCC
Score

No. of positive cases

Positive rate (%)

-

1+

2+

3+

PcMab-47

38

120

32

11

163/201

81.1

47-mG2a

4

134

48

15

197/201

98.0

The intensity of staining was evaluated as -, 1+, 2+, or 3+.
in vivo [43]. As shown in Figure 7A, PcMab-47 did notreact with PODXL-knockout (KO) SAS cells (SAS/hPODXL-KO). To examine the migratory and invasive
abilities of SAS/hPODXL-KO cells, we performed
wound-healing and invasion assays, respectively, but
no significant differences in migration (Figure 7B) andinvasion (Figure 7C) were identified between parental
and SAS/hPODXL-KO cells. We next investigatedwhether PODXL is associated with the growth of OSCC
cell lines in vitro using the MTS assay. The growth of
three SAS/hPODXL-KO cell lines was lower than that ofparental SAS cells (Figure 7D). We further investigatedwhether PODXL affects OSCC tumor growth in vivo bycomparing the growth of SAS and three SAS/hPODXLKO cell lines that were transplanted subcutaneously into
nude mice. As shown in Figure 7E, the growth of SAS/

Figure 5: Immunohistochemical analysis of anti-PODXL antibodies in oral squamous cell carcinomas. Tissue sections(obtained from Case 174) were incubated with 5 μg/mL PcMab-47 (A, F), 0.5 μg/mL of 47-mG2a (B, G), 0.5 μg/mL 47-mG2a-f (C, H), or
control (blocking buffer; D, I) for 1 h at room temperature followed by treatment with an Envision+ kit for 30 min. Color was developed
using 3,3-diaminobenzidine tetrahydrochloride for 2 min, and sections were then counterstained with hematoxylin. (E, J) Hematoxylin &eosin staining. Arrows: endothelial cells. Scale bar = 100 μm.
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Figure 6: Immunohistochemical analysis of anti-PODXL antibodies in normal tongue. Tissue sections of normal tongue

were incubated with 5 μg/mL PcMab-47 (A, D) and 0.5 μg/mL of 47-mG2a (B, E) for 1 h at room temperature followed by treatment with an
Envision+ kit for 30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride for 2 min. Sections were then counterstainedwith hematoxylin. (C, F) Hematoxylin & eosin staining. Arrows: endothelial cells. Scale bar = 100 μm.

Figure 7: Functional analysis of PODXL in vitro and in vivo using PODXL-knockout (KO) oral squamous cell
carcinoma cell lines. (A) Flow cytometry. Cells were treated with 10 μg/mL PcMab-47 followed by secondary antibodies. Black line,
negative control. (B) Evaluation of cell migration. A wound-healing assay was performed to examine the effects of PODXL-KO on SAS
cell migration. Images of wounded cell monolayers taken at 0 and 8 h. The vertical lines indicate the wound edge. (C) Evaluation of cell
invasion using the Transwell invasion assay. The histogram indicates the average of three experiments (values are means ± SEM) and shows
the invasion ratio normalized to the value in parental SAS cells. n.s., not significant. (D) The MTS assay was performed to determine cell
growth. The values are means ± SEM. An asterisk indicates statistical significance between SAS and SAS/hPODXL-KO cells (***P < 0.005,
two-tailed Student’s t-test). (E) In vivo analysis. SAS and SAS/hPODXL-KO cells were injected subcutaneously into female nude mice.
The tumor volume was measured at day 21. The values are means ± SEM. An asterisk indicates statistical significance between SAS andSAS/hPODXL-KO cells (*P < 0.05, two-tailed Student’s t-test).
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hPODXL-KO cells was lower than that of parental SAS
cells.
Furthermore, we investigated the effect of PODXL
on the growth of 3D cells with cancer stem cell-like
properties. The 3D growth of three SAS/hPODXL-KO celllines tended to decrease, and the growth of SAS/hPODXLKO#23 cells was significantly inhibited after incubationfor 72 h, indicating that PODXL has tumorigenic potential
and might be associated with tumorigenicity in vivo
(Figure 8A and 8B). In contrast, there are little effects byanti-PODXL antibodies on 3D cell proliferation in vitro(Figure 8C).

activities against CHO/hPODXL, and might not exert
ADCC activities in vivo.
We also investigated the antitumor activity of 47mG2a and 47-mG2a-f using HSC-2 xenograft models.
47-mG2a and 47-mG2a-f, and mouse IgG (control) were
injected three times (100 μg of the antibodies on days 1,
7, and 14 after cell injections) into the peritoneal cavityof mice. Tumor formation was observed in all groups.
47-mG2a-f significantly reduced tumor development
compared with IgG on day 20 (Figure 11A). Although
47-mG2a also reduced tumor development comparedwith IgG, the result was not significant, indicating thatthe depletion of core fucose is critical for the antitumor
activity of 47-mG2a-f against HSC-2 xenograft models.
The tumor weight of mice in the 47-mG2a-f group was
significantly lower than that in the IgG group (Figure
11B). Conversely, 47-mG2a was not associated with asignificant decrease in tumor weight versus IgG. However,body weight was not significantly different among thethree groups (Supplementary Figure 3A and 3B). The
resected tumors of HSC-2 xenografts are depicted in
Supplementary Figure 3C. No difference was illustrated
among those groups using HE staining (Supplementary
Figure 4).
We further investigated the antitumor activity of 47mG2a and 47-mG2a-f using SAS xenograft models. In thisstudy, we injected 100 μg of the antibodies three times.
As shown in Figure 12A, 47-mG2a-f, but not 47-mG2a,significantly reduced tumor development compared with
IgG on day 20, indicating that the depletion of core fucose
is also critical for antitumor activity of 47-mG2a-f against
SAS xenograft models. Tumor weight was significantly
lower in the 47-mG2a-f group than in the IgG group (Figure
12B). Conversely, 47-mG2a was not linked to decreases intumor weight. Body weight was similar among the threegroups (Supplementary Figure 5A and 5B). The resected
tumors of SAS xenografts are shown in Supplementary
Figure 5C. No difference in HE staining was identified
among the groups (Supplementary Figure 6).
We performed dose-escalation studies using CHO/hPODXL xenograft models (Supplementary Figure7). In this study, we injected 500 μg of the antibodies
twice. As shown in Supplementary Figure 7A, both
47-mG2a and 47-mG2a-f significantly reduced tumor
development compared with IgG on day 16 (P < 0.01).
The resected tumors of CHO/hPODXL xenografts areshown in Supplementary Figure 7B. Tumor weights weresignificantly lower in the 47-mG2a and 47-mG2a-f groups
than in the IgG group (Supplementary Figure 7C). Body
weight was similar among the three groups (data notshown), as were HE staining patterns (Supplementary
Figure 7D). In CHO/hPODXL xenograft models, doseescalation was not necessary because 100 μg of theantibodies twice showed enough anti-tumor activities.
We further performed dose-escalation studies using
HSC-2 xenograft models because we could not observe

ADCC and CDC activities of 47-mG2a and
47-mG2a-f
We investigated whether 47-mG2a and 47-mG2a-f can
induce ADCC and CDC in PODXL-expressing cell lines.
Both 47-mG2a and 47-mG2a-f induced CDC against CHO/hPODXL (Figure 9A), whereas they showed faint ADCC
against CHO/hPODXL cells (data not shown), indicatingthat only CDC is critical for the antitumor activity of
47-mG2a-f against CHO/hPODXL cells.
47-mG2a-f induced ADCC activity against HSC2, whereas 47-mG2a did not (Figure 9B). Furthermore,higher ADCC was observed by 47-mG2a-f against SAS
cells compared with 47-mG2a (Figure 9C). These results
indicate that depletion of core-fucose is important for
inducing ADCC against OSCC cells. Neither 47-mG2a
nor 47-mG2a-f induced CDC in OSCC cell lines (datanot shown), indicating that only ADCC is critical for the
antitumor activity of 47-mG2a-f against OSCC cells.

Antitumor activity of 47-mG2a and 47-mG2a-f
against OSCC xenografts
To study the antitumor effects of 47-mG2a and
47-mG2a-f on primary tumor growth in vivo, CHO/hPODXL cells were subcutaneously implanted into
the flanks of nude mice. 47-mG2a and 47-mG2a-f, andmouse IgG (control) were injected twice (100 μg of theantibodies on days 1 and 9 after cell inoculation) into theperitoneal cavity of mice. Tumor formation was observedin all groups. Both 47-mG2a and 47-mG2a-f significantly
reduced tumor development compared with IgG on
day 16 (Figure 10A). The tumor weights of mice in theboth 47-mG2a and 47-mG2a-f groups were significantly
lower than that in the IgG group (Figure 10B). Body
weight was not significantly different among the threegroups (Supplementary Figure 1A and 1B). The resected
tumors of CHO/hPODXL xenografts are depicted in
Supplementary Figure 1C. No difference was illustrated
among those groups using hematoxylin & eosin (HE)staining (Supplementary Figure 2). Both 47-mG2a and
47-mG2a-f showed the similar results in CHO/hPODXL
xenograft models because those mAbs might exert CDC
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Figure 8: Proliferation of SAS and SAS/hPODXL-KO cells on 3D cultures and the influence of anti-PODXL antibodies
on the 3D cell proliferation. (A) 3D cell proliferation was measured by 3D cell proliferation assay. The graphs show the relative cell

growth in 3D culture at 24, 48, and 72 h. Each bar represents the mean ± SEM. An asterisk indicates statistical significance (**P < 0.01,
Tukey-Kramer’s test). (B) Optical microscopic images of SAS and SAS/hPODXL-KO cells after 72 h incubation. Scale bar, 200 μm. (C)3D cell proliferation treated with or without anti-hPODXL antibodies (47-mG2a and 47-mG2a-f) for 72 h. The values are means ± SEM.

Figure 9: Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of antiPODXL mAbs against PODXL-expressing cell lines. (A) CDC was evaluated using the 51Cr release assay. Target cells wereincubated with [51Cr]sodium chromate (0.1 μCi) for 1 h at 37°C. The 51Cr-labeled cells were incubated with a baby rabbit complement at adilution of 1:4 in the presence of 47-mG2a, 47-mG2a-f, or control mouse IgG (10 μg/mL) for 6 h in 96-well plates. 51Cr release was measured
in the supernatant. (B, C) ADCC was determined using the 51Cr release assay. Target cells such as HSC-2 (B) and SAS (C) were incubatedwith [51Cr]sodium chromate. After washing, 51Cr-labeled target cells were placed in 96-well plates in triplicate. Effector cells and 47-mG2a,47-mG2a-f, or control mouse IgG were added to the plates. After incubation (6, 12, and 18 hrs), 51Cr release was measured in the supernatant.
The values are means ± SEM. *P < 0.05, **P < 0.01 t-test.
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Figure 10: Antitumor activity of 47-mG2a and 47-mG2a-f against CHO/hPODXL. (A) Tumor volume of CHO/hPODXL
xenografts. CHO/hPODXL cells were injected subcutaneously into female nude mice. The indicated antibodies (100 μg/day; 5 mg/kg) wereadministered intraperitoneally 1 and 9 days after cancer cell inoculation. The tumor volume was measured at the indicated time points. The
values are means ± SEM. (B) Tumor weight of CHO/hPODXL xenografts (day 16). The values are means ± SEM. An asterisk indicatesstatistical significance (*P < 0.05, **P < 0.01, Tukey-Kramer’s test).

Figure 11: Antitumor activity of 47-mG2a and 47-mG2a-f against HSC-2 xenografts. (A) Tumor volume of HSC-2 xenografts.

HSC-2 cells were injected subcutaneously into female nude mice. The indicated antibodies (100 μg/day; 5 mg/kg) were administeredintraperitoneally 1, 7, and 14 days after cancer cell inoculation. The tumor volume was measured at the indicated time points. The values aremeans ± SEM. (B) Tumor weight of HSC-2 xenografts (day 20). An asterisk indicates statistical significance (*P < 0.05, Tukey-Kramer’stest).
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Oncotargetsufficient antitumor activity after three injections of 100
μg of the antibodies. In this study, we injected 500 μg of
the antibodies twice. As shown in Figure 13A and 13B,
both 47-mG2a and 47-mG2a-f reduced tumor development
compared with control mouse IgG on day 15 (P < 0.01).
Furthermore, 47-mG2a-f exhibited greater antitumor
activity than 47-mG2a, but it is not significant (Figure
13B). The resected tumors of HSC-2 xenografts areshown in Figure 14A. Tumor weights were significantly
lower in the both 47-mG2a and 47-mG2a-f group than in
the IgG group (Figure 14B). Body weight was similar
among the three groups (data not shown), as were HE
staining patterns (Supplementary Figure 8). These
results indicate that 47-mG2a-f showed higher antitumor activities against HSC-2 due to its higher ADCC
against PODXL-expressing OSCCs, and dose-escalation
was effective for those anti-tumor activities in HSC-2
xenograft models.
We finally performed dose-escalation studies using
SAS xenograft models because we could not observe
sufficient antitumor activity after three injections of 100
μg of the antibodies (Supplementary Figure 9). In thisstudy, we injected 500 μg of the antibodies three times. As
shown in Supplementary Figure 9A, 47-mG2a-f reduced
tumor development compared with control mouse IgG
on day 21 (P < 0.05) and exhibited significantly greater
antitumor activity than 47-mG2a. The resected tumors of
SAS xenografts are shown in Supplementary Figure 9B.

Tumor weight was significantly lower in the 47-mG2a-fgroup than in the IgG group, whereas 47-mG2a did not
reduce tumor weight (Supplementary Figure 9C). Body
weight was similar among the three groups (data notshown), as were HE staining patterns (Supplementary
Figure 9D). These results indicate that 47-mG2a-f showed
higher anti-tumor activities due to its higher ADCC
against PODXL-expressing OSCCs, but dose-escalation
was not sufficient for those anti-tumor activities in vivo.

DISCUSSION
47-mG2a and 47-mG2a-f showed higher binding
activity compared with its original PcMab-47 (Figure 4).
We have sometimes experienced that chimeric antibodies
possesses much higher affinity or lower affinity comparedwith original monoclonal antibodies [34, 44-49]. The
stability of antibodies might be different among constantregions. Although 47-mG2a exhibited ADCC activity
against SAS cells (Figure 9C), this activity is not sufficient
to induce antitumor activity in vivo (Figure 12). Then,
we produced a non-fucosylated version of 47-mG2a(47-mG2a-f) to augment its ADCC activities because nonfucosylated antibodies are known to show higher ADCC
activities [35, 50]. As expected, 47-mG2a-f exhibitedstronger ADCC activities than 47-mG2a against OSCC
cells (Figure 9B and 9C), leading to higher antitumor
activities in HSC-2 and SAS xenograft models (Figure 11

Figure 12: Antitumor activity of 47-mG2a and 47-mG2a-f against SAS xenografts. (A) Tumor volume of SAS xenografts.

SAS cells were injected subcutaneously into female nude mice. The indicated antibodies (100 μg/day; 5 mg/kg) were administeredintraperitoneally 1, 7, and 14 days after cancer cell inoculation. The tumor volume was measured at the indicated time points. The values aremeans ± SEM. (B) Tumor weight of SAS xenografts (day 20). An asterisk indicates statistical significance (*P < 0.05, Tukey-Kramer’s test).
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and 12). In contrast, three injections were not effective
despite a dose of 5 mg/kg, which corresponded to 100
μg/mouse in this study and exceeded the usual dose ofantibody therapy [51, 52]. Then, two injections of 500

μg/mouse, corresponding to 25 mg/kg, resulted in greater
antitumor activities in HSC-2 xenograft models (Figure13), indicating that dose escalation might be necessary
for monotherapy using anti-PODXL antibodies although

Figure 13: Antitumor activity of 47-mG2a and 47-mG2a-f against HSC-2 xenografts (500 μg/day; 25 mg/kg). (A)Comparison of the tumor size and tumor incidence of HSC-2 xenograft in nude mice (day 15). (B) Tumor volume of HSC-2 xenografts.
HSC-2 cells were injected subcutaneously into female nude mice. The indicated antibodies (500 μg/day; 25 mg/kg) were administeredintraperitoneally 1 and 7 days after cancer cell inoculation. The tumor volume was measured at the indicated time points. The values aremeans ± SEM. An asterisk indicates statistical significance (**P < 0.01, Tukey-Kramer’s test).

Figure 14: Antitumor activity of 47-mG2a and 47-mG2a-f against HSC-2 xenografts (500 μg/day; 25 mg/kg). (A)Comparison of tumor size (day 15). (B) Tumor weight of HSC-2 xenografts (day 15). An asterisk indicates statistical significance (**P <0.01, Tukey-Kramer’s test).
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dose-escalation was not sufficient for SAS xenograftmodels (Supplementary Figure 9). Furthermore, we needto combine anti-PODXL antibodies with anti-cancer drugs
or include them in novel antitumor regimens, including
T cells and viruses, to exert antitumor activity against
cancer cells. Previously, we produced chPcMab-47, amouse-human chimeric antibody, which could be applied
to cancer patients [34]. chPcMab-47 exerted ADCC and
CDC activity and showed antitumor activities in mouse
xenograft models. Because 47-mG2a-f showed muchhigher ADCC activity in this study, we also need to
produce core-fucose-deficient chPcMab-47 in the future
study.
Jing et al. reported that high PODXL expression was
significantly associated with worse OS and was predictiveof shorter OS in multiple cancers, especially pancreaticand colorectal cancers [53]. They also revealed thathigh PODXL expression predicted worse DSS and DFS,
although European patients were included in this analysis.
These results suggest PODXL could be a prognostic
factor, and diagnostic tools targeting this protein are
expected. Because the association between PODXL
expression and clinical stage has not been investigatedin OSCC, it was analyzed in the current study. However,
PODXL expression in stages III–IV was not significantly
higher than that in stages I–II in patients with OSCC (datanot shown). Further investigation regarding the clinicalimportance of PODXL expression in more patients with
OSCC is needed.
Taken together, anti-PODXL antibodies could be
useful antibody therapies against PODXL-expressing
OSCCs. PODXL is known to be expressed in kidney,
heart, pancreas, and breast tissues, and it plays importantroles in those tissues [22]. Recently, we successfully
produced cancer-specific mAbs (CasMabs) against human
podoplanin [16, 54]. Using the same methods, we need todevelop CasMabs against PODXL in the near future.

hPODXL-KO cells were produced using CRISPR/Cas9
plasmids (Target ID: HS0000056763) targeting human
PODXL (Sigma-Aldrich Corp., St. Louis, MO, USA).
HSC-2, HSC-3, HSC-4, Ca9-22, HO-1-u-1, SAS and SAS/hPODXL-KO cells were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM; Nacalai Tesque, Kyoto,
Japan), and CHO-K1 and CHO/hPODXL were cultured
in RPMI 1640 medium (Nacalai Tesque), supplemented
with 10% heat-inactivated fetal bovine serum (Thermo
Fisher Scientific Inc.), 100 units/mL penicillin, 100 μg/
mL streptomycin, and 25 μg/mL amphotericin B (Nacalai
Tesque) at 37°C in a humidified atmosphere containing
5% CO2 and 95% air.

Tissues
Cancer tissue microarrays of oral cancers werepurchased from US Biomax (Rockville, MD, USA) and
Cybrdi (Frederick, MD, USA). This study examined 53
patients with oral cancer who underwent surgery at Tokyo
Medical and Dental University. The Tokyo Medical and
Dental University Institutional Review Board reviewed
and approved the use of human cancer tissues. Written
informed consent was obtained for the use of humancancer tissue samples.

Antibodies
PcMab-47, a mouse anti-PODXL mAb (IgG1,
kappa), was developed as previously described [17].
Mouse IgG was purchased from Sigma-Aldrich. To
generate 47-mG2a, appropriate VH and VL cDNAs of mouse
PcMab-47 and CH and CL of mouse IgG2a were subcloned
into pCAG-Ble and pCAG-Neo vectors (Wako Pure
Chemical Industries, Osaka, Japan), respectively. Antibody
expression vectors were transfected into ExpiCHO-S cells
using the ExpiCHO Expression System (Thermo Fisher
Scientific). To generate 47-mG2a-f, antibody expression
vectors were also transfected into PDIS-5 cells using
the ExpiCHO Expression System. Stable transfectantsof chPcMab-47 cells were established previously [34].
CHO-S/chPcMab-47 cells were cultivated in CHO-SSFM II medium (Thermo Fisher Scientific). PcMab-47,
47-mG2a, 47-mG2a-f, and chPcMab-47 were purified
using Protein G-Sepharose (GE Healthcare Bio-Sciences,
Pittsburgh, PA, USA). Goat polyclonal anti-human
PODXL and mouse monoclonal anti-human PODXL
mAb (53D11) were purchased from R&D systems, Inc.
(Minneapolis, MN) and Medical & Biological Laboratories
Co., Ltd. (MBL; Nagoya, Japan), respectively.

MATERIALS AND METHODS
Cell lines
HSC-2 (oral squamous carcinoma from oral cavity),
HSC-3 (oral squamous carcinoma cell line from tongue
with high metastatic potential), HSC-4 (oral squamous
carcinoma cell line from tongue), Ca9-22 (oral squamous
carcinoma from gingiva), HO-1-u-1 (oral squamous
carcinoma from mouth floor), and SAS (oral squamous
carcinoma cell line from tongue) were obtained from theJapanese Collection of Research Bioresources Cell Bank
(Osaka, Japan). CHO-K1 was obtained from the American
Type Culture Collection (ATCC, Manassas, VA). CHO/hPODXL was produced in our previous study [17]. The
CHO-S cell line was purchased from Thermo Fisher
Scientific Inc. (Waltham, MA, USA). PDIS-5 (core fucose
KO CHO-S) cells were established previously [48]. SAS/
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Enzyme-linked immunosorbent assay (ELISA)
47-mG2a and 47-mG2a-f were immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher
Scientific) at 1 μg/mL for 30 min. After blocking using
SuperBlock buffer (Thermo Fisher Scientific) with
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0.5 mM CaCl2, the plates were incubated with biotinlabeled lectins, such as AAL (Vector Laboratories,
Burlingame, CA, USA), PhoSL (J-OIL MILLS, Inc.,Tokyo, Japan) [38], and ConA (Vector Laboratories),
followed by 1:3000 diluted peroxidase-conjugatedstreptavidin (Agilent Technologies, Santa Clara, CA,
USA). The enzymatic reaction was produced using a
1-Step Ultra TMB-ELISA (Thermo Fisher Scientific). The
optical density was measured at 655 nm using an iMark
microplate reader (Bio-Rad Laboratories, Inc., Berkeley,CA).

mAbs for 1 h at room temperature and then treatedusing an Envision+ kit (Agilent Technologies) for 30
min. Color was developed using 3,3-diaminobenzidine
tetrahydrochloride (Agilent Technologies) for 2 min,and sections were then counterstained with hematoxylin(Wako Pure Chemical Industries Ltd.). The intensity ofstaining was evaluated as −, 1+, 2+, or 3+.

Proliferation assay in vitro
Cell proliferation in vitro was measured using MTS
tetrazolium (Cell Titer 96 Aqueous, Promega, Madison,
WI, USA). Cells were plated (500 cells/100 μL/well) in
triplicate in 96-well plates. Cell viability was measured
every 24 h for 96 h. After adding 20 μL of MTS to
the wells followed by a 1-h incubation at 37°C, theabsorbance at 490 nm (reference, 630 nm) was read using
a microplate reader (Power Scan HT, Bio Tek Instruments,
Winooski, VT, USA). The mean absorbance of the 3-well
set was obtained from 0 to 96 h. Statistical significancewas analyzed using the standard Student’s t-test. P-values<0.05 were considered statistically significant.

Lectin microarray
47-mG2a and 47-mG2a-f (100 μL; 31.25–2000 ng/mL) were applied to a lectin array (LecChip ver. 1.0;
GlycoTechnica, Hokkaido, Japan), including triplicate
spots of 45 lectins in each of seven incubation baths on theglass slide. After incubation at 20°C for 17 h, the reaction
solution was discarded. The glass slide was scanned using
a GlycoStation Reader 1200 (GlycoTechnica).

Flow cytometry

Proliferation assay in vivo

Cell lines were harvested via a brief exposure to
0.25% trypsin/1 mM ethylenediaminetetraacetic acid
(Nacalai Tesque). After washing with 0.1% bovine-serum
albumin in phosphate-buffered saline (Nacalai Tesque),cells were treated with primary mAbs for 30 min at 4°C,
followed by treatment with Alexa Fluor 488-conjugatedanti-mouse IgG (1:1000; Cell Signaling Technology,
Danvers, MA, USA), FITC-labeled goat anti-human
IgG (1:1000; Thermo Fisher Scientific), or FITC-labeled
rabbit anti-goat IgG (1:1000; Thermo Fisher Scientific).
Fluorescence data were collected using an EC800 Cell
Analyzer (Sony Corp., Tokyo, Japan).

Five-week-old female BALB/c nude mice werepurchased from Charles River (Kanagawa, Japan). Sevenweek-old mice were used for the in vivo proliferation
assay. Cells (0.3 mL of 1.33 × 108 /mL in DMEM) weremixed with 0.5 mL of BD Matrigel Matrix Growth Factor
Reduced (BD Biosciences, San Jose, CA, USA). A 100
μL suspension (containing 5 × 106 cells) was injectedsubcutaneously into the left flanks of nude mice. The
tumor diameter was measured using calipers, and thetumor volume was calculated using the following formula:volume = W2 × L/2, where W is the short diameter and L is
the long diameter. The mice were euthanized 21 days aftercell implantation. All data were expressed as the mean
± SEM. Statistical analysis was performed using a twotailed Student’s t-test. P-values <0.05 were considered
statistically significant.

Determination of binding affinity using flow
cytometry
HSC-2, SAS, and Ca9-22 cells (2 × 105) were
resuspended in 100 μL of serially diluted PcMab-47, 47mG2a, 47-mG2a-f, or chPcMab-47 (6 ng/mL to 100 μg/mL),followed by the addition of secondary anti-mouse IgG(Cell Signaling Technology) or anti-human IgG (Thermo
Fisher Scientific). Fluorescence data were collected using
a cell analyzer (EC800). KD was obtained by fitting thebinding isotherms using the built-in one-site binding
models in GraphPad PRISM 6 (GraphPad Software, La
Jolla, CA, USA).

Invasion assay
The invasion assay was performed using a
Cytoselect 96-well Collagen Cell Invasion Assay Kit(Cell Biolabs, San Diego, CA, USA) according to the
manufacturer’s instructions. Cells (2.5 × 104) were preincubated in DMEM containing 0.5% FBS for 12 h,
washed with serum-free DMEM, and added to the top
insert of a chamber containing a polycarbonate membranewith 8-μm pores coated with a layer of bovine type I
collagen matrix. The lower chamber was filled withDMEM containing 10% FBS as the chemoattractant.
The assembled chambers were then placed in theCO2 incubator for 20 h. Cells that passed through themembrane pores were lysed and quantitated using a

Immunohistochemical analyses
Histologic sections (4 μm thick) were deparaffinizedin xylene and then rehydrated and autoclaved in citratebuffer (pH 6.0; Agilent Technologies Inc.) for 20 min.
Sections were then incubated with 0.5–5 μg/mL primarywww.oncotarget.com

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Oncotargetfluorescence dye-containing solution and a fluorescenceplate reader. Statistical significance was analyzed using thestandard Student’s t-test. P-values <0.05 were considered
statistically significant.

maximum release. Statistical significance was analyzed
using the standard Student’s t-test. P-values <0.05 wereconsidered statistically significant.

CDC

Wound-healing assay

CDC was evaluated using the 51Cr release assay.
Target cells were incubated with [51Cr]sodium chromate(0.1 μCi) for 1 h at 37°C. The cells were then washedwith 10% FBS/RPMI1640. The 51Cr-labeled cells wereincubated with a baby rabbit complement Cedarlane(Ontario, Canada) at a dilution of 1:4 in the presenceof antibodies (10 μg/mL) for 6 h in 96-well plates.
After the incubation, 51Cr radioactivity was measured
in the supernatants using a gamma counter. The percent
cytotoxicity was calculated using the following formula:% specific lysis = (E – S)/(M − S) × 100, where E is therelease in the test sample, S is the spontaneous release,and M is the maximum release. Statistical significancewas analyzed using the standard Student’s t-test. P-values< 0.05 were considered statistically significant.

Cells were grown to confluence in DMEM
containing 0.5% FBS in 6-well plates. Scratch wounds
were made using 10-μL sterile pipette tips. The cells
were incubated in DMEM containing 10% FBS for theindicated times. Images were captured using an Evolution
MP camera (Media Cybernetics, Rockville, MD, USA).

3D cell proliferation assay
3D cell proliferation in vitro was measured using
the CellTiter-Glo® 3D cell viability assay (Promega)according to the manufacturer’s instructions [55]. Briefly,
the cells were plated (500 cells/100 μL/well) in triplicate
in 96-well ultra low attachment plates (Costar Corning,
Schiphol-Rijk, Netherlands) with DMEM containing 10%
FBS. The 3D cell viability was measured every 24 h for 72
h. The CellTiter-Glo® 3D reagent was added into wells ina 1:1 dilution (100 μL volume in well: 100 μL of reagent)and then the solutions were mixed well by pipetting. Afterincubation for 30 min at 37°C, the luminescent signal wasread using an EnSpire multi-plate reader (Perkin Elmer,
PerkinElmer, Waltham, MA, USA). Images were captured
using an Evolution MP camera (Media Cybernetics). In
addition, to investigate the influence of the antibodies
on the 3D cell proliferation, the cells were plated as
described above with or without the antibodies (30 μg/ml). The proliferation rate was calculated relative to thesignal at 0 h.

Antitumor activity of anti-PODXL antibodies
Five-week-old female BALB/c nude mice werepurchased from Charles River and used in experiments at 7
weeks of age. Cells (0.3 mL of 1.33 × 108 /mL in DMEM)
were mixed with 0.5 mL of BD Matrigel Matrix Growth
Factor Reduced (BD Biosciences). A 100-μL suspension(containing 5 × 106 cells) was injected subcutaneously
into the right flanks of nude mice. After 1 day, 100 μg
or 500 μg of 47-mG2a, 47-mG2a-f, or mouse IgG in 200
μL PBS were injected into the peritoneal cavity of eachmouse. Additional antibodies were injected once weekly
for several weeks. The tumor diameter and tumor volume
were determined as previously described. The mice wereeuthanized 15, 16, 20, or 21 days after cell implantation.
All data were expressed as the mean ± SEM. Statisticalanalysis was performed using the Tukey-Kramer test.
P-values < 0.05 was considered statistically significant.

ADCC
ADCC was examined using the 51Cr-release assay.
Effector cells were prepared as previously described [36].
Mouse splenocytes were harvested from spleens of sixweek-old male SCID mice. Spleens were homogenized in
RPMI 1640 and centrifuged. To deplete red blood cells,
the cell pellet was suspended in red blood cell lysis buffer(Sigma-Aldrich). After washing and re-suspension in 10%
FBS/RPMI1640, splenocytes were used as effector cells.
Target cells were incubated with 0.1 μCi of [51Cr]sodium
chromate at 37°C for 1 h. After three washes with 10%
FBS/RPMI1640, 51Cr-labeled target cells were placed in
96-well plates in triplicate. Effector cells and antibodies(10 μg/ml) were added to the plates (E/T ratio = 100). After6, 12, and 18 hrs of incubation, 51Cr release was measured
in the supernatant (100 μL) from each well using a gamma
counter (PerkinElmer). The percentage of cytotoxicity wascalculated using the following formula: % specific lysis =(E – S)/(M − S) × 100, where E is the 51Cr release in thetest sample, S is the spontaneous release, and M is thewww.oncotarget.com

Abbreviations
AAL Aleuria aurantia lectin
ADCC Antibody-dependent cellular cytotoxicity
CDC Complement-dependent cytotoxicity
DFS Disease-free survival
DMEM Dulbecco’s Modified Eagle’s Medium
DSS Disease-specific survival
EGFR Epidermal growth factor receptor
ELISA Enzyme-linked immunosorbent assay
HE Hematoxylin & eosin
HNC Head and neck cancer
HNSCC
Head and neck squamous cell
carcinoma
LCA Lens culinaris agglutinin
OS Overall survival
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OSCC Oral squamous cell carcinoma
PhoSL Pholiota squarrosa lectin
PSA Pisum sativum agglutinin
R/M Recurrent and/or metastatic.

7. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM,
Cohen RB, Jones CU, Sur R, Raben D, Jassem J, Ove R,
Kies MS, Baselga J, et al. Radiotherapy plus cetuximab forsquamous-cell carcinoma of the head and neck. N Engl J
Med. 2006; 354:567-78.

Author contributions

8. Vokes EE. Induction chemotherapy for head and neckcancer: recent data. Oncologist. 2010; 15 Suppl 3:3-7.

Y.N., M.K., H.H., and Y.K. designed and supervised
the project. S.I, T.O., M.K.K., S.Y., S.A, T.N., M.Y.,
Y.W.C., and S.O. performed the experiments and analyzed
the data. Y.K. and H.H. provided materials, reagents, and/or analysis tools. The manuscript was prepared by S.I. and
Y.K. All authors reviewed the manuscript.

9. Vermorken JB, Mesia R, Rivera F, Remenar E, Kawecki
A, Rottey S, Erfan J, Zabolotnyy D, Kienzer HR, Cupissol
D, Peyrade F, Benasso M, Vynnychenko I, et al. Platinumbased chemotherapy plus cetuximab in head and neckcancer. N Engl J Med. 2008; 359:1116-27.
10. Okano S, Yoshino T, Fujii M, Onozawa Y, Kodaira T, Fujii
H, Akimoto T, Ishikura S, Oguchi M, Zenda S, de Blas
B, Tahara M, Beier F. Phase II study of cetuximab plus
concomitant boost radiotherapy in Japanese patients withlocally advanced squamous cell carcinoma of the head andneck. Jpn J Clin Oncol. 2013; 43:476-82.

ACKNOWLEDGMENTS
We thank Noriko Saidoh, Saori Handa, Yoshimi
Nakamura, and Akiko Harakawa for excellent technicalassistance.

11. Yoshino T, Hasegawa Y, Takahashi S, Monden N, Homma
A, Okami K, Onozawa Y, Fujii M, Taguchi T, de Blas B,
Beier F, Tahara M. Platinum-based chemotherapy plus
cetuximab for the first-line treatment of Japanese patientswith recurrent and/or metastatic squamous cell carcinomaof the head and neck: results of a phase II trial. Jpn J Clin
Oncol. 2013; 43:524-31.

CONFLICTS OF INTEREST
The authors have no conflicts of interest.

FUNDING

12. Ferris RL, Blumenschein G Jr, Fayette J, Guigay J, ColevasAD, Licitra L, Harrington K, Kasper S, Vokes EE, Even C,
Worden F, Saba NF, Iglesias Docampo LC, et al. Nivolumab
for recurrent squamous-cell carcinoma of the head andneck. N Engl J Med. 2016; 375:1856-67.

This research was supported in part by AMED underGrant Numbers: JP17am0301010 (Y.K.), JP17am0101078(Y.K.), and JP17ae0101028 (Y.K.), and by JSPS
KAKENHI Grant Number 17K07299 (M.K.K.) and Grant
Number 16K10748 (Y.K.).

13. Cohen EE, Davis DW, Karrison TG, Seiwert TY, Wong SJ,Nattam S, Kozloff MF, Clark JI, Yan DH, Liu W, Pierce
C, Dancey JE, Stenson K, et al. Erlotinib and bevacizumabin patients with recurrent or metastatic squamous-cellcarcinoma of the head and neck: a phase I/II study. Lancet
Oncol. 2009; 10:247-57.

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