Biochemistry and Biophysics Reports 24 (2020) 100801

Contents lists available at ScienceDirect

Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

A defucosylated anti-PD-L1 monoclonal antibody 13-mG2a-f exerts
antitumor effects in mouse xenograft models of oral squamouscell carcinoma
Junko Takei a, b, Tomokazu Ohishi c, Mika K. Kaneko a, Hiroyuki Harada b, Manabu Kawada c,
Yukinari Kato a, d, *a

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku,
Tokyo, 113-8510, Japanc
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, 18-24 Miyamoto, Numazu-shi, Shizuoka, 410-0301, Japand
New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
PD-L1
Monoclonal antibody
ADCC
CDC
Antitumor activity
Oral cancer

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting
cells and several tumor cells, including melanoma and lung cancer cells. A strong correlation has been reported
between PD-L1 expression in tumor cells and negative prognosis in cancer patients. Previously, we established ananti-PD-L1 monoclonal antibody (mAb), L1Mab-13 (IgG1, kappa), by immunizing mice with PD-L1overexpressing CHO-K1 cells. L1Mab-13 specifically reacts with endogenous PD-L1 in lung cancer cell lines inflow cytometry and Western blot applications, and stains a plasma membrane-like pattern in lung cancer tissues
via immunohistochemical analysis. In this study, we investigated whether L1Mab-13 reacts with oral cancer celllines and exerts antitumor activities. Because L1Mab-13 lacks antibody-dependent cellular cytotoxicity (ADCC)
and complement-dependent cytotoxicity (CDC), we first converted the subclass of L1Mab-13 from IgG1 into IgG2a(13-mG2a), and further produced a defucosylated version (13-mG2a-f) using FUT8-deficient ExpiCHO-S (BINDS09) cells. Defucosylation of 13-mG2a-f was confirmed using fucose-binding lectins, such as Aleuria aurantia andPholiota squarrosa lectins. The dissociation constants (KD) for 13-mG2a-f in SAS and HSC-2 oral cancer cells were
determined via flow cytometry to be 2.8 × 10− 9 M and 4.8 × 10− 9 M, respectively, indicating that 13-mG2a-f
possesses extremely high binding affinity. In vitro analysis demonstrated that 13-mG2a-f showed moderate ADCC
and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 13-mG2a-f significantly
reduced tumor development in SAS and HSC-2 xenografts in comparison to control mouse IgG, even after in­jection seven days post-tumor inoculation. Taken together, these data demonstrate that treatment with 13-mG2a-f
may represent a useful therapy for patients with PD-L1-expressing oral cancers.

1. Introduction
Programmed cell death ligand-1 (PD-L1) is a type I transmembrane
glycoprotein expressed in many tumor types, including melanoma, brain
tumor, lung cancer, breast cancer, gastric cancer, ovarian cancer,
pancreatic cancer, and renal cancer [1–7]. PD-L1 is an immune regula­tory molecule, which limits T cell effector function [8]. Programmed cell
death (PD)-1/PD-L1 association inhibits activated T cell proliferation,
allowing cancer cells to circumvent host immune surveillance [9,10]. A
substantial correlation between PD-L1/L2 expression in cancer cells andpoor prognosis has been reported in several cancers [11–13]. Although
inhibition of PD-1 in patients with tumors is demonstrated to havetherapeutic effects, detecting PD-L1 expression via immunohistochem­
istry would be extremely beneficial in making the clinical determination
to use targeted drugs such as nivolumab or pembrolizumab for cancer
treatment [14,15].
Oral cancers occupy about 2% of all cancer cases worldwide [16].
More than 350,000 individuals are diagnosed as oral cancer every year,
and oral cancers are fatal for about 170,000 of these people. Increas­
ingly, young patients are being diagnosed with oral cancers [17,18].
Oral cancers comprise several histological tumor types, such as* Corresponding author. New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2020.100801
Received 28 July 2020; Accepted 10 August 2020
2405-5808/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Biochemistry and Biophysics Reports 24 (2020) 100801

ELISA
FBS
HNSCC
JCRB
mAb
OSCC
PBS
PDPN
PhoSL
PODXL
SCC

Abbreviations:
AAL
ADCC
ATCC
BSA
CasMab
CDC
CHO
Con A
DMEM
EDTA

Aleuria aurantia lectin
antibody-dependent cellular cytotoxicity
American Type Culture Collection
bovine serum albumin
cancer-specific mAb
complement-dependent cytotoxicity
Chinese hamster ovary
concanavalin A
Dulbecco’s Modified Eagle’s Medium
ethylenediaminetetraacetic acidenzyme-linked immunosorbent assay
fetal bovine serum
head and neck squamous cell carcinoma
Japanese Collection of Research Bioresources Cell Bank
monoclonal antibody
oral squamous cell carcinoma
phosphate-buffered saline
podoplanin
Pholiota squarrosa lectin
podocalyxin
squamous cell carcinoma

primary drug of choice, and it is usually combined with 5-fluorouracil
and docetaxel [21,22]. Other anticancer agents, such as paclitaxel,
methotrexate, and carboplatin can be also used for OSCCs [23], buteffective molecular targeting drugs, including antibody therapies, arelacking.
PD-L1 has been utilized not only as a molecular marker of anti-PD-1
therapy, but also as a molecular target for antibody therapy. Anti-PD-L1
mAbs, such as atezolizumab, durvalumab, and avelumab has been used
for patients with advanced head and neck squamous cell carcinoma(HNSCC) [24]. In 45%–87% of OSCC cases, cancer cells were PD-L1
positive, depending on the cut-off value for positivity and whether
cytoplasmic staining was included as positive [24]. Anti-PD-L1 mAbs
have been mainly used for PD1/PD-L1 blockade, but antitumor activities
byantibody-dependent
cellularcytotoxicity(ADCC)
andcomplement-dependent cytotoxicity (CDC) against oral cancers have notbeen investigated.
In our previous study, we developed a novel anti-human PD-L1
antibody, L1Mab-13 (mouse IgG1, kappa), which is useful for flow
cytometry, Western blot, and immunohistochemical analysis [25]. In
this study, we converted IgG1 subclass L1Mab-13 into IgG2a subclass13-mG2a, and further produced a defucosylated version, 13-mG2a-f,
using FUT8-deficient ExpiCHO-S cells (BINDS-09) [26]. We theninvestigated whether 13-mG2a-f exhibited ADCC, CDC, and antitumor
activities against oral cancers.
2. Materials and methods
2.1. Cell lines
CHO-K1 was obtained from the American Type Culture Collection(ATCC, Manassas, VA). CHO/PD-L1 was previously established [25].
Oral squamous carcinoma cell lines, including SAS (tongue) and HSC-2(oral cavity), were obtained from the Japanese Collection of Research
Bioresources Cell Bank (JCRB, Osaka, Japan). CHO-K1 and CHO/PD-L1
were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan). SAS and HSC-2 were cultured in Dulbecco’s modified Eagle’s
medium (DMEM; Nacalai Tesque, Inc.). The medium is supplemented
with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher
Scientific Inc., Waltham, MA, USA), 100 units/mL of penicillin, 100
μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque,Inc.) at 37 ◦ C in a humidified atmosphere containing 5% CO2.

Fig. 1. Recognition of PD-L1 using anti-PD-L1 mAbs. (A) CHO/PD-L1 cells
were treated with L1Mab-13 and 13-mG2a-f (1 μg/mL), followed by secondary
antibodies. (B) CHO-K1 cells were treated with L1Mab-13 and 13-mG2a-f (1 μg/mL), followed by secondary antibodies. (C) SAS cells were treated with L1Mab13 and 13-mG2a-f (1 μg/mL), followed by secondary antibodies. (D) HSC-2 cells
were treated with L1Mab-13 and 13-mG2a-f (1 μg/mL), followed by secondary
antibodies. The black line represents the negative control.

2.2. Antibodies

squamous cell carcinoma (SCC), adenocarcinoma, mucoepidermoid
carcinoma, and adenoid cystic carcinoma. The most effective treatmentof oral SCC (OSCC), which comprises over 90% of all oral cancers, de­pends upon its clinical stage [19]. Early stages (stage-I and –II) aretreated by surgery or radiotherapy alone. In contrast, advanced stages(stage-III and -IV) require a combination of surgery, radiotherapy, andchemotherapy [20]. For chemotherapy of OSCCs, cisplatin is theAnti-PD-L1 mAb L1Mab-13 (mouse IgG1, kappa) was developed aspreviously described [25]. To generate 13-mG2a, appropriate VH cDNAof mouse L1Mab-13 and CH of mouse IgG2a were subcloned into
pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka,
Japan), and VL and CL cDNAs of L1Mab-13 were subcloned into
pCAG-Neo vector (FUJIFILM Wako Pure Chemical Corporation). The
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Fig. 2. Determination of the binding affinity ofanti-PD-L1 mAbs for oral cancer cells using flow
cytometry. (A) SAS cells were suspended in 100
μl of serially diluted mAbs (0.3 ng/mL - 5 μg/mL),followed by the addition of Alexa Fluor 488-con­jugated anti-mouse IgG, and fluorescence data
were collected. (B) HSC-2 cells were suspended in
100 μL of serially diluted mAbs (0.3 ng/mL - 5
μg/mL), followed by the addition of Alexa Fluor
488-conjugated anti-mouse IgG, and fluorescencedata were collected.

vectors were transfected into BINDS-09 (FUT8-deficient ExpiCHO-S
cells) using the ExpiCHO Expression System [26]. 13-mG2a-f was puri­
fied using Protein G-Sepharose (GE Healthcare Bio-Sciences, Pittsburgh,
PA). Mouse IgG was purchased from Sigma-Aldrich Corp. (St. Louis,MO).

2.5. Enzyme-linked immunosorbent assay (ELISA)
L1Mab-13 and 13-mG2a-f were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific Inc.) at 1 μg/mL for 30
min. After blocking using SuperBlock buffer (Thermo Fisher ScientificInc.) containing 0.5 mM CaCl2, the plates were incubated with biotinlabeled lectins, such as Aleuria aurantia lectin (AAL, fucose binder;
Vector Laboratories, Burlingame, CA, USA) [27], Pholiota squarrosa
lectin (PhoSL, core fucose binder; J-OIL MILLS, Inc., Tokyo, Japan) [28],and Concanavalin A (ConA, mannose binder; Vector Laboratories) [29],followed by 1:3000 diluted peroxidase-conjugated streptavidin (Agilent
Technologies, Santa Clara, CA, USA). The enzymatic reaction was pro­
duced using a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific Inc.).
Optical density was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA, USA).

2.3. Flow cytometry
Cells were harvested by brief exposure to 0.25% trypsin/1 mM eth­ylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Afterwashing with 0.1% bovine serum albumin (BSA) in phosphate-bufferedsaline (PBS), cells were treated with primary mAbs for 30 min at 4 ◦ C
and subsequently with Alexa Fluor 488-conjugated anti-mouse IgG(1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). Fluores­
cence data were collected using an EC800 Cell Analyzer (Sony Corp.,
Tokyo, Japan).

2.6. Animals

2.4. Determination of the binding affinity

All animal experiments were performed in accordance with relevant
guidelines and regulations to minimize animal suffering and distress in
the laboratory. Animal studies for ADCC and antitumor activity wereapproved by the institutional committee for experiments of the Instituteof Microbial Chemistry (Permit number: 2020–007). Mice were moni­
tored for health and weight every 2 or 4 days. Experiment duration was
3 weeks. A bodyweight loss exceeding 25% and a maximum tumor size
exceeding 3000 mm3 were identified as humane endpoints. Mice were

Cells were suspended in 100 μL of serially diluted mAbs (0.3 ng/
mL–5 μg/mL), followed by the addition of Alexa Fluor 488-conjugatedanti-mouse IgG (1:200; Cell Signaling Technology, Inc.). Fluorescencedata were collected using an EC800 Cell Analyzer (Sony Corp.). The
dissociation constant (KD) was calculated by fitting binding isotherms tobuilt-in one-site binding models in GraphPad PRISM 6 (GraphPad
Software, Inc., La Jolla, CA, USA).
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only, and M is maximum fluorescence measured after lysing all cellswith a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4),
and 10 mM of EDTA.
2.8. CDC
To assess cell viability, cells were labeled with 10-μg/mL Calcein AM(Thermo Fisher Scientific, Inc.) and resuspended in the same medium.
The cells (2 × 104 cells/well) were plated in 96-well plates and mixed
with rabbit complement (final dilution 1:10; Low-Tox-M Rabbit Com­
plement; Cedarlane Laboratories, Hornby, Ontario, Canada), anti-PD-L1
antibodies, or control IgG (mouse IgG2a) (Sigma-Aldrich Corp.). After a
6.5-h incubation, the Calcein AM release of supernatant from each well
was measured. Fluorescence intensity was determined as described in
the ADCC part above.
2.9. Antitumor activity of 13-mG2a-f in the xenografts of oral cancers
Thirty-two 6-week-old female BALB/c nude mice were purchasedfrom Charles River (Kanagawa, Japan) and used at 7 weeks of age. SAS
and HSC-2 cells (0.3 mL of 1.33 × 108 cells/mL in DMEM) were mixed
with 0.5 mL BD Matrigel Matrix Growth Factor Reduced (BD Bio­sciences, San Jose, CA, USA). 100 μL of this suspension (5 × 106 cells)
was injected subcutaneously into the left flank. After day 8, 100 μg of 13mG2a-f and control mouse IgG (Sigma-Aldrich Corp.) in 100 μL PBS was
injected intraperitoneally (i.p.) into treated and control mice, respec­tively. Additional antibodies were then injected on days 14 and 21.
Twenty-three days after cell implantation, all mice were euthanized bycervical dislocation, and tumor diameters and volumes were determined
as previously described [30].
Fig. 3. Confirmation of defucosylation of 13-mG2a-f by enzyme-linked immu­
nosorbent assay (ELISA) using lectins. (A) L1Mab-13 and 13-mG2a-f were
immobilized and incubated with biotin-labeled concanavalin A (Con A), fol­
lowed by peroxidase-conjugated streptavidin. OD655 was measured as a func­tion of Con A concentration. (B) L1Mab-13 and 13-mG2a-f were immobilized
and incubated with biotin-labeled Aleuria aurantia lectin (AAL), followed byperoxidase-conjugated streptavidin. OD655 was measured as a function of AAL
concentration. (C) L1Mab-13 and 13-mG2a-f were immobilized and incubatedwith biotin-labeled Pholiota squarrosa lectin (PhoSL), followed by peroxidaseconjugated streptavidin. OD655 was measured as a function of PhoSL
concentration.

2.10. Statistical analyses
All data were expressed as mean ± SEM. Statistical analysis used
ANOVA and subsequently Sidak’s multiple comparisons test for tumor
volume and mouse weight, or Welch’s t-test for ADCC/CDC and tumor
weight using GraphPad Prism 7 (GraphPad Software, Inc.). P < 0.05 wasadopted as a level of statistical significance.
3. Results
3.1. Development and characterization of 13-mG2a-f, a core-fucosedeficient mouse IgG2a-type anti-PD-L1 antibody

euthanized by cervical dislocation, and the death was verified by res­piratory arrest and cardiac arrest.

As mouse IgG2a possesses high ADCC and CDC activities [31], wefirst developed a mouse IgG2a version of L1Mab-13 (mouse IgG1) bysubcloning appropriate VH cDNA of L1Mab-13 and CH of mouse IgG2a
into pCAG-Ble vector, and the light chain of L1Mab-13 into pCAG-Neo
vector. This IgG2a-type of L1Mab-13 is henceforth referred to as
13-mG2a. We additionally produced a core-fucose-deficient type of
13-mG2a, henceforth referred to as 13-mG2a-f, using the BINDS-09 cellline (FUT8-knockout ExpiCHO–S cell line) [26]. We analyzed thesensitivity of 13-mG2a-f in CHO-K1 cells expressing PD-L1 (CHO/PD-L1)
and in OSCC cell lines (SAS and HSC-2) using flow cytometry. BothL1Mab-13 and 13-mG2a-f reacted with CHO/PD-L1 cells (Fig. 1A), but
not with CHO-K1 cells (Fig. 1B). Both mAbs also reacted with SAS cells(Fig. 1C) and HSC-2 cells (Fig. 1D), indicating that 13-mG2a-f demon­
strated high sensitivity and specificity for PD-L1.
We conducted a kinetic analysis of the interactions of L1Mab-13 and13-mG2a-f with SAS and HSC-2 oral cancer cell lines using flow cytom­
etry. The dissociation constant (KD) for L1Mab-13 in SAS cells wasdetermined to be 4.1 × 10− 9 M (Fig. 2A). In contrast, the KD for 13mG2a-f in SAS cells was 2.8 × 10− 9 M. The binding affinity of 13-mG2a-f
in SAS cells was 1.5-fold higher than that of L1Mab-13. Likewise, the KD
for L1Mab-13 against HSC-2 cells was 8.8 × 10− 9 M (Fig. 2B). By
contrast, the KD for 13-mG2a-f in HSC-2 cells was 4.8 × 10− 9 M. The

2.7. ADCC
Six 6-week-old female BALB/c nude mice were purchased fromCharles River (Kanagawa, Japan). After euthanization by cervicaldislocation, spleens were removed aseptically and single-cell suspen­
sions obtained by forcing spleen tissues through a sterile cell strainer(352360, BD Falcon, Corning, New York, NY, USA) using a syringe.
Erythrocytes were lysed with a 10-sec exposure to ice-cold distilledwater. Splenocytes were washed with DMEM and resuspended in DMEM
with 10% FBS and used as effector cells. Target cells were labeled with
10-μg/mL Calcein AM (Thermo Fisher Scientific, Inc.) and resuspended
in the same medium. The target cells (2 × 104 cells/well) were plated in
96-well plates and mixed with effector cells, anti-PD-L1 antibodies, orcontrol IgG (mouse IgG2a) (Sigma-Aldrich). After a 6.5-h incubation, theCalcein AM release of supernatant from each well was measured.
Fluorescence intensity was determined using a microplate reader(Power Scan HT) (BioTek Instruments, Winooski, VT, USA) with an
excitation wavelength of 485 nm and an emission wavelength of 538
nm. Cytolytic activity (as % of lysis) was calculated using the equation:% lysis = (E− S)/(M− S) × 100, where E is fluorescence of combined
target and effector cells, S is spontaneous fluorescence of target cells
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Biochemistry and Biophysics Reports 24 (2020) 100801

Fig. 4. Evaluation of ADCC and CDC activities of 13-mG2a-f in SAS and HSC-2 cells. (A) ADCC activities of 13-mG2a-f and control mouse IgG2a in SAS and HSC-2 cells.
(B) CDC activities by 13-mG2a-f and control mouse IgG2a in SAS and HSC-2 cells. Values are mean ± SEM. Asterisk indicates statistical significance (**P < 0.01, *P <0.05, n.s: not significant, Welch’s t-test).

binding affinity of 13-mG2a-f in HSC-2 cells was 1.8-fold higher than thatof L1Mab-13. The binding affinity of 13-mG2a-f in SAS cells was 1.7-foldhigher than that in HSC-2 cells.
Defucosylation of 13-mG2a-f was confirmed using lectins such as AAL(which binds to fucose) and PhoSL (which binds to core fucose). ConA
(which binds to mannose) was used as a positive control. Both L1Mab-13
and 13-mG2a-f were detected using ConA (Fig. 3A). L1Mab-13, but not
13-mG2a-f, was detected using AAL (Fig. 3B) and PhoSL (Fig. 3C),
demonstrating that 13-mG2a-f was defucosylated.

outstanding, it remained to be seen whether 13-mG2a-f may exert anti­
tumor activity against oral cancer cells in vivo.
3.3. Antitumor activities of 13-mG2a-f in the mouse xenografts of SASoral cancer cells
In the SAS xenograft models, tumor formation in 16 SAS-injected
mice was observed after eight days. These 16 SAS tumor-bearing mice
were then divided into a 13-mG2a-f-treated group and a control group.
On days 8, 14, and 21 after SAS cell injections into the mice, 13-mG2a-f(100 μg) and control mouse IgG (100 μg) were injected i.p. into the mice.
Tumor formation was observed in mice in both treated and control
groups. Tumor volume was measured on days 8, 14, 17, 21, and 23 after
SAS cell injection. 13-mG2a-f-treated mice showed significantly reduced
tumor development on day 17 (P < 0.01), day 21 (P < 0.01), and day 23(P < 0.01) in comparison to IgG-treated control mice (Fig. 5A). Tumor
volume reduction by 13-mG2a-f treatment was 51% on day 23. Tumors
from 13-mG2a-f-treated mice weighed significantly less than tumors
from IgG-treated control mice (41% reduction, P < 0.05; Fig. 5B).
Resected tumors on day 23 are depicted (Fig. 5C). Total body weights
did not significantly differ between the two groups (data not shown).
These results indicate that 13-mG2a-f reduced the growth of SAS xeno­
grafts effectively, even when 13-mG2a-f was injected eight days post-SAScell injections in mice.

3.2. ADCC and CDC activities of 13-mG2a-f in oral cancer cell lines
Because the mouse IgG1 subclass L1Mab-13 does not possess ADCC or
CDC activities, we created a mouse IgG2a subclass mAb, and further
defucosylated it to enhance those activities. In this study, we examined
whether the developed 13-mG2a-f induced ADCC and CDC in PD-L1expressing oral cancer cell lines, such as SAS and HSC-2 cells. 13mG2a-f exhibited higher ADCC (17% cytotoxicity) in SAS cells comparedwith that of control mouse IgG2a treatment (6.6% cytotoxicity; P < 0.05)(Fig. 4A). Similarly, 13-mG2a-f exhibited higher ADCC (8.0% cytotox­
icity) in HSC-2 cells compared with that of control mouse IgG2a treat­ment (2.5% cytotoxicity; P < 0.01) (Fig. 4A).
Furthermore, 13-mG2a-f exhibited higher CDC activity (27% cyto­
toxicity) in SAS cells compared with control mouse IgG2a treatment(7.3% cytotoxicity; P < 0.01; Fig. 4B). Similarly, 13-mG2a-f exhibited
higher CDC activity (15% cytotoxicity) in HSC-2 cells compared with
control mouse IgG2a treatment (4.1% cytotoxicity; P < 0.01; Fig. 4B).
Although ADCC/CDC activities of 13-mG2a-f in oral cancer cells are not5J. Takei et al.

Biochemistry and Biophysics Reports 24 (2020) 100801

Fig. 5. Evaluation of antitumor activity of 13-mG2a-f in SAS xenografts. (A)Tumor volume of SAS xenografts. SAS cells (5 × 106 cells) were injected sub­cutaneously into the left flank. After eight days, 100 μg of 13-mG2a-f or control
mouse IgG in 100 μl PBS were injected i.p. into treated and control mice,
respectively. Additional antibodies were then injected on days 14 and 21.
Tumor volume was measured on days 8, 14, 17, 21, and 23. Values are mean ±SEM. Asterisk indicates statistical significance (**P < 0.01, n.s: not significant,
ANOVA and Sidak’s multiple comparisons test) (B) Tumor weights of SAS xe­
nografts. Tumors of SAS xenografts were resected from 13-mG2a-f and control
mouse IgG groups. Tumor weight on day 23 was measured from excised xe­
nografts. Values are mean ± SEM. Asterisk indicates statistical significance (*P< 0.05, Welch’s t-test). (C) Resected tumors of SAS xenografts from 13-mG2a-f
and control mouse IgG groups on day 23.

Fig. 6. Evaluation of antitumor activity of 13-mG2a-f in HSC-2 xenografts. (A)Tumor volume of HSC-2 xenografts. HSC-2 cells (5 × 106 cells) were injectedsubcutaneously into the left flank. After eight days, 100 μg of 13-mG2a-f orcontrol mouse IgG in 100 μl PBS were injected i.p. into treated and control
mice, respectively. Additional antibodies were then injected on days 14 and 21.
Tumor volume was measured on days 8, 14, 17, 21, and 23. Values are mean ±SEM. Asterisk indicates statistical significance (**P < 0.01, n.s: not significant,
ANOVA and Sidak’s multiple comparisons test) (B) Tumor weights of HSC-2
xenografts. Tumors of HSC-2 xenografts were resected from 13-mG2a-f andcontrol mouse IgG groups. Tumor weight on day 23 was measured from excised
xenografts. Values are mean ± SEM. Asterisk indicates statistical significance(*P < 0.05, Welch’s t-test). (C) Resected tumors of HSC-2 xenografts from 13mG2a-f and control mouse IgG groups on day 23.

3.4. Antitumor activities of 13-mG2a-f in mouse xenografts of HSC-2 oral
cancer cells

4. Discussion
In this study, we investigated whether anti-PD-L1 mAbs are advan­
tageous for the treatment of oral cancers by ADCC/CDC activities, rather
than by neutralization of PD-L1/PD1 interaction, as human PD-L1 does
not react with mouse PD-1. We had previously developed a sensitive andspecific anti-PD-L1 mAb, L1Mab-13 [25], but were unable to investigate
antitumor activity as the IgG1 subclass does not possess ADCC/CDC
activities. Therefore, we converted L1Mab-13 into an IgG2a subclassantibody, and increased ADCC activity via defucosylation. We demon­
strated that 13-mG2a-f exerts ADCC/CDC activities in vitro (Fig. 4), andantitumor activities against oral cancer xenografts in vivo (Figs. 5 and 6).
Importantly, 13-mG2a-f efficaciously reduced the growth of SAS xeno­
grafts (Fig. 5) and HSC-2 xenografts (Fig. 6), even when 13-mG2a-f was
injected eight days after cell implantations into the mice. However, SAS
and HSC-2 tumor volume reduction on day 23 by 13-mG2a-f treatment
only reached 51% and 57%, respectively, indicating that anti-PD-L1
therapy might not be sufficient for solo treatment of most oral can­cers. One potential reason for this weak antitumor activity is low ADCC

In the HSC-2 xenograft models, tumor formation of 16 HSC-2injected mice was observed after eight days. These 16 HSC-2-bearing
mice were then divided into a 13-mG2a-f-treated group and a control
group. On days 8, 14, and 21 after cell injections into the mice, 13-mG2af (100 μg) and control mouse IgG (100 μg) were injected i.p. into themice. Tumor volume was assessed on days 8, 14, 17, 21, and 23. 13mG2a-f-treated mice showed significantly reduced tumor development
on day 17 (P < 0.01), day 21 (P < 0.01), and day 23 (P < 0.01) incomparison to IgG-treated control mice (Fig. 6A). Tumor volume
reduction in 13-mG2a-treated mice was 57% on day 23. Tumors from 13mG2a-f-treated mice weighed significantly less than tumors from IgGtreated control mice (28% reduction, P < 0.05; Fig. 6B). Resected tu­
mors on day 23 are shown in Fig. 6C. Total body weights did not
significantly differ between the two groups (data not shown). These
results indicate that 13-mG2a-f reduced the growth of HSC-2 xenografts
effectively, even when 13-mG2a-f was injected eight days post–HSC–2
cell injections in mice.
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activity (Fig. 4A) and CDC activity (Fig. 4B) of 13-mG2a-f, in spite of thehigh binding affinity in SAS cells (KD: 2.8 × 10− 9 M; Fig. 2A) and HSC-2
cells (KD: 4.8 × 10− 9 M; Fig. 2B). The binding affinity of 13-mG2a-f in
SAS cells was 1.5-fold higher than that of L1Mab-13. Likewise, thebinding affinity of 13-mG2a-f in HSC-2 cells was 1.8-fold higher than thatof L1Mab-13. These results are consistent with our previous observationthat the mouse IgG2a-type mAb 47-mG2a-f also shows a higher affinity
for PODXL than the original PcMab-47 (mouse IgG1), indicating thatfragment crystallizable (Fc) portion of mouse IgG could be important forthe binding affinity for target molecules; or, recombinant mAbs might
possess high purity compared to hybridoma-derived mAbs [32].
We recently developed a sensitive and specific mAb against EGFR
(clone EMab-17, mouse IgG2a), and examined its ADCC/CDC and anti­
tumor activities against SAS and HSC-2 xenografts [30]. In anotherrecent study, HER2 was shown to be expressed in oral cancers, and ananti-HER2 mAb (clone H2Mab-19, mouse IgG2b) showed antitumor ac­
tivity against SAS and HSC-2 xenografts [33]. Further, we previously
investigated whether PODXL may be a therapeutic target in OSCC using
anti-PODXL mAbs [32]. We converted an anti-PODXL mAb of IgG1
subclass (PcMab-47) into a mouse IgG2a-type mAb (47-mG2a) to increase
ADCC. We developed 47-mG2a-f, a core fucose-deficient variant of
47-mG2a to increase its ADCC. In vivo analysis demonstrated that
47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and
HSC-2 xenograft models at a dose of 100 μg/mouse/week administered
three times. Although both 47-mG2a and 47-mG2a-f exhibited antitumor
activity in HSC-2 xenograft models at a dose of 500 μg/mouse/weekadministered twice, 47-mG2a-f also demonstrated higher antitumor ac­
tivity than 47-mG2a, indicating that a core fucose-deficient anti-PODXL
mAb could be profitable for antibody-based therapy against
PODXL-expressing OSCCs.
Targeting multiple targets, such as PODXL, EGFR, HER2, and PD-L1
may be needed for effective therapy to cure oral cancers. Anotherimportant goal is to target cancer-specific antigens using a cancerspecific mAb (CasMab). We previously established CasMab against
PDPN, which is expressed in many cancers, including oral cancers [34].
In xenograft models with HSC-2 cells, a mouse-human chimeric mAb,
chLpMab-23, exerted antitumor activity using human natural killer
cells, indicating that chLpMab-23 may be advantageous for antibody
therapy against PDPN-expressing oral cancers [35]. In the future,
cancer-specific anti-PD-L1 mAbs may also be developed that can reduce
the adverse effects of traditional antibody therapy.

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Funding
This research was supported in part by Japan Agency for Medical
Research and Development (AMED) under Grant Numbers:
JP20am0401013 (Y.K.), JP20am0101078 (Y.K.), and JP20ae0101028(Y.K.).
Acknowledgments
We thank Ms. Saori Handa and Mr. Yu Komatsu (Department of
Antibody Drug Development, Tohoku University Graduate School of
Medicine) for technical assistance of in vitro experiments, and Ms. Akiko
Harakawa (Institute of Microbial Chemistry (BIKAKEN), Numazu, Mi­
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbrep.2020.100801.

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