Received: 5 July 2023

| Revised: 7 November 2023 | Accepted: 5 December 2023

DOI: 10.1111/cas.16046

ORIGINAL ARTICLE

Antipodoplanin antibody enhances the antitumor effects of
CTLA-­4 blockade against malignant mesothelioma by naturalkiller cells
Hiroto Yoneda1 | Atsushi Mitsuhashi1 | Aito Yoshida2 | Hirokazu Ogino1 |Satoshi Itakura2 | Na Thi Nguyen1 | Hiroshi Nokihara1 | Seidai Sato1 |Tsutomu Shinohara3 | Masaki Hanibuchi4 | Shinji Abe2 | Mika K. Kaneko5 |Yukinari Kato5| Yasuhiko Nishioka1,6
1

Department of Respiratory Medicine and Rheumatology, Tokushima University, Tokushima, Japan2

Department of Clinical Pharmacy Practice Pedagogy, Tokushima University, Tokushima, Japan3

Department of Community Medicine for Respirology, Tokushima University, Tokushima, Japan

4

Department of Community Medicine for Respirology, Hematology and Metabolism, Graduate School of Biomedical Sciences, Tokushima University,
Tokushima, Japan
5

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan

6

Department of Community Medicine for Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, Tokushima, Japan

Correspondence
Yasuhiko Nishioka, Department ofRespiratory Medicine and Rheumatology,
Tokushima University, 3-­18-­15 Kuramoto-­cho, Tokushima, Tokushima 770-­8503,
Japan.
Email: yasuhiko@tokushima-u.ac.jp
Funding information
Japan Agency for Medical Research andDevelopment, Grant/Award Number:
JP21am0101078 and JP22ama121008;
Japan Society for the Promotionof Science, Grant/Award Number:
19H03668, 20K07197 and 20K17216

Abstract
Combination immunotherapy with multiple immune checkpoint inhibitors (ICIs) has
been approved for various types of malignancies, including malignant pleural mesothelioma (MPM). Podoplanin (PDPN), a transmembrane sialomucin-­like glycoprotein,
has been investigated as a diagnostic marker and therapeutic target for MPM. We
previously generated and developed a PDPN-­targeting Ab reagent with high Ab-­dependent cellular cytotoxicity (ADCC) and complement-­dependent cytotoxicity(CDC). However, the effects of anti-­PDPN Abs on various tumor-­infiltrating immune
cells and their synergistic effects with ICIs have remained unclear. In the present
study, we established a novel rat–mouse chimeric anti-­mouse PDPN IgG2a mAb(PMab-­1-­mG2a) and its core-­fucose-­deficient Ab (PMab-­1-­mG2a-­f ) to address theselimitations. We identified the ADCC and CDC activity of PMab-­1-­mG2a-­f against the
PDPN-­expressing mesothelioma cell line AB1-­HA. The antitumor effect of monotherapy with PMab-­1-­mG2a-­f was not sufficient to overcome tumor progression in
AB1-­HA-­bearing immunocompetent mice. However, PMab-­1-­mG2a-­f enhanced theantitumor effects of CTLA-­4 blockade. Combination therapy with anti-­PDPN Ab andanti-­C TLA-­4 Ab increased tumor-­infiltrating natural killer (NK) cells. The depletion

Abbreviations: ADCC, antibody-­dependent cellular cytotoxicity; CDC, complement-­dependent cytotoxicity; CTLA-­4, CTL-­associated antigen 4; HER2, human epidermal growth factor
receptor 2; ICI, immune checkpoint inhibitor; IL-­2Rβ, interleukin-­2 receptor β-­chain; MPM, malignant pleural mesothelioma; NK, natural killer; OS, overall survival; PD-­1, programmed
death 1; PD-­L1, programmed death ligand 1; PDPN, podoplanin; Treg, regulatory T cell.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution inany medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
Cancer Science. 2024;115:357–368.	﻿

wileyonlinelibrary.com/journal/cas| 357

YONEDA et al.

of NK cells inhibited the synergistic effects of PMab-­1-­mG2a-­f and CTLA-­4 blockadein vivo. These findings indicated the essential role of NK cells in novel combination immunotherapy targeting PDPN and shed light on the therapeutic strategy in advanced
MPM.
KEYWORDS

CTLA-­4, immune checkpoint inhibitor, malignant pleural mesothelioma, NK cell, podoplanin

1

I NTRO D U C TI O N|

cells in human MPM cell-­bearing SCID mice. In a previous study, we
mainly focused on the ADCC activity of the anti-­PDPN Ab by bind-

Malignant pleural mesothelioma is an aggressive malignant tumor
1–3

mainly caused by environmental asbestos exposure.

The prog-

ing the Fc receptor in NK cells. However, in the actual tumor immune
microenvironment, immunotherapy targets multiple immune cells,

nosis of MPM is extremely poor because of its poor response

including T cells, B cells, NK cells, and Tregs. To evaluate the anti-

to cytotoxic chemotherapy and the difficulty making an earlytumor effects of anti-­PDPN Abs and their interactions with known

diagnosis.

immunotherapies, including ICIs, a preclinical study in immunocom-

A recent study revealed that platinum-­based chemotherapy withpetent conditions is needed.

pemetrexed prolonged the OS of advanced MPM patients comparedWe generated a novel rat–mouse chimeric anti-­mouse PDPN

with platinum monotherapy (12.1 vs. 9.3 months).4 However, be-

IgG2a mAb (PMab-­1-­mG2a) to detect the expression of PDPN in

cause of its insufficient efficacy and the lack of validated second-­

mouse tumor cells. In addition, we also produced a core-­fucose-­line therapy, novel therapeutic strategies for MPM have long been

deficient Ab to improve ADCC activity (PMab-­1-­mG2a-­f ). In the pres-sought.

ent study, we investigated the antitumor effects of an anti-­mouse

Immune checkpoint inhibitors targeting PD-­1, PD-­L1, and

PDPN Ab under immunocompetent conditions and its influence on

CTLA-­4 have shown clinical benefit in various types of malignan-various immune cells in the tumor microenvironment. Furthermore,

cies.

5–7

The phase III CheckMate743 trial showed that combination

the synergistic effects of anti-­PDPN Abs with ICIs, such as PD-­1

immunotherapy with an anti-­PD-­1 Ab (nivolumab) and anti-­C TLA-­4

blockade and CTLA-­4 blockade, were examined to develop a novel

Ab (ipilimumab) results in superior improvements in the OS in unre-combination immunotherapy for MPM.

sectable MPM cases compared with platinum-­based chemotherapy
with pemetrexed (18.1 vs. 14.1 months).8 Combination immunotherapy has shed new light on therapeutic strategies for unresectable
MPM. Thus, the detection of novel therapeutic targets in the tumor
immune microenvironment is required to overcome the poor prog-

2|

M ATE R I A L S A N D M E TH O DS

2.1 | Cell lines

nosis of MPM.
Podoplanin, a transmembrane sialomucin-­like glycoprotein,

The mouse malignant mesothelioma cell line AB1-­HA, a transfectant

was first identified as the regulatory molecule of podocyte mor-with the gene encoding influenza HA into AB1 cells and the mousephology in the kidney.9 The expression of PDPN was also detected

malignant mesothelioma cell line AC29 were purchased from Public

in lymphatic endothelial cells as a marker of lymphangiogene-

Health England. The mouse lung cancer cell line LLC was purchased

sis.10 The PDPN expression has been investigated as a cell sur-from ATCC. Mouse melanoma cell line B16 was kindly provided byface marker for various types of malignancies as well, including

Dr. IJ Fidler (MD Anderson Cancer Center). The mouse fibrosarcoma

oral cancers, colorectal cancers, squamous lung carcinoma, andcell line MCA205 and mouse colon carcinoma cell line MC38 were

11–14

Therefore, PDPN has potential utility as a diagnostic

generously provided by Dr. SA Rosenberg (National Cancer Institute,biomarker and tumor-­associated antigen for immunotherapy in

MPM.

National Institutes of Health). AB1-­HA was maintained in DMEM

MPM.

supplemented with 10% heat-­inactivated FBS, penicillin (100 U/mL),

Several therapeutic Abs targeting tumor-­associated antigens

and streptomycin (50 μg/mL). The other cell lines were maintained inhave been approved for various malignancies, such as rituximab

RPMI-­1640. All cells were cultured at 37°C in a humidified atmos-(anti-­CD20 Ab) and trastuzumab (anti-­HER2 Ab).15,16 These Abs

phere of 5% CO2 in air.

have been known to exert tumor-­killing effects through ADCC and
CDC.17 In our previous studies, we generated a specific Ab targeting
PDPN and investigated its antitumor effects in vitro and in preclin-

2.2 | Reagentsical models. A rat anti-­human PDPN mAb, NZ-­1,18 and a rat–humanchimeric anti-­human PDPN Ab, NZ-­819 and NZ-­12, 20 showed highAnti-­m ouse PD-­1 (clone RMP1-­14) and anti-­m ouse CTLA-­4 (clone

ADCC and CDC activity and enhanced the antitumor effects of NK

4F10) Abs were purchased from BioXCell. Isotype control IgG

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License|358

(rat IgG1, rat IgG2a , and hamster IgG) were also purchased from

2.5 | Immunofluorescence of mouse tumor tissues

BioXCell. PMab-­1, a rat anti-­m ouse PDPN mAb, was developed
as previously described. 21 For the generation of rat–mouse chi-The excised tumor tissues from the model mice were placed intomeric anti-­m ouse PDPN IgG2a mAb (PMab-­1-­m G2a), appropriate

OCT compound (Sakura Finetechnical Co.) and snap-­frozen. Frozen

V H cDNA of rat PMab-­1 was subcloned into pFUSE-­CHIGmG2atissue sections (8 μm thick) were fixed with 4% paraformaldehyde(InvivoGen) and appropriate V L cDNA of rat PMab-­1 and CL

solution in PBS and used for the identification of NK cells, CD8+of mouse kappa chain were subcloned into pCAG-­N eo vec-and CD4 + T cells, and Tregs using a goat anti-­mouse NKp46 (1:100;

tor (FUJIFILM Wako Pure Chemical Corporation), respectively.

R&D Systems), rat anti-­CD8a mAb (1:150, 53–6.7; BD Pharmingen),

Antibody expression vectors were transfected into ExpiCHO-­S

rat anti-­mouse CD4 (1:150, H129.19; BD Pharmingen), and a rabbit(Thermo Fisher Scientific Inc.) using the ExpiCHO Expression

anti-­Foxp3 polyclonal Ab (1:400 dilution; Novus Biologicals), respec-

System (Thermo Fisher Scientific Inc.). The core fucose-­d eficient

tively. To identify NKT cells, rat anti-­mouse CD3 (1:150, H129.19; BD

type of PMab-­1-­m G2a (PMab-­1-­m G2a-­f ) was produced by trans-

Pharmingen), and a rabbit anti-­mouse CD49b polyclonal Ab (1:150

fecting them into FUT8-­d eficient ExpiCHO-­S cells (BINDS-­0 9). 22

dilution, Thermo Fisher Scientific Inc.) were used. Alexa488-­ andAn anti-­m ouse IL-­2Rβ mAb, TM-­β1 (IgG2b), was a gift from Drs.

Alexa594-­labeled secondary Abs (1:250; Thermo Fisher Scientific

Masayuki Miyasaka and Toshio Tanaka (Osaka University, Osaka,Inc.) were used for immunofluorescent detection. Nuclei wereJapan). Anti-­m ouse CD4 (clone GK1.5) and anti-­m ouse CD8 (clone

counter-­stained with DAPI (blue). In each slide, the number of posi-2.43) Abs were purchased from BioXCell.

tive cells was counted under a fluorescent microscope at ×200 magnification. These images were acquired using a BX61 fluorescence

2.3 | Animals
Six-­week-­old male BALB/c mice, C57BL/6J mice, and C.B-­17 SCID

light microscope (Olympus).

2.6 | Flow cytometry

mice were obtained from Charles River Japan Inc. All experiments
were carried out in accordance with the guidelines established by

To examine the surface expression of PDPN in mouse tumor cell

the Tokushima University Committee on Animal Care and Use. At

lines, tumor cells were incubated with PMab-­1-­mG2a or isotype con-the end of each in vivo experiment, the mice were anesthetized withtrol (1 μg/mL) for 30 min. Cells were washed with PBS and incubatedisoflurane and euthanized humanely by cutting the subclavian ar-for 30 min with FITC-­conjugated goat F(ab’)2 fragment anti-­rat IgG

tery. All experimental protocols were reviewed and approved by the(H + L) Ab (Beckman Coulter). AB1-­HA tumor tissue was cut into

animal research committee of The University of Tokushima.

small pieces with scissors and digested by digestion buffer consistingof 1 mg/mL BSA (Sigma-­Aldrich), 1 mg/mL collagenase IV (Thermo

2.4 | In vivo s.c. implantation models of mousetumor cells

Fisher Scientific Inc.), and 100 μg/mL DNase I (Roche) in DMEM
for 40 min of incubation at 37°C, followed by passaging througha 100 μm cell strainer. The cells were incubated with FcR blocking
reagent (BD Biosciences). To examine the surface expression of

AB1-­H A cells or LLC cells (1.0 × 10 6 cells per mouse) suspended

CTLA-­4 and PD-­1 in mouse tumor-­infiltrating immune cells, tumor

in 0.1 mL PBS were s.c. inoculated to the right flank of BALB/c ortissue-­derived cells were stained with FITC-­conjugated anti-­NKp46

C57BL/6J mice, respectively. The tumor size was measured using(BioLegend), FITC-­conjugated anti-­CD8a (BD Biosciences), FITC-­

vernier calipers twice a week, where volume = ab2 2−1 (a, long di-

conjugated anti-­CD4 (BD Biosciences), FITC-­conjugated anti-­F4/80

ameter; b, short diameter). To evaluate the effects of anti-­m ouse

(BioLegend), PE-­Cy7-­conjugated anti-­C TLA-­4 (BioLegend), and PE-­PDPN Ab, the mice were treated with PMab-­1-­m G2a-­f (50 or 100

Cy7-­conjugated anti-­PD-­1 (BioLegend). The stained cells were ana-

or 500 μg per mouse) twice a week. To determine the synergistic

lyzed by flow cytometry using a BD LSRFortessa (BD Biosciences)

effects of anti-­PDPN Ab with PD-­1 blockade or CTLA-­4 blockade

for acquisition and the FlowJo software program (Treestar Inc.) foron tumor growth, the mice were treated twice a week with PMab-­

the analysis.

1-­m G2a-­f (50 μg per mouse), anti-­PD-­1 Ab (200 μg per mouse),anti-­C TLA-­4 Ab (5 μg per mouse), or isotype control IgG by i.p.
injection from day 7 until they became moribund. To deplete NK

2.7 | Preparation of effector cells

cells, the mice were pretreated with anti-­m ouse IL-­2Rβ Ab 4 days
after tumor inoculation. To deplete CD4 + T cells or CD8 + T cells,

Mouse splenocytes were harvested from C.B-­17 SCID mice spleens.

the mice were treated with anti-­m ouse CD4 or CD8 Abs (200 μg

Spleens were homogenized in RPMI-­1640 and centrifuged. To de-per mouse) twice a week from 4 days after tumor inoculation. The

plete red blood cells, the cell pellet was suspended in red blood

mice were killed humanely, and the tumors were resected for fur-cell lysis buffer (Sigma-­Aldrich). After washing and resuspension inther analyses on days 21.

RPMI-­1640, splenocytes were used as effector cells.

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 359

YONEDA et al.

|

YONEDA et al.

2.8 | Antibody-­dependent cellular cytotoxicity

measured using a gamma counter. Percent cytotoxicity was calculated as described above.

Antibody-­d ependent cellular cytotoxicity was determined using
51

Cr release assays. Target cells (AB1-­H A or LLC) were incubatedwith 51Cr-­s odium chromate (3.7 MBq) at 37°C for 1 h. After washing with RPMI-­1640 three times,

2.10 | Statistical analyses

51

Cr-­labeled target cells wereplaced in triplicate in 96-­well plates. Effector cells and anti-­m ouse

The data are presented as the mean ± SE of the mean. The statistical

PDPN Abs (PMab-­1-­m G 2a or PMab-­1-­m G 2a-­f, 10 μg/mL) or control

analyses were carried out using Student's t-­test for unpaired sam-

IgG were added to the plates (effector/target ratio 100). After

ples, the Mann–Whitney U-­test or a one-­way ANOVA, followed by

18 h of incubation,

51

Cr release of the supernatant from eachwell (100 μL) was measured using a gamma counter (PerkinElmer).

Tukey's multiple comparison post-­hoc test, as appropriate. Values ofp < 0.05 were considered to be statistically significant.

Percent of cytotoxicity was calculated using the following formula: % specific lysis = (E − S)/(M − S) × 100, where E is the releasein the test sample, S is the spontaneous release, and M is the
maximum release.

2.9 | Complement-­dependent cytotoxicity

3|

R E S U LT S

3.1 | Evaluating the activity of anti-­mouse PDPN
Abs against mouse MPM cell lines
Initially, we investigated the expression of PDPN in mouse tumor

Complement-­dependent cytotoxicity was evaluated by 51Cr re-cell lines using PMab-­1-­mG2a in flow cytometry (Figure 1A). Highlease assay. Target cells were incubated with 51Cr-­sodium chromate

PDPN expression was identified in the cell membrane of the mouse(3.7 MBq) for 1 h at 37°C. Following this, cells were washed in RPMI-­

mesothelioma cell line AB1-­HA, colon cancer cell line MC38, and1640. The 51Cr-­labeled cells were incubated with baby rabbit com-melanoma cell line B16. To evaluate the antitumor effects of theplement (dilution of 1:64) (Cedarlane) and 10 μg/mL Abs for 6 h inanti-­mouse PDPN Ab against MPM, we next evaluated the ADCC

96-­well plates. After incubation, the supernatant, including 51Cr, was(Figure 1B) and CDC (Figure 1C) activities of PMab-­1-­mG2a and its

F I G U R E 1 Functional analysis of
the rat–mouse chimeric anti-­mouse
podoplanin (PDPN) Ab. (A) Detectionof PDPN on the cell surface of mousetumor cell lines by PMab-­1-­mG2a. (B)Evaluation of the Ab-­dependent cellularcytotoxicity activity of PMab-­1-­mG2a
and PMab-­1-­mG2a-­f using splenocytesof SCID mice against AB1-­HA by an 18 h51
Cr release assay in the presence of Ab(10 μg/mL; effector/target ratio 100). (C)Evaluation of the complement-­dependent
cytotoxicity activity of PMab-­1-­mG2a
and PMab-­1-­mG2a-­f using baby rabbit
complement against AB1-­HA by a 6 h 51Cr
release assay in the presence of Ab (10 μg/
mL; dilution ratio 64). Data are shown as
the mean ± SEM. *p < 0.05 by a one-­way
ANOVA.

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License360

core-­fucose-­deficient Ab, PMab-­1-­mG2a-­f. Splenocytes from SCID

immunohistochemical

analysisoftumor-­infiltrating

NK

cells

mice were used as effector cells to evaluate the NK cell-­dependent(Figure 3C), CD8 T cells (Figure 3D), and CD4 T cells (Figure 3E)

ADCC activity of the anti-­PDPN Ab. Several studies have shown thatwas undertaken in each group. CTLA-­4 is known to dominate thecore-­fucose-­deficient Abs demonstrate high ADCC activities. 23,24 In

functions of various immune cells, including T cells, B cells, antigen-­the present study, PMab-­1-­mG2a did not show mouse splenocyte-­

presenting cells, and NK cells. 25,26 Consistent with recent studies,dependent ADCC activity against PDPN-­expressing mesothelioma

CTLA-­4 blockade significantly increased tumor-­infiltrating NK cells,

cells but did demonstrate high CDC activity in vitro. In contrast,

NKT cells, CD8 T cells, and CD4 T cells (Figures 3F–H and S2A). In

core-­fucose-­deficient PMab-­1-­mG2a-­f enhanced the tumor killing

addition, combination therapy with PMab-­1-­mG2a-­f and CTLA-­4capability of mouse splenocytes by ADCC activity (Figure 1B). The

blockade indicated the further accumulation of NK cells and NKT

ADCC (Figure S1A) and CDC (Figure S1B) activity of anti-­PDPN Abs

cells in AB1-­HA tumor tissue (Figures 3F and S2B). However, theagainst PDPN-­negative tumor cells (LLC) were also evaluated. The

anti-­PDPN Ab did not enhance the effects of CTLA-­4 blockade on

tumor killing capability of PMab-­1-­mG2a and PMab-­1-­mG2a-­f against

tumor-­infiltrating CD8 and CD4 T cells (Figure 3G,H). To examine

PDPN− tumors through ADCC or CDC was extremely weak. These

results indicated that PMab-­1-­mG2a-­f exerted antitumor effects
against PDPN+ MPM through both ADCC and CDC activity.

the target of combined therapy with anti-­PDPN Ab and ICIs, the cellsurface expression of CTLA-­4 and PD-­1 in mouse tumor-­infiltrating
immune cells (Figure S3). The flow cytometry analysis revealed thatsome populations of tumor-­infiltrating NK cells and macrophages

3.2 | Antitumor effects of anti-­mouse PDPN Ab in
mesothelioma-­bearing immunocompetent miceexpressed CTLA-­4, while the majority of T cells did not. Natural killer
cells and macrophages were known to mediate ADCC because of
FcγRIII (CD16) expression.15–17 In contrast, the expression of PD-­1
was observed in various tumor-­infiltrating immune cells, especially

To determine the antitumor effects of the anti-­PDPN Ab on tumor-­in CD4 + T cells.

infiltrating immune cells, we treated AB1-­HA-­bearing BALB/c mice

Given these findings, we focused on NK cells mediating ADCC

with PMab-­1-­mG2a-­f (Figure 2A,B). The lower amount of PMab-­

as the key therapeutic targets for combination immunotherapy with

1-­mG2a-­f (50 μg) tended to suppress tumor progression in vivo,anti-­PDPN Ab and anti-­C TLA-­4 Ab.

whereas higher concentrations (100 μg or 500 μg) failed to demonstrate antitumor effects. In contrast, PMab-­1-­mG2a-­f (50 μg) did
not show antitumor effects against PDPN− LLC tumor tissue in vivo

(Figure S1C). We also investigated the effects of PMab-­1-­mG2a-­f on
tumor immune microenvironment cells by immunohistochemical

3.4 | Natural killer cell dependency
in the synergistic effects of anti-­PDPN Ab andanti-­CTLA-­4 Ab

analysis (Figure 2C–H). The number of NKp46+ NK cells was slightly
increased by treatment with PMab-­1-­mG2a-­f in PDPN+ AB1-­HA

To determine whether or not the synergistic antitumor effects oftumor tissue, while CD8+ or CD4 + T lymphocytes did not accumu-

PMab-­1-­mG2a-­f and CTLA-­4 blockade required tumor-­infiltrating NK

late. These results suggested the potential of PMab-­1-­mG2a-­f to en-cells, we depleted NK cells by treatment with an anti-­IL-­2Rβ Ab (TMβ-­hance the tumor-­killing capacity of NK cells. However, monotherapy1), as described in previous studies (Figure 4A). 27,28 Pretreatment

with PMab-­1-­mG2a-­f seemed to be insufficient for definitive treat-with TMβ-­1 inhibited the antitumor effects of combined immu-

ment in MPM-­bearing mice.

notherapy with PMab-­1-­mG2a-­f and CTLA-­4 blockade in AB1-­HA
tumor-­bearing mice (Figure 4B). Considering the possibility thatTMβ-­1 influenced not only tumor-­infiltrating NK cells but also T cells,

3.3 | Synergistic effects of anti-­PDPN Ab andCTLA-­4 blockade in mesothelioma-­bearing mice

an immunohistochemical analysis was carried out in AB1-­HA tumor
tissue (Figure 4C–E). The NK cells accumulated by the combined
therapy were notably eliminated by TMβ-­1 (Figure 4F), whereas a

Considering the superior clinical outcome of combination immuno-very slight reduction in tumor-­infiltrating CD8 T cells or CD4 T cells

therapy in MPM patients,8 we next focused on combination therapy

was observed (Figure 4G,H). Anti-­C TLA-­4 Abs have been known towith PMab-­1-­mG2a-­f and ICIs. AB1-­HA tumor-­bearing mice wereincrease or decrease tumor-­infiltrating CTLA-­4 + Tregs, depending

treated with PMab-­1-­mG2a-­f and ICIs: CTLA-­4 blockade (Figure 3A)

on the ADCC activity. 29–31 In the present study, CTLA-­4 blockade

and PD-­1 blockade (Figure 3B). Interestingly, combination therapy

treatment increased tumor-­infiltrating Tregs, consistent with a re-with anti-­PDPN Ab and anti-­C TLA-­4 Ab significantly suppressedcent study that used the same clone (Figure S4). 29 Recent studies

tumor progression, whereas the antitumor effects of each mono-

have also revealed that Tregs express IL-­2Rβ.32 TMβ-­1 significantly

therapy were limited (Figure 3A). In contrast, PMab-­1-­mG2a-­f did notdecreased tumor-­infiltrating Tregs in our preclinical model. However,reinforce the antitumor effects of anti-­PD-­1 Ab in AB1-­HA tumor-­

the antitumor effects of combined immunotherapy with an anti-­

bearing mice (Figure 3B).
To

investigate

the

PDPN Ab and CTLA-­4 blockade were inhibited by TMβ-­1, despite amechanisms

by

which

the

anti-­

reduction in Tregs (Figure S4). To evaluate the importance of T cells in

PDPN Ab improved the responses to CTLA-­4 blockade, an

the antitumor effects of combined immunotherapy with anti-­PDPN

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 361

YONEDA et al.

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

YONEDA et al.

|362

F I G U R E 2 Antitumor effects of monotherapy with core-­fucose-­deficient rat–mouse chimeric anti-­mouse podoplanin (PDPN) Ab in
immunocompetent mice. (A, B) Evaluation of the (A) tumor volume and (B) tumor weight of AB1-­HA tumor-­bearing BALB/c mice treatedwith PMab-­1-­mG2a-­f (50, 100, and 500 μg per mouse) beginning 7 days after tumor cell injection. (C–F) Representative images of sections
from AB1-­HA tumors stained for (C) natural killer (NK) cells, (D) CD8 T cells, and (E) CD4 T cells. Tumors were harvested at day 21 from eachgroup studied in (A). Scale bar, 200 μm. (F–H) Quantitative evaluation of the total number of (F) NK cells, (G) CD8 T cells, and (H) CD4 T cells
in the tumors of mice (n = 15 fields per group) is shown in (A). Data are shown as the mean ± SEM. *p < 0.05 by a one-­way ANOVA.

Ab and CTLA-­4 Ab, we also depleted T cells by the administration of

epidermal growth factor receptor reinforced the antitumor effects

anti-­CD4 Ab or anti-­CD8 T cells (Figure S5). Despite the depletion ofof other immunotherapy and enhanced the accumulation of NK cells

T cells, the combination therapy with anti-­PDPN Ab and CTLA-­4 Ab

in tumor foci.33,34 Ramamoorthi et al. elucidated that tumor-­killing

suppressed mouse tumor progression.

effects of anti-­HER2 Ab required CD16 expression in the tumor mi-

Taken together, these findings suggest that NK cells play pivotal

croenvironment.34 According to this evidence, we emphasized theroles in combined immunotherapy with anti-­PDPN Abs and anti-­

ADCC activity in the antitumor effects and the localization of NK

CTLA-­4 Abs.

cells in tumor by treatment with anti-­PDPN Ab. The detailed molecular mechanisms behind ADCC activity-­enhanced NK cell accu-

4|

DISCUSSION

mulation are still unclear and under analysis. The elucidation of themechanisms is required to develop novel combination immunotherapy with ICI.

In the present study, we evaluated the efficacy of anti-­PDPN Abs

Cytotoxicity assays with mouse splenocytes revealed the ADCC

in immunocompetent conditions for the first time. Furthermore, we

and CDC activities of PMab-­1-­mG2a against PDPN-­expressing meso-

discovered the synergistic antitumor effects of anti-­PDPN Ab andthelioma cells. However, monotherapy with PMab-­1-­mG2a showedanti-­C TLA-­4 Ab through NK cell-­dependent mechanisms.

limited therapeutic effects in mesothelioma-­bearing immunocom-

We previously investigated the antitumor effects of an anti-­

petent mice, while tumor-­infiltrating NK cells were increased. To

human PDPN Ab in human MPM cell-­bearing SCID mice.18–20

explain these conflicting results, we focused on the immune check-

Previous studies focused on NK cells as the key effector cells be-point molecules in tumor-­infiltrating NK cells. Natural killer cells arecause NK cells showed high and specific cytotoxicity against PDPN-­

known to express various immune checkpoint molecules, including

expressing tumors treated with anti-­PDPN Abs in vitro. Combination

PD-­1, TIGIT, NKG2A, and CTLA-­4.35,36 Of these, the effects of thetherapy with peritumoral implantation of NK cells and anti-­humanPD-­1/PD-­L1 axis and CTLA-­4 on tumor-­infiltrating NK cells cannot

PDPN Ab significantly suppressed tumor progression in humanbe ignored, given the clinical outcomes of the CheckMate743 trial.8

MPM-­bearing SCID mice, whereas monotherapy with each anti-­

Stojanovic et al. showed that IL-­2-­activated or tumor-­infiltrating

PDPN Ab did not show an antitumor effect.

mouse NK cells expressed CTLA-­4, while splenic NK cells did not.37

These previous studies suggested the potential utility of anti-­

CTLA-­4 expression in NK cells has been reported to suppress γ-­

PDPN Abs as novel immunotherapies against unresectable MPM.

interferon production in response to mature dendritic cells.37 The

However, some limitations to the clinical application of anti-­PDPN

efficacy of CTLA-­4 blockade on tumor-­infiltrating NK cells was

Abs remained. First, the implantation of human or rat NK cells ex-

therefore investigated in recent studies.38,39 Davis-­Marcisak et al.

tracted from peripheral blood or spleen was required to evaluate

revealed the Fc receptor-­independent binding of anti-­C TLA-­4 Ab

the antitumor effects of NZ-­1, NZ-­8, and NZ-­12 in vivo, as mouseand cell surface CTLA-­4 in NK cells.38 Furthermore, the NK cell ac-

NK cells are known to be insufficient as effector cells of ADCC ac-

tivation signature was correlated with an improved overall survival

tivities in these anti-­PDPN Abs. This limitation made it difficult toof advanced melanoma patients treated with ipilimumab. Consistentevaluate the effects of anti-­PDPN Abs on tumor-­infiltrating, host-­

with these recent studies, our preclinical model revealed the syn-derived NK cells. Second, we inoculated human MPM cells in SCID

ergistic antitumor effects of combined immunotherapy with anti-­

mice to investigate the affinity and antitumor effects of anti-­human

CTLA-­4 Ab and anti-­PDPN Ab and its dependency on NK cells.

PDPN Abs in previous studies. The effects of anti-­PDPN Abs on the

In our preclinical model, PMab-­1-­mG2a did not enhance the an-complicated tumor immune microenvironment, including T cells andtitumor effects of PD-­1 blockade, in contrast to CTLA-­4 blockade.

B cells, are thus unclear. In the present study, we established the

Wagner et al. showed that PD-­1-­deficient NK cells have less capa-

novel rat–mouse chimeric anti-­mouse PDPN Ab PMab-­1-­mG2a tobility to migrate and kill tumor cells in vivo than WT.40 They also

overcome these limitations.

revealed the cis-­binding of PD-­1 and PD-­L1 on tumor-­infiltrating

In the present study, PMab-­1-­mG2a-­f increased tumor-­infiltrating

NK cells. This unique function of the PD-­1/PD-­L1 axis on NK cells

NK cells while T cells were not affected (Figure 2). We consider thatpartially explains the poor sensitivity to combined therapy with anti-­

ADCC mediated by anti-­PDPN Ab induced the localization of NK

PD-­1 Ab and anti-­PDPN Ab.

cells in PDPN+ tumors. Natural killer cells, NKT cells and macro-

In the present study, the antitumor effects and the accumu-

phages were reported to play the pivotal role in ADCC by express-

lation of NK cells by PMab-­1-­m G2a-­f were observed only in low

ing CD16. Recent reports revealed that Ab drugs targeting HER2 orconcentration (Figure 2). We considered it is due to the prozone

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 363

YONEDA et al.

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

YONEDA et al.

|364

F I G U R E 3 Synergistic effects of anti-­podoplanin (PDPN) Ab and anti-­CTL-­associated antigen 4 (CTLA-­4) Ab in vivo. (A) Evaluation of thetumor volume of AB1-­HA tumor-­bearing BALB/c mice treated with PMab-­1-­mG2a-­f (50 μg per mouse) and/or αCTLA-­4 Ab (5 μg per mouse)
beginning 7 days after tumor cell injection. (B) Evaluation of the tumor volume of AB1-­HA tumor-­bearing BALB/c mice treated with PMab-­
1-­mG2a-­f (50 μg per mouse) and/or programmed death-­1 (αPD-­1) Ab (100 μg per mouse) beginning 7 days after tumor cell injection. (C–E)
Representative images of sections from AB1-­HA tumors stained for (C) natural killer (NK) cells, (D) CD8 T cells, and (E) CD4 T cells. Tumors
were harvested at day 21 from each group studied in (A). Scale bar, 200 μm. (F–H) Quantitative evaluation of the total number of (F) NK
cells, (G) CD8 T cells, and (H) CD4 T cells in the tumors of mice (n = 15 fields per group) is shown in (A). Data are shown as the mean ± SEM.
*p < 0.05 by a one-­way ANOVA.

effect that high concentration Abs interfere with antigen–Ab in-potential to demonstrate superior synergistic effects with anti-­

teractions. Several reports indicated that excessively high con-

PDPN Abs through the multiple effects of immune cells, including

centrations of Abs inhibit ADCC through prozone effects.41,42 In

the activation of NK cells and the depletion of Tregs.

addition, the prozone-­like effect was also reported in animal models.

43

In summary, we discovered a novel promising treatment by com-

Our preclinical studies indicated that the administration ofbining immunotherapy with an anti-­PDPN Ab and anti-­C TLA-­4 Ab.

higher concentration of anti-­PDPN Ab could suppress the proper

The present study identified the pivotal role of NK cells in novel

antigen–Ab interactions and ADCC activity. Therefore, we usedcombined immunotherapy targeting PDPN. These findings shed

the low concentration of anti-­PDPN Ab in the combined therapy

light on the therapeutic strategy for unresectable MPM.

with anti-­C TLA-­4 Ab.
The present study has several potential clinical implications.

AU T H O R C O N T R I B U T I O N SFirst, we revealed the effects of an anti-­PDPN Ab on the immune

Hiroto Yoneda: Data curation; formal analysis; funding acquisition;

microenvironment of MPM under immunocompetent conditions.

investigation; methodology; visualization; writing – original draft.

Recent studies have shown that the expression of PDPN was de-Atsushi Mitsuhashi: Data curation; formal analysis; investigation;tected in 80%–100% of epithelioid MPM patients.11 Podoplanin ex-

methodology; project administration; validation; visualization; writ-pression is known to correlate with a poor prognosis of various solid

ing – review and editing. Aito Yoshida: Data curation; formal analysis;tumors.14 Furthermore, several studies have revealed the functional

investigation; validation; visualization; writing – review and editing.

effects of PDPN in promoting tumor invasiveness and metastasis

Hirokazu Ogino: Data curation; formal analysis; investigation; meth-by platelet aggregation.44–46 Taken together, these findings suggest

odology; validation; writing – review and editing. Satoshi Itakura:that novel immunotherapy targeting PDPN is needed, especially in

Data curation; formal analysis; investigation; validation; writing

advanced MPMs, considering the insufficient response to current– review and editing. Na Thi Nguyen: Data curation; formal analy-

therapies. The present findings could thus enhance the progresssis; investigation; validation; writing – review and editing. Hiroshi

made in developing PDPN-­t argeting therapies for drug discovery.

Nokihara: Data curation; formal analysis; project administration;

Second, our syngeneic preclinical model enabled the evaluation ofwriting – review and editing. Seidai Sato: Data curation; formal anal-

combined immunotherapy with anti-­PDPN Ab and ICIs. In particular,ysis; writing – review and editing. Tsutomu Shinohara: Supervision;

we identified the potential utility of combined therapy with an anti-­

validation; writing – review and editing. Masaki Hanibuchi: Data

PDPN Ab and anti-­C TLA-­4 Ab as a promising treatment for unre-curation; formal analysis; project administration; validation; writing

sectable MPMs. Finally, the present study indicated the importance– review and editing. Shinji Abe: Data curation; formal analysis; fund-of tumor-­infiltrating NK cells as key effector cells in immunotherapy.

ing acquisition; investigation; methodology; project administration;

However, several limitations associated with the present study

writing – review and editing. Mika K. Kaneko: Resources; supervi-

also warrant mention. First, the efficacy of anti-­human PDPN Abs

sion; writing – review and editing. Yukinari Kato: Conceptualization;

against human MPM in immunocompetent conditions has remained

resources; supervision; writing – review and editing. Yasuhikounclear. To address this limitation, a humanized chimera mouse

Nishioka: Conceptualization; data curation; formal analysis; funding

model should be established in future studies. Second, combined

acquisition; methodology; project administration; visualization; writ-

immunotherapy with anti-­PDPN Ab and anti-­C TLA-­4 Ab might in-

ing – review and editing.

fluence Tregs. A recent study suggested that some clones of CTLA-­4
Abs enhanced the activation of NK cells through the depletion ofTregs by ADCC.

39

AC K N OW L E D G M E N T S

Therefore, in the present study, we used a CTLA-­4

We thank our colleagues at The University of Tokushima, especially

blockade clone known to lack ADCC activity on Tregs. 29 Consistent

A. Tanabe and R. Akutagawa for their technical assistance. This

with the reported study, CTLA-­4 Ab increased the tumor-­infiltrating

study was supported by the Support Center for Advanced Medical

Tregs in our preclinical model, whereas the combined immunotherapy

Sciences, Tokushima University Graduate School of Biomedicalwith anti-­PDPN Ab and anti-­C TLA-­4 Ab showed significant antitu-

Sciences. This research was supported by the Research Support

mor effects. A recent study indicated that the clinically approved

Project for Life Science and Drug Discovery (Basis for Supporting

anti-­C TLA4 Ab ipilimumab has ADCC activity and depletes tumor-­Innovative Drug Discovery and Life Science Research [BINDS]) frominfiltrating Tregs.47 We therefore considered ipilimumab to have the

AMED under Grant Number JP23ama121008.

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 365

YONEDA et al.

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

YONEDA et al.

|366

F I G U R E 4 Role of natural killer (NK) cells in combined immunotherapy with antipodoplanin (PDPN) Ab and anti-­C TL-­associated antigen
4 (CTLA-­4) Ab. (A) Treatment schedule of combined therapy with PMab-­1-­mG2a-­f (50 μg per mouse) and/or αCTLA-­4 Ab (5 μg per mouse) in
AB1-­HA-­bearing mice with the depletion of NK cells by anti-­interleukin-­2 receptor β-­chain (IL-­2Rβ) Ab. (B) Evaluation of the tumor volume
of AB1-­HA tumor-­bearing BALB/c mice treated with PMab-­1-­mG2a-­f (50 μg per mouse) and αCTLA-­4 Ab (5 μg per mouse) beginning 7 days
after tumor cell injection with or without the depletion of NK cells. (C–E) Representative images of sections from AB1-­HA tumors stained for(E) NK cells, (D) CD8 T cells, and (E) CD4 T cells. Tumors were harvested at day 21 from each group studied in (B). Scale bar, 200 μm. (F–H)
Quantitative evaluation of the total number of (F) NK cells, (G) CD8 T cells, and (H) CD4 T cells in the tumors of mice (n = 15 fields per group)is shown in (B). Data are shown as the mean ± SEM. *p < 0.05 by a one-­way ANOVA.

F U N D I N G I N FO R M AT I O NThis study was partly supported by a grant from JSPS KAKENHI
Grant Number 19H03668, a Grant-­in-­Aid for Scientific Research(B) from Japan Society for the Promotion of Science (JSPS) (Y.N.),SPS KAKENHI Grant Number 20 K07197, a Grant-­in-­Aid for
Scientific Research (C) from JSPS (S.A.), SPS KAKENHI Grant
Number 20 K17216, a Grant-­in-­Aid for Early-­C areer Scientists from
JSPS (H.Y.), and from the Japan Agency for Medical Research andDevelopment (AMED) under Grant Numbers JP22ama121008 (Y.K.)
and JP21am0101078 (Y.K.).
C O N F L I C T O F I N T E R E S T S TAT E M E N T
All authors declare no conflict of interest.
E T H I C S S TAT E M E N T
Approval of the research protocol by an institutional review board:
N/A.
Informed consent: N/A.
Registry and the registration no. of the study/trial: N/A.
Animal studies: All experimental protocols were reviewed and approved by the animal research committee of The University ofTokushima, Japan (approval number T30-­130).
ORCID
Yukinari Kato

https://orcid.org/0000-0001-5385-8201

Yasuhiko Nishioka

https://orcid.org/0000-0001-6311-1654

REFERENCES
1. Carbone M, Adusumilli PS, Alexander HR, et al. Mesothelioma: scientific clues for prevention, diagnosis, and therapy. CA Cancer J Clin.
2019;69:402-­429.
2. Mutti L, Peikert T, Robinson BWS, et al. Scientific advances andnew Frontiers in mesothelioma therapeutics. J Thorac Oncol.
2018;13:1269-­1283.
3. Scherpereel A, Wallyn F, Albelda SM, Munck C. Novel therapies formalignant pleural mesothelioma. Lancet Oncol. 2018;19:e161-­e172.
4. Vogelzang NJ, Rusthoven JJ, Symanowski J, et al. Phase III studyof pemetrexed in combination with cisplatin versus cisplatin alone
in patients with malignant pleural mesothelioma. J Clin Oncol.
2003;21:2636-­2644.
5. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint
blockade. Science. 2018;359:1350-­1355.
6. Huang AC, Zappasodi R. A decade of checkpoint blockade immunotherapy in melanoma: understanding the molecular basis for immune sensitivity and resistance. Nat Immunol. 2022;23:660-­670.

7. Ceresoli GL, Mantovani A. Immune checkpoint inhibitors in malignant pleural mesothelioma. Lancet Oncol. 2017;18:559-­561.
8. Baas P, Scherpereel A, Nowak AK, et al. First-­line nivolumab
plus ipilimumab in unresectable malignant pleural mesothelioma(CheckMate 743): a multicentre, randomised, open-­label, phase 3
trial. Lancet. 2021;397:375-­386.
9. Breiteneder-­G eleff S, Matsui K, Soleiman A, et al. Podoplanin,novel 43-­kd membrane protein of glomerular epithelial cells,
is down-­regulated in puromycin nephrosis. Am J Pathol.
1997;151:1141-­1152.
10. Breiteneder-­Geleff S, Soleiman A, Kowalski H, et al. Angiosarcomas
express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.
Am J Pathol. 1999;154:385-­394.
11. Ordóñez NG. D2-­4 0 and podoplanin are highly specific and sensitive immunohistochemical markers of epithelioid malignant mesothelioma. Hum Pathol. 2005;36:372-­380.
12. Kato Y, Fujita N, Kunita A, et al. Molecular identification of Aggrus/
T1alpha as a platelet aggregation-­inducing factor expressed in colorectal tumors. J Biol Chem. 2003;278:51599-­51605.
13. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, Osawa M. Enhanced
expression of Aggrus (T1alpha/podoplanin), a platelet-­aggregation-­
inducing factor in lung squamous cell carcinoma. Tumour Biol.
2005;26:195-­200.
14. Krishnan H, Rayes J, Miyashita T, et al. Podoplanin: an emerging cancer biomarker and therapeutic target. Cancer Sci.
2018;109:1292-­1299.
15. Jiang XR, Song A, Bergelson S, et al. Advances in the assessment
and control of the effector functions of therapeutic antibodies. Nat
Rev Drug Discov. 2011;10:101-­111.
16. Musolino A, Gradishar WJ, Rugo HS, et al. Role of Fcγ receptors
in HER2-­t argeted breast cancer therapy. J Immunother Cancer.
2022;10:e003171.
17. Coleman N, Yap TA, Heymach JV, Meric-­Bernstam F, le X. Antibody-­
drug conjugates in lung cancer: dawn of a new era? NPJ Precis Oncol.
2023;7:5.
18. Kato Y, Kaneko MK, Kunita A, et al. Molecular analysis of the pathophysiological binding of the platelet aggregation-­inducing factor
podoplanin to the C-­t ype lectin-­like receptor CLEC-­2. Cancer Sci.
2008;99:54-­61.
19. Kaneko MK, Kunita A, Abe S, et al. Chimeric anti-­podoplaninantibody suppresses tumor metastasis through neutralization and antibody-­dependent cellular cytotoxicity. Cancer Sci.
2012;103:1913-­1919.
20. Abe S, Kaneko MK, Tsuchihashi Y, et al. Antitumor effect ofnovel anti-­podoplanin antibody NZ-­12 against malignant pleural mesothelioma in an orthotopic xenograft model. Cancer Sci.
2016;107:1198-­1205.
21. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, Sawa Y.
Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human Podoplanin. Acta Histochem
Cytochem. 2012;45:227-­237.

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License| 367

YONEDA et al.

|

22. Kaneko MK, Ohishi T, Takei J, et al. Anti-­EpCAM monoclonal antibody exerts antitumor activity against oral squamous cell carcinomas. Oncol Rep. 2020;44:2517-­2526.
23. Niwa R, Shoji-­H osaka E, Sakurada M, et al. Defucosylated chimeric anti-­CC chemokine receptor 4 IgG1 with enhanced
antibody-­d ependent cellular cytotoxicity shows potent therapeutic activity to T-­cell leukemia and lymphoma. Cancer Res.
2004;64:2127-­2133.
24. Kaneko MK, Ohishi T, Nakamura T, et al. Development ofCore-­Fucose-­deficient humanized and chimeric anti-­human
Podoplanin antibodies. Monoclon Antib Immunodiagn Immunother.
2020;39:167-­174.
25. Oyewole-­Said D, Konduri V, Vazquez-­Perez J, Weldon SA, Levitt
JM, Decker WK. Beyond T-­cells: functional characterization of
CTLA-­4 expression in immune and non-­immune cell types. Front
Immunol. 2020;11:608024.
26. Walker LS, Sansom DM. Confusing signals: recent progress in
CTLA-­4 biology. Trends Immunol. 2015;36:63-­70.
27. Tanaka T, Kitamura F, Nagasaka Y, Kuida K, Suwa H, Miyasaka M.
Selective long-­term elimination of natural killer cells in vivo by ananti-­interleukin-­2 receptor β chain monoclonal antibody in mice. J
Exp Med. 1993;178:1103-­1107.
28. Yano S, Nishioka Y, Izumi K, et al. Novel metastasis model ofhuman lung cancer in SCID mice depleted of NK cells. Int J Cancer.
1996;67:211-­217.
29. Marangoni F, Zhakyp A, Corsini M, et al. Expansion of tumor-­
associated Treg cells upon disruption of a CTLA-­4-­dependent feedback loop. Cell. 2021;184:3998-­4 015.
30. Simpson TR, Li F, Montalvo-­Ortiz W, et al. Fc-­dependent depletion of tumor-­infiltrating regulatory T cells co-­defines theefficacy of anti-­C TLA-­4 therapy against melanoma. J Exp Med.
2013;210:1695-­1710.
31. Sharma A, Subudhi SK, Blando J, et al. Anti-­C TLA-­4 immunotherapy does not deplete FOXP3+ regulatory T cells (Tregs) in humancancers. Clin Cancer Res. 2019;25:1233-­1238.
32. Chinen T, Kannan AK, Levine AG, et al. An essential role for the IL-­2
receptor in Treg cell function. Nat Immunol. 2016;17:1322-­1333.
33. Jin WJ, Erbe AK, Schwarz CN, et al. Tumor-­specific antibody,
Cetuximab, enhances the in situ vaccine effect of radiation in immunologically cold head and neck squamous cell carcinoma. Front
Immunol. 2020;11:591139.
34. Ramamoorthi G, Kodumudi K, Snyder C, et al. Intratumoral deliveryof dendritic cells plus anti-­HER2 therapy triggers both robust systemic antitumor immunity and complete regression in HER2 mammary carcinoma. J Immunother Cancer. 2022;10(6):e004841.
35. Cao Y, Wang X, Jin T, et al. Immune checkpoint molecules in natural killer cells as potential targets for cancer immunotherapy. Signal
Transduct Target Ther. 2020;5:250.
36. Sivori S, Della Chiesa M, Carlomagno S, et al. Inhibitory receptors
and checkpoints in human NK cells, implications for the immunotherapy of cancer. Front Immunol. 2020;11:2156.
37. Stojanovic A, Fiegler N, Brunner-­Weinzierl M, Cerwenka A. CTLA-­4
is expressed by activated mouse NK cells and inhibits NK cell

YONEDA et al.

38.

39.

40.

41.

42.

43.

44.

45.
46.

47.

IFN-­γ production in response to mature dendritic cells. J Immunol.
2014;192:4184-­4191.
Davis-­Marcisak EF, Fitzgerald AA, Kessler MD, et al. Transfer learning between preclinical models and human tumors identifies a
conserved NK cell activation signature in anti-­C TLA-­4 responsive
tumors. Genome Med. 2021;1:129.
Sanseviero E, O'Brien EM, Karras JR, et al. Anti-­C TLA-­4 activates
Intratumoral NK cells and combined with IL15/IL15Rα complexes
enhances tumor control. Cancer Immunol Res. 2019;7:1371-­1380.
Wagner AK, Kadri N, Tibbitt C, et al. PD-­1 expression on mouse
intratumoral NK cells and its effects on NK cell phenotype. iScience.
2022;25:105137.
Jenkins M, Mills J, Kohl S. Natural killer cytotoxicity and antibody-­
dependent cellular cytotoxicity of human immunodeficiency virus-­
infected cells by leukocytes from human neonates and adults.
Pediatr Res. 1993;33(5):469-­474.
Mulgrew K, Kinneer K, Yao XT, et al. Direct targeting of alphavbeta3
integrin on tumor cells with a monoclonal antibody, Abegrin. Mol
Cancer Ther. 2006;5(12):3122-­3129.
Vaidya KS, Oleksijew A, Tucker LA, et al. A "Prozone-­like" effectinfluences the efficacy of the monoclonal antibody ABT-­700
against the hepatocyte growth factor receptor. Pharmacology.
2017;100(5–6):229-­242.
Takeuchi S, Fukuda K, Yamada T, et al. Podoplanin promotes progression of malignant pleural mesothelioma by regulating motility
and focus formation. Cancer Sci. 2017;108:696-­703.
Takemoto A, Takagi S, Ukaji T, et al. Targeting Podoplanin for thetreatment of osteosarcoma. Clin Cancer Res. 2022;28:2633-­2645.
Kunita A, Kashima TG, Morishita Y, et al. The platelet aggregation-­
inducing factor aggrus/podoplanin promotes pulmonary metastasis. Am J Pathol. 2007;170:1337-­1347.
Romano E, Kusio-­Kobialka M, Foukas PG, et al. Ipilimumab-­dependent cell-­mediated cytotoxicity of regulatory T cells ex vivo
by nonclassical monocytes in melanoma patients. Proc Natl Acad Sci
U S A. 2015;112:6140-­6615.

S U P P O R T I N G I N FO R M AT I O N
Additional supporting information can be found online in the
Supporting Information section at the end of this article.

How to cite this article: Yoneda H, Mitsuhashi A, Yoshida A,
et al. Antipodoplanin antibody enhances the antitumor effectsof CTLA-­4 blockade against malignant mesothelioma by
natural killer cells. Cancer Sci. 2024;115:357-368. doi:10.1111/
cas.16046

13497006, 2024, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/cas.16046 by Cochrane Japan, Wiley Online Library on [12/02/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License368