Received: 2 November 2018

| Revised: 30 January 2019 | Accepted: 18 February 2019

DOI: 10.1111/cas.13979

ORIGINAL ARTICLE

Therapeutic efficacy evaluation of radioimmunotherapy with90
Y-­labeled anti-­podoplanin antibody NZ-­12 for mesothelioma
Hitomi Sudo1 | Atsushi B. Tsuji1
Yukinari Kato3,4| Aya Sugyo1 | Tsuneo Saga2| Mika K. Kaneko3 || Tatsuya Higashi1

1Department of Molecular Imaging
and Theranostics, National Institute ofRadiological Sciences, National Institutes
for Quantum and Radiological Science andTechnology (QST-NIRS), Chiba, Japanpressed in malignant mesothelioma. The rat-­human chimeric antibody NZ-­12 has high2

applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the pre-Department of Diagnostic Radiology, Kyoto
University Hospital, Kyoto, Japan3

Department of Antibody Drug
Development, Tohoku University Graduate
School of Medicine, Sendai, Miyagi, Japan
4

New Industry Creation Hatchery
Center, Tohoku University, Sendai, Miyagi,
Japan
Correspondence
Atsushi B. Tsuji, Department of Molecular
Imaging and Theranostics, National Instituteof Radiological Sciences, National Institutes
for Quantum and Radiological Science andTechnology (QST-NIRS), Chiba, Japan.
Email: tsuji.atsushi@qst.go.jp
Funding information
KAKENHI (Grant/Award Numbers:
16K10748, 17K07299, 17K10497,
18H02774, 18K07778), AMED-­CREST(Grant/Award Numbers: JP18gm0710003),
Japan Agency for Medical Research andDevelopment (Grant/Award Number:
JP18am0101078).

Podoplanin is a type I transmembrane sialomucin-­like glycoprotein that is highly exaffinity for human podoplanin and antibody-­dependent cellular cytotoxicity and issent study, we evaluated the in vivo and in vitro properties of radiolabeled NZ-­12 andthe antitumor effect of RIT with 90Y-­labeled NZ-­12 in an NCI-­H226 (H226) malignant
mesothelioma xenograft mouse model. 111In-­labeled NZ-­12 bound specifically to
H226 cells with high affinity, and accumulation was high in H226 tumors but low inmajor organs. RIT with 90Y-­labeled NZ-­12 significantly suppressed tumor growth andprolonged survival without body weight loss and obvious adverse effects. Higher
podoplanin expression levels were observed in human mesothelioma specimens,
suggesting higher tumor accumulation of 90Y-­labeled NZ-­12 in patients comparedwith the H226 tumor xenografts. Our findings suggest that 90Y-­labeled NZ-­12 is apromising RIT agent as a new therapeutic option for malignant mesothelioma thatwarrants further clinical studies to evaluate the dosimetry and efficacy in patients.
KEYWORDS

antibody, malignant mesothelioma, podoplanin, radiation, radioimmunotherapy

1 | I NTRO D U C TI O N

diagnosed at the advanced stage.5 Although newly developed
molecular-­t argeted drugs are expected to improve patient progno-

Malignant mesothelioma is an aggressive tumor with a poor progno1

sis that is classified into three main types based on the histologicalsis, current combination chemotherapy using pemetrexed and cisplatin is reported to prolong the overall survival by only 2.8 months

features: epithelioid, sarcomatoid, and mixed. Median overall sur-compared with cisplatin alone.4 Thus, more effective treatments arevival in patients with malignant mesothelioma is 7 months.3 Surgicalneeded to improve the prognosis. Radioimmunotherapy (RIT) using

resection may be offered to patients with early-­stage disease, andradiolabeled monoclonal antibodies against tumor-­associated an-2systemic chemotherapy is the main treatment for patients withtigens is a selective internal radiation therapy.6 Several preclinicaladvanced-­stage disease.4,5 Most malignant mesothelioma patients

studies demonstrated promising therapeutic outcomes of RIT for

receive chemotherapy, however, because the disease is often

solid tumors, including small cell lung cancer,7,8 pancreatic cancer,9,10

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
Cancer Science. 2019;110:1653–1664.

﻿ wileyonlinelibrary.com/journal/cas

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and breast cancer.11 RIT with antibodies recognizing antigens expressed on mesothelioma cells is a potential therapeutic option for

2.2 | Cell culture
Mesothelioma cell lines, NCI-­H226 (H226, CRL-­5826) and MSTO-­

malignant mesothelioma.
Podoplanin is a transmembrane sialomucin-­like glycoprotein that

211H (211H, CRL-­2081), were obtained from ATCC (Manassas, VA,

is highly expressed in several types of cancer, such as brain tumor,12

USA). The cells were cultured in RPMI-­1640 (Wako Pure Chemical

13-15

squamous cell carcinoma,

16

soft tissue tumors,17

bladder cancer,

and malignant mesothelioma.18,19 Podoplanin is associated with ep-

Industries, Tokyo, Japan) containing 10% FBS (Life Technologies,
Carlsbad, CA, USA) in 5% CO2 at 37°C.

ithelial mesenchymal transition, migration, invasion, and metastasis,20,21 and its expression correlates with the malignancy grade inbrain tumors, lung cancer, and oral cancer.21-23 Several anti-­podoplanin

2.3 | Immunofluorescence staining in vitro

antibodies inhibit cancer metastasis24 and cancer progression12,19 in

H226 and 211H cells were seeded on coverslips, incubated overnight,

mouse tumor models. Therefore, podoplanin is a promising therapeu-

and fixed with 4% paraformaldehyde the next day. Immunofluorescencetic target in various cancers, including malignant mesothelioma.

staining was conducted using anti-­
podoplanin antibody LpMab-­
17

Podoplanin expression is high in cancerous tissues, whereas

as a primary antibody with an Alexa Fluor 594 goat antimouse anti-

expression is low in several normal tissues, such as kidney podo-

body (Molecular Probes, Eugene, OR, USA) as a secondary antibody.

cytes, 25 alveolar type I cells, 26 lymphatic endothelial cells, 27 basal

Coverslips were placed on glass slides using mounting medium withepidermal keratinocytes,15 and mesothelial cells. 28 The physiologi-

DAPI (Vector Laboratories, Burlingame, CA, USA). Fluorescence im-cal expression of podoplanin in normal tissues raises some concern

ages were captured with an exposure time of 50 ms for podoplaninabout the possible risks of toxicity to these normal tissues induced

and 25 ms for DAPI using a fluorescence microscope BX53 (Olympus,

by podoplanin-­targeted anticancer therapy. To address this concern,Tokyo, Japan) and cellSens Standard software (ver. 1.7.1; Olympus).

it is important to select an antibody that recognizes podoplanin expressed on cancer cells only and not on normal cells. We developed
a rat anti-­
podoplanin monoclonal antibody, NZ-­
1, which has the

2.4 | Radiolabeling of antibody

above-­mentioned property, 29 and converted it to a rat-­human chi-Antibodies were conjugated with p-­SCN-­Bn-­CHX-­A′′-­DTPA (CHX-­

meric antibody named NZ-­12.30 These antibodies recognize podo-A′′-­DTPA; Macrocyclics, Dallas, TX, USA) as described previously.36

planin expressed on the surface of cancer cells, such as brain tumor,

Briefly, the antibody (5 mg/mL) was reacted with an equal molar

lung cancer, and malignant mesothelioma, but not in benign tumor

amount of CHX-­A′′-­DTPA in 50 mmol/L borate buffer (pH 8.5) forcells or normal organs.19,31 Therefore, NZ-­12 has potential applica-

16 hours at 37°C. Conjugation ratio of CHX-­A′′-­DTPA to antibody wastion for selective antitumor therapy including RIT against malignant

estimated to be approximately 0.8 as determined by cellulose acetate
electrophoresis. The CHX-­A′′-­DTPA-­conjugated antibody was puri-

mesothelioma.
In the present study, NZ-­12 was labeled with radionuclides In-­111

fied by elution with 0.1 mol/L acetate buffer (pH 6.0) using a Sephadex

and Y-­90 to evaluate the in vitro and in vivo properties as a thera-G-­50 (GE Healthcare, Little Chalfont, UK) column. 111InCl3 (Nihon

nostic pair. Y-­90 is a pure beta emitter with a high energy level (max-Medi-­Physics, Tokyo, Japan) or 90YCl3 (Eckert & Ziegler Radiopharma,

imum energy, 2.3 MeV) and an appropriate half-­life (64.1 hours) for

Berlin, Germany) was incubated in 0.5 mol/L acetate buffer (pH 6.0)

RIT using IgG.32 In-­111, a gamma emitter with 171 keV and 245 keV

for 5 minutes at room temperature. Each was mixed with the CHX-­

and a half-­life of 67.4 hours, is used for diagnostic imaging and do-A′′-­DTPA-­antibody conjugate and incubated for 30 minutes at room

simetry as a surrogate for Y-­90.33,34 The combination of In-­111 andtemperature. The radiolabeled antibody was eluted with 0.1 mol/L ac-

Y-­90 is used in clinical practice (eg, Zevalin)34 in which the 111In-­

etate buffer (pH 6.0) using a Sephadex G-­50 column for purification.

labeled anti-­CD20 antibody is used for patient selection by SPECT/Specific activity of 111In-­labeled NZ-­12, 111In-­labeled control antibody,

CT imaging and the90

Y-­labeled anti-­CD20 antibody is used for RIT ofnon-­Hodgkin's lymphoma. In the present study,

111

In-­labeled NZ-­12

was used for biodistribution and dosimetry studies, and90

Y-­labeled90

Y-­labeled NZ-­12, and 90Y-­
labeled control antibody was approxi-

mately 30, 40, 1100, and 800 kBq/μg, respectively. Radiochemicalyields were approximately 60% for 111In labeling and 35% to 50% for

NZ-­12 was used to evaluate the antitumor effects in a mesothelioma

90mouse model.

rification with a Sephadex G-­50 column.

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

2.5 | Cell binding and competitive inhibition assays

Y labeling, and radiochemical purities were more than 95% after pu-

For cell-­binding assays, H226 and 211H cells (5.0 × 106, 2.6 × 106,

2.1 | Antibodies

1.3 × 106, 6.3 × 105, 3.1 × 105, 1.6 × 105, 7.8 × 104, and 3.9 × 104) in PBS

A rat-­human chimeric antihuman podoplanin antibody, NZ-­12,30 and35

with 1% BSA (Sigma-­Aldrich) were incubated with 111In-­labeled NZ-­12 orwere previ-

control antibody on ice for 60 minutes. After washing, cell-­bound radio-

ously developed. As a control, human IgG1λ was purchased from

activity was measured using a gamma-­counter (ARC-­370M; Aloka, Tokyo,

a mouse antihuman podoplanin antibody, LpMab-­17,
Sigma-­Aldrich (St Louis, MO, USA).

Japan). For competitive inhibition assays, H226 cells (2.0 × 106) in PBS with

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SUDO et al.

1% BSA were incubated with 111In-­labeled NZ-­12 in the presence of vary-

proliferation was evaluated by immunohistochemical staining with a

ing concentrations of intact NZ-­12, CHX-­A′′-­DTPA-­conjugated NZ-­12, or

rabbit anti-­Ki-­67 antibody (SP6; Abcam, Cambridge, MA, USA) and an

intact control antibody (0, 0.5, 0.9, 1.9, 3.8, 7.6, 15.2, and 30.3 nmol/L) on

antirabbit HRP/DAB Detection kit (Abcam) according to the manufac-

ice for 60 minutes. After washing, cell-­bound radioactivity was measured

turer's instructions. Ki-­67 index was calculated by counting the percent-with a gamma-­counter. The dissociation constant was estimated by apply-

age of Ki-­67-­positive tumor cells per >2500 tumor cells in a section withing data to a one-­site competitive binding model using GraphPad Prism

200× magnification (n = 3). Apoptosis was detected using the DeadEnd

software (ver. 7.0d, GraphPad Software, La Jolla, CA, USA).

Colorimetric TUNEL System (Promega, Madison, WI, USA). A tissue array
containing 19 cases of human malignant mesothelioma tissues (13 casesof epithelioid-­type, three cases of sarcomatoid-­type, and three cases of

2.6 | Tumor model

mixed-­type) was obtained from Biomax (Rockville, MD, USA). The tissues

The animal experimental protocol was approved by the Animal

were stained with 4 μg/mL of an anti-­podoplanin antibody LpMab-­17

Care and Use Committee of the National Institute of Radiologicaland an Envision+ system (Agilent Technologies, Santa Clara, CA, USA).

Sciences (13-­1022, 26 May 2016), and all animal experiments were

The nuclei were counterstained with hematoxylin.

conducted in accordance with the Guidelines regarding Animal Careand Handling. H226 cells (3.5 × 106) were s.c. inoculated into malenude mice (BALB/c-­nu/nu, 6 weeks old; CLEA Japan, Tokyo, Japan)

2.10 | External-­beam radiation with X-­rays
When H226 tumor volumes reached approximately 80 mm3, tumors

under isoflurane anesthesia.

(n = 5/dose) were irradiated with 0, 25, and 50 Gy of X-­rays at a rate111

of 4.0 Gy/min using a TITAN-­320 X-­ray generator (Shimadzu, Kyoto,

When H226 tumor volumes reached approximately 80 mm3, miceshield to limit unnecessary radiation exposure. Tumor size and body(n = 5/time-­point), were i.v. injected with 37 kBq of 111In-­labeled NZ-­12weight were measured at least twice a week for 8 weeks after irradia-

or 111In-­labeled IgG1λ in a total of 20 μg antibody adjusted by adding

tion, and tumor volume was calculated using the formula mentioned

2.7 | Biodistribution ofIn-­labeled antibody

Japan). Other parts of the mouse body were covered with a brass

the appropriate amount of unlabeled antibody. The mice were killed byabove. When the tumor volume reached greater than 800 mm3, theisoflurane inhalation at 1 hour, and at 1, 2, 4, and 7 days after injection.

mouse was killed humanely by isoflurane inhalation.

Blood, tumor, and organs of interest were sampled and weighed, andthe radioactivity was measured using a gamma-­counter. The uptake isrepresented as the percentage of injected radioactivity dose per gram

2.11 | Statistical analysisof tissue (% ID/g). The absorbed dose of 90Y-­labeled antibody was esti-

Data were analyzed by ANOVA with Dunnett's multiple comparison

mated using the area under the curve based on the biodistribution data

tests. Log-­rank tests were used to evaluate Kaplan-­Meier survivalof 111In-­labeled antibody and the mean energy emitted per transition of

curves based on the endpoint of tumor volume of 500 mm3. Data

37

Y-­90,−13

1.495 × 10

−1

Gy kg (Bq s)

7

as described previously.

are expressed as means ± standard deviation (SD). The criterion of
statistical significance was P < .05.

2.8 | Radioimmunotherapy with 90Y-­labeled antibody
When H226 tumor volumes reached approximately 80 mm3, the mice
were i.v. injected with intact NZ-­12, 90Y-­labeled NZ-­12 antibody (1.85,
and 3.70 MBq, n = 10/dose) or 90Y-­labeled control antibody (3.70 MBq,n = 10) in a total of 20 μg antibody adjusted by adding the correspond-

3 | R E S U LT S
3.1 | Podoplanin expression analysis in H226 and211H cells

ing unlabeled antibody. Tumor size and body weight were measured at

Two mesothelioma cell lines, H226 (podoplanin-­positive) and 211H

least twice a week for 8 weeks after dosing. Tumor size was measured(podoplanin-­negative), were used in the present study.19 To confirm

using a digital caliper, and tumor volume was calculated using the fol-

podoplanin expression in these cells, they were immunofluores-

lowing formula: tumor volume (mm3) = (length × width2)/2. When thecently stained with an anti-­podoplanin antibody LpMab-­17. Strong

tumor volume reached greater than 800 mm3, the mouse was killedstaining intensity was observed on the cell membranes of H226

humanely by isoflurane inhalation.

cells, but not on 211H (Figure 1).

2.9 | Histological analysis

3.2 | In vitro characterization of antibody

H226 tumors were resected from mice on days 1, 3, and 7 after injection

In cell binding assays with H226 and 211H cells, 111In-­labeled NZ-­12with 3.70 MBq of 90Y-­labeled NZ-­12 and 3.70 MBq of 90Y-­labeled con-bound to H226 cells with a maximum value of 34.5% ± 1.0% at 1 × 107

trol antibody (n = 3/time-­point). The tumors were fixed in 10% neutral-­cells, whereas it did not bind to 211H cells (Figure 2A). 111In-­labeled

buffered formalin and embedded in paraffin. Tumor sections (1-­μm thick)human IgG1λ as an isotype-­matched control did not bind to H226 cells

were deparaffinized and stained with hematoxylin and eosin. Tumor cell

(Figure 2B). From the competitive inhibition assay with H226 cells, the

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SUDO et al.

dissociation constants of intact NZ-­12 and CHX-­A′′-­DTPA-­conjugatedrespectively (Table 2). Among the absorbed doses in major organs, theNZ-­12 were estimated to be 1.1 and 3.0 nmol/L, respectively (Figure 2C),

highest values were observed in the lungs, 14.4 Gy for 90Y-­labeled NZ-­12suggesting that the loss of affinity by the chelate conjugation procedureand 15.9 Gy for 90Y-­labeled control antibody (Table 2).

was very limited. The control antibody IgG1λ did not inhibit the bindingof 111In-­labeled NZ-­12 to H226 cells (Figure 2D). Thus, CHX-­A′′-­DTPA-­
conjugated NZ-­12 was considered acceptable for the following in vivo
experiments, and the human IgG1λ was used as a control.

3.4 | Treatment effects of 90Y-­labeled antibody innude mice bearing H226 tumors
Giving 3.70 MBq of 90Y-­labeled NZ-­12 significantly suppressed H226

3.3 | Biodistribution of 111In-­labeled antibody innude mice bearing H226 tumors
111

Tumor uptake ofIn-­labeled NZ-­12 and control antibody was similar

at 1 hour after injection (2.1 ± 0.3% ID/g of 111In-­labeled NZ-­12 and1.8 ± 0.9% ID/g ofSubsequently,

111

In-­
labeled control antibody) shown in Table 1.

111

In-­labeled NZ-­12 tumor uptake significantly increased

tumor growth during the first 21 days compared with intact NZ-­
12 (0 MBq) and, thereafter, the tumor volumes gradually increased(P < .01, Figure 3A,B). In contrast, no significant tumor suppression
was observed in the groups given 1.85 MBq of 90Y-­labeled NZ-­12 and3.70 MBq of 90Y-­labeled control antibody compared with intact NZ-­12(0 MBq) shown in Figure 3A. Kaplan-­Meier survival curves based on theendpoint of tumor volume of 500 mm3 are shown in Figure 3C. Giving

over time and reached 23.2 ± 0.9% ID/g on day 7, whereas the 111In-­

3.70 MBq of 90Y-­labeled NZ-­12 induced significant survival prolonga-labeled control antibody showed a slight increase and ranged fromtion compared with the other groups (P < .01 vs 0 MBq and 1.85 MBq

6.5 ± 0.5% to 9.0 ± 1.9% ID/g (Table 1). On days 1, 2, 4, and 7, tumor up-of 90Y-­labeled NZ-­12, and P < .05 vs 3.70 MBq of 90Y-­labeled control an-

take was significantly different between 111In-­labeled NZ-­12 and control

tibody), whereas survival did not differ among the groups given 0 MBq

antibody (P < .01, Table 1). Biodistribution of111

In-­labeled NZ-­12 in nor-and 1.85 MBq of 90Y-­labeled NZ-­12 and 3.70 MBq of 90Y-­labeled control

In-­labeled control antibody, al-antibody (Figure 3C). 90Y-­labeled NZ-­12 treatment induced no signifi-

though there were some statistically significant differences in blood, liver,cant body weight reduction compared with the 0-­MBq group, whereas

mal organs was almost similar to that of111

spleen, and muscle (P < .01 or P < .05, Table 1). Based on these results,

giving 3.70 MBq of 90Y-­labeled control antibody induced transient but

the absorbed dose was estimated when In-­111 was replaced with Y-­90.

significant body weight loss during the first 7 days (P < .05, Figure 3D).

The dose absorbed by H226 tumors was 24.8 and 10.6 Gy when injectedwith 3.70 MBq of 90Y-­labeled NZ-­12 and 90Y-­labeled control antibody,

3.5 | Histological analysis of H226 tumors treatedwith 90Y-­labeled NZ-­12
Hematoxylin and eosin staining showed no marked histologicalchanges in H226 tumors treated with intact NZ-­12 (0 MBq), as shownin Figure 4. Sections of H226 tumors treated with 3.70 MBq of 90Y-­
labeled NZ-­12 showed a few small necrotic foci on days 1 and 3, andexpansion of the necrotic area and hemorrhage were observed on day
7, in contrast to untreated tumors and tumors treated with intact NZ-­
12 (0 MBq) shown in Figure 4. Next, to evaluate the effects on cellproliferation, Ki-­
67-­
immunostaining was conducted. Cell proliferation did not change significantly in tumors treated with intact NZ-­12(0 MBq), whereas 3.70 MBq of 90Y-­labeled NZ-­12 reduced cell proliferation (Ki-­67-­positive cells) in a time-­dependent way (Figure 5), andthe reduction was statistically significant on days 3 and 7 comparedwith untreated tumors and tumors treated with intact NZ-­12 (0 MBq).
In contrast, few apoptotic cells, as determined by TUNEL staining,were observed in any of the groups (data not shown).

3.6 | Podoplanin expression in human malignant
mesothelioma specimens
Human malignant mesothelioma specimens containing three main histological types, epithelioid, sarcomatoid, and mixed, were immunostained
F I G U R E 1 Immunofluorescence staining of H226
(podoplanin-­positive) and 211H (podoplanin-­negative) cells
using anti-­podoplanin antibody LpMab-­17 (red). The nuclei werecounterstained with DAPI (blue)

with an anti-­podoplanin antibody LpMab-­17. A positive staining signal
was observed both in the cytoplasm and in plasma membranes of tumor
cells and tended to be stronger compared with H226 tumors (Figure 6).
Table 3 provides a summary of the immunohistochemical staining data.

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SUDO et al.

F I G U R E 2 In vitro characterizationof radiolabeled antibody NZ-­12. A, Cellbinding assay of 111In-­labeled NZ-­12with H226 (black circles) and 211H cells.
(white circles). B, Cell binding assay of111In-­labeled control antibody (IgG1λ) with
H226. C, Competitive inhibition assay for
intact NZ-­12 (black circles) and CHX-­A′′-­
DTPA-­conjugated NZ-­12 (white circles)with H226 cells. D, Competitive inhibition
assay for control antibody (IgG1λ)

Positive podoplanin staining on human specimens was found in 90% ofof podoplanin,19 and the frequency of high podoplanin expression is

all types of malignant mesothelioma specimens: 92% (12/13 cases) of

similar to or higher than that of other mesothelioma-­associated an-epithelioid-­type, 67% (2/3 cases) of sarcomatoid-­type, and 100% (3/3

tigens such as calretinin, thrombomodulin, cytokeratin 5, WT1, andcases) of mixed-­type.

mesothelin. 28,38,39 Therefore, podoplanin is a promising immunological target for diagnosis and therapeutics in malignant mesothelioma.

3.7 | Treatment effects of external-­beam radiation
with X-­rays in nude mice bearing H226 tumors

We previously developed a rat-­human chimeric anti-­podoplanin antibody, NZ-­12, which has high specificity and high binding affinity
for podoplanin expressed on mesothelioma cells.30,31 In the present

In mice irradiated with 25 and 50 Gy, H226 tumor volumes increased

study, the potential of radiolabeled NZ-­12 as a RIT agent was evalu-

during the first 14 days and thereafter significantly decreased com-

ated in a mesothelioma mouse model.

pared with the control (0 Gy) group (P < .01, Figure 7A). Tumor re-

Immunofluorescence staining analysis of podoplanin expression

duction was observed until day 28, and tumor volumes graduallyin mesothelioma cell lines showed that high expression of podo-

increased thereafter. Grade of regrowth was more pronounced in theplanin was observed on the membrane of H226 cells, but not on

25-­Gy radiation group, and tumor size differed significantly between

211H cells, which is consistent with the findings of flow cytometricthe 25-­Gy and the 50-­Gy groups from days 35 to 56 (P < .01 fromanalysis in a previous study.19 Thus, H226 as a positive control anddays 35 to 45, P < .05 from days 49 to 56, Figure 7A). The radiation

211H as a negative control were considered suitable for the sub-dose of 25 Gy corresponds to the estimated absorbed dose when in-sequent in vitro studies. In vitro cell binding and competitive inhi-

jected with 3.70 MBq of 90Y-­labeled NZ-­12, as mentioned above. No

bition assays showed that 111In-­labeled NZ-­12 bound specifically tobody weight reduction was observed in any of the groups (Figure 7B).

podoplanin-­expressing H226 cells with high affinity, and the affinity
loss after the labeling procedures was limited, indicating that radio-

4 | D I S CU S S I O Nlabeled NZ-­12 is acceptable for the following in vivo experiments.
Biodistribution studies in H226 tumor-­bearing mice showed thattumor uptake of 111In-­labeled NZ-­12 was significantly higher than

Malignant mesothelioma is an aggressive tumor with a very poorthat of 111In-­labeled control antibody. By replacing 111In with a ther-

prognosis.1 More effective treatments must be developed to improve

apeutic radionuclide 90Y, 3.70 MBq of 90Y-­labeled NZ-­12 showed athe prognosis. Malignant mesothelioma shows high expression levels

stronger tumor growth suppression effect and prolonged survival

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1h

TA B L E 1 Biodistribution of 111In-­labeled antibody in nude mice bearing
H226 tumors

Day 1

Day 2

Day 4

Day 7

46.4 ± 3.8**

20.8 ± 2.8**

20.7 ± 1.1

14.9 ± 1.4**

11.6 ± 2.0

Brain

1.3 ± 0.4

0.7 ± 0.1

0.7 ± 0.1

0.4 ± 0.1

0.4 ± 0.1

Lung

14.0 ± 2.7

9.2 ± 0.8

9.4 ± 0.6

7.1 ± 1.5*

5.5 ± 0.9

Anti-­podoplanin antibody NZ-­12
Blood

Liver

10.8 ± 1.3**

6.7 ± 0.6

7.6 ± 0.4*

6.8 ± 0.8

5.3 ± 0.7

Spleen6.6 ± 0.6**

6.0 ± 0.4**

6.3 ± 0.5**

5.3 ± 1.0

6.3 ± 0.6**

Pancreas

2.5 ± 0.5

2.3 ± 0.4

1.9 ± 0.2

1.6 ± 0.3*

1.3 ± 0.2

Stomach1.7 ± 0.3

2.4 ± 0.6

1.9 ± 0.2

1.9 ± 0.4

1.5 ± 0.3

Intestine

3.8 ± 0.7

2.5 ± 0.3

2.3 ± 0.3

2.0 ± 0.3

1.5 ± 0.2

Kidney11.4 ± 1.3

7.5 ± 0.8

7.3 ± 1.0

6.5 ± 0.8

4.7 ± 0.7

Muscle0.7 ± 0.1

1.4 ± 0.1

1.3 ± 0.1**

1.2 ± 0.1**

1.0. ± 0.1

Bone

3.3 ± 0.7

2.5 ± 0.4

2.6 ± 0.3

2.2 ± 0.4

2.2 ± 0.6

H226
tumor

2.1 ± 0.3

12.0 ± 1.3**

18.0 ± 3.3**

22.4 ± 2.0**

23.2 ± 0.9**

Control antibody
Blood

40.8 ± 2.4

25.3 ± 0.9

24.3 ± 1.6

20.4 ± 2.8

13.9 ± 1.2

Brain

1.2 ± 0.2

0.8 ± 0.2

0.8 ± 0.2

0.7 ± 0.2

0.6 ± 0.3

Lung

14.9 ± 1.3

10.2 ± 0.2

9.9 ± 0.7

9.2 ± 0.8

6.3 ± 0.6Liver

7.7 ± 0.8

7.1 ± 0.6

5.8 ± 0.9

6.9 ± 0.9

6.6 ± 1.3

Spleen4.6 ± 0.2

4.0 ± 0.3

4.5 ± 0.5

5.3 ± 0.7

4.2 ± 0.7

Pancreas

2.3 ± 0.2

2.4 ± 0.3

2.3 ± 0.2

2.2 ± 0.2

1.7 ± 0.4

Stomach1.8 ± 0.2

2.4 ± 0.3

2.1 ± 0.4

2.2 ± 0.5

1.6 ± 0.5

Intestine

3.5 ± 0.5

2.3 ± 0.3

2.4 ± 0.4

2.2 ± 0.3

1.5 ± 0.1

Kidney11.8 ± 1.1

7.7 ± 0.4

7.2 ± 1.0

6.5 ± 0.9

4.8 ± 0.4

Muscle0.9 ± 0.1

1.7 ± 0.5

1.8 ± 0.3

1.8 ± 0.5

1.2 ± 0.1Bone

3.0 ± 0.3

2.3 ± 0.2

2.3 ± 0.3

2.5 ± 0.3

1.6 ± 0.5

H226
tumor

1.8 ± 0.3

6.5 ± 0.5

7.0 ± 0.6

8.7 ± 2.0

9.0 ± 1.9

Data are expressed as percentage of injected dose per gram (% ID/g) and as mean ± SD.
**P < .01.
*P < .05, vs control antibody.

with statistically significant differences compared with giving intact NZ-­12 and 3.70 MBq of
1.85 MBq of90

90

TA B L E 2

Estimated absorbed dose (Gy) of 90Y-­labeled antibody

Y-­labeled control antibody, whereas

Y-­labeled NZ-­12 had no significant effect. Histologicalanalysis showed an increase in necrotic foci and a reduced numberof proliferating (Ki-­67 positive) cells in H226 tumors treated with
3.70 MBq of90

Y-­labeled NZ-­12. These histological alterations are

frequently observed in tissues damaged by radiation.

40,41

Brain

Anti-­podoplanin antibody
NZ-­12Control antibody

1.85 MBq

3.70 MBq

3.70 MBq

0.5

1.1

1.3

Our find-

Lung

7.2

14.4

15.9

ings suggest that 90Y-­labeled NZ-­12 has the potential to treat malig-

Liver

5.9

11.8

10.6

nant mesothelioma patients.

Spleen

4.7

9.5

7.3

Pancreas

1.5

3.1

3.5

Stomach

1.5

3.1

3.4

Intestine

1.9

3.8

3.8

Kidney

6.0

12.0

12.0

Muscle

0.9

1.8

2.5

Radiation therapy for malignant mesothelioma patients is generally used as adjuvant therapy to prevent recurrences after surgicalresection of early-­stage mesothelioma and symptom palliation.5 The
standard dose of radiation therapy for preventing recurrences andsymptom palliation is 21 Gy in three fractions or 20 Gy in four to five
fractions according to the clinical practice guidelines for malignant
mesothelioma.5 Prophylactic irradiation may prolong the recurrence-­
free period, although it did not confer a statistically significant

Bone

0.9

1.8

2.5

H226 tumor

12.4

24.8

10.6

| 1659

SUDO et al.

F I G U R E 3 Therapeutic efficacy of90Y-­labeled antibodies in nude micebearing H226 tumors. A, Tumor growth
curves after injection with 90Y-­labeled
NZ-­12 and control antibody. Data indicate
mean and SD (n = 10). **P < .01 vs 0 MBq
of 90Y-­labeled NZ-­12 (intact NZ-­12 only).
B, Representative images of mice at day
21 after injection with 90Y-­labeled NZ-­12and control antibody. Dashed circles
indicate tumors. C, Kaplan-­Meier survival
curves based on the endpoints of tumor
volume of 500 mm3. **P < .01, 0 MBq
and 1.85 MBq of 90Y-­labeled NZ-­12 vs3.70 MBq of 90Y-­labeled NZ-­12; *P < .05,
3.70 MBq of 90Y-­labeled control antibody
vs 3.70 MBq of 90Y-­labeled NZ-­12. D,
Body weight changes after injection with90
Y-­labeled NZ-­12 and control antibody.
Data indicate mean and SD (n = 10).
*P < .05 vs 0 MBq of 90Y-­labeled NZ-­12

difference compared with unirradiated patients.5 Palliative radiation

expected to be low, although further dosimetry studies in malig-

therapy provides significant pain relief in approximately 50% of ma-nant mesothelioma patients are required.

lignant mesothelioma patients.5 In our dosimetry analysis, the absorbed dose to H226 tumors injected with 3.70 MBq of

90

Giving 3.70 MBq of 90Y-­labeled NZ-­12 delayed tumor growth inY-­labeled

the H226 mesothelioma tumor model, whereas tumor volume re-NZ-­12 was estimated to be 24.8 Gy, suggesting that it is potentially

duction was observed in mice irradiated with X-­rays of 25 Gy, whichapplicable for recurrence prevention and palliation.

corresponds with the absorbed dose when injected with 3.70 MBq

The safety of RIT with 90Y-­labeled NZ-­12 is important whenof 90Y-­labeled NZ-­12, as mentioned above. Buras et al48 reported

considering clinical application. In the present study, givingthat the antitumor effect by RIT is limited, especially in radioresis-

3.70 MBq of 90Y-­labeled NZ-­12 induced no significant body weighttant tumors, compared with external-­beam radiation therapy as aloss and no visible adverse effects, such as diarrhea and dyspnea.

result of lower radiation dose rates of RIT. Similar findings were ob-

Most mesothelioma cells spread into the diaphragm, chest wall,

tained in our previous RIT studies.7,9 H226 is a radioresistant celland mediastinum with an irregular shape and are close to criti-

line 49 and, thus, the antitumor effect of RIT using 90Y-­labeled NZ-­12cal structures such as the spinal cord, liver, esophagus, heart,for H226 tumors was considered less effective than external-­beam

Therefore, external-­b eam radiation to mesothelioma

radiation therapy at the corresponding doses. However, 90Y-­labeledin the thorax and mediastinum has substantial technical limita-

NZ-­12 suppressed tumor growth and, therefore, further efforts as

tions.42,43 The most common toxicities in patients treated withdescribed below, may improve the therapeutic effect to achieve

external-­b eam radiation for thoracic cancer are symptomatic ra-complete remission in malignant mesothelioma patients.

and lungs.

42

diation pneumonitis,44

and the mean dose to the lungs for 20%

Currently, RIT is applied to hematological malignancies such as

risk of radiation pneumonitis is 20 Gy.44 The estimated absorbed

lymphoma, but not to solid tumors, because most solid tumors aredose in mouse lungs injected with 3.70 MBq of 90Y-­labeled NZ-­

more radioresistant.50 To exert the full potential of RIT, radiolabeled

12 was 14.4 Gy, suggesting a low risk of toxicity to the lungs inantibodies must be specifically delivered in tumoricidal doses topatients. The absorbed doses of the other organs are also consid-

tumors (eg, 30-­50 Gy for radiosensitive tumors such as hematopoi-

ered tolerable because these absorbed doses are lower than the

etic tumors, and 80-­100 Gy for radioresistant tumors such as mosttolerance doses of those organs based on the findings of external-­

solid tumors51), whereas radiation doses to healthy tissues must be

This is supported by several previous

limited.50 Several potential strategies may improve the efficacy ofstudies using 90Y-­labeled antibodies, which showed no severe tox-

RIT with 90Y-­labeled NZ-­12. One approach is repeated dosing of 90Y-­

beam radiation studies.

45,46

10,47

icities induced by similar absorbed doses.

Therefore, the risklabeled NZ-­12, which can give H226 tumors approximately 25 Gyof radiation-­induced toxicity from RIT with 90Y-­labeled NZ-­12 iswith no adverse effects, at least in mice. A simple calculation shows

1660

|

SUDO et al.

F I G U R E 4 Histological analysis of H226 tumors after injection
with 0 MBq (intact NZ-­12 only) and 3.70 MBq of 90Y-­labeled NZ-­12.
Hematoxylin-­and-­eosin-­stained sections of H226 tumors at days
1, 3, and 7 after injection. White arrowheads indicate necrosis andblack arrowheads indicate hemorrhagethat a regimen of four repeated doses can provide approximately
100 Gy, which could be effective for solid tumors.51 As mentioned
above, RIT generally shows lower anticancer effects compared withexternal-­beam radiation therapy and, thus, further cycles of dosing
may be needed. Moreover, clinical studies for dosimetry in mesothelioma patients are required because the biodistribution of radiolabeled NZ-­12 likely differs between mouse and human. In fact,
the present immunohistochemical staining analysis showed higher
podoplanin expression in human malignant mesothelioma specimens
compared with H226 tumors, suggesting that 90Y-­labeled NZ-­12
would likely accumulate more in tumors and deposit higher energy
to tumors in patients than in H226 tumor xenografts. Second, aradiosensitizer could be combined with 90Y-­labeled NZ-­12. Somechemotherapeutic agents such as pemetrexed and gemcitabineF I G U R E 5 Tumor cell proliferation analysis by Ki-­67
immunostaining. A, Ki-­67-­stained sections of H226 tumors atdays 1, 3, and 7 after injection with 0 MBq (intact NZ-­12 only) and3.70 MBq of 90Y-­labeled NZ-­12. B, Quantification of proliferating(Ki-­67 positive) cells. Data are mean and SD. **P < .01, *P < .05

have radiosensitizing effects.52,53 Pemetrexed is recommended
as first-­line chemotherapy for malignant mesothelioma5 and is thethe therapeutic effect because NZ-­12 is reported to induce po-

first candidate as a radiosensitizer with RIT against mesothelioma.

tent antibody-­dependent cellular cytotoxicity against podoplanin-­

Third, repeated dosing with intact NZ-­12 after RIT could enhance

expressing tumors.30 NZ-­12 is a chimeric antibody and is expected| 1661

SUDO et al.

F I G U R E 6 Podoplanin expression
analysis in human mesothelioma
specimens. A, Representative imagesof immunohistochemical staining with
LpMab17. B, High magnification imagesof (A)
to have a low risk of immunogenicity, suggesting that NZ-­12 is suit-

NZ-­12 has the potential to induce cytotoxic effects and suppress

able for repeated dosing over a long period of time. Taken together,tumor growth for a long time. Further preclinical studies are needed

the triple combination of90

Y-­labeled NZ-­12, pemetrexed, and intact

to evaluate the efficacy and safety of these therapeutic options.

1662

|

SUDO et al.

F I G U R E 7 Therapeutic efficacy of external-­beam radiation with X-­rays in nude mice bearing H226 tumors. A, Tumor growth curves after
irradiation with X-­rays. Data indicate mean and SD (n = 5). **P < .01 and *P < .05 vs 0 Gy. ++P < .01 and +P < .05, 25 Gy vs 50 Gy. B, Body
weight changes after irradiation with X-­rays. Data indicate mean and SD (n = 5)
TA B L E 3 Summary of immunohistochemical analysis of
podoplanin in human mesothelioma specimenspart by KAKENHI 18K07778 (HS), 17K10497 (AS), 18H02774 (ABT),
17K07299 (MKK), and 16K10748 (YK); by AMED JP18am0101078(YK); and by AMED-­CREST JP18gm0710003 (ABT).

NumberPositive (%)

Epithelioid13

12 (92.3)

Sarcomatoid

3

2 (66.7)

C O N FL I C T S O F I N T E R E S T

Mixed

3

3 (100.0)

Authors declare no conflicts of interest for this article.

Total

19

17 (89.5)

Although the present study showed the potential of 90Y-­labeled

ORCID
https://orcid.org/0000-0003-2726-288X

NZ-­12 as an RIT agent for the treatment of mesothelioma, there are

Atsushi B. Tsuji

some limitations. First, 90Y-­labeled NZ-­12 delayed tumor growth,

Tsuneo Saga

https://orcid.org/0000-0001-7801-9316

Yukinari Kato

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

but further preclinical studies are needed to evaluate whether
complete remission of malignant mesothelioma can be achieved, as
mentioned above. Second, in the analysis of podoplanin expression,
the number of human mesothelioma specimens analyzed for podoplanin expression was small and, thus, expression analyses using alarger number of specimens are needed. Third, further clinical studies are required to evaluate the dosimetry of radiolabeled NZ-­12 inmalignant mesothelioma patients for prediction of the therapeutic
effects and safety.
In summary, giving 3.70 MBq of 90Y-­labeled anti-­p odoplaninantibody NZ-­
12 significantly suppressed tumor growth without body weight loss in a mesothelioma mouse model H226.
Considering the higher expression levels of podoplanin in humanmalignant mesothelioma specimens compared with H226 tumors,
RIT with 90Y-­labeled NZ-­12 is a promising potential therapeutic option to provide greater clinical benefit to malignant mesothelioma
patients.

AC K N OW L E D G M E N T S
We thank Yuriko Ogawa and Naoko Kuroda for technical assistancefor the animal experiments and the staff in the Laboratory Animal
Sciences section for animal management. This work was supported in

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How to cite this article: Sudo H, Tsuji AB, Sugyo A, et al.
Therapeutic efficacy evaluation of radioimmunotherapy with90

Y-­labeled anti-­podoplanin antibody NZ-­12 for mesothelioma.

Cancer Sci. 2019;110:1653–1664. https://doi.org/10.1111/
cas.13979