cells
ArticlePreclinical Evaluation of Podoplanin-Targeted
Alpha-Radioimmunotherapy with the Novel Antibody
NZ-16 for Malignant Mesothelioma
Hitomi Sudo 1 , Atsushi B. Tsuji 1, * , Aya Sugyo 1 , Mika K. Kaneko 2 , Yukinari Kato 2,3 , Kotaro Nagatsu 4 ,Hisashi Suzuki 4 and Tatsuya Higashi 1
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Citation: Sudo, H.; Tsuji, A.B.; Sugyo,A.; Kaneko, M.K.; Kato, Y.; Nagatsu,
K.; Suzuki, H.; Higashi, T. Preclinical
Evaluation of Podoplanin-Targeted
Alpha-Radioimmunotherapy with theNovel Antibody NZ-16 for Malignant
Mesothelioma. Cells 2021, 10, 2503.
https://doi.org/10.3390/cells10102503
Academic Editors: Gary S. Goldberg,
Ester Martín-Villar andWolfgang Sauerwein
Received: 6 August 2021
Accepted: 10 September 2021
Published: 22 September 2021

Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in*

Department of Molecular Imaging and Theranostics, Institute for Quantum Medical Science (iQMS),
National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage,
Chiba 263-8555, Japan; sudo.hitomi@qst.go.jp (H.S.); sugyo.aya@qst.go.jp (A.S.);
higashi.tatsuya@qst.go.jp (T.H.)
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,
2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan; k.mika@med.tohoku.ac.jp (M.K.K.);
yukinarikato@med.tohoku.ac.jp (Y.K.)
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980-8575, Japan
Department of Advanced Nuclear Medicine Science, Institute for Quantum Medical Science (iQMS),
National Institutes for Quantum and Radiological Science and Technology (QST), 4-9-1 Anagawa, Inage,
Chiba 263-8555, Japan; nagatsu.kotaro@qst.go.jp (K.N.); suzuki.hisashi@qst.go.jp (H.S.)
Correspondence: tsuji.atsushi@qst.go.jp; Tel.: +81-43-382-3704

Abstract: The prognosis of advanced mesothelioma is poor. Podoplanin (PDPN) is highly expressed inmost malignant mesothelioma. This study aimed to evaluate the potential alpha-radioimmunotherapy(RIT) with a newly developed anti-PDPN antibody, NZ-16, compared with a previous antibody, NZ-12.
Methods: The in vitro properties of radiolabeled antibodies were evaluated by cell binding and competitive inhibition assays using PDPN-expressing H226 mesothelioma cells. The biodistribution of
111 In-labeled antibodies was studied in tumor-bearing mice. The absorbed doses were estimated basedon biodistribution data. Tumor volumes and body weights of mice treated with 90 Y- and 225 Ac-labeled
NZ-16 were measured for 56 days. Histologic analysis was conducted. Results: The radiolabeled NZ-16
specifically bound to H226 cells with higher affinity than NZ-12. The biodistribution studies showed
higher tumor uptake of radiolabeled NZ-16 compared with NZ-12, providing higher absorbed doses
to tumors. RIT with 225 Ac- and 90 Y-labeled NZ-16 had a significantly higher antitumor effect than
RIT with 90 Y-labeled NZ-12. 225 Ac-labeled NZ-16 induced a larger amount of necrotic change andshowed a tendency to suppress tumor volumes and prolonged survival than 90 Y-labeled NZ-16. Thereis no obvious adverse effect. Conclusions: Alpha-RIT with the newly developed NZ-16 is a promisingtherapeutic option for malignant mesothelioma.
Keywords: molecular radiotherapy; improved efficacy; tumor volume reduction; prolonged survival;
actinium-225

published maps and institutional affiliations.

1. Introduction

Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Malignant mesothelioma is an aggressive tumor that arises primarily in the pleuralor peritoneal mesothelial surfaces [1]. Surgical resection is only offered to patients withearly-stage disease [1,2]. Most patients reach advanced-stage disease before diagnosis,
and thus the primary treatment is systemic chemotherapy [1,2]. The prognosis is poorand the median overall survival of patients who undergo chemotherapy is approximately
12 months [2]. Therefore, the development of more effective treatments for unresectable
malignant mesothelioma is strongly desired.
Mesothelioma is classified into three types, epithelioid, sarcomatoid, and biphasic,
based on histological characteristics [1,2]. There are several markers for the epithelioid sub-Cells 2021, 10, 2503. https://doi.org/10.3390/cells10102503

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type, such as calretinin, WT-1, cytokeratin 5, and ERC/mesothelin [3,4]. Those markers do
not express in the sarcomatoid subtype, but podoplanin (PDPN) is overexpressed in morethan 80% of all types [5,6]. PDPN is a type I transmembrane sialomucin-like glycoprotein
expressed in kidney podocytes, alveolar type I cells, and lymphatic endothelial cells [7].
High expression of PDPN in tumors is associated with epithelial–mesenchymal transition,
migration, invasion, and metastasis [8,9]. Several preclinical studies have demonstrated
that anti-PDPN antibodies inhibit cancer metastasis [10] and cancer progression [11,12].
Therefore, PDPN is a promising therapeutic target for malignant mesothelioma.
Radioimmunotherapy (RIT) is a selective internal radiation therapy in which highaffinity antibodies against tumor-associated antigens are used to transport radionuclides totumors [13]. In clinical practice, RIT for hematologic malignancies such as non-Hodgkin’s
lymphoma utilizes anti-CD20 antibodies conjugated with β-emitters, 90 Y or 131 I, andthe overall response rates are high, reaching 60–80%, with a complete remission rate of
15–40% [13,14]. The clinical efficacy of existing RIT for solid tumors, however, remains low,
mainly due to the low radiosensitivity of solid tumors. Overcoming the radioresistance isnecessary to enhance the clinical efficacy of RIT.
The clinical efficacy of α-particle emitters in the treatment of solid cancer was recently
demonstrated [15]. α-Particle emitters have a greater linear energy transfer comparedwith β-emitters and deposit more energy into tumor cells, which results in greater DNA
damage to the cells [16]. Actinium-225 is an α-particle-emitting radionuclide that generates
a total of four α-particles in the decay chain [17]. The half-life of 225 Ac is appropriate forthe pharmacokinetics of antibodies. Therefore, RIT with 225 Ac is expected to improve thetherapeutic efficacy of RIT treatment for solid tumors.
A previous study reported that 90 Y-labeled anti-PDPN antibody NZ-12 suppresses
tumor growth in a mesothelioma model cell line NCI-H226 (H226); unfortunately, complete
remission was not achieved [6]. To improve the therapeutic effect of RIT with an anti-PDPN
antibody, we newly developed an anti-PDPN antibody, NZ-16, having a different constantregion than NZ-12. NZ-16 has a higher affinity than NZ-12 for H226 mesothelioma cells
and is, therefore, expected to deliver more radionuclides to the tumors. In the present study,
we first compared the in vitro and in vivo properties of NZ-12 and NZ-16 radiolabeledwith 111 In. After confirming that NZ-16 has more favorable binding properties than NZ-12,
the antitumor effects of 225 Ac-labeled NZ-16 were compared with those of 90 Y-labeled
NZ-16 in an H226 mesothelioma mouse model.
2. Materials and Methods
2.1. Antibody
A rat–human chimeric anti-human PDPN antibody, NZ-12, was previously generated [18]. To generate the novel chimeric anti-human PDPN antibody NZ-16, the appropriate heavy chain variable domain of a rat NZ-1 antibody [19] and heavy chain constantdomain of human IgG1 were subcloned into the pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan), and the light chain variable domain of a rat NZ-1
antibody and human lambda light chain constant domain were subcloned into pCAGBle vectors (FUJIFILM Wako Pure Chemical Corporation). The vectors were transfected
into ExpiCHO-S cells using the ExpiCHO Expression System (Thermo Fisher ScientificInc., Waltham, MA, USA). NZ-16 was purified using Protein G-Sepharose (GE Healthcare
BioSciences, Chicago, PA, USA).
2.2. Cell Culture
Mesothelioma cell line NCI-H226 (H226, CRL-5826) was obtained from ATCC (Manassas,
VA, USA). The cells were cultured in RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation)
containing 10% fetal bovine serum (Thermo Fisher Scientific Inc.) in 5% CO2 at 37 ◦ C.

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2.3. Radiolabeling of Antibodies
For radiolabeling of radiometals 111 In, 90 Y, and 225 Ac, antibodies are necessary to
be conjugated with a chelator. The present study employed p-SCN-Bn-DOTA (DOTA,
Macrocyclics, Dallas, TX, USA). All radiolabeled antibodies were conjugated with DOTA,
but DOTA was abbreviated; namely, we presented them like 111 In-, 90 Y-, and 225 Ac-labeled
antibodies. Antibodies were conjugated with DOTA as described previously [20]. Briefly,
antibodies (5 mg/mL) were reacted with four equal molar amounts of DOTA in 50 mM
borate buffer (pH8.5) for 16 h at 37 ◦ C. The conjugation ratios of DOTA to antibodies were
estimated to be approximately 2.8 each as determined by radio-thin-layer chromatography with 80% methanol. The DOTA-conjugated antibodies were purified by elution with
0.1 M acetate buffer (pH 6.0) using a Sephadex G-50 column (GE Healthcare BioSciences).
111 InCl (Nihon Medi-Physics, Tokyo, Japan) or 90 YCl (Perkin Elmer, Waltham, MA, USA)33
was incubated in 0.5 M acetate buffer (pH 6.0) for 5 min at room temperature. Each was
mixed with the DOTA-antibody conjugate and incubated for 60 min at 37 ◦ C. Radiolabelingof the antibody with 225 Ac was conducted as previously described [20]. 225 AcNO3 (OakRidge National Laboratory, Oak Ridge, TN, USA) dissolved in 0.2 M optima grade HCl(Thermo Fisher Scientific Inc.) was added to 2 M tetramethylammonium acetate (Tokyo
Chemical Industry, Tokyo, Japan) and 150g/L L-ascorbic acid (MilliporeSigma, St. Louis,
MO, USA), and the solution was incubated for 5 min at room temperature. The solution was then mixed with the DOTA-conjugated antibodies and incubated for 60 min at
37 ◦ C. The radiolabeled antibodies were purified using an Amicon Ultra centrifugal filter(Merck Millipore, Darmstadt, Germany), and the purified antibodies were analyzed byradio-thin layer chromatography. The specific activity of 111 In-labeled NZ-12, 111 In-labeled
NZ-16, 90 Y-labeled NZ-16, and 225 Ac-labeled NZ-16 was approximately 4.9 ± 2.5, 9.7 ± 4.5,
662.3 ± 151.8, and 0.6 ± 0.1 kBq/µg, respectively. The radiochemical yield was approximately 40% for 111 In-labeled NZ-12, 50–80% for 111 In-labeled NZ-16, 90% for 90 Y-labeled
NZ-16, and 25% for 225 Ac-labeled NZ-16. The radiochemical purities were greater than
95% after purification.
2.4. Cell Binding and Competitive Inhibition Assays
For the cell binding assays, H226 cells (1.0 × 107 , 5.0 × 106 , 2.5 × 106 , 1.3 × 106 ,6.3 × 105 , 3.1 × 105 , 1.6 × 105 , and 7.8 × 104 ) in phosphate-buffered saline with 1% bovine
serum albumin (MilliporeSigma) were incubated with 111 In-labeled NZ-12 or NZ-16 antibodies on ice for 60 min. After washing, cell-bound radioactivity was measured using a
gamma counter (Wizard2 Automatic Gamma Counters, PerkinElmer, Waltham, MA, USA).
For competitive inhibition assays, H226 cells (1.0 × 106 ) in phosphate-buffered saline with
1% bovine serum albumin were incubated with 111 In-labeled NZ-12 or NZ-16 in the presenceof varying concentrations of intact NZ-12, intact NZ-16, DOTA-conjugated NZ-12, or DOTAconjugated NZ-16 antibodies (0, 0.02, 0.07, 0.2, 0.7, 2.0, 6.1, 18.2, and 54.5 nmol/L) on ice for
60 min. After washing, cell-bound radioactivity was measured with a gamma counter. The
dissociation constant was estimated by applying data to a one-site competitive binding modelusing GraphPad Prism 8 software (ver. 8.4.3, GraphPad Software, La Jolla, CA, USA).
2.5. Tumor Model
The animal experimental protocol was approved by the Animal Care and Use Committee of the National Institutes for Quantum and Radiological Science and Technology(13–1022, 26 May 2016), and all animal experiments were conducted according to theinstitutional guidelines regarding animal care and handling. H226 cells (5 × 106 ) were
subcutaneously inoculated into male nude mice (BALB/c-nu/nu, 4 weeks old, CLEA Japan,
Tokyo, Japan) under isoflurane anesthesia.
2.6. Biodistribution of Radiolabeled Antibodies
When tumor volumes reached approximately 50 mm3 , mice (n = 4–5/time-point), wereintravenously injected with 111 In-labeled NZ-12 (37 kBq, n = 4/time-point), 111 In-labeledCells 2021, 10, 2503

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NZ-16 (37 kBq, n = 5/time-point), or 225 Ac-labeled NZ-16 (3.7 kBq, n = 5/time-point) ina total of 40 µg of antibody adjusted by adding the corresponding unlabeled antibodies.
The mice were euthanized by isoflurane inhalation at 1, 2, 4, or 7 days after injecting the111 In-labeled antibodies and 4 days after injecting the 225 Ac-labeled NZ-16. Blood was
obtained from the heart, and the tumor, brain, liver, spleen, intestine, kidney, and muscle
were dissected and weighed. Radioactivity was measured using a gamma counter withan energy window of 150–350 keV for 111 In and 200–300 keV for 225 Ac. The uptake isrepresented as a percentage of the injected dose (radioactivity) per gram of tissue (% ID/g).
2.7. Dosimetry
As described previously [21], the absorbed doses of the 90 Y- and 225 Ac-labeled antibodies were estimated using the area under the curve based on the biodistribution data of
the 111 In-labeled antibodies and the mean energy emitted per transition of Y-90, 1.495 ×
10−13 Gy kg (Bq s)−1 [22] and that of Ac-225 and all the daughter nuclei with corrections
for branching, 4.6262 × 10−12 Gy kg (Bq s)−1 [22]. The absorbed dose of bone marrow
was based on the blood data, considering a red-marrow-to-blood activity ratio of 0.4 [23].
Radiation weighting factors of 1 and 5 were used for Y-90 and Ac-225, respectively, as
recommended by the Medical Internal Radiation Dose Committee [24]. The estimatedabsorbed dose is expressed as Sv when considering the radiation weighting factors.
2.8. Radioimmunotherapy with 90 Y- and 225 Ac-Labeled Antibody
The mice were intravenously injected with intact NZ-16 (0 megabecquerel (MBq), n = 5),
90 Y-labeled NZ-16 (3.7 MBq, n = 5), or 225 Ac-labeled NZ-16 (11,1 and 18.5 kBq, n = 5) antibodies at

a total of 40 µg of antibody adjusted by adding the corresponding unlabeled antibodies. Tumor
sizes and body weights were measured at least twice a week for 8 weeks after administration.
Tumor size was measured using a digital caliper, and tumor volume was calculated according to
the following formula: tumor volume (mm3) = (length × width2)/2. When the tumor volume
reached greater than 800 mm3 or body weight loss was more than 20% compared with that at
day 0, the mouse was euthanized humanely by isoflurane inhalation.
2.9. Histologic Analysis
H226 tumors were resected from mice on days 1, 3, and 7 post injection with intact NZ-16(0 MBq, n = 3/time-point), 90 Y-labeled NZ-16 (3.7 MBq, n = 3/time-point), or 225 Ac-labeled NZ16 (18.5 kBq, n = 3/time-point). The tumors were fixed in 10% neutral-buffered formalin andembedded in paraffin. The tumor sections (1 µm thick) were deparaffinized and stained withhematoxylin and eosin. Tumor cell proliferation was evaluated by Ki-67-immunohistochemical
staining with a rabbit anti-Ki-67 antibody (SP6, Abcam, Cambridge, MA, USA) and ananti-rabbit HRP/DAB Detection Kit (Abcam) according to the manufacturer’s instructions.
The Ki-67 index was calculated by counting the percentage of Ki-67-positive tumor cells
per >2500 tumor cells in a section with 200 × magnification (n = 3).
2.10. Statistical Analysis
Data are expressed as the means ± standard deviation. Statistical analysis was performed using GraphPad Prism 8 software (ver. 8.4.3). Cell binding data and tumor volumedata were analyzed by two-way ANOVA. Ki-67 staining data were analyzed by one-way
ANOVA with Tukey’s multiple comparison post hoc test. Uptake data of radiolabeled
antibodies were analyzed by unpaired t-test. Log-rank tests were used to evaluate Kaplan–
Meier survival curves based on a tumor volume endpoint of 300 mm3 . p < 0.05 wasconsidered statistically significant in all experiments.
3. Results
3.1. In Vitro Characterization of the Antibodies
To confirm the reactivity of NZ-16 in H226 cells, immunofluorescence staining was conducted. A strong intensity was observed on the cell membranes of H226 (Figure S1). In the cellwas considered statistically significant in all experiments.
3. Results
3.1. In vitro characterization of the Antibodies
Cells 2021, 10, 2503

15
To confirm the reactivity of NZ-16 in H226 cells, immunofluorescence staining5 ofwasconducted. A strong intensity was observed on the cell membranes of H226 (Figure S1).
In the cell binding assays with H226, 111In-labeled NZ-16 showed significantly higher spe111In-labeledcific binding
than
NZ-12 (pNZ-16< 0.01).showed
The maximum
valueswerespecific29.6 ± 3.7%
for
111 In-labeledbinding
assayswith H226,
significantly
higherbinding
7
7 (Figure 1a,b). The specific
NZ-16
at
1.0
×
10
cells
and
22.9
±
3.3%
for
NZ-12
at
1.0
×
10
111
than In-labeled NZ-12 (p < 0.01). The maximum values were 29.6 ± 3.7% for NZ-16 at
111In- and7225Ac-labeled NZ-16 (Figure S2). The
binding
notsignificantly
differ
1.0
× 107didcells
and22.9 ± 3.3%
for between
NZ-12 at 1.0
× 10 (Figure 1a,b). The specific binding
111
results
ofthecompetitive
inhibition
assay
are
shown
in Figure
1c,d.
The binding
affinities
did not significantly differ between In- and 225
Ac-labeled
NZ-16(FigureS2). The
results(KDthe) ofcompetitive
intact NZ-12
and DOTA-conjugatedwereestimated
to beaffinities
1.7 and 5.7ofinhibition
assay are shownNZ-12
in Figure
1c,d.
The binding(KDnM,) ofrespectively(Figure
1c).
Those
of
intactNZ-16
and
DOTA-conjugated
NZ-16
were
estiintact NZ-12 and DOTA-conjugated NZ-12 were estimated to be 1.7 and 5.7 nM, respectively
mated tobeThose
1.8 andnM,
respectively(Figure 1d). These
results
that
DOTA(Figure1c).
of 2.7
intactNZ-16
and DOTA-conjugatedNZ-16
wereindicate
estimatedto thebe 1.8andconjugation
procedure
decreasedtheaffinity
of
NZ-16
for
PDPN
to
a
lesserextentthan
2.7 nM, respectively (Figure 1d). These results indicate that the DOTA conjugation procedurethat of NZ-12.
decreased
the affinity of NZ-16 for PDPN to a lesser extent than that of NZ-12.

1. In vitro
vitro characterization
characterization ofof radiolabeled
radiolabeled antibodies
antibodies NZ-12
NZ-12 andand NZ-16.
NZ-16. (a)(a) Cell
Cell binding
binding
Figure 1.
111
111 In-labeled
assay ofH226
cells.
(b)(b)
CellCellbindingassay
of 111ofIn-labeled
NZ-16
with H226
assay
of 111In-labeled
In-labeledNZ-12
NZ-12withwith
H226
cells.
binding
assay
NZ-16
with
cells. (c)Competitive
inhibition
assay forintact(white(whitecircles)circles)
and DOTA-conjugated
NZ-12
H226
cells.
(c) Competitive
inhibition
assay
for NZ-12
intact NZ-12
and DOTA-conjugatedNZ-12 (black circles) with H226 cells. (d) Competitive inhibition assay for intact NZ-16 (white circles)
and DOTA-conjugated NZ-16 (black circles) with H226 cells.

3.2. Biodistribution of 111 In-Labeled Antibodies in Nude Mice Bearing H226 Tumors
The biodistribution of 111 In-labeled NZ-12 and NZ-16 in H226 tumor-bearing miceis shown in Table 1 and Figure 2. 111 In-Labeled NZ-12 and NZ-16 gradually cleared fromthe blood and accumulated in the H226 tumors. The uptake of 111 In-labeled NZ-16 innormal organs tended to be higher than that of 111 In-labeled NZ-12, except in the liver(Table 1). The uptake of 111 In-labeled NZ-16 in the liver was significantly lower than thatof NZ-12 on days 1 and 2 post injection (p < 0.05 for day 1, p < 0.01 for day 2). The uptakeof 225 Ac-NZ-16 in normal organs was significantly higher than that of 111 In-labeled NZ-16(p < 0.01 in the brain, lung, spleen, and muscle, p < 0.05 in the intestine, Table 2). Tumor

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uptake of 111 In-labeled NZ-16 tended to be higher than that of NZ-12 over the observation
period (Figure 2). The maximal tumor uptake of 111 In-labeled NZ-16 was significantly
higher than that of 111 In-labeled NZ-12 on day 4 post injection (15.1 ± 2.4% ID/g for NZ-16
and 10.0 ± 0.4% ID/g for NZ-12; p < 0.01, Figure 2). The tumor uptake of 225 Ac-NZ-16 on
day 4 was higher than that of 111 In-labeled NZ-12 and NZ-16 (p < 0.01, Figure 2).
Table 1. Biodistribution of 111 In-labeled antibodies in H226 tumor-bearing mice.
Day 1

Day 2

Day 4

Day 7

18.0 ± 1.5
0.4 ± 0.1
6.2 ± 0.4
15.2 ± 2.1
9.9 ± 0.7
2.0 ± 0.3
12.1 ± 0.3
0.9 ± 0.1

13.8 ± 1.5
0.4 ± 0.0
5.0 ± 1.0
14.1 ± 1.1
9.0 ± 1.4
1.6 ± 0.2
9.5 ± 0.6
0.9 ± 0.1

8.6 ± 2.3
0.3 ± 0.1
3.6 ± 1.0
10.8 ± 1.0
8.6 ± 0.9
1.0 ± 0.2
6.4 ± 1.0
0.9 ± 0.2

5.1 ± 0.5
0.2 ± 0.1
2.1 ± 0.3
7.3 ± 0.9
5.4 ± 1.4
0.6 ± 0.1
3.7 ± 0.7
0.3 ± 0.1

21.8 ± 2.9
0.6 ± 0.1
7.7 ± 1.3
11.3 ± 3.5 *7.5 ± 0.8
2.6 ± 0.6
7.9 ± 1.2 **1.2 ± 0.2

19.7 ± 2.6 **0.6 ± 0.1
6.9 ± 0.7 *9.3 ± 0.9 **9.7 ± 0.7
2.2 ± 0.2
7.8 ± 0.7
1.2 ± 0.3 *13.6 ± 1.4 *0.5 ± 0.1
7.0 ± 2.7
7.9 ± 2.3
8.0 ± 0.9
1.9 ± 0.4
7.0 ± 0.7
1.1 ± 0.1

8.2 ± 4.0
0.3 ± 0.2
3.4 ± 1.4
7.1 ± 1.6
8.1 ± 3.1 *1.0 ± 0.4
4.9 ± 1.6
0.7 ± 0.4

NZ-12
Blood
Brain
Lung
Liver
SpleenIntestine
KidneyMuscle
NZ-16
Blood
Brain
Lung
Liver
SpleenIntestine
Cells
2021, 10, x FOR PEER REVIEW
KidneyMuscle7 of 16

Data areData
indicatedas the percentage of injected dose per gram (% ID/g) and as the mean ± standard deviation. * p < 0.05, ** p < 0.01 vs.
are indicated as the percentage of injected dose per gram (% ID/g) and as the mean ± standard deviation. ** p < 0.01,
NZ-12. * p < 0.05 vs. 111In-NZ-16 (Table 1).

225AcFigure 2. Tumor uptake of 111In-labeled111(N = 4), and 111In- labeled NZ-16 (N = 5)
Figure 2. Tumor uptake of NZ-12
In-labeled
NZ-12 (N = 4), and 111andIn- labeled
NZ-16 (N = 5) andlabeled NZ-16 (N = 5). Data indicate the mean and standard deviation (N = 4–5). ** p < 0.01.
225 Ac-labeled NZ-16 (N = 5). Data indicate the mean and standard deviation (N = 4–5). ** p < 0.01.

3.3. Dosimetry
The absorbed doses were estimated on the basis of the biodistribution studies when
In-111 was replaced with Y-90 or Ac-225. Table 3 shows the estimated absorbed doses
when the radiation-weighted factor was not considered. The absorbed doses of radiolabeled NZ-16 for tumors and organs tended to be higher than those of radiolabeled NZ12, except for in the liver and kidney, although the difference between the two antibodies
was not statistically significant (Table 3). The tumor-absorbed doses of 225Ac-labeled NZ12 and NZ-16 were 60-fold greater than those of 90Y-labeled NZ-12 and NZ-16, respectively (Table 3).
Table 3. Estimated absorbed dose (Gy/MBq) for 90Y- and 225Ac-labeled NZ-12 and NZ-16 based on the biodistribution dataof 111In-labeled antibodies, not considering a radiation weighting factor.

Y

Ac

90

Brain

NZ-12
0.1 ± 0.0

225

NZ-16
0.2 ± 0.0NZ-12
6.4 ± 0.5NZ-16
9.7 ± 0.8

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Table 2. Biodistribution of 225 Ac-labeled NZ-16 in H226 tumor-bearing mice.
Day 4
12.4 ± 0.8
2.0 ± 0.4 **9.9 ± 1.1 **10.5 ± 1.5
11.9 ± 1.0 **2.7 ± 0.6 *7.9 ± 1.0
3.6 ± 1.1 **

Blood
Brain
Lung
Liver
SpleenIntestine
KidneyMuscleData are indicated as the percentage of injected dose per gram (% ID/g) and as the mean ± standard deviation.
** p < 0.01, * p < 0.05 vs. 111 In-NZ-16 (Table 1).

3.3. Dosimetry
The absorbed doses were estimated on the basis of the biodistribution studies when
In-111 was replaced with Y-90 or Ac-225. Table 3 shows the estimated absorbed doses when
the radiation-weighted factor was not considered. The absorbed doses of radiolabeled NZ-16
for tumors and organs tended to be higher than those of radiolabeled NZ-12, except for in
the liver and kidney, although the difference between the two antibodies was not statisticallysignificant (Table 3). The tumor-absorbed doses of 225 Ac-labeled NZ-12 and NZ-16 were60-fold greater than those of 90 Y-labeled NZ-12 and NZ-16, respectively (Table 3).
Table 3. Estimated absorbed dose (Gy/MBq) for 90 Y- and 225 Ac-labeled NZ-12 and NZ-16 based on the biodistribution dataof 111 In-labeled antibodies, not considering a radiation weighting factor.
90 Y

Brain
Lung
Liver
SpleenIntestine
KidneyMuscleBone marrow a
Tumor

225 Ac

NZ-12

NZ-16

NZ-12

NZ-16

0.1 ± 0.0
1.7 ± 0.1
4.6 ± 0.1
3.2 ± 0.1
0.5 ± 0.0
3.1 ± 0.1
0.3 ± 0.0
1.8 ± 0.1
3.0 ± 0.2

0.2 ± 0.0
2.4 ± 0.2
3.5 ± 0.2
3.1 ± 0.1
0.7 ± 0.0
2.6 ± 0.1
0.4 ± 0.0
2.5 ± 0.1
3.8 ± 0.2

6.4 ± 0.5
84.6 ± 4.7
239.0 ± 7.3
168.7 ± 9.4
25.8 ± 1.1
156.9 ± 4.7
15.5 ± 0.9
89.4 ± 4.0
174.6 ± 158.0

9.7 ± 0.8
124.2 ± 11.4
185.5 ± 13.5
172.1 ± 36.4
37.1 ± 2.0
140.0 ± 9.5
21.1 ± 1.7
126.1 ± 6.1
236.0 ± 108.6

Data indicate the mean ± standard deviation. a The absorbed doses of bone marrow were estimated based on the blood uptake (Table 1),
considering a red-marrow-to-blood activity ratio of 0.4. There is no significant difference between NZ-12 and NZ-16.

The relative biologic effect (RBE) was determined by calculating the absorbed doses fromtreatments with 90Y- and 225Ac-labeled NZ-16 without considering the radiation-weighted
factor (Table 4). The dose absorbed by tumors treated with 3.7 kBq of 90Y-labeled NZ-16 was
5.7-fold higher than that of tumors treated with 11.1 kBq of 225Ac-labeled NZ-16 (Table 4).
With regard to safety, the absorbed doses considering radiation weighting factorsof 1 for 90 Y and 5 for 225 Ac are shown in Table 5. The absorbed dose to bone marrowfrom treatment with 3.7 MBq of 90 Y-labeled NZ-16 was higher than that from treatment
with 11.1kBq of 225 Ac-labeled NZ-16 (Table 5). The absorbed doses to tumors and organs
injected with 18.5 kBq of 225 Ac-labeled NZ-16 were higher than those of tumors injectedwith 3.7 MBq of 90 Y-labeled NZ-16 (Table 5).

Cells 2021, 10, 2503

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Table 4. Estimated absorbed doses (Gy) from the treatment dose of 90 Y- and 225 Ac-labeled NZ-16, not considering a radiation
weighting factor.

Brain
Lung
Liver
SpleenIntestine
KidneyMuscleBone marrow a
Tumor

3.7 MBq 90 Y

11.1 kBq 225 Ac

18.5 kBq 225 Ac

0.7
8.8
12.8
11.4
2.7
9.7
1.5
8.1
14.9

0.1
1.4
2.1
1.9
0.4
1.6
0.2
1.4
2.6

0.2
2.3
3.4
3.2
0.7
2.6
0.4
2.3
4.4

a The absorbed doses of bone marrow were estimated based on the blood uptake (Table 1), considering a red-marrow-to-blood activity

ratio of 0.4.

Table 5. Estimated absorbed doses (Sv) from the treatment dose of 90Y- and 225Ac-labeled NZ-16 using radiation weighting factors a.

Brain
Lung
Liver
SpleenIntestine
KidneyMuscleBone marrow b
Tumor

3.7 MBq 90 Y

11.1 kBq 225 Ac

18.5 kBq 225 Ac

0.7
8.8
12.8
11.4
2.7
9.7
1.5
8.1
14.9

0.5
6.9
10.3
9.6
2.1
7.8
1.2
7.0
13.1

0.9
11.5
17.2
15.9
3.4
13.0
2.0
11.7
21.8

a The radiation weighting factors of 1 for 90 Y and 5 for 225 Ac. b The absorbed doses of bone marrow were estimated based on the blood

uptake (Table 1), considering a red-marrow-to-blood activity ratio of 0.4.

3.4. Treatment Effects of Radiolabeled Antibodies in Nude Mice Bearing H226 Tumors
Marked antitumor effects were observed in mice treated with 90 Y- and 225 Ac-labeled
NZ-16 (p < 0.01, vs. 0 MBq, Figure 3a). Treatment with 3.7 MBq of 90 Y-NZ-16 reduced tumor
volume from day 7 to day 21 post injection, and thereafter the tumor volume gradually
increased (p < 0.01 vs. 0 MBq, Figures 3a ande S3). In the group injected with 11.1 kBq of
225 Ac-NZ-16, the tumor volume increased during the first 10 days, and thereafter decreased
until day 42 (p < 0.01 vs. 0 MBq, Figure 3a). In the group injected with 18.5 kBq of 225 AcNZ-16, tumor growth was suppressed during the first 28 days, and thereafter the tumor
volume decreased until the end of the observation period (p < 0.01 vs. 0 MBq and 3.7 MBqof 90 Y-NZ-16, Figure 3a).
Kaplan–Meier survival curves based on a tumor volume endpoint of 300 mm3 areshown in Figure 3b. Injection with 3.7 MBq of 90 Y-labeled NZ-16, 11.1 kBq 225 Ac-labeled
NZ-16, and 18.5 kBq of 225 Ac-labeled NZ-16 significantly prolonged survival comparedwith the 0-MBq groups (p < 0.01). At the end of the observation period, all mice treatedwith 11.1 kBq and 18.5 kBq of 225 Ac-NZ-16 were defined as surviving, and survival in
the 90 Y-treatment group was 60%. No statistically significant difference in survival was
detected among the three groups treated with the radiolabeled antibodies (Figure 3b).
The three radiolabeled treatments induced temporary weight loss. Body weight loss
never exceeded 20% compared with that at day 0, however, which is the criterion forhumane euthanasia (Figure S4a). No obvious damage was detected in the spleen, kidney,
liver, or bone marrow in mice treated with 90 Y- or 225 Ac-labeled NZ-16 (Figure S4b).

ells 2021, 10, x FOR PEER REVIEW

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225

3. Therapeutic
efficacy
Y- andAc-labeled antibodies
in in
H226
tumor-bearing
mice (Nmice= 5/treatmentgroup).
225Ac-labeled
Figure 3.Figure
Therapeutic
efficacyof 90ofY- andantibodies
H226
tumor-bearing(N = 5/treatmentgroup).
(a) Tumor growth curves of individual mice after injection with 90 Y-90or 225 Ac-labeled
NZ-16 (thin lines). Bold lines indicate
225
(thin
lines).
Bold
lines
indicate(a) Tumor growth curves of individual mice after injection with Y- or Ac-labeled NZ-16
the mean. (b) Kaplan–Meier survival curves based on a tumor volume endpoint of 300 mm3 . ** p < 0.01, vs. 0 MBq NZ-16.
the mean. (b) Kaplan–Meier survival curves based on a tumor volume endpoint of 300 mm3. ** p < 0.01, vs. 0 MBq NZ-16.

3.5. Histologic Analysis of H226 Tumors Treated with 90Y- and 225Ac-Labeled NZ-16

Tumors treated with 0 MBq of NZ-16 (intact NZ-16 only) were composed of s
nests of epithelial cells and some mitotic cells (Figure 4, upper panels). Sections of H
tumors treated with 3.7 MBq of 90Y-labeled NZ-16 showed a few small necrotic foci on

Cells 2021, 10, 2503

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3.5. Histologic Analysis of H226 Tumors Treated with 90Y- and 225Ac-Labeled NZ-16
Tumors treated with 0 MBq of NZ-16 (intact NZ-16 only) were composed of solid
nests of epithelial cells and some mitotic cells (Figure 4, upper panels). Sections of H226
tumors treated with 3.7 MBq of 90 Y-labeled NZ-16 showed a few small necrotic foci on
Cells 2021, 10, x FOR PEER REVIEW
11 of 16
day 1, and expansion of the necrotic area was observed on days 3 and 7 (Figure 4, middle
225
panels). In the tumors treated with 18.5 kBq of Ac-NZ-16 on day 1, more necrotic fociwere observed compared with the tumors treated with 90 Y-NZ-16, and extensive necrosis
andpostinjection
injection(Figure(Figure4,4,lower
lowerpanels).
panandlymphocyte
lymphocyteinfiltration
infiltrationwere
were observed
observed on day 33 post
els).
On
day
7,
the
tumor
cells
decreased
andpartialreplacementofnecrotic
tumor
cells
On day 7, the tumor cells decreased and partial replacement of necrotic tumor cells
by
225
225
byfibrous
fibroustissuewasobserved
Ac-NZ-16-treatedtumors
tumors(Figure(Figure4,4,lower
lowerpanels).
panels).
tissuewasobservedinin Ac-NZ-16-treatedFigureFigure4.4.Hematoxylin
Hematoxylinandandeosin-stained
eosin-stainedsections
sectionsofofH226
H226tumors
tumorstreatedtreatedwith
with0 0MBq
MBq(intact(intactNZ-16
NZ-16
9090
225
225
only),
3.7
MBqof
Y-labeled
NZ-16,
and
18.5
kBq
of
Ac-labeled
NZ-16
at
days
1,
3,
only), 3.7 MBq of Y-labeled NZ-16, and 18.5 kBq of Ac-labeled NZ-16 at days 1, 3,andand7 7after
after
injection.
injection.Arrowheads
Arrowheadsindicate
indicatenecrosis.
necrosis.N,N,necrosis;
necrosis;T,T,tumor;
tumor;F,F,fibrous
fibroustissue;
tissue;bars,
bars,50
50μm.
µm.
225
Treatment
Ac-labeled
Treatmentwith
with3.7
3.7MBqMBqofof9090Y-labeled
Y-labeledNZ-16
NZ-16andand18.5
18.5kBqkBqof
of 225Ac-labeledNZ-16
NZ-16
significantly
reducedtheproliferationof(Ki-67-positive)
tumor
cells
comparedwithtusignificantly reduced the proliferation of (Ki-67-positive) tumor cells compared withtumorsmors
treated
0 MBq
NZ-16(intact
NZ-16)
on days
1–7 post
injection(p < 0.01,
Figuretreatedwithwith
0 MBq
NZ-16
(intact
NZ-16)
on days
1–7 post
injection(p < 0.01,
Figure5a,b).
90Y225Ac-NZ-16,
225and5a,b).
A apoptotic
few apoptotic
cells wereobserved
in tumors
A few
cells wereobserved
in tumors
treatedtreatedwith 90with
Y- andAc-NZ-16,
but nobut
no apoptosis
was observed
the 0-MBq(Figureapoptosis
was observedin thein
0-MBqgroupgroup(FigureS5). S5).

CellsCells2021,
2021,10,102503, x FOR PEER REVIEW

11
12 ofof1516

Figure5.5. Tumor
Tumor cell
cell proliferation analysis
H226
tuFigure
analysis using
using Ki-67
Ki-67immunostaining.
immunostaining.(a)(a)Ki-67-stained
Ki-67-stained
H226
90Y-labeled
90
NZmors
at
days
1,
3,
and
7
after
injection
with
0
MBq
(intact
NZ-16only),
3.7
MBqof
tumors at days 1, 3, and 7 after injection with 0 MBq (intact NZ-16 only), 3.7 MBq of Y-labeled
225Ac-labeled
225 Ac-labeled
NZ-16.
Bar, 50Bar,
μm50
(b)µm
Quantificationof proliferating(Ki-67 positive)
16, andand
18.518.5
MBqMBqNZ-16,
NZ-16.
(b) Quantificationof proliferating(Ki-67cells(N=
3/group).
Data
represent
the
mean
and
standard
deviation.
**
p
<
0.01.
is
a
figure.
positive) cells (N = 3/group). Data represent the mean and standard deviation. ** p < 0.01. is aSchemes
figure.
follow the same formatting.
Schemes follow the same formatting.

Discussion
4.4.Discussion
Ananti-PDPN
anti-PDPNantibody,
antibody,NZ-16,
NZ-16,waswasnewly
newlydeveloped
developedfrom
fromthetheparental
parentalantibody
antibodyAn
NZ-1. The
The constant region
differs
fromthat
in NZ-12,
which
was
NZ-1.
region ofofthe
theNZ-16
NZ-16heavy
heavychain
chain
differs
fromthat
in NZ-12,
which
evaluated
as a asradioimmunotherapeutic
agentagent
in a previous
studystudy[6]. NZ-16
has a higher
wasevaluateda radioimmunotherapeutic
in a previous[6]. NZ-16
has aaffinityand showed
highertumor
uptakeinina aPDPN-expressing
H226 mesothelioma
mesothelioma
higheraffinityand showed
highertumor
uptakePDPN-expressing H226
mouse
mousemodel
modelthan
thanNZ-12.
NZ-12.Therefore,
Therefore,RIT
RITwith
withNZ-16
NZ-16waswasexpected
expectedtotobe
bemoremoreeffective
effective
andY-Y-andandNZ-16
NZ-16waswasselected
selectedfor
forfurther
furtherevaluation.
evaluation.As
Asexpected,
expected,9090
and225225Ac-labeled
Ac-labeledNZ-16
NZ-16
showedshowedsignificant
significantantitumor
antitumoreffects
effectsinintumor-bearing
tumor-bearingmice,
mice,compared
comparedwith
with9090Y-labeled
Y-labeled
NZ-12
NZ-12[6].
[6].The
Thesignificantly
significantlyhigher
highereffectivenesseffectivenessofof225225Ac-labeled
Ac-labeledNZ-16
NZ-16compared
comparedwith
with9090Y-labeled NZ-16 (p < 0.01) suggests that α-RIT with NZ-16 is a promising therapy for
Y-labeled NZ-16 (p < 0.01) suggests that α-RIT with NZ-16 is a promising therapy formalignantOurfindings
areareencouraging
andandwarrant
furtherstudies
towardmalignantmesothelioma.
mesothelioma.
Ourfindings
encouraging
warrant
furtherstudies
toclinicalapplications.
ward clinicalapplications.
PDPN
PDPNisishighly
highlyexpressed
expressedin
inmanymanytypes
typesofofcancer,
cancer,suchsuchasasbrainbraintumors
tumors[11],[11],squasquamous
mouscellcellcarcinoma
carcinoma[25],[25],soft
softtissue
tissuetumors
tumors[26],[26],andandbladderbladdercancer
cancer[27].
[27].AApreliminary
preliminary
study
radiolabeled NZ-16
NZ-16highly
highlyboundbound
PDPN-expressing
LN-319gliomastudyshowed
showed radiolabeled
to toPDPN-expressing
LN-319gliomacells
cells and showed high tumor uptake. RIT with 225 Ac-labeled NZ-16 is applicable for theCells 2021, 10, 2503

12 of 15

treatment of such cancers, although further preclinical studies in these cancer models
are required.
Our pathologic analysis showed that 225 Ac-labeled NZ-16 induced a larger extent of
necrotic change in tumor tissues compared with 90 Y-labeled NZ-16, although the extent
of apoptotic cell death and reduction in proliferating cells were similar. α-Emitters canprovide a large amount of energy and induce irreparable damage to cells, resulting in morecell death, manifested as apoptosis or necrosis, compared with β-emitters [13]. Our findings
revealed that 225 Ac-labeled NZ-16 more frequently induced necrosis than apoptosis. This
result is consistent with previous reports of RIT with α-emitters that apoptotic change
is not often observed in solid tumors [28]. Further studies with various cancer types areneeded to evaluate whether α-particle-induced cell death is depending on the cancer type.
In the present study, the estimated tumor absorbed doses following treatment with
3.7 MBq of 90 Y-labeled NZ-16 and 11.1 kBq of 225 Ac-labeled NZ-16 were 14.9 and 2.6 Gy,
respectively (Table 4). Our finding indicates that the RBE of 225 Ac-labeled NZ-16 is 5.7,
which is similar to the recommended amount for α-emitters by the Medical Internal
Radiation Dose Committee [24]. The treatment with 225 Ac-labeled NZ-16 was markedly
more effective than that with 90 Y-labeled NZ-16, suggesting that the real RBE is greaterthan 5.7. To estimate the RBE by another method, we referred to the results of X-ray
treatment against H226 tumors in a previous report [6]. The efficacy of 11.1 kBq of 225 Aclabeled NZ-16 was almost equivalent to that of 50 Gy of X-ray radiation, and the RBE wascalculated to be 19.2 for 225 Ac relative to X-rays. The efficacy of 3.7 MBq of 90 Y-labeled
NZ-16 was equivalent to 25 Gy of X-ray irradiation, and the RBE was calculated to be 1.7
for 90 Y relative to X-rays. Taken together, the RBE for 225 Ac to 90 Y was calculated to be 11.3.
The RBE values for the cell-killing effects of α-emitters are reported to be three to five on
the basis of in vivo experiments [24]. Our previous study with α-emitting 211 At-MABG
also showed that the RBE was approximately three [28]. Compared with other α-emitting
compounds, the calculated RBE for 225 Ac-labeled NZ-16 is quite high. The reason for thisdiscrepancy is not clear at present, but the increased RBE might depend on the tumor type.
Further studies are necessary to estimate a more accurate RBE for 225 Ac-labeled NZ-16 to
predict the therapeutic efficacy and safety in patients.
The clinical safety of RIT with NZ-16 must be evaluated. Bone marrow is generally
the dose-limiting tissue in RIT. The limiting absorbed doses in the bone marrow are 6–9 Sv
in rodents and 4.5 Sv in humans [23,29]. We provided the estimated absorbed doses of
90 Y- and 225 Ac-labeled NZ-16, considering a radiation weighting factor of one for 90 Y andfive for 225 Ac [24] in Table 5. The dose to the bone marrow was 8.1 SvRBE1 for 3.7 MBq of
90 Y-labeled NZ-16 and 7.0 Sv
225 Ac-labeled NZ-16. These doses would
RBE5 for 11.1 kBq ofbe acceptable in rodents; treatment-related mortality and toxicity to the bone marrow
were not observed in the present study, although there was a temporary decrease in bodyweight. The doses, however, are greater than the limiting dose in humans of 4.5 Sv. The
injected dose to patients should thus be decreased. The biodistribution of drugs, including
antibodies, however, is generally not identical between humans and animals. Clinicaldosimetry studies are needed to determine a safe injected radioactive dose for humans.
In mesothelioma patients, most mesothelioma cells spread into the diaphragm, chest
wall, and mediastinum; radiotherapy is therefore limited due to the high risk of injury
to the lungs and surrounding organs [30]. Radiation pneumonitis is the most common
toxicity in patients treated with radiation for cancers in the thorax [31]. The mean dose to
the lungs for a 20% risk of radiation pneumonitis is 20 Sv [31]. Our dosimetry showed thatthe absorbed doses of 90 Y- and 225 Ac-labeled NZ-16 in the lungs were lower than 20 Sv.
The spleen, liver, and kidneys would also be tolerable because these doses were lower than
the tolerated doses in humans [31]. Therefore, the risk of radiation-induced toxicity from
RIT with radiolabeled NZ-16 is expected to be low.
As a SPECT imaging agent for dosimetry and treatment monitoring, 111 In-labeled
antibodies are a suitable surrogate for 225 Ac-labeled antibodies [32]. In the present study,
uptake of 225 Ac-labeled NZ-16 in most organs tended to be higher than that of 111 In-labeledCells 2021, 10, 2503

13 of 15

NZ-16, and the RBE of 225 Ac-labeled NZ-16 on tumor growth suppression was greater thanfive. Taken together, the dosimetry of 225 Ac-labeled NZ-16 could be underestimated by
111 In-labeled surrogate imaging. Although the present study revealed no severe damage inmice treated with 225 Ac-labeled NZ-16, further studies, including dosimetric assessmentsof toxicity in the therapeutic use of α-RIT for solid tumors, are needed to determine theappropriate dose of 225 Ac-labeled NZ-16 for first-in-human studies with high confidence.
The present study has several limitations. First, 225 Ac-labeled NZ-16 did not achievecomplete remission. Further strategies are needed to improve the antitumor effect. Fractionated therapy with 225 Ac-labeled NZ-16 is promising. Two fractions of 225 Ac-lintuzumab
produced complete remission in patients with hematologic malignancies [33]. The therapeutic effects of RIT might be enhanced by combining them with chemotherapeutic agents.
Pemetrexed, as a first-line chemotherapy for mesothelioma, has a radiosensitizing effectand might therefore be particularly effective with 225 Ac-labeled NZ-16 [34]. Second, ourdosimetry in mice cannot be directly applied to determine the appropriate dose for malignant mesothelioma patients. Clinical imaging studies with 111 In-labeled NZ-16 are required
to guarantee the safety of therapeutic treatments. These studies would promote the possible
clinical application of 225 Ac-labeled NZ-16 for malignant mesothelioma. Third, the present
study employed only one PDPN-expressing mesothelioma cell line. Unfortunately, anothermesothelioma cell line with high PDPN expression is not available. There is a need togenetically develop PDPN-expressing mesothelioma cell lines and evaluate the efficacy ofradiolabeled NZ-16 in the future.
5. Conclusions
The novel anti-PDPN antibody NZ-16 has a higher binding affinity and higher tumor
uptake compared with the previous antibody NZ-12. Treatment with 225 Ac-labeled NZ16 showed a potent antitumor effect without obvious adverse effects in a mesothelioma
mouse model. RIT with 225 Ac-labeled NZ-16 is a promising potential therapeutic option
for malignant mesothelioma patients.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/cells10102503/s1, Supplementary Methods, Figure S1: Immunofluorescence staining of H226
cells using anti-PDPN antibody NZ-12 and NZ-16 (Green), Figure S2: Cell binding assay of 111 Inand 225 Ac- labeled NZ-16 with H226 cells (1.3 × 106 cells), Figure S3: Photos of mice treated with
0 MBq, 3.7 MBq of 90 Y-labeled NZ-16, and 225 Ac-labeled NZ-16 at Day 56, Figure S4: Side effects after
treatments, Figure S5: TUNEL-stained H226 tumors at days 1, 3, and 7 after injection with 0 MBq(intact NZ-16 only), 3.7 MBq of 90 Y-labeled NZ-16, and 18.5 MBq of 225 Ac-labeled NZ-16.
Author Contributions: Conceptualization, H.S. (Hitomi Sudo) and A.B.T.; methodology, H.S.
(Hitomi Sudo), K.N., H.S. (Hisashi Suzuki) and A.B.T.; validation, H.S. (Hitomi Sudo) and A.B.T.; formal analysis, H.S. (Hitomi Sudo); investigation, H.S. (Hitomi Sudo), A.S. and A.B.T.; resources,
Y.K. and M.K.K.; data curation, H.S. (Hitomi Sudo); writing—original draft preparation, H.S.
(Hitomi Sudo); writing—review and editing, A.B.T.; visualization, H.S. (Hitomi Sudo); supervision, T.H.; project administration, T.H.; funding acquisition, H.S. (Hitomi Sudo), A.S., A.B.T., M.K.K.
and Y.K. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported in part by KAKENHI 18K07778 (H.S.), 21K07688 (H.S.), 21K07230(A.S.), 18H02774 (A.B.T.), 21K07168 (M.K.K.), and 19K07705 (Y.K.), and in part by the Japan Agency
for Medical Research and Development (AMED) under Grant Numbers JP21am0401013 (Y.K.) andJP21am0101078 (Y.K.), and 21ym0126002 (T.H.).
Institutional Review Board Statement: The animal experimental protocol was approved by the
Animal Care and Use Committee of the National Institutes for Quantum and Radiological Scienceand Technology (13-1022, 26 May 2016), and all animal experiments were conducted according to theinstitutional guidelines regarding animal care and handling.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from thecorresponding author.

Cells 2021, 10, 2503

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Acknowledgments: We thank Yuriko Ogawa, Naoko Kuroda, and Akihito Abe for technical assistance and the staff in the Laboratory Animal Sciences section for animal management.
Conflicts of Interest: The authors declare no conflict of interest.

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