1336®
Verification of the effect of human allogeneic iPS cell-derived gene-engineered NK cells (eNK cells HLCN061) on mesothelioma
Seiji Matsumoto1), Yoshiko Fujita1), Kumiko Goto2), Yuka Sato2), Fusako Nishigaki2), Yasuyuki Higashi2), Hironobu Kimura2), Kouichi Tamura2)
1) Department of Thoracic Surgery, Hyogo Medical University, Japan
2) HEALIOS K.K., Japan

EGFR Expression (%)

Tumor volume (mm3)Background

2500

EGFR Fluorescence intensity

Malignant pleural mesothelioma is a malignant tumor
that is caused by exposure to asbestos. Because it is arare cancer, there are few drugs that can be used to treat
it. Healios is developing iPSC-derived NK cells (eNKcells) (HLCN061) engineered with NKG2D, DAP10,IL-15, CD16, CCL19, and CCR2B genes for thetreatment of refractory solid tumors. This study
evaluated the antitumor effect of eNK cells against
human malignant pleural mesothelioma cell lines.

1500

NK比 1： 0100

NK比 1： 180

NK比 1： 540

Cell viability(%)
生存率（％）(%)
Cytotoxicity
障害率（％）

NK比 1： 320110100

1000

Cetuximab(ng/ml)

NK比 1： 310024608 10 12 14 16 18 201234567820

1101001000601n=310100

1000

Cetuximab(ng/ml)

10000
n=3H226
LDH (%) ET比 0.510060604040202000.001

00.010.1

110WST-8 (%) ET比 1LDH (%) ET比 1
WST-8 (%) ET比 0.5808060604040202000.001

1000.1110100Cetuximab(ug/ml)

Conclusions

n=3H28LDH (%) ET比 0.5

LDH (%) ET比 0.5100100202060604040202000.001

0WST-8 (%) ET比 1

Cytotoxicity (%)

40WST-8 (%) ET比 0.58080

Cell viability(%)
生存率（％）

40

Cytotoxicity (%)

viability(%)
Cell生存率（％）

Cytotoxicity (%)

60

WST-8 (%) ET比 1

細胞障害率（％）

2060

LDH (%) ET比 1

WST-8 (%) ET比 0.5808000.001W ST -8 (%) E T 比 1

00.01

n=3

100

00.01

0.1

1

10Cetuximab(ug/ml)

100
n=3Fig.5 RNA-seq showed increased
expression of granulysin, IL2RB,
cathepsin W

LDH (%) ET比 0.5100100

WST-8 (%) ET比 0.580

80

細胞障害率（％）

40

細胞障害率（％）

40Cathepsin WGranzymeA

00.1

10060

IL2RB40

LDH (%) ET比 18056

NK比 1： 3
NK比 1： 5

10000

100Fig.1 Antitumor effects of eNK®
cells on human malignant pleural
mesothelioma cell lines in vitro.

49

NK比 1： 1

H2452

6042

Granulysin

80

LDH (%)
WST-8 (%)

35

20

Cetuximab(ug/ml)

H28

28

n=3

LDH (%) ET比 1

T:E ratio

Cell生存率（％）
viability(%)

viability(%)
Cell
生存率（％）

40100

T:E ratio

2110000

NK比 1： 0H2052

9 10 1114

Cytotoxicity (%)

060

viability(%)
Cell生存率（％）

1000

NK比 1： 5Cytotoxicity (%)

201020障害率（％）(%)
Cytotoxicity

201000100

NK比 1： 3

Cetuximab(ng/ml)

viability(%)
Cell
生存率（％）

40100

細胞障害率（％）

viability(%)
Cell 生存率（％）

30

7H226 %WST-8細胞障害率（％）

Cytotoxicity (%)

60

10Cetuximab(ng/ml)

NK比 1： 0

50
40020

n=380

00.1

細胞障害率（％）

2040

細胞障害率（％）

30

60

6080

4080

WST-8 (%)

100

50

100viability(%)
Cell生存率（％）

211H

WST-8 (%)

Cytotoxicity (%)

H2052

LDH (%)

04000.1

NK比 1： 1

LDH (%)

500eNK® cells 1x107cells/dose
intratumorally 3 times weekly(near tumor administration)
Intraperitoneal administration of
5 µg/dose of IL-2 for 0-4 weeks,
5 days/week in the IL-2 groupeNK ® cells with or without IL-2
treatment inhibited subcutaneoustumor growth

Day60

10000

100Results

eNK/IL2

NK比 1： 5

H226 %LDHMethods

eNK®cells

1000

NK比 1： 0

NK比 1： 1

00.1

コントロール
Untreated
eNK

H2052 %WST-860

The antitumor effect of eNK cells on humanmalignant pleural mesothelioma cell lines in vitro
was evaluated using the Viability/Cytotoxicity
Multiplex Assay Kit (WST-8/LDH).
In vivo, immunodeficient mice were subcutaneously
inoculated with human malignant pleural
mesothelioma cell lines. After subcutaneous tumor
formation, eNK cells were inoculated into or near
the tumor, the size of the subcutaneous tumor wasmeasured, and the tumor volume was comparedwith that of an untreated group. After the study
was completed, the tumors were removed and thetumors in the untreated group and the eNK celladministered group were analyzed using RNA-seq.

2000

Fig.2 Expression of EGFR on mesothelioma cell lines
All human malignant pleural mesothelioma cell lines express EGFR,
H226 has the highest expression intensity
H2052 %LDH

Fig.4 Antitumor effects of eNK®
cells on human malignant pleural
mesothelioma cell lines (H226)
in vivo

0.01

0.1

1

10

Cetuximab(ug/ml)

100
n=3

The antitumor effect of eNK cells against human malignant pleural
mesothelioma cell lines was confirmed in vitro. In a mouse subcutaneous tumor
model, the eNK cell treatment group suppressed tumor growth compared to theuntreated group (p≦0.05). RNA-seq showed increased expression of granulysin,
IL2RB, cathepsin W, etc. in subcutaneous tumors treated with eNK cells.

20000123456T:E ratio

789 10 11Fig.3 The combination of eNK ® cells and anti-EGFR antibody (cetuximab)In H226, which has the highest EGFR expression intensity, the combination of eNK ® cells andanti-EGFR antibody (cetuximab) in H226, which has the highest EGFR expression intensity.

Corresponding author: Seiji Matsumoto
smatumo@hyo-med.ac.jp

AACR Annual Meeting 2024
April, 2024 San Diego, CA, USA