HLCN061: An "Off-the -Shelf" Gene Engineered Human iPSC-Derived NK Cell Product for the Treatment of Solid Tumors
Hironobu Kimura, Yuma Fukutani, Norihiro Tsuneyoshi, Kodai Saito, Makoto Hayashi, Yosuke Yamazaki, Yusuke Torisawa, Tetsuya Sakurai, Sono Watanabe, Kotoko Miyata, Yuka Sato, Kumiko Goto, Yoichi Naritomi, Tohru Ozaki, Masashi Yamada,Kenji Kurachi, Tomonori Hosoya, Yuriko Takeno, Fusako Nishigaki and Kouichi Tamura.
Kobe Research Ins�tute, HEALIOS K.K., Kobe, Japan.

5 mmGene engineered hiPSCs (clone 18H5) can be differentiated into NK cells.
(A) NK cell differentiation method. (B) Expression of NK markers and transgenes on clone 18H5
derived NK cells (HLCN061).

0.5% Agar + RPMI1640 + 5% FBS
60H

92+IL-15 -IL-15 +IL-15 -IL-15

iNK

90+IL-15 -IL-15 +IL-15 -IL-15

eNK

iNK

1.E+05eNK

1.E+04
1.E+03
1.E+02eNK

primary NK iNK
050

100

150

200

eNK

(male mice)
1.E+06eNK(female mice)

1.E+05

1.E+0401030

20

1.E+08⑦ Feature of HLCN061

1.E+07
1.E+06eNK

1.E+05eNK(female mice)

1.E+04
1.E+0305(A)Surface markers expression on A549weeks

HLCN061 (eNK) exhibited prolonged survival both in vitro and in vivo.
(A) HLCN061(eNK) proliferated and showed higher viability compared to non-engineered iPSC derived
NK (iNK) without IL-15 supplement. The cells were cultured in NK medium without IL-15 for 4 days. (B)
HLCN061(eNK) demonstrated longer persistence than primary NK or iNK cells in peripheral blood (upto 7 days), and was detected in the lung until 39 weeks after transplantation in immune deficient mice.
The eNKs were transplanted at day 0 and detected by qPCR with a human Alu sequence primer set ateach time point.

ICAM1: isotype(D)

CD155

CD112

: w/o IFN-γ(E)

IL-15 (pg/ 1x10^6 cells)

2D2
3C3
4A3
10F8
11C5
14F12
18H5
19F10
20A2
20G72000
1000

CCL19 (pg/ 1x10^6 cells)

Clone
4A3
6G8
11C5
18H5
19E1219F10

NK cell population
Low
Low
MediumHighLow
High

Yield
Low
Low
Low
High
Low
Low

Table 2. Copy number of transgenes
Copy numberTransgeneIL-1523CCL193CCR2CD16 (F176V)
1NKG2D
2DAP102Clone 18H5 was selected as a starting material of HLCN061.
Clinical grade hiPSCs were electroporated with constructs containing theexpression cassettes of CD16 (F176V), NKG2D, DAP10, CCR2B, IL-15,CCL19, and PiggyBac transposase genes, controlled under human EF1apromoter. Single cell clones were isolated from the antibiotics-resistantpopulation by limiting dilution. The isolated clones were evaluated fortransgene expression (upper panel) and NK cell differentiation capacity(Table 1). Copy number of transgenes integrated in clone 18H5 are listed
in Table 2.

CD56

2.E+00iNK+Cmab

0.E+00

0No antibody

Anti-NKG2D

No antibody

0Anti-NKG2D50

6.E+00Cmab
eNK
eNK+NmabeNK+Cmab

4.E+002.E+000.E+00

100

Nmab

150

050

100

150

Time (hours)

Time (hours)

eNK

HLCN061 (eNK) exhibited superior anti-tumor activity compared to iNK in IFN-� secretion (A)and ADCC (B).
(A) HLCN061(eNK) demonstrated NKG2D dependent anti-tumor activity. A549 cells were co-culturedwith either iNK or eNK in the presence or absence of anti-NKG2D antibodies. The concentration ofIFN-� in the medium was measured by ELISA. The data are presented as mean ± standard deviation(n=3). (B and C) A549-GFP spheroids were co-cultured with iNK or eNK in the presence or absence ofanti-EGFR antibodies (Cmab: Cetuximab, Nmab: Necitumumab). Green fluorescence intensity wasmeasured with Incucyte. The data are presented as mean ± standard deviation (n=3).

⑤ Migration

Green : A549
Red : eNK

Green+/Red+

7.E+05
6.E+05iNK

5.E+05iNK+RS504393
eNK
eNK+RS504393

4.E+05
3.E+05
2.E+05
1.E+050.E+000510152025

Time (hours)

HLCN061 (eNK) migrated into A549-GFP spheroids via CCL2-CCR2B axis.
A549-GFP spheroids were co-cultured with iNK or eNK labeled with Vybrant DiD. The migration ofiNK or eNK was evaluated by measuring the overlapped area of green and red fluorescence signals
by timelapse imaging (A). To confirm the contribution of CCR2B in the migration of eNK, the CCR2B
inhibitor, RS504394, was added to the assay. The overlapped area values at each time point wereplotted (B). The data are presented as mean ± standard deviation (n=6).

2010: w/o IFN-γ
50

2.0E+06

2540

10No antibody

Anti-ICAM1(A)Manufacturing Process
3D Perfusion Bioreactor SystemiPSC Sphere
Formation

500 mL Single
Use Bioreactor

50No
Antibody

20eNK0102030

**
**
** 40

Day after tumor inoculation ［days］

HPC
Differentiation

NK cell
Differentiation

Induction

Induction(B)1.E+11NK cellExpansion

1040PBMC

2.0E+060.0E+00

50

1560

K562

3.0E+06

20

IgG

80

PBMC

4.0E+06

1.0E+06

Anti-HLA-E

No
Antibody

IgG

Anti-HLA-E

3 L Single Use
Bioreactor

Perfusion System with
Automatic Medium exchanger
Waste

Feed

PBMC+IFN-γ

batch-1
1.E+10batch-2
batch-3eNKbioreactor

1.E+09＊Illustration of Perfusion System (adopted from homepage of SATAKE

Effector dead(UL)

Target dead
(UR)

(C)

(D)

Dns = UR/LR (PBMC only)

GRANZYME B

2025
NK cell differentiation (day)3035100

80

604020Specific cytotoxicity =PERFORIN

00.10.3131030

E/T ratio

De = UR/LL (NK only)
Target live(LR)15

Non-specific death of Target cells
Effector Noise

Effector live(LL)

10MultiMix )100

PBMC+IFN-γ

5.0E+06⑩ Manufacturing

20: w/o IFN-γ: w IFN -γ

p < 0.05*30

HLCN061:1.25×10^6(n=7)
HLCN061:2.5×10^6(n=7)
HLCN061:5.0×10^6(n=7)
HLCN061:1.0×10^7(n=7)

6.0E+06

HLCN061 (eNK) exhibited dose-dependent anti-tumor activities in an orthotopic lung cancer
mouse model.
Orthotopic lung cancer mice were generated by intravenously injecting H1975-luc (A) or HCC827-luc(B) on day 0. On day 13 for H1975 or day 7 for HCC827 transplanted mice, HLCN061 wasadministered at the indicated dose (cells/mouse) three times per week for two weeks. Luciferase
activity was recorded at each time point using In Vivo Imaging System. The data are presented asmean ± standard deviation; *p < 0.05, **p < 0.01 (Steel's multiple compare test between HBSS andHLCN061 treated samples at day 34 or 40).

403020HBSS (control; n=7)

7.0E+06

Day after tumor inoculation ［days］

30

eNK

35

108.0E+06

400Primary NK: w IFN-γ⑧ Safety of HLCN061

primary NK(B)(C)HLCN061 (eNK) demonstrated a resistance to the reduction of cytotoxicity against HLA-E
expressing cells.
(A) IFN-� treatment up-regulated the expression of ICAM1 and HLA-E on A549 cells. (B) IFN-�
treatment of target cells reduced cytotoxicity of primary NK cells, but not eNK. The data arepresented as mean ± standard deviation (n=3). (C) ICAM1 expression contributed to theenhancement of eNK cytotoxicity against A549 treated with IFN-�. The data are presented as mean± standard deviation (n=3). (D) NKG2A was not expressed on eNK. (E) There was limited
contribution of HLA-E/NKG2A axis to the reduction of cytotoxicity in primary NK. The data arepresented as mean ± standard deviation (n=3). The p-values were calculated using Student's t-test.

K562

8.E+0530

primary NK0(A)Table 1. NK cell differentiation capacity

4.E+00Cmab
iNK
iNK+NmabTotal GFP fluorescenceintensity (GCU x μm2)

3000

Nmab

3.0E+06n.s.

SYTOX Green

CCR2B

4000

6.E+00

0: w IFN-γ

4045

NKG2A

8.E+00Cytotoxicity (% of lysis)

NKG2D

5000

iNK

① Clone selection
CD16

6000

Green + Red Object Area (μm2)

IL-15
Cytokine for improved
survival and persistence

7000

(C)

: w/o IFN-γ

500: w IFN-γ
eNK

Primary NK

HLA-E

4.57%

8.E+00Total GFP fluorescenceintensity (GCU x μm 2)

CCR2B
Receptor for improved
migration into solid tumors

(B)

8000

IFN-� concentration(pg/mL)

PiggyBac transposon system wasused to integrate transgenes(A)(B)

10 15 20 25 30 35 40dayshours(male mice)cytotoxicity (% of lysis)

0.594

1.E+06

1.E+07

cytotoxicity (% of lysis)

Viability (%)

196

1.E+07

4.0E+06

0.0E+00

Lung

Peripheral blood

Alu copies/100 ng gDNA

98

1.50(B)100

Alu copies/100 ng gDNA

296H

HLCN061 demonstrated recruitment of dendritic cells.
Dendritic cells (DCs) were obtained by differentiating PBMC derived CD14+ cells. The DCs werelabeled with Vybrant DiO. The labeled DC, iNK and eNK were co-cultured separately in agar (A). The
timelapse images were captured by Incucyte (B).

In vivo (NOG mice)
Alu copies/10 ng gDNA(A)

HLCN061:1.25×10^6(n=8)
HLCN061:2.5×10^6(n=8)
HLCN061:5.0×10^6(n=8)
HLCN061:1.0×10^7(n=8)

1.0E+06③ Survival and persistence
In vitro (NK medium without IL15)

Total Flux ［p/s］

NKG2A

9.0E+06

Cytotoxicity (% of lysis)

NKG2D④ In vitro cytotoxicity
NKG2D/DAP10
Receptor for enhanced
recognition and cytotoxicity

36HHBSS (n=8)

5.0E+06

Cell number

CCR2

67.9%

CCL19
Chemokine release torecruit host immune cells

DC0H

Total Flux ［p/s］

CD16

HLCN061(≥200 μm)

(B)

1.0E+07

6.0E+06

DC

Gene engineered hiPSC
derived NK cell (HLCN061)

High affinity CD16 (176F/V)
Receptor for enhanced ADCC

(A)

7.0E+06

eNK

iNK

CD56

5~6 weeks

(B)

eNK

CD56

Clinical gradecGMP MCB

2 weeks

CD56

1 weekiNKcytotoxicity (% of lysis)

Gene engineered
single hiPSC clone

NK differentiation & expansion

HPC differentiation

CD56

iPSC
sphereCD56(B)

HLCN061
Clinical grade
hiPSC

(A)

⑨ In vivo cytotoxicity

Green : DC labeled with Vybrant DiO

CD56

(A)

proliferation

NK cells are part of the innate immune system andcontribute to the elimination of virus-infected cells,
stressed cells, and tumor cells. Unlike T cells, NK cells
do not require prior sensitization to target and do notcause GVHD in the allogeneic setting. Therefore, NKcells are considered as a good candidate for off-theshelf products in adoptive cell therapy. Currently,
peripheral blood or cord blood is used as the main
source of NK cells for immunotherapy. However, theseNK cells are heterogeneous, and their efficacy depends
on the donor. In contrast, iPSC derived NK cells can be
standardized for quality control. Moreover, iPSCs havethe advantage of ease of gene modification to generate
the desired cell. We developed HLCN061, which is an
NK cells derived from genetically engineered human
iPSC (hiPSC), expressing molecules that enhance antitumor activity, persistence, tumor homing, and hostimmune cell recruitment for the treatment of solidtumors.

⑥ Recruiting② NK differentiation

Background

UR-((Dns*LR)+(De*LL))
LR+UR((Dns*LR)+(De*LL))

Cell trace Violet

HLCN061 (eNK) did not respond to PBMCs from a healthy donor.
PBMCs were labeled with CellTrace Violet and treated with or without IFN-�� The PBMCs were cocultured with primary NK cells or eNK for 4 hrs. Dead cells were stained by CYTOX Green. The specificcytotoxicity was calculated using the formula indicated above. K562 cells were used as a positivecontrol. The data are presented as mean ± standard deviation (n=3).

Manufacturing of HLCN061 (eNK) using 3D perfusion bioreactor system.
(A) The manufacture process of HLCN061 using 3D perfusion bioreactor system with automatic
medium exchanger. (B) Cell numbers were monitored at each time point during the manufacture
process of three batches. This process can manufacture fifty billion cells of HLCN061 per 3 Lbioreactor on average. (C) The manufactured HLCN061 expressed major NK cell markers. (D)The manufactured HLCN061 demonstrated cytotoxicity against A549 cells. A549 cells were cocultured with eNK at indicated E/T ratio and cytotoxicity was measured by LDH assay. The data
are presented as mean ± standard deviation (n=3).

Conclusion• We established a gene engineered hiPSC clone expressing IL-15, NKG2D/DAP10, CD16 (F176V), CCR2B and CCL19.
• The gene engineered hiPSC derived NK cells (HLCN061) demonstrated enhanced persistence, cytotoxicity, migration, and dendritic cell recruitmentcompared to non-engineered cells.
• The HLCN061 demonstrated resistance to dysfunction of NK cell caused by IFN-� treated cancer cells.
• The HLCN061 did not show any response to normal PBMCs.
• The HLCN061 exhibited anti-tumor activities in the orthotopic lung cancer model mice.
• We developed a manufacture process using 3D perfusion bioreactor system that can manufacture 100 billion cells with two 3 L bioreactors.