Clinical grade genetically engineered hypoimmunogenic
human induced pluripotent stem cell line
Norihiro Tsuneyoshi, Mitsuhiro Iwasaki, Hidetaka Murai, Kodai Saitoh, Sono Watanabe, Yudai Hasegawa, Yuriko Takeno,
Kumiko Goto, Yuka Igarashi, Fusako Nishigaki, Tomonori Hosoya, Hironobu Kimura and Kouichi Tamura(Kobe Research Institute, HEALIOS K.K.)

Abstract:

Gene Editing Procedure for Healios UDC

We have generated universal donor cells (UDC), an iPSC clone inwhich HLA-A, HLA-B, HLA-C and RFXANK have been knockedout, while HLA-G, PD-L1, PD-L2 and iCasp9 have been ectopicallyexpressed. After single-cell cloning, an iPSC clone having biallelic

HEALIOS
Universal Donor Cells

HLA Class I/II KO(intermediate)

Clinical grade MCB

frame shift mutation at the 4 gene loci as well as expressing all

PD-L2

PD-L1

the 4 factors was selected for further application. RAIS (Rapid」

Amplification of Integration Sites) method unveiled that donoriCasp9DNAs have been integrated in the genome at total 32 locations.
T cell response against the UDC-derived cells was diminished.

Knock-out
HLA class Ia
HLA class II

Furthermore, the UDC showed no vulnerability against NK cellTransgene
HLA-G
PD-L1
PD-L2
iCaspase9

cytotoxicity. Master Cell Bank (MCB) of the UDC iPSC clone

HLA-G

showed negative in mycoplasma, bacterial and endotoxin tests.

• Off-the-shelf, scalable and cost-efficientG-banding karyological test with the MCB showed normal• Address broadest population with single producthuman karyotype. No deleterious mutation that could increase• Enhanced level and duration of efficacy

tumorigenicity risk was found in the MCB compared to parental
iPSC by Cancer Panel Amplicon-seq data. Our UDC could be used

Established clinical grade universal donor cell line in 2020, and established MCB in 2021

15Knock-out HLA class Ia and class II

9Characteristics Test for UDC

Category

NK cell cytotoxicity assay, and Macrophage phagocytosis

Test

Escape from immune cells T cell response (in vitro)
CD8+ T cell cytotoxicity (in vitro, in vivo)
NK cell cytotoxicity (in vitro)
Macrophage phagocytosis (in vitro)
Safety SwitchResponse to Rapamycin (in vitro, in vivo)

Knockouts

Genomic DNA sequenceCell surface expression of HLA
Off-target mutation

UDC

HLA-A

HLA-B

HLA-C

HLA-DR,DQ,DP
Assay Protein

HLA expression was analyzed by FACS using iPSC-derived CD45+ cells.
Cells were treated with 50ng/mL IFN-g for 2 days before FACS analysis.

IgG control

Diminished expression of cell surface HLA molecules

2Transgenes

WGS short-reads SNV/indel and RNA-seq at iPSC
Karyotype, G-band

Passage +24
Passage +26

121210106644

THP-1 cell line was used as
HLA mismatch control ofTarget cells.

220Effector T THP-1cell only

Parental
06E

UDC
9G11

Effector T THP-1
cell only

Parental
06E

UDC
9G11

1:1
Effector : Target

HLA-mismatched CD3+ T cells were co-cultured with iPSC-derived CD45+ cells (Mitomycin C treated) for 7 days.
Responded T cells (CD8+ T cell and CD4+ T cell respectively) were measured with Click-iT Plus EdU Flow Cytometry
Assay Kit (Thermo Fisher Scientific).

CD8+ T cell cytotoxicity assay (in vitro)

Dimerization

InduceApoptosis

Ratio (live / total)

iCaspase9Frequency of SYTOX positive
dead cells in target (%)

Cell Viability (ATP assay)
Parental iPSC
UDC0.8Anti-proliferation effect0.60.4Apoptosis0.2*) Ref: M. Saito et al. Int J Hematol. 112, 300-306 (2020)

Quality Control Test for MCB

Karyotype, G-band

46,XY

20

Sterility

Sterile15

Endotoxin< 0.50 EU/mL

Mycoplasma

negative

Virus

negative

FCM of iPSC markers

Pass(SSEA-4, TRA-1-60, TRA-1-81, OCT-4)(99.8%, 99.2%, 99.6%, 98.3%, respectively)

Alkaline phosphatase staining

Pass

FCM of transgenes

Pass

Parental iPSC
UDC502.5:1

15:1

10:120:1

Effector cell：Target cell

(HLA-G: 100.0%, PD-L1: 99.5%, PD-L2: 96.5%)

To verify the function of Rapamycin inducible Caspase 9, cells were treated with Rapamycin for 24 hours since the next day ofcell seeding in 96-well plate, and then assayed using CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega).

The iPSC-derived CD45+ cells were co-cultured with Primed CD8+ T cells (primed by Parental iPSC-derived CD45+ cells)
for 3 hours. The dead cells of iPSC-derived CD45+ cells were measured by FACS analysis with SYTOX staining.

Apoptosis was induced by Rapamycin treatment

4Not vulnerable against HLA mismatch CD8+ T cells

8Function of iCasp9

Pass

Trilineage differentiation potential

Pass

Cancer Panel Amplicon-seq

Pass

12iCaspase9 Safety Switch (in vivo assay)

CD8+ T cell cytotoxicity assay (in vivo)

UDC and iPSC Collaborations

140

Supplying UDC and iPSC to several companies and academic institutions(>10 facilities) and evaluating their potential for various diseases

2.00E+07

120
100

Total Flux ［p/s］

Tumor regression ratio (%)

PositionDirection Transgene
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx

Results

1:1

0.003 0.01 0.03 0.1
0.3
Rapamycin conc. (nM)

Chromosome
Chr10
Chr12
Chr13Chr14
Chr15
Chr15
Chr15
Chr16
Chr16
Chr17
Chr18
Chr20
Chr21
Chr22
ChrX
ChrX

Quality Control Test

100.00

Position
Direction Transgene
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxx
reversexxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxxforward
xxxx
xxx,xxx,xxx
reversexxxx

11iCaspase9 Safety Switch (in vitro assay)

1.0

The iPSC-derived CD45+ cells (CellTrace Violet-labeled)were co-cultured with Macrophages (CFSE-labeled) for
2 hours. The phagocytosing ratio were measured byFACS analysis.

Total 32 integration sites were identified by RAIS*) method

Diminished T cell response to almost T cell only level7

Rapamycin

Chromosome
Chr1
Chr1
Chr3
Chr3
Chr3
Chr3
Chr4
Chr5
Chr5
Chr6
Chr6
Chr7
Chr7
Chr7
Chr9
Chr10

Parental iPSC
UDC81:1Effector : Target

1.20%

Integration sites of transgenes

80Expression of transgenes was observed for over half-year culture

5%CD4+ T cell

14PD-L2Transgenes (HLA-G, PD-L1, and PD-L2) expression was analyzed by FACS
using undifferentiated UDC. UDC were passaged once a week.

K562

10%

10

EdU positive cell ratioin T cells (%)

Passage +22

UDC

Not vulnerable against NK cells and macrophage phagocytosis

T cell response assay (in vitro)

Passage +20

HLA class I/II KO

15%

Effector : Target(Macrophage)

The iPSCs (Calcein-labeled) were co-cultured withNK cells for 2 hours. The dead cells of iPSC weremeasured by FACS analysis with SYTOX staining.

Tumorigenicity risk

Parental iPSC

K562 06E
1 :6B75 9G1110 : 1
5:1
Effector cell : Target cell
(NK cells)

Differentiation potential (in vitro, in vivo)
iPSC marker

Passage +3Mechanism

100Trilineage differentiation
potential

CD8+ T cell

315

Cell surface expression of transgenes
Integration sites

Transgenes are expressed on cell surface of UDC

PD-L12056HLA-G20%25Lysis (%)

ParentaliPSC

30

Phagocytosing ratio
in macrophages (%)

for a future starting material of regenerative therapy.

80604020Parental iPSC with Primed T cells
UDC with Vehicle
2.00E+06

UDC with Primed T cells

021 days
21 days
21 days
21 days
Vehicle i.p. RPM i.p. Vehicle i.p. RPM i.p.
injection injection injection injection
06E
Parental

9G11UDC

2.00E+05
050

100

150

Hour after iPSC-derived HPC inoculation ［hours］

iPSC
Cells were transplanted into NOG mouse to make teratoma formation (Day 1).
Rapamycin had been injected (8 mg/kg i.p.) every day for 21 days since 3 weeks later (Day 22-42).
At Day 71, the tumor mass was measured.

The iPSC-derived CD45+ cells (additionally Luciferase gene was introduced) were injected into NSG mouse, and Primed CD8+
T cells were administered at 1 hour later. Luminescence was confirmed by IVIS system for immune response evaluation.

UDC-derived tumor was eliminated by Rapamycin treatment

UDC-derived cells could escape from T cell reaction

07/2022

Exercise of Exclusive Option for aLicense Agreement with STEMAXON

09/2022

12/2022Signed License Agreement withRxCell

Signed Material Transfer Agreementwith Sumitomo Pharma