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A novel iPSC-derived CAR-invariant natural killer T (iNKT) cell therapy platform forhematologic malignancies and solid tumors
Akane Urakami1, Tomokuni Shigerura1, Makoto Kondo1, Mari Isomi1, Yuko Kokubu1, Ken-ichi Suzuki1, 3, Haruhiko Koseki2
1. BrightPath Biotherapeutics Co., Ltd., Cell Technology Laboratories, Kawasaki, Japan, 2. Riken, Center for integrative Sciences, Yokohama, Japan,
3. Contact: suzuki_k@brightpathbio.com

10080% Cytosis

80% Cytosis

60
4060
40

SSEA-4

TRA1-60

CD19-CAR

SSEA-420048

1216HER2-CAR283236040

E/T=5%Cytosis

40Phenotype analysis of CD19-CAR and HER2-CAR iNKT cells
showed CAR-iNKT cells have similar phenotypic properties toiPSC-derived non-transduced iNKT cells

Resultsa) CD19-CAR iNKTCD45 gate00481216202428

32036323640481216

E/T=3100

10080

80

60
40

2024283236Hr

E/T=100481216202480

60
400280481216202402860
4060
4001216202428

3204812Hr

1620242832HrCD3/CD28 stimulation

NKG2DNostimulation
No stim
CD3/CD28 stimulated
CD3/CD28
stimulation

4×10 4
3×10 42×10 40PD-10048121620Unmodified
iNKT
iNKT
CD19CAR-iNKT iNKTCD19-CAR

4×10 3

2×10 3

CD19CAR-iNKT

FSC
NKG2D

PD-1

FSC

CD45

No
stimulation
No stim
CD3/CD28
stimulated
CD3/CD28
stimulation

4030
20328

32

IFN-γ concentration of iNKT cells
culture supernatants weremeasured by ELISA.
CD3/CD28 stimulation: iNKT cells
were treated with anti- CD3 andanti-CD28 antibodies for 24 hours.
Antigen stimulation: iNKT cells
were mixed with indicated target
tumor cells for 48 hours.

Unmodified
iNKTiNKT
CD19CAR-iNKT iNKTHER2-CAR

21CD4

CD3

NKG2D

CD4PD-1

FSC
NKG2DiNKTHER2-CAR iNKT0U2OS ET=1

K562 E/T=5Conclusion

FSC

CD56

CD8a
CD3CD56

CD45

CD8a

FSC

HER2-CAR iNKTVɑ24Jɑ18

Unmodified iNKT

7050K562 E/T=5480
60

NALM6 E/T=1

Antigen stimulation

90IFNʏ(pg/mL)

CD424Hr0iNKT

CD3/CD28 stimulation

CD56

CD8aVɑ24Jɑ18CD3Vɑ24Jɑ18iNKT-derived iPSC was prepared by reprogramming expanded
iNKT cells from human PBMC. Next, iPSCs were engineered toexpress CD19-CAR or HER2-CAR by targeting the adenoassociated virus integration site-1 locus using genome editing.
CAR-introduced iPSCs can differentiate to CAR-iNKT cells underfeeder cell free culture conditions.

40

6×10 3

5×10 40FSC

CAR-iNKT

Unmodified iNKTHER2-CAR iNKT

10FSC

CAR-HSPCCD45

2860

Antigen stimulation

IFNʏ(pg/mL)

IFNʏ(pg/mL)

FSCCD56CD4b) HER2-CAR iNKTDifferentiation
to HSPC

Differentiation
to HSPC
CAR-iPSC

CD324E/T=1a) CD19-CAR iNKTIFNʏ(pg/mL)

CD45

FSC

SSC

CD19-CAR iNKTSSC

iPSC

CD8aVɑ24Jɑ18FSC

SSC

FSC

SSC

CAR
transduction20

1×10 4

FSC

Reprogramming

16Interferon-γ release from CAR-iNKT cellsb) HER2-CAR iNKTCD19-CAR or HER2-CAR

1220

6×10 4

Unmodified iNKT88020208410080

40Hr

E/T=210080040Hr

E/T=50Unmodified iNKTHER2-CAR iNKT6020Hr100E/T=0.51002020K562(HER2-)

28

E/T=17×10 4Strategy for development of CAR-iNKT from iNKT
derived-iPSCsiNKTNKT gateU-2OS(HER2+)

2440%Cytosis

CAR positive cells were measured by flow cytometry using target antigen (rhCD19 or
rhHER2) followed by detection antibody treatment. These data were derived fromindependent experiment.

2060%Cytosis

TargetHER2-CAR

16Hr% Cytosis

CD31220b) HER2-CAR iNKTCountVɑ24Jɑ18CD45

CD19-CAR

HER2-CAR

CD45% Cytosis

CD3iNKTFSCCountVɑ24Jɑ18iNKTCD43CD19-CAR

CD4588020CD4341006000Hr80%Cytosis

K562(CD19-)Count

CD3124Hr100HSPC20% Cytosis

CountCountNALM6(CD19+)

Cytotoxic activities of iNKT cells
against tumor cells were
measured by real-time cell
Unmodified iNKT analyzer xCELLigence.
CD19-CAR iNKT In case of using non-adherent
cell line (NALM6 or K562) as atarget, xCELLigence IMT Assay
Kit (anti-CD29 or anti-CD9) were
Unmodified iNKT used to tethering the cells to theCD19-CAR iNKT wells of an electronic microtiterplate.

E/T=0.2

E/T=1% Cytosisx

TRA1-60

Gene targetingCD1d is highly expressedon Monocyte and MoDCTarget

0HSPC

Invariant NKT (iNKT) cells

Class I peptide/HLA
complex is presented
on cDC1a) CD19-CAR iNKT

20CD45• iNKT cells are a rare subset of innate lymphocytes thatbridge innate and adaptive immune response.
• HLA-independent TCR: No risk of GvHD.
• Providing clinical durability through indirect anti-tumor
effect by activating host endogenous T cells, dendritic
cells and NK cells, and reprograming pro-tumor myeloid
cells in the tumor microenvironment.
• Direct cytotoxic effect via NK receptors and/or
endogenous TCRs

iPSC

CD19-CAR iNKT and HER2-CAR iNKT cells showed target-specificanti-tumor effects in vitro
100CountCountCountiPSC

HER2CAR-iNKT

CD34Advantages of iNKT over NK / αβT / γδT cells

CD19-CAR iNKTCD31• Use of iPSC derived from iNKT cells is an ideal strategy
to realize clinical scale production of functional iNKTcells from such a rare population.
• A Phase 1 study of the iPSC derived non-transduced(unmodified) iNKT cells is currently ongoing in patientswith head and neck squamous cell carcinoma.

CountFollowing the success of autologous chimeric antigen
receptor (CAR)-T cells in hematologic malignancies,
allogeneic CAR-transduced cells have been developedwith various immune cells including induced pluripotent
stem cell (iPSC)-derived NK cells and T cells. We havedeveloped a novel platform of first-in-class iPSC-derived
CAR-invariant natural killer T (iNKT) cells. To demonstratethat CAR-transduced iPSC-derived iNKT cells provide anovel platform for effective cancer immunotherapy, thekilling activities of CD19-CAR or HER2-CAR-transduced
iPSC-derived iNKT cells were investigated in this first setof studies.

Production of CD19-CAR and HER2-CAR
iNKT cells
differentiated from iPSCs in feeder cell-free culture system
without losing CAR expression

CD34

iPSC derived iNKT cells

FSC

Introduction

Copies of this e-Poster obtained through QR codes arefor personal use only and may not be reproduced
without written permission of the authors

PD-1Unmodified iPSCs and CAR transduced iPSCs were differentiated into iNKT cells by thesame differentiation protocol. a) and b) data were derived from independent experiment.

• This study showed the first successful delivery of a CAR construct
into iPS cells that differentiate precisely into iNKT cells with enhanced
cytotoxicity.
• iPSC-derived CAR-iNKT cells are demonstrated to become a novel
allogeneic cell therapy platform.
Disclosure: Urakami A.: Employee of BrightPath Biotherapeutics