#315 iPSC-derived CAR-iNKT cells targeting HER2 show prolonged tumor control and promote durable survival in atumor xenograft model.
*E-mail Contact:urakami_a@brightpathbio.com
zhang_l@brightpathbio.com

Akane Urakami1, Koichiro Shioya1, Tomokuni Shigeura1, Tomio Matsumura1, Yuji Mishima1, Momoko Okoshi2, Haruhiko Koseki2, Lilin Zhang1
1. BrightPath Biotherapeutics Co., Ltd., Cell Technology Laboratories, Kawasaki, Japan, 2. Riken, Center for Integrative Medical Sciences, Yokohama, Japan.

PBMC

ReprogrammingiNKTB

CD45+ gated

CAR-iPS-iNKTCD450013100

13100

1unmodified

310IFN-γ

10000

5000

5000

5000

0000131013100

1310HER2-CAR

Day29Perforin

20000

10000

100

unmodified

15000

3HER2-CAR

Day21

HER2-CAR

15000

1Unmodified

15000

Day35

10000

0

13100

1unmodified

HER2-CAR

310Day410

HER2-CAR

13100

unmodified

1310

HER2-CAR

Day48

CC

Immobilized rhHER-2 concentration（µg/mL）

Unmodified or HER2-CAR-iPS-iNKT cells were cultured on recombinant HER2His protein coated plate for 24, 48 or 72 hrs. Culture supernatant were assessedby LEGENDplex™ Human CD8/NK Panel.

Day55
Day62

Introduction of CAR increased killing and cytokine
production of iPS-iNKT cells in vitroA

E/T=1100

E/T=2100

HER2-CAR

40

80

60
40

60
40

20

20

20

00006121824
Hr

30

3642IL-2

600

4806121824Hr

30

364248

IL-6

7000
5000
4000
3000
2000

100

1000

000.5

125IFN-γ

3000

2000
1500
1000
500
0

E/T ratio

125125Granzyme B

80000

2500

0.50.512 18 24 30 36 42 48
Hr

TNF-α

60000

1200

50000

100040000

80030000

60020000

40010000

20000251

NSG-hIL15/hIL7KI mice were i.p. inoculated with 2M SK-OV-3 expressing luciferase cells (SK-OV-3-luc) on day 0. Lactatedringer (vehicle), 10M iPS-iNKT cells (Unmodified) or 10M HER2-CAR-iPS-iNKT cells (HER2-CAR) were i.p. administered toeach group mice on day 7, 11, 14 and 18. A; Tumor burden of SK-OV-3-luc bearing mice was acquired as bioluminescenceimaging once a week. B; Spider plot of total flux and body weight ratio based on day 6 body weight were shown eachgroups. Time points of administration are indicated by red arrows. C; Survival rate (n = 6) for SK-OV-3-luc bearing mice wasshown as Kaplan–Meier survival curves. Survival estimates were assessed by log-rank tests.
*: P<0.05, **: P<0.01.

Conclusions

25Perforin

1600
1400

160.5

70000

0.501800
1600
1400
1200
1000
800
600
400
200
0

6000

500

E/T=510080

Unmodified

60

B

HER2-CARA. Schematic strategy to generate CAR-iPS-iNKT. B. Phenotypic analysis of iPS-iNKTcells with no gene editing (Unmodified) or HER2-CAR introduced iPS-iNKT cells(HER2-CAR) by Flow cytometry.

3200000Vehicle

Day14020000

300

HER2-CAR

125000400

Unmodified

010TNF-αB

A
Day6500

HER2-CAR

unmodified

CD45+CD3+Vα24Jα18+ gated

NKG2D

1000

025000

CD56
CD3600200200Vα24Jα181500

2000

unmodified

Differentiation/Expansion

CAR-iPS

800
400

80

Gene editing

NKT-iPS

4000%Cytosis

NKT specificexpansion

6000

CAR-iPS-iNKT shows strongly prolonged survival with its target specificanti-tumor activity without body weight loss for a long-term

2000

1000

8000

IL-10

2500

1200

10000

Cytokine production (pg/mL)

A

12000

IL-4

1400%Cytosis

Generation of CAR-iPS-iNKT cells

IL-2%Cytosis

Invariant natural killer T (iNKT)
cells are a rare subset of T
lymphocytes that express aninvariant TCR which recognizes
glycolipids presented by themonomorphic MHC like moleculeCD1d. iNKT cells can directly killtumor cells through TCR, while
also indirectly exert antitumor
activities by prompting dendritic
cell maturation,priming tumor-specific CD8+T cells, and reprograming pro-tumor
myeloid cells. iNKT cells do not induce graft-versus-host disease,which makes them an ideal cell source for “off-the-shelf” cell
product. However, the rarity of iNKT cells in human blood poses achallenge in manufacturing large quantities of iNKT cells. Induced
pluripotent stem cells (iPSCs) offer a promising strategy toovercome this hurdle by their potential to generate thousands ofdoses of iNKT cells from a single manufacturing campaign. In thepresent study, taking advantage of gene editing technology, weestablished a CAR-iPS cell line and differentiated it into CAR-iPSiNKT cells with enhanced in vitro and in vivo anti-tumor activities.

Cytokine production (pg/mL)

Introduction

CAR signal enhanced production of Th1 cytokines
to promote immune response whereas decreased
immune suppressive cytokine

This study demonstrated that differentiated CAR-iPS-iNKT cells derived from CAR-iPSC
showed potent anti-tumor effects and promoted survival in a tumor xenograft model.
Results from in vitro analysis suggest that target-specific killing activity of HER2-CAR-iPSiNKT cells were derived from increased secretion of Th1 and inflammatory cytokines andNK phenotype such as NKG2D for direct killing.
These findings suggest that iPSC-derived CAR-iNKT cells would be a novel allogeneic celltherapy platform because of enhanced innate and adapted immunity.

0.5

125

Unmodified or HER2-CAR-iPS-iNKT cells were co-cultured with HER2 expressing
tumor cell line SK-OV-3. A. Cytotoxic activities were measured by real-time cellanalyzer xCELLigence. B. Cytokine secretion into culture supernatant after 48hr ofco-culture were measured by LEGENDplex™ Human CD8/NK Panel. IL-4 and IL10 were not detected.

Acknowledgements
We would like to thank Dr. Mari Isomi for contributing setting up the in vivo studies and collecting data, Dr. Masashi
Matsuda at RIKEN for supporting in vivo study environments. We are grateful to all the past and current lab members
for their work and contribution.