There was a trend that higher expression of CAR resulted in higher ligand-independent activation and

lower CD19-specific activity. Especially, the CD19-CAR T cells with CD8 hinge domain were highly

activated with CD19-independent manner. The excessive non-specific activation lowered the cytokine

secretion and the cytotoxic activities against CD19-expressing tumor cells.

Because of the nature of CAR construct by artificial synthesis, the exact mechanism of ligand-

independent activation by CD8 hinge domain is still unclear. However, it is desired to select the optimal

design of CAR constructs which shows high antigen-specific activities with low non-specific activities

for the safe and effective CAR-gene modified T cell therapy. A number of ongoing and planned clinical

trials in the world will navigate the future direction toward the best CD19-CAR design.

In order to determine the optimal CAR for clinical use, we compared 4 CARs that contained scFv combinations of VL-VH or VH-

VL and the linker sequences of variable region.

A; Vector constructs used in this study.

B; Antigen-non-specific activation by measuring the CD25 and CD69 expression level of CAR-transduced T cells.

C; CD19-antigen specific activation of CAR-transduced T cells by Intracellular cytokine secretion analysis.

D; Immunophenotype analysis of CAR-transduced T cells.

NGMC, non gene modified cells; CM, central memory; EM, effector memory, TdEM, terminally differentiated effector memory.

Highly activated CAR-T cells showed lower antigen specific reactivity of intracellular

cytokine secretion activity. Finally we chose the order of VL-VH (LH1) flanked by CD28

extracellular domain and CD28 co-stimulatory molecules as our CAR design.

NYESO1-G50-TCR_β chain 2A NYESO1-G50-TCR_α chainNY-ESO1-TCR

LH1-28zL

SVL ECD CD3ζ-ICDVH TM ICD

Lin

ker

LH2-28zL

SVL ECD CD3ζ-ICDVH TM ICD

Lin

ker

HL1-28zL

SVL ECD CD3ζ-ICDVH TM ICD

Lin

ker

HL2-28z ECD CD3ζ-ICDTM ICDL

SVLVH

Lin

ker

L

SVL CD3ζ-ICDVH TM ICD

hCD8α-

hingeFMC63_8a

Lin

ker

Anti CD19 scFv; FMC63 CD28

0

2

4

6

8

10

01

%

copy

CD69陽性率(CD8+)

LH1

LH2

HL1

HL2

FMC63_8a

NYESO1-TCR

NGMC

0

10

20

30

40

50

60

70

80

0 1 2 3 4

Exp

ressio

n %

Copies/cell

CD25

0

5

10

15

20

25

0 1 2 3 4

Exp

ressio

n %

Copies/cell

CD69

Activation levels were as follows

“CD8a-hinge（control）>>HL2 >HL1>>LH1=LH2”

LH LH

CAR-B construct represented higher CD25 activation

without ligand mediated stimulation. Consequently, higher

CD25 activation resulted in the reduction of the naive T-

cell populations.

Ligand independent non-specific activation were analyzed among TCR- and CAR-

transduced T cells.

A; Vector construct and experimental procedure used in this study.

B; Flow cytometric analysis of CD25 expression level in TCR- or CAR-transduced

T cells.

C; Relative CD25 expression level of TCR- or CAR-transduced T cells compared

to the non gene modified T cells.

D; Immuno phenotype analysis of TCR- or CAR-transduced T cells.

NGMC, non gene modified cells; CM, central memory; EM, effector memory, TdEM,

terminally differentiated effector memory.

Adoptive immunotherapy using the Chimeric

Antigen Receptor (CAR) gene-modified T cells is a

promising strategy to treat patients with malignancy

and autoimmune diseases. The latest results of

CD19 CAR-T immunotherapy clinical trials have

demonstrated impressive potential in a range of B-

lymphoid malignancies. In spite of the recent great

success, serious adverse events occurred after

infusion of CAR T cells including “on-target off-

organ” activation, and cytokine release syndrome

has been observed in a number of CAR T-cell

therapies as a result of excessive T-cell activation.

To improve the efficacy and safety of CAR- T cells,

selection of the target tumor-associated antigen,

that are expressed only on tumor cells, and the

suitable CAR constructs showing the optimal T cell

activities are essential, thereby minimizing the risk

of side effects.

In this study, aiming to select the optimal design

of CD19 CAR for effective and safe immunotherapy,

using anti-CD19 antibody clone FMC63, we

performed detail analysis of non-specific activation

of CD19 CAR-T cells caused by the design of scFv

(e.g.the leader sequences, the order of VH and VL,

the spacer sequences between VH and VL, and the

extracellular-spacer domains).

Background

Conclusions/Discussions

[ Disclosure ]

Sachiko Okamoto: Takara Bio Inc., Employment, Yasunori Amaishi: Takara Bio Inc., Employment, Zheng Pei: Takara Bio Inc., Employment, Hideto Chono: Takara Bio Inc., Employment, Junichi Mineno: Takara Bio Inc., Membership on Board of Directors.

Evaluation of hinge

Okamoto et al., poster presentation at the ASGCT 18th Annual Meeting, May 13, 2015, New Orleans, Louisiana, USA.

Evaluation of scFv; FMC63 VH/VL and linker sequences

0%

20%

40%

60%

80%

100%

NG

MC

TC

R

CA

R-A

CA

R-B

NG

MC

TC

R

CA

R-A

CA

R-B

TC1800 TC3300

Day11

Donor A Donor B

Immunophenotype

0%

20%

40%

60%

80%

100%

NG

MC

TC

R

CA

R-A

CA

R-B

NG

MC

TC

R

CA

R-A

CA

R-B

TC1800 TC3300

Day18

Donor A Donor B

CD45RA+ CCR7+ Naive CD45RA- CCR7+ CM CD45RA- CCR7- EM CD45RA- CCR7+ TdEM

D

Fig. 3. Analyses of ligand independent non-specific activation of TCR or CAR engineered T cells.

0

1

2

3

4

5

6

7

8

0 0.5 1 1.5 2

Donor A R

ela

tiv

e C

D2

5 e

xp

res

sio

n le

ve

l

NGMC CAR-B CAR-A TCR

proviral copy number (copies/cell)

Relative CD25 expression level

compared to non-gene modified cells

0

2

4

6

8

10

12

14

16

18

0 0.5 1 1.5 2 2.5

Donor B

proviral copy number (copies/cell)

C

L

S CAR-BhIgG-CL

SpacerCD3ζ-ICD

CD28

TM

L

S CAR-AhIgG-CL

Spacer CD3ζ-ICDCD28

TM

L

S NYESO1-G50-TCR_β chain 2AL

S NYESO1-G50-TCR_α chain

Day 0

RN/OKT3

IL-2

Transduction

Day4 Day5 Day10 Day11

Proviral copy number

analysis

CAR

staining

【PBMC transduction schedule】

【CAR and TCR Retrovirus Vector】

CAR-A

CAR-B

TCR

CAR stain

Donor

A

Donor

B

CAR-ANGMC TCR NGMC CAR-B

NY-ESO1 tetramer stain

CAR or TCR

CD

25

in

CD

8+

ce

lls

A B

Non-specific activation of CAR-T cells

Detail analysis of non-specific activation of CD19 CAR-T cell caused by CD19 CAR design

for effective and safe CAR-T cell immunotherapy

Sachiko Okamoto, Yasunori Amaishi, Zheng Pei, Hideto Chono and Junichi Mineno CDM Center, Takara Bio Inc., Otsu, Shiga, Japan

CD28

ECD CD3ζ-ICDanti-CD19-scFv TM ICDFMC63_28

CD3ζ-ICDTM ICDFMC63_8aCD8ahingeanti-CD19-scFv

CD3ζ-ICDTM ICDFMC63_CL hIgG-CLanti-CD19-scFv

activation-marker expression

CD69

Immunophenotype

0

20

40

60

80

0 1 2 3

Exp

ressio

n %

copies/cell

0

5

10

15

20

25

0 1 2 3

copies/cell

CD25

0%

20%

40%

60%

80%

100%

CD45RA/CCR7

CD45RA+ CCR7+ Naive CD45RA- CCR7+ CM

CD45RA- CCR7- EM CD45RA- CCR7+ TdEM

FMC63_28 FMC63_8a

FMC63_CL NGMC

Exp

ressio

n %

0

20

40

60

80

0 1 2 3

0

20

40

60

0 1 2 3

IFNγ TNFα

Copies/cell Copies/cell

Cytotoxicity

(CD19+ Raji cells)

Intracellular cytokine secretion

(CD19+ Raji cells)

FMC63_28 FMC63_8a FMC63_CL NGMC

0

20

40

60

80

100

120

10 3 1

% L

ysis

E/T ratio

CAR construct having CD8α hinge represented higher

CD25 and CD69 activation without ligand mediated

stimulation. Consequently, highly activated CAR-T cells

with CD8α hinge showed lower antigen specific reactivity

of intracellular cytokine secretion activity and cytotoxity

activity.

Three kinds of hinge sequence were compared with the same backbone of scFv

and intracellular signal domain.

A; Vector constructs used in this study. Hinge sequence were chosen from CD28,

CD8α or human immunoglobulin G constant region of light chain (hIgG-CL).

B; Antigen-non-specific activation by measuring the CD25 and CD69 expression

level of CAR-transduced T cells compared to the non gene modified T cells (left

panel) and Immunophenotype analysis (right panel).

C; CD19-antigen specific activation of CAR-transduced T cells. Intracellular

cytokine secretion analysis (left panel) and cytotoxity assay of calsein labeled

tumor killing assay (right panel).

C

A

B

Fig. 4. Evaluation of hinge sequences flanked by scFv of clone FMC63.

IFN

γ

0

10

20

30

40

50

60

0 1 2 3 4

0

10

20

30

40

50

60

70

0 1 2 3 4

0

2

4

6

8

10

0

%

copy

CD69陽性率(CD8+)

LH1

LH2

HL1

HL2

FMC63_8a

NYESO1-TCR

NGMC

TN

Fα

Copies/cell Copies/cell

0

2000

4000

6000

8000

10000

12000

14000

16000

0 1 2 3 4

0

2000

4000

6000

8000

10000

12000

14000

0 1 2 3 4

expression % MFILH

LH

LH

LH

76.9 79.1 8072.6 78 77.8

70.276.1 78.2

61.869.6 72.5

46.2 5161.1

81.3

1.3 1.3 1.1

1.91.5 1.3

2.92.1 1.4

4.82.7 1.9

17.214.6

7.7

1.14.6 5.3 4.85.4

5 57.3

6.3 5.2

8.57 6.4

14.8 13.9 10.3

4.717.2 14.3 14.1

20 15.5 15.9 19.7 15.5 15.224.9 20.8 19.2 21.7 20.5 20.9

12.9

0%

20%

40%

60%

80%

100%

copy 2.14 1.25 0.74 2.50 1.31 0.77 1.45 0.92 0.46 1.88 1.30 0.62 1.72 1.29 0.66 0

virus LH1 LH2 HL1 HL2 FMC_8a NGMC

immunophenotype

0

5

10

0 1

%

copy

CD69陽性率(CD8+)

LH1

LH2

HL1

HL2

FMC63_8a

NYESO1-TCR

NGMC30

40

50

60

70

80

90

100

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Naiv

e %

Copies/cell

Naive rate

CD45RA+ CCR7+ Naive CD45RA- CCR7+ CM CD45RA- CCR7- EM CD45RA- CCR7+ TdEM

LH

C

A B

D

Fig. 5. Evaluation of scFv; FMC63 VH/VL order and linker sequences.

Fig. 1. Overview of CD19-CAR T gene therapy

Fig. 2. Chimeric antigen receptors

215