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 regions of variable regions.
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; Immuno phenotype 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.
Anti CD19 scFv; FMC63 CD28
Takara CD19-CAR Design (MS3-LH1-28z)
ψ L
SVL ECD CD3ζ-ICDVH TM ICD
Lin
ker
MSCV LTRLTR
hEF1α 5’ UTR
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).
PBMCs from healthy donors were stimulated with anti-CD3 monoclonal antibody
(OKT3) together with recombinant fibronectin fragment (RetroNectin®) and transduced
with CAR-retroviral vectors and further expanded. The resultant CAR-T cells were
assessed for their non-specific activation via expression analyses of activation markers
CD25 and CD69. CD19 ligand specific activation was analyzed by intracellular
cytokine secretion such as IFN-γ and TNF-α in the presense of CD19 positive Raji
cells.
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
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
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 4E
xp
ressio
n %
Copies/cell
CD69
Activation levels were as follows
“CD8a-hinge(control)>>HL2 >HL1>>LH1=LH2”
LH LH
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.
Background
Evaluation of hinge Evaluation of scFv; FMC63 VH/VL and linker
sequences
Methods
Best CAR design based on the analyses of ligand-
independent activation of CD19-CAR T cells
Hideto Chono, Yasunori Amaishi, Zheng Pei, Sachiko Okamoto 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. CAR
construct having CD28 extracellular domain showed the
lowest non-specific activation of CAR-T cells.
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.
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
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
PO-51
Fig. 3. Outline of experimental procedures
[ Disclosure ]
Hideto Chono: Takara Bio Inc., Employment, Yasunori Amaishi: Takara Bio Inc., Employment,
Zheng Pei: Takara Bio Inc., Employment, Sachiko Okamoto: Takara Bio Inc., Employment, Junichi Mineno: Takara Bio Inc., Membership on
Board of Directors.
Chono et al., poster presentation at the JSGT Annual Meeting, July 24, 2015, Osaka, Japan
Conclusions/Discussions
C
A