5ʼ LTR V ʼ ζ H L C SD SA CAR (e.g.19z1) scFv-CD28-4-1BB H VL ʼ … · construction (i.e....
5
5ʼ LTR V H EC V L TM C IRES dsRED 3ʼ LTR SD SA Ψ + 5ʼ LTR V H EC V L TM C IRES hrGFP 3ʼ LTR SD SA Ψ + CD3ζ CD8 CD28 4-1BB scFv-CD3ζ CAR (e.g.19z1) scFv-CD28-4-1BB CCR (e.g.P28BB) Mock 19z1 P28BB 19z1 + P28BB T Cell Groups dsRED hrGFP CD8 CD4 a b CD8α Leader C
Transcript of 5ʼ LTR V ʼ ζ H L C SD SA CAR (e.g.19z1) scFv-CD28-4-1BB H VL ʼ … · construction (i.e....
5ʼ LTR VH EC VL TM C IRES dsRED 3ʼ LTR SD SA
Ψ+
5ʼ LTR VH EC VL TM C IRES hrGFP 3ʼ LTR SD SA
Ψ+
CD3ζ CD8 CD28 4-1BB
scFv-CD3ζ CAR
(e.g.19z1)
scFv-CD28-4-1BB CCR
(e.g.P28BB)
Mock
19z1
P28BB
19z1 + P28BB
T Cell Groups
dsRED
hrG
FP
CD8
CD4
a
b
CD8α Leader
C
t.moogan
Pencil
t.moogan
Pencil
IL-2
(p
g/m
L)
Moc
kHz1
P28BB
Hz1
+ P
28BB
0
2000
4000
6000
8000
10000
Moc
kLz
1
P28BB
Lz1
+ P28
BB0
200
400
600
800
1000
IL-1
3 (
pg
/mL
)
a 19z1 CAR
e
b Hz1 CAR
c Mz1 CAR
d Lz1 CAR
Moc
k
19z1
P28BB
19z1
+ P
28BB
0
500
1000
1500
IL-1
3 (
pg
/mL
)
Moc
kHz1
P28BB
Hz1
+ P
28BB
0
2000
4000
6000
8000
GM
-CS
F (
pg
/mL
)
IFN!
(pg
/mL
)
Moc
kHz1
P28BB
Hz1
+ P
28BB
0
2000
4000
6000
8000
10000
Moc
kHz1
P28BB
Hz1
+ P
28BB
0
20
40
60
80
TN
F!
(p
g/m
L)
Moc
kM
z1
P28BB
Mz1
+ P
28BB
0
2000
4000
6000
8000
GM
-CS
F (
pg
/mL
)
Moc
kLz
1
P28BB
Lz1
+ P28
BB0
2000
4000
6000
8000
GM
-CS
F (
pg
/mL
)
Moc
kHz1
P28BB
Hz1
+ P
28BB
0
200
400
600
800
1000
IL-1
3 (
pg
/mL
)
Moc
kM
z1
P28BB
Mz1
+ P
28BB
0
2000
4000
6000
8000
10000
IFN!
(pg
/mL
)
Moc
kM
z1
P28BB
Mz1
+ P
28BB
0
200
400
600
800
1000
IL-1
3 (
pg
/mL
)
Moc
kM
z1
P28BB
Mz1
+ P
28BB
0
2000
4000
6000
8000
10000
IL-2
(p
g/m
L)
Moc
kM
z1
P28BB
Mz1
+ P
28BB
0
20
40
60
80
TN
F!
(p
g/m
L)
Moc
kM
z1
P28BB
Mz1
+ P
28BB
0
100
200
300
400
500
TN
F!
(p
g/m
L)
Moc
kLz
1
P28BB
Lz1
+ P28
BB0
2000
4000
6000
8000
10000
IFN!
(pg
/mL
)
Moc
kLz
1
P28BB
Lz1
+ P28
BB0
200
400
600
IL-2
(p
g/m
L)
CD19+PSMA+Empty
PSCA+PSMA+
Empty
PSCA+PSMA+
Empty
PSCA+PSMA+
Empty
Moc
k
19z1
P28BB
19z1
+ P
28BB
0
5000
10000
15000
IL-2
(p
g/m
L)
Moc
k
19z1
P28BB
19z1
+ P
28BB
0
100
200
300
400
500
TN
F!
(p
g/m
L)
Moc
k
19z1
P28BB
19z1
+ P
28BB
0
500
1000
1500
TN
F!
(p
g/m
L)
Moc
k
19z1
P28BB
19z1
+ P
28BB
0
5000
10000
15000
IFN!
(pg
/mL
)M
ock
19z1
P28BB
19z1
+ P
28BB
0
2000
4000
6000
8000
GM
-CS
F (
pg
/mL
)
TN
F!
(p
g/m
L)
Moc
kHz1
P28BB
Hz1
+ P
28BB
0
100
200
300
400
500
Moc
kLz
1
P28BB
Lz1
+ P28
BB0
20
40
60
80
TN
F!
(p
g/m
L)
Moc
kLz
1
P28BB
Lz1
+ P28
BB0
100
200
300
400
500
TN
F!
(p
g/m
L)
PSCA+PSMA+
Empty
0102
103
104
105
<FITC-A>
0
102
103
104
105
<APC-A>
0.38 100
00
0 102 103 104 105<PE-A>
0
102
103
104
105<APC-A>
1.84 97.5
0.550.0810 102 103 104 105
<PE-A>
0
102
103
104
105
<APC-A>
0 0
99.80.22
0102
103
104
105
<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
99
0102
103
104
105
<APC-A>
0
50K
100K
150K
200K
250K
SSC-A
98
0 102 103 104 105<FITC-A>
0
50K
100K
150K
200K
250K
SSC-A
99.7
0 102 103 104 105<FITC-A>
0
50K
100K
150K
200K
250K
SSC-A
99.3
0 102 103 104 105<FITC-A>
0
50K
100K
150K
200K
250K
SSC-A
99
0 102 103 104 105<PE-A>
0
102
103
104
105
<APC-A>
95.5 1.73
0.12.71
PC3-CD19-GFP/Luc
PC3-PSMA-GFP/Luc
PC3-PSMA-CD19-GFP/
Luc
0102
103
104
105
<FITC-A>
0
50K
100K
150K
200K
250K
SSC-A
0
0102
103
104
105
<PE-A>
0
102
103
104
105
<APC-A>
100 0.031
00.031
0102
103
104
105
<PE-A>
0
102
103
104
105
<APC-A>
0 0
0.066100
0102
103
104
105
<PE-A>
0
102
103
104
105
<APC-A>
0 0
0100
PC3-Empty PC3-PSCA-GFP/Luc
PC3-PSCA-PSMA-GFP/
Luc
hrGFP
PSMA
SSC
hCD1
9
PSMA
PSCA
Supplemental Figure 1: CAR and CCR vector design and expression via transduction of primary human T cells. (a) CARs were generated by using first generation CAR construction (i.e. chimeric receptors that supplies solely an activation signal upon scFv binding) that fuses heavy and light chains of immunoglobulin variable domains to the CD8 transmembrane domain; this is fused to cytosolic signaling domains of CD3ζ. By using an Internal Ribosomal Entry Site (IRES) to enable bicistronic expression, CAR expression can be easily detected by correlation with dsRED fluorescence (data not shown). The CCR was generated by fusing an scFv to extracellular, transmembrane and signaling domains of CD2814; this was fused to a 4-1BB cytosolic signaling domain.20 By again using an IRES, CCR expression can be correlated with expression
of hrGFP (data not shown). Abbreviations: LTR – Long Terminal Repeat, SD – Splice Donor site, SA – Splice Acceptor site, VH or VL – Variable Heavy or Light domains, respectively, EC – Extracellular domain, TM – Transmembrane domain, C – Cytosolic domain, IRES – Internal Ribosomal Entry Site, dsRED - Discosoma sp. Red fluorescent protein, hrGFP – Human Recombinant Green Fluorescent Protein (b) Representative transduction efficiencies of primary human T cells using these retroviral vectors (or mock transduction). Supplemental Figure 2: Enhanced cytokine secretion and BclXL expression induced by T cell stimulation through both a CAR and a CCR. (a) Untransduced T cells or T cells transduced with 19z1 and/or P28BB were stimulated with either untransduced PC3 cells (Empty) or CD19+PSMA+ PC3 cells. Cytokine expression was analyzed using Luminex technology. Error bars represent standard deviation from the mean of 2 biological replicates. (b-d) Untransduced T cells or T cells transduced with Hz1 (b), Mz1(c), and Lz1(d) anti-PSCA CARs, and/or P28BB CCRs were stimulated with empty or PSCA+PSMA+ PC3 cells. Cytokine expression was analyzed using Luminex technology. Error bars represent standard deviation from the mean of 2 biological replicates. (e) Western blot analysis for BclXL expression in cellular lysates of untransduced T cells or T cells transduced with 19z1 and/or P28BB, after T cells were stimulated with
CD19+PSMA+ target cells for 24 hours. Total amount of Akt was used as a loading control. Supplemental Figure 3: Generation of prostate tumor cells expressing the Green Fluorescent Protein/Firefly Luciferase fusion protein (GFP/Luc) and tumor antigens. Untransduced PC3 cells (Empty) were transduced first with retrovirus encoding GFP/Luc and subsequently transduced with retroviruses encoding CD19 , PSMA, PSCA, or a combination of two. Cells were purified by double sorting the cells with a BD FACSAria using high purity sort collection settings (low flow rate and high cell exclusion rate) for GFP/Luc, CD19, PSMA, and/or PSCA.
