administration of tumor-specific cytotoxic t lymphocytes ......bollard et al, jit 2004 making lmp1...
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CM. Bollard, G Dotti, S Gottschalk, E Liu, A Sheehan, M Mims, H Liu, AP. Gee,
MK. Brenner, HE. Heslop, CM. Rooney
Administration of Tumor-Specific Cytotoxic T Lymphocytes Engineered to Resist TGF-ß to Patients with EBV-Associated Lymphomas
• Significant failure rate of therapy for advanced stage or recurrent disease
• Long-term side effects of chemotherapy and
radiation
• EBV antigens expressed by up to 40% of lymphomas are potential targets for T cell immunotherapy
Rationale of Immunotherapy for EBV-positive Lymphoma
EBV Infected B cells
EBV +ve Lymphoma Cell
TGFb
EBV Specific Cytotoxic T Lymphocytes (CTL) Control EBV Infection in vivo
LMP1
LMP2A
Lytic EBNA 3 LMP 2 LP LMP 1 EBNA 2 EBNA1
PBMC
CTL
• LMP1 and LMP2A are potential CTL targets
Hodgkin R-S Cell/NHL Cell
LMP1 and LMP2A-specific CTL For Hodgkin and non-Hodgkin Lymphoma
EBNA1
LMP1
LMP2A
Bollard et al, JIT 2004
Making LMP1 and LMP2 Immunodominant Antigens
Gottschalk et al, Blood 2004 and Leen et al, JIT 2007
LMP-specific Cytotoxic T
Lymphocytes (CTL)
Ad5f35 LMP1-I-LMP2
PBMC IL-2
Lymphoblastoid cell line (LCL) Dendritic
Cells
Ad5f35 LMP2
OR
-10%
0%
10%
20%
30%
40%
50%
60%
70%
80%
Ratio 40:1 Ratio 20:1 Ratio 10:1 Ratio 5:1
% S
peci
fic
Lysi
s
AutoLCLAlloLCLPHABlasts
FC109Y08.133
CD8
ILL
100
101
102
103
104
101
102
103
104
0.03% 1.03%
18.96% 79.98%
FC109Y08.130
CD8
CLG
100
101
102
103
104
101
102
103
104
0.02% 0.19%
16.83% 82.95%
FC109Y08.131
CD8
FLY
100
101
102
103
104
101
102
103
104
0.17% 15.32%
19.48% 65.03%
FC109Y08.132
CD8
YLL
100
101
102
103
104
101
102
103
104
0.01% 2.95%
16.53% 80.50%
15.32% 0.19% 1.03% 2.96%
CD8
FLY-A2 LMP2 tetramer
CLG-A2 LMP2 tetramer
ILL-A29 LMP2 tetramer
YLL-A2 LMP1 tetramer
LMP1 & LMP2–Specific Activity in LMP-CTL from a Hodgkin Disease Patient
Relapsed Disease Arm (n=21)
• No toxicity • 11 CR (1 also given Rituximab) (includes 1PRCR)
• 2 very good partial responses (up to 36 mths) • 8 progressive disease (2-8 wks) Median clinical response: 1.5y
(range: >6 to >40 mths)
CR PR
n =21
Patients with disease at CTL infusion
Clinical Responses post LMP-CTL
Bollard et al, JCO 2013 in press
CR PR
n =21
X X X X X X X X
CR PR
NR
50% Disease Free Survival at 2 years
Clinical Responses post LMP-CTL in Patients with Active Disease
Year
Pro
port
ion d
isease-f
ree
0 1 2 30
0.2
0.4
0.6
0.8
1
Alascer
ALCI
P=0.744
LMP1/2-CTL study
LMP2-CTL study
Figure 4. Immune Reconstitution
LMP1
SFC
per
2x105
LMP2
SFC
per
2x105
LMP1
SFC
per
2x105
LMP2
SFC
per
2x105
Responders Non-Responders
Immune Reconstitution of LMP1 and LMP2-specific T cells in Patients Treated with
LMP1/2-CTL
Responders Non-
responders
Figure 4C. Immune Reconstitution
0
20
40
60
pre-infusion post-infusion
Survivin
SF
C p
er
2x
10
5 c
ell
s
0
10
20
30
40
50
60
70
pre-infusion post-infusion
PRAME S
FC
pe
r 2
x1
05 c
ell
s
0
20
40
60
pre-infusion post-infusion
Survivin
0
20
40
60
80
100
120
pre-infusion post-infusion
MAGEA4
0
20
40
60
pre-infusion post-infusion
PRAME
0
20
40
60
80
100
120
pre-infusion post-infusion
MAGE A4
Evidence of Epitope spreading in Responding Patients Treated with LMP1/2-
CTL
Can we make LMP-CTL resistant to the inhibitory effects of TGF-ß
secreted by Lymphoma cells?
• Inhibits CTL proliferation
• Inhibits cytotoxicity
- perforin
• Inhibits cytokine production
- IFN
TGFb Effects on CTL
Creating a Mutant TGFb Receptor II
Stop codon 597
Wild type
Receptor
Truncated TGFß Receptor II
Dominant Negative Receptor (DNR)
Transmembrane domain
Bollard et al, Blood 2002
MoMLV MoMLV
U3 R U5 U3 R U5
NcoI/BamHI
SD PBSQ
SA
+
TM domain
SFG:DNR
Retroviral vector SFG
DNR
Bollard et al, JIT 2004, Bollard et al, Blood 2002, Foster et al, JIT 2008
Rendering LMP-specific T cells Resistant to TGFb
Ad5f35 LMP1-I-LMP2
PBMC IL-2
EBV-LCL
DC
DNR-transduced LMP CTLs
SFG:DNR
• EBV+ malignant cells EBER and/or LMP1
and/or LMP2 positive Lymphoma cells
• Patients with relapsed Hodgkin Disease or NHL including after allogeneic SCT
Study Eligibility
CD4 and CD8 T cells are DNR-transduced
CD8 FITC
TG
FB
RII
PE
100
101
102
103
104
100
101
102
103
104
81.32%16.26%
2.39%0.03%
NON Trans
CD8 FITC
TG
FB
RII
PE
100
101
102
103
104
100
101
102
103
104
76.84%10.68%
10.19%2.29%Trans
n=6 CTL lines
XX
0
20000
40000
60000
80000
100000
120000
140000
Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6
Tra
ns
ge
ne
co
py n
um
be
r in
10
0n
g D
NA
1%
25% 28%
0%
5%
10%
15%
20%
25%
30%
35%
CD45RA+CD62L+
CD45RA-CD62L+
CD45RA-CD62L-
17%
4%
11%
0%
2%
4%
6%
8%
10%
12%
14%
16%
18%
20%
TGFβ+ CD3+ TGFβ+ CD3+ CD4+
TGFβ+ CD3+ CD8+
n=6 CTL lines
DNR-transduced CD4 and CD8 T cells are Predominantly Effector Memory
DNR-Transduced CTL are
LMP-specific
EBV-LCL
• 5 females and 3 males
• EBV+ HL
7 – relapsed post autologous SCT
1 – relapsed post allogeneic SCT
• Two previously treated with LMP-CTL alone
• All refused additional chemotherapy
Patients Studied
0.1
1
10
100
1000 CTLs
0.1
1
10
100
1000
10000 CTLs
DNR-transduced T-cells Persist in vivo for 5-23 months
Patient 1
Co
py n
um
be
rs in
10
3 n
g D
NA
0
Patient 3
0
• No dose limiting toxicity
• TGFb-resistent LMP-CTL may beneficial in EBV+ Lymphoma
• DNR-trans LMP-CTL persist up to 3 years
• Now plan to explore the use of DNR-CTL in other TGFb-secreting cancers
Conclusions