Download PD-L1 Interaction

Enhancement Of T-Cell Immunity To
Osteosarcoma By Modulation Of
Programmed Death Receptor Pathway
Pooja Hingorani, Danielle Lussier, Joseph Blattman
Overview
• T cell tolerance and immune inhibitory
pathways in osteosarcoma
• Review our data
Immune tolerance in osteosarcoma
• Loss of Class I HLA expression in osteosarcoma tumors related
to worse overall survival*
• CTLA4 polymorphisms are associated with higher risk of
developing osteosarcoma**
• Metastatic osteosarcoma expresses T cell Ig/mucin molecule 3
(TIM3), which in other tumor settings inhibits the function of
infiltrating CTL, leading to tumor progression #
• B7-H3 expression, a co-inhibitory protein involved in tumor
immune escape from T cells, inversely correlates with CTL
infiltration in human osteosarcoma, and is indicative of poor
prognosis in osteosarcoma patients @
*Tshukahara et al., Cancer Sci 2006; **Liu et al, DNA Cell Bio 2011; #Shang et al., Oncol Lett 2013; @ Wang et al., Plos One 2013
Relative expression of PDL1 in osteosarcoma
Shen J K et al. Cancer Immunol Res 2014;2:690-698
©2014 by American Association for Cancer Research
Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene
expression.
Shen J K et al. Cancer Immunol Res 2014;2:690-698
©2014 by American Association for Cancer Research
Characterization of the origins of metastases.
Shen J K et al. Cancer Immunol Res 2014;2:690-698
©2014 by American Association for Cancer Research
Specific Aims
• Evaluate expression of PD-L1 on tumor cells and PD-1 on TILs in patient
samples and metastatic osteosarcoma cell line (in vitro and in vivo)
• Evaluate the functional ability of TILs infiltrating metastatic tumors
• Evaluate the effect of PD-L1 blockade on development and progression of
pulmonary metastases in vivo
• Evaluate the effect of combinational therapies (PD-L1 antibody +
chemotherapy; PD-L1 antibody + ipilimumab)
PD-L1 is expressed in human metastatic osteosarcoma
Primary Osteosarcoma
50um
50um
Metastatic Osteosarcoma
50um
50um
PD-L1
50um
50um
50um
50um
Isotype
12/ 16 (75% ) of metastatic tumors had some PD-L1 expression
PD-1 is expressed on human metastatic TILs
Primary Osteosarcoma
Metastatic Osteosarcoma
50um
50um
50um
50um
50um
50um
50um
PD-1
50um
50um
50um
50um
50um
50um
50um
Isotype
-13/16 (81%) of metastatic tumors had PD-1+ TILs
-In 11/16 (70%) metastatic tumors, PD-1 expression on TILs correlated
with PD-L1 expression on tumor cells
PD-L1 expression is increased in K7M2 osteosarcoma
cells
Pre Implantation
% of Maximum
1848
Post Tumor
4073
30%
PD-L1
TILs from lung metastases are PD-1+
Percentage of PD1+CD8+ Cells are High in Osteosarcoma
*
TIL
100
87%
PD-1
6%
%PD1+CD8+
%PD1+
SPLEEN
13%
94%
80
60
40
20
0
SPLN CD8
TIL CD8
SPLEEN CD8 TIL CD8
CD8
Early Disease Late Disease
Healthy
Lung 50 µm
Lung 50 µm
Lung 50 µm
PD1
µm
5050µm
50 µm
50 µm
Isotype
% change%
inchange
TNF production
of CD8+ cells
in TNF production
of CD8+ c
TILs are functionally impaired in the presence
ofonlytumor
TIL only
TIL
TIL+Antigen TIL+Antigen
in
IL2 production
by CD8+ cells
% change%
inchange
IL2 production
by CD8+ cells
cells
nge%inchange
IFNy Production
in IFNy Production
with or without
with orantigen
without antigen
TIL only
TIL only
TIL+AntigenTIL+Antigen
0
4
2
0
-
+
SPLN CD8 SPLN
TIL
CD8
PD-L1+
+tumor
K7M2
-
+
Ag
2
1
0
*
3
2
1
0
SPLN CD8
- TIL CD8
+CD8
SPLN
TIL PD-L1CD8
TIL CD8
TIL CD8Cytotoxicity
LDH
PD-L1+
+tumor
+tumor
+tumor lysate+tumor lysate
4T1
K7M2
- TIL
+CD8Ag
TIL
CD8
5
4
4
3
2
1
0
4T1
*
*
2
1
0
-
20
10
Tumor
APC NP
APC P
+
TIL
CD8
SPLN
CD8
PD-L1+
+tumor
K7M2
Tumor
APC
Tumor Lysate APC
30
*
3
TIL CD8
SPLN CD8
assay
PD-L1+tumor
+tumor lysate+tumor lysate
40
0
5
%TNF+
*
6
3
%IL-2+
*
*
%TNF+CD8+
2
8
4
%IL2+CD8+
4
*
%IL2+CD8+
6
*
4
% Cytotoxicity
8
10
%IFNγ+
%IFNy+CD8+
10
*
%TNF+CD8+
TIL only
TIL only
TIL+AntigenTIL+Antigen
-
+
Ag
TILPD-L1CD8
TIL
CD8
TIL CD8
+tumor
+tumor
4T1 lysate +tumor lysate
TIL only
TIL+Antigen
TIL only
PD-L1 blockade restores function to TILs
*
C.
TIL+Antigen
8
6
*
4
2
TNF+CD8+indiv
TIL CD8Ab
PD-L1
TIL CD8
Control
TIL only
TIL+Antigen
%TNF+
%TNF+CD8+
with in vivo mAb
*
*
10
8
6
4
*
4
2
0
Control
TIL CD8
PD-L1
TIL CD8 Ab
with in vivo mAb
*
2
0
0
B.
*
6
*
%IL-2+
%IL2+CD8+
%IFNγ+
%IFNy+CD8+
A.
Control
TIL CD8
PD-L1
Ab
TIL CD8
with in vivo mAb
urvival of K7M2 injected mice treated with PD-L1 monoc
Blockade of PD-1:PD-L1 significantly enhances survival
in metastatic osteosarcoma
model
PD-L1
blockade (n=9)
Control (n=10)
PD-L1 Ab
Percent Survival
Percent survival
100
50
P=0.0005
0
0
50
100
Days post
implant
Days
post implant
150
30 day PD-L1 antibody treatment does not significantly
Survival proportions: Survival of 30 day treatment
improve survival compared to 15 day treatment
PD-L1 (30day)
PD-L1 (15day)
Control
Percent survival
100
50
0
0
50
Days post implant
Is immune escape occurring?
100
PD1+CD8+antibody
decrease after 30day
treatment
PD-L1
treatment
changes
inhibitory receptor
CTLA4+CD8+ no change in expression after 30day trea
expression
*
15
CTLA4+CD8+
60
40
20
0
*
LAG3+CD8+ no difference after 30 day treatment
80
untreated
10
5
0
treated
40
LAG3+CD8+
PD-1+CD8+
100
30
20
10
0
untreated
treated
untreated
treated
PD-L1 antibody treated osteosarcoma is resistant to
additional treatment when injected into naïve mice.
Survival proportions: Survival of reimplanted alone
Reimplant
50
0
0
20
40
60
80
PDL1 MFI
PD-L1
Days post implant
*
2000
PD-L1 MFI
Percent survival
% of Maximum
Reimplant/PD-L1
100
1500
1000
500
0
Control
Reimplanted
Combinational
PD-L1
antibody
coupled with
Survival
proportions:
Survivaltreatment
of PDL1 vs chemo
conventional chemotherapy
Chemo + PDL1
Chemo
PD-L1
Control
Percent survival
100
50
0
0
50
100
Days post implant
The combination improves survival as compared to chemotherapy alone but
is similar to PD-L1 antibody alone.
After reinjection
Combinational
PD-L1
antibody
coupled
with CTLA-4
K7M2 after CTLA4/PDL1 treatment
Survival proportions: Survival of PDL1 vs CTLA4
antibody treatment improves survival
CTLA4+PDL1
CTLA4
PD-L1
Control
Percent survival
Percent survival
100
50
0
0
50
Days post implant
100
CTLA4+PDL1
Control
100
50
0
0
20
40
Days after reimplantation
60
In conclusion
• PD-1/PD-L1 interaction induces immune tolerance in
metastatic osteosarcoma
• PD-L1 blockade improves survival but not 100%
• Resistance to therapy develops by up-regulation of alternative
immune escape pathways
• Combinational immune therapies may have the highest
impact on disease control in metastatic osteosarcoma
Acknowledgements
•
•
•
•
•
•
•
Joseph Blattman
Danielle Lussier
John Johnson
Lauren O’Neill
Lizbeth Nieves
Megan McAfee
Susan Holechek
• Paul Dickman
• Patients and families