Age at primary
Case # Biopsy (year)
Age at metastasis
Biopsy (year)
Primary Tumor
site
Metastasis
site
1
7
9
R radius
2
8
9
3
14
18
4
12
5
6
Biopsy
decalcification
Metastasis
decalcification
PD-L1 metastatic PD-1+ TIL metastatic
expression
expression
L iliac bone & soft tissue No
No
+
+
L distal femur
Mediatinum, thymus
Yes
No
+
+
R distal femur
Lung
No
No
+
-
14
R distal femur
Lung
Yes
No
++
++
14
15
L distal femur
Lung
No
Yes
-
-
13
14
R femur
Lung
No
No
-
+
7
12
15
L proximal tibia
Lung
Yes
No
+
++
8
10
10
L humerus
Lung
Yes
Yes
-
-
9
15
21
R femur
Lung
Yes
No
-
+
10
10
12
R shoulder
Lung
No
No
+
++
11
9
10
R distal femur
Lung
Yes
No
++
++
12
15
15
N/A
Lung
Yes
Yes
+
+
13
15
21
N/A
Lung
Unknown
No
+
+
14
8
9
N/A
Lung
Unknown
Yes
+
+
15
16
16
N/A
Lung
No
No
++
++
16
16
18
N/A
Lung
No
No
++
+
Primary Osteosarcoma
A.
Metastatic Osteosarcoma
50um
50um
Data 1
50um
PD-L1
C.
50um
Isotype
B.
50um
50um50um
80
60
40
20
0
r2 = 0.732
0
10
20
30
Mean # of PD-L1+ cells/field
50um
PD-1
50um
50um
Mean # of CD8+ cells/field
50um
50um
Isotype
40
Percent
survival
%Survival
100
50
P<0.0001
0
0
10
20
30
40
Days
post
implant
Days
post
implantation
50
1848
Tumor
C.
SPLEEN
4073
3030%
%
PD-L1
B. Healthy
6%
87%
94%
13%
CD8
Percentage of PD1+CD8+ Cells are High in Osteosa
50 µm
Early Disease
Late Disease
Lung 50 µm
Lung 50 µm
D.
PD1
50 µm
50 µm
*
100
%PD1+CD8+
CD8
%PD1+
Lung
TIL
PD-1
% of Maximum
A.
Pre Implantation
50 µm
Isotype
80
60
40
20
0
SPLN CD8
SPLEEN
TIL CD8
TIL
C.
TIL only
TIL+Antigen
2
1
TIL + 4T1
K7M2 lysate
TIL+ K7M2
PD-L1-/-
TIL + 4T1
TIL + K7M2
PD-L1 shRNA
TIL
TIL+
K7M2
TIL +
tumor lysate
TIL + 4T1
K7M2 lysate
Cytotoxicity
LDH assay
TIL+
TIL+ K7M2
TIL +
K7M2
D.
PD-L1-/-
tumor lysate
*
*
40
20
0
TIL + K7M2
0
2
0
% Cytotoxicity
3
TIL
%TNF+PD-1+CD8+
*
*
4
*
4
80
TIL only
TIL+Antigen
60
*
5
6
TIL +
tumor lysate
*
TIL only
TIL+Antig
8
TIL + 4T1
TIL + 4T1
K7M2 lysate
TIL+ K7M2
PD-L1-/-
TIL + 4T1
B.
TIL + K7M2
PD-L1 shRNA
TIL+
K7M2
TIL
0
TIL + K7M2
5
*
TIL + K7M2
PD-L1 shRNA
* TNF with controls
*
TIL
*
10
TIL
%IFNy+PD-1+CD8+
*
TIL + K7M2
*
15
10
TIL
*
%IL2+PD-1+CD8+
A.
K7M2
4T1
4T1
K7M2
K7M2
PD-L1 shRNA
K7M2
4T1 Nonpuls
PD-L1 blockade (n=9)
Control (n=10)
A.
PD-L1 Ab
%Survival
Percent survival
100
50
P=0.0005
Number of Mets in PDL1 mAb treated
0
0
Area of largest pulmonary met
Control
50
100
PD-L1 mAb
1000ug post
Days
150
80
60
40
20
0
60
Control
PD-L1 mA
40
3
# of metastases
Number of pulmonary mets
Volume of largest
mm
metastasis
(mm3)
Days post implantationimplant
B.
α-PD-L1 Ab
Control
K7M2 implantable Model
20
0
Control
α-PD-L1
Ab
K7M2 implantable
Model
TIL+Antigen
C.
8
6
*
4
2
TNF+CD8+indiv
0
TIL CD8Ab
PD-L1
with in vivo mAb
TIL only
TIL+Antigen
%TNF+
%TNF+CD8+
*
*
10
8
6
*
4
*
2
0
TIL CD8
Control
B.
*
6
*
%IL-2+
%IL2+CD8+
%IFNγ+
%IFNy+CD8+
A.
*
4
2
0
Control
TIL CD8
PD-L1
TIL CD8 Ab
with in vivo mAb
TIL CD8
Control
TIL CD8
PD-L1
Ab
with in vivo mAb