43
WIN 2015 Symposium Radiation and immunology: a new therapeutic partnership
WIN 2015 Symposium
Radiation and immunology: a
new therapeutic partnership
Dr. Ralph Weichselbaum
Conflict of Interest
• Nothing to Disclose
Limitations of Radiotherapy
• Radiotherapy cannot be given in high doses to large normal tissue volumes for very large primary tumors or widespread metastasis
• Normal tissue complications are common following high curative doses in some sites
• Need newer therapies for effective control of radioresistant histology e.g. Glioblastoma, large (T4), and micro and gross metastasis. Even with local control metastasis will defeat the purpose of treatment
• Immunotherapy?
Courtesy of Joseph K. Salama, M.D. and Karl Farrey
Immunodeficiency Abrogates the Anti-tumor Effect of RT. (Blood, Vol. 114, Issue 3, 589-595, July 16, 2009)
Day 16
20Gy
CFSE 2C transfer
How Can RT Induce Antigen-specific T cells Priming?
DLN
SP
No RT RT
2C CFSE
Day 0
5x105 B16-SIY
Harvest Draining
Lymph Nodes, Spleen
5 d
Increase antigen availability
Reduced suppression
Increase positive cytokines
or other molecules
Tolerance may be reversed?
Can RT increase the Cross-priming
Capacity of Intratumoral DCs?
3 days
20 Gy
CD11c+ from tumor
Proliferation
Assay
2C T cells
0
5000
10000
15000
20000
25000
***
T c
ell
Pro
life
ration (
cpm
)
14 days
B16-SIY
Functional capacity of tumor infiltrating DCs
directly ex vivo
CD4+ (Non-Treg)
WT N
o RT
WT R
T
0
100000
200000
300000
400000
To
tal #
CD
4+
Fo
xP
3-
CD8+ T cell
WT N
o RT
WT R
T
0
500000
1000000
1500000
2000000
2500000
To
tal #
CD
8+
CD90+ Lympocytes
WT N
o RT
WT R
T
0
2×1006
4×1006
6×1006
To
tal
# C
D9
0+
Ly
mp
ho
cy
tes
] ]
No R
T
20 G
y
Local RT Increases CD8+ T cell Infiltration
Day 7 Post-RT Analysis of Tumor Infiltrating Lymphocytes (TIL)
Local Chemokine Expression
24hrs
0 10 20 300
500
1000
1500
2000
2500
WT WT RT
WT WT Control
***
Days post RT
Tum
or
Volu
me (
mm
3)
0 10 20 300
500
1000
1500
2000
2500
WT KO RT
WT KO Control
***
Days post RT
Tum
or
Volu
me (
mm
3)
0 5 10 15 20 250
500
1000
1500
2000
2500 WT WT RT
WT WT no RT
IFNAR KO WT RT
IFNAR KO WT no RT
Days post tumor RT
Tu
mo
r V
olu
me (
mm
3) Interpretation: Type I interferon
signaling on hematopoietic cells
is required for tumor reduction
following RT
Type I Interferon is Essential for RT
Radiation & Interferon Induction
Kim et. al. Blood 2012
What are we actually modeling?
No T cell response
Existing T cell
Response
Generate Systemic cell Response
Quantitativly Enhance Existing T cell
Response
Qualitatively Enhance Existing T
cell Response IR
Anti-PD-1
Anti-PD-L1
Anti-CTLA4
Anti-PD-1
Anti-PD-L1
Anti-CTLA4
How to Improve the Radiation Response?
• Strategies that induce host Interferon production
• Immune Modulating Antibodies Anti-CTLA-4 (Ipilimumab) Anti-PD-1 / Anti-PD-L1 OX40
Blockade of PD-1 or CTLA-4 Signaling in Tumor Immunotherapy
N Engl J Med. 2012 Jun 28;366(26):2517-9.
Immunologic Correlates of the
Abscopal Effect in a Patient with
Melanoma
Michael A. Postow, M.D., Margaret K. Callahan, M.D., Ph.D., Christopher A. Barker, M.D., Yoshiya Yamada, M.D.,
Jianda Yuan, M.D., Ph.D., Shigehisa Kitano, M.D., Ph.D., Zhenyu Mu, M.D., Teresa Rasalan, B.S., Matthew Adamow,
B.S., Erika Ritter, B.S., Christine Sedrak, B.S., Achim A. Jungbluth, M.D., Ramon Chua, B.S., Arvin S. Yang, M.D.,
Ph.D., Ruth-Ann Roman, R.N., Samuel Rosner, Brenna Benson, James P. Allison, Ph.D., Alexander M. Lesokhin, M.D.,
Sacha Gnjatic, Ph.D., and Jedd D. Wolchok, M.D., Ph.D.
N Engl J Med 2012; 366:925-931 March 8, 2012
16
Case Report #1:
Postow, NEJM, 2012
9.5 Gy x 3
Clinical Observations Radiation & anti-CTLA-4
Radiation-Induced Equilibrium Is a Balance
between Tumor Cell Proliferation and
T Cell–Mediated Killing
2 weeks
before SBRT
4months 9 months 10 months
A: Early responses but relapse
after SBRT
0 day 2 months 22 months
after SBRT
Three major outcomes after SBRT as monitored by MRI
Hurthle cell
thyroid
B: Cure or not
Melanoma
(20Gyx1)
(20Gyx1)
before SBRT Post SBRT 1 month Post SBRT 11 months
Post SBRT 25 months Post SBRT 49 months relapse Post SBRT 57 months
RT-induced dominancy can be relapse for years
(1)
(6) (5) (4)
(3) (2)
R D Schreiber et al. Science 2011;331:1565-1570
T cell – Tumor Cell Interactions
The specificity and
magnitude of the T cell
response against tumor
antigens determines the
outcome between tumor
suppression/elimination and
tumor outgrowth.
Immunotherapy, or
modification of existing or
induced (vaccination) T cells
responses, can push this
interaction in the direction of
tumor elimination.
Ablative RT Can Suppress Local Tumor
Regrowth and Induce Stable Disease
Tubo
0 10 20 300
200
400
600
15Gy
RT(n=23)Ctrl (n=5)
Days after tumor challenge
Tu
mo
r V
olu
me (
mm
3)
B16-SIY
0 10 20 300
200
400
600RT (n=29)
25Gy
Ctrl (n=5)
Days after tumor challenge
Tu
mo
r V
olu
me
(m
m3)
A B
Liang H, Deng LF et al. The Journal of Immunology 2013 190 (11) 5874-5881.
7-8 days later
NR
R
21 days post IR
R Dormant (D)
Partial Response (PR)
Remove tumor collagenase Clonogenic assay
Non-RT control
7 days
7 days
0, 2, 5 or 10 Gy
0 2 4 6 8 100
100
200
300
Non-RT controlNon-responsive (NR)responsive (R)
Days post RT
Tu
mo
r V
olu
me (
mm
3)
5 Gy 10Gy0.0
0.1
0.2
0.3
0.4Non-RT
NR
R
Perc
en
tag
e o
f su
rviv
al c
ells
7 days post RT
no-IR 2Gy 5Gy 10Gy0.0
0.2
0.4
0.6
0.8
1.0
1.2Non-RT PR D
p=0.057
Fractio
n o
f su
rviv
al cells
21 days post RT
Radio-sensitivity of Cancer Cells was not
Correlated with the Efficacy of RT
A B C
5 Gy 10Gy0
10
20
30
40
Non-RT
NR
R
Perc
enta
ge o
f su
rviv
al c
ells
0Gy 2Gy 5Gy 10Gy0
20
40
60
80
100
Non-RT
PR
S
Perc
enta
ge o
f su
rviv
al c
ells
Ki-67 TUNEL CD8
are
a+
CD8 a
rea
+
Ki67
0.0
0.5
1.0
1.5
2.0
2.5 **
Rela
tive a
popto
sis
Apoptosis co-localized with CD8+ cells
Host immune responses, not the radiosensitivity of cancer cells,
correlate with efficacy of RT
PD-L1 Blockade Breaks the Equilibrium of Stable
Disease to Favor Tumor Regression
0. 15. 26. 36. 49.0
50
100
150
200
Ctrl
anti-PD-L1
anti-PD-L1
Days post RT
Tu
mo
r V
olu
me
(m
m3)
*
Ctrl D D+PDL-1
0
50
100
150
2003T3KB (Control)3T3NKB
RT
*
*****
IFN+
cells / 2
x10
5 D
LN
s
0 10 20 30 40 50 600
25
50
75
100
RT
RTanti-PD-L1
Days after RT
Tu
mo
r-b
ea
rin
g (
%)
A B
C
Galon et al, Science. 2006. 313(5795):1960-4
Infiltration of T cells:
A better predictor for colorectal cancer patients survival
PD-1/PD-L1 Checkpoint
27
Objective Response
Objective-Response
Rate
Melanoma 26/94 28%
Non–small-cell lung Cancer
14/76 18%
Renal-Cell Cancer
9/33 27%
Clinical Activity of Anti–PD-1 Antibody in the Efficacy Population
N Engl J Med 2012; 366:2443-2454
N Engl J Med 2012; 366:2443-2454
Association between Pretreatment Tumor PD-L1 Expression and Clinical Response
Irradiation and anti–PD-L1 treatment synergistically
promote antitumor immunity in mice
0 10 2 10 3 10 4 10 5
PE-A
0
20
40
60
80
100
% o
f M
ax
CD11c+ CD11b+Gr1+ CD11+F4/80+ CD45-
0 102 103 104 105
<PE-A>
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105
<PE-A>
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105
<PE-A>
0
20
40
60
80
100
% o
f M
ax
CD8+T CD4+T
0 102 103 104 105
<PE-A>
0
20
40
60
80
100
% o
f M
ax
0 102 103 104 105
<PE-A>
0
20
40
60
80
100
% o
f M
ax
Isotype
Non-RT
RT
PD-L1
PD-1
A
B
Iso type
Non-RT
RT
Expression of PD-L1 and PD-1 in the tumor microenvironment
0 5 10 150
100
200
300
400Naive (n=4)RT+PD-L1 (n=4)
Days after tumor rechallenge
Tu
mo
r V
olu
me
(m
m3)
A B C D
0 5 10 15 20 250
100
200
300
400
500
600
700control
RT
PD-L1
RT+ PD-L1
Days post RT
Vo
lum
e(m
m3)
0 10 20 30 400
200
400
600
800 CtrlPD-L1RTRT+PD-L1
***
**
TUBO
Days after tumor challenge
Tu
mo
r V
olu
me (
mm
3) MC38
0 10 20 30 400
200
400
600CtrlPDL-1RTRT+PDL-1
*
***
Days after tumor challenge
Tu
mo
r V
olu
me (
mm
3)
Anti-PD-L1 blockade synergizes with Radiation
CD8+ T Cells are Required for Combination
Therapy with IR and PD-L1 Blockade
0 10 20 30 400
200
400
600
800 CtrlPD-L1RTRT+PD-L1RT+PD-L1+CD8
*
Days after tumor challenge
Tum
or
Volu
me (m
m3)
Ctrl
PD-L1
RT
PD-L1
RT+
0
20
40
60
803T3KB3T3NKB
IFN+
cells / 2
x105 D
LN
s *****
**anti-PD-L1
9 days post RT
A B
B
0 102 103 104 105
<PerCP-Cy5-5-A>: CD11b
0
102
103
104
105
<F
ITC
-A>:
Gr1 15.1
0 102 103 104 105
<PerCP-Cy5-5-A>: CD11b
0
102
103
104
105
<F
ITC
-A>:
Gr1 5.71
0 102 103 104 105
<PerCP-Cy5-5-A>: CD11b
0
102
103
104
105
<F
ITC
-A>:
Gr1 3.96
0 102 103 104 105
<PerCP-Cy5-5-A>: CD11b
0
102
103
104
105
<F
ITC
-A>
: G
r1 0.269
Isotype Ctrl αPD-L1 IR IR+αPD-L1
Gr-
1
CD11b
A
15.1% 5.71% 3.96% 0.27%
+
Gr1+
CD11b
+
F4/80+
CD11b
T+
CD8T+
CD4
0
10
20
30Day 10 after IR
***
***
% o
f C
D45
+ce
lls
+
Gr1+
CD11b
+
F4/80+
CD11b
T+
CD8 T+
CD4
0
10
20
30
40 Isotype ctrlPD-L1IRIR+PD-L1
Day 3 after IR
**
% o
f C
D45
+ c
ells
IR and PD-L1 blockade induce the reduction of MDSCs
Gr1 CD8α Caspase3
Iso
typ
e
Ctr
l G
r-1
CD11b
Isotype Ctrl IR+αPD-L1 IR+αPD-L1+αCD8 C D
0 102 103 104 105
<PE-Cy7-A>: CD11b
0
102
103
104
105
<P
E-A
>: G
r-1 19.9
0 102 103 104 105
<PE-Cy7-A>: CD11b
0
102
103
104
105
<P
E-A
>: G
r-1 0.545
0 102 103 104 105
<PE-Cy7-A>: CD11b
0
102
103
104
105
<P
E-A
>: G
r-1 10.2
19.9% 0.55% 10.2%
A
0
20
40
60
80IR+PD-L1Isotype Ctrl
**
Inte
rcellu
lar
dis
tan
ce b
etw
een
Gr1
+ a
nd
CD
8+
T (
m)
B IR
+ α
PD
-L1
Gr1 CD8 Merge
Isoty
pe Ctrl
PD-L1
IR
+
CD8
PD-L1+
IR
+
CD8
0
5
10
15
20
25
** **
% (
MD
SC
s in
CD
45
+ T
ILs
)
Figure 5
A
B C D
Gr1+ alone Gr1+ +Naïve CD8+T isotype αTNF-α αIFN-γ αTNF-α+αIFN-γ
Gr1+ + activated CD8+T
0 20 40 60 80 100 1200
10
20
30
40TNF-IFN-
Concentration (ng/ml)
An
nexin
V+
MD
SC
(%)
0 102 103 104 105
<APC-A>: Annexin-V
0
102
103
104
105
<F
ITC
-A>:
Ly6C
94 5.96
00
0 102 103 104 105
<APC-A>: Annexin-V
0
102
103
104
105
<F
ITC
-A>:
Ly6C
57.5 42.5
00
0 102 103 104 105
<APC-A>: Annexin-V
0
102
103
104
105
<F
ITC
-A>:
Ly6C
77.2 22.8
00
0 102 103 104 105
<APC-A>: Annexin-V
0
102
103
104
105
<F
ITC
-A>:
Ly6C
61.3 38.7
00
0 102 103 104 105
<APC-A>: Annexin-V
0
102
103
104
105
<F
ITC
-A>:
Ly6C
79.5 20.5
00
0 102 103 104 105
<APC-A>: Annexin-V
0
102
103
104
105
<F
ITC
-A>:
Ly6C
87.4 12.6
00
alone+
Gr1
T+
+ naive CD8
+
Gr1
rat IgG1
-TNF
-IFNg
-IFNg
+-TNF
0
20
40
60
Gr1++ activated CD8+T
*** *****
***
An
nexin
V+ M
DS
C(%
)
Annexin-V
Ly-6
C
5.96% 12.6% 42.5% 22.8% 38.7% 20.5%
Figure 6 0 10 20 30 400
200
400
600 non-IR
IR
IR+1A8
**
Days after tumor challenge
Tu
mo
r V
olu
me(m
m3)E F
0 5 10 15 20 25 30 350
100
200
300
400
500
600
700
Isotype Ctrl IR+PD-L1
IR+PD-L1+TNFRhIgG
*
TNFRhIgG
Days after tumor challenge
Tu
mo
r V
olu
me (
mm
3)
Day 10 after RT
+
Gr1+
CD11b
+
F4/80+
CD11b
+
CD11c B+
B220 T+
CD8 T+
CD4
0
20
40
60
*
% o
f to
tal s
ple
nocyt
es
Day 3 after RT
+
Gr1+
CD11b
+
F4/80+
CD11b
+
CD11c B+
B220 T+
CD8 T+
CD4
0
20
40
60
IRIR+PD-L1
Isotype ctrlPD-L1
% o
f to
tal s
ple
nocyt
es
Supplementary Fig. 4
B7-H1 PD-1
B7-H1
IR
CD8+ T cells Myeloid cells
Tumor cells MDSCs
TNFα
Antigen
Antigen
Schematic of proposed mechanism for tumor destruction
induced by IR and PD-L1 blockade
Questions to be answered:
• What is mechanism behind the synergistic combination of PD-L1/ PD-1 blockade?
• Does radiation provide a window for anti-PD-L1/PD-1 axis inhibitors?
• What is the best sequence/timing for RT and Ab? • Does RT induce PD-L1 directly? If so, what is the mechanism? • Study the effect of RT and anti-PD-L1 in several different tumor
models? (e.g. B16.SIY, MC38, Autochthonous Tumor Models) • What changes in tumor microevironment after the combination
of radiation and anti-PD-L1/PD-1? A) Immune infiltration B) cytokine/chemokine profile
• Is anti-PD-1 superior to anti-PD-L1 when combined with radiation?
The IRDS is expressed by primary breast cancer and a wide variety of other human tumors
in a manner resembling Nu61/SCC61.
Weichselbaum R R et al. PNAS 2008;105:18490-18495
IRDS-positive clones can be selected by tumor microenvironment
due to their resistance to the host defense systems
Khodarev, Roizman, Weichselbaum, Clin Cancer Res 2012
Acknowledgements
Weichselbaum Lab
Radiation and Cellular Oncology
Liufu Deng
Hua “Laura” Liang
Mike Beckett
Helena Mauceri
Byron Burnette
Nikolai Khodarev
Tom Darga
Fu Lab. – Department of Pathology/Immunology
Yang-Xing Fu, M.D., Ph.D
