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8/3/2016
1
Radiation therapy to ignite an anti-cancer immune response
Silvia Formenti, M.D.
Weill Cornell Medical College
New York Presbyterian Hospital
New York, NY
DISCLOSURES
Principal Investigator: NCI R01CA161891-01 Immunomodulation of breast cancer via TLR7 agonist IMQ and RT DOD BC100481 / W81XWH-11-1-0530 Multi-Team Award (MTA) Radiation-Induced Vaccination to Breast Cancer 13-A0-00-001870-01 Breast Cancer Research Foundation Targeting key inhibitory pathways to improve radiation-induced vaccination in breast cancer NIH 1 S10 RR027619-01 Preclinical Research Irradiator
Consultant/Speaker: Bristol Myers Squibb, Varian, Elekta, Janssen, Regeneron, GlaxoSmithKline, Eisai, Dynavax, Astra Zeneca
Why are abscopal effects of radiation so rare?
8/3/2016
2
• 1969-2014: 46 abscopal cases
• Median dose 31 Gy; median follow up 17.5 months
• Only one case single dose SBRT (2.1%): all others fractionated RT (wide range of dose and fractions)
• ~ 13% were VMAT/SBRT
• NON ABLATIVE DOSES
IMMUNOSUPPRESSION DOMINATES IN ESTABLISHED TUMORS
Vesely MD, 2011, Annu.Rev.Immunol 29:235-71
How to enable the pro-immunogenic effects of radiation:
Overcoming immunosuppressive
microenvironment of established tumors
- enhancing cross priming
- blocking immune checkpoints
c)
Overcoming RT immunosuppressive effects
- overcoming RT-induced
immunosuppression (adenosine,TGFb)
- mitigating RT-induced lymphopenia
8/3/2016
3
BALB/C mice injected at two separate sites with the syngeneic mammary carcinoma 67NR cell line
R
“primary” L
“secondary” X RT
20 Flt3-L (0.5mg/kg) RT
2 Gy
0 67NR
5x104 or 105
each sides,
primary R and secondary L
Day:
Ionizing radiation stimulates anti-tumor immunity by generating an in situ
vaccine: combination with immunotherapy uncovers the abscopal effect
GM-CSF: 125 mg/m2
Daily X 14 days
RT: 3.5 GyX10
27% ABSCOPAL ORR
1 2 3 4 5 6 7 8 90
50
100
150
250
500
750
1000
IFNδ
(pg/
mL)
Strain
RT
α-CD1d
BALB/c WT NKT-/-
-
--
+
- - - - +
+
+
- +
++ -
-
-
-
+
- - -
- +
++
α-CTLA4
AH-1-A5
pMCMV
***
***
**
*
**g
AH1
Generation of anti-tumor T cell responses requires
tumor irradiation + CTLA-4 blockade
Demaria et al., Clin Cancer Res 2005
Jim Allison
8/3/2016
4
Clinical study design to test for abscopal responses
Abscopal response
IJROBP 2004; Lancet Oncology 2009
-Either a prospective randomized trial (IT+ RT versus IT) -Or a trial of radiation with an immunotherapy proven ineffective when used alone
Limited objective response rate to CTLA-4 Blockade (without and with chemo) in NSCLC
Reference Stage Study Design # PTS OR
Zatloukal et al ASCO 2009
LOCALLY ADV/METS
-TREMELIMUMAB (15 mg/kg) VERSUS BSC 87 4.5% (2 PRs)
Lynch et al JCO 2012
Stage III/IV Carbo/Taxol vs Carbo/T with Ipi (10mg/kg) Carbo/T and Ipi sequential (10mg/kg)
204 NS PFS
No CRs in either studies
Patient with Refractory Metastatic NSCLC
Progressing after 3 lines of chemo
and chest RT: Multiple lung, bone
and liver metastasis
RT to one liver met 6 Gy X 5 ( TD 30 GY)
Ipilimumab, 3 mg/Kg, after first RT q3 weeks, X 4 cycles Golden et al Cancer Immunology Research, 2014
8/3/2016
5
Same patient, response to RT+ ipilimumab
Clinical and radiological CR at one year:
currently NED at 36 m
NYU S14-00208 Ipilimumab and localized RT in chemo-refractory metastatic NSCLC
-100
0
100
200
Ch
ange
in
No
n-
Irra
dia
ted
Le
sio
ns
fro
m B
ase
line
…
Evaluable Patients
39 patients, Response rates (CR + PR): Intent to treat = 18 %
Pts completing 4 Ipi = 33%
0 5 10 150
50
100
Months
% S
urvi
val
CR/PR/SD
PD
Median follow-up: 12 months Log-rank test: p = 0.0161 HR = 9.174 Median survival: CR/PR/SD = not reached PD = 9 months
8/3/2016
6
Same patient: PDL-1 up-regulation as a marker for the
induction of an effective anti-tumor T cell response
CD8 (brown) Ki67 (red)
Other
CD8+
CD8+ Ki67+
Ki67+
Demaria and Stack, (PerkinElmer)
12.1% of CD8 T cells are Ki67+
How to enable the pro-immunogenic effects of radiation:
Overcoming immunosuppressive effects of RT
- overcoming RT-induced
immunosuppression (adenosine,TGFb)
- mitigating RT-induced lymphopenia
Erik Wennerberg
AACR 2016, 4033
RT-induced adenosine
8/3/2016
7
Day: 0 12
Tumor inoculation (right flank, s.c.)
TSA or MCA38
14
20 Gy RT
11 17 20 18
• Isolation and phenotyping of
intratumoral immune cells • Peptide re-stimulation of
tumor-draining lymph node cells
Anti-CD73 mAb (TY/23), i.p. injections
Tumor progression
and
survival
BALB/c WT
or
PHARMACOLOGICAL ADENOSINE BLOCKADE
Adenosine-blockade promotes intratumoral infiltration of CD8α+ activated DCs, reduces Treg infiltration
while promoting CD8+ T cell infiltration
IgG
2a
anti-CD73
20Gy
+ IgG
2a
20Gy
+ anti-
CD730
20
40
60
CD
8a+
DC
s (%
of D
Cs)
**
Anti-CD73 Ab Anti-CD73 Ab Anti-CD73 Ab
IgG
2a
anti-C
D73
20 Gy
+ IgG
2a
20 Gy
+ anti-
CD
730
5
10
15
Tre
gs
(% o
f CD
4+
T c
ells
)
*
2A3
TY/23
20 Gy
+ 2A3
20 Gy
+ TY/2
30
20
40
60
80
CD
8+ T
cel
ls (
% o
f T c
ells
)
Combined RT and anti-CD73 treatment delays tumor progression and prolongs survival
0 10 20 30 400
500
1000
1500
Days after tumor inoculation
Tum
or v
olum
e (m
m3 )
iso ctrl
αCD73
20 Gy + iso ctrl
20 Gy + αCD73
*
0 10 20 25 30 35 40 45 50 55 60 65 700
50
100
Days after tumor inoculation
Perc
ent s
urvi
val
iso ctrl
αCD73
20 Gy + iso ctrl
20 Gy + αCD73 **
Modulation of adenosine generation and/or uptake in the TME may facilitate the immunogenic effect of radiation
8/3/2016
8
Inhibition of
DC activation
TDLN
Inhibition of T cell
effector function
TGFb activation by radiation-induced ROS hinders
priming of anti-tumor T cells
TGFb
LAP
Anti-TGFβ (1D11)
Tum
or
volu
me
(mm
3)
± SE
M
Sham +
Iso
Sham +
1D11
RT +
1D11
RT +
Iso
Lun
g m
etas
tase
s
60
80
20
40
0
Days post tumor cells injection
12 14 16 18 20 22 24 26 28
500 400 300 200 100
0
1000
600 700 800 900 ***
***
***
Therapeutic synergy of radiation and TGFb blockade
11/18 First Fresolimumab+RT to liver 2/8/12 Second Freso+RT to breast skin Response: irSD, 28% reduction, no new lesions
59 F with metastatic Triple
Negative Breast Cancer
4th line therapy 18 months after diagnosis:
RT+ Fresolimumab
Anti-TGFbeta + RT: 22 patients, <10% ORR
RESULTS
8/3/2016
9
Increased PDL-1 and PDL-2 expression on tumor
and myeloid cells by RT and TGFb blockade
Vanpouille –Box, Cancer Research 2015
Days post tumor cells injection 0 10 20 30 40 50 60 70
n=19/gp
% S
urv
ival
20
0
40
60
80
100
Sham + Isotype Sham + 1D11 Sham + a-PD1 1D11 + a-PD1
RT + Isotype RT + 1D11 RT + a-PD1 RT + 1D11 + a-PD1
N=19/group
Days post tumor cells injection 12 14 16 18 20 22 24 26 28 30 10
500 400 300 200 100
0
600 700 800 900
Tum
or
volu
me
(mm
3)
± SE
M
PD-1 blockade extends survival in mice
treated with radiation and TGFb blockade
TCRp-ERK in CD4+ T cells
In vitro anti-PD-1 (pembrolizumab) partially restores TCRp-ERK /p-AKT
PD-1-
- + - +
TCRp-AKT in CD4+ T cells PD-1+ PD-1- PD-1+
Basis for in vitro PD-1 patient selection marker for combination therapies
8/3/2016
10
The delicate balance of cross-presentation
Need for sufficient naïve T cells
8-cm tumor, 60 Gy/30 fractions modeled with Pinnacle™ radiation planning system
Radiation doses to circulating cells (DCC) analyzed using MatLab™
A single radiation fraction delivered 0.5 Gy to 5% of circulating cells,
after 30 fractions 99% of circulating blood had received ≥0.5 Gy
Circulating lymphocytes : D10 = 3 Gy D50 = ~2 Gy D90 = ~.5 Gy
Naïve T cells are the most radiosensitive
Impact of Number of fractions, Dose rate, Target Size
Yovino et al Cancer Invest. 2013
High dose rate Small, superficial fields Hypo-fractionated RT
8/3/2016
11
Conclusions • RT- induced signaling effects interacts with multiple immunological
pathways, including adenosine, TGF-b,PD-1 etc.
• Success of combination of anti-CTLA-4 and radiation in metastatic NSCLC was independent from PD-L1 expression/blockade. Conversely, effectiveness of blocking TGFb likely depends on overcoming PD-L1 expression
• Hypo-fractionated, short courses of RT to a small target to avoid
lymphopenia is likely to be key to the success of RT and immunotherapy
When combined with Immunotherapy what is the best Radiation Source,
Dose, Fractionation?
Looking at the ICD of Protons, Deuterons, and Helium
• RARAF Columbia
• Prep Cells for the Track Segment Charged-Particle Accelerator
• Allows for irradiation of particles with Linear Energy Transfer (10-200KeV/mm).
http://raraf.org/tracksegment.html
8/3/2016
12
At 35kEV, 5Gy of deuterons ~20 Gy x-rays
0
0.5
1
1.5
2
2.5
3
0 Gy (XRAY) 5 Gy (XRAY) 20 Gy (XRAY) 5 Gy Deuterons 25KeV(24.7)
5 Gy Deuterons 35KeV(33.6)
5 Gy Deuterons 50KeV(50.6)
HM
GB
1 [R
FU]
Treatment
HMGB1 Release vs Particle Irradiation
(Unpublished data)
Fractionated but not single dose
RT elicits an abscopal response
in combination with anti-CTLA-4
Dewan et al. Clin Cancer Res. 2009
d0
TSA
d35
d12
d14 d13
d14 d17 d20
d12
20Gy
3x8 Gy
d2
TSA 9H10 – 200 ug/mouse – i.p. 0.0
0.2
0.4
0.6
0.8
Primary tumor weight
Tum
or w
eigh
t (g)
0.0
0.2
0.4
0.6
0.8
Tum
or w
eigh
t (g)
Secondary tumor weight
IR: 9H10:
0 -
0 +
20x1 -
20x1 +
8x3 -
8x3 +
IR: 9H10:
0 -
0 +
20x1 -
20x1 +
8x3 -
8x3 +
0/5
0/5 0/5
0/5 0/5
4/5
2/5
0/5 0/5 0/5 0/5
0/5
Differentially expressed Immune Response genes in at least one of 4 comparisons(>2-fold, Paired T-test p-value<0.05) are displayed as normalized to 0Gy control within each set of three samples.
4.0
1.0
0.25
SD-4hr
SD-24hr
MF-4hr
MF-24hr
Cytokines & Chemokines
SD-4hr
SD-24hr
MF-4hr
MF-24hr
Interferon related genes
interferon type 1 pathway
SD – 20 Gy x1
MF – 8 Gy x3
d0
TSA
d14
d14 d13
4h 24h
d12
20Gy
8 Gy X 3
Balb/c 5-6 wo
Claire Vanpouille-Box
N. Coleman & M. Aryankalayil NIH Radiation Oncology Branch
8/3/2016
13
IFN-I pathway activation in Hypo-Fx tumors
Haller et al. 2007. Cytokine & Growth Factor Reviews 18 (2007) 425–433
Response to Interferon- type I 0
1
2
3
4
5
6
7
8
9
IFN
b1
Mx1
Oas
1a
Oas
1b
Oas
l1
Oas
2
Oas
l2
Oas
3
ISG
15
IRF
7
ifit
1
ifit
2
ifit
3
ifi4
4
ifih
1
ifi2
04
IFN
g
Ccl
5
CxC
l10
Ccl
2
Ccl
7
n-f
old
(0G
y)
20 Gy - 4h20Gy - 24h3x8Gy - 4h3x8Gy - 24h
IFN-I ISG OAS genes ISRE Cytokines Chemokines
No abscopal response demonstrated!
Reject Second Challenge
But concurrent second tumors continue to grow!
8/3/2016
14
Side-by-side comparison of 30 Gyx1, 8 Gyx1 and 8 Gyx3 with Immune Checkpoint Blockade
d0
TSA
d14 d17 d20
d12 30 or 8Gy
d2
9H10 – 200 ug/mouse – i.p. TSA
3x8 Gy
d12 d13 d14
n=7/group
Follow-up
Balb/c 6wo
0Gy 30Gy 8Gy 3x8Gy 9H10
Group 1 X
Group 2 X X
Group 3 X
Group 4 X X
Group 5 X
Group 6 X X
Group 7 X
Group 8 X X
(Unpublished data)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
0Gy0Gy+9H108Gy8Gy+9H10
30Gy30Gy+9H103x8Gy3x8Gy+9H10
***
*
n.s.
n.s.
**
Tumor growth curves - Irradiated site - 8Gyx1 8Gyx1+9H10
30Gyx1 30Gyx1+9H10
8Gyx3+9H10 8Gyx3
T-test
30Gy irradiated tumors regrew faster compared to 8Gy x 3
30Gyx1+9H10 8Gyx3+9H10
12 14 16 18 20 22 24 26 28 30 32 340
50
100
150
200
250
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
50
100
150
200
250
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
Single Fractionated
8/3/2016
15
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
1600
Days post -TSA cells implantationTu
mor
Vol
ume
(mm
3)
Abscopal site (non irradiated)
12 14 16 18 20 22 24 26 28 30 32 340
200
400
600
800
1000
1200
1400
Days post -TSA cells implantation
Tum
or V
olum
e (m
m3)
***
0Gy
0Gy+9H10
8Gy
8Gy+9H10
30Gy
30Gy+9H10
3x8Gy
3x8Gy+9H10
8Gyx1 8Gyx1+9H10
30Gyx1 30Gyx1+9H10
8Gyx3+9H10 8Gyx3
T-test
Survival
0 10 20 30 40 50 60 70 800
20
40
60
80
100
Days post -TSA cells implantation
Per
cent
sur
viva
l
0Gy0Gy+9H108Gy8Gy+9H10
30Gy30Gy+9H103x8Gy3x8Gy+9H10
Endpoints: -Abscopal response -Survival
8 Gy X 3 superior to single fraction 8/30 Gy
Dose and fractionation and RT source
• When combined with ICB tumor hypo-fractionated regimens are required for abscopal effects
• High LET radiation more likely to induce ICD
8/3/2016
16
S. Formenti M.D. E. Golden M.D.,Ph.D. J. Kang M.D., Ph.D. John Ng, M.D. Wen Shen, Ph.D
C. Vanpouille-Box Ph.D. Karsten Pilones Ph D. M. Kerimian, N.P.
Radiation and Immunity Research Team
S. Demaria M.D.
S. Chandrasekhar, MS Erik Wennerberg, Ph .D.
Our patients