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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?

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• 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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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!

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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

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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

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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