Combining radiotherapy with in metastatic

Silvia C Formenti, M.D. New York University School of Medicine and Langone Medical Center

WIN 2014 Symposium • 23-24 June • Paris • France Disclosures

. P.I. DOD Multi-Team Award . P.I. BCRF grant . P.I NIH R01 . 1 S10 RR027619-01 (NIH) . P.I. peer-reviewed Varian research grant (No personal salary support) . Scientific Advisory Board of Bristol Meyers Squibb, Oncoimmunology Division

Fresolimumab trial: drug gifted by /Aventis (FDA-IND) NSCLC trial: drug gifted by Bristol Myers Squibb (FDA-IND)

WIN 2014 Symposium • 23-24 June • Paris • France How do standard anti-cancer treatments interfere with this landscape ?

Dunn et al, Nature Immunology 2002 Koebel et al, Nature 2007 WIN 2014 Symposium • 23-24 June • Paris • France IN SITU VACCINATION HYPOTHESIS RT

TUMOR

CD4 T cell

IL-12

CD8 T cell

WIN 2014 Symposium • 23-24 June • Paris • France Formenti & Demaria, IJROBP 2004; Lancet Oncology 2009 Cross‐priming of anti‐tumor T cells: immunogenic cell death

CRT HMGB-1! TLR4 ! IL-1 ! ! TUMOR DDC C

P2RX7 CD4 T cell ATP

Tumor cell lysis IL-12 CD8 CTL activation T cell IL-2 IFN

CRT, “eat me” signal, translocation to membrane induced by ER stress (Obeid et al., Nat Med 2007, 13:54-61)

ATP released by dying cells by autophagy binds to P2RX7 purinergic receptor leading to inflammasome activation and IL-1 production (Ghiringhelli et al., Nat Med 2009, 15:1170)

HMGB-1, a damage associated molecular pattern (DAMP) binds to TLR4 to promote cross-presentation of tumor-derived antigens (Apetoh et al., Nat Med 2007, 13:1050) WIN 2014 Symposium • 23-24 June • Paris • France In vitro assay for RT-induced ICD (Golden et al. OncoImmunology 2014)

Encouse Golden

WIN 2014 Symposium • 23-24 June • Paris • France Oncoimmunology, in press Abscopal Effect Latin ab (position away from) and scopus (mark or target) *Mole RJ. Whole body irradiation - radiology or medicine? Br J Radiol 1953; 26:234

RELEVANCE TO METASTATIC CANCER

Abscopal response

WIN 2014 Symposium • 23-24 June • Paris • France Why are abscopal effects of radiation rarely observed in the clinic?

WIN 2014 Symposium • 23-24 June • Paris • France IMMUNOSUPPRESSION DOMINATES IN ESTABLISHED TUMORS

Vesely MD, 2011, Annu.Rev.Immunol 29:235-71 WIN 2014 Symposium • 23-24 June • Paris • France Immunotherapy strategies to combine with radiation

Priming phase : . FLT-3L/GM-CSF . TLR agonists

Effector phase: . Anti-CTLA4 . Anti-PD-1 . Anti-TGF

WIN 2014 Symposium • 23-24 June • Paris • France Hypothesis: Ionizing radiation can stimulate anti-tumor immunity –by generating an in situ vaccine - and combination with immunotherapy may uncover this effect

Day: 0 20 67NR RT Flt3-L (0.5mg/kg) 5x104 or 105 2 Gy each sides,

primary R and secondary L

R L “primary” “secondary” non-irradiated X RT

BALB/C mice injected at two separate sites with the syngeneic mammary carcinoma 67NR cell line WIN 2014 Symposium • 23-24 June • Paris • France RT+Flt3-L : systemic anti-cancer effects irradiated non-irradiated

WIN 2014 Symposium • 23-24 June • Paris • France Abscopal Effect is tumor specific and abrogated in nude mice

WIN 2014 Symposium • 23-24 June • Paris • France NYU 0258 Abscopal trial RT+GM-CSF in metastatic solid tumors

DAY 1 8 15 22 29 36 42 49-56 Week 1, 2 Week 4, 5 RT RT

Week 2, 3 Week 5, 6 GM-CSF GM-CSF

Within 2 weeks from study entry: Baseline measurements End of Week 3 Week 7-8 CT and PET Assess clinical response Assess clinical response and CT/PET response

RT 3.5GyX10

GM-CSF 125 g/m2

WIN 2014 Symposium • 23-24 June • Paris • France NYU 0258 Abscopal trial RT+GM-CSF in metastatic solid tumors

Cycle 1 Baseline 5 yr F/U 7/12/2007 4/4/2007 7/28/2012

WIN 2014 Symposium • 23-24 June • Paris • France Lancet Oncology 2009 Abscopal effect 10/37 (27%) Abscopal effect and survival, 37 patients (NYU 02-58)

A.

Abscopal responders likely to be patients already more immunocompetent WIN 2014 Symposium • 23-24 June • Paris • France ASTRO 2012 4T1 mouse model of metastatic breast cancer

Lung metastases Anti-CTLA-4 mAb 9H10

Primary tumor

Experimental Endpoints: - Primary Tumor growth - Lung metastasis - Immune response evaluation WIN 2014 Symposium • 23-24 June • Paris • France WIN 2014 Symposium • 23-24 June • Paris • France Characterization of T cell-tumor cell interactions by intravital two-photon laser scanning microscopy

Michael Dustin

Injection of RT RT 4T1 tumor cells

day 0 13 1415 16 18 22

2-p intravital imaging ‘Short term’ ‘Long term’ WIN 2014 Symposium • 23-24 June • Paris • France Radiation up-regulates ICAM-1 and Rae1 on 4T1 cancer cells in vivo

CD8 T CELLS TUMOR CELLS (day 16) NONE RT+9H10

TUMOR

SPLEEN NKG2D

TDLN

CD69 WIN 2014 Symposium • 23-24 June • Paris • France Ruocco et al JCI 2012 Blocking NKG2D abolishes immune-mediated tumor inhibition by combination of RT+anti-CTLA-4

Ruocco et al JCI 2012 WIN 2014 Symposium • 23-24 June • Paris • France Radiation-induced Rae-1/NKG2D interaction is required for stable immunological synapse of tumor and T cells

TCR

NKG2D CD8 RAE-1

CTLA-4

ICAM-1

MHC-I

Anti-CTLA-4 CD8 LFA1

Clinical translation: NKG2D and its ligand MHC class I chain-related protein A (MICA)

WIN 2014 Symposium • 23-24 June • Paris • France Ruocco et al., J Clin Iinvest 2012 12 ongoing trials testing RT and Ipilimumab:

At NYU: - randomized trial -NSCLC Phase II trial

WIN 2014 Symposium • 23-24 June • Paris • France Postov et al 2012 Patient with 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 WIN 2014 Symposium • 23-24 June • Paris • France Golden et al Cancer Immunology Research, 2014 Metastatic NSCLC: Response to RT+ipilimumab

WIN 2014 Symposium • 23-24 June • Paris • France Comparison of SCV metastasis at diagnosis (2010) and after RT and Ipilimumab (2013) CD8 2010 2013 200

150

100

H&E 50 0

200 Fox-P3

150

100 cells/HPF

CD8 50

0

2.5

2.0

1.5 TIA-1 1.0

0.5 Ratio CD8/FoxP3 cells/HPF 0.0 2010 2013 WIN 2014 Symposium • 23-24 June • Paris • France Cancer Imm. Research, 2014 Clinical and radiological CR at one year ( currently NED at 22 months from tx)

WIN 2014 Symposium • 23-24 June • Paris • France Pre-clinical to clinical testing of RT and immunotherapy in metastatic cancer

. Flt3LPhys, 2004) (Demaria et al., Int J Radiat Oncol Biol . NYU 02-58 Proof of principle abscopal trial

2005; Matsumura et al., J Immunol 2008; Pilones et . anti-CTLA-4al., Clin Cancer Res (Demaria 2009; Dewanet al., Clin et al., Cancer Clin CancerRes . open trial in met melanoma Res 2009; Ruocco et al., J Clin Invest 2012) (NCT01689974) . open trial in met NSCLC . Developing trial of in met breast cancer

2012) . TLR7-agonist (Dewan et al. Clin Cancer Res . open trial in met BC (NCT01421017)

2012) . anti-TGFBouquet et al Clin Cancer Res . open trial in met BC (NCT01401062)

WIN 2014 Symposium • 23-24 June • Paris • France Preclinical efficacy of low dose CTX when added to IMQ/RT

Adams et al Clin Can Res 2012 WIN 2014 Symposium • 23-24 June • Paris • France 3 ARMS IN METASTATIC BREAST CANCER

Imiquimod (IMQ) a synthetic, TLR-7 agonist Aldara is an IMQ topical 3 arms study in metastatic BC cream FDA approved for treatment of superficial With skin mets: RT + IMQ basal cell carcinoma, CTX+RT+IMQ actinic keratosis, and external genital warts Without skin mets: CTX+ RT

CTX 200mg i.v. I week before RT

WIN 2014 Symposium • 23-24 June • ParisAdams• France Clin Can Res, 2012 TLR7 agonist Imiquimod: Abscopal response

Baseline vs post-tx photo of RT + Imiquimod) Baseline vs post-tx photo of Control area (NO Imiquimod)

Response at a distant site of disease

10/2012 01/2013 03/2013

WIN 2014 Symposium • 23-24 June • Paris • France NCI-R01 Immunomodulation of breast cancer via TLR7 agonist IMQ and RT, 2011-15 Immunotherapy strategies to combine with radiation

Priming phase : . FLT-3L/GM-CSF, . TLR agonists

Effector phase: . Anti-CTLA4 . Anti-PD-1, . Anti-TGF

WIN 2014 Symposium • 23-24 June • Paris • France Inhibition of

CD4 T cell Immunosuppression CD8 T cell effector T cells

Activation of latent TGF Induction of regulatory T cells

Treg Treg Treg Inhibition of Treg TC DC maturation

Cross-priming

TAA Release

TDLN Decreased DDR TAA Uptake Anti‐TGFβ by DC Cross-priming Latency Associated Peptide Anti-tumor immunity TGF‐ WIN 2014 Symposium • 23-24 June • Paris • France RT synergizes with TGF neutralizing Ab to in immunocompetent models of breast and lung tumors, but not in the immunocompromised model 4T1

1000 13C4 1D11 800  RT + 13C4 6Gyx5 RT + 1D11 600 4T1  400

Tumor Volume (mm3) Tumor Volume 

200 4T1 model in nude mice 0 12 14 16 18 20 22 24 26 28

1200 1100 1000 900  800 700 6Gyx5 600 LLC 500  400 Tumor Volume (mm3) Tumor Volume 300 200 6Gyx5 100 0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Day

16 14 12 10 6Gyx5 1/10 complete regression 8 TSA 6  Tumor Volume 4 Normalized to day 13 2 4/7 complete regression 0 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Days post tumor inoculationWIN 2014 Symposium • 23-24 June • Paris • France 4T1 5x6Gy d0 d13 d14 d15 d16 d17 d21 d28

Tumor growth and survival

BALB/c mice d12 d14 d16 d18 d20 d22 d24 d26 200 µg/mouse 1D1 1D1 1D1 1D1 1D1 1D1 1D1 1D1 1 1 1 1 1 1 1 1

**

) * 3 *** *** *** (mm

** volume

metastases

Tumor Lungs

Days post inoculation

WIN 2014 Symposium • 23-24 June • Paris • France CLINICAL TRIAL IN METASTATIC BC

WIN 2014 Symposium • 23-24 June • Paris • France Accrued 22 patients:11 per arm Comparison of OS and PFS based on fresolimumab dose ( arm A=10mg, arm B=1 mg)

WIN 2014 Symposium • 23-24 June • Paris • France Fresolimumab and radiation (7.5 GyX3) to one lesion Patient #2

11/10/11 before tx 12/19/11 a month after first TX 2/27/12 Last PET/CT 11/18/11 First Fresolimumab+RT to liver 2/8/12 Second Freso+RT to breast skin Response reported as irSD, 28% reduction, no new lesions,

WIN 2014 Symposium • 23-24 June • Paris • France Slide content not available for publication

WIN 2014 Symposium • 23-24 June • Paris • France CONCLUSIONS

Ionizing radiation can convert the tumor into an in situ vaccine

Promising partner with immunotherapy: localized/predictable toxicity approved, standard modality accessible at any cancer center easy to standardize for a consortium trial

WIN 2014 Symposium • 23-24 June • Paris • France RT and Immunity Team

MH Barcellos-Hoff Ph.D. Encouse Golden M.D.,Ph.D. Mike Dustin Ph.D. Sandra Demaria M.D.

Claire Vanpouille-Box Ph.D. Karsten Pilones Ph D. Keith DeWyngaert, Ph.D. Maria Fenton-Kerimian, N.P. WIN 2014 Symposium • 23-24 June • Paris • France Our patients