8/3/2016. Why are abscopal effects of radiation so rare? Radiation therapy to ignite an anti-cancer immune response DISCLOSURES

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1 8/3/216 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 Consultant/Speaker: Bristol Myers Squibb, Varian, Elekta, Janssen, Regeneron, GlaxoSmithKline, Eisai, Dynavax, Astra Zeneca Principal Investigator: NCI R1CA Immunomodulation of breast cancer via TLR7 agonist IMQ and RT DOD BC1481 / W81XWH Multi-Team Award (MTA) Radiation-Induced Vaccination to Breast Cancer 13-A Breast Cancer Research Foundation Targeting key inhibitory pathways to improve radiation-induced vaccination in breast cancer NIH 1 S1 RR Preclinical Research Irradiator Why are abscopal effects of radiation so rare? 1

2 8/3/ : 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, 211, Annu.Rev.Immunol 29: How to enable the pro-immunogenic effects of radiation: Overcoming immunosuppressive microenvironment of established tumors - enhancing cross priming - blocking immune checkpoints Overcoming RT immunosuppressive effects - overcoming RT-induced immunosuppression (adenosine,tgfb) - mitigating RT-induced lymphopenia c) 2

3 IFNδ g (pg/ml) 8/3/216 Ionizing radiation stimulates anti-tumor immunity by generating an in situ vaccine: combination with immunotherapy uncovers the abscopal effect Day: 67NR 5x1 4 or 1 5 each sides, primary R and secondary L 2 RT 2 Gy Flt3-L (.5mg/kg) RT R primary X L secondary BALB/C mice injected at two separate sites with the syngeneic mammary carcinoma 67NR cell line RT: 3.5 GyX1 GM-CSF: 125 mg/m 2 Daily X 14 days 27% ABSCOPAL ORR 1 * 75 Generation of anti-tumor T cell responses requires ** 5 *** tumor irradiation CTLA-4 blockade AH ** *** AH-1-A pmcm Jim Allison Strain BALB/c WT NKT -/- α-ctla RT α-cd1d Demaria et al., Clin Cancer Res 25 3

4 8/3/216 Clinical study design to test for abscopal responses -Either a prospective randomized trial (IT RT versus IT) -Or a trial of radiation with an immunotherapy proven ineffective when used alone Abscopal response IJROBP 24; Lancet Oncology 29 Limited objective response rate to CTLA-4 Blockade (without and with chemo) in NSCLC Reference Stage Study Design # PTS OR Zatloukal et al ASCO 29 Lynch et al JCO 212 LOCALLY ADV/METS Stage III/IV -TREMELIMUMAB (15 mg/kg) VERSUS BSC % (2 PRs) Carbo/Taxol vs Carbo/T with Ipi (1mg/kg) Carbo/T and Ipi sequential (1mg/kg) 24 NS PFS No CRs in either studies Progressing after 3 lines of chemo and chest RT: Multiple lung, bone and liver metastasis Patient with Refractory Metastatic NSCLC RT to one liver met 6 Gy X 5 ( TD 3 GY) Ipilimumab, 3 mg/kg, after first RT q3 weeks, X 4 cycles Golden et al Cancer Immunology Research, 214 4

5 Change in Non- Irradiated Lesions from Base line % Survival 8/3/216 Same patient, response to RT ipilimumab Clinical and radiological CR at one year: currently NED at 36 m NYU S14-28 Ipilimumab and localized RT in chemo-refractory metastatic NSCLC 39 patients, Response rates (CR PR): Intent to treat = 18 % Pts completing 4 Ipi = 33% 2 1 CR/PR/SD PD Evaluable Patients Months Median follow-up: 12 months Log-rank test: p =.161 HR = Median survival: CR/PR/SD = not reached PD = 9 months 5

6 8/3/216 Same patient: PDL-1 up-regulation as a marker for the induction of an effective anti-tumor T cell response CD8 (brown) Ki67 (red) Demaria and Stack, (PerkinElmer) Other CD8 CD8 Ki67 Ki % 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 RT-induced adenosine Erik Wennerberg AACR 216, 433 6

7 Tumor volume (mm 3 ) CD8a DCs (% of DCs) Tregs (% of CD4 T cells) Percent survival CD8 T cells (% of T cells) 8/3/216 PHARMACOLOGICAL ADENOSINE BLOCKADE Isolation and phenotyping of intratumoral immune cells Peptide re-stimulation of BALB/c 2 Gy RT tumor-draining lymph node cells WT Day: Tumor progression and survival Tumor inoculation (right flank, s.c.) TSA or MCA38 Anti-CD73 mab (TY/23), i.p. injections or Adenosine-blockade promotes intratumoral infiltration of CD8α activated DCs, reduces Treg infiltration while promoting CD8 T cell infiltration Anti-CD73 Ab Anti-CD73 Ab Anti-CD73 Ab 6 ** 15 * IgG2a anti-cd73 2Gy IgG2a 2Gy anti-cd73 IgG2a anti-cd73 2 Gy IgG2a 2 Gy anti-cd73 2A3 TY/23 2 Gy 2A3 2 Gy TY/23 Combined RT and anti-cd73 treatment delays tumor progression and prolongs survival 15 iso ctrl αcd Gy iso ctrl 2 Gy αcd iso ctrl αcd73 2 Gy iso ctrl 2 Gy αcd73 ** 5 * Days after tumor inoculation Days after tumor inoculation Modulation of adenosine generation and/or uptake in the TME may facilitate the immunogenic effect of radiation 7

8 Tumor volume (mm3) ± SEM Lung metastases 8/3/216 TGFb activation by radiation-induced ROS hinders priming of anti-tumor T cells Anti-TGFβ (1D11) LAP TGFb TDLN Inhibition of DC activation Inhibition of T cell effector function Therapeutic synergy of radiation and TGFb blockade 1 *** 9 *** *** Days post tumor cells injection Sham Iso Sham 1D11 RT Iso RT 1D11 RESULTS Anti-TGFbeta RT: 22 patients, <1% ORR 59 F with metastatic Triple Negative Breast Cancer 4 th line therapy 18 months after diagnosis: RT Fresolimumab 11/18 First FresolimumabRT to liver 2/8/12 Second FresoRT to breast skin Response: irsd, 28% reduction, no new lesions 8

9 Tumor volume (mm3) ± SEM % Survival 8/3/216 Increased PDL-1 and PDL-2 expression on tumor and myeloid cells by RT and TGFb blockade Vanpouille Box, Cancer Research 215 PD-1 blockade extends survival in mice treated with radiation and TGFb blockade Sham Isotype Sham 1D11 Sham a-pd1 1D11 a-pd Days post tumor cells injection RT Isotype RT 1D11 RT a-pd1 RT 1D11 a-pd n=19/gp N=19/group Days post tumor cells injection In vitro anti-pd-1 (pembrolizumab) partially restores TCR p-erk /p-akt TCR p-erk in CD4 T cells TCR p-akt in CD4 T cells PD-1- PD-1 PD-1- PD Basis for in vitro PD-1 patient selection marker for combination therapies 9

10 8/3/216 The delicate balance of cross-presentation Need for sufficient naïve T cells 8-cm tumor, 6 Gy/3 fractions modeled with Pinnacle radiation planning system Radiation doses to circulating cells (DCC) analyzed using MatLab Circulating lymphocytes : D1 = 3 Gy D5 = ~2 Gy D9 = ~.5 Gy A single radiation fraction delivered.5 Gy to 5% of circulating cells, after 3 fractions 99% of circulating blood had received.5 Gy Naïve T cells are the most radiosensitive Impact of Number of fractions, Dose rate, Target Size High dose rate Small, superficial fields Hypo-fractionated RT Yovino et al Cancer Invest

11 8/3/216 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 (1-2KeV/mm). 11

12 HMGB1 [RFU] Tumor weight (g) Tumor weight (g) 8/3/216 At 35kEV, 5Gy of deuterons ~2 Gy x-rays 3 HMGB1 Release vs Particle Irradiation Gy (XRAY) 5 Gy (XRAY) 2 Gy (XRAY) 5 Gy Deuterons 25KeV 5 Gy Deuterons 35KeV 5 Gy Deuterons 5KeV (24.7) (33.6) (5.6) Treatment (Unpublished data) Primary tumor weight Fractionated but not single dose RT elicits an abscopal response in combination with anti-ctla-4 TSA d TSA d2 d12 2Gy 9H1 2 ug/mouse i.p. d14 d17 d2 d35 d12 d13 d14 3x8 Gy IR: 9H1: /5 /5 - /5 /5 /5 2x1 - /5 2x1 Secondary tumor weight /5 /5 /5 8x3 - /5 4/5 8x3 2/5 Dewan et al. Clin Cancer Res. 29. IR: 9H1: - 2x1-2x1 8x3-8x3 Cytokines & Chemokines SD-4hr Balb/c 5-6 wo TSA d d14 2Gy d12 d13 d14 8 Gy X 3 4h 24h Interferon related genes SD-24hr MF-4hr MF-24hr SD-4hr SD-24hr MF-4hr MF-24hr interferon type 1 pathway Claire Vanpouille-Box N. Coleman & M. Aryankalayil NIH Radiation Oncology Branch SD 2 Gy x1 MF 8 Gy x3 4. Differentially expressed Immune Response genes in at least one of 4 comparisons(>2-1. fold, Paired T-test p-value<.5) are.25 displayed as normalized to Gy control within each set of three samples. 12

13 n-fold (Gy) IFNb1 Mx1 Oas1a Oas1b Oasl1 Oas2 Oasl2 Oas3 ISG 15 IRF 7 ifit1 ifit2 ifit3 ifi44 ifih1 ifi24 IFNg Ccl5 CxCl1 Ccl2 Ccl7 8/3/216 IFN-I pathway activation in Hypo-Fx tumors Gy - 4h 2Gy - 24h 3x8Gy - 4h 3x8Gy - 24h Response to Interferon- type I Haller et al. 27. Cytokine & Growth Factor Reviews 18 (27) IFN-I OAS genes ISRE ISG Cytokines Chemokines Reject Second Challenge But concurrent second tumors continue to grow! No abscopal response demonstrated! 13

14 /3/216 Side-by-side comparison of 3 Gyx1, 8 Gyx1 and 8 Gyx3 with Immune Checkpoint Blockade TSA TSA 9H1 2 ug/mouse i.p. d d2 d14 d17 d2 Follow-up Balb/c 6wo d12 3 or 8Gy d12 d13 d14 3x8 Gy n=7/group Gy 3Gy 8Gy 3x8Gy 9H1 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) Tumor growth curves - Irradiated site - 8Gyx1 8Gyx19H ** 4 2 *** Gy 3Gy n.s. Gy9H1 3Gy9H1 n.s. 8Gy 3x8Gy 8Gy9H1 3x8Gy9H1 * Gyx Gyx Gyx19H Gyx39H1 T-test Single 3Gyx19H1 25 Fractionated 8Gyx39H Gy irradiated tumors regrew faster compared to 8Gy x 3 14

15 Percent survival /3/216 Abscopal site (non irradiated) 8Gyx1 8Gyx19H Gy Gy9H1 8Gy 8Gy9H1 3Gy 3Gy9H1 3x8Gy 3x8Gy9H1 *** Gyx Gyx Gyx19H Gyx39H1 T-test Survival Endpoints: -Abscopal response -Survival Gy Gy9H1 8Gy 8Gy9H1 3Gy 3Gy9H1 3x8Gy 3x8Gy9H1 8 Gy X 3 superior to single fraction 8/3 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 15

16 8/3/216 Radiation and Immunity Research Team Our patients S. Formenti M.D. S. Demaria 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. Erik Wennerberg, Ph.D. S. Chandrasekhar, MS M. Kerimian, N.P. 16