Convergence of radiation and immunogenic signaling pathways

Transcription

1 Convergence of radiation and immunogenic signaling pathways Silvia Formenti, M.D. Weill Cornell Medical College New York Presbyterian Hospital New York, NY

2 Principal Investigator: DISCLOSURES Consultant/Speaker: Bristol Myers Squibb, Varian, Elekta, Janssen, Regeneron, GlaxoSmithKline, Eisai, Dynavax, Astra Zeneca 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

3 Radiation-induced immunogenic cell death and cross-priming Need for sufficient naïve T cells

4 Radiotherapy and immune-mediated rejection: a balancing act TGFb ActivinA (PS3-63) Adenosine PDL-1 T regs IFN-I ATP NKG2D-Ligands Teff chemokines Formenti & Demaria, J Natl Cancer Inst 213

5 Radiation as a generator of T cells: overcoming immunosuppression in the TME (adenosine, TGFb) Role of the PD-1/PDL-1 pathway in resistance to treatment with RT and TGFb neutralization RT re-positioning of anti-ctla-4 in NSCLC : dose fractionation and technique for the abscopal effect

6 Oncoimmunology, 214 CRT In vitro assay for RT-induced ICD HMGB1 ATP Encouse Golden

7 Blockade of adenosine generation improves recruitment and maturation of DCs and tumor response to SBRT Erik Wennerberg AACR 433 Session 27 RT ATP CD39 CD73 ADO TUM Treg TUM Tumor Ag A2AR ATP DC activated DC

8

9 Does adenosine blockade improve radiation-induced anti-tumor immunity? PHARMACOLOGICAL ADENOSINE BLOCKADE BALB/c WT 2 Gy RT Isolation and phenotyping of intratumoral immune cells Peptide re-stimulation of tumor-draining lymph node cells Day: Tumor progression and survival Tumor inoculation (right flank, s.c.) TSA or MCA38 Anti-CD73 mab (TY/23), i.p. injections or A2AR-inhibitor (SCH58261, daily i.p. Injections) A2AR-DEFICIENT MICE C57BL/6 WT / Adora2A -/- 2 Gy RT Isolation and phenotyping of intratumoral immune cells Peptide re-stimulation of tumor-draining lymph node cells Day: Tumor inoculation (right flank, s.c. MCA38)

10 CD8a+ DCs (% of DCs) Adenosine-blockade promotes intratumoral infiltration of CD8α+ activated DCs Anti-CD73 Ab (TY/23) A2AR inhibitor (SCH58261) A2AR KO mice (ADORA2A -/- ) 6 ** 25 4 CD8a+ DCs (% of DCs) 4 2 IgG2a anti-cd73 2Gy + IgG2a 2Gy + anti-cd PBS A2ARi 2 Gy 2 Gy + A2ARi CD8a+ DCs (% of DCs) WT ADORA2A -/- WT + 2 Gy ADORA2A -/- + 2Gy

11 CD73-blockade reduces radiation mediated Treg infiltration while promoting CD8+ T cell infiltration Anti-CD73 Ab (TY/23) Anti-CD73 Ab (TY/23) 15 8 Tregs (% of CD4+ T cells) 1 5 * CD8+ T cells (% of T cells) IgG2a anti-cd73 2 Gy + IgG2a 2 Gy + anti-cd73 2A3 TY/23 2 Gy + 2A3 2 Gy + TY/23

12 Combined RT and anti-cd73 treatment delays tumor progression and prolongs survival Tumor volume (mm 3 ) iso ctrl αcd73 2 Gy + iso ctrl 2 Gy + αcd73 * Percent survival Days after tumor inoculation iso ctrl αcd73 2 Gy + iso ctrl 2 Gy + αcd73 ** Days after tumor inoculation Modulation of adenosine generation and/or uptake in the TME following tumor tx may facilitate the immunogenic effect of radiation

13 TGFb activation by radiation-induced ROS hinders priming of anti-tumor T cells Anti-TGFβ (1D11) TDLN LAP TGFb Inhibition of DC activation Inhibition of T cell effector function

14 Tumor volume (mm3) ± SEM Lung metastases Therapeutic synergy of radiation and TGFb blockade 1 9 *** *** Days post tumor cells injection *** Sham + Iso Sham + 1D11 RT + Iso RT + 1D11

15 Fresolimumab and Radiotherapy in Metastatic Breast Cancer Fresolimumab: 1 or 1 mg/kg IV RT: 7.5 Gy x 3, weeks 2 and 7 Blood: weeks, 2, 5 and 15 Response assessed at week 15 (PET/CT) NCT14162, supported by DOD Breast Cancer Research Program- Multi- Team Award (PI S. Formenti)

16 Increase in tumor-specific CD8 T cells in patients treated with radiotherapy + fresolimumab 5/9 HLA-A2+ patients showed increase/induction of survivin-specific CD8 T cells by tetramer analysis

17 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 Fresolimumab+RT to liver 2/8/12 Second Freso+RT to breast skin Response: irsd, 28% reduction, no new lesions

18 Comparison of OS and PFS based on fresolimumab dose Accrued 22 patients:11 per arm fresolimumab dose ( arm A=1mg, arm B=1 mg)

19 BACK TO THE MOUSE Is adaptive immune resistance limiting tumor response to radiotherapy + TGFb blockade?

20 Increased PDL-1 and PDL-2 expression on tumor and myeloid cells by RT and TGFb blockade Vanpouille Box, Cancer Research 215

21 Tumor volume (mm3) ± SEM % Survival PD-1 blockade extends survival in mice treated with radiation and TGFb blockade Sham + Isotype Sham + 1D11 Sham + a-pd1 1D11 + a-pd1 RT + Isotype RT + 1D11 RT + a-pd1 RT + 1D11 + a-pd Days post tumor cells injection n=19/gp N=19/group Days post tumor cells injection

22 PD-1 blockade delays tumor recurrence after radiation and TGFb blockade Tumor volume (mm3) ± SEM Tumor volume (mm3) ± SEM RT RT alone Tumor volume (mm3) ± SEM RT+an -TGFb RT + 1D11 1 Local RT + multiple ITs may be required in established 5 tumors RT+an + a-pd1 -PD-1 RT+an + 1D11 -TGFb+an + a-pd1 -PD Days post tumor cells injec on Tumor volume (mm3) ± SEM Days post tumor cells injec on Vanpouille-Box, Cancer Research 215

23 Poster #4987, session 26 Elevated baseline levels of PD-1 and PD-L1 expression and reduced TCR signaling in breast cancer patients (SCNP) PD-L1 in CD4+ T Cells PD-L1 in Monocytes PD-1 in CD4+ T Cells OX-4 in CD4+ T Cells Single cell network profiling at baseline: 7 healthy donors and 15 BC patients (antitgfb/rt trial)

24 Reduced TCR signaling in PD-1+ CD4+ and CD8+ T cells vs PD-1- T cells PD-1 : TCR p-erk CD4+ - CD PD-1 : TCR p-akt CD4+ CD Healthy donors Colors represent different donors Breast cancer patients

25 In vitro anti-pd-1 (pembrolizumab) partially restores TCR p-erk /p-akt TCR p-erk in CD4+ T cells PD-1- TCR p-akt in CD4+ T cells PD-1+ PD-1- PD Basis for in vitro PD-1 patient selection marker for combination therapies

26 Radiation as a generator of T cells: overcoming immunosuppression in the TME (adenosine, TGFb) Role of the PD-1/PDL-1 pathway in resistance to treatment with RT and TGFb neutralization RT re-positioning of anti-ctla-4 in NSCLC : dose fractionation and technique for the abscopal effect

27 1 * 75 tumor irradiation + CTLA-4 5 blockade *** AH1 ** 1 Generation of anti-tumor T cell responses requires IFNδ g (pg/ml) ** *** AH-1-A pmcm Jim Allison Strain BALB/c WT NKT -/- α-ctla RT α-cd1d Demaria et al., Clin Cancer Res 25

28 RT and anti-ctla-4 drive oligoclonal expansion of CD8 TILs CDR3 region of B chain =unique identifier Clonality vs % CD8 Percentage of annotated T-cells Control CD8 CD4! -CTLA-4 RT RT+! -CTLA4 Clonality.4 RT+anti-CTLA-4.3 Anti-CTLA-4.2 RT.1 r=.8944 p< % CD8 Pilones et al., AACR 215

29 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

30 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 LOCALLY ADV/METS -TREMELIMUMAB (15 mg/kg) VERSUS BSC % (2 PRs) Lynch et al JCO 212 Stage III/IV Carbo/Taxol vs Carbo/T with Ipi (1mg/kg) Carbo/T and Ipi sequential (1mg/kg) 24 NS PFS No CRs in either studies

31 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

32 Same patient, response to RT+ ipilimumab

33 Clinical and radiological CR at one year: currently NED at 36 m

34 Change in Non-Irradiated Lesions from Base line (%) 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% CR/PR/SD PD % Survival Evaluable Patients Months Median follow-up: 12 months Log-rank test: p =.161 HR = Median survival: CR/PR/SD = not reached PD = 9 months

35 NYU S14-28 Ipilimumab and localized RT in chemo-refractory metastatic NSCLC TCR repertoire changes in PBMC (baseline versus day 22) 6 ** * 15 * ** Number of contracted clones PD SD CR+PR Number of expanded clones PD SD CR+PR N = N = Adaptive Biotechnologies ImmunoSEQ platform

36 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+ Ki % of CD8 T cells are Ki67+ Demaria and Stack, (PerkinElmer)

37 Radiation Dose, Fractionation Technique

38 Primary tumor weight Fractionated but not single dose RT elicits an abscopal response in combination with anti-ctla-4 Tumor weight (g) /5 /5 /5 /5 /5 TSA d TSA d2 9H1 2 ug/mouse i.p. d14 d17 d2 d35. IR: 9H1: - + 2x1-2x1 + 8x3-4/5 8x3 + Secondary tumor weight d12 2Gy.8 /5 d12d13d14 3x8 Gy Tumor weight (g) /5 /5 /5 /5 2/5 Dewan et al. Clin Cancer Res. 29. IR: 9H1: - + 2x1-2x1 + 8x3-8x3 +

39 Cytokines & Chemokines SD-4hr TSA d 4h 24h SD-24hr MF-4hr MF-24hr Balb/c 5-6 wo d14 2Gy Interferon related genes d12d13 d14 SD-4hr 8 Gy X 3 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 x Differentially expressed Immune Response genes in at least one of 4 comparisons(>2- fold, Paired T-test p-value<.5) are displayed as normalized to Gy control within each set of three samples.

40 IFNb1 Mx1 Oas1a Oas1b Oasl1 Oas2 Oasl2 Oas3 ISG 15 IRF 7 ifit1 ifit2 ifit3 ifi44 ifih1 ifi24 IFNg Ccl5 CxCl1 Ccl2 Ccl7 n-fold (Gy) IFN-I pathway activation in irradiated 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

41 The delicate balance of cross-presentation Need for sufficient naïve T cells

42 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

43 Impact of Number of fractions, Dose rate, Target Size High dose rate Small, superficial fields Hypo-fractionated RT Yovino et al Cancer Invest. 213

44 Conclusions RT- induced signaling 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 may be key to the success of RT and immunotherapy

45 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.