Induction of immune response by combining TLR activation and radiotherapy

Transcription

1 Induction of immune response by combining TLR activation and radiotherapy Peter Huber Molecular & Radiation Oncology dkfz/university hospital Heidelberg

2 Nothing to disclose

3 Physical Dose Precision Meningioma H&N Esophagus Anal cancer Mesothelioma Sarcoma

4 Clinically radioresistance associated with expression of pro-survival signaling Specific drugs EGFR Cetuximab, Erlotinib VEGF Bevacizumab, Sunitinib, HIF1-alpha (HIF) Prolyl Hydroxylase Inhibitor (PHI) HDAC SAHA, Valproic acid PDGF SU6668, Imatinib, Sunitinib, Cox2 Celecoxib PI3K, Akt, Enzastaurin mtor Rapamycin, Everolimus, Integrins Cilengitide, S-247 TGF-beta CD44 CTGF TGF-beta RKI TGF-beta RKI CTGF AB

5 Rationales for combined radiation and targeted drug therapy VEGF, PDGF, TGFß, COX2, Integrins, CTGF, PI3K, Akt, EGFR, HDAC Biomaterials Cancer Cell Cancer Letters Cancer Res Clin Cancer Res Drug Res Updates IJROBP J Exp Med Mol Cell Neoplasia Oncogene Oncotarget Oncoimmunology PNAS Radiat Oncol Radiat Res Radiother Oncol Strahlenther Onkol Endothelium Tumor PDGFR Sutent VEGF PDGF VEGFR PDGFR P P PI3K P AKT Apoptosis EGFR Avastin Cilengitide/S-247 v 3 P P Rapamycin EGF EGFR Erbitux Iressa/Gefitinib

6 Radiotherapy hits tumor cells and non-tumor cells and causes adaptive responses of the immune regulation Hallmarks of Cancer, Hanahan und Weinberg, Cell 2000, 2011

7 Potential Immune Therapies and where Radiotherapy affects the Cancer Immunity Cycle

8 Radiation and Cancer Therapy Abscopal but desirable: The contribution of immune responses to the efficacy of radiotherapy Kroemer and Zitvogel, Oncoimmunology 2012 Radiation Oncology

9 Abscopal Radiotherapy Effects- Case Reports Radiation Oncology

10 After Radiotherapy 3x9.5Gy Postow et al, NEJM 2012 Radiation Oncology

11 Immunotherapy and the cancer immunoediting hypothesis Cancer Therapy: Shift the balance from Escape to Equilibrium to Elimination PD1/CTLA4

12 Role of RT in induction of the antitumor immune response Tumor smaller More T cells More Antigens/Damage-associated molecular pattern molecules/damps More Chemokines Kalbasi et al JCI 2013

13 Proimmunogenic and immunosuppressive effects of radiotherapy Radiation Dose dependence Upregulated by RT TGF-ß Treg?? M2polarization Upregulated by RT IFN- FAS MHC1 ICAM-1 VCAM-1 TNF- HMGB1 CXCL16 Treg modifications Formenti et al, JNCI 2013 Schaue and McBride, 2012

14 Low dose (~2 Gy) irradiation programs macrophages to promote antitumor immunity

15 In human pancreatic carcinoma: Low dose irradiation induces T cell invasion, vascular normalization and promotes antitumor immunity Klug et al Cancer Cell, 2013

16 Radiotherapy in 1899 Basal cell carcinoma = basalioma

17 Tolle Like Receptor 7 (TLR7) agonist Imiquimod (Aldara ) approved for basalioma Resiquimod (imidazoquinolinamine analog) Gardiquimod (imidazoquinolinamine analog) Approved as topical cream in Dermatology Warts Actinic keratosis C 14 H 16 N 4 3-(2-Methylpropyl)-3,5,8- triazatricyclo[ ,6]trideca-1(9),2(6),4,7,10,12-hexaen-7-amine

18 Toll-like receptors family of pattern recognition receptors, at least 12 members recognize various pathogen-associated molecular patterns (PAMPs) e.g. TLR 4: LPS, TLR 5: flagellin, TLR 7+8: viral ssrna located on the cell surface (bacterial antigens) or within endosomes (viral antigens, TLR 7+8) expressed on cells of both the innate immune system (e.g., monocytes, macrophages, dendritic cells, NK cells) and the adaptive immune system (e.g., T cells) intracellular signaling cascade via MyD88 and NFκB ligand binding (=antigen recognition) results in activation of immune cells Schön and Schön. Oncogene 27,

19 Crosstalk TLR agonist - increased inflammatory response - NFkB signaling activation - release of tumor antigens photons

20 NFkB activation by the TLR ligand is dependent on MyD88 fold activation [mean SEM THP1 x Blue Human myloid Wang et al. Oncogene 27, TLR7 agonist 3M-011 (imidazoquinolinamine analog)

21 3M- 011 Toll-like receptor 7/8 Concept Dendritic cell Immature CD8(+)- T-lymphocyte Cytotoxic CD8(+)- T-lymphocyte MHC TCR NK cell Cell-mediated cytotoxicity photon irradiation Tumor antigen Necrosis tumor cell Schölch et al, Oncotarget 2015

22 TLR7 agonist (3M-011) activates NK cells indirectly via monocyte-derived IL-6 In vitro: human PMBC stimulated HT29 lysis Lysis assay 3M-011 Schölch et al, Oncotarget 2015

23 Syngeneic murine colon carcinoma CT26 CT26 on BalbC mice s. c. - most tumors in the combination group disappeared Schölch et al, Oncotarget 2015

24 Tumor complete responese and rechallenge 80 Complete remissions [%] Primary CR Rechallenged No TUMORS If primary CR 0 control 3M x 2Gy photons 3M x 2Gy photons Mice in complete remission rechallenged with increasing numbers of CT26 cells on the non-irradiated contralateral side, none of the animals in remission showed subsequent signs of tumor growth Adaptive immunity? Schölch et al, Oncotarget 2015

25 The treatment combination TLR7 agonist (3M-011) + RT induces a tumor-specific T-cell response ELISPOT assay of splenocytes derived from CT26-bearing BALB/c mice Schölch et al, Oncotarget 2015

26 Syngeneic murine pancreatic carcinoma sc TLR7 agonist 3M-011+RT Panc02 in C57Bl/6 mice s.c. Schölch et al, Oncotarget 2015

27 Orthotopic pancreatic cancer Panc02 mouse model Primary tumor of the pancreras Perivascular mesenteric metastases

28 TLR7 agonist 3M-011+RT Schölch et al, Oncotarget 2015

29 Orthotopic panc02 model TLR7 agonist 3M-011+RT Tumor weight on d12 Peritoneal/Mesenterial metastases on d12 Combination of 3M-011 and irradiation significantly decreases primary tumor weight and local metastases Schölch et al, Oncotarget 2015

30 Tumor size monitored in vivo via FDG-PET TLR7 agonist 3M-011+RT Orthotopic panc02 tumors: PET uptake standard uptake value (SUV) control 3M RT RT 3M RT Schölch et al, Oncotarget 2015

31 Depletion of NK1.1 cells and CD8a(+) cells Depletion was performed using monoclonal antibodies against NK1.1 or CD8a, respectively Controls received isotype control antibodies Success of depletion was routinely assessed by cytometric analysis: CD8a-Alexa Fluor Kontrolle 18,66% 0,00% 73,36% 7,98% NK1.1-Alexa Fluor 488 CD8a-AlexaFluor NK1.1 depl ,77% 0,00% 89,14% 0,09% NK1.1-Alexa Fluor 488 CD8a-Alexa Fluor CD8a depl ,32% 0,00% 88,51% 11,16% NK1.1-Alexa Fluor 488

32 Orthotopic Pancreatic Ca Schölch et al, Oncotarget 2015

33 In 3M-011 monotherapy the therapeutic effect is mediated both by NK cells and cytotoxic CD8 T-lymphocytes Cytotoxic CD8 T-lymphocytes are the main effector cells in combined radio-immunetherapy Tumor weight Schölch et al, Oncotarget 2015

34 The CD11c.DOG mouse Transgenic mouse, construct: DOG: diphteria toxin receptor, ovalbumin, green flourescent protein. Mice express DTR under control of CD11 Promoter. When injected with diphteria toxin, all CD11c+ cells (mainly dendritic cells) can be eradicated in vivo

35 3M-011 requires CD11c + dendritic cells for any tumor inhibition CD11c + dendritic cells mediate >50% of antitumor effects of the treatment combination (via d NK and T-cell priming) Tumor weight reduction Schölch et al, Oncotarget 2015

36 3M-011 shifts the intratumoral and systemic cytokine equilibrium to a pro-inflammatory state Serum cytokines upon TLR7 agonist 3M-011 Schölch et al, Oncotarget 2015

37 3M-011 shifts the intratumoral and systemic cytokine equilibrium to a pro-inflammatory state Intratumoral cytokines upon TLR7 agonist 3M-011+RT [pg/ml] SEM Kontrolle 3M-011 RT 3M-011+RT Schölch et al, Oncotarget 2015

38 CT26 CRC/Balb C Mouse Panc-02/Bl6 Mouse Orthotopic/sc Pancreatic sc Colon Ca Model Local and Distant Response Schölch et al, Oncotarget 2015 Schölch et al, OncoImmunology 2015 Radiation Oncology

39 Radiation Oncology

40 NF-kB in vitro IFNa in vivo DSR 6434 is a specific TLR 7 agonist an induces specific immune activation in vitro and in vivo Colon and sarcoma Radiation Oncology

41 Systemic administration of R848 (Resiquimod) leads to activation of T and B lymphocytes, induction of cytokine expression, and increased long-term survival in EG7 tumor-bearing mice- dependent on CD 8 T cells B and T cell lymphoma Radiation Oncology Dovedi et al, Blood 2013

42 Low-dose cyclophosphamide improves local control and reduces recurrence of tumors treated with RT and topical imiquimod Imiquimod 5% cream (Aldara) Radiation Oncology Dewan et al, CCR 2012

43 Abscopal effect Topical imiquimod synergizes with local radiotherapy in inhibition of the irradiated tumor and of a tumor outside of the radiation field Local control Dewan et al, CCR 2012 Radiation Oncology

44 Radiation and Immune Therapy Combination TLR Agonist Wattenberg et al, Rad Res 2014

45 Preclinical studies radiotherapy+ immunotherapy Dovedi et al (Illidge TM): Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 Blockade. Cancer Res 2014 Mondini M et al (Deutsch E): Synergy of radiotherapy and a cancer vaccine for the treatment of HPV-associated head and neck cancer. Mol Cancer Ther 2015 Radiation Oncology

46 Cinical Trials Radiotherapy+ Immunotherapy > 200 ongoing Radiation Oncology Kalbasi et al JCI 2013 Vacchelli et al, Oncoimmunology, 2013 Checkpoint inhibitor Anti-CTLA-4 Antibody Ipilumumab Ipilimumab + 8Gy in patients with bone metastatic prostate cancer (progressed after docetaxel, phase 3 trial. Kwon et al, Lancet Oncol 2014 Jun;15(7):700 12) No difference in overall survival but signs of activity

47 DSR 6434 potentiates radiation effects in CT26 colorectal cancer Radiation Oncology

48 Long-term surviving mice treated with radiation and R848 are protected against subsequent tumor rechallenge by the induction of tumor-specific memory CD8cells. Radiation Oncology