1. MAXIMISING ANTI-TUMOUR IMMUNE PRIMING WITH REOVIRUS ONCOLYTIC VIROTHERAPY Alan Melcher Professor of Clinical Oncology and Biotherapy University of Leeds

2. OVERVIEW Oncolytic viruses as immunotherapy. Reovirus. Systemic delivery. Melanoma and glioma. Enhancing immunovirotherapy with other immunotherapy – GM-CSF, checkpoint blockade.

3. Immune system (anti-viral immunity) is ‘bad’ Oncolytic viruses were initially developed as direct cytotoxic agents

4. Oncolytic viruses increasingly recognised as immunotherapy

5. Reovirus is a dsRNA virus which targets cells with activation of the ras pathway. Previous lab work: In mouse and human pre-clinical systems reovirus is effective by direct and immune-mediated effects, alone or in combination with chemotherapy or radiotherapy. Almost everyone exposed to reovirus in childhood and so NAB positive – potentially a problem for systemic delivery. REOVIRUS

6. REO13 TRIAL Patients with colorectal cancer metastatic to the liver, planned for radical surgery. One cycle of single agent i.v. reovirus (5 daily treatments), 1 – 5 weeks before surgery. Endpoints - immune response and analysis of blood, tumour and normal liver.

7. REO13 BRAIN STUDY Phase 1b translational trial. Preoperative intravenous reovirus. Recurrent high grade primary or metastatic brain tumours. Can intravenous virus access tumours in the brain in patients (almost all OV for brain tumours has been by i.t. injection). Similar design to REO13, administering a single iv infusion of reovirus, prior to resection. 9 patients treated - tumour positive by IHC for reovirus in all but 1 patient.

8. REO13 BRAIN – TUMOUR IHC IHC for reovirus protein (brown): High grade glioma

9. IHC for reovirus protein (brown): metastatic brain melanoma metastatic brain adenocarcinoma reovirus RNA by ISH (blue) reovirus protein (red) co-staining for RNA and protein (yellow) IHC for reovirus protein (brown): metastatic brain adenocarcinoma

10. CONCLUSIONS FROM REO13 AND REO13 BRAIN Reovirus is delivered preferentially to tumours after intravenous infusion, despite the presence of anti-virus NAB at baseline. ‘Functional’ virus is associated with cells; plasma virus is neutralised. Replication-competent virus can be selectively retrieved from tumour at the time of surgery (colorectal cancer and ? brain). Systemic reovirus can cross the blood brain barrier to access primary and secondary tumours within the brain.

11. FATE OF REOVIRUS FOLLOWING INTRAVENOUS DELIVERY IN MICE Consistent with REO13 data

12. Cells pre-loaded with reo/NAB complexes and co-cultured with B16 targets CD11b + CELLS DELIVER REOVIRUS/NAB COMPLEXES TO TUMOUR CELLS IN VITRO FOR PRODUCTIVE TUMOUR CELL INFECTION

13. GMCSF INCREASES CD11b CELLS/REOVIRUS DELIVERY TO TUMOURS IN VIVO Pre-immunized tumour-bearing mice, treated +/- GM-CSF 1 cycle GM-CSF/reo

14. PRE-CONDITIONING WITH GM-CSF FOLLOWED BY REOVIRUS TREATMENT CURES MICE BEARING 3 DAY TUMOURS - THE THERAPY IS ONLY EFFECTIVE IN REOVIRUS-IMMUNIZED MICE

15. Mock depleted CD8 depleted CD4 depleted NK depleted Neutrophil depleted Monocyte/neutrophil depleted IN VIVO THERAPY WITH REO/GMCSF IN PRE-IMMUNE MICE IS DEPENDENT ON NK CELLS AND MONOCYTES, BUT NOT T CELLS OR NEUTROPHILS Antibody depletion of: Ly-6C+ve cells - expressed on neutrophils and monocytes Ly-6G+ve - expressed on neutrophils

16. GM-CSF INCREASES FcγR1 RECEPTOR EXPRESSION ON CD11b+ MOUSE CELLS FcγR1 expression on CD11b cells

17. IMMUNE MEDIATED TUMOUR CELL KILLING IN VITRO BY GM-CSF- TREATED LYMPH NODE CELLS/SPLENOCYTES IS DEPENDENT UPON Fc RECEPTORS LN/splen LN/splen + GM-CSF + reo/NAb LN/splen + GM-CSF + reo/NAb + anti-FcγR

18. PROPOSED MECHANISM FOR GM-CSF CONDITIONING TO ENHANCE SYSTEMIC REOVIRUS THERAPY

19. DOES REOVIRUS/NAB DATA APPLY TO HUMANS? In mice, protective cell carriage by monocytes/macrophages is key in the context of pre-immune reovirus/GM-CSF therapy. Does the same apply to human cell carriage? Can human monocytes hitchhike reovirus/NAB complexes? human monocyte Mel-624 fully neutralised reovirus reovirus + reo-immune serum + Mel-624 + monocyte ?

20. REOVIRUS-ANTIBODY COMPLEXES

21. DELIVERY OF REO/NAB COMPLEXES VIA HUMAN MONOCYTES Reo/NAB complexes CD14 + cells + reo/NAB complexes % Mel-624 cell death

22. POSSIBLE FcγR INVOLVEMENT

23. REOVIRUS AND REO-NAB COMPLEXES ACTIVATE HUMAN MONOCYTES

24. HUMAN DATA TO DATE Reovirus forms IgG complexes with serum from treated patients containing high levels of NAB. Reovirus/NAB complexes can be handed off by monocytes to tumour cells for direct oncolysis. Reovirus/NAB complexes activate monocytes. What is mechanism of reovirus-NAB hitchhiking? What is effect of GM-CSF? Consistent with mouse model to date.

25. NEXT TRIAL - REO13 MELANOMA GM-CSF Reovirus +/- GM-CSF prior to planned resection of advanced melanoma. Patients will be pre-immunised against the virus.

26. HOW WIDELY APPLICABLE IS REOVIRUS/NAB/GM-CSF THERAPY? If generally applicable, suggests repeat OV treatments in the context of high NAB could be beneficial. Tested other models: Reovirus sensitive/resistant Brain models Integration with standard of care (chemotherapy/radiotherapy for high grade glioma)

27. 5d Established Intra-cranial B16 Tumours Treated with 3 Cycles of GM-CSF/REO 3 Cycles GM-CSF/REO GM-CSF REO THERAPY WITH GM-CSF/REO IN PRE-IMMUNE MICE IN MELANOMA REOVIRUS-SENSITIVE BRAIN METASTASIS MODEL

28. THERAPY WITH GM-CSF/REO IN PRE-IMMUNE MICE IN MELANOMA REOVIRUS-RESISTANT BRAIN METASTASIS MODEL Established Intra-cranial B16.OVA Tumours Treated with GM-CSF/REO

29. 5d Established intra-cranial GL261 Tumours Treated with 3 Cycles of GM-CSF/REO THERAPY WITH GMCSF/REO IN PRE-IMMUNE MICE IN GLIOMA MODEL

30. REOVIRUS/GM-CSF ENHANCES CHEMORADIATION THERAPY FOR GLIOMA Trial: Addition of reo/GM-CSF to standard of care chemoradiotherapy for high grade glioma

31. CHECKPOINT BLOCKADE – AN ADDITIONAL ROUTE TO MAXIMISING REOVIRUS IMMUNOVIROTHERAPY?

32. Checkpoint inhibitors block the brakes of the immune system. Antibodies against eg CTLA-4, PD-1, PD-L1. Already standard of care in melanoma, but also active in lung cancer, bladder cancer. Most effective when a T cell response is already present against the tumour, which checkpoint blockade can enhance. Might OV ‘light the fire’ in tumours where baseline T cell response is weak? We don’t understand how/where checkpoint blockade works in patients.

33. RATIONALE FOR COMBINING REOVIRUS WITH CHECKPOINT INHIBITORS Reovirus upregulates PD-L1 on tumour cells, including glioma stem cells

34. NK and T cells (within PBMC) upregulate PDL-1 in response to reovirus Supports rationale of combining reovirus with PD-1/PD-L1 axis blockade RATIONALE FOR COMBINING REOVIRUS WITH CHECKPOINT INHIBITORS

35. RATIONALE FOR REOVIRUS/CHECKPOINT BLOCKADE REO13 BRAIN – SYSTEMIC REOVIRUS UPREGULATES TUMOUR PD-L1 ON GBM IN PATIENTS reovirus protein (brown) PD-L1 (brown) – trial patient control patient

36. PD-1 BLOCKADE IMPROVES THERAPY WITH INTRATUMOURAL REOVIRUS IN MELANOMA

37. CHECKPOINT BLOCKADE ENHANCES SYSTEMIC REOVIRUS/GM-CSF THERAPY IN GLIOMA PBS REO/GM-CSF anti-CTLA-4+anti-PD-1 REO/GM-CSF+anti-PD-1 REO/GM-CSF+anti-CTLA-4 REO/GM-CSF+anti-PD-1+anti-CTLA-4 Trial proposal: reovirus/GM-CSF/checkpoint blockade first line for relapsed high grade glioma

38. CONCLUSIONS Systemic reovirus can access tumours in patients in the liver and brain, despite the presence of anti-virus NAB at baseline. GMCSF enhances reovirus therapy in pre-immune mice, in flank and brain tumour models. Reovirus forms complexes with NAB from treated patients’ serum. Human monocytes can hand-off reovirus/NAB complexes to tumour cells. Reovirus upregulates the PD-1/PD-L1 axis. Checkpoint blockade enhances both intratumoural and systemic reovirus therapy. Further translational and therapeutic trials will address maximising reovirus oncolytic virotherapy using combination immunotherapies.

39. Liz Ilett Fiona Errington Rob Berkeley Emma West Karen Scott Ailsa Rose Jen Kingston Lizzie Appleton Sam Turnbull Gemma Migneco Gina Scott Giles Toogood Rob Adair Susan Short Paul Chumas Simon Thomson Acknowledgements Richard Vile Tim Kottke Jill Thompson Kevin Harrington Vickie Roulstone Hardev Pandha Rob Hoeben Diana van den Wollenberg Aat Mulder Matt Coffey (Oncolytics) Jerry Nuovo