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ONCOLYTIC VIRUSES BIOTHERAPEUTICS AGAIN 73,000 CANADIANS WILL DIE THIS YEAR GENES CONTROL ALL ASPECTS OF GROWTH Hours Weeks Decades Normal Cell Growth and Death Cell Death (controlled by “cell death” genes) Cell Divides Tumour Cell Growth Cigarette smoke, UV, pollutants, bad luck Immortalized cells form tumour Cell Death Genes Mutated Cell Divides And Divides And Divides And Divides……. CHEMOTHERAPY CANCER TARGETED THERAPIES Our bodies are made of billions and billions of cells Cell Genetic Material Virus u VIRUS MULTIPLIES Viruses are parasites and can only reproduce inside of cells Cellular Antiviral Programs… neighbourhood blockwatch! • Interferon (IFN) is secreted by infected cells • Alerts neighbouring cells to presence of virus • Leads to death of infected cells, prevents growth of surrounding cells and raises their defenses against infection IFN IFN IFN Immunology 101 Note : The immune system can also be trained to recognize tumors! Antiviral defense: The “Achilles Heel” of Cancer Normal Cell Mutations in individual genes Tumour Cell Some of the same genes that control cell growth/death are also involved in cellular anti-viral programs ~70% of Cancer cells have defects in anti-viral programs !!! Virus Cancer Oncolytic Viruses : A targeted approach to Cancer therapy Because cancers have defects in antiviral responses, this makes it possible to create viruses that replicate in and specifically kill cancer cells! In addition to antiviral defects, the typical tumor cell… • Grows rapidly and generates tumors with leaky vasculature • Expresses high levels of enzymes involved in nucleic acid metabolism (eg. Thymidine kinase or TK) • Has Hyperactive growth receptor pathways (eg. EFGR, Ras) Vaccinia Virus 1. Large double-stranded DNA poxvirus ~ 200 kbp 2. Replicates exclusively in the cytoplasm of cells 3. Can’t recombine with cellular DNA in the nucleus 4. Large amount of genes which can be removed or replaced to accomodate transgenes 5. One of the best studied viruses known to man Vaccinia Virus engineered from live vaccine Given to > 100 million healthy children world-wide 1800 1980 The JX-594 Oncolytic Vaccinia strain thymidine kinase vaccine strain genome GM GM-CSF payload lac-Z lac-Z marker engineered product: JX-594 B18R mutation 1. Viral gene expressing the cellular equivalent of Thymidine kinase has been removed => dependence on high TK levels provided by tumor cells 2. Mutation in B18R gene required to overcome IFN-mediated antiviral response => no consequence in tumor cells have defects in this pathway 3. Added GM-CSF transgene to stimulate an anti-tumor immune response Vaccinia Virus (JX-594) cancer targeting & three-pronged MOA EGFR IFN JX JX X T K NORMAL X E 2F EGFR IFN GM JX JX JX JX ras JX JX JX JX JX JX TK E2F GM JX JX JX JX JX JX JX JX GM JX JX JX GM GM JX JX JX JX GM GM GM JX JX JX JX JX GM JX JX JX JX GM JX JX JX GM GM JX JX JX JX JX JX JX JX JX JX JX JX CANCER JX JX GM JX JX ONCOLYTIC VIRUSES GROW IN AND KILL TUMOURS Phase 1: RECIST tumor response in HCC patient Baseline 4 cycles Phase 2: RECIST responses in HCC tumors located at periphery of cirrhotic liver Baseline Week 8 Week 8 Baseline Baseline Day 12 ONCOLYTIC VIRUSES SELF AMPLIFYING DOSING JX-594 activity Amplification, spread, cell killing within human tumors Stanford Bio-Imaging Center: (S Thorne - Jennerex virus labeled green) Pharmacokinetic Profile: Waves of 1˚+ 2˚ Vaccinia Spread in Human Cancer Patients Hours, post administration Hours, post administration 108 4.5 x 107 genome 107 106 105 0 2 4 6 8 10 12 14 16 18 20 22 24 Days, Post JX594 injection Genomes/ 5L Whole Blood Genomes/ 5L Whole Blood 01201C1 1011 1010 8.8 x 109 genome 109 108 107 106 105 0 2 4 6 8 10 12 14 16 18 20 22 24 Days, Post JX594 injection Days post tumour injection with vaccinia virus ONCOLYTIC VIRUSES MINIATURE “BIOLOGICAL BATTLESHIPS” ATTACK TUMOURS IN MULTIPLE WAYS VIRUS ENTERS THROUGH LEAKY VASCULATURE (24 Hr PI) Oncolytic Virus Initial Sites of Infection in Mouse Tumour Tumor Vasculature CD31-red Steve Thorne University of Pittsburgh Virus-GFP-green Tumour Vasculature Infection in Patients Treated IV in Ottawa VV positive tumor VV infected vessel VV positive tumor VV negative stroma Necrotic response in distant non-injected tumor Baseline Pt 1304 Day 5 Week 8 Immunity and OV therapy Colon Cancer tumour Challenge with Colon Cancer cells IV VSV “Cured” mouse wait 7 months Mice reject Tumours! Long-term Survivors Disease-Free after Vaccinia Phase I Clinical Trial Metastatic Melanoma Pre-treatment 32 year-old woman: • Refractory, widespread met • Complete tumor regressions: • Injected • Distant dermal, chest (surg) • Disease-free 1.5+ years 75 year-old man: • Multiple met sites (n=24) • Complete tumor regressions: • Injected • Distant dermal • Disease-free 3+ years After vaccinia JX-594: novel 3-pronged mechanism-of-action Replication & GM-CSF expression dependent primary MOA: 1. Infection & cell lysis leads to complementary: 2. Immune response stimulation 3. Vascular disruption JX 594 Clinical Activity Ph 1 long-term survival: many cancer types + + renal melanoma lung melanoma + melanoma melanoma patients + thyroid liver thymic melanoma colon colon melanoma melanoma colon Survival (years) 3-4 months life expectancy >/= 8 months survival (1) Based on data from ~43 8-mo evaluable patients to date. 15 long-term Survivors(1) 7 cancer types Intra-hepatic injection of JX-594 for Phase II clinical trial Promising Ph 2 survival in advanced liver cancer Superior to internal & historical controls, including sorafenib Lancet Oncology 2009 (n=226) Hep007 2010 (n=22) % patients IV response summary (n=6) (n=4) (n=4) Full dose (n=9)* *Note: Response rate assessment incomplete IV delivery: biopsy-proven cancer-specific targeting Colon cancer glandular structures = infected (IHC+) & evolving necrotic tumor tissue Clinical data so far with JX-594 and other oncolytic viruses suggest that significant therapeutic responses can be obtained in a subset of patients But What do we do when tumors resist infection with OVs? 1. Negative single strand RNA virus of the Rhabdovirus family 2. Small genome, 5 gene products (N, P, M, G, L) 3. Potent cytolytic VSV M protein -plays a role in virion budding -causes cell rounding and induces cell death -interacts with nuclear export machinery prevent expression of cellular antiviral genes VSVD51 : Mutation in M protein at methionine 51 prevents interaction with nuclear machinery => Sensitivity to antiviral signaling eg. Interferon Tumor Cells V V V V Highly Sensitive Normal Cell V V V V V V V V V V V V V V V V V V = Antiviral defense pathway V V Moderately Sensitive V Resistant Can we complement the defects of VSVD51 in resistant tumors using a chemical complementation strategy? Tumor Cells V V V V Highly Sensitive Normal Cell V V V V Drugs? V V V V V V V V V Resistant V V V V V = Antiviral defense pathway V V Moderately Sensitive V Resistant High Throughput screen identifies novel “virus sensitizer” or VSe drugs In vitro validation of VSe compounds Screen design 4T1 mouse breast cancer cells Library Compounds 4h pre-treat Controls (SAHA/DMSO) Add control (media) Add VSVD51at low MOI (0.03) 40h incubation Add Alamar Blue (fluorescent viability dye) 2h incubation Measure fluorescence Calculate normalized viability ratio (VSV-treated/Control) for each drug => Low ratio indicates viral sensitizer Identification of VSe candidates VSe1 increases viral replication in cancer but not normal cells Up to > 1000-fold increase in virus in cancer cells ! VSe1 represses VSVD51-induced genes Overall # of genes affected by VSe1 = 111 VSe1 enhances VSVd51 efficacy in a resistant CT26 syngenic colon tumor model VSe1 + VSVD51 VSVD51 PBS VSe1 enhances VSVd51 replication in human clinical samples immune cells shutting off tumor blood supply tumor-targeting antibodies virus infection & cell lysis Acknowledgements Bell/Atkins lab Dr. John Bell Dr. Harry Atkins Dr. Fabrice LeBoeuf Dr. Markus Vaha-Koskela Heather MacTavish Theresa Falls Julie Cox Alanah Kemp Nicayla Keath Jad Farah Institutions OHRI University of Ottawa McMaster University Funding Agencies FRSQ OICR Lichty Lab Dr. Brian Lichty Frances Lai Auer Lab Dr.Rebecca Auer Lisa Mackenzie Jennerex Biotherapeutics David Kirn Caroline Breitbach Tae Ho Hwang Theresa Hickman Adina Peluso Kelley Parato Ann Moon Manijeh Daneshmand HTS Facility (McMaster) Jenny Wang Jan Blanchard Ryan Brown Dr.Eric Brown