INTRATUMORAL REGULATED EXPRESSION OF IL-12 AS A GENE THERAPY APPROACH TO IMMUNOTHERAPY John Nemunitis 1 , John A. Barrett 2 , Francois Lebel 2 , Thomas D. Reed 3 , E. Antonio Chiocca 4 , Antonio M. Omuro 5 , Larry Norton 5 , Jonathan Lewis 2 1 Mary Crowley Cancer Research Centers, Dallas, TX, United States, 75201 2 ZIOPHARM Oncology Inc., Boston, MA, United States, 02129 3 Intrexon Corporation, Germantown, MD, United States, 20876 4 Brigham and Woman’s Hospital, Harvard Medical School, Boston, MA, United States, 02115 5 Memorial Sloan-Kettering Cancer Center, New York, NY, United States, 10065 Exhibit 99.2 |
Background & Rationale IL-12 in Glioma • Tumors escape the immune system through the process of immunoediting. Thus, restoration of the immune system’s ability to detect the tumor should result in improved treatment outcomes. • Localized IL-12 administration has been shown to have antitumor activity that is mediated by direct tumor cell cytotoxicity, and enhancement of immuno-regulatory activities including activation of anti-tumor natural killer (NK) cells, CD4 + T cells and CD8 + T cells. |
Background & Rationale IL-12 in Glioma • Roy & Kranz (University of Illinois) IL-12. Journal of Immunology, 2000, 165: 7293–7299. Model SV11 Transgenic mouse administered recombinant mIL-12 i.c. Findings Increased survival infiltration of activated CD8 and CD4 T cells. • Vom Berg & Becher (University of Zurich) J Exp Med, 2013, 210: 2803-2811. Model: GL261 transduced to constitutively express IL-12 i.c. Findings: Increased survival combination of IL-12 + CTLA4 elicits decrease Tregs while increasing Teff. • Dimeco & Olivi Johns Hopkins School of Medicine & Istituto Nazionale Tumori, Milan, Italy) J Neurosurg 2000, 92:419–427, Model: Rat 9L gliosarcoma cells expressing IL-12. Findings Local delivery of IL-12 in rat brain prolongs survival in animals challenged i.c. with a malignant glioma cells. • Sonabend & Lesniak (University of Chicago). Anti-Cancer Drugs 2008, 19:133–142. Model: GL-261 orthotopic glioma model. Findings: Synergy in survival with locally administered pmIL-12/PPC + biodegradable carmustine • Markert & Whitley (University of Alabama) Journal of Virology 2012,86: 5304–5313 Model: 4C8 glioma cells orthotopic B6D2F1 mouse. Findings: Hsv mutant M002 expressing IL-12 demonstrated prolonged survival vs. control • Liu & Yu (Cedars Sinai Medical Center) Cancer Gene Therapy (2002) 9, 9–15 Model: GL-26 orthotopic mouse. Ad5-mIL-12 Findings: Survival was significantly prolonged in Ad-mIL-12–treated animals with increased CD4+ and CD8+ T- cell infiltration ( : : : |
 1. The Switch Components: The RTS® gene program includes 2 receptor protein fusions: VP16-RXR and Gal4-EcR. They form unstable and unproductive heterodimers in the absence of any ligand. 2. The Inducible Promoter: A customizable promoter to which basal transcription proteins are recruited and the target gene is transcribed. 3. The Activator Ligand (veledimex): An ecdysone analog, diacylhydrazine-based small molecule functions as an activator. In the presence of the ligand, the protein heterodimer changes to a stable conformation and binds to the inducible promoter. EcR RXR VP16 Gal4 Basal Transcription Proteins Activator Ligand RXR VP16 Gal4 EcR Inducible Gene Program Inducible Gene Program RheoSwitch Therapeutic System® (RTS®) is a 3-component transcriptional regulator Inducible Gene Regulation: RheoSwitch Therapeutic System ® |
 IL-12 Production is Modulated by Activator Ligand in HT 1080 Cells 5 On Off On AL=75nM 5 |
 Dose-Dependent Increase in Expression of Tumor IL-12 mRNA & IL-12 Protein in Response to Veledimex 10 2 10 10 4 10 5 10 6 0 2 4 6 8 10 12 14 RTS gDNA Tumor Veledimex Level 10 0 10 1 10 2 10 3 0 2 4 6 8 10 12 14 Time (days) Time (days) Vehicle/Vehicle Vehicle/Veledimex 150 mg/m Ad (1e10) + Veledimex 15 mg/m Ad (1e10) + Veledimex 30 mg/m Ad (1e10) + Veledimex 75 mg/m Ad (1e10) + Veledimex 150 mg/m 2 Tumor IL-12 Protein Level IL-12 RNA 0.1 1 10 0 2 4 6 8 10 12 14 Time (days) 10 0 10 1 10 2 10 3 10 4 0 2 4 6 8 10 12 14 Time (days) 2 2 2 2 3 |
 Ad-RTS-mIL-12 + Veledimex Increases Tumor CD8 + & CD4 + While Decreasing CD4 + Fox P3 + TILs in the 4T1 Syngeneic Mouse Vehicle Ad-RTS-mIL-12 1 x 10 10 vp + Veledimex 150 mg/m 7 CD8 + CD4 + CD4 + Fox P3 + 2 |
 Dose-Dependent Anti-Tumor Activity of Ad-RTS- mIL-12 + Veledimex (AL) in Murine 4T1 Model Start of treatment Tumor volume reached 100-200 mm 3 0 4 8 12 16 20 24 28 32 0 5 10 15 Vehicle/Vehicle Vehicle/Ad-RTS-mIL12 Vehicle/Veledimex 15 mg/m 2 Ad-RTS-mIL12 + AL 15 mg/m 2 Ad-RTS-mIL12 + AL 30 mg/m 2 Ad-RTS-mIL12 + AL 75 mg/m 2 Ad-RTS-mIL12 + AL 150mg/m 2 Time (Days) |
• High expression of IL-12 mRNA in tumors, tightly controlled by veledimex dose • Tumor biopsies show increased tumor infiltrating lymphocytes in both injected and systemic non-injected lesions • Serious adverse events are mechanism-based and consistent with immunotherapy (Fever, N&V, leukopenia, increased LFTs, hyponatremia, cytokine release response) •Serious adverse events reversed within days after stopping veledimex dosing •Subjects who have had IL-12 expression turned “off” have been redosed, and IL-12 turned “on” again Clinical Observations to Date 9 We can control gene expression to achieve a systemic immune response We have seen systemic and fully reversible toxicity |
 Higher Veledimex Levels Normal and in GL261 Orthotopic Glioma Mouse Brains Veledimex levels at 24 hr posttreatment 0 1000 2000 3000 4000 5000 6000 450 mg/m 2 /day 1200 mg/m 2 /day |
 Effects of Ad-RTS-mIL-12 + Veledimex (AL) in the Orthotopic GL261 Mouse Normal Mouse Vehicle BID x 14 Ad-RTS-mIL-12 1x10 10 vp + AL 450 mg/m /day BID x14 Treatment For 14 days Day 74 (end of study) Control Day 20 2 |
 Ad-RTS-mIL-12 + Veledimex (in Chow) Results in Increased Survival in the GL261 Orthotopic Glioma Mouse Model Veledimex administered ad lib in chow from Day 4 to EOS at ~ 675 mg/m /day Ad-RTS-mIL12 administered on Day 5 0 20 40 60 80 0 20 40 60 80 100 Vehicle/Vehicle Vehicle/AD (5e9) Vehicle/Activator chow (1000) Activator chow + AD (5e9) Activator chow + AD (1e9) Activator chow + AD (1e8) Time (Days) 2 |
 Ad-RTS-mIL-12 + Veledimex (AL) Results in Increased Survival When Compared to Control in the GL261 Orthotopic Glioma Mouse Model Ad-RTS-mIL12 administered on Day 5 ; Veledimex (mg/m 2 ) administered BID for 14 days from Day 5; 0 20 40 60 80 0 10 20 30 40 50 60 70 80 90 100 maisine/saline maisine/vector Veledimex (AL)/saline AL 150/day + AD 1e10 AL 300/day + AD 1e10 AL 450/day + AD 1e10 AL 675/day + AD 1e10 AL 1200/day + AD 1e10 dexamethasone 6 bidx14 bevacizumab 30 biwkx3 temozolamide 300Qdx5 Time (Days) |
 Ad-RTS-mIL-12 + veledimex significantly reduces brain cancer stem cells in GL-261 Orthotopic Glioma Model Nestin levels (marker for cancer stem cells) inverse correlation with survival (Pearson r= 0.92) 0 10 20 30 Vehicle/Vehicle Vehicle/Ad-RTS-mIL-12 Vehicle/Veledimex Veledime x 450 mg/m /day Veledime x 600 mg/m /day Veledime x 1200 mg/m /day Nestin 2 2 2 |
15 Conclusions • Ad-RTS-mIL-12 + veledimex PO exhibits controllable systemic immune activation in human subjects with melanoma and breast cancer. • Veledimex exhibits dose-related increases in plasma and brain tissue exposure with no accumulation in brain. • Ad-RTS-mIL-12 (1x10 10 vp) + veledimex PO improves survival over temozolomide, dexamethasone and bevacizumab. • Ad-RTS-mIL-12 + veledimex significantly reduces brain cancer stem cells • These findings support the utility of localized, regulatable IL- 12 production as an approach for the treatment of malignant glioma in human subjects. |