Expanded Phase 1 study of intratumoral Ad-RTS-hIL-12 plus oral veledimex: Tolerability and survival in recurrent glioblastoma. E. Antonio Chiocca 1 , John Yu 2 , Surasak Phuphanich 2 , Rimas Vincas Lukas 3 , Priya Kumthekar 4 , Yijun Yang 5 , Qiang (John) Zhou 5 , Jill Y. Buck 5 , Alicia Deary 5 , Hongliang Cai 5 , John A. Barret 5 , Laurence JN Cooper 5 , Francois M. Lebel 5 1 Brigham and Women's Hospital, Boston, MA; 2 Cedars-Sinai Medical Center, Los Angeles, CA; 3 University of Chicago, Chicago, IL; 4 Northwestern Memorial Hospital, Chicago, IL; 5 ZIOPHARM Oncology, Inc, Boston, MA Background: Glioblastoma (GBM) is an aggressive brain tumor afectng ~74,000 people worldwide an- nually. Recurrent GBM patents have a median OS (mOS) of 6-7 months. OS in patents who have failed temozolomide, bevacizumab or equivalent salvage chemotherapy, is ~3-5 months. New therapies are ur- gently needed. Ad-RTS-hIL-12 (Ad) is a novel gene therapy expressing IL-12 under the control of an oral actvator ligand, veledimex (V), through the RheoSwitch Therapeutc Sys tem ® . Intratumoral administraton of Ad results in targeted tumor cytotoxicity and inducton of sys temic T cell memory. Ad + V is a treat- ment strategy to extend the IL-12 therapeutc window. Methods: In a multcenter Phase I dose escalaton trial and expansion cohort, subjects with recurrent or progressive Grade III or IV glioma undergoing resec- ton were injected intratumorally with Ad 2 x 10 11 viral partcles (vp) and daily oral V for 15 doses, begin- ning prior to surgery. The primary endpoint is safety and tolerability of Ad + V; secondary endpoints in- clude OS. Results: 25 subjects were dosed in 3 dose escalaton cohorts: 20 mg (n = 7), 30 mg (n = 4), and 40 mg (n = 6) and an expansion cohort of 20 mg (n = 8). Results show V crossed the blood brain-barrier with 35±5% of plasma levels detected in the brain tumor. The 20 mg dose (n = 15) had beter drug com- pliance (86%) than the 30 mg (63%) or 40 mg (52%) cohorts and the 20 mg cohort shows beter survival (mOS 12.7 months) compared to other cohorts. The frequency of related ≥Grade (G)3 AEs in the 20 mg cohort was signifcantly lower: 20% in 20mg, 50% in 30mg and 40 mg. In the 20 mg cohort, the most fre- quent AEs were transient mild fulike symptoms seen in 12/15, G3 cytokine release syndrome in 2/15, G3 elevated ALT/AST in 1/15 and G3 lymphopenia in 3/15. All AEs reversed promptly upon discontnuing V. Conclusions: Overall, Ad + 20 mg V is well tolerated; toxici tes were predictable and reversible upon dis- contnuing V. There is a correlaton between V dose, BBB penetraton and drug related AEs. The tolerabil- ity and encouraging survival observed to date warrant further inves tgaton in a pivotal trial. A stereotac- tc arm and a pediatric trial in difuse intrinsic pontne glioma patents are planned. RheoSwitch Therapeutc System ® (RTS ® ) Gene Switch is a 3-Component Transcriptonal Regulator 1. The Switch Components: The RTS ® gene program includes 2 receptor protein fusions: VP16-RXR (co-actvaton partner, CAP) and Gal4-EcR (ligand-inducible transcripton factor, LTF). In the absence of ligand, the LTF binds to the inducible promoter and does not form a stable interacton with the CAP. 2. The Inducible Promoter: A customizable promoter to which basal transcripton proteins are recruited and the target gene is transcribed. 3. The Actvator Ligand (veledimex): An ecdysone analog, diacylhydrazine -based small molecule, functons as an actvator. In the presence of the ligand, a conformatonal change in the LTF leads to a stable, high-afnity interacton with the CAP. Ad 2X10 11 vp 20 mg Cohort (N=15) 30 mg Cohort (N=4) 40 mg Cohort (N=6) Total (N=25) Age in Years Mean (Min, Max) 45.93 (26, 68) 59.75 (43, 74) 47.67 (36, 58) 48.56 (26, 74) Gender Male : Female 10 : 5 2 : 2 4 : 2 16 : 9 Recurrence (n) 1 st 2 nd 3 rd or more 4 5 6 1 2 1 2 2 2 7 9 9 Prior Lines of Treatment (mean) 2.2 3.0 2.5 2.4 Grade at Study Entry HGG Grade III Glioblastoma 2 13 0 4 0 6 2 23 KPS at Screening ≥ 90 ≥ 70 and < 90 9 6 3 1 2 4 14 11 IDH Status WT mutated TBD 8 5 2 3 0 1 5 1 0 16 6 3 Total Steroid Use in mg Day 0 Median (Min, Max) 4 (0, 10) 7 (0, 14) 10 (0, 14) 8 (0, 14) Total Steroid Use in mg Days 1-3 Median (Min, Max) 18 (0, 44) 36 (0, 44) 8 (0, 40) 18 (0, 44) Total Steroid Use in mg Days 0-14 Median (Min, Max) 48 (0, 140) 106 (0, 136) 35.5 (10, 102) 54 (0, 140) Veledimex Dosing Compliance 84% 63% 58% 73% As of 24 May 2017, 25 subjects enrolled in Group 1 Dose Escalaton. Follow-up is ongoing. Subject Characteristcs Adenovirus serotype 5 NR Overall Survival: 20 mg vs 30 & 40 mg Cohorts Tight transcriptonal control of the RTS ® gene switch by veledimex regulates recombinant IL-12 protein expression and downstream endogenous IFN- in a dose-related manner There is a strong correlaton between veledimex dose, BBB penetraton, IL-12 and IFN- producton Safety  Related AEs were tolerable, predictable, and rapidly reversible upon discontnuing veledimex  Severity and frequency of CRS correlated with veledimex dose, serum IL-12 and IFN- levels, and was always reversible upon holding veledimex  Neurologic adverse events were relatvely mild and transient. The were no drug-related deaths Efcacy  Survival appears to correlate with cellular immune modulaton  Median overall survival at 20 mg of veledimex is maintained at 12.5 months and contnues to compare favorably to historical controls  Concurrent steroid use during frst 15 days dampens the survival beneft of IL-12 driven immune actvaton 20 mg N=15 30 mg N=4 40 mg N=6 Total N=25 Related Adverse Events (AEs) Related AEs 12 (80%) 4 (100%) 5 (83%) 21 (84%) Related ≥ Grade 3 AEs 7 (47%) 3 (75%) 4 (67%) 14 (56%) Related Serious Adverse Events (SAEs) Cytokine release syndrome 2 (13%) 1 (25%) 2 (33%) 5 (20%) Thrombocytopenia 2 (13%) 0 1 (17%) 3 (12%) LFT increased, Leukopenia, Neutropenia, Py- rexia, and Aseptc meningits Each term 1 (7%) 0 0 Each term 1 (4%) Related ≥ Grade 3 AEs That Occurred in ≥ 5% of Subjects Lymphopenia 6 ( 40%) 3 ( 75%) 3 (50%) 12 ( 48%) AST increased 1 ( 7%) 0 2 ( 33%) 3 ( 12.%) Cytokine release syndrome* 2 (13%) 1 (25%) 3 (50%) 6 (24%) Headache 2 ( 13%) 0 0 2 ( 8%) Hyponatremia 1 ( 7%) 0 1 ( 17%) 2 ( 8%) Thrombocytopenia 2 ( 13%) 0 0 2 ( 8%) Related ≥ Grade 3 Neurological AEs Headache 2 (13%) 0 0 2 (8%) Brain edema 0 1 ( 25%) 0 1 (4%) Confusional state 0 0 1 (16.7%) 1 (4%) Aseptc meningits 1 (6.7%) 0 0 1 (4%) Cytokine Release Syndrome* 20 mg N=15 30 mg N=4 40 mg N=6 Total N=25 Grade 2 4 (27%) 2 (50%) 2 (33%) 8 (32%) Grade 3 2 (13%) 1 (25%) 3 (50%) 6 (24%) IL-12 (pg/mL) IFN- (pg/mL) Mean Min Max Mean Min Max Grade 2 60.2 3.6 198.7 43.5 <8.0 194.7 Grade 3 101.7 4.7 >200.0 213.2 <8.0 370.7 Abstract Biological propertes of IL-12 “Master Regulator” Study Design Patents scheduled for SOC resecton Patents not scheduled for resecton Ad 2x10 11 vp Cohort 1: 20 mg v Cohort 2: 40 mg v Cohort 3: 30 mg v Expansion Cohort: 20 mg v Safety Cytokine Release Syndrome* and Cytokine Levels RTS ® Switch Responds to the Presence/Absence and Dose of Veledimex in Recurrent or Progressive GBM Patents  Tumor veledimex levels are ~40% of plasma levels  Based on the mouse model, the 20 mg dose is projected to generate 30-fold higher level of IL-12 and IFN- in the tumor N=25 * ZIOPHARM CRS Working Defniton Peripheral Blood CD8+/FOXP3 Rato at 14-28 Days Afer Viral Injectons Sugges ts Correlaton With Survival Centrally processed samples. N= 11 Deceased Alive Phase 1 Study: Updated Results R= 0.84 GBM Patent With Grade 3 CRS* With Rapid Reversal 0 2 4 6 8 10 12 14 16 18 20 22 24 Time in Months from Dosing 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Survival probability Censored 30 & 40 mg (N=10) 20 mg (N=15)  Median OS (mOS) is at 12.5 months with a mean follow up of 9.2 months (range: 1.8, 23.4)  6 of 15 subjects alive at 20 mg veledimex as of 24 May 2017 0 2 4 6 8 10 12 14 16 18 20 22 24 Time in Months from Dosing 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Survival probability Censored >100 mg (N=4) >10 - <=100 mg (N=7) <=10 mg (N=4) Dexamethasone Impact on Survival at 20 mg Veledimex Dexamethsone Use Days 0-14 Alive Deceased mOS Lower bound Upper bound ≤10 mg 4 0 Not reached Not reached Not reached 11-100 mg 2 5 12.5 Not reached Not reached ≥100 mg 0 4 8.0 1.8 12.7