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Ad-RTS-hIL-12 (Ad) is a novel gene therapy expressing IL-12 via the RheoSwitch Therapeutic System(®) gene switch under control of an oral activator ligand, veledimex (V). We previously reported on an open label Phase I trial describing biological activity of recombinant IL-12 with downstream IFN-γ and activation of the immune system. We provide an update on the intratumoral injections of Ad (2x10(11)virus-particles) + V for patients with recurrent GBM (rGBM) in Group 1 (G1) (craniotomy, n=31) and initial results for Group 2 (G2) (stereotactic administration n=7). In G1, the V 20-mg cohort mOS increased to 12.7 months with mean follow-up of 12.9 months. 20-mg V in G1 showed fewer toxicities and higher V compliance (84%) compared with higher-doses of V (30 and 40-mg) with 75% and 67%, respectively. These data are encouraging compared to historical data that predict mOS of 5 to 8 months. An additional cohort at V 10-mg (n=6) was well tolerated, but subtherapeutic, with a mOS of 7.6 months (mean follow-up 6.7 months). There was an association between V dose level, blood-brain-barrier penetration, and drug-related adverse events (AEs) with increased TEAEs observed above V 20-mg. Subgroup analyses across all cohorts did not detect statistically significant differences including extent of resection or IDH mutation status. Subjects (20-mg V) who received a cumulative dose of ≤10mg of dexamethasone during the first 15 days of treatment showed improved OS versus >100mg of dexamethasone, suggesting corticosteroid-mediated blunting of the IL-12 dependent immune-mediated therapeutic effect. In the G2 20-mg V cohort, similar cytokine levels and reversible AEs were observed compared to G1; follow up is ongoing and mOS will be presented. Based on these results and the best risk-benefit profile, the 20-mg V dose level was chosen for further investigation. Combination with an immune checkpoint inhibitor in rGBM is underway.

Modulating the tumor microenvironment with immuno-stimulatory therapy maybe an attractive approach for the treatment of glioma. We took advantage of IL-12 biology utilizing a replication-incompetent adenovirus (Ad) engineered to conditionally express mouse or human IL-12 (Ad-RTS-mIL-12 or Ad-RTS-hIL-12), via our RheoSwitch Therapeutic System® (RTS®) gene switch, tightly controlled by the oral activator ligand veledimex (V). Previous human and mouse studies demonstrated that V crosses the blood-brain-barrier (BBB), enabling controlled IL-12 expression. Ad-RTS-mIL-12 injected into orthotopic GL-261 glioma at the optimal dose of 5x109 viral particles (vp) +veledimex (Ad+V) elicited dose-related increases in local IL-12 and downstream IFN-gamma production. Cytokines within the tumor were ~20 times higher than serum and elicited sustained increases within glioma of cytotoxic T cells and macrophages concomitant with reduction in regulatory T cells (Tregs) resulting in enhanced survival where ~65% of mice receiving Ad+V 10-30mg/m2/day were tumor free 80 days after immunotherapy (end of study). Ad+V 10mg/m2/day was the optimal therapeutic index. Using allometric modeling, mouse Ad+V 10mg/m2/day translated into ~20mg V for a 70kg subject and supports Ad-RTS-hIL-12 up to 5x1012vp. Based on these results, a phase 1 trial in recurrent glioma studying local hIL-12 production using Ad-RTS-hIL-12 2x1011vp +V 10-40mg QDx14po. was initiated. Interim results demonstrated that V20mg elicited the optimal therapeutic index. Results at 20mg (N=15) demonstrated that V crossed the BBB with 35 ± 5% of plasma levels in tumor and peak serum levels of hIL-12 and IFN-gamma at 27 and 36pg/ml. Increase in circulating cytotoxic-T cell/Tregs ratio at days 14-28 correlated with an increase in the median overall survival of 12.5mo. Our working hypothesis tested in the mouse that IL-12 activates the immune response leading to mouse anti-tumor effects now appears to be validated in humans.

Successful immunotherapy is dependent on the regulation of an immune response concomitant with modulation of the tumor microenvironment. To study this hypothesis, we utilized a replication-incompetent adenovirus engineered to conditionally express mouse IL-12 (Ad-RTS-mIL-12), via our RheoSwitch Therapeutic System® (RTS®) gene switch which is tightly controlled by the oral activator ligand veledimex (V) in combination with the chemotherapeutic CCNU (lomustine, alkylating nitrosourea) in a GL-261 orthotopic glioma mouse model. The effects of Ad-RTS-mIL-12+veledimex (Ad+V) alone Ad 5x109vp+V 3-30mg/m2QDx14po or concomitantly with CCNU 9-18mg/m2QDx5ip were studied. Mice without treatment succumb to disease progression by day 39 (median 22 days). Ad+V 3mg/m2 monotherapy prolonged median overall survival (mOS) to 37 days. Eighty days after immunotherapy (end of study), ~65% mice receiving Ad +V 10-30mg/m2/day survived. CCNU 9mg/m2 failed to improve mOS while 18mg/m2 increased mOS to 36 days. Ad+V 3mg/m2 combined with CCNU 9mg/m2 failed to improve mOS while Ad+V 3mg/m2+CCNU 18mg/m2 increased survival with 60% surviving at study end. Ad+V 10mg/m2+CCNU 9mg/m2 failed to increase mOS over monotherapy while Ad+V 10mg/m2+CCNU 18mg/m2 resulted in 100% survival at end of study. Ad+V 30mg/m2+CCNU (9-18mg/m2) did not further improve survival with increased clinical signs over monotherapy that recovered on discontinuance of dosing. Ad+V increased tumor IL-12 (80 pg/mg) which was ~15-times greater than that of plasma while CCNU alone or in combination with Ad+V did not affect tumor and serum cytokines. Within the tumor, Ad+V elicited a dose-related increase in cytotoxic T cells and macrophages and decrease in regulatory T cells consistent with immune-mediated anti-tumor effects. Ad+V+CCNU did not further alter the tumor immune profile over Ad+V monotherapy. In summary, the controlled local immunostimulation with IL-12 combined with CCNU, warrants further investigation in GBM.

Glioblastoma (GBM) is an aggressive brain tumor affecting ~74,000 people worldwide annually. Recurrent GBM patients have a median overall survival (mOS) of 6–7 months. OS in patients who have failed temozolomide, bevacizumab or equivalent salvage chemotherapy, is ~3–5 months. New therapies are urgently needed. Ad-RTS-hIL-12 is a novel gene therapy expressing IL-12 under the control of an oral activator ligand, veledimex, via the RheoSwitch Therapeutic System® gene switch. Intratumoral administration of Ad-RTS-hIL-12 results in targeted tumor cytotoxicity and induction of systemic T cell memory. Ad-RTS-hIL-12 + veledimex is a treatment strategy to extend the IL-12 therapeutic window. In a multicenter, Phase I dose escalation trial and expansion cohort of subjects with recurrent or progressive Grade III or IV glioma undergoing resection, Ad-RTS-hIL-12 + veledimex was well tolerated; toxicities were predictable and reversible upon discontinuing veledimex with a correlation between veledimex dose, BBB penetration and drug related AEs to assigned dose level. The most common adverse reactions were related to cytokine exposure in the form of fever, decrease in peripheral lymphocytes and platelets, and elevation of liver transaminases. Emerging biomarker data appear to show that serum CD8+/FOXP3 ratio might represents a putative predictor of response. Subjects treated with 20mg of veledimex had a mOS of 12.5 months and when given ≤10mg of dexamethasone during the first 15 days of treatment, overall survival was improved, possibly suggesting a negative effect of steroids on immunostimulation. The tolerability and encouraging survival observed to date warrant additional clinical studies of this controlled immunogene therapy.