Hongliang Cai

Plasma Pharmacokinetics of Veledimex, a Small‐Molecule Activator Ligand for a Proprietary Gene Therapy Promoter System, in Healthy Subjects

Major obstacles to developing effective immunotherapy are the ability of tumors to escape the immune system and the toxicity associated with systemic administration. To overcome these challenges, a gene delivery platform technology, RheoSwitch Therapeutic System (RTS), has been developed to enable the regulated expression of a target gene, Ad‐RTS‐IL‐12, administered intratumorally, where IL‐12 expression is controlled via the administration of an oral activator ligand, veledimex. Pharmacokinetics in healthy human subjects indicated that veledimex plasma exposure increased with increasing dose after single‐ and multiple‐dose administration in Labrasol slurry and F‐22 capsule formulations. No apparent formulation or sex‐related difference in veledimex pharmacokinetics (PK) was observed. Minimal or no plasma accumulation of veledimex was observed after once‐daily oral administration for 14 days. Veledimex steady state in plasma was reached after 5 daily doses. Food consumption prior to veledimex administration prolonged and enhanced absorption with no impact on the elimination rate and extent of metabolism of veledimex, resulting in significantly increased systemic exposure to veledimex and its 2 major circulating metabolites. Overall, veledimex was well tolerated and exhibited a PK profile supportive of once‐daily dosing. For enhanced efficacy, veledimex should be taken under fed conditions to ensure optimal absorption and sufficient systemic exposure.

Regulated intratumoral expression of IL-12 using a RheoSwitch Therapeutic System ® (RTS ® ) gene switch as gene therapy for the treatment of glioma

The purpose of this study was to determine if localized delivery of IL-12 encoded by a replication-incompetent adenoviral vector engineered to express IL-12 via a RheoSwitch Therapeutic System® (RTS®) gene switch (Ad-RTS-IL-12) administered intratumorally which is inducibly controlled by the oral activator veledimex is an effective approach for glioma therapy. Mice bearing 5–10-day-old intracranial GL-261 gliomas were intratumorally administered Ad-RTS-mIL-12 in which IL-12 protein expression is tightly controlled by the activator ligand, veledimex. Local tumor viral vector levels concomitant with veledimex levels, IL-12-mRNA expression, local and systemic cytokine expression, tumor and systemic flow cytometry and overall survival were studied. Ad-RTS-mIL-12+veledimex elicited a dose-related increase in tumor IL-12 mRNA and IL-12 protein and discontinuation of veledimex resulted in a return to baseline levels. These changes correlated with local immune and antitumor responses. Veledimex crossed the blood–brain barrier in both orthotopic GL-261 mice and cynomolgus monkeys. We have demonstrated that this therapy induced localized controlled production of IL-12 which correlates with an increase in tumor-infiltrating lymphocytes (TILs) leading to the desired biologic response of tumor growth inhibition and regression. At day 85 (study termination), 65% of the animals that received veledimex at 10 or 30 mg/m2/day were alive and tumor free. In contrast, the median survival for the other groups were: vehicle 23 days, bevacizumab 20 days, temozolomide 33 days and anti-PD-1 37 days. These findings suggest that the controlled intratumoral production of IL-12 induces local immune cell infiltration and improved survival in glioma, thereby demonstrating that this novel regulated immunotherapeutic approach may be an effective form of therapy for glioma.

509. Regulated Expression of IL-12 as Gene Therapy Concomitant with Blockade of PD-1 for Treatment of Glioma

The utility of immunotherapy in the treatment of glioma may be improved through combination therapies that enhance cytotoxic immune-activation while concomitantly reducing immunosuppression. We provide data in mice to support evaluation of combining controlled local interleukin 12 (IL-12) administration and blockade of programmed cell death protein 1 (PD-1) in humans. To circumvent challenges surrounding the uncontrolled activation, we have implemented clinical trials using a replication-incompetent adenovirus engineered to conditionally express IL-12 (Ad-RTS-IL-12), via our RheoSwitch Therapeutic System® (RTS®) gene switch. When directly injected intra-tumorally in pre-clinical or clinical studies, IL-12 expression is “off” when devoid of the activator ligand (veledimex, V) and IL-12 production is turned “on” (in a dose-dependent manner) by oral administration of veledimex. PD-1 inhibitors using therapeutic monoclonal antibodies (mAbs) demonstrate an ability to reverse tumor immunosuppression and are effective in the treatment of some cancers. In the present pre-clinical study we assessed the effects of Ad-RTS-mIL-12+veledimex (Ad+V) alone, Ad 5×109 viral particles (vp) + V 10-30mg/m2/day for 14 days or in combination with the antiPD-1-specific mAb RMP1-14 (antiPD-1, 7.5 & 15.0 mg/m2 for 4/day for 5 days i.p.) in the orthotopic GL-261 mouse model. All mice without treatment succumb to disease progression by Day 35. Eighty days after immunotherapy, 70-80% receiving Ad +V monotherapy survived, 30-40% receiving antiPD-1 monotherapy survived and 100% receiving Ad +V 30 mg/m2 + antiPD-1 15.0 mg/m2 combination survived. There was an increase in tumor IL-12 (100 pg/mg) which was 15-times greater than that of plasma peak 5 days after Ad +V. Furthermore, the combination of Ad +V+antiPD-1 sustained peak IL-12 levels in tumor which was associated with a 100-150% increase of activated T cells in the spleen compared with the minimal changes observed with either immunotherapy alone. In addition, there was an additive reduction in regulatory T cells (FOXP3) compared with monotherapies. In summary we demonstrate that controlled local immunostimulation with IL-12 combined with inhibition of PD-1 is an attractive approach for the treatment of glioma. Since both Ad-RTS-IL-12 and mAb blocking PD-1 are clinically available, these data provide impetus for evaluating this combination immunotherapy in humans.

IMST-07. LOCAL REGULATED IL-12 EXPRESSION AS AN IMMUNOTHERAPY FOR THE TREATMENT OF PONTINE GLIOMA

John A Barrett, Ph.D. jabarrett@ziopharm.com • Glioma in the pontine region accounts for ~15% of pediatric brain tumors with a grim median survival time of <1 year. Immunotherapy may be an attractive approach for the treatment of pontine glioma, however, the lack of defined tumor antigens with homogeneous expression that may be safely targeted has handicapped this approach. • We are conducting clinical trials to recruit and stimulate the endogenous immune response by local delivery of a replication-incompetent adenovirus engineered to conditionally express IL-12, via our RheoSwitch Therapeutic System® (RTS®) gene switch (Ad-RTS-IL-12). • Previous studies in supratentorial syngeneic mouse tumor models demonstrated that Ad-RTS-mIL-12 + veledimex elicited a dose-related increase in tumor IL-12 mRNA resulting in IL-12 protein and downstream IFN- which correlated with increased anti-tumor effects and survival. In ongoing clinical trials with Ad-RTS-hIL-12 + veledimex within recurrent supratentorial gliomas, we observed encouraging improvement in overall survival and toxicity profile that is readily manageable by stopping veledimex.Copy and paste your text content here, adjusting the font size to fit into the textbox.

Abstract A049: Demonstration of systemic antitumor immunity via intratumoral regulated expression of IL-12 in advanced breast cancer and melanoma patients

Immunotherapy is an attractive approach to cancer treatment. Localized controlled production of IL-12 in a tumor generates both local and systemic antitumor immune responses. We have developed an adenoviral vector, Ad-RTS-IL-12 (AD), administered intratumorally under control of the RheoSwitch Therapeutic System® (RTS®) expression platform. Gene expression and subsequent IL-12 protein production is tightly controlled by oral administration of the small molecule activator ligand veledimex (AL). We have shown on mechanism biologic activity and safety with this system in syngeneic mouse studies of melanoma and breast cancer (BC). Those studies demonstrated an AL dose-related increase in tumor IL-12 mRNA and IL-12 protein expression. In addition, a return to baseline IL-12 mRNA and IL-12 protein expression was observed on cessation of AL. These changes correlate with a local and systemic immune and antitumor response. Subsequently, two open label, phase 2 trials evaluating the safety of inducible IL-12 expression in heavily pretreated subjects with recurrent/metastatic breast or melanoma cancer with surface accessible lesions were performed. The results of these studies showed biologic activity with an acceptable therapeutic index. Treatment with Ad-RTS-hIL-12 + veledimex resulted in an increase in IL-12 and downstream IFN-gamma production and was followed by a rapid increase in IL-10 and IP-10 (indicating IL-12 biologic activity). Tumor lesion results from these two maximum tolerated dose studies are encouraging. In the breast cancer study, 12 subjects were administered Ad-RTS-IL-12 + veledimex. A total of 16 non-injected lesions in 7 breast cancer subjects were evaluated. Of the 16 non-injected lesions, 4 increased in diameter >20%, 6 increased in diameter 0-20% with observed decreases in lesion diameters of 1 lesion 12%, 2 lesions 30-49% and 3 lesions 50-100%. Injected lesions were not evaluated in this study. In the melanoma study, 26 subjects were administered Ad-RTS-IL-12 + veledimex. A total of 70 non-injected lesions in 19 melanoma subjects were evaluated. Of the 70 lesions, 23 increased in diameter >20%, 30 increased in diameter 0-20% with observed decreases in lesion diameters of 7 lesions 10% to 19%, 4 lesions 20-29%, 4 lesions 30-49%, and 3 lesions 50-100%. In the same 19 melanoma subjects, a total of 33 injected lesions were evaluated. Of the 33 lesions, 11 increased in diameter >20%, 9 increased in diameter 0-20% with observed decreases in lesion diameters of 3 lesions 10% to 19%, 1 lesion 25%, 4 lesions 30-49% and 5 lesions 50-100%. Most common ≥ Grade 3 treatment emergent adverse events in BC and melanoma included neutropenia and hyponatremia (16% each), hypotension, cytokine release syndrome, AST increase (11% each), dehydration, fatigue, pyrexia (8% each). All TEAEs and SAEs ≥ Grade 3 reversed rapidly upon discontinuation of veledimex In conclusion, regulated and controllable IL-12 expression using Ad-RTS-hIL-12 + veledimex in all advanced breast cancer and melanoma patients is promising with an acceptable and rapidly reversible adverse experience profile upon discontinuation of veledimex. The administration of Ad-RTS-hIL-12 + veledimex has shown biologic activity and warrants further clinical investigation. Citation Format: Francois Lebel, John A. Barrett, Hongliang Cai, John Miao, Suma Krishnan, Laurence JN Cooper. Demonstration of systemic antitumor immunity via intratumoral regulated expression of IL-12 in advanced breast cancer and melanoma patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A049.

Abstract B091: Demonstration of systemic antitumor immunity via intratumoral regulated expression of IL-12 as a gene therapy approach to treatment of cancer

Immunotherapy is an attractive approach for cancer therapy. We have developed a replication-incompetent adenovirus engineered to express IL-12 (Ad-RTS-IL-12), via our RheoSwitch Therapeutic System® (RTS®) gene switch, injected directly into a tumor. IL-12 expression is “off” devoid of the activator ligand, veledimex, while IL-12 production is turned “on” (in a dose-dependent manner) by oral administration of veledimex. Veledimex had good bioavailability in mice (> 53%) following oral administration with apparent terminal half-life of 6-8 hours. In normal mice with intact blood brain barrier (BBB), dose-related increases in veledimex exposure were exhibited in brain tissue with approximately 1% plasma exposure in cerebral spinal fluid, which thereby demonstrated that veledimex crossed the BBB in mice. Mechanistic studies in numerous syngeneic mouse tumor models with Ad-RTS-mIL-12+veledimex have demonstrated a dose-related increase in tumor IL-12 mRNA and IL-12 protein expression. Discontinuation of veledimex resulted in a return to baseline IL-12 mRNA and protein expression. The ability to treat tumors at different physical locations derived from the same tumor lineage was studied in melanoma, colon cancer and glioma syngeneic mouse models. In a B16 mouse melanoma model, 1e6 melanoma cells were placed in both flanks. When the tumors reached 100mm3 one tumor was administered Ad-RTS-mIL-12 (1e10vp) + veledimex administered at ~675mg/m2/day ad lib in the chow). The results of this study showed > 90% tumor growth reduction in both the treated and untreated tumors when compared to control. In the second study, 1e6 CT26-LUC colon cancer cells were placed in the right flank with tumor growth monitored using bioluminescence imaging. On Day 15 Ad-RTS-mIL-12 (1e10vp) was administered intratumorally + veledimex at ~675mg/m2/day ad lib in the chow. On Day 43, 60% were tumor free and remaining mice had smaller tumors. When these mice were rechallenged with CT26-LUC cells in the contralateral flank no tumor growth was observed versus age matched controls for > 21days (end of study). In the glioma study, 1e5 GL-261 cells were implanted in the brain and 5 days later mice were treated with a single cycle of Ad-RTS-mIL-12 (1e10vp) administered intratumoral + veledimex 450mg/m2/day, po. QDx14. Vehicle cohorts all succumbed to the disease by Day 30. The 12 surviving animals from the treatment group on Day 100 were reinoculated with 1e5 GL-261 cells at the same site and survival monitored for an additional 80 days vs. age matched controls. We found >95% of the pretreated animals survived for an additional 80 days (end of study) vs. ~40% of the control animals. Similar results have been observed in patients with metastatic melanoma and breast cancer where nontreated lesions were also observed to decrease in size In summary, these results demonstrate this novel regulated immunotherapeutic approach may be an effective form of therapy for both primary and metastatic tumors with the same tumor lineage in melanoma, colon cancer and glioma. Further studies are planned in other tumor populations. Citation Format: John A. Barrett, Tim Chan, Hongliang Cai, John Miao, Suma Krishnan, Francois Lebel. Demonstration of systemic antitumor immunity via intratumoral regulated expression of IL-12 as a gene therapy approach to treatment of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B091.

Abstract 3647: Treatment of glioblastoma through the controlled localized production of IL-12 by the RheoSwitch Therapeutic System® Platform

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Challenges in developing immunotherapies against glioblastoma include the immune-privileged status of the CNS and the physiological processes that contribute to the suppression of immune responses in the brain. The localized controlled production of IL-12 in the tumor may increase cytotoxic T cell infiltrate and subsequently reduce tumor vascularity and tumor volume, resulting in a prolongation of survival. We have developed an adenoviral vector, Ad-RTS-mIL-12 (AD), administered intratumorally under the control of the RheoSwitch Therapeutic System® (RTS®) expression platform. Gene expression and subsequent IL-12 protein production is controlled by the oral administration of the small molecule activator ligand veledimex (AL). We have shown the the safety and biologic activity of this system in both nonclinical and clinical studies in melanoma and breast cancer. Based upon these favorable results we chose to study the activity of RTS-mIL-12 delivered intratumorally by an adenoviral vector-based approach in an orthotopic murine glioma model. The ability of AL to cross the blood brain barrier was assessed in both naive and orthotopic GL-261 tumor bearing C57B6 mice. The results in mice demonstrated that AL brain tissue levels increased from 25325 to 47868 ng*h/g in naive mice at doses of 225- 450 mg/m2 p.o. administered on either QD or BID schedules while, as anticipated, the levels of AL in brain tissue increased in the tumor bearing mice. At 450 mg/m2/day BID p.o. increased brain tissue AL levels in GL261 mice increased by approximately 6 fold from 324±51 ng/g in normal mice to 1950±573 ng/g at Day 3 and were sustained through 14 days of continuous dosing (1150±212 ng/g). Based on these findings we assessed the effects of AD + AL on survival in the orthotopic GL261 syngeneic mouse glioma model where each animal received 1 x 105 GL261 glioma cells via intracranial injection on Day 0. In this study AD was administered intratumorally at 1 x 1010 vp/animal on Day 5. AL was administered via gavage at doses of 450-1200 mg/m2/day for 14 consecutive days on a BID schedule. In addition, bevacizumab 30mg/m2 biwkx3 and dexamethasone 6mg/m2/day BID x14 days were studied. AD + AL demonstrated a dose-related increase in survival benefit without exhibiting an adverse safety profile. At Day 100 with (study termination), 45, 50 and 68% of the animals that received doses of 450, 900 or 1200mg/m2/day, respectively, were alive. In contrast, the median survival in the vehicle control groups was 18 days, while in the bevacizumab and dexamethasone groups the median survival was 22 and 24 days, respectively. Additional studies in this model are ongoing to determine the optimal dose and schedule. This novel regulated immunotherapeutic approach could potentially be translated into an effective clinical regimen for the treatment of glioblastoma. Citation Format: John A. Barrett, Hongliang Cai, Francois Lebel, Kay R. Meshaw, Tami Zmetra, Jonathan Lewis. Treatment of glioblastoma through the controlled localized production of IL-12 by the RheoSwitch Therapeutic System® Platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2014-3647