RheoSwitch Therapeutic System Research Articles

CTIM-20. FINAL RESULTS OF CONTROLLED IL-12 MONOTHERAPY AND IN COMBINATION WITH PD-1 INHIBITOR IN ADULT SUBJECTS WITH RECURRENT GLIOBLASTOMA

Abstract Ad-RTS-hIL-12(Ad) is a gene therapy candidate conditionally expressing IL-12 under the transcriptional control of veledimex(V) acting via the RheoSwitch Therapeutic System® gene switch. Veledimex plasma and tumor PK demonstrated a dose-response relationship and crossing the BBB. PD-1+ T-cells were increased in tumor biopsy samples after treatment with Controlled-IL-12 in a phase-l study. This finding was the rationale for conducting 2 trials of Controlled-IL-12 in combination with PD-1-inhibitor to enhance T-cell-mediated anti-tumor effects. Data from two completed phase-I studies (presented in SNO2020), and an ongoing phase-II study of Controlled-IL-12 with cemiplimab study for the treatment of recurrent glioblastoma (rGBM) will be discussed. Ziopharm has conducted 3 phase-I (NCT02026271/NCT03679754 (monotherapy), NCT03636477 (combination with nivolumab)) and one phase-ll (NCT04006119) multicenter, open-label, single-arm trial in subjects with rGBM is evaluating Ad (single intratumoral injection, 2 x 1011-viral-particles, Day0) with oral V dosing (20mg, Days 0-14) with cemiplimab infusions (350 mg IV) on Days -7, 15, then Q3W. Systemic biomarkers (serum cytokines, and immune-activation-markers), local effects (tumor cytokines, T-cell immunobiology, pathology), neoepitope, and imaging will be assessed. Subject characteristics (Controlled IL-12 monotherapy (n=75); combination (Controlled IL-12 with anti-PD-1s) (n=61) were consistent across all 3 studies. Safety profiles were comparable between monotherapy and in combination with anti-PD-1s. Adverse reactions (ARs) after nivolumab or cemiplimab were consistent with labeling of anti-PD-1s. ARs related to Controlled IL-12 were all manageable and reversible with no synergistic toxicities in combination with anti-PD-1s. Increases in serum cytokine levels and pathology findings consistent with immune-mediated anti-tumor effect were observed in subjects who received Controlled-IL-12 monotherapy and in combination with anti-PD-1s. Final survival data and results from neoepitope analysis will be presented. Further investigation is warranted to understand the impact of monotherapy vs. combination, concurrent steroids use and unifocal vs. multifocal disease on overall survival in subjects with rGBM receiving Controlled-IL-12.

ATIM-18. SURVIVAL OF SUBJECTS WITH RECURRENT GLIOBLASTOMA RECEIVING INTRA-TUMORAL ADMINISTRATION OF IL-12 MANAGED WITH LOW-DOSE DEXAMETHASONE

Abstract Ad-RTS-hIL-12 (Ad) is a novel gene therapy, conditionally expressing IL-12 via the RheoSwitch Therapeutic System® (RTS®) gene switch under control of an oral activator ligand, veledimex (V). We previously reported results from 51 subjects (NCT02026271 and NCT03679754) describing biological activity of controlled IL-12, safety and survival data. Previously, subjects who received Ad+V (20 mg) managed with low-dose dexamethasone in the Main study achieved a mOS of 17.8 months, which is approximately twice the anticipated survival compared to historical controls. The mechanism of action of Ad+V is based on controlled secretion of recombinant IL-12 (measured in peripheral blood as a surrogate for intra-tumor-production), downstream upregulation of endogenous IFN-g (measured in peripheral blood), and an increase in the “cytoindex” (ratio of circulating CD8+ T cells to FoxP3+ regulatory T cells), an emerging biomarker of overall survival. Herein we provide an update of subject characteristics, survival and biomarker analysis from the ongoing Phase 1 and expansion substudy. assessing safety and tolerability of local, inducible IL-12 by single intratumoral injection of Ad (2 x 1011 viral particles) + V (20 mg PO QD x15 doses Days 0–14) in subjects, including a subset receiving low-dose corticosteroids (≤20 mg cumulative dexamethasone Days 0–14). Drug-related toxicities were predictable, dose-related, and promptly reversible upon discontinuation of V with no drug-related deaths. Biomarker studies related to production of IL-12 and IFN-g, as well as cytoindex remain encouraging. As of 04Jun19, mOS in the Expansion substudy had not yet been reached (patient enrollment occurred from September 2018-February 2019). Most subjects (65%) received low-dose dexamethasone (cumulative ≤20mg Days 0–14); initial impact of this and other subject characteristics on survival will be presented.

ATIM-25. PD-1 INHIBITION CAN BE COMBINED WITH IL-12 IN SUBJECTS WITH RECURRENT GLIOBLASTOMA

Abstract Monotherapy with intratumoral Ad-RTS-hIL-12 (Ad), a novel gene therapeutic conditionally expressing IL-12 under the transcriptional control of oral veledimex (V, 20 mg) acting via the proprietary RheoSwitch Therapeutic System® (RTS®), was shown in a phase 1 Main study (NCT02026271) to elicit a sustained intra-tumoral activated cytotoxic T-cell response with co-expression of PD-1. Additionally, the Main study showed improved median overall survival (mOS), compared to historical controls, in subjects with recurrent glioblastoma (rGBM) receiving Ad + V. Herein, we report updated findings from on an ongoing open label, dose-escalation Phase 1 substudy (NCT03636477) evaluating safety and tolerability of local, controlled IL-12 plus nivolumab in adult subjects with rGBM. Ad was administered by single intratumoral injection (2 x 1011 viral particles) on Day 0 plus V (10 and 20 mg) PO QD x 15 with nivolumab (1 and 3mg/kg) IV on Days -7, 15, then Q2W. Subjects have been accrued into three cohorts and follow-up is ongoing. Data from all three cohorts regarding dose escalation of V and nivolumab will be presented. The initial safety profile during V dosing period was similar to Ad+V monotherapy with adverse reactions being dose-related and rapidly reversible upon discontinuation of V. And those adverse reactions during the follow on nivolumab dosing were tolerable and manageable and consistent with nivolumab labeling, with no synergistic toxicities, and drug-related deaths. In the first two cohorts (where data is available), combination therapy improved the biomarker “cytoindex” (ratio of circulating CD8+ T cells to FoxP3+ regulatory T cells). (In the Main study, cytoindex correlated with overall survival). Controlled IL-12 production using Ad+V with nivolumab is a rational combination with initial data consistent with immune-mediated anti-tumor effects with a favorable safety profile. Further phase 2 investigation of Ad+V plus a checkpoint inhibitor in rGBM is planned.

Evaluation of controlled IL-12 in combination with a PD-1 inhibitor in subjects with recurrent glioblastoma.

2020 Background: Ad-RTS-hIL-12 (Ad) is a novel gene therapy candidate conditionally expressing IL-12 under the control of veledimex (V) acting via the proprietary RheoSwitch Therapeutic System (RTS) gene switch with a therapeutic window. Intratumoral Ad + oral V monotherapy (Phase 1 study, NCT02026271 ) resulted in a new sustained intra-tumor influx of activated cytotoxic T cells, consistent with an immune-mediated anti-tumor effect improving median overall survival (mOS) of subjects with recurrent glioblastoma (rGBM). This correlated with an increased circulating CD8+/FoxP3+ T cell ratio (“cytoindex”), an emerging biomarker for mOS. PD-1 expression on infiltrating T cells at biopsy after Ad+V, supports combining controlled IL-12 with a PD-1 inhibitor to further augment T-cell-mediated anti-tumor effects. The rationale is also supported by increased OS (100% combo vs 63% for Ad+V vs 40% for anti-PD-1) in mice bearing GL-261 glioma. Methods: An ongoing open label, dose-escalation Phase 1 trial (NCT03636477) is evaluating safety and tolerability of local, controlled IL-12 with nivolumab (nivo) in adult subjects with rGBM. Ad was administered by single intratumoral injection (2 x 1011 viral particles, Day 0) plus V (10-20 mg) PO QD x 15 with nivo (1-3mg/kg) IV on Days -7, 15, then Q2W. Results: Safety data revealed a similar profile as Ad +V monotherapy. Adverse reactions (ARs) during follow-on nivo dosing were consistent with anti-PD-1 reports. ARs were manageable and reversible with no synergistic toxicities. Nivo alone did not alter peripheral IL-12 levels (median baseline (before anti-PD-1) 0.9 pg/mL; Day 0 1 pg/mL) increasing to 5.5 pg/mL on Day 3. Nivo alone increased peripheral T cells (CD3+CD8+ median baseline 23%; Day 0 26%) and Ad+V elevated peripheral CD3+CD8+ to 31% at Day 14. Nivo alone decreased regulatory T cells (FoxP3 baseline 1.5% vs Day 0 0.8%). Ad+V decreased these to 0.3% (Day 14). Combination therapy improved the cytoindex (baseline 15; Day 0 29; Day 14 80). Conclusions: Controlled IL-12 production using Ad + V with nivo is a rational combination with initial data consistent with immune-mediated anti-tumor effects with a favorable safety profile, warranting continued investigation in rGBM. Clinical trial information: NCT03636477.

Evaluation of controlled IL-12 as monotherapy in subjects with recurrent GBM.

2053 Background: Interleukin-12 (IL-12), a master regulator of the immune system, results in anti-tumor responses in preclinical models, but safe use requires tightly controlled production. It was conditionally produced in Ph1 “main” study (NCT02026271) in subjects with recurrent glioblastoma (rGBM) using a replication-incompetent adenovirus modified to express IL-12 under transcriptional control of the proprietary RheoSwitch Therapeutic System (Ad-RTS-hIL-12, Ad) regulated by dose of veledimex (V). Monotherapy resulted in sustained intra-tumor influx of activated cytotoxic T cells, consistent with immune-mediated anti-tumor effect, improving overall survival (OS). This correlated with increased circulating CD8+/FoxP3+ T-cell ratio (“cytoindex”), an emerging biomarker of OS. While widely used with neurosurgery, dexamethasone (dex) blunts response to immunotherapies, nevertheless median mOS of subjects who received 20mg V of 12.7 mo (n=15) at 13.1 mo follow-up. However, subanalysis (n=6) showed low-dose dex (total ≤20 mg) during V dosing improved mOS (17.8 mo). We report a 36 subject substudy in rGBM with limited dex, total rGBM treated (n=70+). Methods: Ongoing Phase 1 substudy (NCT03679754) assesses safety and tolerability of local, inducible IL-12 by single intratumoral injection of Ad (2 x 1011 viral particles) + V (20 mg PO QD x15 doses Days 0-14) in subjects not receiving dex 4 wks prior to Ad. Results: As of 03Jan19, the majority of new subjects received low-dose dex (total ≤20mg Days 0-14). The initial impact of dex on mOS will be reported. As in the main study, Ad+V 20 mg respectively increased (median) serum IL-12 and downstream IFN-g from Days 0-3: 0.8 to 8.8 pg/mL and 0 to 8.6 pg/mL. Between Days 0-14, there was net increase in cytoindex (from 20 to 46). The safety profile was similar to the main study with the main adverse reaction (AR) being mild to moderate cytokine release syndrome (CRS) characterized by flu-like symptoms. No grade 4 CRS was noted; all ARs were manageable and reversable upon holding V. Conclusions: Local, controlled IL-12 production using the Ad + V platform in subjects with rGBM safely activates the immune system and when dex is limited, appears to further improve mOS, which warrants continued investigation. Clinical trial information: NCT03679754.

Regulated intratumoral expression of IL-12 using a RheoSwitch Therapeutic System\xae (RTS\xae) 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.

IMMU-33. CONTROLLED LOCAL EXPRESSION OF IL-12 AS GENE THERAPY CONCOMITANT WITH SYSTEMIC CHEMOTHERAPY IMPROVES SURVIVAL IN GLIOMA

AbstractSuccessful 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.

Expanded phase I study of intratumoral Ad-RTS-hIL-12 plus oral veledimex: Tolerability and survival in recurrent glioblastoma.

2044 Background: Glioblastoma (GBM) is an aggressive brain tumor affecting ~74,000 people worldwide annually. Recurrent GBM patients have a median OS (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 (Ad) is a novel gene therapy expressing IL-12 under the control of an oral activator ligand, veledimex (V), through the RheoSwitch Therapeutic System. Intratumoral administration of Ad results in targeted tumor cytotoxicity and induction of systemic T cell memory. Ad + V is a treatment strategy to extend the IL-12 therapeutic window. Methods: In a multicenter Phase I dose escalation trial and expansion cohort, subjects with recurrent or progressive Grade III or IV glioma undergoing resection were injected intratumorally with Ad 2 x 1011 viral particles and daily oral V for 15 doses, beginning prior to surgery. The primary endpoint is safety and tolerability of Ad + V; secondary endpoints include OS. Results: 25 subjects were dosed in 3 dose escalation 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 better drug compliance (86%) than the 30 mg (63%) or 40 mg (52%) cohorts and the 20 mg cohort shows better survival (mOS 12.7 months) compared to other cohorts. The frequency of related ≥Grade (G)3 AEs in the 20 mg cohort was significantly lower: 20% in 20mg, 50% in 30mg and 40 mg. In the 20 mg cohort, the most frequent AEs were transient mild flu-like 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 discontinuing V. Conclusions: Overall, Ad + 20 mg V is well tolerated; toxicities were predictable and reversible upon discontinuing V. There is a correlation between V dose, BBB penetration and drug related AEs. The tolerability and encouraging survival observed to date warrant further investigation in a pivotal trial. A stereotactic arm and a pediatric trial in diffuse intrinsic pontine glioma patients are planned. Clinical trial information: NCT02026271.

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.

Effect of controlled intratumoral viral delivery of Ad-RTS-hIL-12 + oral veledimex in subjects with recurrent or progressive glioma.

2052Background: The prognosis of subjects with glioblastoma multiforme (GBM) after first recurrence is extremely poor with a median overall survival (OS) of approximately 90 - 160 days. New therapies are needed, thus we developed an adenoviral vector, Ad-RTS-IL-12 (AD), administered intratumorally under the control of the RheoSwitch Therapeutic System (RTS) expression platform. Gene expression and IL-12 protein production is tightly controlled by activator ligand veledimex (V) and in our mouse model of glioma we have consistently shown a dose-response and survival benefit compared to temozolamide, bevacizumab, and a PD-1 inhibitor. Methods: In a multicenter Phase I dose escalation trial, subjects with recurrent or progressive Grade III or IV glioma undergoing resection were injected intratumorally with AD 2x1011viral particles and daily oral V for 15 doses, beginning on Day 0, prior to surgery. The study primary endpoint is safety and tolerability of AD+V with secondary endpoints including OS. Results: In...

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.

Abstract OT1-01-05: Phase 1b/2 study of intratumoral Ad-RTS-hIL-12 + veledimex in patients with chemotherapy-responsive locally advanced or metastatic breast cancer

Abstract Background: Immune-based strategies involving T-cell activation have recently shown significant activity in multiple tumor types. The presence of immune elements in breast cancers has prognostic and predictive impact. Thus, strategies that optimize the interplay between a breast cancer and the effected individual's immune system may be therapeutic. Interleukin-12 (IL-12), a pro-inflammatory cytokine, reverses immune escape mechanisms induced by myeloid derived suppressor and dendritic cells which, in turn, improves the function of activated CD8+ T cells and promotes tumor stroma collapse. Because tumor neoantigens may be generated in response to chemotherapy, IL12-mediated immune modulation may be optimal in patients with chemotherapy-sensitive metastatic breast cancer. Ad-RTS-hIL-12 (Ad) is a novel gene therapy candidate expressing IL-12 under the control of an orally-administered activator ligand, veledimex (V) through the proprietary RheoSwitch Therapeutic System® (RTS). Trial Design: Open-label, phase 1b/2, single-arm, single-center study of Ad+V in women with stable or responsive disease after ≥ 12-weeks of 1st or 2nd-line chemotherapy. Eligible patients will be placed on chemotherapy-holiday and enter the immunotherapy phase, consisting of a single cycle of Ad administered intratumorally (Day 1), along with V (80 mg QDx7). HER2-directed antibody therapy may be continued during the immunotherapy phase for women with HER2- disease. Key Eligibility Criteria: Women ≥18 years with histologically-confirmed locally advanced or metastatic breast cancer of any subtype who have achieved a partial response (PR) or stable disease (SD) to 1st or 2nd-line chemotherapy are eligible. Exclusion criteria include use of immunosuppressive drugs, compromised immune function, autoimmune disorder, or brain metastases. Specific Aims: To evaluate the safety and tolerability of Ad+V immunotherapy in eligible women. Secondary endpoints include 12 week overall response rate, 12 week disease control rate and the impact of treatment on exploratory immune biomarkers. Statistical Methods: Safety and efficacy will be evaluated separately for HER2-/HER2+ patients. Tumor response will be evaluated by RECIST v1.1 at 6 and 12 weeks. To ensure safety, stopping rules defined by grade 3/4 adverse events and12-week progression rate were adopted. Target Accrual: Up to 40 patients, including up to 8 patients (20%) with HER2+ disease. Summary: Ad+V is a novel gene therapy which controls local expression of IL-12 and may induce tumor stroma collapse and stimulation of an anti-cancer T cell immune response. The ability to regulate the production of IL-12 by modulating V dosing may result in an improved therapeutic index in combination with standard of care. The data from this study will directly inform future studies. Study Contact (Clinical Trials.gov: NCT02423902). Citation Format: McArthur HL, Page D, Proverbs-Singh T, Solomon S, Hudis C, Norton L, Patil S, Barrett JA, Lebel F. Phase 1b/2 study of intratumoral Ad-RTS-hIL-12 + veledimex in patients with chemotherapy-responsive locally advanced or metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-01-05.

A Synthetic Biology Rheoswitch Therapeutic System® for the Controlled Local Expression of IL-12 as an Immunotherapy for the Treatment of Cancer

Tumors escape the immune system through the process of immunoediting. Restoration of the immune system’s ability to detect the tumor should result in improved therapeutic outcome. A gene delivery platform technology, RheoSwitch Therapeutic System® (RTS®), has been developed to enable the controlled, regulated expression of a target gene which locally delivers the desired therapy to patients. A replication-incompetent adenoviral vector, administered intratumoral, is engineered to express IL‑12 (Ad-RTS-IL-12) under control of the RheoSwitch Therapeutic System, where IL-12 expression is controlled via the administration of an oral activator ligand (veledimex). In the presented preclinical studies we have demonstrated that the oral administration of veledimex resulted in a dose-related increase in tumor veledimex levels. The increase in tumor veledimex levels in combination with Ad-RTS-mIL-12 resulted in a dose-related increase in the IL-12 mRNA (switch on) leading to dose-related increases in IL-12p70 in the tumor with minimal increase in serum IL-12. The increase in tumor IL-12 correlated with an increase in tumor CD8+ cytotoxic T cells and a concomitant decrease in regulatory T cells (Tregs) in the tumor microenvironment. Importantly, Ad-RTS-mIL-12 + veledimex elicited dose-related decreases in tumor growth rate with no significant change in body weight in both breast and melanoma syngeneic mouse models. When veledimex was discontinued, expression of IL-12 mRNA returned to baseline concomitant with tumor IL-12 levels. In summary, these results highlight the potential of a synthetic biology-based approach for cancer treatment, safely leveraging the full anticancer potential of some immunomodulators without the serious side-effects associated with systemic injection.

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

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

Abstract 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.

IMPS-03INTRATUMORAL REGULATED EXPRESSION OF IL-12 AS A GENE THERAPY APPROACH TO TREATMENT OF GLIOMA

Immunotherapy is an attractive approach for glioma 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 (in a dose-dependent manner) by oral administration of veledimex. 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. These changes correlated with a local and systemic immune and anti-tumor response. Veledimex crossed the blood-brain-barrier in both naive and orthotopic GL-261 mice with increased brain tissue level of ∼6 fold observed in tumor bearing vs. normal mice (1950 ± 573 and 324 ± 51ng/g). Based on these findings the effects of Ad-RTS-mIL-12(5e9vp) + veledimex were studied in the following cohorts; dexamethasone, bevacizumab, temozolamide and CD279 (PD-1 inhibitor). Ad-RTS-mIL-12 + veledimex demonstrated a dose-related increase in survival without significant adverse events. At Day 92 (study termination), 50% of the animals that received veledimex at 100mg/m2/day for 14 consecutive days were alive and tumor free with peak tumor IL-12 at Day 3 of ∼240pg/mg. In contrast, the mean survival for the other groups were: vehicle 18d, dexamethasone 24d, bevacizumab 25d, temozolamide 40d and CD279 38d demonstrating that this novel regulated immunotherapeutic approach may be an effective form of therapy for glioma. A Phase 1 study of Ad-RTS-hIL-12 + veledimex in patients with recurrent or progressive malignant glioma are stratified into two groups: resection plus localized injection or stereotactic injection (3D-located) of Ad-RTS-hIL-12 (single intratumoral injection) + veledimex 14 days (oral) has initiated. The primary endpoint is the safety and tolerability of Ad-RTS-hIL-12 + veledimex with secondary endpoints of MTD, immune response, objective response rate, disease progression, progression-free and overall survival.

Abstract B25: Controlled production of a bispecific antibody by a genetically modified stem cell triggers T cell activation and cytolysis in non-small cell lung carcinoma

Abstract Bispecific antibodies and their derivatives can induce potent cytolytic activity through activation of T cells which target tumor cells. Cell linking moieties (CLM) represent engineered, bispecific antibodies consisting of two scFv. Previous studies have shown that the systemic distribution and pharmacokinetic profile of these agents has limited their utility for many target and effector combinations. We hypothesize that a genetically-modified, allogeneic cellular product could be developed to express such molecules leading to the inducible release at the tumor site following systemic delivery and thereby providing spatial and temporal control of the CLM distribution to increase the number of target-effector combinations. The RheoSwitch Therapeutic System® (RTS®) is a gene switch activated by a small molecule ligand that has been demonstrated clinically to control the timing and extent of gene expression. The RTS® platform has three essential elements: (1) switch components (co-activation partner and a ligand-controllable transcription factor); (2) activator ligand (veledimex); and (3) inducible promoter. Sans ligand, the switch complex is inactive and inhibits gene expression. By adding ligand, the switch components form an active complex, prompting gene expression in a dose-dependent manner. Endometrial regenerative cells (ERC) are a mesenchymal-like stem cell with a high replicative potential. We demonstrate that genetically modified ERC (GM-ERC) can be generated with high efficiency via nucleofection, lipofection or transduction with adenovirus, adeno-associated virus (AAV) or lentivirus. GM-ERC expressing a CLM construct comprised of anti-CD3-anti-EGFR bispecific antibody (CLM) under constitutive expression secretes up to 300 ng/ml of CLM as evaluated by ELISA. Addition of GM-ERC cells, supernatants from GM-ERC, or purified CLM to co-cultures of unstimulated peripheral blood mononuclear cells and EGFR+ A549 lung carcinoma cells led to T cell activation as measured by cytokine secretion (IL-2 and IFN-γ) and upregulation of CD69, CD25, and EGFR-specific cell-mediated cytotoxicity of A549 cells in vitro. CLM-expressing ERC were effective in coculture killing assays at doses as low as 1% of A549 target cells. Expression of CLM under the inducible promoter (RTS-CLM) yielded effective control of CLM secretion and killing activity, with greater than 80% cytotoxicity against A549 at 48-72 hours of cocultures with ERC-RTS-CLM, A549 cells and T cells when in the presence of veledimex (ligand), but not vehicle control. The data demonstrate that GM-ERC can secrete a bispecific antibody that induces T cell mediated killing in vitro, and that this activity can be controlled using an RTS® expression vector in combination with an activator ligand. This provides evidence supporting the feasibility of the cytolytic activity of CLM-secreting GM-ERC as an allogeneic cell therapy product, a novel and promising approach for the therapy of lung carcinoma and other EGFR+ malignancies. Citation Format: Amy Wesa, Yumei Xiong, Tim Chan, Jeff Rosenbloom, Srinivas Rengarajan, Paul Szymanski, John A. Barrett, Francois Lebel, Farzad Haerizadeh, Richard Einstein. Controlled production of a bispecific antibody by a genetically modified stem cell triggers T cell activation and cytolysis in non-small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B25.

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

Abstract 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 naïve 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

Regulated intratumoral expression of IL-12 as a basis for combination therapy in melanoma

Background Major obstacles for the development of immunotherapeutics are the ability of tumors to escape the immune system coupled with toxicity associated with systemic administration. To overcome these challenges, we have developed an adenoviral vector, Ad-RTS-IL-12 (AD), administered intratumorally under control of the RheoSwitch Therapeutic System gene expression platform. Expression of IL-12 mRNA and IL-12 protein is tightly regulated by the oral administration of a small molecule activator ligand, veledimex (AL).

Abstract P5-02-01: Regulated intratumoral expression of IL-12 in combination with cytotoxic agents as a strategy for the treatment of metastatic breast cancer

Abstract Major obstacles for the development of immunotherapeutics are the ability of tumors to escape the immune system coupled with toxicity associated with systemic administration. To overcome these challenges, we have developed an adenoviral vector, Ad-RTS-IL-12 (AD), administered intratumorally (IT) under control of the RheoSwitch Therapeutic System® (RTS) technology platform. Expression of IL-12 mRNA and IL-12 protein is tightly regulated by the oral administration of a small molecule activator ligand, veledimex (AL). We have previously demonstrated a concentration-related increase in IL-12 mRNA concomitant with increase in expression of IL-12 protein in HT1080 cells transduced with AD and incubated with AL. Removal of AL from the media resulted in a return to baseline IL-12 expression within 48 hours. Results from the subcutaneous 4T1 syngeneic BALB/c mouse mammary tumor model demonstrated an AL dose-related increase in tumor IL-12 mRNA and IL-12 protein expression with the maximum IL-12 tumor protein level of 280 ng/mg achieved at 150 mg/m2 AL + 1e10 vp AD. In addition, a return to baseline IL-12 mRNA and IL-12 protein expression was observed on cessation of AL. The effect of AD + AL on tumor growth rate was evaluated in a subcutaneous 4T1 syngeneic BALB/c mouse mammary tumor model. A single intratumoral injection of 1e10 vp AD combined with oral administration of AL (15, 30, 75 or 150 mg/m2) on a Q1Dx5 schedule led to significant AL dose-related tumor growth inhibition with tumor size reduction of 27%, 37%, 57% and 60%, respectively when compared to vehicle on Day 33. Neither AD or AL alone had an effect on tumor growth rate relative to vehicle control. No change in clinical signs or body weight was observed when compared to vehicle alone. Tumor growth inhibition correlated with an increase in tumor IL-12 levels, a decrease in Tregs, and an increase in cytotoxic T cells. Results from a phase 1 study in melanoma have shown dose-related production of IL-12 and clinical activity in injected and non-injected lesions at 100 and 160 mg of AL. Cytotoxic agents at low doses have been shown to prime the immune system and combination with immunotherapy may augment tumor specific T-cell immune responses resulting in enhanced efficacy. In the 4T1 mouse mammary tumor model Ad-RTS-mIL12 (1e10vp) + AL (30 mg/m2) combined with palifosfamide (40 or 120 mg/m2 QD IP for 3 days) significantly inhibited tumor growth (∼71-90% vs. control) concomitant with increased median survival when compared to the single agents alone. Based on these findings in a multicenter, open-label, randomized, phase 2 study evaluating the safety and efficacy of AD + AL alone or in combination with cytotoxic agents in subjects with recurrent/metastatic breast cancer with accessible tumor(s) is ongoing. This study assesses AD + AL administered as a monotherapy and a combination therapy. In the monotherapy arm, AD is administered IT on Day 1 and AL 140 mg is administered orally on either a Q1Dx7 or QODx14 schedule. In the combination arm, palifosfamide 120 mg/m2 IV Q1Dx3 is be administered with AD + AL QODx14 (28 day cycle). Preliminary data show that AD + AL is safe and well tolerated. Preclinical and clinical data will be presented. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-02-01.