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Ad-RTS-hIL-12 (Ad) is a gene therapy candidate for intratumoral (IT) delivery that conditionally expresses IL-12 (IL-12) under the transcriptional control of orally administered veledimex (V) acting via the RheoSwitch Therapeutic Systemâ gene switch. Increased PD-1 expression in samples of rGBM following Ad+V (ASCO 2020) supports combination immunotherapy with a PD-1 inhibitor. Phase 1 trials in rGBM of Ad+V as monotherapy and in combination with PD-1 inhibitor revealed encouraging safety and survival data. This phase 2 trial (NCT04006119) in adults with rGBM is evaluating safety and efficacy (overall survival) of Ad + V with pre/post-operative cemiplimab-rwlc (cemi) 350 mg IV, Days -7, 15, then Q3W; Ad single IT injection (2 x 1011 viral particles, day of resection (Day 0) /craniotomy); and V (20 mg PO, Days 0–14). Longitudinal sampling of serum assessed IL-12 and endogenous cytokines production. Anti-tumor effects were described upon preliminary review. Serial MRI evaluated tumor response. Follow-up described overall survival. Initial safety data (51 unique adverse reactions in 28 subjects) appeared similar to Ad+V and the cemi label, respectively, being manageable without synergistic toxicities and generally reversible. Of 35 SAEs reported in 18 subjects, 10 SAEs in 7 subjects were related to Ad+V. IL-12 and IFN-g levels increased after Ad+V administration peaking on Day 3 at 34 ± 11 pg/mL and 13 ± 5 pg/mL (mean ± SEM), respectively. Immune-mediated anti-tumor effects were noted, including a post-treatment biopsy to rule out progression which demonstrated an immune infiltrate consistent with pseudoprogression and loss of tumor cell heterogeneity suggesting immunoediting. Enrollment is anticipated to be completed in 2Q2020 with follow-up ongoing and initial survival data will be presented. V crossed the blood-brain barrier to produce functional IL-12. Controlled IL-12 therapy and cemi is a rational combination with initial data consistent with immune-mediated anti-tumor effects with a favorable safety profile.

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.

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.

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.

DIPG, which is the leading cause of pediatric brain cancer death with no effective treatment, has neither a highly immunosuppressive nor inflammatory tumor microenvironment (TME). Therefore, eliciting a pro-inflammatory TME may provide therapeutic benefit. We previously demonstrated in adults with recurrent glioblastoma that loco-regional delivery of interleukin 12 administered under the control of the proprietary transcriptional switch RheoSwitch Therapeutic Systemâ (RTSâ) delivered via a replication-incompetent adenovirus (“Controlled IL-12”) turned “cold” tumors “hot” for up to 5.8 months (Sci Transl Med. 2019;11(505)) and seemed to improve median overall survival as compared with historical controls (SNO 2020). A multicenter, phase I/II, open-label study (NCT03330197) is determining the safety and tolerability of Ad (2 x 1011 viral particles) administered by stereotactic intratumoral injection (Day 0) and 14 daily (Days 1 to 14) V doses (10 or 20 mg, body surface area adjusted). The first DIPG subject enrolled was in April 2020 with completion of the first cohort (arm 1, n=3) enrollment anticipated by September 2020. The first subject has tolerated treatment well with no SAEs during the evaluation period. Endogenous serum cytokines increased (including IFN-g 11.4 pg/mL, Day 3), consistent with V crossing the blood-brain barrier and activating the RTSâ switch to conditionally produce recombinant IL-12. Other biomarkers include plasma PK and circulating DNA. Follow-up is ongoing and enrollment is proceeding. Since development of effective immunotherapy for DIPG likely depends on eliciting a tumor-specific effector immune response, Controlled IL-12 is a promising immunotherapy candidate. The first DIPG subject shows encouraging data on safety, tolerability, serum cytokines and early signs consistent with a clinical response. After completion of dose-escalation, the study may be expanded up to 30 patients, which will be considered the phase II component of the study.

A published clinical trial of veledimex (V)-regulatable interleukin-12 (IL-12) gene therapy (“Controlled IL-12”) under the control of a transcriptional switch (RheoSwitch Therapeutic Systemâ, RTSâ) as monotherapy in recurrent glioblastoma (rGBM) showed sustained infiltration of activated T cells within the tumor months after treatment (Sci Transl Med. 2019;11(505)). These T cells demonstrated up-regulation of immune checkpoint signaling, providing a rationale for combination therapy with the PD-1 inhibitor, nivolumab (nivo). We report interim findings following completion of enrollment (with follow-up ongoing) for a multi-institutional, open label, dose-escalation phase 1 trial (NCT03636477) evaluating safety and tolerability of loco-regional Controlled IL-12 in combination with nivo in adults with rGBM. Replication-incompetent adenovirus coding for RTS-IL-12 (Ad) was administered during surgery by free-hand injection into the tumor and periphery (2 x 1011 viral particles, Day 0) accompanied by V (10 or 20 mg) PO QD x 15 (Days 0 to 14) in combination with nivo (1 or 3 mg/kg) IV on Days -7, 15, then Q2W. Twenty-one subjects were treated (Cohort 1: V 10 mg, nivo 1 mg/kg, n=3; Cohort 2: V 10 mg, nivo 3 mg/kg, n=3; and Cohort 3: V 20 mg, nivo 3 mg/kg, n=3 & 12 in expansion). Safety data were comparable to Ad+V monotherapy. Adverse reactions (ARs) during follow-on nivo dosing were consistent with nivo labeling. ARs were manageable and generally reversible with no synergistic toxicities. Updated overall survival findings will be presented. Baseline and post-treatment histochemical staining and multiplex immunofluorescence analyses for a subgroup of subjects will be discussed. The safety of this combination immunotherapy has been established, leading to a currently accruing phase 2 clinical trial of loco-regional Controlled IL-12 gene therapy in combination with the PD-1 inhibitor cemiplimab-rwlc (NCT 04006119).

3040 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. This phase 1 trial (NCT02026271) is the first to evaluate the safety and tolerability of Ad-RTS-hIL-12 (Ad) under transcriptional control with veledimex (V) in adults with grade III or IV gliomas. Methods: Multicenter, phase 1, open-label, 3 + 3 dose escalation study of Ad (a single intratumoral injection, 2 × 1011 viral particles, Day 0) with oral V dosing (Days 0 to 14) of 10, 20, 30, and 40 mg in subjects with rGBM. Results: 38 subjects were treated (resection group: V 10 mg (n = 6); 20 mg (n = 15); 30 mg (n = 4); 40 mg (n = 6); and, stereotactic group: V 20 mg, n = 7). The adverse event profile of Ad+ V, was predictable and controllable, with the main adverse reactions (ARs) being mild to moderate. All ARs were manageable and reversible upon withholding V. We observed increased peak (mean ± SEM) serum recombinant IL-12 and downstream endogenous IFN-g: V 10mg 21.4 ± 11.7 pg/mL and 14.6 ± 7.1 pg/mL; V 20 mg 25.8 ± 7.1 pg/mL and 57.0 ± 26.5 pg/mL; V 30 mg 65.7 ± 45.5 pg/mL and 60.7 ± 50.0 pg/mL; V 40mg 108.8 ± 41.0 pg/mL and 167.5 ± 70.9 pg/mL, V 20mg (stereotactic) 25.1 ± 7.2 pg/mL and 69.8 ± 48.5 pg/mL, respectively. In the V 20 mg cohort , there was an increase in tumor-associated T cells (CD3+CD8+%) from pre-Ad (mean ± SEM) 0.6 ± 0.4 to biopsy (~5 mons) 6.3 ± 5.0 and production of IFN-g 97.2 ± 85.3 pg/g (n = 2). Median overall survival (mOS) in the V 20 mg cohort (resection, n = 15) was 12.7 mons (mean follow-up, 13.1 mons). Subjects with unifocal disease (n = 6) who received low-dose (≤ 20mg total) dexamethasone during active dosing (Days 0-14) had an mOS of 17.8 mons. Tumor response data will be presented. Conclusions: Results of Controlled IL-12 in rGBM are promising, with V-dependent and proportional increases in IL-12 and IFN-g resulting in immune activation, with a favorable safety profile and encouraging survival. The 20 mg V dose is the recommended phase 2 dose. Controlled IL-12 is being evaluated in a monotherapy substudy (n = 36, V 20 mg) and two combination studies with immune checkpoint inhibitors for rGBM. Clinical trial information: NCT02026271 .

2510 Background: Monotherapy with intratumoral Ad-RTS-hIL-12 (Ad), a gene therapeutic conditionally expressing IL-12 under the transcriptional control of oral veledimex (“Controlled IL-12”), was shown in a phase 1 study (NCT02026271) to elicit a new and sustained intra-tumoral infiltration of T cells with co-expression of PD-1. We report updated findings following completion of enrollment (with follow-up ongoing) for a phase 1 substudy (NCT03636477) evaluating safety and tolerability of local, Controlled IL-12 in combination with nivolumab (nivo) in adults with recurrent glioblastoma (rGBM). Methods: Multicenter, open label, dose-escalation phase 1 trial to evaluate safety and tolerability of local, Controlled IL-12 with nivo in adult subjects with rGBM. Ad was administered by single intratumoral injection (2 x 1011 viral particles, Day 0 at time of resection) plus V (10 or 20 mg) PO QD x 15 with nivo (1 or 3 mg/kg) IV on Days -7, 15, then Q2W. Results: 21 subjects were treated (Cohort 1: V 10 mg, nivo 1 mg/kg, n = 3; Cohort 2: V 10 mg, nivo 3 mg/kg, n = 3; and Cohort 3: V 20 mg, nivo 3 mg/kg, n = 3 + 12 expansion). Safety data were similar to Ad+V monotherapy. Adverse reactions during follow-on nivo dosing were consistent with anti-PD-1 labeling, manageable, and generally reversible with no synergistic toxicities. Focusing on the 20mg V cohort (recommended phase 2 dose), serum IL-12 mean ± SEM (screening, 0.4 ± 0.1 pg/mL; Day 0, 0.6 ± 0.1 pg/mL), increased after Ad+V to 8.7 ± 3.3 pg/mL on Day 3. Similarly, serum IFN-g levels did not increase due to nivo alone (screening, 0 ± 0 pg/mL; Day 0, 0 ± 0 pg/mL), increasing after Ad+V to 6.2 ± 2.3 pg/mL on Day 7. Additionally, nivo alone did not significantly increase circulating T cells (CD3+ CD8+%) (paired differences comparison, Day 0 to screening) 3.1%, p =0.13, whereas Ad+V significantly increased peripheral T cells (Day 28 - Day 0) 3.6%, p =0.02. Pseudoprogression followed by a decrease in size (SPD) has been shown as evidenced by serial MRIs in a subgroup of subjects. Preliminary overall survival findings will be presented. Conclusions: Controlled IL-12 with PD-1 inhibition is a rational combination with initial data consistent with immune-mediated effects, a favorable safety profile, and early evidence of anti-tumor effects. An additional phase 2 study combining Controlled IL-12 with cemiplimab-rwlc in adults with rGBM is ongoing. Clinical trial information: NCT03636477 .

2564 Background: Interleukin-12 (IL-12) results in anti-tumor responses in preclinical models but requires tightly controlled production to achieve safety and elicit immune system activation to realize efficacy. A phase 1 “main study” (NCT02026271) enrolled subjects with Grade III or IV gliomas who at the time of resection received intratumoral administration of a replication-deficient adenovirus expressing IL-12 under control of a transcriptional switch (Ad-RTS-hIL-12, Ad) regulated by veledimex (V), referred to as “Controlled IL-12”. It was anticipated that dexamethasone (dex), a lymphocytotoxic corticosteroid used to control edema, might diminish response to immunotherapies. We report updated findings from a substudy of subjects who were dex-free during the 4 weeks prior to Ad administration. Methods: Multicenter, phase 1 substudy (NCT03679754) that assesses safety and tolerability of Controlled IL-12 by local injection (Day 0, time of resection) of Ad (2 x 1011 viral particles) + V (20 mg PO QD x15 doses, Days 0-14) in subjects that were bevacizumab naïve and not receiving dex 4 weeks prior to Ad. Results: 36 subjects were treated. Of the 36, a majority received low-dose corticosteroids (≤ 20 mg dex total during V) as compared with the main study (75% vs 40%). More subjects in the substudy as compared with the main study had multifocal vs. unifocal disease (39% vs 7%). The safety profile was similar for both. Adverse reactions were mild to moderate and were manageable and reversable upon withholding V. Activation of the switch in both the main study and substudy (V 20 mg; n = 51) resulted in increased mean peak values (Day 0-28) of serum IL-12 (25.8 vs. 20.4 pg/mL) and IFN-g (57.0 vs. 39.5 pg/mL). Initial median overall survival (mOS) (unifocal, ≤ 20 mg dex cumulative, n = 20) was 16.2 (8.9, 18.5) mons (mean follow-up 12.3 mons) (Neuro Oncol 2019; 21 [suppl_6]:vi5). mOS including the impact of dex and key subject characteristics from the two studies (n = 51) will be updated and tumor response data will be provided. Conclusions: Monotherapy with Controlled IL-12 resulted in sustained increase in serum recombinant IL-12 and downstream endogenous IFN-g. There is evidence of immune-mediated anti-tumor effects which is associated with increased mOS as compared with historical controls. Follow up will investigate the adverse impact of dex, as well as the effect of additional subject characteristics ( e.g., unifocal vs. multifocal disease) on mOS. Clinical trial information: NCT03679754 .

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.

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.

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.

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.

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.