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.

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 .

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 10 11 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 .

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.

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.

INTRODUCTION: We conducted a single-institution phase 1 trial of a dendritic cell immunotherapy targeting glioblastoma stem-like cells for patients with either newly diagnosed or recurrent glioblastoma. Following gross total or near-gross total resection, patients who consented to participate in this trial underwent leukapheresis for isolation of peripheral blood mononuclear cells, which were then differentiated into dendritic cells in culture, pulsed with lysate derived from an allogeneic glioblastoma stem-like cell line, and administered by intradermal injection weekly x 4 weeks, then every other month until disease progression or vaccine depletion. Patients with newly diagnosed glioblastoma were treated with standard-of-care chemoradiation, with vaccine injections beginning 1 week following the completion of radiation therapy. Patients with recurrent disease received no other disease-directed therapy while on trial. From December 2013 to February 2018, 38 patients enrolled in this trial -- 12 patients with newly diagnosed glioblastoma and 26 with recurrent disease. Median age 57 (range 19–77), median KPS 80 (range 70–100), 66% male. Survival functions were estimated using the Kaplan-Meier method. For newly diagnosed patients, median Time-to-Progression (TTP) was 8.86 mo, and median Overall Survival (OS) was 21.1 mo. For patients with recurrent glioblastoma, median TTP was 3.14 mo and median OS was 12.0 mo. Treatment was well-tolerated with no related grade 3/4 toxicities. As of this analysis, 7 patients are still alive, and 2 patients in the newly diagnosed glioblastoma cohort are still progression-free. Immune response studies and tumor antigen profiling are ongoing. CONCLUSION: Consistent with other previously completed dendritic cell immunotherapy trials, this phase 1 trial demonstrates improved TPP and OS for patients with either newly diagnosed or recurrent glioblastoma compared to historical controls. Ongoing efforts include characterizing and expanding the subset of patients who most benefit from immunotherapy.

Exosomes can mediate a dynamic method of communication between malignancies, including those sequestered in the central nervous system and the immune system. We sought to determine whether exosomes from glioblastoma (GBM)-derived stem cells (GSCs) can induce immunosuppression. We report that GSC-derived exosomes (GDEs) have a predilection for monocytes, the precursor to macrophages. The GDEs traverse the monocyte cytoplasm, cause a reorganization of the actin cytoskeleton, and skew monocytes toward the immune suppresive M2 phenotype, including programmed death-ligand 1 (PD-L1) expression. Mass spectrometry analysis demonstrated that the GDEs contain a variety of components, including members of the signal transducer and activator of transcription 3 (STAT3) pathway that functionally mediate this immune suppressive switch. Western blot analysis revealed that upregulation of PD-L1 in GSC exosome-treated monocytes and GBM-patient-infiltrating CD14+ cells predominantly correlates with increased phosphorylation of STAT3, and in some cases, with phosphorylated p70S6 kinase and Erk1/2. Cumulatively, these data indicate that GDEs are secreted GBM-released factors that are potent modulators of the GBM-associated immunosuppressive microenvironment.

Ad-RTS-hIL-12 (Ad) is a novel gene therapy expressing IL-12 via the RheoSwitch Therapeutic System® gene switch under control of an oral activator ligand, veledimex (V). We previously reported on an open label Phase I trial describing biological activity of recombinant IL-12 with downstream IFN-γ and activation of the immune system. We provide an update on the intratumoral injections of Ad (2x1011virus-particles) + V for patients with recurrent GBM (rGBM) in Group 1 (G1) (craniotomy, n=31) and initial results for Group 2 (G2) (stereotactic administration n=7). In G1, the V 20-mg cohort mOS increased to 12.7 months with mean follow-up of 12.9 months. 20-mg V in G1 showed fewer toxicities and higher V compliance (84%) compared with higher-doses of V (30 and 40-mg) with 75% and 67%, respectively. These data are encouraging compared to historical data that predict mOS of 5 to 8 months. An additional cohort at V 10-mg (n=6) was well tolerated, but subtherapeutic, with a mOS of 7.6 months (mean follow-up 6.7 months). There was an association between V dose level, blood-brain-barrier penetration, and drug-related adverse events (AEs) with increased TEAEs observed above V 20-mg. Subgroup analyses across all cohorts did not detect statistically significant differences including extent of resection or IDH mutation status. Subjects (20-mg V) who received a cumulative dose of ≤10mg of dexamethasone during the first 15 days of treatment showed improved OS versus >100mg of dexamethasone, suggesting corticosteroid-mediated blunting of the IL-12 dependent immune-mediated therapeutic effect. In the G2 20-mg V cohort, similar cytokine levels and reversible AEs were observed compared to G1; follow up is ongoing and mOS will be presented. Based on these results and the best risk-benefit profile, the 20-mg V dose level was chosen for further investigation. Combination with an immune checkpoint inhibitor in rGBM is underway.

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.

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

2059 Background: A hallmark of glioblastoma is the high incidence of tumor recurrence, thought to be triggered by cancer stem cells. These tumorigenic cells are resistant to irradiation and chemotherapeutic agents. The target antigen, CD-133, was chosen because it has been reported as a cancer stem cell antigen overexpressed in glioblastoma tumors and associated with shorter survival. Recent clinical trials suggest that the mean overall survival for these patients is roughly 5-9 months, emphasizing the important unmet medical need in this disease requiring additional strategic approaches. Dendritic cell immunotherapies such as ICT-121 could provide benefit to patients by educating their immune systems to induce the formation of cytotoxic T cells that attack tumor cells bearing the target antigen. In addition to immediate attack on tumor cells present at dosing, a long-term memory response effective against tumor recurrence might be induced. Immunotherapy, such as ICT-121, that targets cancer stem cells could be an important treatment for this disease. Methods: This Phase I multi-center trial of ICT-121 targeting CD133 was designed to assess safety and tolerability (primary endpoint) and to monitor overall survival and progression-free survival (secondary endpoints). ICT-121 is comprised of autologous dendritic cells that are loaded with two HLA-A2 restricted epitopes of the CD133 antigen. CD133 is overexpressed on glioblastoma cancer stem cells. The HLA-A2 patients that had undergone resection for recurrence of glioblastoma were treated with ICT-121 once a week for 4 weeks during the induction phase and then once every 2 months during the maintenance phase until disease progression, death, ICT-121 depletion or discontinuation. Results: A total of 20 patients were treated and eight of these patients are still alive. Immune response data with cytokine mRNA expression demonstrated a response to the CD133 epitopes. A total of 20 patients were treated and eight of these patients are still alive. Conclusions: The results from this Phase I trial suggest that ICT-121 is both safe and well-tolerated with an immune response seen in a subset of patients. Clinical trial information: NCT02049489.

Abstract Background: NK cells play a crucial role in the antitumor immune response and are involved in controlling tumor formation, progression and metastases. Glioblastoma (GBM) is the most devastating brain tumor, associated with very poor prognosis. While immunotherapy emerged as a promising approach for anti cancer therapy, GBM appears immune resistant. Here, we studied the role of NK cells in targeting glioblastoma and possible mechanisms of NK immune evasion. Methods: NK cells were isolated from freshly resected GBM tissue, matching peripheral blood and the blood of healthy controls. Flow cytometry was used to characterize the cells and their ability to produce cytokines, and chromium release assay was perfumed to assess their cytotoxicity. In vitro assays were performed by co-culturing GBM stem cells (GSCs) with healthy donor NK cells. Results: We found that whereas NK cells were abundant in primary GBM tissue and could efficiently target GBM stem cells (GSCs), GBM infiltrating NK cells (TiNKs) displayed an abnormal phenotype with downregulation of many activating receptors including CD16, NKG2D, DNAM, NKp30, NKp46, 2B4 and NKG2C and upregulation of inhibitory proteins such as PD-1. This inhibitory phenotype was associated with impaired NK cell function when compared with NK cells isolated from the peripheral blood of patients and healthy donors. GSC-NK cell-cell contact resulted in release of TGF-β by GCSs, which in turn led to NK dysfunction through constitutive activating of the p-Smad pathway. TGF-β activation, in turn, is partially mediated by the matrix metalloproteases MMP-2 and MMP-9, secreted by GSCs upon contact with NK cells and enhanced upon TGF-β exposure. We demonstrated that inhibition of the TGF-β axis, in particular by the small molecule inhibitor, galunisertib, can prevent GSC-induced NK cell dysfunction but is unable to inactivate the p-Smad pathway, thus, cannot reverse existing dysfunction. Conclusions: Our results indicate that NK-GBM cross-talk plays an important role in tumor escape and highlight the importance of developing future adoptive transfer therapies with the intent of limiting tumor escape from antitumor immunity. Note: This abstract was not presented at the meeting. Citation Format: Hila Shaim, Abdullah Alsuliman, Konrad Gabrusiewicz, Jun Wei, John Yu, Rafet Basar, May Daher, Lucila Kerbauy, Mayela Mendt, Muharrem Muftuoglu, Li Li, Enli Liu, Nobuhiko Imahashi, Sonny Ang, Young Gi, Pinaki Banerjee, David Marin, Richard Champlin, Elizabeth Shpall, Amy Heimberger, Katayoun Rezvani. TGF-β is a key mediator of NK cell dysfunction in gliolastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2949. doi:10.1158/1538-7445.AM2017-2949

2052 Background: 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 the first cohort, consisting of 4 males and 3 females (ages 32-58), it was observed that V crossed the blood-brain barrier with 30±6% of the V plasma level in the resected tumor sample (17±6 ng/ml vs. 5±2 ng/ml). Peak serum IL-12 was observed at Day 3 (mean 23 pg/ml) and peak IFNg (mean 32 pg/ml) on Day 7. Peripheral blood samples showed an increase in CD8 T cells and an increase in the ratio of CD8/CD4 and CD8/FoxP3. AD+V was well tolerated with expected toxicities promptly reversing with discontinuance of V. Neurotoxicities were minimal and manageable. To date, 7/7 subjects are alive with 5 subjects having a follow-up of > 90 days and 3 > 160 days posttreatment. Conclusions: Intratumoral regulated IL-12 expression using AD+V in a salvage population with advanced and recurrent glioma has an acceptable and rapidly reversible safety profile. The intracranial administration of AD followed by oral V is clinically feasible and suggests possible benefit vs. historical control and provides a strong rational for dose-escalation as reviewed and approved by the independent Data Safety Monitoring Board. Clinical trial information: NCT02026271.

We previously reported 7 patients with prolonged survival > 5 years from a phase I study of 16 newly diagnosed GBM patients (Neuro-Oncology 15(suppl3), 71, IT-015, 2013) and expression of four ICT-107 targeted antigens in pre-immunotherapy tumors correlated with prolonged OS and PFS in GBM patients (Cancer Immunol Immunother. 2013 Jan;62(1):125-35. ICT-107 is an autologous immunotherapy consisting of patient dendritic cells pulsed with six tumor associated peptides (TAA) peptides derived from AIM-2, TRP-2, HER2/neu, IL-13Ra2 (overexpressed on CSC), gp100, MAGE1 administered intradermal three times at two week intervals to HLA-A1 and/or HLA-A2 positive GBM patients after a standard therapy with concurrent Temozolomide and radiation therapy. With 10-year follow up, we identified 3(19%) of 16 the GBM patients were disease free over 8 years (PFS 9.2+, 8.3+, 8.1+) and 6 patients (KPS 90%, age range 34-62 years old at age of diagnosis) survived > 7.7 years (range 7.7- 9.6 years). There were 2 HLA-A1+ and 4 HLA-A2+ and their TAA expressed at least 5 peptides including antigens overexpressed on CSC in this group. One patient with PFS >9.2 years is working full time with good quality of life, showed immune response 2.5 (>1.5 fold increase IFNg production after administration by tetramer/FACS). Six (38%) of 16 GBM patients are presently alive > 7.7 years and only one patient received active treatment for recurrence. While long term remission and survival was seen in this 16 GBM patient cohort, median PFS was 16.9 months, 5-year PFS was 37.5% (95%:15.4-60), median OS was 38.4 months and 5-year OS rate was 50% (95% CI: 24.5-71). It is unprecedented to have 19% long term remission > 8 years in this group from ICT-107 immunotherapy. This correlated with cancer-stem-associated expression, and a trend toward greater CD8 T cell cytokine responses.

Detailed confirmatory testing and analysis verifies and strengthens the association between clinical outcomes and immune response of HLA-A2+ patients enrolled in a randomized phase 2 trial of ICT-107. 77 HLA-A2+ patients, randomized 2:1, received ICT-107 (autologous DCs incubated with 6 synthetic peptide CTL epitopes targeting GBM tumor/stem cell-associated antigens, including the four HLA-A2-restricted antigens HER-2, TRP-2, gp100, and IL-13Rα2) or matching control (un-incubated DC). Multimer testing was performed on a subset of these patients. The pioneering analysis heuristic of fusion metrics used in conjunction with Monte Carlo simulation was used to identify multimer immune responders. P-values between dependent variables and multiple overall survival (OS) or progression-free survival (PFS) metrics was performed using log-rank test and Fisher’s exact test. HLA-A2+ patients consistently continued to show evidence of immune response being associated with both OS and PFS. Multimer immune responders independently confirmed the ELISpot immune responder associations between assignment group (p=0.0308), and initial OS and PFS (p=0.0043 and 0.0352, respectively). Combining ELISpot and multimer responders strengthened or maintained associations with all OS and PFS metrics. Notable significant associations were determined when data was stratified by treatment group in both treatment and placebo subgroups, leading to speculation of the possible positive effects of DCs alone. This finding supports changing the placebo in the Phase III study from dendritic cells to PBMCs. The robust associations identified between OS and PFS with immunologic response, explored using both multimer and ELISpot analysis to determine immune response with fusion metrics in a Monte Carlo setting, provide support for the efficacy of ICT-107 to induce peptide-specific T cell responses in HLA-A2+ patients.

BACKGROUND: Dendritic cell vaccines are a promising treatment for glioblastoma. The glioblastoma stem-like cell subpopulation is refractory to standard chemotherapy and radiation treatment, but may be susceptible to immunotherapeutic targeting. METHODS: This single-institution phase I trial is designed to assess the safety, tolerability, and potential efficacy of an autologous dendritic cell vaccine pulsed with lysate derived from a glioblastoma stem-like cell line. Patients with newly diagnosed glioblastoma (Cohort A) receive standard radiation and temozolomide in addition to vaccine. Patients with up to third glioblastoma recurrence (Cohort B) receive vaccine alone, although patients previously treated with bevacizumab are allowed to continue bevacizumab on study. Prior to enrollment, patients must undergo a qualifying gross total or near-gross total resection. Treatment consists of a Vaccine Induction Phase (weekly vaccines x 4 – for Cohort A, given upon completion of radiation), followed by a Vaccine Maintenance Phase (vaccine every 8 weeks until supply depleted). Imaging is performed during Week 4 of the Induction phase and every 8 weeks thereafter until progression. The primary objective of safety and tolerability includes adverse event grading per NCI CTC. Clinical assessment includes progression-free survival at 6 months and overall survival. Immunological testing to assess cytotoxic T-lymphocyte response will be performed at predetermined intervals. Goal enrollment is 20 patients in each cohort. Stopping rules for safety and efficacy using Bayesian sequential design will be followed. RESULTS: To date, 18 patients (7 new GBM, 11 recurrent GBM) have enrolled in this trial. No adverse events related to vaccine have been noted. CONCLUSION: This single-institution phase I dendritic cell vaccine trial builds upon our previous research efforts by specifically targeting the glioblastoma stem-like cell subpopulation that is resistant to standard radiation and chemotherapy. Accrual is proceeding rapidly, and results will be published when available.

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning.

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.

INTRODUCTION: Exosomes secreted by cancer cells have pleiotropic functions, and can promote autocrine signaling to distant cells. Elucidating the mechanistic modulation of the immune system by these exosomes provides insight into potential biomarkers for detection, recurrence, and response, and identifies potential new therapeutic targets. METHODS: Exosomes were isolated from human glioblastoma stem cells (GSC) and fibroblasts (control) using differential centrifugation. Fluorescent-labeled exosomes were co-cultured with human peripheral blood mononuclear cells (PBMCs). Flow cytometry and confocal microscopy methods were utilized to determine the ability of immune cells to uptake exosomes and to evaluate subsequent intracellular trafficking. The exosomal protein and RNA content was analyzed by mass spectrometry and Nanostring Counter System, respectively. The phenotypic and functional skewing of the monocyte lineage was analyzed after exposing these cells to exosomes. The cytokine array was used to analyze the cytokines generated following treatment of human normal monocytes with exosomes. RESULTS: The GSC-secreted exosomes were preferentially absorbed by CD14+ monocytes and Gr-1+ derived myeloid cells isolated from healthy volunteers and/or glioblastoma patients. When activated, CD4+ and CD8+ T cells could also uptake GSC-secreted exosomes. Confocal microscopy revealed that only monocytes could internalize GSC-secreted exosomes but not fibroblast-secreted exosomes. The exposure to GSC-secreted exosomes induces a phenotypic change in monocytes and prevents them from undergoing apoptosis. GSC-secreted exosomes, but not the fibroblast-secreted exosomes, increased expression of CD163, CD206, and decreased expression of MHC class II. Monocytes treated with GSC-secreted exosomes release IL-6, IL-1RA, CCL3, and CCL4 when compared to cells exposed to fibroblast exosomes. GSC-secreted exosomes contained distinct protein composition in contrast to fibroblast-secreted exosomes that may affect the anti-tumor function of monocyte-derived macrophages. CONCLUSIONS: Monocytes demonstrated a preferential uptake of GSC-secreted exosomes which then induced a glioma-supportive M2 phenotype. GSC-secreted exosomes can be a contributing factor in the M2 skewing within glioma microenvironment.

BACKGROUND: Clinical outcomes were associated with immune response of HLA-A2+ patients enrolled in a randomized phase 2 trial of ICT-107. METHODS: 124 patients (77 HLA-A2+), randomized 2:1, received ICT-107 (autologous DCs incubated with 6 synthetic peptide CTL epitopes targeting GBM tumor/stem cell-associated antigens MAGE-1, HER-2, AIM-2, TRP-2, gp100, and IL-13Rα2) or matching control (un-incubated DC). Immune response was determined by a functional ELISPOT assay pre- and post-treatment (ICT-107 30.5% vs. 15.5% in controls). Determination of p-values and significance between dependent variables and overall survival (OS) or progression-free survival (PFS) was performed using log-rank test. Fisher's exact tests were performed for association between extended overall survival (EOS: OS > 22.2 months) and extended progression-free survival (EPFS: PFS > 16.5 months) and the same variables. RESULTS: HLA-A2+ patients showed evidence of immune response being associated with both OS and PFS. After determining HLA-A2 ELISPOT responders (responders) using a comprehensive scoring system, responders had a median OS of 23.1 months versus 13.7 for non-responders (p = 0.0673). Similarly, responders had an EOS percentage of 52% versus 29% non-responders (p = 0.0615). Further, responders had a significantly higher percentage of EPFS (41% versus 15%, p = 0.0259). When censored to 7.5 months post-treatment, immune response was found to significantly improve OS (median 17.6 months versus 13.7, p = 0.0018). Level of IL-12 production can be linked to survival. High IL-12 producers had a median OS of 20.6 compared to 15.4 for low producers (p = 0.0515). Within the treatment group, the difference was larger, with medians of 23.3 months versus 15.2 (p = 0.0654), and high producers had a significantly higher percentage of EOS (54% versus 31%, p = 0.0426) and EPFS (43% versus 19%, p = 0.0293). CONCLUSIONS: The associations identified between clinical outcomes of OS and PFS with immunologic response provide support for the efficacy of ICT-107 in HLA-A2+ patients due to a biologic T cell response.