Schrecengost, Randy S.

Abstract GB13 is a novel biologic therapy in the final stages of preclinical development for the treatment of glioblastoma (GBM) and H3 K27-altered diffuse midline glioma (DMG) that has received Orphan Drug Designation by the Food and Drug Administration for treatment of malignant gliomas. GB13 targets a specific tumor-restricted receptor of interleukin 13 called interleukin 13 receptor alpha 2 (IL13Rα2). IL13Rα2 is highly expressed in malignant gliomas and, importantly, is not expressed on non-cancerous cells of the brain. GB13 consists of an engineered proprietary mutant of IL13, which binds exclusively to IL13Rα2, and is fused to a highly cytotoxic exotoxin molecule. In vitro data demonstrate that GB13 potently kills IL13Rα2+ cancer cells while sparing non-cancerous cells. These characteristics make GB13 an attractive agent for safe and efficacious targeting of tumor cells expressing IL13Rα2 in upcoming clinical trials. In the current studies, we employed patient-derived GBM and DMG cell and animal models to provide key conclusions that support GB13 as an effective treatment option for gliomas in the clinic. First, we demonstrated that single acute infusions of GB13 by enhanced delivery (CED) is effective at decreasing orthotopic tumor volumes and increasing animal survival and that a long-term infusion can more significantly extend animal survival. Second, we determined that IL13Rα2 is requisite for GB13 activity as tumors that do not express the receptor are insensitive to the cytotoxic effects of GB13, demonstrating a precise and clear biomarker for clinical trial patient selection. Finally, we determined that GB13 works in concert with clinical standard-of-care radiation therapy to increase cell death, compared to radiation alone. Based on this work, we and our clinical collaborators are confident that the current work confirms GB13 has many important characteristics as an exciting therapy, which continues development towards a Phase I clinical trial for both GBM and pediatric DMG.

Abstract Diffuse midline glioma with H3 K27 alterations (DMG) is the most aggressive primary malignant brain tumor in the pediatric population. Radiation therapy (RT) is the standard-of-care; however, it has not demonstrated a significant improvement in overall survival or stalled disease progression, and children usually succumb to disease within 12-15 months of diagnosis. Consequently, finding efficient treatments for DMG remains one of the most critical unmet needs in modern neuro-oncology. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface receptor upregulated in ~80% of DMG versus normal brain, posing a potentially promising therapeutic target. Immunotherapies harnessing IL-13Rα2 to selectively delivery cytotoxic payloads, such as pseudomonas exotoxin A (PE), to tumor cells have been previously demonstrated to be safe in DMG patients and efficacious in preclinical models of DMG with high IL-13Rα2 expression using convection-enhanced delivery (CED). Furthermore, there is evidence that PE-based therapies can sensitize cells to RT. In this study, we utilized in vitro and in vivo models of DMG to evaluate clinically-relevant combination treatments of RT and the IL-13/PE immunotoxin GB-13 (IL13.E13K-PE4E). GB-13 improved the effectiveness of low-dose RT in multiple DMG cell lines by promoting caspase activation, which boosted apoptotic cell death. Intratumoral administration of GB-13 via chronic CED decreased tumor burden and prolonged survival in both DMG patient-derived orthotopic xenograft and genetically engineered mouse models. When GB-13 and RT were administered concurrently, this impact became even more prominent, informing future preclinical and clinical investigation of a potentially efficacious therapeutic approach in a subset of DMG.

Abstract INTRODUCTION High grade gliomas (HGG) are devastating diseases with largely unchanged survival outcomes despite decades of research. Recent studies suggest the interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is selectively upregulated in up to 80% of HGG, including glioblastoma (GBM) and diffuse midline gliomas (DMG) harboring H3K27 alterations. Immunotoxins targeting IL-13Rα2 have been demonstrated as safe and have shown some benefit for patients with HGG in previous phase I/II and III clinical trials. We hypothesized that by using GB-13 (IL13.E13K-PE4E), a novel peptide-toxin that binds IL-13Rα2 with high specificity and possesses a Pseudomonas exotoxin moiety, we would enhance the anti-tumor effects of this immunotherapy for HGG in vitro and in vivo while decreasing off-target toxicity. METHODS We examined the pharmacological effects of GB-13 in multiple patient-derived cell lines and rodent models of HGG. GBM and DMG lines were used to confirm IL-13Rα2 expression and sensitivity towards GB-13. Tumor naïve rats were evaluated for toxicity, and orthotopic PDX mice were used to monitor tumor size and survival following loco-regional infusion of GB-13. RESULTS GB-13 induced a potent cytotoxic response strongly predicated on IL-13Rα2 expression in vitro. No treatment-related adverse effects were noted after 7-day continuous intracranial infusion of GB-13 in tumor naïve rats. Further, in IL-13Rα2-upregulated orthotopic PDX mice, direct intratumoral administration of GB-13 via convection-enhanced delivery abrogated tumor growth and prolonged survival. CONCLUSIONS Given these promising results as well as the critical need for novel therapies in CNS malignancies, we are progressing to human trials using GB-13 targeting recurrent HGG. Ongoing safety studies in tumor-bearing animals will be able to define dose levels for the initial adult study-arm and the following pediatric study-arm. In this Phase 1 clinical trial, we hypothesize that loco-regional infusion of GB-13 will safely enhance tumor clearance by causing selective killing of IL-13Rα2-upregulated HGG cells.

Abstract High-grade gliomas (HGG) encompass a large proportion of malignant tumors within the central nervous system. Despite advances in our understanding of underlying disease mechanisms, the prognosis remains dismal and efficacious therapies are lacking. As such, there is a dire, unmet, gap in clinical practice for treating this devastating disease. Here, we performed convection-enhanced delivery (CED) of GB-13 (also known as IL13.E13K-PE4E), a tumor-specific immunotoxin, into the mouse brain in an effort to assess safety and efficacy. Fifty-five nude mice were inoculated with cells from 3 distinct patient-derived HGG cell lines (low, medium and high IL-13Rα2 expression). After tumor size reached a pre-determined threshold, mice underwent stereotactic cannula placement into the tumor followed by a single 40-min ramped infusion (rate 0.2-0.8 ul/min) of GB-13 (volume of infusion 20 ul) at concentrations ranging from 5 to 50 ug/ml. Tumor progression was monitored semiweekly and animals were euthanized at the indication of progressive neurologic deficit. All animals tolerated the infusions without exhibiting any neurological changes. GB-13 decreased tumor burden and prolonged survival in a manner strongly associated with IL-13Rα2 expression. While no survival benefit was observed in animals harboring IL-13Rα2-low expressing HGG, IL-13Rα2-medium and -high animals lived significantly longer after GB-13 infusion than vehicle-treated animals (median survival prolongation >25 days). Postmortem examination of the brains revealed no morphological changes beyond the site of the cannula tract. While GB-13 decreased cell proliferation and increased the number of apoptotic cells, neuronal cell density in ipsilateral brain regions was retained and no monocyte infiltrate was evidenced following GB-13 exposure. These findings indicate that a single therapeutic infusion of GB-13 administered by CED is well tolerated and underscore the potential of IL-13Rα2-targeted therapies in a subset of HGG with increased IL-13Rα2 expression.

High-grade gliomas (HGG) are devastating diseases in children and adults. In the pediatric population, diffuse midline gliomas (DMG) harboring H3K27 alterations are the most aggressive primary malignant brain tumors. With no effective therapies available, children typically succumb to disease within one year of diagnosis. In adults, glioblastoma (GBM) remains largely intractable, with a median survival of approximately 14 months despite standard clinical care of radiation and temozolomide. Therefore, effective therapies for these tumors remain one of the most urgent and unmet needs in modern medicine. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface transmembrane protein upregulated in many HGGs, including DMG and adult GBM, posing a potentially promising therapeutic target for these tumors. In this study, we investigated the pharmacological effects of GB-13 (also known as IL13.E13K-PE4E), a novel peptide–toxin conjugate that contains a targeting moiety designed to bind IL-13Rα2 with high specificity and a point-mutant cytotoxic domain derived from Pseudomonas exotoxin A. Glioma cell lines demonstrated a spectrum of IL-13Rα2 expression at both the transcript and protein level. Anti-tumor effects of GB-13 strongly correlated with IL-13Rα2 expression and were reflected in apoptosis induction and decreased cell proliferation in vitro. Direct intratumoral administration of GB-13 via convection-enhanced delivery (CED) significantly decreased tumor burden and resulted in prolonged survival in IL-13Rα2-upregulated orthotopic xenograft models of HGG. In summary, administration of GB-13 demonstrated a promising pharmacological response in HGG models both in vitro and in vivo in a manner strongly associated with IL-13Rα2 expression, underscoring the potential of this IL-13Rα2-targeted therapy in a subset of HGG with increased IL-13Rα2 levels.

Abstract High grade gliomas are devastating diseases. In the pediatric population, diffuse midline gliomas with the H3K27M mutation (H3K27M DMG) are the most aggressive primary malignant brain tumors. With no effective therapies available, children typically succumb to disease within one year of diagnosis. In adults, glioblastoma (GBM) remains a death sentence despite standard clinical care. Therefore, effective therapies for these tumors remain one of the most urgent and unmet needs in modern medicine. Interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is a cell-surface transmembrane protein upregulated in H3K27M DMG and GBM versus normal brain tissue, posing a potentially promising therapeutic target for both tumors. In this study, we investigated the pharmacological effects of a novel peptide-toxin conjugate, IL13.E13K-PE4E (also known as GB-13), that contains a targeting moiety designed to bind IL-13Rα2 with high specificity and a point-mutant cytotoxic domain derived from Pseudomonas exotoxin A. We demonstrated that IL13.E13K-PE4E was a potent killer of cultured H3K27M DMG and GBM cells in vitro. Intratumoral administration of IL13.E13K-PE4E via convection-enhanced delivery (CED) decreased tumor burden and prolonged survival in both H3K27M DMG and GBM murine xenograft models. Furthermore, we observed enhanced drug tissue retention and volume of distribution after CED, suggesting IL13.E13K-PE4E is capable of covering the target area and remaining at the site of infusion long enough to impart therapeutic effects. In summary, administration of IL13.E13K-PE4E demonstrated a potent pharmacological response in H3K27M DMG and GBM models both in vitro and in vivo in a manner strongly associated with IL13Rα2 expression, underscoring the potential of IL13Rα2 targeted therapy in a subset of these tumors.