Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics

Abstract

Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.

Fig. 1.

Adenovirus-mediated expression of the therapeutic cytotoxin mhIL-13-PE in vitro and in vivo. (A) Diagram showing the mechanism of Ad-mediated regulated therapeutic cytotoxin expression. Ad.mhIL-4.TRE.mhIL-13-PE expresses mhIL-4 and mhIL-13-PE under the control of the bidirectional TRE promoter, which is activated by rtTA2^sM2 in the presence of Dox (ON state). In the OFF state, the transactivator is unable to induce transgene expression, which is further repressed by tTS^kid. (B) Cell viability in control COS-7 cells and human GBM cells (IN859 and U251) infected with control Ad.mhIL-4.TRE.mhIL-13 or therapeutic Ad.mhIL-4.TRE.mhIL-13-PE. *P < 0.05 versus OFF transgene expression state; Student's t test. Images show expression of IL13Rα2. (C) Colocalization of therapeutic transgenes (arrows) in the mouse brain 7 d after injection of Ad.mhIL-4.TRE.mhIL-13-PE.

Fig. 2.

Cytotoxicity of Ad.mhIL-4.TRE.mhIL-13-PE and the protein formulations hIL-13-PE and mhIL-13-PE in human glioma cells, astrocytes, and neurons. Human GBM U251 cells (A) and human NPC-derived astrocytes and neurons (B) were infected with control Ad.mhIL-4.TRE.mhIL-13, therapeutic Ad.mhIL-4.TRE.mhIL-13-PE, or as a control an Ad expressing the reporter gene LacZ (Ad.TRE.LacZ). Transgene expression was activated using 1 μg/mL Dox. Cells were also incubated in the presence of 1 μg/mL hIL-13-PE or mhIL-13-PE. Graphs show cell viability as determined by flow cytometric analysis of Annexin V and propidium iodide-stained cells. Insets show representative dot plots. *P < 0.05 versus Ad.TRE.LacZ or mock; one-way ANOVA followed by Tukey's test.

Fig. 3.

Intracranial administration of Ad.mhIL-4.TRE.mhIL-13 leads to tumor regression and long-term survival in the absence of neurotoxicity. (A) Nude mice were implanted with human U251 cells in the striatum and 5 d later they were treated with either a single intratumoral injection of control vector Ad.mhIL-4.TRE.mhIL-13 or therapeutic vector Ad.mhIL-4.TRE.mhIL-13-PE, in combination with Ad.TetON, or as controls they received saline or Ad.0. Animals were fed Dox chow. (B) Kaplan–Meier survival curves of nude mice bearing intracranial U251. *P < 0.05 versus saline, ^P < 0.05 versus control Ad.mhIL-4.TRE.mhIL-13; Mantel log-rank test. (C) Naïve wild-type BALB/c mice were intracranially injected with saline, control vector Ad.mhIL-4.TRE.mhIL-13, or therapeutic vector Ad.mhIL-4.TRE.mhIL-13-PE and Ad.TetON. Animals were fed Dox chow. Seven days postvector delivery, neuropathological analysis was assessed by Nissl staining and immunocytochemistry using antibodies against tyrosine hydroxylase (TH) and myelin basic protein (MBP). (Scale bar, 200 μm.) The arrows indicate the injection site.

Fig. 4.

Efficacy and neurotoxicity of hIL-13-PE and mhIL-13-PE protein formulations. (A and B) Intracranial administration of hIL-13-PE protein formulation does not lead to long-term survival and induces severe neurological toxicity. (A) Nude BALB/c mice were implanted with human U251 cells in the striatum and 5 d later they were treated with a single intratumoral injection of saline or 0.2 or 1 μg of hIL-13-PE protein. *P < 0.05 versus saline; Mantel log-rank test. (B) Naïve wild-type BALB/c mice were intracranially injected with saline or 0.2 or 1 μg of hIL-13-PE protein. Three days postdelivery, mice were euthanized due to severe neurological deficits, and neuropathological analysis was assessed by Nissl staining and immunostaining of TH and MBP. Note the severe local neurotoxicity of both doses of hIL-13-PE when injected into the normal brain parenchyma (arrows). (C and D) Intracranial administration of mhIL-13-PE protein formulation induces antitumor efficacy and leads to dose-dependent neurological toxicity. (C) Nude BALB/c mice bearing intracranial human U251 glioma were treated with a single intratumoral injection of saline or mhIL-13-PE protein. *P < 0.05 vs. saline; Mantel log-rank test. (D) Naïve wild-type BALB/c mice were intracranially injected with saline or mhIL-13-PE protein. Neuropathological analysis was performed 3 or 7 d postdelivery, depending on the toxicity. Note the severe local neurotoxicity of the high dose of mhIL-13-PE protein in the normal brain parenchyma (arrows). (Scale bars, 200 μm.)

Fig. 5.

Efficacy and neurotoxicity of therapeutic Ads in alternative intracranial GBM models. (A) Rag1^−/− mice were implanted with human primary GBM12 cells in the striatum and 5 d later they were treated with a single intratumoral injection of control (Ad.mhIL-4.TRE.mhIL-13) or therapeutic vector (Ad.mhIL-4.TRE.mhIL-13-PE), in combination with Ad.TetON. As controls they received saline or an empty Ad (Ad.0). (B) Immune-competent C57/B6 mice were intracranially implanted with GL26 cells expressing IL13Rα2 (GL26-H2) and treated 7 d later with the Ads. Animals were fed Dox chow. *P < 0.05 versus saline, ^P < 0.05 versus control Ad.mhIL-4.TRE.mhIL-13; Mantel log-rank test. (C and D) Neuropathology was assessed in moribund mice and long-term survivors (120 d) by Nissl staining and immunostaining of TH, MBP, F4/80 (macrophages/activated microglia), and major histocompatibility complex II (MHCII). The arrows indicate the injection site. Insets show higher-magnification microphotographs of the areas indicated with a white square. (Scale bars, 1,000 μm.) Note complete tumor regression in long-term survivors and the absence of neuropathological or inflammatory side effects.

Fig. 6.

Gene therapy-mediated delivery of mhIL-13-PE leads to antitumor efficacy and long-term survival in the absence of neurological toxicity. The diagram depicts the mechanism of action of the therapeutic strategy. The targeting of IL-13α2 receptor overexpressed in glioma cells has been approached by constructing an Ad encoding the truncated form of Pseudomonas exotoxin fused to a mutated form of human IL-13 (mhIL-13, IL-13.E13K), which has higher affinity for the glioma-associated IL13Rα2 receptor and negligible binding to the physiological IL13/IL4R. Binding of mhIL-13-PE to IL13Rα2 promotes its internalization into glioma cells. Domain II (PE II) mediates the translocation of the toxin into the endosomes by endocytosis. Once in the endosomes, furin mediates proteolytic cleavage that activates the catalytic domain III (PE III). Due to the low pH of the endosome, the processed fragment of the toxin is translocated to the cytosol and inhibits protein synthesis, leading to glioma cell death. The absence of IL13Rα2 protects normal cells from mhIL-13-PE cell death. The safety of our approach has been further enhanced by coexpressing a mutated form of IL-4 (mhIL-4, IL-4.Y124D) encoded in the therapeutic Ad which acts as an antagonist of the physiological receptor comprising the IL-13α1R and IL-4αR chains. Secreted IL-4 would inhibit the already negligible binding of mhIL-13-PE to normal cells, without affecting the binding of the mhIL-13-PE to GBM cells.