Novel anti–brain tumor cytotoxins specific for cancer cells

Abstract

The vast majority of brain cancers (gliomas) express a receptor (R) for interleukin 13 (IL13). In order to achieve specific targeting of the IL13R in gliomas, we have mutagenized human (h) IL13. The mutation was made to alter IL13 interaction with the shared functional IL13/4 normal tissue receptor, but not with the glioma-associated receptor. We have thus produced hIL13.E13K (glutamic acid at position 13 changed to lysine) and fused it to derivatives of Pseudomonas exotoxin A. The hIL13.E13K-based cytotoxins are less active on normal cells and thus less toxic, and are better antitumor agents compared with the cytotoxins containing nonmutagenized hIL13.

Mutants of Interleukin 13 with Altered Reactivity toward Interleukin 13 Receptors

“…The plasmids were constructed as described (24). BL21(DE3) Escherichia coli, which carries the T7 RNA polymerase gene in an isopropyl-1-thio-␤-galactopyranoside-inducible form, was used as the host for recombinant protein expression.…”

“…By using the alignment data, we have begun rational mutational studies of hIL13 to identify regions of hIL13 needed for its interaction with the components of the shared receptor. We have previously reported a mutant form of hIL13, hIL13.E13K, which not only has an impaired signaling through the shared receptor but also has enhanced avidity toward the IL13 cancer-associated receptor (24). Position 13 of hIL13 is, according to the sequence alignment data, equivalent to position 9 in hIL4.…”

Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics

“…To achieve sustained expression of GBM-specific mhIL-13-PE within the tumor microenvironment, we constructed a regulatable, bidirectional Ad expressing the mhIL-13-PE cytotoxin under the control of the tetracycline-inducible promoter system. This vector also expresses a mutated form of human IL-4, IL4.Y124D (mhIL-4), which blocks the putative binding of mhIL-13-PE to the physiological IL4R/IL13R without affecting its binding to the glioma-associated IL13Rα2 (21,22). We also constructed a control vector expressing mhIL-4 and mhIL-13 (without the PE) under the control of the tetracycline-responsive (TRE) promoter.…”

“…We hypothesized that a regulatable adenoviral vector (Ad) encoding mhIL-13 fused to Pseudomonas exotoxin would provide long-term local expression leading to an effective cytotoxic response in IL13Rα2-expressing GBM cells, with minimal neurotoxicity toward the normal surrounding brain parenchyma. To further increase safety, the vector encodes mutated human IL-4 [mhIL-4, or IL-4.Y124D (21,22)] that binds and blocks the physiological receptor IL4R/IL13R without interacting with IL13Rα2, thereby inhibiting any potential binding of mhIL-13-PE to normal brain cells and subsequent neurotoxicity.…”

“…To restrict the targeted toxin exclusively to IL13Rα2 + -glioma cells, a mutated form of IL-13 was engineered [IL-13.E13K, or mhIL-13 (21)], which was previously shown to bind to the GBMassociated IL13Rα2 with higher affinity than the native hIL-13, exhibiting negligible binding to the physiological receptor IL4R/ IL13R (21,22). We hypothesized that a regulatable adenoviral vector (Ad) encoding mhIL-13 fused to Pseudomonas exotoxin would provide long-term local expression leading to an effective cytotoxic response in IL13Rα2-expressing GBM cells, with minimal neurotoxicity toward the normal surrounding brain parenchyma.…”