Novel immunotherapies using recombinant cytotoxins have been introduced in the treatment of Glioblastoma multiforme (GBM) with encouraging clinical results. Recombinant cytotoxins are fusion proteins consisting of a carrier targeted to plasma membrane molecules specifically over-expressed in GBM, and a derivative of bacterial toxins, such as Pseudomonas exotoxin A (PE). We previously generated an interleukin 13 (IL13)-based cytotoxin, hIL13-PE38QQR, by fusing the C-terminal end of IL13 to the N-terminal end of a derivative of PE, PE38QQR. We also uncovered that GBM cells, but not normal brain, over-express IL13Rα2, a restricted receptor for the cytokine, in contrast to another IL13 receptor, IL13Rα1/IL4α, which is predominantly expressed in normal tissues. Our structure-function relationship analysis of IL13 has demonstrated that IL13 has two distinct receptor-binding regions: (i) the N-terminal region responsible for IL4α affinity, with Glu13 as a hot spot (Site I), (ii) the C-terminal sequence, which provides both IL13Rα1 and IL13Rα2 affinity, with Lys105 as a hot spot (Site II). We therefore proposed that (i) the key amino acid mutations of Site I receptor binding region should diminish IL13 affinity towards the IL13Rα1/IL4α on normal cells and minimize toxicity of variant IL13-based cytotoxins, (ii) exposure of Site II by generation of IL13-based cytotoxins through the fusion of C-terminus of the toxin to the N-terminus of IL13 should increase the binding affinity to IL13Rα2 on GBM cells. IL13 can be fused to the C-terminus of another toxin, Diphtheria toxin (DT) through its N-terminus. In the present study, we have generated a novel variant IL13 molecule, IL13QM, by replacing four key amino acids for IL13Rα1/IL4Rα and IL13Rα2 binding. Both wild type and variant IL13-based cytotoxins were fused to the C-terminus of DT derivative, DT390, expressed in E. Coli and purified by FPLC. Site I mutations of IL13 resulted in loss of its ability to stimulate proliferation of TF1 lymphoblast cells, which express IL13Rα1/IL4Rα signaling receptor, but not IL13Rα2. However, a 100-fold molar excess of IL13QM neutralized cytotoxicity of IL13-PE38QQR on IL13Rα2-expressing GBM cells as efficiently as wild type IL13. Furthermore, both DT-IL13 and DT-IL13QM demonstrated effective and specific cell killing of GBM cell lines. DT-IL13 and DT-IL13QM were not only 10-15 fold more potent than IL13-PE38QQR in GBM cell killing, but also less cytotoxic on cells expressing IL13Rα1/IL4Rα, even at high concentrations of the cytotoxins, implying a potential broad therapeutic window for these cytotoxins. Interestingly, we found that DT-IL13TM cytotoxin, in which three mutations were made in Site I of IL13 only, had even larger therapeutic window. Our results show for the first time that DT-IL13QM and DT-IL13TM are potent therapeutics which may provide selective cytotoxicity with decreased toxicity in patients with GBM.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA