Abstract 5525: Targeting the IL13Rα2 tumor-restricted biomarker with a novel ligand delivery system
Authors: Nguyen Van , Hollingsworth Charlotte , Debinski Waldemar , Mintz Akiva 
Published: February 2011

Abstract
Our objective is to create an optimized ligand-based delivery system that targets the IL13Rα2, a tumor-restricted plasma membrane receptor that is overexpressed on greater than 70% of Glioblastoma Multiforme (GBM) patients. In addition, IL13Rα2 is overexpressed in a variety of other cancers, including melanoma, adenocarcinoma, ovarian cancer and renal cell carcinoma.
Based on our prior experience identifying functionally relevant “hotspot” amino acid mutations of IL13, we designed and produced a novel IL13Rα2-Targeted Quadruple Mutant of IL-13 (TQM13) that we hypothesized will bind with a heightened affinity towards the tumor-restricted IL13Rα2 via a K105R mutation, but not to the physiologically abundant IL13Rα1/IL4Rα heterodimer as a result of the E13K/R66D/S69D mutations. In addition, we created a matching negative control, IL13R-Binding Null mutant (BN13, IL13.E13K/R66D/S69D/K105A) in which we disrupted IL13Rα1 and IL13Rα2 binding (via a K105A mutation).
We expressed these proteins in an E. coli expression system, denatured, refolded and purified them via Nickel-based affinity chromatography. To assess functional activity towards the physiologically abundant IL13Rα1/IL4Rα, we measured the IL13-dependent proliferation of TF-1 premyeloid leukemia cells after exposure to TQM13, BN13 and wtIL13. As expected, wtIL13 interacted and caused TF-1 cell proliferation at doses as low as 1 ng/ml, in contrast to TQM13 and BN13, which did not proliferate TF-1 cells even at 1000 ng/ml. We initially assessed binding towards the tumor-restricted IL13Rα2 using an IL13-cytotoxin neutralization assay and found that only TQM13 (and wtIL13) specifically bound/saturated the IL13Rα2 binding site and prevented the IL13Rα2-targeted cytotoxin from killing the cells. To further confirm binding specificity, we radioiodinated TQM13 using the IODO-GEN method and demonstrated that 125I-TQM13, but not 125I-BN13, bound with a strong affinity to GBM cells and GBM tumor specimens expressing IL13Rα2, but not normal brain. Importantly, in a preliminary in vivo biodistribution study, 125I-TQM13 preferentially bound to IL13Rα2-expressing tumors at a 3-to-1 ratio in comparison to background muscles.
In conclusion, we have successfully generated and radioiodinated an optimized, biomarker-targeted IL13 derivative that demonstrates specific binding activity towards the tumor-associated IL13Rα2 in vitro and favorable biodistribution in vivo, but does not bind the physiological IL13Rα1/IL4Rα. Thus, we demonstrate for the first time that combining a functional mutation that targets IL13Rα2 with multiple mutations that disrupt binding to the physiological receptor results in an attractive molecular transporter for diagnostic and therapeutic agents targeting IL13Rα2-expressing tumors.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5525.