Abstract 3639: In vivo targeting of a Glioblastoma Multiforme restricted biomarker
Authors: Nguyen Van , Conyers Jesse M. , Zhu Dongqin , Hantgan Roy R. , Debinski Waldemar , Mintz Akiva 
Published: June 2012

Abstract
Our objective is to develop an optimized ligand-based delivery system that targets IL13Rα2, a tumor-restricted plasma membrane receptor overexpressed on greater than 70% of Glioblastoma Multiforme (GBM) patients but not present in normal brain tissues. 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 a novel IL13Rα2-Targeted Quadruple Mutant of IL-13 (TQM13) that we hypothesized will bind with a heightened affinity towards the tumor-restricted IL13Rα2 but not to the physiologically abundant IL13Rα1/IL4Rα heterodimer. In addition, we created a matching negative control, IL13R-Binding Null mutant (BN13, IL13.E13K/R66D/S69D/K105A) with disrupted binding to both IL13Rα1 and IL13Rα2. We successfully expressed these proteins in an E. coli expression system, denatured, refolded and purified them via Nickel-based affinity chromatography. Our experimental goals were to confirm the ligand secondary structure and assess its functional activity in vitro and in vivo.
The α-helical-enriched secondary structure of TQM13 was confirmed to be highly similar to that of wild-type IL13 via circular dichroism spectroscopy measurement. TQM13 demonstrated high affinity towards the tumor-restricted IL13Rα2 in a surface plasmon resonance (Biacore) study (Kd ≈ 3 nM versus Kd ≈ 800 nM for BN13). To further confirm binding specificity, we radioiodinated TQM13 using the IODO-GEN method and demonstrated that 125I-TQM13, but not 125I-BN13, bound to the tumor-restricted IL13Rα2 via electrophoretic mobility shift assay. However, as demonstrated by surface plasmon resonance, TQM13 did not exhibit binding activity to the physiologically abundant IL13Rα1/IL4Rα heterodimer, in contrast to wild-type IL13. Importantly, in an in vivo biodistribution study, 125I-TQM13 bound to IL13Rα2-expressing tumors at a 2-to-1 ratio in comparison to background muscles only one hour after intravenous delivery of radiolabeled ligands. The tumor-to-muscle binding ratio improved to 5-to-1 at four hours after injection and 7-to-1 at the 24-hour timepoint. In contrast, 125I-BN13 did not bind to IL13Rα2-expressing tumors as its activity remained similar to background muscles at 24 hours after injection
In conclusion, we have successfully generated an optimized, biomarker-targeted IL13 derivative and demonstrated for the first time via independent biomolecular studies its secondary structure and specific binding towards the tumor-associated IL13Rα2 in vitro and in vivo, but not towards the physiologically abundant IL13Rα1/IL4Rα heterodimer. This novel ligand is therefore suitable to deliver high doses of diagnostic and therapeutic radioactivity specifically to GBMs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3639. doi:10.1158/1538-7445.AM2011-3639