Abstract 1650: Combinatorial targeting of GBM: A means of therapeutically overcoming tumor heterogeneity

Our goal is to therapeutically exploit Glioblastoma Multiforme (GBM) restricted biomarkers that are not expressed in normal brain. Since GBMs are highly heterogeneous tumors, targeting any single biomarker will likely not be relevant to all GBMs. Thus, we hypothesize that utilizing a combinatorial approach that targets two GBM-restricted biomarkers (IL13Rα2 and EphA2) can successfully deliver high doses of targeted therapy to nearly all GBM patients. IL13Rα2 and EphA2 are membrane-associated receptor biomarkers that are independently expressed and each present in the majority of GBMs. We therefore created high-affinity ligands that separately target each biomarker. To target IL13Rα2, we created a novel IL13Rα2-Targeted Quadruple Mutant of IL13 (TQM13; IL13.E13K.R66D.S69D.K105R) based on our prior work that identified functional “hotspot” mutations. Recombinant TQM13 was expressed in an E. coli expression system and purified via Nickel-based affinity chromatography. Binding to the tumor-associated IL13Rα2 was confirmed with Biacore binding analysis. Disruption of binding to the physiologically abundant IL13Rα1/IL4Rα heterodimer was confirmed by TF1 proliferation assay. To target EphA2, we genetically fused its high-affinity ligand, EphrinA1, to the constant domain of human IgG1 (Fc1) and expressed it in a CHO expression system. Binding to the EphA2 biomarker was confirmed via ELISA and autoradiography. Each ligand was separately radiolabeled utilizing the IODOGEN method. Binding of each ligand to 10 human GBM tumor specimens was measured via autoradiography. TQM13 demonstrated high affinity towards the GBM-restricted IL13Rα2 (KD∼2nM), but did not bind/activate the physiologically abundant IL13Rα1/IL4Rα heterodimer. We confirmed functionality of purified EphrinA1.Fc1 by eliciting cell rounding and EphA2 activation in U251MG cells. We investigated the potential of each ligand to bind a series of 10 human GBM specimens either alone or in combination by performing autoradiography with 125 I-TQM13 and 125 I-EphrinA1.Fc1. 125 I-TQM13 demonstrated specific binding towards 8/10 specimens (5/10 moderate/strong binding). 125 I-EphrinA1.Fc1 specifically bound 10/10 specimens (7/10 moderate/strong binding). When both ligands were co-incubated on the same specimens, there was an additive binding and all specimens bound the TQM13/EphrinA1 mixture (9/10 strong binding, 1/10 moderate binding). Neither ligand significantly bound normal brain. Importantly, we found that even in subpopulations of GBM cells that did not express IL13Rα2 still expressed EphA2. We successfully created two ligands that separately target attractive GBM-restricted biomarkers and demonstrated that utilizing them in combination targeted all tested GBM samples and subpopulations of GBM cells. We anticipate that these ligands can therefore be used to deliver targeted therapeutics selectively 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 1650. doi:10.1158/1538-7445.AM2011-1650