Abstract 4141: A heptapeptide specific for Interleukin 13 receptor alpha 2 homes to the receptor-positive human GBM xenografts

Introduction: One of our goals is to identify specific therapeutic delivery and imaging agents for Glioblastoma multiforme (GBM), a malignant primary brain tumor. Interleukin 13 receptor alpha 2 (IL-13Rα2), a cell membrane receptor is over-expressed in >75% of GBM patients and is a target for various therapeutic/imaging approaches. Here, we explored whether peptides could be identified that bind IL-13Rα2. Smaller compounds may offer better uptake by tumors from circulation or better distribution when used with convection-enhanced delivery for intracranial tumors. Methods: To isolate the peptides binding to IL-13Rα2, we screened a cyclic M13 heptapeptide phage display library, Ph.D-C7C (New England Biolabs) with a diversity of 1.2 × 109 peptides. The phage library was repetitively bio-panned using GBM cell lines which over-express and which do not express IL-13Rα2. The peptide against IL-13Rα2, Peptide#1, isolated from the library, was synthesized in disulfide-constrained and linear forms, with or without biotin attached. ELISA, fluorescent and confocal microscopy was used to determine the binding of the peptide phages and synthetic peptides. Human G48a GBM subcutaneous and intracranial xenografts were induced in athymic nude mice. Peptide-biotin-streptavidin-Cy5.5 conjugates were injected intravenously. Results: We have selected three different peptide phage clones binding IL-13Rα2 using various ELISA and cell binding experiments. Peptide#1 phage demonstrated the highest affinity and specificity towards IL-13Rα2. The results obtained with the synthetic peptides reproduced those with peptide phages. Furthermore, an excess of IL-13, unlike antibody to the receptor, did not block the peptide's binding to IL-13Rα2. The affinity of Peptide#1 was found to be in high nanomolar, low micromolar range. Fluorescent binding experiments confirmed that the Peptide#1 binds to IL-13Rα2-positive GBM cells. Peptide#1 is internalized by GBM cells as indicated by confocal microscopy experiments on live U-251 MG GBM cells. We next demonstrated readily detectable binding and retention of Peptide#1 (linear) and Peptide#1 (disulphide) vs. the control scrambled peptides by the IL-13Rα2-expressing G48a subcutaneous human GBM xenografts using non-invasive Near Infrared Fluorescence (NIF) imaging. Peptide#1 bound to the GBM tumors in both dose-dependent and time-dependent manner and was retained up to at least 72 hr. Preliminary experiments also indicate that Peptide#1 targets and bi