Abstract 5519: Development of ‘multiple-specificity’ vectors for targeted delivery to intracellular organelles of the glioblastoma multiforme tumor cells

Abstract

Abstract Most anti-cancer therapeutics have defined targets such as proteins, enzymes or DNA which are localized in distinct intra-cellular compartments like nucleus, mitochondria and lysosomes. We reasoned that direct delivery of therapeutics to these sub-cellular compartments will lead to increased specificity, efficacy and less toxicity for these therapeutics. To this end, we have designed a true multiple-specificity delivery vehicle targeting the IL-13Rα2 receptor for transporting such therapeutics to the nucleus of GBM cells. Glioblastoma multiforme (GBM) is a primary brain tumor of poor prognosis and Interleukin 13 receptor alpha 2 (IL-13Rα2), a plasma membrane receptor that is over-expressed in over 75% of GBM patients. The construct for the targeted vector to the nucleus was made using a modified native ligand, IL-13.E13K, domain II (D2) of Pseudomonas exotoxin A (PE) and SV40 T antigen nuclear localization signal sequence (NLS) [IL-13.E13K-D2-NLS]. Domain II of PE contains a site of proteolytic cleavage and is responsible for the translocation of the toxin, and any other protein fused to it, from the endosome to the cytosol. We also produced a construct consisting of IL-13.E13K-D2 as a control protein which will predominantly localize in the cytosol. The recombinant proteins were produced in BL21 (λDE3) E. coli cells and purified by the Fast Protein Liquid Chromatography system to >90% purity. The binding of these proteins to IL-13Rα2 receptor on U-251 MG GBM cells was confirmed by, e.g., neutralization of IL-13 based cytotoxin cell killing. Next, we labeled these proteins and monitored their localization in GBM cells. For this, we first labeled the proteins at the carboxyl groups of primary amines using EDC-Sulfo-NHS labeling technique. The labeled proteins were then provided to live U-251 MG GBM cells and localization observed using confocal microscopy. The IL-13.E13K-D2-NLS protein showed nuclear localization whereas the control IL-13.E13K-D2 protein was prominently localized in the cytosol of cells. To get more clear pictures, we repeated the above experiment with proteins labeled at primary amines with Alexa-fluor 488; we observed similar localization of the proteins as described above. We also tried another labeling technique wherein we labeled the proteins at primary amines with biotin and then used streptavidin-HRP and tyramide-alexa fluor 488 signal amplification method as a more optimal technique. We repeated the intracellular localization experiments and found readily detectable nuclear localization of IL-13.E13K-D2-NLS and cytoplasmic/perinuclear localization of IL-13.E13K-D2. Thus, we have generated mutliple-specificity vector for nuclear delivery targeting the IL-13Rα2 in cancer cells and which can be further used for radiolabeling and/or conjugation to various chemotherapeutics. 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 5519.