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Abstract
Abstract The human epidermal growth factor receptor (Her) family plays an important role in cell survival and proliferation, and is implicated in oncogenesis. Overexpression of Her2/neu is associated with aggressive disease and poor prognosis. Herceptin, the only anti-Her2/neu, humanized monoclonal antibody approved by FDA for therapy of mammary carcinoma is currently and successfully prescribed for treatment of Her2/neu positive cancer. However, in 40-60% of all patients with Her2/neu-overexpressing tumors, Herceptin has little or no effect. The goal of this study was to demonstrate the construction and characterization of various Herceptin-based immunotoxins, loaded with a highly cytotoxic recombinant gelonin (rGel) molecule. Utilizing a humanized anti-Her2/neu scFv (4D5), we generated 2 orientation constructs: 4D5/rGel and rGel/4D5. We also generated a conventional chemical conjugate with full-length Herceptin (Herceptin/rGel conjugate), and we generated a unique fusion of anti-Her2/neu IgG containing 2 rGel molecules (Aglyco-Herceptin/rGel). This is the first report of the preparation of active full-length antibody-toxin construct from E.coli. In vitro studies showed that all immunotoxins retained the affinity and specificity of the original antibody, as well as the biological activity of rGel toxin. Against the Her2/neu overexpressing cancer cell lines, all immunotoxins demonstrated specific cytotoxic effects, even against Herceptin-resistant breast cancer cells. Against ovarian (SKOV3) cells, the Herceptin/rGel conjugate, Aglyco-Herceptin/rGel and rGel/4D5 fusion constructs demonstrated the best in vitro efficacy (IC50 of 0.28nM, 12.19nM and 1.57nM for each construct respectively vs 70.86 nM for 4D5/rGel). rGel/4D5 outperformed 4D5/rGel demonstrating improved internalization efficiency and increased intracellular delivery of rGel. All the immunotoxins displayed similar minimal exposure times to target cells for optimal cytotoxic activity. All constructs demonstrated a distinct cell-killing efficiency time course. Functional stability studies of the constructs showed that all remained biologically active after incubation in human plasma. Efficacy studies of the various constructs against SKOV3 tumor xenografts are ongoing and will be reported. Based on our in vitro results, the bivalent full-length antibody-toxin immunotoxins were superior to the corresponding recombinant monovalent constructs. Among monovalent constructs, the rGel/4D5 fusion was superior in vitro overall to 4D5/rGel. Different orientations of antibody and toxin can promote optimal cell internalization and toxicity of the immunotoxin, and may be valuable considerations for further construct optimization. Research conducted, in part, by the Clayton Foundation for Research. 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 1759. doi:10.1158/1538-7445.AM2011-1759
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