Abstract
Abstract The EphA2 receptor is over-expressed on the surface of many different human tumors, and has been shown to internalize upon ligand or antibody binding. The fully human monoclonal antibody anti-EphA2 antibody was developed by phage display methods and specifically binds both human and rodent EphA2 receptors. The type II ribosome-inactivating protein gelonin was genetically engineered with an extra cysteine residue to facilitate chemical cross-linking with targeting proteins. The fully human anti-EphA2 antibody, anti-EphA2 antibody, was conjugated to the recombinant gelonin (rGel) using the heterobifunctional cross-linker SPDP and partially reduced rGel. The conjugated antibody (anti-EphA2 antibody/rGel) was separated from free antibody and rGel by gel-permeation and affinity chromatography. The final material was composed of antibody+1 rGel (predominant) and antibody+2 rGel (minor) and was essentially free of contaminating rGel or unreacted anti-EphA2 antibody. The anti-EphA2 antibody/rGel conjugate was assessed for cell-free protein synthesis inhibitory activity and found to be similar to free rGel. The conjugate proved specifically cytotoxic to the EphA2 expressing PC-3, MDA-MB-231 and CT-26 cell lines, with IC50 values of 3.8, 6.1 and 11 ng/ml, respectively. The Targeting Index for the anti-EphA2 antibody/rGel conjugate (vs. rGel) was high, with values of 1909, 306 and 75 for PC-3, PC-3-MDR and MDA-MB-231, respectively. Confocal microscopy showed that the anti-EphA2 antibody/rGel conjugate was specifically and rapidly internalized. The maximal intracellular concentration was achieved 4 hours after exposure. Minimal contact time studies showed specific cytotoxicity with only 1 hour exposure while optimal cytotoxicity was achieved with an exposure time of 24 hours. Of special interest, the anti-EphA2 antibody/rGel conjugate demonstrated no significant loss in specific cytotoxicity against Dox-resistant (MDR expressing) cell lines derived from either MDA-MB-231 or PC-3 cells compared to their parental cell lines. These data suggest that the development of MDR-resistant phenotypes may not impair the successful utilization of rGel-based targeted therapeutics. These results support the continued evaluation of the rGel antibody conjugate technology. Research conducted, in part, by the Clayton Foundation for Research. 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 4399.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have