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Impaired apoptosis is often a key element in tumor development. Therefore, drugs mimicking prosurvival antagonists offer promise as cancer therapeutics. When ABT-737, a BH3-only mimetic, was added to KB3-1 human cervical adenocarcinoma cells, we noted an induction of an endoplasmic reticulum (ER) stress response and the dislocation of ER luminal proteins, including chaperones, to the cell cytosol. Furthermore, when immunotoxin (antibody–toxin chimeric molecule) and ABT-737 combinations were added to cells, there was enhanced toxin-mediated inhibition of protein synthesis, consistent with enhanced translocation of toxin to the cytosol. A similar enhancement was not seen with thapsigargin, suggesting that ER stress alone was not responsible for enhanced translocation. Cytosol preparations from ABT-737–treated but not from thapsigargin-treated cells revealed the presence of greater amounts of processed 37-kDa toxin fragment compared with the addition of immunotoxin alone. As early as 4 hours after the addition of ABT-737 and immunotoxin, there was release of mitochondrial cytochrome c and activation of caspase-3/7 indicating that the combination caused apoptotic cell death. These results were reflected in decreased cellular ATP levels that were noted with combinations of ABT-737 and immunotoxin but not with either agent alone or with combinations of thapsigargin and immunotoxin. We conclude that ABT-737 increases ER permeability, promoting the dislocation of toxin from the ER to the cytosol resulting in early apoptotic cell death. These mechanistic insights suggest why this class of BH3-only mimetic synergizes in a particular way with Pseudomonas exotoxin–based immunotoxins. Mol Cancer Ther; 13(6); 1655–63. ©2014 AACR.

Dysregulation of the epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of malignancies including glioblastoma and various epithelial cancers. Oncogenesis occurs from overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287-302 loop from the extracellular domain is exposed completely on EGFRvIII (deletion of exons 2-7), partially exposed on some cancers but cryptic on normal cells expressing WT levels of EGFR. We report on the generation of antibodies to this loop. A peptide corresponding to amino acids 286-303 was synthesized and then coupled chemically to Keyhole Limpet Hemocyanin. After immunizations, sera were assayed for reactivity to the peptide. Mice with high titers were used for hybridoma production. Purified antibodies were isolated from hybridoma supernatants, while V-regions were cloned and sequenced. Receptor binding was characterized using ELISA and flow cytometry. Monoclonal antibodies (n=7) reactive for the 287-302 loop were characterized further. Each one reacted with EGFRvIII but not EGFR WT. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-468 and A431 cells but not to non-cancerous WI-38 cells. Because of its unusual binding characteristics, a recombinant immunotoxin was generated from 40H3, which proved to be potently cytotoxic for MDA-468, A431 and F98-EGFRvIII-expressing cells. We conclude that immunization with a peptide corresponding to a cryptic EGFR epitope can produce cancer-binding antibodies. By way of example, the 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be developed as a therapeutic agent for clinical use. Citation Format: Eric Chun Hei Ho, Antonella Antignani, Robert Sarnovsky, David J FitzGerald. Characterization of monoclonal antibodies generated to the 287-302 amino acid loop of the human epidermal growth factor receptor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B099. doi:10.1158/1535-7163.TARG-19-B099