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PDF - 65K, Loading controls of the cytosol and membrane fractions for the western blot shown in Fig 4A.

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.

Background: The dysregulation of epidermal growth factor receptor (EGFR) has been implicated in the oncogenesis of various malignancies including glioblastoma and some epithelial cancers. Oncogenesis occurs from the overexpression of EGFR, often linked to gene amplification or receptor mutagenesis. The 287–302 loop in the extracellular domain is exposed completely on EGFR variant III (EGFRvIII), partially exposed on some cancers but cryptic on normal cells. We report on the generation of antibodies to this loop.Methods: The 286–303 peptide was 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 enzyme-linked immunosorbent assay and flow cytometry. A recombinant immunotoxin was generated from the 40H3 antibody and its cytotoxic activity characterized on relevant cancer cell lines.Results: Seven monoclonal antibodies were generated to the 287–302 loop and characterized further. Each one reacted with EGFRvIII but not wild-type EGFR. Based on reactivity with the immunizing peptide, antibodies were mapped to one of three subgroups. One antibody, 40H3, also exhibited binding to MDA-MB-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 cytotoxic to MDA-MB-468, A431 and F98npEGFRvIII expressing cells.Conclusions: Immunization with a peptide corresponding to a cryptic epitope from EGFR can produce tumor cell-binding antibodies. The 40H3 antibody was engineered as a cytotoxic recombinant immunotoxin and could be further developed as a therapeutic agent.

Background A feature of many B-cell tumors is a surface-expressed immunoglobulin (sIg). The complementarity-determining regions (CDRs) of the sIg, termed the ‘idiotype’, are unique to each tumor. We report on a phage selection strategy to generate anti-idiotype therapeutics that reacts with sIg CDR3 sequences; the MEC1 B-cell tumor line was used as proof of concept. Methods To create a mimetic of the MEC1 idiotype, CDR3 sequences from heavy and light chains of the sIg were grafted into a single chain variable fragment (scFv) framework scaffold. Using the Tomlinson I phage library of human scFvs, we enriched for binders to MEC1 CDR3 sequences over unrelated CDR3 sequences. Results By ELISA we identified 10 binder phages. Of these, five were sequenced, found to be unique and characterized further. By flow cytometry each of the five phages bound to MEC1 cells, albeit with different patterns of reactivity. To establish specificity of binding and utility, the scFv sequences from two of these binders (phages 1 and 7) were converted into antibody-toxin fusion proteins (immunotoxins) and also cloned into a human IgG1 expression vector. Binders 1 and 7 immunotoxins exhibited specific killing of MEC1 cells with little toxicity for non-target B-cell lines. The full-length antibody recreated from the binder-1 scFv also exhibited specific binding. Conclusion Our results establish the utility of using engrafted CDR3 sequences for selecting phage that recognize the idiotype of B-cell tumors.

Cell deletion approaches to pain directed at either the primary nociceptive afferents or second-order neurons are highly effective analgesic manipulations. Second-order spinal neurons expressing the neurokinin 1 (NK1) receptor are required for the perception of many types of pain. To delete NK1+ neurons for the purpose of pain control, we generated a toxin–peptide conjugate using DTNB-derivatized (Cys0) substance P (SP) and a N-terminally truncated Pseudomonas exotoxin (PE35) that retains the endosome-release and ADP-ribosylation enzymatic domains but with only one free sulfhydryl side chain for conjugation. This allowed generation of a one-to-one product linked by a disulfide bond (SP-PE35). In vitro, Chinese hamster ovary cells stably transfected with the NK1 receptor exhibited specific cytotoxicity when exposed to SP-PE35 (IC50 = 5 × 10−11 M), whereas the conjugate was nontoxic to NK2 and NK3 receptor-bearing cell lines. In vivo studies showed that, after infusion into the spinal subarachnoid space, the toxin was extremely effective in deleting NK1 receptor-expressing cells from the dorsal horn of the spinal cord. The specific cell deletion robustly attenuated thermal and mechanical pain sensations and inflammatory hyperalgesia but did not affect motoric capabilities. NK1 receptor cell deletion and antinociception occurred without obvious lesion of non–receptor-expressing cells or apparent reorganization of primary afferent innervation. These data demonstrate the extraordinary selectivity and broad-spectrum antinociceptive efficacy of this ligand-directed protein therapeutic acting via receptor-mediated endocytosis. The loss of multiple pain modalities including heat and mechanical pinch, transduced by different populations of primary afferents, shows that spinal NK1 receptor-expressing neurons are critical points of convergence in the nociceptive transmission circuit. We further suggest that therapeutic end points can be effectively and safely achieved when SP-PE35 is locally infused, thereby producing a regionally defined analgesia.

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

Immunotoxins are antibody-toxin fusion proteins under development as cancer therapeutics. In early clinical trials, immunotoxins constructed with domains II and III of Pseudomonas exotoxin (termed PE38), have produced a high rate of complete remissions in Hairy Cell Leukemia and objective responses in other malignancies. Cholera exotoxin (also known as cholix toxin) has a very similar three-dimensional structure to Pseudomonas exotoxin (PE) and when domains II and III of each are compared at the primary sequence level, they are 36% identical and 50% similar. Here we report on the construction and activity of an immunotoxin made with domains II and III of cholera exotoxin (here termed CET40). In cell viability assays, the CET40 immunotoxin was equipotent to tenfold less active compared to a PE-based immunotoxin made with the same single-chain Fv. A major limitation of toxin-based immunotoxins is the development of neutralizing antibodies to the toxin portion of the immunotoxin. Because of structure and sequence similarities, we evaluated a CET40 immunotoxin for the presence of PE-related epitopes. In western blots, three-of-three anti-PE antibody preparations failed to react with the CET40 immunotoxin. More importantly, in neutralization studies neither these antibodies nor those from patients with neutralizing titers to PE38, neutralized the CET40-immunotoxin. We propose that the use of modular components such as antibody Fvs and toxin domains will allow a greater flexibility in how these agents are designed and deployed including the sequential administration of a second immunotoxin after patients have developed neutralizing antibodies to the first.

Background Triple-negative breast cancers (TNBCs) are typically more aggressive and result in poorer outcomes than other breast cancers because treatment options are limited due to lack of hormone receptors or amplified human epidermal growth factor receptor 2 (HER2). Many TNBCs overexpress the epidermal growth factor receptor (EGFR) or manifest amplification of theEGFRgene, supporting EGFR as a therapeutic target. While EGFR-directed small molecule inhibitors have shown limited effectiveness in clinical settings, use of EGFR as a mechanism of delivering enzymatic cytotoxins to TNBC has not been demonstrated. Methods Using the single-chain variable fragment (scFv) of the 806 antibody that binds only cells with overexpressed, misfolded, or mutant variants of the EGFR, a recombinant immunotoxin was engineered through gene fusion withPseudomonas aeruginosaExotoxin A (806-PE38). The potency of 806-PE38 on reducing TNBC cell growth in vitro and in xenograft models (n ≥ 6) was examined for six TNBC cell lines. All statistical tests were two-sided. Results 806-PE38 statistically significantly reduced the viability of all tested TNBC lines, with IC50values below 10 ng/mL for three of six cell lines, while not affecting cells with wild-type EGFR (IC50>300 ng/mL). Systemic treatments with 806-PE38 vs vehicle resulted in statistically significantly reduced tumor burdens (806-PE38 mean = 128 mm(3)[SD = 46 mm(3)] vs vehicle mean = 749 mm(3)[SD = 395 mm(3)], P = .001) and increased median survival (806-PE38 median = 82 days vs vehicle median = 50 days,P= .01) in a MDA-MB-468 TNBC mouse xenograft. Deletion of the catalytic residue eliminated both cytotoxic activity in vitro and the reduction in tumor burden and survival (P= .52). Conclusions These data support the further development of the 806-PE38 immunotoxin as a therapeutic agent for the treatment of patients with EGFR-positive TNBC. Follow-up experiments with combination therapies will be attempted to achieve full remissions.