We use bacterial toxins as killing agents to eliminate cancer cells. To accomplish this, we modify the toxin so it no longer binds via its own cell-binding domain and substitute in place of the binding domain a monoclonal antibody. The antibody is chosen to bind cancer cells preferentially over normal cells. These toxin-antibody molecules are called immunotoxins. Immunotoxins are promising but imperfect anticancer agents. Our goal is to understand the interaction of various toxins with eukaryotic cells and use this information to design better agents for treating cancer. To study interactions, we add toxins to mammalian cells and study the pathway of death. In tracking the killing of cancer cells by immunotoxins, we made the observation that cells grown to high density are resistant to killing. We wish to understand this phenomenon and determine its relevance for cancer therapy in general. A convenient and potentially useful way to study cell-killing pathways is to use RNA interference to identify pathways that participate in toxin delivery to the cytosol - where it acts. Recently, we initiated studies with a newly described toxin from V cholera, called Vibrio Cholera Exotoxin (CET). This toxin is related to the exotoxin from Pseudomonas, exhibiting about 50% identity in selected domains (domains II and III). However, antibodies that neutralize the exotoxin from Pseudomonas do not neutralize CET, despite the close similarity. Truncated versions of Pseudomonas Exotoxin (PE) have been fused with antibody fragments to produce potent cytotoxic agents termed recombinant immunotoxins. These agents are targeted to kill cancer cells based on the binding specificity of the antibody fragment. Potency is derived from the enzymatic nature of the toxin as it translocates to the cytosol, ADP-ribosylates elongation factor 2 and terminates the synthesis of new cellular protein. Most prior investigations reported that PE and PE-immunotoxins kill cells via apoptosis. Here we report that PE and PE immunotoxin inhibit protein synthesis and cell growth of colon cancer cell lines but do not provoke an apoptotic response. However, the addition of the BH3-only mimetic, ABT-737 in combination with the immunotoxin produces a profound apoptotic response in these cells that neither agent alone can achieve. Tissue culture data for ABT-737 activity has now been confirmed in xenograft models confirming this approach as a viable approach for overcoming resistance to immunotoxin action. We are conducting whole genome screens using RNAi agents to silence all human genes. Immunotoxin is then added to RNAi-treated cells with the goal of identifying genes that inhibit immunotoxin action. These inhibitory genes are identified because, when silenced, cells display greater sensitivity to immunotoxin action. Likewise we conduct large scale drug screens: again to identify and inhibit gene products that reduce the effectiveness of immunotoxin action. These are likely to include gene products that interfere with cell killing and might also be useful in reversing some forms of drug resistance to chemotherapy. Our overarching goal is to make our immunotoxin program more effective by eliminating the cellular barriers to targeted therapy.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Onda, Masanori; Ghoreschi, Kamran; Steward-Tharp, Scott et al. (2014) Tofacitinib suppresses antibody responses to protein therapeutics in murine hosts. J Immunol 193:48-55
Antignani, Antonella; Sarnovsky, Robert; FitzGerald, David J (2014) ABT-737 promotes the dislocation of ER luminal proteins to the cytosol, including pseudomonas exotoxin. Mol Cancer Ther 13:1655-63
Hollevoet, Kevin; Antignani, Antonella; Fitzgerald, David J et al. (2014) Combining the antimesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cells. J Immunother 37:8-15
Liu, Xiu-Fen; Xiang, Laiman; FitzGerald, David J et al. (2014) Antitumor effects of immunotoxins are enhanced by lowering HCK or treatment with SRC kinase inhibitors. Mol Cancer Ther 13:82-9
FitzGerald, David J (2014) Targeted diphtheria toxin to treat BPDCN. Blood 124:310-2
Haso, Waleed; Lee, Daniel W; Shah, Nirali N et al. (2013) Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood 121:1165-74
Mattoo, Abid R; FitzGerald, David J (2013) Combination treatments with ABT-263 and an immunotoxin produce synergistic killing of ABT-263-resistant small cell lung cancer cell lines. Int J Cancer 132:978-87
Liu, Xiu Fen; FitzGerald, David J; Pastan, Ira (2013) The insulin receptor negatively regulates the action of Pseudomonas toxin-based immunotoxins and native Pseudomonas toxin. Cancer Res 73:2281-8
Wei, Hui; Xiang, Laiman; Wayne, Alan S et al. (2012) Immunotoxin resistance via reversible methylation of the DPH4 promoter is a unique survival strategy. Proc Natl Acad Sci U S A 109:6898-903
Kreitman, Robert J; Tallman, Martin S; Robak, Tadeusz et al. (2012) Phase I trial of anti-CD22 recombinant immunotoxin moxetumomab pasudotox (CAT-8015 or HA22) in patients with hairy cell leukemia. J Clin Oncol 30:1822-8

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