David J. FitzGerald, Ph.D.
My lab develops antibody-based therapeutics for treating cancer. Our approach is to use antibodies or antibody fragments to deliver toxins for the purpose of killing cancer cells. Antibody-toxin molecules are called "immunotoxins". When immunotoxin treatment is less than 100% effective, we then add chemotherapy of various kinds to enhance killing. Along with our collaborators, discovering the best combinations of chemotherapy and immunotoxin has been the main goal of the lab for the past several years. Several particular combinations are under investigation for use in the clinic to treat patients with various malignancies.
1) antibodies, 2) toxins, 3) cancer therapeutics, 4) cell biology, 5) immunotoxins
Toxin-Based Treatment of Cancer and Vaccine Development
Bacterial protein toxins are profoundly damaging for mammalian cells. Interestingly, a subset of these toxins interact with very well-conserved and universally expressed cellular proteins. Among the toxins exhibiting these characteristics are Pseudomonas exotoxin (PE) and diphtheria toxin (DT), both of which inhibit protein synthesis. By removing either toxin's binding domain and replacing it with sequences encoding recombinant antibodies or ligands that bind cell surface receptors, it has been possible to make novel fusion proteins termed "recombinant immunotoxins".
Immunotoxins are targeted to kill cancer cells expressing particular surface antigens or receptors. PE binds and enters cells using the low-density lipoprotein receptor-related protein (LRP) as its surface receptor. The toxin is delivered to an acidic endosomal compartment where it is cleaved by a furin-like protease to generate two large fragments. The N terminal fragment (28 kD) of PE is comprised primarily of the toxin's binding domain, while the C terminal fragment (37 kD) has the ADP-ribosylating activity and is translocation-competent. Translocation requires the reduction of the disulfide bond linking the two fragments and also the presence of a KDEL-like sequence at the C terminus.
Currently, we are trying to understand the mechanism of cell-mediated toxin reduction. Our preliminary data indicate that furin-cleaved toxin must be unfolded before reductants can gain access to a key disulfide bond linking cyteines 265 and 287. Several projects are under way that use toxin sequences for the development of therapeutic agents. To produce Pseudomonas vaccine for cystic fibrosis patients, we are using nonlethal PE as an immunogenic carrier for the C terminal loop of pilin. Neuropeptides, including substance P, conjugated to truncated PE are being used to target neurons involved in the transmission of chronic pain signals.
Investigations into the pathogenic mechanism of Pseudomonas aeruginosa have been extended to include: studies of pilin-mediated adhesion to epithelia, the secretion of exoproducts such as amino peptidase (PA2939) and the regulation of gene expression by small non-coding RNAs.
Collaborators on these projects include Guojun Bu, Washington University, St. Louis; Marion McKee, ATCC; Susan Gottesman, NIH; Mike Vasil, University of Colorado, Denver; Ralf Hertle, Institute for Microbiology, Turbingen, Germany; Mike Iadarola, NIH; and Randall Mrsny, Trinity Biosystems, Inc.
Selected Key Publications
Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol.J. Biol. Chem. 265: 20678-85, 1990. [ Journal Article ]
Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein.J. Cell Biol. 129: 1533-41, 1995. [ Journal Article ]
- Blood. 90: 2020-6, 1997. [ Journal Article ]
ABT-737 overcomes resistance to immunotoxin-mediated apoptosis and enhances the delivery of pseudomonas exotoxin-based proteins to the cell cytosol.Mol. Cancer Ther. 9: 2007-15, 2010. [ Journal Article ]
ABT-737 promotes the dislocation of ER luminal proteins to the cytosol, including Pseudomonas exotoxin.Mol. Cancer Ther. 13: 1655-63, 2014. [ Journal Article ]
Dr. Fitzgerald is chief of the Biotherapy Section, Laboratory of Molecular Biology, in the NCI. He received his Ph.D. in 1982 from the University of Cincinnati Medical School, Department of Microbiology. The focus of his Ph.D. dissertation was the endocytic uptake of Pseudomonas exotoxin. He has a longstanding interest in bacterial toxins and their use as therapeutic agents.
|Antonella Antignani Ph.D.||Staff Scientist|
|Eric Chun Hei Ho Ph.D.||Postdoctoral Fellow (Visiting)|
|Robert Sarnovsky||Research Biologist|
|Nathan C. Simon Ph.D.||Postdoctoral Fellow (CRTA)|