11 months ago

2002 Scientific Report

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  • Institute
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anthracis. The three

anthracis. The three proteins of the exotoxin secreted by the organism are protective antigen (PA), lethal factor (LF), and edema factor (EF). The PA63 fragment forms a heptameric complex on the cell surface that is capable of binding with the 90 kDa LF protein to form lethal toxin (LeTx). It is known that macrophages are particularly sensitive to LF: at low concentrations, LF stimulates TNF-α and IL-1β; at high concentrations, LF causes the death of macrophages and the release of cytokines into the bloodstream. The symptoms of systemic anthrax are inducible by injection of LeTx alone in animal models; it is likely that death is caused by cytokine-induced shock. Because of its rarity, anthrax is not often included in differential diagnosis; in cases of inhalational anthrax, the diagnosis is rarely made until the patient is moribund. Antibiotic treatment should be initiated at the earliest stage of infection. By the time characteristic symptoms appear, the bacteria are already multiplying rapidly in the bloodstream and have produced massive amounts of toxin. Killing the bacteria cannot eliminate the toxin, and its effects result in death of the host despite antibiotic treatment at this point. Therefore, we hypothesize that a high-affinity neutralizing monoclonal antibody to LF would be a potentially useful reagent for the treatment of anthrax infection, in combination with the use of antibiotics. In collaboration with the laboratories of Nicholas Duesbery and Han-Mo Koo at VARI, we have developed two panels of mAbs against protective antigen and lethal factor. Our major goal of producing these mAbs is to find those that have properties of specifically blocking the biological functions of LF and to further evaluate those antibodies as potential agents for the treatment of anthrax infection. We have characterized their biological neutralization properties to PA and LF by in vitro bioassays and found a particular anti-LF mAb that has strong neutralizing activity to LF: at a low molar ratio to LF, it shifts the effective concentration (EC50) over 500-fold in the macrophage cell line J774A.1 (Figure 1). Our results also showed that this antibody specifically recognizes the binding site of LF to PA and blocks their binding, preventing the formation of lethal toxin (Figure 2). We will further test this mAb in vivo on a variety of animals models (including mouse, rat, and rabbit) to observe if it neutralizes LF in vivo and protects the animal from death caused by challenges with either purified LeTx or live bacteria. Meanwhile, we have also epitope-mapped this mAb using the phagedisplay technique, and a few synthetic small peptides based on the epitope mapping information are being tested to determine if they could be LF antagonists with biological functions similar to those of LF-neutralizing mAbs. Our overall goals in this project are to characterize the in vivo neutralizing activities of anti-LF mAbs and/or synthetic peptides as LF antagonists for their suitability as passive protection for animals, including their potential clinical application to treat anthrax infection. Figure 1. Macrophage cell survival Figure 2. Gel shifting assay. Lane 1: molecular weight marker 66 kDa; lane 2: PA63 alone; lane 3: PA63 plus anti-LF mAb; lane 4: LF alone; lane 5: LF plus mAb; lane 6: PA63 plus LF; lane 7: PA63, LF, and mAb. 13

External Collaborators Lonson Barr, Michigan State University, Lansing Milton Gross, Department of Veterans Affairs Medical Center – University of Michigan Medical Center, Ann Arbor Yi Ren, Royal Mary Hospital, Hong Kong University Wei-cheng You, Beijing Institute for Cancer Research, People’s Republic of China Dong-zheng Yu, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing, People’s Republic of China Publications Hay, Rick V., Brian Cao, R. Scot Skinner, Ling-Mei Wang, Yanli Su, James H. Resau, George F. Vande Woude, and Milton Gross. 2002. Radioimmunoscintigraphy of tumors autocrine for human Met and hepatocyte growth factor/scatter factor. Molecular Imaging 1(1): 56–62. Qian, Chao-Nan, Xiang Guo, Brian Cao, Eric J. Kort, Chong-Chou Lee, Jindong Chen, Ling-Mei Wang, Wei-Yuan Mai, Hua-Qing Min, Ming-Huang Hong, George F. Vande Woude, James H. Resau, and Bin T. Teh. 2002. Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma. Cancer Research 62(2): 589–596. From left to right: Zhao, Edgar, Kalbfleisch, Cao 14

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