Because the protective antigen (PA) component of anthrax toxin is a major anthrax antigen, development of anthrax vaccines focuses on improving methods for producing PA. An avirulent strain of B. anthracis, termed BH450, has been engineered to be sporulation deficient and to lack extracellular proteases. Proteins such as PA secreted from this strain are expected to be more easily purified with a higher yield in a homogeneous form. An earlier candidate vaccine that is modified to be less sensitive to proteases is now in a clinical trial. A candidate conjugate vaccine that induces antibodies to a sugar termed anthrose that is unique to the anthrax spore surface and a few other rare bacteria. Evaluation of this conjugate is underway. Another approach to immunological defense against anthrax uses passive administration of monoclonal antibodies. In addition to the successful development of PA neutralizing chimpanzee/human monoclonal antibodies (mAbs) reported previously, work during this period in collaboration with Dr. Purcell (LID, NIAID) was extended to isolation and characterization of mAbs against the anthrax toxin enzymatic moieties, edema factor (EF) and lethal factor (LF). In this work, we produced a high affinity EF mAb EF13D. EF13D could neutralize edema toxin (ET)-mediated cAMP responses in cells and protected mice from systemic ET-mediated lethality. The antibody epitope was mapped to domain IV of EF. EF13D was able to compete with calmodulin (CaM) for EF binding and displaced CaM from EF-CaM complexes. The EF-mAb binding affinity (0.05-0.12 nM) was 50 to 130-fold higher than that reported for EF-CaM. In this collaborative work, we have also isolated chimpanzee/human Fabs reactive with LF and converted them into complete MAbs with human gamma-1 heavy-chain constant regions. In macrophage cytotoxicity assays, two of these MAbs, LF10E and LF11H, neutralized anthrax lethal toxin (LT). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a EC50 of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC50 of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although these anti-EF and LF neutralizing mAbs could potentially be used alone, it is expected that even more efficient and broader protection against anthrax toxin could be gained when combining them with anti-PA mAbs in the prophylaxis and treatment of anthrax infection. Our studies on vaccination of newborns against anthrax were continued during this period in collaboration with the laboratory of Dr. Matzinger (NIAID). Extensive studies were completed on the role of adjuvant, route and dose in the immunization of newborn mice against anthrax protective antigen. A major discovery in these studies was that an absence of toxin neutralization titers in mice did not always correlate with absence of protection, so that some mice lacking measurable anti-PA antibodies of any kind were protected against anthrax toxin challenge. We are currently investigating the ability to immunize newborn mice with PA in the presence of anti-PA maternal antibodies for a better understand of newborn immunization responses. In the area of small molecule therapeutics against the anthrax toxins we have continued our collaboration with Panthera BioPharma in the development of new LF protease inhibitors. Three rounds of animal testing helped to drive the identification and optimization of candidate inhibitors, leading to synthesis of two compounds within a particular inhibitor subset that are the most potent in vivo inhibitors of LF yet reported. Also in the area of small molecule therapeutics of anthrax, in collaboration with Dr. Bugge (NIDCR), a high throughput screen was initiated with the Chemical Genomics Group (C. Austin, NHGRI) using the lethal factor fusion protein to beta-lactamase as a reporter of the anthrax toxin internalization process. In this work, we identified several small molecules capable of obstructing the process of anthrax toxin internalization. Among these compounds, diphyllin and niclosamide not only protected RAW264.7 macrophages and CHO cells from anthrax lethal toxin, but also protected these cells from diphtheria toxin. We further demonstrated that these compounds blocked the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor but not the related TEM8 receptor, suggesting that these molecules may interfere with the endosomal acidification process. Because our work showed that CMG2 is the major physiological relevant anthrax toxin receptor in vivo, and TEM8 only plays a minor role, these compounds have potential to block anthrax toxin action in vivo. In our continuing efforts to develop modified anthrax toxin proteins as anti-tumor agents, we demonstrated that the agents shown to work well by intra-tumoral injection also work when administered systemically to mice. Studies in cell culture have helped to show that the efficacy for melanoma of the particular combination that contains native lethal factor depends on expression of the cell surface metalloproteinases and possession of a mutation that activates the same MAP-kinase pathway that the lethal toxin inactivates. Most striking was the finding that combining the PA variant requiring activation by matrix metalloproteases with the native lethal factor produced high potency toward a variety of solid tumors in mice. Examination of the molecular basis of this broad specificity showed it to be due to inhibition of tumor angiogenesis rather than direct action on the tumor cells. Through collaboration with Dr. Frankel (Scott &White Memorial Hospital, Cancer Research Institute), we have expanded on these initial findings by showing that this MMP-activated lethal toxin reduces endothelial proangiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, our data suggest that inhibition of the c-jun NH(2)-terminal kinase and p38, but not extracellular signal-regulated kinase-1/2, pathways is significant in the antiangiogenic activity of the MMP-activated lethal toxin. In this collaborative work, we further demonstrated that this MMP-activated anthrax lethal toxin could potently inhibit tumor angiogenesis in an orthotopic model of anaplastic thyroid carcinoma, and improve long-term survival that was comparable to that produced by the multi-kinase inhibitor Sorafenib. Collectively, these results support the clinical development of the MMP-activated lethal toxin for the treatment of solid tumors.
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