Recombinant Anthrax Toxin Receptor-Fc Fusion Proteins Produced in Plants Protect Rabbits against Inhalational Anthrax

Abstract

Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ϳ50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

Anthrax lethal and edema toxins in anthrax pathogenesis

“…Several additional human and chimpanzee monoclonal antibodies against PA, LF and EF are in clinical development (for review see [90]). Recently, a fusion of the extracellular domain of human CMG2 and human IgG Fc fragment (CMG2-Fc) was shown to be effective in neutralizing PA and protecting rabbits from B. anthracis infection [91]. One potential advantage of CMG2-Fc is that deliberately engineered B. anthracis strains having variants of PA that evade individual neutralizing anti-PA monoclonal antibodies would remain subject to inhibition by CMG2-Fc.…”

Improving the Anti-Toxin Abilities of the CMG2-Fc Fusion Protein with the Aid of Computational Design

“…This fusion may extend the biological half-life of CMG2 [14]. CMG2-Fc has high in vitro toxin-neutralizing potency and can protect rabbits against lethal pulmonary infection [14].…”

Plant-Derived Monoclonal Antibodies for Prevention and Treatment of Infectious Disease

“…CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with Bacillus anthracis spores at a dose of 2 mg/kg of body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later (69).…”

“…The results also demonstrate that plant production is an appropriate technology for generating nonglycosylated MAbs and could be useful in meeting the production challenge involved in the treatment of inhalation anthrax in humans. An additional plant-derived immunotherapeutic utilized a recombinant fusion protein comprised of a fusion of a human receptor for anthrax toxins (capillary morphogenesis protein 2 [CMG2]) and the Fc of human IgG1, for long-circulating half-life and immune effector cell interaction (69). CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with Bacillus anthracis spores at a dose of 2 mg/kg of body weight administered at the time of challenge.…”