Novel antitoxin agents

Many toxin-mediated diseases are treated using antitoxin therapies. Typically, antitoxins are the antisera obtained from large animals immunized with inactivated toxin. More recently, antitoxin therapies for some toxin-mediated diseases, such as our treatment for Shiga toxins, contain antitoxin monoclonal Abs (mAbs). Antisera and mAbs can be difficult to produce economically at scale, usually require long development times and often have problematic quality control, shelf-life and safety issues.

The Department of Infectious Disease and Global Health is developing a novel alternative antitoxin platform employing VHH-based neutralizing agents (VNAs) that would radically change current approaches to antitoxin therapies. The department’s antitoxin strategy should permit rapid development and commercialization of safe, effective antitoxin products with low development and production costs and long shelf lives. VNAs can incorporate multiple VHHs that can neutralize more than one toxin with the same biomolecule. The VNA antitoxin platform can be enhanced by co-administration of an anti-tag effector antibody (efAb) to promote toxin clearance. VNAs can also be delivered effectively using gene therapy approaches. For details see VHH-based neutralizing agents.

We have developed and successfully tested VNAs for efficacy in animal models of exposures from numerous toxins and infections with toxin-mediated diseases (see Publications below). For example, we have developed VNAs that are effective against three different serotypes of Botulinum neurotoxin (BoNT/A, /B and /E) including a single hexavalent VNA that targets all three toxins. In collaborations with Dr. Nicholas Mantis and Dr. Stephen Leppla, VNAs have been developed for ricin, anthrax protective antigen, anthrax lethal factor and anthrax edema factor. In addition, VNAs targeting both Shiga toxins, Stx1 and Stx2, have been shown to protect mice from lethal exposures to both toxins and to protect pigs from lethal infections of enterohemorrhagic E. coli (EHEC). Other VNAs that neutralize both Clostridium difficile toxins, TcdA and TcdB, protect mice from exposures to both of these toxins and protect mice, guinea pigs and gnotobiotic pigs from C. difficile infections. In some cases, the VNAs have also been successfully tested in animal models employing adenovirus gene therapy in collaboration with Dr. David Curiel.

Another application of VHH-based agents for toxin therapy is in the development of Botulism antidotes. For this application, VHHs are identified that neutralize the protease activity of BoNTs that have already intoxicated motor neurons to cause muscle paralysis. These VHH agents would be delivered by a vehicle such as an atoxic BoNT ‘Trojan Horse’. We are seeking to identify motifs that can be fused to the protease-neutralizing VHH that promote the intraneuronal destruction of the pathogenic protease to permanently cure patients with botulism. For details, see Botulism antidotes.

Publications

  1. Moayeri M, Tremblay JM, Debatis M, Dmitriev IP, Kashentseva EA, Yeh AJ, Cheung GY, Curiel DT, Leppla S, Shoemaker CB. 2016. Adenoviral expression of a bispecific VHH-based neutralizing agent targeting protective antigen provides prophylactic protection from anthrax in mice. Clin Vaccine Immunol. CVI.00611-15.
  2. Herrera C, Tremblay JM, Shoemaker CB, Mantis NJ. 2015. Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies. J Biol Chem. 2015 Sep 22. pii: jbc.M115.658070. [Epub ahead of print]
  3. Moayeri M, Leysath CE, Tremblay JM, Vrentas C, Crown D, Leppla SH, Shoemaker CB. 2015. A Heterodimer of a VHH Antibody that Inhibits Anthrax Toxin Cell Binding Linked to a VHH that Blocks Oligomer Formation is Highly Protective in an Anthrax Spore Challenge Model. J Biol Chem. 290:6584-95. PMCID:PMC4358291
  4. Yang Z, Schmidt D, Liu W, Li S, Shi L, Sheng J, Chen K, Yu H, Tremblay JM, Chen X, Piepenbrink KH, Sundberg EJ, Kelly CP, Bai G, Shoemaker CB, Feng H. 2014. “A novel multivalent, single-domain antibody targeting TcdA and TcdB prevents fulminant Clostridium difficile infection in mice”. J. Inf. Dis. Sep 15;210(6):964-72.
  5. Rudolph MJ, Vance DJ, Cheung J, Franklin MC, Burshteyn F, Cassidy MS, Gary EN, Herrera C, Shoemaker CB, Mantis N. 2014. Crystal Structures of Ricin Toxin’s Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-neutralizing Single Chain Antibodies. J Mol Biol. 426(17):3057-68. PMCID:PMC4128236.
  6. Herrera C, Vance DJ, Eisle L, Shoemaker CB, Mantis N. 2014. Differential Neutralizing Activities of a Single Domain Camelid Antibody (VHH) Specific for Ricin Toxin’s Binding Subunit (RTB). PLoS ONE. Jun 11;9(6):e99788. PMCID:PMC4053406.
  7. Kaliberov SA, Kaliberova LN, Buggio M, Tremblay JM, Shoemaker CB, Curiel DT. 2014. Adenoviral targeting using genetically incorporated camelid single variable domains. Lab. Invest. 94(8):893-905. PMCID:PMC4157633.
  8. Mukherjee J, Dmitriev i, Debatis M, Tremblay JM, Beamer G, Kashentseva EA, Curiel DT, Shoemaker CB. 2014. Prolonged prophylactic protection from botulism with a single adenovirus treatment promoting serum expression of a VHH-based antitoxin protein. PLoS ONE. Aug 29;9(8):e106422. PMCID:PMC4149568.
  9. Barrera D, Rosenberg J, Chiu J, Chang YN, Debatis M, Ngoi SM, Chang J, Shoemaker CB, Oyler G, Mayfield S. 2014. Algal chloroplast produced camelid VHH anti-toxins are capable of neutralizing botulinum neurotoxin. Plant Biotech. J. 13:117-24. PMCID:PMC4620920
  10. Sheoran AS, Dmitriev IP, Kashentseva EA, Cohen O, Mukherjee J, Debatis M, Shearer J, Tremblay JM, Beamer G, Curiel DT, Shoemaker CB, Tzipori S.. 2014. Adenovirus vector expressing Stx1/2-neutralizing agent protects piglets infected with E. coli O157:H7 against fatal systemic intoxication. Infect Immun. 83:286-91. PMCID: PMC4288880
  11. Vance DJ, Tremblay JM, Mantis NJ, Shoemaker CB. 2013. Stepwise engineering of heterodimeric single domain camelid VHH antibodies that passively protect mice from ricin toxin. J. Biol. Chem. Dec 20;288(51):36538-47. PMCID:PMC3868766.
  12. Tremblay JM, Mukherjee J, Leysath CE, Debatis M, Ofori K, Baldwin K, Boucher C, Peters R, Beamer G, Sheoran A, Bedenice D, Tzipori S, Shoemaker CB. 2013. A single VHH-based toxin neutralizing agent and an effector antibody protects mice against challenge with Shiga toxins 1 and 2. Infect Immun. Dec;81(12):4592-603. doi: 10.1128/IAI.01033-13. Epub 2013 Sep 30. PMCID: PMC3837998
  13. Mukherjee J, Tremblay JM, Leysath CE, Ofori K, Baldwin K, Feng X, Bedenice D, Webb RP, Wright PM, Smith LA, Tzipori S, Shoemaker CB. 2012. A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model. PLoS ONE. 7(1):e29941. PMCID: PMC3253120
  14. Kuo CL, Oyler GA, Shoemaker CB. 2011. Accelerated neuronal cell recovery from Botulinum neurotoxin intoxication by targeted ubiquitination. PLoS ONE. 6(5):e20352. PMCID: PMC3101245
  15. Tremblay JM, Kuo CL, Abeijon C, Sepulveda J, Oyler G, Hu X, Jin MM, Shoemaker CB. 2010. Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases. Toxicon. 56:990-8. PMCID: PMC2946066
  16. Sepulveda J, Mukherjee J, Tzipori S, Simpson LL, Shoemaker CB. 2010. Efficient serum clearance of Botulinum neurotoxin achieved using a pool of small antitoxin binding agents. Infection and Immunity. 78:756-63. PMCID: PMC2812214