Development and application of VHH-based therapeutic agents

The Shoemaker lab in the Department of Infectious Disease and Global Health develops therapeutic biomolecules based on single domain antibody (sdAb) binding agents that are engineered into multiple scaffolds. The sdAb agents derive from the VH region of heavy-chain-only Abs (VHHs) obtained from alpacas immunized with the target antigen. VHH agents are small proteins (14 kDa) that are produced economically at high levels, more stable to temperature and pH extremes than conventional antibodies, and commonly block (neutralize) the function of targets to which they bind. We have extensively refined our VHH discovery platform such that we typically obtain a large and diverse pool of high affinity binding agents to each biomolecule that we ‘target’ in our search for novel therapeutics. From our pool of VHHs, we typically identify VHHs that most potently neutralize the target functions. Where possible we identify neutralizing VHHs that bind at different (non-overlapping) sites on the target. These VHHs are then engineered in various ways to improve their in vivo efficacy and their commercial properties. The VHH agents also have value as research reagents and for diagnostic tests.

VHH-based neutralizing agents (VNAs)

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For most of our therapeutic strategies, we produce ‘multispecific’ proteins in which we genetically link two (or more) VHHs that each neutralize the therapeutic target to create VHH-based neutralizing agents (VNAs) (see Mukherjee et al). The different VHH components preferentially neutralize target functions at different sites. VNAs consisting of two different neutralizing VHHs have demonstrated dramatically improved in vivo efficacy in numerous toxin models (see Novel antitoxin agents), as well as other disease models such as Anti-infective VNAs. (publications pending).

In many cases, we link neutralizing VHHs to more than one target such that a single VNA can target more than one pathogenic biomolecules, such as two different toxins produced by the same pathogen (see Tremblay et al and Yang et al), for improved therapeutic benefit. We have successfully targeted and neutralized three different botulism serotypes with a single hexaspecific VNA (publication pending).

VNAs have proven to be ideal for gene therapy approaches. For example, in collaboration with the Curiel lab at WUSTL, we have produced adenovirus vectors that lead to the expression of therapeutic serum VNA levels in treated animals and protection from exposures to toxins (see Mukherjee et al) or toxin-mediated disease pathogens (see Sheoran et al and Moayeri et al). In collaboration with Curevac, we demonstrated antitoxin efficacy in mice treated with packaged mRNA gene therapy agents (publication pending).

Effector Ab (efAb) for enhancedin vivo efficacies

Our VNA technology offers the novel capability for therapeutic efficacy to be substantially enhanced by the co-administration (or co-expression in gene therapy) of an effector antibody (efAb) to provide the effector functions of conventional antibody Fc domains. The efAb is an antibody recognizing a small peptide ‘tag’ motif that is engineered at one or more sites within each VNA. As shown in the figure (above), by including two tags on a bispecific VNA, the target protein becomes ‘decorated’ by up to four efAb Fc domains, and this valency promotes rapid serum clearance of the target protein (see Sepulveda et al). Employing co-administered efAb has been shown to improve antitoxin efficacy in several animal models of intoxication (see Mukherjee et al, Tremblay et al and Vance et al). The efAb itself can be engineered to possess the Fc isotype(s) required to most effectively achieve desired therapeutic benefits. In addition, the same efAb can be engineered for the animal model needed for efficacy testing, or it can be ‘humanized’ for applications in people. Importantly, the same efAb can be used for all VNA therapies since the same tag motif is incorporated into all VNAs.

Other applications of VHHs

VHHs, as high affinity and highly specific binding agents, have numerous additional potential applications beyond their use in VNAs. One application that has shown great promise is to employ VHHs that neutralize the protease of Botulinum neurotoxins (BoNTs) in the development of Botulism antidotes, agents that can ‘reverse’ the paralysis of botulism patients. Secondly, we have ongoing collaborations with numerous labs to develop VHHs for diagnostic application. Thirdly, VHHs are proving to be valuable as research tools. For example, because VHHs tend to recognize protein conformations, they can be used to probe for different conformations exhibited by the same target protein. Finally, VHHs make excellent reagents in basic research, particularly as capture or detection agents for native proteins.

Publications

  • Vrentas CE, Moayeri M, Keefer AB, Greaney AJ, Tremblay J, O'Mard D, Leppla SH, Shoemaker CB. 2016. A diverse set of single-domain antibodies (VHHs) against the anthrax toxin lethal and edema factors provides a basis for construction of a bispecific agent that protects against anthrax infection. J Biol Chem. 2016 Aug 18. pii: jbc.M116.749184. [Epub ahead of print]
  • Schmidt DJ, Beamer G, Tremblay JM, Steele JA, Kim HB, Wang Y, Debatis M, Sun X, Kashentseva EA, Dmitriev IP, Curiel DT, Shoemaker CB, Tzipori S. 2016. A Tetraspecific VHH-based Neutralizing Antibody Modifies Disease Outcome in Three Animal Models of Clostridium difficile Infection. Clin Vaccine Immunol. Jul 13. pii: CVI.00730-15.
  • 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.
  • 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]
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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
  • 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
  • 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
  • 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
  • Maass, DM, Sepulveda, J, Pernthaner, A, Shoemaker, CB. 2007. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J Immunol Methods. 324:13-25. PMCID: PMC2014515