Development of recombinant human anti-diphtheria toxin neutralizing antibodies for diphtheria therapy

Abstract

Diphtheria is an infectious disease caused by toxigenic Corynebacterium spp. that produce diphtheria toxin (DT). Diphtheria is a significant health problem in countries with poor immunization coverage or disrupted immunization programs. Even in countries where the disease is well controlled, there is a need to maintain a stockpile of therapeutic diphtheria antitoxin (DAT) for management of sporadic or imported cases. Currently, diphtheria is still treated with equine sera in the same way it was treated more than 100 years ago by Emil von Behring. Antibodies against DT have been generated from immune and naive human antibody gene libraries by phage display. The panning was performed on unnicked DT once classically with DT immobilized on a microtiterplate and once with biotinylated-DT in panning in solution. Among both panning strategies, 660 monoclonal antibodies were selected. The first screening was either for binding of the antibodies against diphtheria toxin or for neutralization of diphtheria toxin in a cell-based assay. In total, 439 antibodies were further characterized in scFv-Fc format regarding neutralization in a cell-based in vitro neutralization assay. In this assay, 290 scFv-Fc antibodies showed neutralization potency. The best 35 neutralizing scFv-Fc were sub-cloned into IgG1 format. Three best neutralizing lead candidates were selected, one against each of the three domains of DT. The IgG targeting the receptor binding domain is ewe372-D4 and has a neutralization potency of 446 IU/mg. ewe375-H4 has a neutralization potency of 20.4 IU/mg and binds the catalytic domain and ewe372-F6 binds the translocation domain with a neutralization potency of 2.25 IU/mg. In comparison, neutralization potency of equine DAT is approximately 50 IU/mg. In this study, seven antibodies with a higher neutralization potency then equine DAT were developed, and they all bind the receptor binding domain of diphtheria toxin. In the next step, experiments in a non-lethal guinea pig model were conducted to test in vivo protection potency. Possible prospects include further development and testing of these antibodies for clinical application as therapeutic agents against diphtheria toxin.