Journal Of Molecular Biology
To develop an antibody (Ab) therapeutic against Staphylococcal Enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a "Class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed "Class M" Abs. A systematic engineering approach was applied to affinity mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) revealed that the Class T epitope on SEB overlapped with the T-cell receptor (TCR) binding site, whereas other evidence suggested that the Class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ~50-fold more potent toxin-neutralizing efficacy than the best Class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two Class M binding sites and two Class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that Class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bispecific Ab engineering. Copyright 2019. Published by Elsevier Ltd.