Neutralization of Clostridium difficile toxin B with V(H)H-Fc fusions targeting the delivery and CROPs domains

An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C. difficile immunotherapeutics, we previously isolated several single-domain antibodies (V(H)Hs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing V(H)Hs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 V(H)Hs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as K(D) = 70 pM, none of the V(H)Hs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with V(H)H-V(H)H dimers and to a much greater extent with V(H)H-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several V(H)H-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other V(H)H-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the V(H)Hs described here are effective at toxin B neutralization when formatted as bivalent V(H)H-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site.