Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B

Abstract

Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C . difficile –secreted exotoxins—toxin A (TcdA) and toxin B (TcdB)—that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC 50 ) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4–1.4E and U3—bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and “kisses” the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C . difficile . The structural insights regarding both the “native” conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti– C . difficile toxin therapeutics.

Identification of the C. sordellii lethal toxin receptor elucidates principles of receptor specificity in clostridial toxins

“…Initial analysis of the dataset revealed that approximately 50% of the SEMA6A dimers were bound to TcsL using this sample preparation approach. Consequently, 2D and 3D classification resulted in two homogenous datasets corresponding to the TcsL- Figure 3 The architecture of TcsL is similar to previously-determined structures of other clostridial toxins (8,(22)(23)(24) of SEMA6A, which is buried in a TcsL hydrophobic pocket formed by C1433, I1434, Y1471, A1486 and I1493. Indeed, expression of a SEMA6A M109D in SEMA6A knockout cells does not make the cells more sensitive to TcsL, in contrast to wild-type SEMA6A (Fig.…”

Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B

“…1b). In addition, some studies reported that several immunoproteins including bezlotoxumab, an FDA-approved human monoclonal antibody for treating CDI 45 , may have distinct neutralizing activities to TcdB1 and TcdB2 28,46 (and potentially to other TcdB subtypes), which could be vital to clinical antitoxin treatment for CDI. In some cases, TcdB subtyping together with MLST analysis could be a helpful procedure when choosing appropriate immunoproteins.…”

In Vitro Production and Immunogenicity of a Clostridium difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine

“…To date, vaccine development for the prevention of CDI has primarily focused on the toxins Toxin A TcdA and Toxin B TcdB produced by vegetative cells during the infection process [16][17][18]. However, attention has more recently been directed toward the spore of C. difficile as it is the primary agent of transmission and persistence within the gut [19].…”