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Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027.

ABSTRACT: Clostridium difficile infection (CDI) is a leading cause of hospital-acquired diarrhea. In recent decades, the emergence of the "hypervirulent" BI/NAP1/027 strains of C. difficile significantly increased the morbidity and mortality of CDI. The pathogenesis of CDI is primarily mediated by the action of two toxins, TcdA and TcdB, with TcdB being the major virulent factor in humans. In this report, we describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) that potently neutralize TcdB from the BI/NAP1/027 strains (e.g., TcdBUK1). The most effective DARPin, D16, inhibits TcdBUK1 with a 50% effective concentration (EC50) of 0.5 nM, which is >66-fold lower than that of the FDA-approved anti-TcdB antibody bezlotoxumab (EC50, ∼33 nM). Competitive enzyme-linked immunosorbent assays (ELISAs) showed that D16 blocks interactions between TcdB and its receptor, chondroitin sulfate proteoglycan 4 (CSPG4). The dimeric DARPin U3D16, which pairs D16 with DARPin U3, a disrupter of the interaction of TcdB with Frizzled 1/2/7 receptor, exhibits 10-fold-to-20-fold-enhanced neutralization potency against TcdB from C. difficile strains VPI 10463 (laboratory strain) and M68 (CF/NAP9/017) but identical activity against TcdBUK1 relative to D16. Subsequent ELISAs revealed that TcdBUK1 did not significantly interact with Frizzled 1/2/7. Computation modeling revealed 4 key differences at the Frizzled 1/2/7 binding interface which are likely responsible for the significantly reduced binding affinity.IMPORTANCE We report the engineering and characterization of designed ankyrin proteins as potent neutralizers of TcdB toxin secreted by a hypervirulent ribotype 027 strain of Clostridium difficile We further show that although TcdB toxins from both ribotype 027 and VPI 10461 interact efficiently with TcdB receptors CSPG4 and Pvrl3, TcdB027 lacks significant ability to bind the only known physiologically relevant TcdB receptor, Frizzled 1/2/7.

SUBMITTER: Peng Z

PROVIDER: S-EPMC6796971 | biostudies-other | 2019 Oct

REPOSITORIES: biostudies-other

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Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027.

Peng Zeyu Z Simeon Rudo R Mitchell Samuel B SB Zhang Junjie J Feng Hanping H Chen Zhilei Z

mSphere 20191002 5

<i>Clostridium difficile</i> infection (CDI) is a leading cause of hospital-acquired diarrhea. In recent decades, the emergence of the "hypervirulent" BI/NAP1/027 strains of <i>C. difficile</i> significantly increased the morbidity and mortality of CDI. The pathogenesis of CDI is primarily mediated by the action of two toxins, TcdA and TcdB, with TcdB being the major virulent factor in humans. In this report, we describe the engineering of a panel of designed ankyrin repeat proteins (DARPins) th ...[more]

PMID: 31578248

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Project description: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 (EC50) 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.

Project description:PurposeHuman epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application.MethodsG3 DARPins tagged with hexahistidine (His6) or with histidine glutamate (HE)3 and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with (125)I, or with (111)In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE)3-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts.ResultsFor both isotopes, (HE)3-G3 had significantly lower liver uptake than His6-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His6-G3 and untagged G3. (HE)3-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from (111)In-(HE)3-G3 was better maintained in tumours and cleared faster from serum than radioactivity from (125)I-(HE)3-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, (111)In-labelled and (125)I-labelled (HE)3-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of radioactivity with (111)In-(HE)3-G3. Preadministration of trastuzumab did not affect the uptake of (HE)3-G3 by HER2-positive tumours.ConclusionRadiolabelled DARPin (HE)3-G3 is a versatile radioligand with potential to allow the acquisition of whole-body HER2 scans on the day of administration.

Dataset Information

Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027.

Publications

Designed Ankyrin Repeat Protein (DARPin) Neutralizers of TcdB from Clostridium difficile Ribotype 027.

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