Broadly Neutralizing Non-Antibody Protein For Treating Clostridium Difficile Infection
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abstract
Clostridium difficile infection (CDI) is the leading cause of infectious diarrhea in hospitalized patients, with anestimated annual cost to the United States of between $750 million and $3.2 billion. The pathology of CDI iscaused by the toxins secreted by the bacteria. Recently, two anti-C. difficile toxin antibodies, actoxumab andbezloxumab, that bind to and neutralize C. difficile toxins A and B, respectively, have achieved success for CDItreatment in a phase II clinical trial. However, only bezloxumab demonstrated efficacy in the subsequent phaseIII clinical trial. The neutralization potency of antibody is strongly epitope-dependent. Since both actoxumaband bezlotoxumab were engineered using toxins from laboratory C. difficile strain VPI 10463, both showedsignificantly reduced neutralization potencies against some recently emerged hypervirulent strains of C.difficile. Thus, there is an urgent need to develop broadly effective C. difficile toxins neutralizer. We propose toengineer a non-antibody protein, designed ankyrin repeat protein (DARPin), that is able to bind to andneutralize toxins A and B from a broad range of C. difficile strains. DARPins represent a versatile class ofbinding proteins that have been engineered to bind diverse targets with up to picomolar affinity. Furthermore,DARPin can be very efficiently expressed in E. coli (accounting for >50% of all E. coli proteins) and very easilypurified due to its high thermostability. In this project, we will first use bacteriophage display to isolate DARPinsthat are able to bind to toxins from different strains of the C. difficile (Aim 1) and then subject the selectedDARPins to an anti-toxin functional screen in cultured cells to identify toxin-neutralizing DARPins (Aim 2). Thepotency of the selected DARPins against a panel of emerging and clinically relevant strains of C. difficile will beanalyzed and be further optimized by directed evolution (Aim 3). Successful completion of this study will yieldan arsenal of high-potency toxin-neutralizing DARPins. These broadly neutralizing anti-toxin DARPins canpotentially be fused to Fc and used as antibody therapy for CDI, or be formulated for oral administration todirectly neutralize C. difficile toxin(s) in the gut. The approach of neutralizing bacterial virulence factors withDARPins should also offer a new treatment paradigm for other bacterial infection.
