B. thuringiensis crystal proteins as powerful next-generation anthelmintics

Univ Of Massachusetts Med Sch Worcester, Worcester MA

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Abstract

New treatments are urgently needed for parasitic gastrointestinal nematodes (GINs; hookworms, whipworms, roundworms) that infect ~1.5 billion people and cause significant morbidity in children, pregnant women, and working adults. Mass drug administration with albendazole has not been curative, and there are limitations with additional approaches involving ivermectin and the experiment drug emodepside. Controlling GINs remains a critical unmet medical need. For over 60 years, Bacillus thuringiensis crystal (Cry) proteins have been safely and massively used to combat insect pests that vector disease and damage crops. Pioneering work has shown that some Cry proteins (e.g., Cry5Ba) target nematodes and are anthelmintics. Orally administered Cry5Ba is highly effective against GINs in rodents, dogs, sheep, pigs, and horses and against all three major human GIN classes. The goal here is to develop Cry protein anthelmintics to meet World Health Organization (WHO) Target Product Profiles (TPPs) for GIN anthelmintics and perform key studies leading to first in human trials. Anthelmintic Cry proteins can be delivered via a simple-to-produce, inexpensive, safe, and a scalable form of anthelmintic Cry protein called IBaCC for Inactivated Bacteria with Cytosolic Crystal(s). Currently, six Cry proteins are known anthelmintics. The means to study all three GIN classes in rodents, to measure synergy of Cry protein combinations, to produce hybrid (domain swapped) Cry proteins with increased activity, and to produce IBaCC in commercially relevant yields via industry collaborators are successfully in hand. These advancements will be leveraged here to engineer Cry protein therapies to meet the WHO's TPP to target all three GIN classes with high efficacy, low dose, and low cost and to advance Cry protein anthelmintics to the clinic. In Aim 1, it is hypothesized that the WHO TPP will be achieved by identifying synergy among all pairwise combinations of six anthelmintic Cry proteins in vitro against all three classes of GINs. All synergistic combinations will be tested in vivo in rodents against all three GIN classes at a Step 1 TPP of 5 mg/kg. Fit for purpose formulation strategies for improved gastric resistance and anthelmintic efficacy in rodent models vs. the three GINs will also be tested in this aim. In Aim 2, it is hypothesized that the WHO TPP will be achieved by swapping domains among the six anthelmintic Cry proteins to generate hybrid Cry proteins. These hybrid proteins will be tested in vitro against all three GIN classes for superior efficacy and then in vivo in rodents against all three GIN classes at the Step 1 TPP. Combining Cry proteins (1) and swapping domains among Cry proteins (2) are evolutionary strategies seen in nature to increase Cry protein efficacy and spectrum of activity. In Aim 3, the best Cry protein combinations will be tested against multi-drug-resistant dog hookworms in vitro and in vivo in order to select a single lead candidate for GLP-toxicological studies as a prelude to first in human trials. The results will identify optimal combinations of Cry proteins and hybrid Cry proteins that meet the recommended WHO TPP, with safety studies in place for progression of a lead anthelmintic to clinical trials.