Development of the Gastrobody platform to combat Clostridium perfringens toxins

Antibodies are proteins that can bind specific targets such as bacteria, viruses or toxins to stop them causing disease. Antibodies are not widely used in agriculture because, like nearly all proteins in the diet, they are quickly destroyed by the stomach and small intestine. We have generated "gastrobodies" by engineering a protein from soybeans to bind new targets. Unlike antibodies or other protein scaffolds, gastrobodies are highly resistant to the harsh digestive enzymes of the gut. Here we will test the ability of gastrobodies to protect against bacterial disease. Clostridium perfringens is a bacterium that causes a wide range of diseases affecting farmers, including in pigs, cows, sheep and chickens. These effects are caused by toxins released by the bacterium. We will establish gastrobodies that can stop these bacterial toxins in the gut from causing damage. We will focus on the toxins that cause necrotic enteritis in chickens, which has increased dramatically following the banning of antibiotics in animal feed and costs billions of pounds per year worldwide. We aim to establish the cost-effective production of gastrobodies, so that they can be simply added to animal feed and then recognise specific targets within the digestive system. These goals should set the stage for broad use of gastrobodies for gut health, with relevance to many agricultural and medical diseases.

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the gastrointestinal (GI) tract. We found that two scaffold proteins (nanobody and nanofitin), previously developed to surpasss the resistance of antibodies, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Therefore we engineered gastrobodies, a protein scaffold derived from soybean trypsin inhibitor. The gastrobody scaffold is highly resistant to the challenges of the GI tract, including digestive proteases, bile acids and pH 2. Through computational prediction and library selection, we identified binders to Clostridium difficile toxins, giving nanomolar affinity and enzyme inhibitory activity. Here we will identify binders to neutralize toxins from Clostridium perfringens. Toxins from C. perfringens cause a range of diseases, including in pigs, cows, sheep and humans. We will establish the high yield production of gastrobodies in an industrially relevant fungal host. We will determine the distribution and pharmacokinetics of gastrobodies and determine their effectiveness in protecting against necrotic enteritis. Necrotic enteritis has increased substantially in chickens following the banning of antibiotics in animal feed. By impairing animal health and growth, this disease costs billions of pounds per year globally. This project should establish the potential and scalability of the gastrobody platform, towards establishing a new path for therapeutic intervention in the GI tract.