Abstract
Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI’s evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.
Highlights
Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract
A major fraction of new drugs approved by the U.S Food and Drug Administration (FDA) are monoclonal antibodies, but it is long known that antibodies are rapidly digested and inactivated in the adult stomach[15]
Encouraged that soybean trypsin inhibitor (SBTI) is stable in gastrointestinal conditions and heat-resilient, we explored the evolvability of this candidate scaffold protein, to generate what we term a gastrobody. β-trefoil fold proteins tend to share similar tertiary structure, but have low sequence identity, except for some inward-facing hydrophobic residues[40]
Summary
Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. There have been extensive protein engineering efforts to develop different antibody formats or antibody mimetics (e.g. nanobodies, DARPins, affibodies), with tremendous therapeutic and diagnostic potential[16]. These scaffolds have generally been optimized for performance at neutral pH and are rapidly destroyed in the GI tract[15]. Nanofitins (affitins)[17,18] and nanobodies[12,19,20] are leading protein scaffolds that have been engineered to enhance their use by oral administration
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