Pre-clinical proof of principle for orally delivered Th17 antagonist miniproteins

Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics. In the related work, proteins were computationally designed to bind IL-23R or IL-17A. Site-directed saturation mutagenesis libraries were generated based on the sequences of sub-optimal minibinders, transformed into yeast for surface display, and selected for high-affinity binding to each target, optionally after pre-treatment of the library with simuated intestinal fluid. Next generation sequencing analysis of the naive and selected pools was used to determine the fitness of each mutation for affinity and stability.