introduction
IL-23R and IL-17 are key therapeutic targets for autologous inflammatory diseases, especially in the treatment of inflammatory bowel disease (IBD) and psoriasis, and inhibition of IL-23 and IL-17 signaling pathways can significantly reduce inflammatory responses. However, there are many limitations to existing antibody therapies. Only about 30% of patients with IBD achieve remission after treatment with anti-IL-23 monoclonal antibodies (e.g., Stelara), and about 20% of initial responders gradually lose response due to the development of anti-drug antibodies. In addition, systemic immunosuppression increases the risk of malignancy and serious infection. Due to the large size and poor permeability of the antibody molecules, they cannot be taken orally and can only be injected intravenously or subcutaneously, which is inconvenient and stress-prone for patients. Antibodies are also relatively expensive to produce and transport due to the need to produce them in a mammalian expression system, undergo a complex purification process to achieve purity suitable for injection, and refrigerated storage and transportation. The study "Preclinical proof of principle for orally delivered Th17 antagonist miniproteins" reported by Cell on June 26 aims to develop orally available IL-23R and IL-17 antagonist small proteins to address the therapeutic needs of autologous inflammatory diseases. Through computational design, the research team successfully designed IL-23R and IL-17 microbinding proteins with low picomoletic affinity and high stability at antibody levels, which not only effectively block cell signaling, but also exhibit strong resistance under heat, acid, and proteolysis conditions. The designed IL-23R and IL-17 small protein binders not only have the advantages of low cost and ease of manufacturing, but also can achieve therapeutic effects through the oral route. In a mouse model of colitis, oral IL-23R microbinding protein is even more effective than clinical anti-IL-23 antibodies. This study demonstrates the ability of denewed-engineered microbinders for oral administration to cross the intestinal epithelial barrier and reach therapeutic targets, with the advantages of high potency, intestinal stability, and ease of manufacturing, providing a new potential modality for oral biologics. Overall, this study demonstrated the great potential of oral IL-23R and IL-17 antagonist small proteins in the treatment of autologous inflammatory diseases, demonstrated their feasibility and advantages as oral biologics, and provided an important reference for the development and application of related drugs in the future.
Autoinflammatory diseases are diseases caused by abnormalities of the autoimmune system, such as inflammatory bowel disease (IBD) and psoriasis. The study found that the cytokines IL-23 and IL-17 play a key role in the pathogenesis of these diseases. IL-23 is produced by antigen-presenting cells and promotes the differentiation and maintenance of T helper type 17 (Th17 cells), while IL-17 is produced by circulating Th17 cells and tissue-resident innate lymphocytes (ILCs) and γδT cells, which have a strong pro-inflammatory effect. Therefore, targeting and inhibiting the signaling pathways of IL-23 and IL-17 has become an important strategy for the treatment of autoinflammatory diseases.
Although existing antibody therapies (e.g., Stelara, a monoclonal antibody against IL-23) have shown some clinical efficacy, these therapies have many limitations, such as high cost, safety risks, unsustainable efficacy, and inconvenient administration (injectable). Therefore, this study aims to develop IL-23R and IL-17 antagonist microproteins that can be orally administered orally through computational design, which not only have high affinity for antibody levels, but also have extremely high stability and low-cost manufacturing advantages, so as to provide a novel, more convenient and safe treatment modality for the treatment of autoinflammatory diseases.
The Computational Design and Screening research team first used computational design methods to design IL-23R and IL-17 microbinding proteins with low nanomolar affinity and high stability. This process starts with the crystalline or cryo-electron microscopy (cryo-EM) structure of the protein of interest, combined with information from key binding sites (hotspots), to optimize the amino acid sequence and conformation of the protein using the Rosetta Molecular Modeling Suite. Eventually, the researchers obtained thousands of potential microbinding proteins and transferred the genes encoding these proteins into yeast cells to screen for the optimal binding proteins through multiple rounds of fluorescence-activated cell sorting (FACS).
The microbinding proteins screened by in vitro biochemical characterization were expressed and purified in Escherichia coli, and the researchers quantitatively determined the binding affinity and inhibitory potency of these proteins by biolayer interference (BLI) and cell signaling inhibition assays. The results showed that these microbinding proteins all bind to their target proteins with low nanomolar affinity and exhibited extremely high stability under thermal, acidic, and proteolytic conditions.
In vivo experiments validated in a mouse model of colitis, the research team evaluated the efficacy of oral administration of IL-23R microbinding protein. The results showed that the oral IL-23R microbinding protein was superior to the anti-IL-23 antibody used in clinical practice in relieving the symptoms of colitis in mice. In addition, through pharmacokinetic and biodistribution studies, it was found that these microbinding proteins have good pharmacokinetic properties and distribution characteristics in rats, and are able to cross the intestinal epithelial barrier and reach target tissues.
Through computational design, the research team successfully designed IL-23R and IL-17 microbinding proteins with high affinity and high stability. Among them, IL-23R microbinding proteins 23R-1 and 23R-2 contain 55 and 54 amino acid residues, respectively, forming three helical bundle structures, which can effectively bind IL-23R. IL-17 microbinding proteins 17-1, 17-2, and 17-3 contain 43 and 61 amino acid residues, respectively, and can effectively bind IL-17A.
In vitro experiments showed that the designed microbinding protein could bind its target protein with low nanomolar affinity. In circular dichroism (CD) experiments, the denaturation temperature (Tm) of IL-23R microbinding proteins 23R-1 and 23R-2 exceeded 95 °C and extremely high chemical denaturation midpoint concentrations (5 M Gdn for 23R-1, > 6 M for 23R-2), respectively. IL-17A microbinding protein 17-1 has a Tm of approximately 90 °C and a chemical denaturation midpoint of 4 M. IL-17A microbinding protein 17-2 is the least stable with a Tm of about 70 °C and a chemical denaturation midpoint of 2 M.
Gastrointestinal Stability of IL-23R Microbinding Proteins in Vitro and in vivo (Credit: Cell) Proteolytic Stability in Vitro: Each microbinding protein or V565 nanobody as a control (oral nanobody currently under development for IBD) was digested at 37°C for up to 24 h in simulated intestinal fluid (SIF) or simulated gastric juice (SGF). The results showed that some microbinding proteins were more stable in SGF or SIF, while some were degraded in a short period of time. Residual Binding Activity: Samples digested by SIF and SGF were diluted to a microbinding protein concentration of 10 nM at various time points and their residual binding activity against human IL-23R was measured by BLI (Biolayer Interferometry). The results showed that some of the microbinding proteins still retained their binding ability to IL-23R after SGF and SIF digestion. In vivo pharmacokinetic study in rats: In healthy rats, a single dose of 20 mg/kg of 23R-72 or 23R-91 was administered orally in PBS or gastrointestinal protective vehicle (GPV) formulation, and microbinding protein concentrations in serum and target tissues were measured after 6 hours. The results showed that these microbinding proteins were concentrated at concentrations of 50-100 nM in small intestinal contents, 40-200 nM in small intestinal tissues, and at lower concentrations of 2-20 nM in colon tissues, regardless of PBS or GPV formulations. No significant concentrations of microbinding proteins were detected in rat serum. Serum concentration time curve: A single dose of 23R-91 at 140 mg/kg was administered orally in healthy rats, and the concentration of microbinding protein in serum was measured at different time points. The results showed that the microbinding protein reached a peak serum concentration of 73 nM after 15 minutes, followed by a rapid decline with a half-life of approximately 15 minutes, and concentrations below the detection limit after 6 hours.
In a mouse model of colitis, oral administration of IL-23R microbinding protein showed significant efficacy in alleviating colitis symptoms. Pharmacokinetic studies have shown that these microbinding proteins have good pharmacokinetic properties and distribution characteristics in rats, and are able to cross the intestinal epithelial barrier and reach target tissues. The specific data are as follows: a single oral dose of 20 mg/kg of 23R-91 is produced at concentrations of 50-100 nM and 40-200 nM in intestinal tissue and contents in healthy rats, respectively, and at concentrations of 2-20 nM in colonic tissue. In addition, after administration of a single dose of 140 mg/kg of 23R-91, its concentration in rat serum peaked at 73 nM after 15 minutes and then decreased rapidly with a half-life of approximately 15 minutes.
The research team also evaluated the immunogenicity of the microbinding proteins. 23R-91 is highly soluble and stable, and its immunogenicity is expected to be low. In human clinical trials, oral administration of 23R-91 did not elicit a strong antimicrobial response. Further bioinformatics analysis revealed that the 23R-91 fragment had a low binding affinity for a variety of major histocompatibility complex (MHC) class II molecules, which was associated with low immunogenicity.
The study diagram (Credit: Cell) shows great potential as oral biologics for IL-23R and IL-17 antagonist microproteins developed through computationally designed microproteins. Compared with traditional antibody therapies, these microproteins not only have the advantages of high potency, low cost, and ease of manufacturing, but also can be easily administered through the oral route, greatly improving the treatment experience of patients. This study provides a new idea for the treatment of autoinflammatory diseases and demonstrates the broad application prospect of computationally engineered proteins in the field of biomedicine. In the future, with the in-depth development of more preclinical and clinical studies, these microbinding proteins are expected to bring safer, effective and convenient treatment options to the majority of patients.
bibliography
Berger S, Seeger F, Yu TY, Aydin M, Yang H, Rosenblum D, Guenin-Macé L, Glassman C, Arguinchona L, Sniezek C, Blackstone A, Carter L, Ravichandran R, Ahlrichs M, Murphy M, Pultz IS, Kang A, Bera AK, Stewart L, Garcia KC, Naik S, Spangler JB, Beigel F, Siebeck M, Gropp R, Baker D. Preclinical proof of principle for orally delivered Th17 antagonist miniproteins. Cell. 2024 Jun 23:S0092-8674(24)00631-7. doi: 10.1016/j.cell.2024.05.052. Epub ahead of print. PMID: 38936360.https://doi.org/10.1016/j.cell.2024.05.052
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