De novo development of proteolytically resistant therapeutic peptides for oral administration

Abstract

The oral administration of peptide drugs is hampered by their metabolic instability and limited intestinal uptake. Here, we describe a method for the generation of small target-specific peptides (less than 1,600 Da in size) that resist gastrointestinal proteases. By using phage display to screen large libraries of genetically encoded double-bridged peptides on protease-resistant fd bacteriophages, we generated a peptide inhibitor of the coagulation Factor XIa with nanomolar affinity that resisted gastrointestinal proteases in all regions of the gastrointestinal tract of mice after oral administration, enabling more than 30% of the peptide to remain intact, and small quantities of it to reach the blood circulation. We also developed a gastrointestinal-protease-resistant peptide antagonist for the interleukin-23 receptor, which has a role in the pathogenesis of Crohn’s disease and ulcerative colitis. The de novo generation of targeted peptides that resist proteolytic degradation in the gastrointestinal tract should help the development of effective peptides for oral delivery.

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Fig. 1: Genetic encoding of double-bridged peptides by protease-resistant phage.
Fig. 2: Panning of double-bridged peptide libraries under protease pressure.
Fig. 3: Characterization and size reduction of F3, the most stable peptide.
Fig. 4: Structure analysis and impact of chemical bridges on stability.
Fig. 5: Stability and oral administration in mice.
Fig. 6: Stable IL-23R antagonists.

Data availability

The main data supporting the results of this study are available within the paper and its Supplementary Information. The data used to make the graphs in the figures are provided as Supplementary Information. Next-generation sequencing source data are available from figshare at https://doi.org/10.6084/m9.figshare.11921139.v2. Coordinates of the two X-ray structures have been deposited in the Protein Data Bank with accession numbers 6TWB and 6TWC.

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Acknowledgements

We thank B. Mangeat from the Ecole Polytechnique Fédérale de Lausanne (EPFL) Gene Expression Core Facility for help with next-generation sequencing, A. Reynaud, D. Hacker, L. Durrer and S. Quinche from the EPFL Protein Production and Structure Core Facility for help with protein expression and crystallization, and E. Simeoni, I. Desbaillets, G. Ferrand and C. Waldvogel of the EPFL Center of Phenogenomics (CPG) for help with mouse experiments. The financial contributions from the Swiss National Science Foundation grants (project grant 157842, NCCR Chemical Biology and project grant 169526) and the EPFL are gratefully acknowledged.

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X.-D.K. and C.H. conceived the strategy for proteolytic phage display with double-bridged peptides. X.-D.K. established the phage selection procedure, cloned the libraries, performed the phage selections against FXIa, synthesized and characterized the peptides, expressed and purified FXIa and determined the X-ray structures. J.M. expressed and purified IL-23R, performed the phage selections against IL-23R and characterized the peptides. V.C. identified the d-proline mutant. F.P. collected X-ray data and analysed the structures. L.A.A. performed the NMR study. X.-D.K., K.D. and C.H. wrote the manuscript with help from all authors.

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Correspondence to Christian Heinis.

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J.M., X.-D.K. and C.H. are inventors on a patent protecting the IL-23R antagonists.

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Supplementary information

Supplementary Information

Supplementary results, methods, figures and tables.

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Supplementary Dataset 1

Source data for the figures.

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Kong, XD., Moriya, J., Carle, V. et al. De novo development of proteolytically resistant therapeutic peptides for oral administration. Nat Biomed Eng 4, 560–571 (2020). https://doi.org/10.1038/s41551-020-0556-3

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