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EP-4735043-A1 - PRO-POLYPEPTIDES AND METHODS OF USING AND DESIGNING THE SAME

EP4735043A1EP 4735043 A1EP4735043 A1EP 4735043A1EP-4735043-A1

Abstract

Provided are pro-polypeptides for delivery of a polypeptide of interest (POI) to a biological target. In some embodiments, the pro-polypeptides comprise the POI and a masking structure linked to the POI through one or more protease-cleavable sites. Such pro-polypeptides comprise a stably-folded tertiary structure that masks the POI from the biological target, and where cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target. Aspects of the present disclosure further include computer-implemented methods of designing such pro-polypeptides. Also provided are nucleic acids, cells and methods that find use in producing the pro-polypeptides of the present disclosure, as well as methods of using the pro-polypeptides, e.g., to deliver a POI to a biological target in a subject in need thereof.

Inventors

  • DEGRADO, WILLIAM
  • SCHNAIDER, Lee
  • LARWOOD, David
  • BHATTACHARYA, Sagar
  • JO, Hyunil
  • CHEN, Yuda
  • TACKIE-YARBOI, Ethel

Assignees

  • The Regents of the University of California

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1 . A pro-polypeptide for delivery of a polypeptide of interest (POI) to a biological target, wherein the pro-polypeptide comprises: the POI; and a masking structure linked to the POI through one or more protease-cleavable sites, wherein the pro-polypeptide comprises a stably-folded tertiary structure that masks the POI from the biological target, and wherein cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target.
  2. 2. The pro-polypeptide of claim 1 , wherein the masking structure is linked to the POI through one or more flexible protease-cleavable sites.
  3. 3. The pro-polypeptide of claim 1 or claim 2, wherein the pro-polypeptide comprises two or more POIs.
  4. 4. The pro-polypeptide of any one of claims 1 to 3, wherein the stably-folded tertiary structure is selected from the group consisting of: a three-helix bundle, a single-chain helixloop-helix, a four-helix bundle, and an Alpha-Beta protein bundle.
  5. 5. The pro-polypeptide of claim 4, wherein the pro-polypeptide comprises two or more of the stably-folded tertiary structures flexibly linked together.
  6. 6. The pro-polypeptide of any one of claims 1 to 5, wherein the pro-polypeptide is a multimeric assembly.
  7. 7. The pro-polypeptide of claim 6, wherein the pro-polypeptide comprises a quaternary structure comprising a multimeric assembly.
  8. 8. The pro-polypeptide of claim 7, wherein the quaternary structure comprises a dimeric assembly.
  9. 9. The pro-polypeptide of claim 8, wherein the dimeric assembly comprises two helix-loop- helix motifs.
  10. 10. The pro-polypeptide of any one of claims 1 to 9, wherein the biological target is a cell membrane.
  11. 11 . The pro-polypeptide of any one of claims 1 to 10, wherein the biological target is a protein.
  12. 12. The pro-polypeptide of any one of claims 1 to 11 , wherein the POI is selected from a cell-penetrating peptide (CPP), an antimicrobial peptide, a cytotoxic peptide, an anticancer peptide, an endosomal escape peptide, a cellular localization peptide, a peptide hormone, a membrane-associating peptide, a lytic peptide, and a pore-forming peptide.
  13. 13. The pro-polypeptide of any one of claims 1 to 12, wherein a ligand for radioimaging or therapy is appended onto the POI.
  14. 14. The pro-polypeptide of any one of claims 1 to 12, wherein the POI comprises or is substituted with a ligand for radioimaging or therapy.
  15. 15. The pro-polypeptide of any one of claims 1 to 14, wherein the protease-cleavable site is cleavable by a protease expressed on the surface of a cell.
  16. 16. The pro-polypeptide of any one of claims 1 to 15, wherein the protease-cleavable site is cleavable by a protease secreted by a cell.
  17. 17. The pro-polypeptide of any one of claims 1 to 14, wherein the protease-cleavable site is cleavable by an intracellular protease.
  18. 18. The pro-polypeptide of any one of claims 15 to 17, wherein the cell is a cancer cell.
  19. 19. The pro-polypeptide of any one of claims 15 to 18, wherein the cell is an immune cell.
  20. 20. The pro-polypeptide of any one of claims 1 to 19, wherein the protease-cleavable site is cleavable by a protease present in a tumor microenvironment.

Description

PRO-POLYPEPTIDES ND METHODS OF USING AND DESIGNING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63/524,593, filed June 30, 2023, which application is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under R35 GM122603 awarded by the National Institutes of Health. The government has certain rights in the invention. SUMMARY Provided are pro-polypeptides for delivery of a polypeptide of interest (POI) to a biological target. In some embodiments, the pro-polypeptides comprise the POI and a masking structure linked to the POI through one or more protease-cleavable sites. Such pro-polypeptides comprise a stably-folded tertiary structure that masks the POI from the biological target, and where cleavage of the one or more protease-cleavable sites decreases the conformational stability of the tertiary structure leading to release of the POI and availability of the POI to interact with the biological target. Aspects of the present disclosure further include computer-implemented and/or rational design methods of designing such pro-polypeptides. Also provided are nucleic acids, cells and methods that find use in producing the pro-polypeptides of the present disclosure, as well as methods of using the pro-polypeptides, e.g., to deliver a POI to a biological target in a subject in need thereof. BRIEF DESCRIPTION OF THE FIGURES FIG. 1A-1 D: Schematic illustrations of an approach for pro-polypeptide design according to some embodiments of the present disclosure. FIG. 2A-2D: Schematic illustrations of an approach for pro-polypeptide design according to some embodiments of the present disclosure. FIG. 3A-3D: Schematic illustrations of a single scaffold to mask a POI from several POI families. FIG. 4A-4H: Schematic illustrations of various quaternary and tertiary scaffolds which may be designed/implemented to mask the same POI. FIG. 5A-5E: Schematic illustrations and confocal microscopy images relating to a proof- of-concept pro-polypeptide designed according to the methods of the present disclosure. FIG. 6A-6C: Schematic illustration of structural blocks of a useful design according to some embodiments. FIG. 7A-7C: Schematic illustration of a variation of the design shown in FIG. 6 in which the pro-polypeptide does not include a cargo/payload. FIG. 8: Schematic illustration of a uPA-cleavable pro-polypeptide in which the POI is an antimicrobial peptide, and minimum inhibitory concentration (MIC) data demonstrating that the masking/unmasking mechanism works as intended. FIG. 9: Schematic illustration of a neutrophil elastase-cleavable pro-polypeptlde In which the POI is an antimicrobial peptide, and minimum inhibitory concentration (MIC) data demonstrating that the masking/unmasking mechanism works as intended. FIG. 10: Schematic illustration of a pro-polypeptide and masking/unmasking mechanism according to embodiments of the present disclosure. DETAILED DESCRIPTION Before the pro-polypeptides and methods of the present disclosure are described in greater detail, it is to be understood that the pro-polypeptides and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the pro-polypeptides and methods will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the pro-polypeptides and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the pro-polypeptides and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the pro-polypeptides and methods. Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary s