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US-12617856-B2 - Protease-cleavable substrates and methods of use thereof

US12617856B2US 12617856 B2US12617856 B2US 12617856B2US-12617856-B2

Abstract

Isolated polypeptides that include a cleavable moiety that is a substrate for at least one protease (e.g., MT-SP1 and/or an MMP) and isolated polypeptides that include a substrate that has a first cleavable moiety cleavable by a first protease and a second cleavable moiety cleavable by a second protease are disclosed. Activatable molecules including the isolated polypeptides are disclosed. Methods of making and using the isolated polypeptides and activatable molecules including the isolated polypeptides in a variety of therapeutic, diagnostic, and prophylactic applications are disclosed.

Inventors

  • Olga Vasiljeva
  • Michael B. Winter

Assignees

  • CYTOMX THERAPEUTICS, INC.

Dates

Publication Date
20260505
Application Date
20250317

Claims (20)

  1. 1 . An isolated polypeptide comprising a substrate, wherein the substrate comprises a sequence selected from the group consisting of SEQ ID NOs: 13, 163, 164, 177, 178, 191-193, 690, and 691, wherein the isolated polypeptide comprises a first linking peptide (LP1)N-terminal to the sequence, and a second linking peptide (LP2)C-terminal to the sequence.
  2. 2 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 12.
  3. 3 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 11.
  4. 4 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 223.
  5. 5 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 120.
  6. 6 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 121.
  7. 7 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 132.
  8. 8 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 138.
  9. 9 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 144.
  10. 10 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 150.
  11. 11 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 693.
  12. 12 . The isolated polypeptide of claim 1 , wherein the substrate comprises SEQ ID NO: 695.
  13. 13 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 13, 163, 164, 177, 178, 191-193, 690, and 691, wherein the isolated polypeptide is an activatable molecule and further comprises an active moiety (AM) that specifically binds a target.
  14. 14 . The isolated polypeptide of claim 13 , wherein the AM is coupled to the substrate.
  15. 15 . The isolated polypeptide of claim 14 , wherein the AM is coupled directly to the substrate.
  16. 16 . The isolated polypeptide of claim 14 , wherein the AM is coupled indirectly to the substrate via a linking peptide.
  17. 17 . The isolated polypeptide of claim 13 , wherein the AM is a therapeutic macromolecule.
  18. 18 . The isolated polypeptide of claim 13 , wherein the AM is a cytokine or a chimeric antigen receptor.
  19. 19 . The isolated polypeptide of claim 13 , wherein the substrate comprises SEQ ID NO: 12.
  20. 20 . The isolated polypeptide of claim 13 , wherein the substrate comprises SEQ ID NO: 11.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 19/100,493, filed Jan. 31, 2025, which is a 35 U.S.C. 371 National Phase Entry Application of PCT/US2023/071310, filed Jul. 31, 2023, which claims the priority benefit of U.S. Provisional Application No. 63/370,026 filed Aug. 1, 2022, the contents of which are incorporated herein in their entireties by reference thereto. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jul. 27, 2023 is named CYTX-095-PCT_SL.xml and is 562,378 bytes in size. TECHNICAL FIELD The present disclosure generally relates to polypeptides that include a substrate comprising one or more cleavable moieties, each of which is cleavable by a protease, and to methods of making and using the polypeptides and activatable molecules in a variety of therapeutic, diagnostic, and prophylactic applications. BACKGROUND Proteases are enzymes that catalyze the hydrolysis of peptide bonds between amino acid residues. Some proteases are known to break specific peptide bonds based on the presence of a particular amino acid sequence within a protein. Proteases occur naturally in all organisms and are involved in a variety of physiological reactions from simple degradation to highly regulated pathways. Some proteases break specific peptide bonds based on the presence of a particular amino acid sequence within a protein while some amino acid sequences are resistant to cleavage by particular proteases. Accordingly, there exists a need to identify new substrates for proteases and to use these substrates in a variety of therapeutic, diagnostic and prophylactic applications. SUMMARY OF THE INVENTION In one aspect, the present disclosure provides an isolated polypeptide comprising a substrate, wherein the substrate comprises a first cleavable moiety (CM1) cleavable by at least a first protease and a second cleavable moiety (CM2) cleavable by at least a second protease, and wherein the CM1 comprises the amino acid sequence of PXGL, wherein X is W, Y, F, R, K, Q, A, or M. In one aspect, the present disclosure provides an isolated polypeptide comprising a substrate, wherein the substrate comprises a first cleavable moiety (CM1) cleavable by at least a first protease and a second cleavable moiety (CM2) cleavable by at least a second protease, and wherein the CM1 comprises the amino acid sequence of PXGL, wherein X is W, Y, F, R, K, Q, or M. In some embodiments, the CM2 comprises the amino acid sequence of RS. In some embodiments, the CM1 comprises the amino acid sequence of PWGL (SEQ ID NO: 2). In some embodiments, the substrate comprises the amino acid sequence of RSPWGLN (SEQ ID NO: 10), RSPWGL (SEQ ID NO: 90), PWGLRS (SEQ ID NO: 48), PWGLSGKS (SEQ ID NO: 150), or PWGLRSN (SEQ ID NO: 9). In some embodiments, the CM2 comprises the amino acid sequence of GRS. In some embodiments, the CM2 comprises the amino acid sequence of SGRS (SEQ ID NO: 31), SGRSNI (SEQ ID NO: 32), or LSGRSNI (SEQ ID NO: 26). In some embodiments, the CM1 comprises the amino acid sequence of PWGL (SEQ ID NO: 2). In some embodiments, the substrate comprises the amino acid sequence of GPWGLSGRSNI (SEQ ID NO: 11), PWGLSGRS (SEQ ID NO: 12), GRSPWGLL (SEQ ID NO: 13), APMGLKHLSGRSNI (SEQ ID NO: 14), or GPYGLSGRSNI (SEQ ID NO: 15). In some embodiments, the CM2 comprises the amino acid sequence of PRS. In some embodiments, the CM2 comprises the amino acid sequence of APRS (SEQ ID NO: 34). In some embodiments, the CM1 comprises the amino acid sequence of PWGL (SEQ ID NO: 2). In some embodiments, the substrate comprises an amino acid sequence of APRSPWGL (SEQ ID NO: 16), PWGLPRS (SEQ ID NO: 17), PRSPWGL (SEQ ID NO: 313) or PRSPWGLL (SEQ ID NO: 18). In some embodiments, the CM2 comprises the amino acid sequence of SRS. In some embodiments, the CM2 comprises the amino acid sequence of HQSRS (SEQ ID NO: 28). In some embodiments, the substrate comprises the amino acid sequence of PWGLSRS (SEQ ID NO: 19), PFGLSRS (SEQ ID NO: 20), APMGLKHDHQSRS (SEQ ID NO: 21) or DHQSRSAPMGLKH (SEQ ID NO: 22). In some embodiments, the substrate comprises the amino acid sequence of KPRGL (SEQ ID NO: 664). In some embodiments, the substrate comprises the amino acid sequence of KPRGLN (SEQ ID NO: 23) or KPRGLF (SEQ ID NO: 24). In some embodiments, the substrate comprises the amino acid sequence of PYGLSGRS (SEQ ID NO: 151). In some embodiments, the substrate comprises the amino acid sequence of PFGLSGRS (SEQ ID NO: 152). In some embodiments, the substrate comprises the amino acid sequence of PRGLSGRS (SEQ ID NO: 153). In some embodiments, the substrate comprises the amino acid sequence of PAGLSGRS (SEQ ID NO: 687). In some embodiments, the substrate comprises the amino acid sequence of LSGRSPWGL (SEQ ID NO: 177). In some embodiments, the substrate comprises the amino acid se