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KR-20260066183-A - MATRIX METALLOPROTEASE-CLEAVABLE AND SERINE OR CYSTEINE PROTEASECLEAVABLE SUBSTRATES AND METHODS OF USE THEREOF

KR20260066183AKR 20260066183 AKR20260066183 AKR 20260066183AKR-20260066183-A

Abstract

The present invention relates to a polypeptide comprising, in general, at least a first cleavable moiety (CM1) which is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) which is a substrate for at least one serine protease (SP) or at least one cysteine protease (CP); an activable antibody; other larger molecules comprising such polypeptide comprising at least CM1 which is a substrate for at least one MMP protease and at least CM2 which is a substrate for at least one SP protease or at least one cysteine protease (CP); and methods for preparing and using such polypeptide comprising at least CM1 which is a substrate for at least one MMP protease and at least CM2 which is a substrate for at least one SP protease or at least one cysteine protease (CP) in various therapeutic, diagnostic, and prophylactic indications.

Inventors

  • 바실리예바 올가
  • 윈터 마이클 비.

Assignees

  • 싸이톰스 테라퓨틱스, 인크.

Dates

Publication Date
20260512
Application Date
20191205
Priority Date
20181206

Claims (20)

  1. A cleavable polypeptide comprising a cleavable moiety (CM) comprising amino acid residues 11 to 17 of SEQ ID NO. 39, wherein the cleavable polypeptide further comprises an epitope that binds to a target, and the cleavable polypeptide further comprises a masking moiety (MM) that reduces the binding of the epitope to the target.
  2. In claim 1, the cleavable polypeptide comprising amino acid residues 10 to 17 of SEQ ID NO. 39.
  3. A nucleic acid molecule encoding the cleavable polypeptide of claim 1.
  4. A vector containing a nucleic acid molecule according to paragraph 3.
  5. A method for producing a polypeptide containing a cleavable site (CM), comprising the step of culturing a cell containing a vector according to claim 4 under conditions that induce the expression of the polypeptide.
  6. A method for preparing a polypeptide containing a cleavable moiety (CM), comprising the following steps: (a) a step of culturing cells comprising a nucleic acid construct encoding a cleavable polypeptide according to claim 1 to express said cleavable polypeptide; and (b) A step of recovering the above-mentioned cleavable polypeptide.
  7. A nucleic acid molecule encoding a cleavable polypeptide according to paragraph 2.
  8. A vector containing a nucleic acid molecule according to paragraph 7.
  9. A method for producing a polypeptide containing a cleavable site (CM), comprising the step of culturing a cell containing a vector according to claim 8 under conditions that induce expression of the polypeptide.
  10. A method for preparing a polypeptide containing a cleavable moiety (CM), comprising the following steps: (a) a step of culturing cells comprising a nucleic acid construct encoding a cleavable polypeptide according to paragraph 2 to express said cleavable polypeptide; and (b) A step of recovering the above-mentioned cleavable polypeptide.
  11. A polypeptide comprising a cleavable polypeptide according to claim 1, and an antibody that binds to a target or an antigen-binding fragment (AB) thereof.
  12. A polypeptide comprising a cleavable polypeptide according to paragraph 2, and an antibody that binds to a target or an antigen-binding fragment (AB) thereof.
  13. A polypeptide according to claim 11, wherein the antigen-binding fragment is selected from the group consisting of Fab fragment, F(ab') 2 fragment, scFv, scAb, dAb, single-domain heavy-chain antibody and single-domain light-chain antibody.
  14. In claim 11, a polypeptide in which the above AB is connected to the above CM.
  15. In claim 11, a polypeptide in which the above AB is connected to the above CM through a linking peptide.
  16. A nucleic acid molecule encoding a polypeptide according to paragraph 11.
  17. A vector containing a nucleic acid molecule according to paragraph 16.
  18. A method for producing a polypeptide containing a cleavable moiety (CM), comprising the step of culturing cells containing a vector according to claim 17 under conditions that induce expression of the polypeptide.
  19. A method for preparing a polypeptide containing a cleavable moiety (CM), comprising the following steps: (a) a step of culturing cells comprising a nucleic acid construct encoding a cleavable polypeptide according to claim 11 to express said polypeptide; and (b) A step of recovering the polypeptide.
  20. A cleavable polypeptide according to claim 1, wherein the epitope binding to the target is an antigen-binding domain.

Description

Matrix Metalloprotease-Cleavable Substrates and Serine or Cysteine Protease-Cleavable Substrates and Methods of Use Thereof Cross-reference regarding related applications This application claims priority to U.S. provisional application No. 62/776,409 filed December 6, 2018 and No. 62/778,062 filed December 11, 2018, the contents of which are incorporated herein by reference in their entirety. Technology field The present invention generally involves at least one matrix metalloprotease: At least a first cleavable moiety (CM1) that is a substrate for MMPs and at least one serine protease The invention relates to a polypeptide comprising at least a second cleavable moiety (CM2) that is a substrate for SP and/or at least one cysteine protease (CP), an activable antibody comprising at least CM1 that is a substrate for at least one MMP protease and CM2 that is a substrate for at least one SP protease and/or at least one CP protease, and other larger molecules and a method for preparing and using such polypeptide comprising at least CM1 that is a substrate for at least one MMP protease and CM2 that is a substrate for at least one SP protease and/or at least one CP protease in various therapeutic, diagnostic and prophylactic indications. Reference to the sequence list The “Sequence List,” which was electronically submitted concurrently with this application in a computer-readable format (CFR) via EFS-Web under the filename “CYTX-058-PCT_ST25” in accordance with 37 C.F.R. § 1.821, is incorporated herein by reference. An electronic copy of the Sequence List was created on November 26, 2019, and has a disk size of 159 kilobytes. Proteases are enzymes that break down proteins by cleaving peptide bonds between amino acid residues. Proteases occur naturally in all organisms and are involved in a wide range of physiological responses, from simple degradation to highly regulated pathways. Some proteases are known to break specific peptide bonds based on the presence of specific amino acid sequences within a protein. Therefore, it is necessary to identify new substrates for protease and utilize these substrates for various therapeutic, diagnostic, and prophylactic indications. In an embodiment of the present invention, an isolated polypeptide comprising a tandem substrate is provided herein, wherein the tandem substrate comprises at least a first cleavable moiety (CM1) which is a substrate for at least one matrix metalloproteinase (MMP) and at least a second cleavable moiety (CM2) which is a substrate for at least one serine protase (SP) or cysteine protase (CP), and CM1 comprises the amino acid sequence AHGL or PRQV, and the N-terminal to C-terminal arrangement of the tandem substrate is CM1-CM2 or CM2-CM1. In some embodiments, CM1 of the isolated polypeptide comprises an amino acid sequence selected from the group consisting of ALAHGLF (SEQ No. 1), ALAHGL (SEQ No. 52), LAHGLF (SEQ No. 50), LAHGL (SEQ No. 53), and AHGLF (SEQ No. 51). In some embodiments, CM1 of the isolated polypeptide comprises an amino acid sequence selected from the group consisting of HVPRQV (SEQ No. 8) and VPRQV (SEQ No. 60). In some embodiments, the isolated polypeptide of the present disclosure comprises CM1 and CM2 connected by a linking peptide. In some embodiments, CM1 and CM2 of the isolated polypeptide are directly connected to each other. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 comprising a substrate for a CP enzyme, wherein the CP enzyme is legumain. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 comprising a substrate for an SP enzyme selected from the group consisting of urokinase, matriptase, and neutrophil elastase. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 comprising a substrate for an SP enzyme selected from the group consisting of urokinase, matrixtase, and neutrophil elastase, and a substrate for a CP enzyme, wherein the CP enzyme is legumine. In some embodiments, the isolated polypeptide of the present disclosure comprises CM1 comprising a substrate for an MMP enzyme selected from the group consisting of MMP2, MMP9, or MMP14. In some embodiments, the isolated polypeptide of the present disclosure comprises CM2 having an amino acid sequence selected from the group consisting of SGR, LSGR (SEQ No. 73), ARG, PRS, TFVH (SEQ No. 141), AAN, SAN, and GPTN (SEQ No. 152). In some embodiments, the isolated polypeptide of the present disclosure comprises an amino acid sequence selected from the group consisting of SGR, LSGR (SEQ No. 73), LSGRS (SEQ No. 72), LSGRSD (SEQ No. 71), LSGRSA (SEQ No. 110), LSGRSDN (SEQ No. 70), LSGRSAN (SEQ No. 109), LSGRSDNH (SEQ No. 20), LSGRSGNH (SEQ No. 78), LSGRSDNP (SEQ No. 90), LSGRSDNI (SEQ No. 84), LSGRSANI (SEQ No. 108), LSGRSANP (SEQ No. 114), LSGRSDYH (SEQ No. 86), LSGRSDTH (SEQ No. 92), LSGRSDQH (SEQ No. 96), LSGRSDIH (SEQ No. 100), and LSGRSDDH (SEQ No. 104). Includes CM2. In s