Search

EP-4740938-A1 - PREPARATION METHOD FOR KLK1 FUSION PROTEIN

EP4740938A1EP 4740938 A1EP4740938 A1EP 4740938A1EP-4740938-A1

Abstract

A method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1), a fusion protein comprising KLK1, a pharmaceutical composition comprising the fusion protein, and a use of the pharmaceutical composition in treating or preventing diseases.

Inventors

  • SHU, CHUTIAN

Assignees

  • ALEBUND PHARMACEUTICALS (HONG KONG) LIMITED

Dates

Publication Date
20260513
Application Date
20240701

Claims (20)

  1. A method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1), comprising Step (1): introducing a recombinant vector comprising a nucleic acid molecule encoding a fusion protein into a suitable protein expression system, wherein the fusion protein comprises a masking peptide, a first polypeptide and a second polypeptide, the masking peptide, the first polypeptide and the second polypeptide are linked directly or covalently via a linker, and a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease; b) the first polypeptide comprises KLK1 protein or a variant thereof; and c) the second polypeptide is used to extend the half-life of the first polypeptide.
  2. The method according to claim 1, further comprising Step (2): mixing the fusion protein obtained in Step (1) with a protease to perform a specific enzymatic cleavage reaction, thereby removing the masking peptide.
  3. The method according to claim 2, wherein the fusion protein obtained in Step (1) is purified before performing the specific enzymatic cleavage reaction in Step (2).
  4. The method according to any one of the preceding claims, wherein the masking peptide is selected from the group consisting of: an enterokinase (EK) recognition sequence, a thrombin recognition sequence, a blood coagulation factor Xa recognition sequence, a tobacco etch virus (TEV) protease recognition sequence, an SUMO protease recognition sequence, and a sortase recognition sequence.
  5. The method according to claim 4, wherein the masking peptide is an EK recognition sequence.
  6. The method according to claim 5, wherein the EK recognition sequence consists of an amino acid sequence as shown in SEQ ID NO: 4 (DDDDK) or SEQ ID NO: 5 (VDDDDK).
  7. The method according to any one of the preceding claims, wherein the first polypeptide is human KLK1 protein or a variant thereof.
  8. The method according to claim 7, wherein the first polypeptide comprises an amino acid sequence having at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence as shown in SEQ ID NO: 2, and yet retains the function or activity of KLK1.
  9. The method according to claim 8, wherein an amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, or SEQ ID NO: 122.
  10. The method according to any one of the preceding claims, wherein the second polypeptide is an Fc domain or albumin.
  11. The method according to claim 10, wherein the Fc domain is derived from human IgG Fc domain.
  12. The method according to claim 11, wherein the Fc domain is derived from human IgG1 Fc domain.
  13. The method according to claim 11, wherein the Fc domain is derived from human IgG4 Fc domain.
  14. The method according to claim 12, wherein the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as shown in SEQ ID NO: 11.
  15. The method according to claim 14, wherein the Fc domain comprises the amino acid sequence as shown in SEQ ID NO: 11; or the Fc domain consists of the amino acid sequence as shown in SEQ ID NO: 11.
  16. The method according to claim 13, wherein the Fc domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the amino acid sequence as shown in SEQ ID NO: 12.
  17. The method according to claim 16, wherein the Fc domain comprises the amino acid sequence as shown in SEQ ID NO: 12; or the Fc domain consists of the amino acid sequence as shown in SEQ ID NO: 12.
  18. The method according to any one of claims 10 to 17, wherein the Fc domain comprises mutation(s) at one, two, three, four, five or more amino acid positions.
  19. The method according to claim 18, wherein the mutations are M252Y, S254T, and T256E.
  20. The method according to claim 19, wherein the Fc domain comprises an amino acid sequence as shown in SEQ ID NO: 13, or the Fc domain consists of the amino acid sequence as shown in SEQ ID NO: 13.

Description

FIELD OF THE INVENTION The present application relates to the technical field of biomedicines, and specifically, to a method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1) (for example, KLK1-Fc fusion protein), a fusion protein comprising KLK1, a pharmaceutical composition comprising the fusion protein, and use thereof in treating or preventing diseases. RELATED ART The kallikrein-kinin system (also known as "KKS") is a complex endogenous multi-enzyme system consisting of kininogen, kallikrein (also referred to as "KLK" or "kininogenase"), and kinin, which is involved in the blood pressure regulation, the glucose metabolism, and the physiological functions of the kidney and the nervous system. KLK can catalyze the cleavage of kininogen, to produce biologically active kinin. KLK can be divided into plasma KLK and tissue KLK. There are great differences between the substrates, molecular weights and functions of the plasma KLK and the tissue KLK. For example, the plasma KLK is encoded by KLKB1 gene located on chromosome 4q34-35, and no other known paralogues are found; and the tissue KLK is encoded by KLK1-KLK15 genes located on chromosome 19q13, and 15 known paralogues (KLK1-KLK15) are found. KLK1 is the only member of the tissue KLK family that can produce bioactive kinin. KLK1 can relax isolated arteries, have vasodilation, anti-inflammation, and cell repair effects and reduce the apoptosis. At present, the commercially available KLK1 is mainly extracted from human urine or porcine pancreas, and is an endogenous protein. Because of the complex production process, short half-life (for example, Kailikang® has a half-life of less than 4 h in human), high administration frequency (for example, Kailikang® is intravenously injected once a day for consecutive 21 days according to the clinical instructions), high cost and difficult quality control, the use is restricted to some extent. Therefore, there is obviously an urgent unmet need in clinic to develop a KLK1 protein product with prolonged half-life, lower frequency of administration, and good production cost and quality controllability. SUMMARY OF THE INVENTION In an aspect, the present application provides a method for preparing a fusion protein comprising tissue kallikrein 1 (KLK1). The method comprises Step (1): introducing a recombinant vector comprising a nucleic acid molecule encoding a fusion protein into a suitable protein expression system. The fusion protein comprises a masking peptide, a first polypeptide and a second polypeptide. The masking peptide, the first polypeptide and the second polypeptide are linked directly or covalently via a linker, and, a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease;b) the first polypeptide comprises KLK1 or a variant thereof; andc) the second polypeptide is used to extend the half-life of the first polypeptide. In certain embodiments, the method further comprises Step (2): mixing the fusion protein obtained in Step (1) with a protease to perform a specific enzymatic cleavage reaction, thereby removing the masking peptide. In certain embodiments, the fusion protein obtained in Step (1) is purified before performing the specific enzymatic cleavage reaction in Step (2). In certain embodiments, the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5. In certain embodiments, an amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122. In certain embodiments, the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. In another aspect, the present application provides a fusion protein, comprising a masking peptide, a first polypeptide and a second polypeptide. The masking peptide, the first polypeptide and the second polypeptide are linked directly or covalently via a linker, and a) the masking peptide has an amino acid sequence that can be specifically recognized and cleaved by a protease;b) the first polypeptide comprises KLK1 or a variant thereof; andc) the second polypeptide is used to extend the half-life of the first polypeptide. In certain embodiments, the amino acid sequence of the masking peptide is as shown in SEQ ID NO: 5. In certain embodiments, the amino acid sequence of the first polypeptide is as shown in SEQ ID NO: 2, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121 or SEQ ID NO: 122. In certain embodiments, the amino acid sequence of the second polypeptide is as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. In certain embodiments, the amino acid sequence of the KLK1 fusion protein is as shown in any one of SEQ ID NOs: 36-55, SEQ ID NOs: 100-109, SEQ ID NOs: 111-113, and SEQ ID NOs: 125-127. In another aspect,