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EP-4741498-A1 - MUTANT OF IMMUNOGLOBULIN-DEGRADING ENZYME IDEE

EP4741498A1EP 4741498 A1EP4741498 A1EP 4741498A1EP-4741498-A1

Abstract

Provided are a coding sequence of an immunoglobulin-degrading enzyme and a polypeptide encoded thereby. The function of the polypeptide at least includes the function of the immunoglobulin-degrading enzyme IdeE.

Inventors

  • WANG, ZHENG
  • XU, YUNXIA
  • ZHENG, Leilei

Assignees

  • Shanghai Bao Pharmaceuticals Co., Ltd.

Dates

Publication Date
20260513
Application Date
20241108

Claims (18)

  1. An immunoglobulin-degrading enzyme, wherein the immunoglobulin-degrading enzyme does not cause bacterial, fungal, or cellular autolysis during induced expression.
  2. The immunoglobulin-degrading enzyme according to claim 1, wherein the absence of bacterial, fungal, or cellular autolysis means that during expression in a bacterial, fungal, or cellular host, a OD 600 value of the bacterial, fungal, or cellular host does not decrease after exceeding 40, 50, 60, 70, or 80; preferably, during expression in a bacterial, fungal, or cellular host, a OD 600 value of the bacterial, fungal, or cellular host does not decrease after exceeding 70; more preferably, during expression in a bacterial, fungal, or cellular host, a OD 600 value of the bacterial, fungal, or cellular host does not decrease after exceeding 80.
  3. The immunoglobulin-degrading enzyme according to claim 1 or 2, wherein the immunoglobulin-degrading enzyme comprises or consists of: (a) the amino acid sequence set forth in SEQ ID NO: 2; or (b) an amino acid sequence having one or more amino acid substitutions, additions, and/or deletions compared with the amino acid sequence set forth in SEQ ID NO: 2.
  4. The immunoglobulin-degrading enzyme according to any one of claims 1-3, wherein the immunoglobulin-degrading enzyme is derived from Streptococcus equi ssp. equi.
  5. An immunoglobulin-degrading enzyme, comprising or consisting of: (a) the amino acid sequence set forth in SEQ ID NO: 1 or 2; or (b) an amino acid sequence having one or more amino acid substitutions, additions, or deletions compared with the amino acid sequence set forth in SEQ ID NO: 1 or 2.
  6. The immunoglobulin-degrading enzyme according to claim 5, wherein the immunoglobulin-degrading enzyme is derived from Streptococcus equi ssp. equi.
  7. The immunoglobulin-degrading enzyme according to claim 5, wherein the immunoglobulin-degrading enzyme does not cause bacterial, fungal, or cellular autolysis during expression.
  8. The immunoglobulin-degrading enzyme according to claim 7, wherein the absence of bacterial, fungal, or cellular autolysis means that during expression in a bacterial, fungal, or cellular host, a OD 600 value of the bacterial, fungal, or cellular host does not decrease after exceeding 40, 50, 60, 70, or 80; preferably, during expression in a bacterial, fungal, or cellular host, a OD 600 value of the bacterial, fungal, or cellular host does not decrease after exceeding 70; more preferably, during expression in a bacterial, fungal, or cellular host, a OD 600 value of the bacterial, fungal, or cellular host does not decrease after exceeding 80.
  9. The immunoglobulin-degrading enzyme according to claim 1 or 5, wherein an immunoglobulin is intracellularly expressed or extracellularly expressed.
  10. A nucleotide sequence encoding the immunoglobulin-degrading enzyme according to any one of claims 1-9, preferably, comprising or consisting of: (a) the nucleotide sequence set forth in SEQ ID NO: 3; (b) a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence homology to the nucleotide sequence set forth in SEQ ID NO: 3; or (c) a nucleotide sequence having one or more nucleotide substitutions, additions, and/or deletions compared with the nucleotide sequence set forth in SEQ ID NO: 3.
  11. An expression vector, comprising the nucleotide sequence according to claim 10.
  12. A host cell, comprising the nucleotide sequence according to claim 10 or the expression vector according to claim 11, wherein the host cell is preferably a bacterial cell or a fungal cell; the bacterial cell is preferably an Escherichia coli cell, or the fungal cell is a yeast cell.
  13. A method for intracellular expression of the immunoglobulin-degrading enzyme according to any one of claims 1-9, comprising the following steps: (a) selecting a single colony of the host cell according to claim 12; (b) culturing a seed culture; (c) culturing the seed culture in a fermenter; (d) inducing expression of the immunoglobulin-degrading enzyme; and optionally (e) collecting the immunoglobulin-degrading enzyme.
  14. A composition, comprising: the immunoglobulin-degrading enzyme according to any one of claims 1-9; and optionally a pharmaceutically acceptable carrier or excipient.
  15. The composition according to claim 14, further comprising: an antibody or a protein comprising an Fc fragment, wherein a target of the antibody is preferably selected from the following group: a cell surface protein, a cytokine, a hormone, an enzyme, an intracellular messenger, an intercellular messenger, and an immune checkpoint inhibitor.
  16. The composition according to claim 14 or 15, further comprising: a viral vector drug and/or a drug capable of reducing a blood IgG level, wherein the viral vector drug is preferably selected from the following group: an oncolytic virus, a gene therapy virus, and a viral vector vaccine; the drug capable of reducing the blood IgG level is preferably selected from the following group: an FcRn antibody and an Fc fragment variant with high affinity for FcRn.
  17. A kit, comprising: (1) the immunoglobulin-degrading enzyme according to any one of claims 1-9; and (2) one or more selected from the following group: (a) a pharmaceutically acceptable carrier or excipient; (b) an antibody or a protein comprising an Fc fragment; and/or (3) a viral vector drug selected from an oncolytic virus, a gene therapy virus, and a viral vector vaccine; and/or (4) a drug capable of reducing a blood IgG level selected from a neonatal Fc receptor (FcRn) antibody and an Fc fragment variant with high affinity for FcRn.
  18. A kit, comprising a kit A and a kit B, wherein, the kit A comprises the immunoglobulin-degrading enzyme according to any one of claims 1-9, and the kit B comprises one or more selected from the following group: (1) a pharmaceutically acceptable carrier or excipient; (2) an antibody or a protein comprising Fc; and/or (3) a viral vector drug; and/or (4) a drug capable of reducing a blood IgG level, wherein the viral vector drug is selected from an oncolytic virus, a gene therapy virus, and a viral vector vaccine; the drug capable of reducing the blood IgG level is selected from an FcRn antibody and an Fc fragment variant with high affinity for FcRn.

Description

PRIORITY CLAIM This application claims priority to Chinese Patent Application No. 202311508868.8 filed on November 13, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD The present disclosure relates to the field of biotechnology, and in particular to mutants of an immunoglobulin-degrading enzyme and expression thereof. BACKGROUND TECHNOLOGY Immunoglobulin G (IgG) is the main antibody component in serum, and accounts for about 75% of serum immunoglobulins. It mainly serves a protective function in immune defense by effectively preventing infectious diseases. Beyond its protective role, IgG exhibits disease associations. In some autoimmune diseases, IgG antibodies react with self-antigens, and IgG can induce acute transplant rejection in organ transplantation. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is an extracellular cysteine protease produced by the human pathogen S. pyogenes. IdeS catalyzes a single proteolytic cleavage in the lower hinge region of the heavy chain across all human IgG subclasses. IdeS efficiently cleaves IgG into Fc and F(ab')2 fragments through a two-stage enzymatic mechanism. In the first stage, one (first) IgG heavy chain is cleaved to produce a single-cleaved IgG (scIgG) molecule with a non-covalently bound Fc molecule. The scIgG molecule is an intermediate product that retains the remaining (second) heavy chain of the original IgG molecule. In the second stage, the second heavy chain is cleaved by IdeS to release a F(ab')2 fragment and a homodimeric Fc fragment, which facilitate Group A Streptococcus (GAS) evasion of antibody-mediated phagocytosis and cellular responses, thereby attenuating host immune clearance of GAS pathogens. IdeE is derived from Streptococcus equi ssp. equi, an equine pathogen (Jonas Lannergard, Bengt Guss, FEMS Microbiol Lett., 2006, 262: 230-235). The two enzymes, IdeE and IdeS, cleave IgG at exactly the same position. The cleavages are highly reproducible and specific, and have a very similar substrate range. Given the applications of the immunoglobulin-cleaving enzymes described above, there is substantial demand for immunoglobulin-degrading enzymes and the large-scale production thereof. CONTENT OF THE INVENTION In some aspects of the present disclosure, provided is an immunoglobulin-degrading enzyme that reduces or eliminates bacterial, fungal, or cellular autolysis during induced expression. In some embodiments, the immunoglobulin-degrading enzyme of the present disclosure does not cause bacterial, fungal, or cellular autolysis. In some embodiments, during expression of the immunoglobulin-degrading enzyme of the present disclosure, OD600 value of the bacteria, fungi, or cells does not decrease after exceeding 40, 50, 60, 70, or 80; preferably, during expression of the immunoglobulin, OD600 value of the bacteria, fungi, or cells does not decrease after exceeding 70; more preferably, during expression of the immunoglobulin, OD600 value of the bacteria, fungi, or cells does not decrease after exceeding 80. In some embodiments, during expression of the immunoglobulin-degrading enzyme of the present disclosure (e.g., 4 h, 8 h, 12 h, 16 h, 24 h, or longer following induced expression), OD600 value of the bacteria does not decrease after exceeding 40, 50, 60, 70, or 80. In some aspects of the present disclosure, provided is an immunoglobulin-degrading enzyme, which includes or consists of: (a) an amino acid sequence set forth in SEQ ID NO: 2; or(b) an amino acid sequence having one or more amino acid substitutions, additions, and/or deletions compared with SEQ ID NO: 2. In some embodiments, the immunoglobulin-degrading enzyme of the present disclosure includes or consists of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the immunoglobulin-degrading enzyme includes or consists of the amino acid sequence set forth in SEQ ID NO: 2, wherein the encoding sequence reduces or eliminates bacterial, fungal, or cellular autolysis during expression. In some embodiments, the immunoglobulin-degrading enzyme does not cause bacterial, fungal, or cellular autolysis during induced expression. In some embodiments, the immunoglobulin-degrading enzyme of the present disclosure is derived from Streptococcus equi ssp. equi. In some embodiments, the immunoglobulin-degrading enzyme of the present disclosure is intracellularly expressed or extracellularly expressed. In some aspects of the present disclosure, provided is a nucleotide sequence encoding the immunoglobulin-degrading enzyme of the present disclosure. In some embodiments, the nucleotide sequence encoding the immunoglobulin-degrading enzyme of the present disclosure includes or consists of: (a) a nucleotide sequence set forth in SEQ ID NO: 3; or(b) a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence homology to the nucleotide sequence