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CN-115947868-B - CXCL-BPI fusion protein and use thereof

CN115947868BCN 115947868 BCN115947868 BCN 115947868BCN-115947868-B

Abstract

The invention discloses CXCL-BPI fusion protein for treating gram-negative bacterial infection, encoding nucleic acid, an expression preparation method and application of the CXCL-BPI fusion protein in preparing a pharmaceutical composition for treating gram-negative bacterial infection. The CXCL-BPI fusion protein comprises human ELR+CXC chemotactic factors and human BPIN end domain functional fragments, has the dual functions of the ELR+CXC chemotactic factors and BPI, has the functions of combining LPS, directly killing gram-negative bacteria and chemotactic cell migration, has the function of promoting phagocyte to guide and combine with the phagocyte of the gram-negative bacteria, and has the action mechanism capable of overcoming the drug resistance of the gram-negative bacteria.

Inventors

  • CHEN JINDONG
  • Yang Miqing
  • XING CHAO
  • LI JINCHAO
  • CAO WEI
  • CAO BIN
  • AN YUNQING

Assignees

  • 安君(北京)基因科技有限责任公司
  • 厦门联合安金生物工程有限公司

Dates

Publication Date
20260512
Application Date
20220928

Claims (14)

  1. 1. A CXCL-BPI fusion protein comprising a human ELR+CXC chemokine and a human BPI N-terminal domain functional fragment, wherein the human ELR+CXC chemokine is selected from the group consisting of human CXCL1, human CXCL2, human CXCL3, human CXCL5, human CXCL6, human CXCL7 and human CXCL8, wherein human CXCL8, human CXCL1, human CXCL2, human CXCL3, human CXCL5, human CXCL6 and human CXCL7 are the sequences shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively, wherein the human BPI N-terminal domain functional fragment is a human BPI 1-233 fragment, wherein human BPI 1-233 is the sequence shown as SEQ ID NO:10, Wherein the human ELR+CXC chemokine is the N-terminal domain of the fusion protein and the human BPI N-terminal domain functional fragment is the C-terminal domain of the fusion protein, Wherein said human elr+cxc chemokine and said human BPI N-terminal domain functional fragment are joined by a linker.
  2. 2. The CXCL-BPI fusion protein of claim 1, wherein the linker is selected from GPPSGSGGGSGGG (SEQ ID NO: 8) and GGGSGGGSGGG (SEQ ID NO: 9).
  3. 3. A nucleic acid encoding the CXCL-BPI fusion protein of any one of claims 1-2.
  4. 4. The nucleic acid of claim 3, comprising, in order from 5 'to 3', a 5 'end adaptor sequence, a signal peptide coding sequence, a human elr+cxc chemokine coding sequence, a linker coding sequence, a human BPI N-terminal domain functional fragment coding sequence, and a 3' end adaptor sequence.
  5. 5. The nucleic acid according to claim 3, wherein the coding sequence of human ELR+CXC chemokines is the sequence shown in SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 or SEQ ID NO. 19, and the coding sequence of the N-terminal domain functional fragment of human BPI is the sequence shown in SEQ ID NO. 22.
  6. 6. Use of the CXCL-BPI fusion protein of any one of claims 1-2 for the preparation of a pharmaceutical composition for the treatment of a gram negative bacterial infection.
  7. 7. An expression vector for expressing the CXCL-BPI fusion protein of any one of claims 1-2.
  8. 8. The expression vector of claim 7, comprising a nucleic acid encoding the CXCL-BPI fusion protein of any of claims 1-2.
  9. 9. The expression vector of claim 8, which is selected from the group consisting of high-efficiency expression vectors pSCm-CXCL1-BPI, pSCm-CXCL2-BPI, pSCm-CXCL3-BPI, pSCm-CXCL5-BPI, pSCm-CXCL6-BPI, pSCm-CXCL7-BPI, and pSCm-CXCL8-BPI.
  10. 10. A pharmaceutical composition comprising the CXCL-BPI fusion protein of any one of claims 1-2 and a pharmaceutically acceptable carrier.
  11. 11. A host cell comprising an expression vector stably transfected or transformed with a nucleic acid comprising a fusion protein encoding the CXCL-BPI of any of claims 1-2.
  12. 12. A method of making the CXCL-BPI fusion protein of any one of claims 1-2, comprising culturing the host cell of claim 11 under conditions suitable for expression of the CXCL-BPI fusion protein, harvesting the expressed CXCL-BPI fusion protein, and optionally further purifying the expressed CXCL-BPI fusion protein.
  13. 13. Use of the CXCL-BPI fusion protein of any of claims 1-2 or the pharmaceutical composition of claim 10 in the manufacture of a medicament for use in a method of treating a gram-negative bacterial infection, the method comprising administering a therapeutically effective amount of the CXCL-BPI fusion protein of any of claims 1-2 or the pharmaceutical composition of claim 10 to a subject suffering from a gram-negative bacterial infection.
  14. 14. The use of claim 13, wherein the method further comprises administering an antibiotic compound to a subject suffering from a gram-negative bacterial infection prior to, concurrently with, or after administering a therapeutically effective amount of the CXCL-BPI fusion protein of any one of claims 1-2 or the pharmaceutical composition of claim 10.

Description

CXCL-BPI fusion protein and use thereof Technical Field The invention relates to the field of biological medicine. In particular, the invention relates to CXCL-BPI fusion proteins and nucleic acids encoding the same, methods of expression and preparation thereof, which are useful for the treatment of gram-negative bacterial infections, and the use thereof for the preparation of pharmaceutical compositions for the treatment of gram-negative bacterial infections. The CXCL-BPI fusion protein comprises a human ELR+CXC chemokine and a human BPI N-terminal domain functional fragment, has the dual functions of the ELR+CXC chemokine and the BPI, has the functions of combining LPS, directly killing gram-negative bacteria and chemotactic cell migration, has the function of promoting phagocyte to guide and combine with the gram-negative bacteria, and has the action mechanism capable of overcoming the drug resistance of the gram-negative bacteria. Background Chemokines (Chemokines) are a family of small molecular weight cytokines that have chemotactic effects on cells, especially leukocytes, and chemokine receptors (Chemokine Receptors, CKRs) are a family of G protein-Coupled Receptor Superfamily (GPCRs) that mediate the function of chemokines. Chemokines mediate the migration of immune cells to sites of infection and inflammation, and activate immune cells to participate in immune responses and inflammatory responses. Phagocytes such as granulocytes, monocytes/macrophages of higher animals have phagocytic sterilization function, and are important components of nonspecific immunity of organisms. Chemokines and phagocytes play an important role in the immunity of the body against bacterial infections, but do not have specific mechanism of action characteristics that promote phagocytic bacteria like specific IgG antibody opsonization (Oposonization). Chemokines are divided into four subfamilies CC, CXC, CX C and C according to their arrangement of two cysteines near the N-terminus, with the CXC subfamilies being divided into ELR+CXC chemokines and non-ELR+CXC chemokines according to whether the first Cys is preceded by an ELR (Glu-Leu-Arg) Motif structure (Motif). IL-8, CXCR1 and CXCR2 (also known as IL-8RA and IL-8 RB) and their ELR+CXC chemokine ligands (Ligands) were discovered in the beginning of the nineties to the twenties of 1986, CXCR1 and CXCR2 are G-protein coupled receptors (GPCRs) expressed primarily on neutrophils, monocytes/macrophages and endothelial cells, and the ligands of CXCR1 and CXCR2 constitute the ELR+CXC chemokine family. Seven elr+cxc chemokines are known to be Gro- α (also known as CXCL 1), gro- β (CXCL 2), gro- γ (CXCL 3), ENA-78 (CXCL 5), GCP-2 (CXCL 6), NAP-2 (CXCL 7) and IL-8 (CXCL 8), the primary functions of which are to major chemotactic neutrophils (which also have chemotactic effects on monocytes/macrophages) and promote angiogenesis, wherein all seven chemokines (CXCL 1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL 8) mediate signals by binding to CXCR2, while CXCL6 and CXCL8 and the like also mediate signals by binding to CXCR1 (which all contribute to redundancy of the activation pathway). IL-8 (CXCL 8) is a representative member of the CXC subfamily and has the main functions of chemotaxis and activation of neutrophils, promoting neutrophil phagocytosis and also has a certain chemotaxis and activation of monocytes/macrophages. See, e.g., ,[Hughes,et al.,FEBS J.(2018)285(16):2944][Bacon,et al.,J.Interferon Cytokine Res.(2002)22(10):1067][ Bi Huijuan et al, journal of immunology (2010) 26 (12): 1091] [ Baggiolini et al, adv immunol. (1993) 55:97]. The bactericidal/permeation enhancing protein (Bactericidal/permaability-INCREASING PROTEIN, BPI) is a cationic antibacterial protein which is first found in human polymorphonuclear neutrophils by Weiss et al in 1978 and is composed of 456 amino acid residues, the molecular weight of which is about 55KD, and the protein structure is composed of an N-terminal domain and a C-terminal domain which are connected through a connector, and researches prove that the N-terminal functional fragments BPI 1-199 and BPI 1-193 (respectively, the 1-199 amino acid residue substrate segment of the N-terminal domain and the 1-193 amino acid residue substrate segment of the C-terminal truncated 6 amino acids) have the functions of binding Lipopolysaccharide (LPS) and lipoid A (Lipid A) which are equivalent to human natural BPI and bind gram-negative bacteria (GRAM NEGATIVE Bacterium, GNB or G-bacteria for short) and increasing the permeability of susceptible GNB to directly kill GNB. XOMA corporation research and development of recombinant human BPI N-terminal functional fragment from nineties of the twentieth centuryRBPI 21) and a plurality of clinical trials are carried out, but because the BPI sterilization needs to be maintained for a longer time to maintain a higher concentration, the rBPI 21 has the factors of short half-life in vivo, large therapeu